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doi.org/10.26434/chemrxiv.12110214.v2

Evaluation of Traditional Ayurvedic Preparation for Prevention andManagement of the Novel Coronavirus (SARS-CoV-2) Using MolecularDocking ApproachDharmendra Kumar Maurya, Deepak Sharma

Submitted date: 02/05/2020 • Posted date: 04/05/2020Licence: CC BY-NC-ND 4.0Citation information: Maurya, Dharmendra Kumar; Sharma, Deepak (2020): Evaluation of TraditionalAyurvedic Preparation for Prevention and Management of the Novel Coronavirus (SARS-CoV-2) UsingMolecular Docking Approach. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12110214.v2

Since the emergence of novel Coronavirus (SARS-CoV-2) infection in Wuhan, China in December 2019, ithas now spread to over 205 countries. The ever-growing list of globally spread corona virus-19 disease(COVID19) patients has demonstrated the high transmission rate among human population. Although 12 newdrugs are being tried for management of COVID19, currently there are no FDA approved drugs or vaccines toprevent and treat the infection of the SARS-CoV-2. Considering the current state of affairs, there is an urgentunmet medical need to identify novel and effective approaches for prevention and treatment of COVID19 byre-evaluating the knowledge of traditional medicines and repurposing of drugs. Here, we used moleculardocking approach to explore the beneficial roles of an array of phytochemicals and active pharmacologicalagents present in the Indian herbs (Tulsi, Haldi, Giloy, Black pepper, Ginger, Clove, Cardamom, lemon, andAshwagandha) which are widely used in the preparation of Ayurvedic medicines in the form of Kadha tocontrol various respiratory disorders such as cough, cold and flu. The evaluation was made based on thedocking scores calculated by AutoDock Vina. Our study has identified an array of phytochemicals present inthese herbs which have significant docking scores and potential to inhibit different stages of SARS-CoV-2infection as well as other Coronavirus target proteins. Molecular docking also indicated that, thephytochemicals present in these herbs possess significant anti-inflammatory property. Overall our studyprovides scientific justification in terms of binding of active ingredients present in different plants used inKadha preparation with viral proteins and target proteins for prevention and treatment of the COVID19. Thispreparation can boost individual’s immunity and inhibit the viral severity by interfering at different stages ofvirus multiplication in the infected person.

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Evaluation of traditional ayurvedic preparation for prevention and

management of the novel Coronavirus (SARS-CoV-2) using molecular docking

approach

Dharmendra Kumar Maurya 1, 2,* and Deepak Sharma1, 2

1Radiation Biology & Health Sciences Division, Bhabha Atomic Research Centre,

Anushaktinagar, Mumbai 400 085, India

2Homi Bhabha National Institute, Anushaktinagar, Mumbai-400094, India

*Corresponding authors

Dharmendra Kumar Maurya, Ph.D

Scientific Officer/F

Free Radical Biology Section, Radiation Biology & Health sciences Division

Bhabha Atomic Research Centre, Mumbai 400085, Maharashtra, INDIA

Email: [email protected], [email protected]

Tel: +91-22-25590407

mailto:[email protected]


Abstract

Since the emergence of novel Coronavirus (SARS-CoV-2) infection in Wuhan, China in

December 2019, it has now spread to over 205 countries. The ever-growing list of globally

spread corona virus-19 disease (COVID19) patients has demonstrated the high transmission

rate among human population. Although 12 new drugs are being tried for management of

COVID19, currently there are no FDA approved drugs or vaccines to prevent and treat the

infection of the SARS-CoV-2. Considering the current state of affairs, there is an urgent unmet

medical need to identify novel and effective approaches for prevention and treatment of

COVID19 by re-evaluating the knowledge of traditional medicines and repurposing of drugs.

Here, we used molecular docking approach to explore the beneficial roles of an array of

phytochemicals and active pharmacological agents present in the Indian herbs (Tulsi, Haldi,

Giloy, Black pepper, Ginger, Clove, Cardamom, lemon, and Ashwagandha) which are widely

used in the preparation of Ayurvedic medicines in the form of Kadha to control various

respiratory disorders such as cough, cold and flu. The evaluation was made based on the

docking scores calculated by AutoDock Vina. Our study has identified an array of

phytochemicals present in these herbs which have significant docking scores and potential to

inhibit different stages of SARS-CoV-2 infection as well as other Coronavirus target proteins.

