Phytocompound screening, antioxidant activity and molecular docking studies of pomegranate seed: a preventive approach for SARS-CoV-2 pathogenesis

A global hazard to public health has been generated by the coronavirus infection 2019 (COVID-19), which is spreading quickly. Pomegranate is a strong source of antioxidants and has demonstrated a number of pharmacological characteristics. This work was aimed to analyze the phytochemicals present in ethanolic pomegranate seed extract (PSE) and their in vitro antioxidant potential and further in-silico evaluation for antiviral potential against crystal structure of two nucleocapsid proteins i.e., N-terminal RNA binding domain (NRBD) and C-terminal Domain (CTD) of SARS-CoV-2. The bioactive components from ethanolic extract of PSE were assessed by gas chromatography-mass spectroscopy (GC–MS). Free radical scavenging activity of PSE was determined using DPPH dye. Molecular docking was executed through the Glide module of Maestro software. Lipinski’s 5 rule was applied for drug-likeness characteristics using cheminformatics Molinspiration software while OSIRIS Data Warrior V5.5.0 was used to predict possible toxicological characteristics of components. Thirty-two phytocomponents was detected in PSE by GC–MS technique. Free radical scavenging assay revealed the high antioxidant capacity of PSE. Docking analysis showed that twenty phytocomponents from PSE exhibited good binding affinity (Docking score ≥ − 1.0 kcal/mol) towards NRBD and CTD nucleocapsid protein. This result increases the possibility that the top 20 hits could prevent the spread of SARS-CoV-2 by concentrating on both nucleocapsid proteins. Moreover, molecular dynamics (MD) simulation using GROMACS was used to check their binding efficacy and internal dynamics of top complexes with the lowest docking scores. The metrics root mean square deviation (RMSD), root mean square fluctuation (RMSF), intermolecular hydrogen bonding (H-bonds) and radius of gyration (Rg) revealed that the lead phytochemicals form an energetically stable complex with the target protein. Majority of the phytoconstituents exhibited drug-likeness with non-tumorigenic properties. Thus, the PSE phytoconstituents could be useful source of drug or nutraceutical development in SARS-CoV-2 pathogenesis.


Collection of Punica granatum and extract preparation
In month of February, fresh fruits of cultivated Punica granatum plant, var.Jalore seedless were collected from Jodhpur, Rajasthan, India.Pomegranate fruits were identified, and submitted at Pharmacognosy Department, Integral University, Lucknow (File No. IU/PHAR/HRB/14/08).Pomegranate seeds were separated, rinsed with ddH 2 O, dried in the shade, and then ground into a powder using a Bajaj grinder.A coarse powder was steeped in 95 percent ethanol for three days at 25 °C in order to remove any soluble components.The filtrate was separated from the supernatant using a Whatman No. 1 filter membrane, and the ethanol was evaporated using a Rotatory evaporator.To obtain the semi-dried form of pomegranate seed extract, the resultant semi-solid paste was further evaporated on a water bath.All procedures were followed as per guidelines: https:// www.biome dcent ral.com/ getpu blish ed/ edito rial-polic ies# resea rch+ invol ving+ plants.

GC-MS based analysis
Gas chromatography-mass spectroscopy is an ideal technique for phytoconstituents characterization viz. to predict their name, structure, formula and retention time.As per previous study, RTX-5 MS capillary column (Restek) was used to chemically characterize the 95% ethanolic PSE with the help of GC-MS-QP2010 Plus system (Shimadzu, Japan) 12 .GC separates the various constituents into their individual components with their variable retention times and mass spectrophotometer identifies the components.A chromatogram of relative abundance against retention time was produced by the software, coupled to the mass spectrophotometer.Comparisons between the mass spectra of known and unknown components were conducted by NIST08 and WILEY8 chemistry libraries.It was possible to determine the components' names, molecular weights, m/z values, and structures.

