*Corresponding Author:
G. Prakash Yoganandam
Department of Pharmacognosy, College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences, (Govt. of Puducherry), Gorimedu, Puducherry 605006, India
E-mail: [email protected]
Date of Received 29 June 2021
Date of Revision 24 April 2022
Date of Acceptance 12 September 2022
Indian J Pharm Sci 2022;84(5):1203-1209  

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In 2019, a new strain of Coronavirus disease was identified in Wuhan, China. Specific therapies are unavailable and investigations regarding Coronavirus disease treatment are lacking. The present study aimed to assess bioactive compounds found in medicinal plant Phyllanthus amarus as potential Coronavirus disease main protease inhibitors, using a molecular docking study. Molecular docking was performed using Autodock 4.2. Coronavirus disease main protease was docked with several compounds, and docking was analysed by Autodock 4.2, Lopinavir was used as standard for comparison. The binding energies obtained from the docking of 6LU7 with native ligand, lopinavir, Phyllanthin, Hypophyllanthin, Hexanedioic acid, bis (2-ethylhexyl) ester, Benzeneethanamine,3,4-dimethoxy-n-((pentafluoro phenyl) methylene), Phenethylamine, 2-methoxy-alpha.-methyl-4,5-(methylenedioxy), Diisooctyl adipate, P-Tert-Octylresorcinol, 4-methyl-2,5-dimethoxy phenethylamine, 3-(2,4-dimethoxy-phenyl)-2-formylaminopropionic acid, ethyl ester, Rutin, Quercetin and Niranthin were -6.08, -4.44, -4.14, -2.32, -4.23, -4.07, -2.5, -4.66, -4.14, -3.38, -7.46, -6.28 and -4.62 kcal/mol, respectively. Therefore, lopinavir may represent potential treatment option and Rutin and Quercetin appeared to have the best potential to act as Coronavirus disease main protease inhibitors. Thus all the compounds have shown significant binding energy and potent inhibitory effect against Coronavirus disease main protease. However, further research is necessary to investigate their potential medicinal use.


Coronavirus disease, main protease, 6LU7, Phyllanthus amarus, phytoconstituents, molecular docking

Coronavirus Disease (COVID-19) is an ongoing pandemic of COVID-19 which is caused by Severe Acute Respiratory Syndrome-Corona Virus-2 (SARS-CoV-2). The outbreak of COVID-19 was first identified in Wuhan, china, in December 2019[1]. It is easily transmissible and spread worldwide. Common symptoms are including fever, muscle and body aches, cough, sore throat, loss of smell and shortness of breath. Symptoms may appear 5-6 d after infection but it may range from 2-14 d. Table 1 shows COVID-19 outbreak updates.

Place Total cases Recovered Total deaths
World wide 5036 815 1990 057 326 251
India 106 750 42 298 3303
America 1576 950 364 263 93 858
England 248 818 344 35 341
Italy 226 699 129 401 32 169
Spain 278 803 196 958 27 778
France 180 809 62 563 28 239
China 82 965 78 244 4634
Russia 308 705 85 392 2972
Pakistan 45 898 13 101 985
Tamilnadu 12 448 4895 84
Puducherry 18 - 1

Table 1: Covid-19 Outbreak Updates on May 20, 2020.

At present, specific therapies such as vaccine, specific antiviral drugs for COVID-19 are not available and antiviral drugs for COVID-19 are not available and acquired immunodeficiency syndrome patients, for the treatment of COVID-19-infected patients[2]. Other reported antiviral treatments form human pathogenic Corona Viruses (CoV) include nucleoside analogues, neuraminidase inhibitors, remdesivir, umifenovir (arbidol), Tenofovirdisoproxil (TDF) and Lamivudine (3TC)[2]. However, the measures that have been implemented remain limited to preventive and supportive therapies, designed to prevent further complications and organ damage. To treat COVID-19 infections, some preliminary studies have investigated potential combinations that include anti-malarial drug chloroquinone and anti-HIV vaccines. The Main protease (Mpro)/3 Chymotrypsin Like protease (3CLpro) from COVID-19 represent a potential target for the CoV replication inhibition[2]. It was observed that genome of CoV encodes two proteins ppla and pplb which are involved in spike, membrane, envelop nucleoprotein, replicase and polymerase activity of viruses. This function is performed by Mpro/3CLpro[3].

The Mpro has 3 structural domains; domain I (residues 8-101) and domain II (residues 102-184) both have beta barrel motifs representing chymotrypsin catalytic domain and domain III (residues 185-200) with a helical structure participates in dimerization of protein and active enzyme production. Mpro is considered to be a suitable target for viral inhibitor development as an approach towards SARS. At present, natural compounds have served humans as cheaper and safer drug candidates against several diseases[4,5]. Most of the herbal drugs having antiviral properties which are used as supportive treatment for COVID-19 patients. Phyllanthus amarus (P. amarus) (Keezhkainelli) is one of the herbal drugs has got hepatoprotective properties, antioxidant and anti-inflammatory properties[6] and also antiviral properties against Hepatitis B virus and reverse transcriptase of retroviruses[7,8]. From the above light of information, it was thought worth to investigate the phytoconstituents of methanolic extract of P. amarus as potential inhibitor for COVID-19 Mpro.