Molecular docking also indicated that, the phytochemicals present in these herbs possess

significant anti-inflammatory property. Overall our study provides scientific justification in

terms of binding of active ingredients present in different plants used in Kadha preparation

with viral proteins and target proteins for prevention and treatment of the COVID19. This

preparation can boost individual’s immunity and inhibit the viral severity by interfering at

different stages of virus multiplication in the infected person.

Introduction

Viral infections cause a wide spectrum of human diseases which appear with mild, severe or

life-threatening symptoms and underlie major clinical and socio-economic problems

worldwide. Human Coronaviruses (CoVs), including severe acute respiratory syndrome

Coronavirus (SARS-CoV), middle east respiratory syndrome Coronavirus (MERS-CoV) and

2019 novel Coronavirus (SARS-CoV-2) have caused three recent major global epidemics with

significant morbidity and mortality (Paules et al., 2020). Presently, there are no specific drugs

or vaccines approved for SARS-CoV-2. In light of the high infectivity rate of SARS-CoV-2 and

lack of treatment options, World Health Organization (WHO) has declared it as a global

emergency and researchers from all-over the world are trying to find possible cure for this

disease called Corona Virus Disease or COVID19. Phylogenetic analysis shows that SARS-CoV-

2 has very high nucleotide sequence identity with SARS-CoV-1 (79.7%) (Zhou et al., 2020). The

envelope and nucleocapsid proteins of SARS-CoV-2 are two evolutionarily conserved regions,

with sequence identities of 96% and 89.6%, respectively. CoVs are enveloped viruses with a

positive-sense single-stranded RNA genome and contain at least four structural proteins:

Spike (S) protein (trimeric), envelope (E) protein, membrane (M) protein, and nucleocapsid

(N) protein. The Spike protein promotes host attachment and virion-cell membrane fusion

during infection. Therefore, Spike proteins play a crucial role in determining the host range

and tissue tropism. Zoonosis is common among CoVs and they can be transmitted from one

animal species to another, animals to humans, and humans to humans (Lim et al., 2019).

Therapeutic interventions against CoVs can either activate the host defence machinery and

immune system or block viral life cycle events including transmission, cell binding, enzymes

involved in synthesis of the viral components, replication and assembly (Wu et al., 2020). In

search of therapeutics against CoVs, researchers are using following three broad strategies;

(i) Test existing broad-spectrum anti-viral drugs, (ii) in silico screening of molecular databases

to identify the lead molecules against viral or host proteins and (iii) rational drug design based

on the genomic information and pathological characteristics of COVID19 (Wu et al., 2020).

Among these, repurposing approach will shorten the time and reduce the cost as compared

to other strategies (Wu et al., 2020). Apart from the above strategies, alternative approaches

including traditional and herbal medicines may also have significant potential for

management of COVID19 both as prophylaxis and therapeutic purpose.

Ayurvedic medicines and their extracts are used for treatment of viral diseases for a very long

time (Arora et al., 2010; Alleva et al., 2010). Ayurvedic medicines are used in Indian

subcontinent since the Vedic period dating back to more than 2000 years (Saini, 2016). An

important Ayurvedic method for enrichment of active pharmacological agents from herbs

involves preparation of Kadha (decoction) for oral consumption. According to Panchvidh

Kashyapam described in the Charak Samhita (an ancient text on Ayurveda), there are five

https://jamanetwork.com/searchresults?author=Catharine+I.+Paules&q=Catharine+I.+Paules

prescribed ways to consume medicinal herbs and plants (Shingadiya et al., 2016). These

includes: i) Swaras (juicing), ii) Kwath (decoction), iii) Kalka (in paste form), iv) Hima (an herb

induced concoction), v) Phant (an herb-infused concoction). The decoction (Kadha) from a

mixture of spices and herbs is considered to be one of the oldest forms of medicine invented

by humans. Kadha is prepared from dry or less juicy ingredients like spices and herbs. The

Ministry of AYUSH (Ayurvedic, Yoga and Naturopathy, Unani, Siddha and Homeopathy),

Government of India, has recently recommended the use of Kadha for boosting the immunity

and reducing inflammation during COVID 19 crisis (The ministry of AYUSH, 2020).