Free radical scavenging activity of PSE
The free radical scavenging capacity of PSE was assessed by 2, 2-diphenyl-2-picryl-hydrazyl (DPPH) according to a previously described procedure 13 .Briefly, 2 mL of 0.1 mM DPPH solution made in methanol was added to various concentrations of PSE prepared in 1% DMSO and adjusted to 3 mL using DPPH solution.The mixtures were shaken and allowed for 30 min at RT. Absorbance was determined at 517 nm with the help of a UV-Vis double beam spectrophotometer (Systronics, 2203).Low absorbance value shows high free radical scavenging activity.DPPH scavenging capacity (% inhibition) = [(Ac−As)/Ac)*100]; Ac, absorbance of the control reaction; As, absorbance of PSE.

Standard precision (SP) for Glide Docking
Following the preparation of the ligands and proteins, the glide SP flexible ligand mode of Schrödinger Maestro Release 2020-2 was used to dock the ligands with the proteins in 10 poses each 14 .The OPLS3e force field was used in the glide SP flexible docking analysis.Poses with least energy were selected, and the Glide score was used to determine the final score.The lowest glide score and the best-docked orientation for each ligand were documented.The docking scores of phytocomponents, which represent the binding energies, were then used to organize them.

Interpreting and visualizing docking results
Data in the form of glide energy and glide score were produced following the glide docking study with SP mode.The top-ranking compounds were sorted based on Glide score to identify the appropriate binding interaction complex.The 2D interactions with the best poses, such as hydrogen bond contacts, wander Waal interactions, and hydrophobic interactions, were also displayed and analysed using SP visualizer as a ligand interaction tool.With the aid of the software LigPlot + v.2.2, the binding pattern of the best docking results was further examined.

Molecular dynamics simulation
Following the docking studies, lead compounds obtained from docking analysis i.e. 1. 3.alpha.,12.beta.-Dihydroxy-bisnor-5,7-cholenicacid with NRBD protein and 2. ethyl 5-oxo-2-pyrrolidinecarboxylate with CTD domain of SARS-CoV-2 were used for MD simulation study to check their binding efficacy and internal dynamics of both complex 15 .The GROMACS (Version 2023.2) was utilized to perform MD simulation.CHARMM27 force field and transferable intermolecular potential 3P (tip3p) water model was applied to conduct MD simulation of both complexes 16 .SwissParam was used to generate force field parameters for the phytochemicals.Na and Cl ions were used to balance complex charges.Simulations were run at a pressure of 1 bar and a temperature of 300 K.The steepest descent algorithm was used to reduce each system in 5000 steps.The Particle-Mesh-Ewald (PME) summation was used to calculate electrostatic interactions 17 .By running 1000 kJ/mol nm 2 position restraint simulations for 100 ns in the NVT and NPT ensembles, the systems were brought to equilibrium.A 100 ns no restraint production run was simulated using equilibrated systems.Root mean square deviation, root mean square fluctuations, radius of gyration, and hydrogen bond occupancy are some of the post-MD analyses that were carried out.Utilizing the XMGrace program, the entire plot was created 18 .

Potential toxicity study
When developing pharmaceutically active compounds, the preliminary knowledge of the physico-chemical and potential toxicological molecular characteristics of constituents needs to be optimized.Predicting various phytocomponent features at an early stage is essential for lead research and development.Software application OSIRIS Data Warrior V5.2.1 was used to forecast physico-chemical and toxicological molecular features like mutagenic, drug-likeness, tumorigenic, reproductive, and irritating effects 20 .