Materials and Methods

Ligand preparation:

The bioactive constituents used for docking were obtained from methanolic extract of P. amarus such as as Phyllanthin, Hypophyllanthin, Hexanedioic acid, bis (2-ethylhexyl) ester, Benzeneethanamine,3,4-dimethoxy-n-(pentafluorophenyl) methylene), Phenethylamine, 2-methoxy-alpha.-methyl-4,5-(methylenedioxy), Diisooctyladipate, P-Tert-Octylresorcinol, 4-methyl-2,5-dimethoxy phenethylamine, 3-(2,4-dimethoxy-phenyl)-2-formylamino-propionic acid, ethyl ester, Rutin, Quercetin and Niranthin.

Ligands can be sketched using tools like Chemsketch or Marvinsketch. While selecting the ligand, the Lipinski rule of 5 should be applied (Table 2). The rule is very important for drug development where the pharmacologically active lead structure is optimized stepwise for increased activity and selectivity as well as drug like properties.

Compounds Molecular weight Log P No. of atoms H-bond donors H-bond acceptors Violations
Std 628.81 5.69 46 4 9 2
L1 418.53 3.92 30 0 6 0
L2 430.5 3.5 31 0 7 0
L3 370.57 7.36 26 0 4 1
L4 359.29 3.75 25 0 3 0
L5 209.25 1.2 15 2 4 0
L6 370.57 6.84 26 0 4 1
L7 222.33 4.5 16 2 2 0
L8 195.26 1.34 14 2 3 0
L9 281.31 1.55 20 1 6 0
L10 610.52 -1.06 43 10 16 3
L11 302.24 1.68 22 5 7 0
L12 432.51 3.95 31 0 7 0

Table 2: Lipinski Rule of 5 for the Docked Phytoconstituents.

Protein preparation:

To investigate the phytochemical analogs of P. amarus against SARS-CoV-2 virus, we have selected COVID-19 3CLpro/Mpro (Protein Data Bank (PDB) ID: 6LU7)[9]. The structure of protein was obtained from PDB (https://www.rcsb.org/), in PDB format (fig. 1). PDB is an archive for the crystal structures of biological macromolecules, worldwide[10].


Fig. 1: Target protein (6LU7) for molecular docking.

Active sites for 6LU7:

THR24, THR26, PHE140, ASN142, GLY143, CYS145, HIS163, HIS164, GLU166, HIS172.

Molecular docking studies:

Molecular docking was carried out for standard drug Lopinavir (anti-viral drug) and 12 phytochemical constituents of methanolic extract of P. amarus. The phytochemical analogs were docked with COVID-19 3CLpro/Mpro (PDB ID: 6LU7) by using Autodock software with default settings and the grid box was defined based on trial and error and carried out in normal mode (fig. 2). The crystal structure of protein was obtained from protein data bank. The structures of phytochemical constituents were sketched by using Marvinsketch and the structures were converted into Three Dimensional (3D) images and saved as PDB format. Analysis of docking results was done with Autodock Software and the results are compared with the standard drug and shown in Table 3.


Fig. 2: Interaction of compounds with amino acid residues of Mpro COVID-19; (A): Phyllanthin; (B): Hypophyllanthin, (C): Hexanedioic acid, bis (2-ethylhexyl) ester, (D): Benzeneethanamine, 3, 4-dimethoxy-n-[(pentafluoro phenyl) methylene], (E): Phenethylamine, 2-methoxy-alpha.-methyl-4,5-(methylenedioxy), (F): Diisooctyladipate, (G): P-Tert-Octylresorcinol, (H): 4-methyl-2,5-dimethoxy phenethylamine, (I): 3-(2,4-dimethoxy-phenyl)-2-formylamino-propionic acid, ethyl ester, (J): Rutin, (K): Quercetin, (L): Niranthin, (M): Lopinavir.

Compounds Binding energy (kcal/mol) Ligand efficiency Inhibitory constant (µM) Vanderwaals desolvation energy Intermolecular energy
Std -6.08 -0.13 34.74 -10.38 -10.56
L1 -4.44 -0.15 559.12 -8.2 -8.32
L2 -4.14 -0.13 919.66 -6.62 -6.53
L3 -2.32 -0.09 19.87 -8.01 -7.99
L4 -4.23 -0.17 794.62 -5.84 -6.02
L5 -4.07 -0.27 1.03 -4.62 -4.97
L6 -2.5 -0.1 14.82 -8.04 -8.16
L7 -4.66 -0.27 384.34 -5.91 -5.85
L8 -4.14 -0.3 920.17 -3.69 -5.34
L9 -3.38 -0.17 3.34 -6.03 -6.06
L10 -7.46 -0.17 3.4 -9.15 -9.25
L11 -6.28 -0.29 24.87 -6.48 -6.58
L12 -4.62 -0.15 407.56 -8.1 -8.2

Table 3: Molecular Docking Analysis of Selected Phytoconstituents against 6LU7.