During viral infections, inflammation is part of the body’s immune response to reduce

infection, limit viral replication and transmission, reduce tissue injury and kill infected cells.

Acute inflammation is beneficial and it is followed by healing and regeneration. However,

CoVs are known to manipulate host machinery and subvert the immune system leading to

chronic inflammation (Chen et al., 2017; Takeuchi and Akira, 2007). The induction of pro-

inflammatory cytokines and chemokines in the host during SARS-CoV infection acts as a

double-edged sword which not only activates host immune response for viral clearance but

also aggravates tissue injury and organ toxicity during clinical evolution of the disease (Gu and

Korteweg, 2007; Tisoncik et al., 2012). CoVs infect cells using their spike proteins by making

interaction with cognate receptors present on the host cell surface. The spike proteins of

SARS-CoV and MERS-CoV attach to the cellular receptor angiotensin-converting enzyme 2

(ACE2) and dipeptidyl peptidase 4 (DPP4) receptor respectively for their entry inside the cells

(Gu and Korteweg, 2007; Wang et., 2013). SARS-CoV-2 spike protein has higher affinity

towards human ACE2 as compared to that of SARS-CoV-1 (Zhou et al. 2020). Upon infecting

the host cells, CoVs manipulate host machinery for new virus production and also elicit

inflammatory response in the host due to tissue injury (Gu and Korteweg, 2007; Wang et.,

2013). Clinical investigation of critically ill patients infected with SARS-CoV-2 have shown high

concentration of cytokines and chemokines in human plasma, suggesting that cytokine storm

was associated with disease severity, multi-organ failure and mortality (Huang et al., 2020).

Cyclooxygenase-2 (COX-2), phospholipase A2 (PLA2), NF-κB-inducing kinase (NIK), and

interleukin-1 receptor associated kinase (IRAK) are important druggable targets involved in

SARS-CoV-2 induced inflammatory response and can be used to screen anti-inflammatory

molecules.

In the present study, the phytochemicals and active pharmacological ingredients found in

different herbs used in making Kadha were docked with different viral proteins (such as viral

capsid spike, proteases, NSP polymerase), host cell receptors & proteases (such as human

ACE2 and furin) as well as pro-inflammatory mediators (such as COX2, PLA2, IRAK-4 and NIK

proteins). Our study predicted that many of these phytochemicals possess significant affinity

towards functional region of viral proteins including spike, proteases, nucleoproteins and

polymerase as well as host surface receptors. These phytochemicals also showed significant

binding affinity for the functional region of different inflammatory mediators. Our findings

indicate that regular consumption of this ayurvedic Kadha in consultation with the ayurvedic

practitioner may significantly boost the host immunity and also help in the prevention of viral

infection and pathogenicity and reduce disease-severity in the infected individuals.

Materials and Methods

Phytochemicals name of commonly used herbs in making Kadha: Some of the commonly

used herbs in the preparations of Kadha includes, Tulsi (Ocimum sanctum), Haldi (Curcuma

longa), Giloy (Tinospora cordiofolia), Black pepper (Piper nigrum), Ginger (Zingiber officinale),

Clove (Syzygium aromaticum), Cardamom (Elettaria cardamomum), lemon (Citrus limon), and

Ashwagandha (Withania somnifera). A list of 108 phytochemicals present in herbs that are

used in preparation of Kadha or similar drink were collected from literature. Phytochemicals

found in Ocimum sanctum (Oleanolic acid, Ursolic acid, Rosmarinic acid, Eugenol, Carvacrol,

Linalool, Beta-caryophyllene, Estragole, Eugenic acid, Apigenin, Cirsimaritin, Isothymusin,

Isothymonin, Vicenin, Orientin and Cirsilineol) (Pattanayak et al., 2020), Curcuma longa