GC-MS analysis of pomegranate seed extract
Traditional herbal medicines provide vital role in health sectors by improving various acute and chronic conditions without or minimal toxic effect.It is essential to identify the chemical nature and their medicinal properties of phytocomponents present in traditional herbs.This method might be able to prove experimentally the traditional use of medicinal plants.Small group of acids, hydroxyl acids, amino acids, fatty acids, sugars, alcohols, sterols, catecholamines, toxins and drugs are among the small molecular metabolites that are ideal for GC-MS-based metabolomics.These compounds are frequently chemically modified to make them volatile enough for gas chromatography 21 .While MS uses a plot of the ion signal as a function of the mass-to-charge ratio to determine the elemental profile, chemical makeup, or structural characteristics of chemical compounds, GC is utilized to distinguish between volatile and thermally stable alternatives in a sample.Natural occurring volatile components are investigated using by GC-MS analysis and those having semi-or non-volatile metabolites can be induced into volatile through derivatization and then can be investigated using GC-MS.Using effective derivatization techniques involving silylation, alkylation or acylation reactions, a wide range of semi-and nonvolatile metabolites, including sugars, sugar alcohols, sugar phosphates, organic acids, lipids, flavonoids, peptides, amino acids, amides, amines, long-chain alcohols, and alkaloids can also be studied 22,23 .In this GC-MS analysis, silylation processes was employed to detect and identify semi-volatile and non-volatile components of pomegranate seed extract.The chromatogram of GC-MS analysis showed thirty-two peaks representing the presence of thirty-two components in pomegranate seed alcoholic extract.Figure 1 represents the total ion current (TIC) chromatogram of the pomegranate seed extract.The identified phytochemicals from alcoholic extract of native pomegranate seed, molecular weight, percent peak area and their retention time described in Table 1.The results revealed that the major constituents were found to be 2-Furancarboxaldehyde, 5-(hydroxymethyl) (22.78%),Cyclopentane, 1-acetyl-1,2-epoxy-(11.36%), 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl (11.2%), n-Hexadecanoic acid (6.55%), 3-Deoxy-d-mannoic lactone (5.74%), Oleic acid (5.06%), 2,3-dihydroxypropyl  All other components were identified in trace amount.In an earlier study, hexane: ethanoic (3:1) extracts of pomegranate seed oil was analyzed by GC-MS, which identified 23 compounds, the majority of which belonged to fatty amides and fatty acids 24 .Another study has shown that pomegranate seed oils include polyunsaturated long-chain fatty acids as one of the primary components 25 .Approx.thirty-eight phytochemicals were found in the Pomegranate fruit extract, with the majority containing alkaloids, fatty amides, and indazole derivatives 26 .Fatty acids, terpenes, heterocyclic compounds, and flavonoids types of compounds were found in the pomegranate leaf extract 27 .In a similar study, GC-MS analysis revealed the presence of twenty-three chemicals in pomegranate peel extract, with predominantly secondary alcohols, organosulfur compounds, and fatty acid derivatives 28 .In the current study, GC-MS analysis identified thirty two phytocompounds, with the majority containing pyranones, fatty acids, alkaloids and phytosterols (Table 1).Numerous studies have demonstrated the antiviral effects of plant metabolites such pyranone, fatty acid ketone, alkaloid, polyphenol, phytosterol and fatty acid ester against a variety of viruses [28][29][30][31] .Based on these studies, the reported phytocomponents in PSE were further assessed for their potential as virtual antiviral agents utilizing Glide Docking in SP mode with Schrödinger Maestro Release 2020-2.