Results and Discussion

Computational biology means in silico investigation which is an essential arm of biotechnology focused at enhancing a deeper insight of bimolecular interactions in order to address cellular disease pathogenesis while having immense contribution towards design and development of possible therapeutic candidates[11]. Indeed, this technique has assisted in identifying lead compounds for various diseases[12].

The exponential increase in cases and mortality of COVID-19 has called for a need to develop drugs to treat this infection. Using in silico and molecular docking approaches, this study investigated the inhibitory effects of Pradimicin A, Lamivudine, Plerixafor and Lopinavir against SARS-CoV-2 Mpro[13]. Recent study on Spondias mombim L molecular docking of the studied enzymes with chlorogenic acid, lutein and zeaxanthin were carried out using PatchDock. SMEAF had remarkable enzyme inhibitory effects against Phosphodiesterase-5 (PDE-5), arginase, Angiotensin I-Converting Enzyme (ACE), cholinesterase, Monoamine Oxidase A (MAO), Ecto-5’ Nucleotidase (E-NTDase), tyrosinase and stimulated Sodium (N+)-Potassium (K+) Adenosine-Triphosphatase (ATPase) activities[14].

On the other hand, the emergence and sporadic spread of COVID-19 has called for the urgent identification of novel drug and repurposing of existing available chemotherapeutic agents against this pandemic. Corona viruses have a long history of infecting animals and humans and causing respiratory, digestive, liver and central nervous system diseases in them[15]. Currently, COVID-19 has emerged in the human population and is a potential threat to global health, worldwide[15]. The virus polyprotein encodes two proteases, the 3CLpro (Mpro) and a papain-like protease; both are vital targets for drug discovery platforms against coronaviruses. Accordingly, the Mpro is the most probable antiviral candidate due to its crucial role in self-maturation and ensuing development of polyproteins[16].

However, no approved drug currently exists to treat the disease. The currently available drugs for COVID-19 treatment primarily act on the Mpro. The aim of this study was to examine the phytoconstituents of medicinal plant P. amarus that may be used to inhibit the COVID-19 infection pathway. Table 2 shows that Lipinski’s rule of five for tested compounds. As per docking score obtained in energy kcal/mol as shown in Table 3. The highest docking score was obtained for the derivative L10 with score of -7.46 kcal/mol followed by compound L11 with score of -6.28 kcal/mol. Our study revealed that all the phytoconstituents present in P. amarus had satisfactory binding affinity and potent inhibitory effect for the SARS-CoV-2 Mpro (fig. 3). The docking analysis in the present study showed the inhibition potential of several compounds, ranked by affinity (ΔG); Rutin (L10)>Quercetin (L11)>Lopinavir (Std)>P-Tert-Octylresorcinol (L7)>Niranthin (L12)>Phyllanthin (L1)>Benzeneethanamine, 3,4-dimethoxy-n-((pentafluoro phenyl) methylene) (L4)>Hypophyllanthin (L2)>4-methyl-2,5-dimethoxy phenethylamine (L8)>Phenethylamine, 2-methoxy-alpha.-methyl-4,5-(methylenedioxy) (L5)>3-(2,4-dimethoxy-phenyl)-2-formylamino-propionic acid, ethyl ester (L9)>Diisooctyladipate (L6)>Hexanedioic acid, bis (2-ethylhexyl) ester (L3). Table 4 shows that hydrogen bond formation and its distance and amino acid involved in H-bond interactions. However, further research is necessary to investigate the potential uses of the phytoconstituents of P. amarus plant.


Fig. 3: Binding mode of compounds in SARS COV2 mainprotease; (A): Phyllanthin, (B): Hypophyllanthinin, (C): Niranthin, (D): Lopinavir.

Compounds No. of hydrogen bonds formed Amino acid involved in hydrogen bond interactions Distance between donor and acceptor (A°)
Std 0 - -
L1 0 - -
L2 0 - -
L3 2 THR198 2.811
L4 1 GLN110 2.783
L5 2 THR199;TYR239 2.985; 2.978
L6 1 ASN238 2.996
L7 1 TYR239 2.61
L8 0 - -
L9 1 GLN110 2.577
L10 1 GLN107 2.694
L11 0 - -
L12 0 - -

Table 4: Hydrogen Bond Formation and its Distance and Amino Acid involved in H-Bond Interactions.