(Curcumin, Demethoxycurcumin, Bisdemethoxycurcumin, Ar-turmerone, Alpha-turmerone,

Beta-turmerone, Atlantone, Cyclocurcumin, Calebin A , Trans-Ferulic acid, Vanillin and Vanillic

acid) (Li et al., 2011), Tinospora cordiofolia (Magnoflorine, Berberine, Choline, Jatrorrhizine,

Beta-Sitosterol, Tinosporide, Tinosporaside, Cordifolioside A, Tinocordioside, Cordioside,

Tinocordifolioside andTinocordifolin) (Sharma et al., 2019), Piper nigrum (Piperine,

Piperamide, Piperamine, Pipericide, Sarmentosine, Sarmentine, Brachyamide B,

Dihydropipericide, N-Formylpiperidine, Guineensine, Pentadienoylpiperidine, Tricholein,

Trichostachine, Piperettine, Piperolein B, Retrofractamide A, Chavicine, Isochavicine,

Isopiperine, Nerolidol, β-caryophyllene and Piperic acid) (Damanhouri and Ahmad, 2014),

Zingiber officinale (6-gingerol, 6-shogaol, 6-paradol, Zingiberene, Bisabolene, 1-

dehydrogingerdione, 6- gingerdione, 10-gingerdione, 4-gingerdiol, 6-gingerdiol, 10-

gingerdiol, Citral and Eucalyptol)(Bhattarai et al., 2018; Prasad et al., 2015), Syzygium

aromaticum (beta-caryophyllene, Vanillin, Eugenol, Acetyl eugenol, Crategolic acid, Eugenin,

Methyl salicylate, Kaempferol, Rhamnetin, Eugenitin, Oleanolic acid, Stigmasterol,

Campesterol, Gallic acid and Flavonol glucosides) (Cortés-Rojas et al., 2014), Elettaria

cardamomum (Protocatechualdehyde, Protocatechuic acid, Alpha-terpinyl acetate, 1,8-

cineole, Linalool, Linalyl acetate, Limonene, 4-terpineol andGeraniol) (Noumi et al., 2018),

Citrus limon (Eriodictyol, Quercetin, Hesperetin, Phloroglucinol, Umbelliferone, Vitamin C

(Vandercook and Stephenson, 1966; Rangel et al., 2011) and Withania somnifera (Withaferin

A, Somniferine, Choline, Anaferine, Withanolide A, Withanolide B, Withanone and

Withanolide) (Sangwan et el., 2004). 3D structures of these different phytochemicals were

downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov) in structure-data file (SDF).

Preparation of Ayurvedic Kadha: There are specific Ayurvedic methods to prepare the Kadha.

In India, variants of standard Kadha are also prepared using different combinations of herbs

depending on the severity of disease / ailment and availability of ingredients. The most

common ingredients are Tulsi leaves (10-15 leaves or ¼ teaspoon powder), Ginger (2-5g

rhizome or ¼ teaspoon powder), Clove (4-5 pieces), Black pepper (4-5 pieces), Cardamom (4-

5 pieces), Ashwagandha (2-5g raw or ¼ teaspoon powder) and Giloy (2-5g raw or ¼ teaspoon

powder). To make the Kadha, these herbs are boiled in 200 ml water for 5-10 minutes, and

jaggery or honey is added to make it sweet. The preparation is filtered and mixed with ¼

teaspoon of lemon juice. In case, if all ingredients are not available, it can be prepared using

locally available ingredients.

Protein structures: In order to study the mode of interaction of different phytochemicals with

various proteins including SARS-CoV-2, SARS-CoV-1, other important proteins and receptors

found on virus and host cells, molecular docking was performed. We have used following PDB

ID’s 6LU7 (SARS-CoV-2 main protease), 6m3m (SARS-CoV-2 nucleocapsid), 6vww (SARS-CoV-

2-NSP15 Endoribonuclease), 6vyo (SARS-CoV-2 RNA binding domain), 6w02 (SARS CoV-2-

NSP3), 6w4b(SARS-CoV-2-NSP9 replicase), 2ajf (ACE2 and SARS-CoV spike), 4mds (SARS-CoV

3CLpro) and 5mim (proprotein convertase; furin). In order to study the interaction of different

phytochemicals with different pro-inflammatory mediators like COX2, PLA2, NIK and IRAK-4,

we have used following PDB ID’s, 5f1a, 4uy1, 4dn5 and 2nrurespectively. All the protein

structures were retrieved from protein data bank (www.rcsb.org) and cleaned using UCSF

Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the

University of California.