Antioxidant activity of PSE
DPPH radical scavenging activity of PSE is depicted as percent DPPH scavenging effects in Fig. 2. Results showed that the percent DPPH scavenging activity was found to be 24.3,45.9, 59.5, 75.9 and 86.5% at 10, 25, 50, 75 and 100 µg/mL of PSE.This study suggested that free radical scavenging activity of DPPH radical was increased in a dose-dependent manner.The antioxidant capacity might be due to the high content of phenolic and flavonoid components in the ethanolic extract of PSE.Pomegranate seeds are a byproduct in the production of pomegranate juice.Pomegranate seeds possess potent antioxidants, and anti-inflammatory chemicals such as vitamin E, sterols, and phenols, plethora of fatty acids and natural estrogens 32 .The details of all Glide E energy, Gibbs binding free energy and docking scores of identified constituents with their respective NRBD and CTD proteins are mentioned in Tables 2 and 3.As shown in Table 2, twenty one phytocomponents (compounds' serial numbers 1-11, 13-15 and 17-23) and standard drug hydroxychloroquine displayed good binding affinity (Docking score > − 1.00 kcal/mol) towards the NRBD nucleocapsid protein of SARS-CoV-2.Whereas, compounds' serial numbers 12 and 16 displayed mild binding affinity (Glide score < − 1.00 kcal/mol).Two compounds exhibited positive docking score, while remaining compounds and ivermectin did not exhibit any binding interaction suggesting little/no affinity towards proteins (Table S1).More negative values of the Docking score which imitate binding free energy indicating stronger binding between ligand-protein interactions.The highest docking score was displayed by 3.alpha.,12.beta.-dihydroxy-bisnor-5,7-cholenicacid with NRBD nucleocapsid protein (Docking score = − 7.697).In case of CTD nucleocapsid protein (Table 3), 19   S2).Alternatively, the standard compound hydroxychloroquine displayed a negative Docking score (− 3.831 kcal/mol).This study confirms the good binding affinity of hydroxychloroquine against both NRBD and CTD nucleocapsid protein, representing efficient inhibitor of SARS-CoV-2 in comparison to ivermectin.
In ligand-protein complex, they bind with each other generally through non-covalent interactions viz.electrostatic, van der Waals forces, π-π interaction, and hydrophobic interaction 33 .The hydrophobic interactions increase the significance of protein binding as molecules become more lipophilic.But as evidenced by the interactions of numerous polar and nonpolar phytocomponents with specific proteins, many hydrophilic molecules are also electrostatically linked with protein molecules through ionic interactions and/or hydrogen bonds (Tables 2 and 3).As represented in Tables 2 and 3, twenty-one and eighteen hits, respectively had a good glide score > − 1.0 kcal/mol because of electrostatic attraction and hydrogen bonds between the hydroxyl groups of the ligand and various amino acid residues.As per results of Tables 2 and 3, the top twenty hits of pomegranate seeds with NRBD and CTD nucleocapsid proteins could be used to develop antiviral drugs against SARS-CoV-2 pathogenesis.The 2-D interaction of the amino acids Gln84, Val73, Pro74, Ile75, Thr136, Gly70, Glu137, Pro163, Thr77, Glu161, Leu162, Gly165, Thr166, Ser79, and Asn76 of a best-docked complex between 3.alpha.,12.beta.-Dihydroxy-bisnor-5,7-cholenicacid and SARS-CoV-2 NRBD protein is shown by the LigPlot (Fig. 3A).The O-group of ligand molecule is connected through three H-bonds with bond length 2.81 Å, 3.33 Å and 3.20 Å with N-group of Leu162, Gly165 and Thr166 amino acid residues of protein molecule.While, amino acid residues Gln84, Val73, Pro74, Ile75, Thr136, Gly70, Glu137, Pro163, and Thr77 were linked to the ligand through hydrophobic contact.Figure 3B illustrates the interaction between the amino acid residues Phe274, Phe286, Val270, Trp301 and Ala264 of CTD domain of SARS-CoV-2 and ethyl 5-oxo-2-pyrrolidinecarboxylate molecule of pomegranate seed.In this complex, all amino acid residues are linked with ligand through hydrophobic interaction.Figure 3C  Different groups of phytochemicals such as Pyranone, Cyclopentane, Fatty acid ketone, Aromatic heterocycle (alkaloid), Amine, Glucopyranoside, Lactone derivative, Ketone, Sterol, Steroid, Vitamin E, Fatty acid ester and β-sitosterol (phytosterol) and their derivatives were identified as active constituents in PSE (Table 1).These constituents were selected as antiviral agents for in silico binding interaction towards NRBD and CTD nucleocapsid proteins of the SARS-CoV-2 and interestingly, these components showed greater affinity towards their targeted protein.Pyrones or pyranones are a class of heterocyclic chemical compounds which display chemotherapeutic potentials especially antibacterial and anticancer activity 34 .Cyclopentane or C pentane is a highly flammable alicyclic hydrocarbon which derivatives have also been shown the potent activity against hepatitis C and dengue viruses 35 .Oleic acid Ketones are cellular energy-sustaining endogenous compounds that also have drug-like signalling properties that influence immune response, metabolism, and epigenetics 36 .Aromatic heterocyclic compounds are analogous to benzene which has been proven to be more effective for treating a number of infectious and life-threatening disorders.It demonstrates a broad range of biological effects, including antiviral, antibacterial, anti-diabetic, anti-cancer, anti-inflammatory, and antifungal properties 37 .Amines, derived from ammonia, is an organic compound containing nitrogen atoms with a lone pair of electrons.Various amino acid and peptide-based antiviral agents have been developed as protease inhibitors in clinical practice 38 .An earlier patent investigation found that administering compositions comprising vitamin E, tocopherol, or a tocopherol derivative orally, subcutaneously, intramuscularly, intravenously, or intraperitoneally suppressed viral and retroviral reproduction 39 .Phytosterols are phytosteroids, similar to cholesterol, that are used by plants as structural elements in their biological membranes.Previous research has demonstrated the antioxidant, antibacterial, and antifungal properties of phytosterol found in the bark of Norway spruce Picea abies 40 .Punica granatum seed is a good source of antioxidants because of its high polyphenolic and flavonoids and vitamin C content 41 .As a result, it possesses both therapeutic as well as preventive properties from disease progression.Thus, natural chemicals found in pomegranate seeds could be used as preventive and/or therapeutic candidates in the drug discovery process based on these earlier results and current virtual in silico analysis.

Molecular dynamics simulation analysis of ligand-protein complex
RMSD analysis was evaluated with c-alpha atom of protein and ligand fit on protein, demonstrating the stable interaction between protein and ligand.A good RMSD value depends on the protein and ligand having the best binding interactions and the lowest binding energy 42 .As shown in the Table 4 and Fig. 4A of RMSD trajectory, SARS-CoV-2 nucleocapsid protein NRBD C-alpha atoms were ranging between 0.117 and 4.879 nm, while ligand alpha.,12.beta.-Dihydroxy-bisnor-5,7-cholenicacid RMSD were ranged between 0.086 and 7.718 nm.Considering this data, the SARS-CoV-2 NRBD-alpha.,12.beta.-Dihydroxy-bisnor-5,7-cholenicacid complex was notably stable instead of rotational movement of alpha.,12.beta.-Dihydroxy-bisnor-5,7-cholenicacid in binding pocket of NRBD protein.RMSD analysis of SARS-CoV-2 CTD-ethyl 5-oxo-2-pyrrolidinecarboxylate complex was also found in stable configuration with protein C-alpha atom RMSD ranging between 0.09 and 3.607 nm with ligand RMSD between 0.132 and 3.251 nm (Table 4 and Fig. 5A).Moreover, RMSF C-alpha atom trajectory was analyzed for assessment of secondary structure fluctuation during the 100 ns MD simulation run.The RMSF values were ranging between 1.206 and 3.973 nm and 0.609-3.898nm for C-alpha of SARS-CoV-2 nucleocapsid NRBD and SARS-CoV-2 nucleocapsid CTD proteins, respectively (Table 4, Figs.4B and 5B).The radius of gyration (Rg) describes how atoms in a protein are distributed along its axis and how compact a molecule is at its center of mass.Rg for both ligands i.e. alpha.,12.beta.-Dihydroxy-bisnor-5,7-cholenicacid and ethyl 5-oxo-2-pyrrolidinecarboxylate were found below the 0.45 and 0.28 nm, respectively (Figs. 4C and 5C).The number of hydrogen bonds between protein and ligand were calculated for each frame of the simulation.In the SARS-CoV-2 nucleocapsid protein NRBD-alpha.,12.beta.-Dihydroxy-bisnor-5,7-cholenicacid complex the number of H-bonds were found maximum seven in number and most of the time frame was resonated between 2 and 4 bond (Fig. 4D).This resonated conformation made this complex notably stable throughout 100 ns MD simulation.In case of SARS-CoV-2 nucleocapsid CTD-ethyl 5-oxo-2-pyrrolidinecarboxylate complex, maximum five H-bonds were found in the complex while most of the time frame was resonating between 2 and 4 H-bonds (Fig. 5D).pomegranate seed constituents.Lipinski should only have committed one violation for an oral active compound 43 .Interestingly, 15 pomegranate seed phytoconstituents exhibited no violation and only one violation was exhibited by remaining five constituents.Standard drugs, ivermectin displayed two of Lipinski's violations, while hydroxychloroquine exhibited zero violation.The percentage of medications that are absorbed represents the oral absorption, or the number of drugs that enter the bloodstream through the portal vein from the gastrointestinal lumen 44 .The results of activity spectra for substances reveals that the drug availability in the gut lumen is more than > 50% (Table 5; Column 1).TPSA has the potential to serve as effective molecular descriptor in the investigation of drug transport qualities like blood-brain barrier penetration and intestinal absorption.It can also be used as a reliable predictor of the bioavailability of medicinal compounds to the cells.TPSA is the aggregate of polar atoms (oxygen, nitrogen) and their corresponding hydrogen atoms' contributions to a molecule's surface area, which would be generally van der Waals 45 .Molecules which are unable to penetrate cell membranes often have polar surface areas > 160 2. It typically takes a PSA of a molecule 90 2 to pass across the BBB and subsequently act on receptors in the central nervous system.It's interesting to note that all phytoconstituents, as shown in Table 5, had TPSA values lower than 160, indicating adequate intestinal wall absorption.The phytoconstituents of pomegranate seeds also adhered to the other characteristics of Lipinski's rule of 5.The potential toxicity and drug-likeness of the phytocomponents found in pomegranate seeds are shown in Table 6.Except for cyclopentane, 1-acetyl-1,2-epoxy-Heptanoic acid, 6-oxo-1-O-hexyl-d-glucitol, fucosterol, and hydroxychloroquine, the results showed that all phytocomponents are safe to use and have no known toxicity in terms of mutagenic, tumorigenic, adverse effects on reproduction, and irritation.The benefits and cons of various components were only partially disclosed by the current computational analysis of pomegranate seed contents; nonetheless, the bulk of phytocomponents showed strong antiviral activity and drug-like qualities.

Conclusions
GC-MS analysis of PSE showed thirty-two phytoconstituents of different groups such as pyranone, cyclopentane, fatty acid ketone, aromatic heterocycle (alkaloid), amine, glucopyranoside, lactone derivative, ketone, sterol, vitamin E, fatty acid ester and β-sitosterol (phytosterol) and their derivatives.These elements might be the underlying factor for antioxidant activity of PSE.The top twenty hits of PSE phytoconstituents displayed strong binding affinity with NRBD and CTD nucleocapsid protein of SARS-CoV-2.Remarkably, the majority of the phytoconstituents showed drug-like characteristics without any indication of toxicity.Based on this study, top twenty phytocomponents of pomegranate seed may be used as valued source for drug or nutraceutical development.
More specifically based on Docking score and MD simulation analyzes, top two compounds, ethyl 5-oxo-2-pyrrolidinecarboxylate (a class of alpha amino acids derivatives) and 3.alpha.,12.beta-dihydroxy-bisnor-5,7-cholenic acid (phytosteroid derivative), could be explored further for their antiviral activity.These findings, however, urge for more refinement and confirmation through preclinical research to combat SARS-CoV-2 pathogenesis.

Figure 1 .
Figure 1.Total ion chromatogram, showing the intensities of all mass spectral peaks belonging to the same scan of the identified compounds from pomegranate seed extract (95% ethanolic).Y-axis is total ion current; X-axis is retention time.

Figure 2 .
Figure 2. Antioxidant activity of ethanolic extract of PSE using DPPH free radical scavenging method.Values are shown as mean ± SD of three independent experiments.*p < 0.05 as compared to control.

Table 1 .
The identified phytoconstituents and molecular weight (MW), molecular formula (MF), retention time (RT), %Area, and nature of compounds from PSE through GC-MS technique. S.

Table 3 .
Docking score, Glide E model, Gibbs binding free energy, Glide energy, interacting amino acids and 2-D interaction diagram of the docked ligand-protein complex of nineteen PSE phytocomponents and standard drug with CTD; PDB ID: 6WJI of SARS-CoV-2 nucleocapsid protein using glide SP module of Schrödinger.

Table 5 .
PASS analysis and physicochemical properties of major active components of top twenty hits from pomegranate seed components and reference drugs.Note: a Percentage absorption was calculated as: % Absorption = 109−[0.345× Topological Polar Surface Area].b Topological polar surface area (defined as a sum of surfaces of polar atoms in a molecule).c Logarithm of compound partition coefficient between n-octanol and water.

Table 6 .
Druglikeness and toxicity calculation of top twenty hits from pomegranate seed components as well as reference drugs.
N, no toxicity; L, low toxicity; H, high toxicity.