Molecular docking: PyRx virtual screening tools was used for preparation of the input files

and performing molecular docking using Vina wizard. For preparation of protein input files,

all water molecules, ligands and ions were removed from *.pdb files. The polar hydrogens

were added to protein structure and prepared files were saved in *.pdbqt format. The

molecule’s energy was minimized using energy minimization tools of PyRx virtual screening

tools and ligands were saved in *.pdbqt format after adding polar hydrogens for further

docking process. All docking results were sorted by the binding energy. 2D interaction of the

ligand and protein was visualized using Discovery Studio Visualizer. Region-specific docking

was performed against SARS-CoV-2 main protease and spike protein as well as for human

ACE2 & Furin protease. Following AutoDock Vina docking parameters such as (center_x = -

16.69, center_y = 27.23, center_z = 68.46, size_x = 36.65, size_y = 42.12, size_z = 50.40),

(center_x = 190.45, center_y = 197.88, center_z = 260.72, size_x = 61.32, size_y = 41.03, size_z

= 43.79), (center_x = 6.68, center_y = -2.42, center_z = 48.2, size_x = 37.03, size_y = 70.29,

size_z = 55.72) and (center_x = 32.41, center_y = -37.97, center_z = -11.64, size_x = 71.93,

size_y = 55.05, size_z = 47.46) were used for SARS-CoV-2 main protease (PDB ID: 6LU7), SARS-

CoV-2 spike (PDB ID: 6VXX), human ACE2 (PDB ID: 2AJF) and furin (PDB ID: 5MIM) respectively.

Other proteins from SARS-CoV-2, SARS-CoV and host anti-inflammatory mediators were

blindly docked using complete protein structure.

Results

Our molecular docking study revealed that different phytochemicals found in the traditional

Ayurvedic Kadha, may have high binding affinity (the lowest binding energy) with various viral

and host macromolecular targets and other human pro-inflammatory mediators and

proteins. Table 1 shows the list of the phytochemicals which showed significant binding

affinity (≤-7.5 kcal/mole) with SARS-CoV-2 main protease, spike protein, human ACE2 and

furin proteins.

Table 2 shows the predicted binding of top 12 phytochemicals and native ligand N3, against

SARS-CoV-2 main protease and their different protein residues which are involved in the

interaction. These molecules are Somniferine A, Tinosporide, Tinocordioside, Orientin,

Flavonol glucoside, Withanolide, Apigenin, Cyclocurcumin, Withanolide B, Kaempferol,

Withanone and Withaferin A. Their predicted binding energies are -8.6, -8.6, -8.1, -8.1, -8.0, -

8.0, -7.8, -7.8, -7.8, -7.7, -7.6 and -7.6 kcal/mole respectively. All these molecules share same

binding site with the well-known inhibitor, N3 and their binding energy is also comparable

(Fig 1).

SARS-CoV-2 spike protein interacts with the host ACE2 receptor present on the surface of the

host cells for their entry. Table 3 shows the binding of top 12 phytochemicals with SARS-CoV-

2 spike (PDB ID: 6VXX) and the different residues involved in their interaction. These

molecules are, Withanolide B, Withanolide, Withaferin A, Withanone, Somniferine A, Beta-

sitosterol, Pipericide, Crategolic acid, Retrofractamide A, Ursolic acid, Piperolein B and

Tinosporide. Their predicted binding energies are -8.7, -8.4, -8.2, -8.2, -7.9, -7.8, -7.6, -7.6, -

7.5, -7.5, -7.5, -7.5, -7.4 and -7.4 kcal/mole respectively. These phytochemicals were found to

share same binding pocket in the target proteins (Fig 2 a).

Table 4 shows the binding of top 10 phytochemicals with human ACE2 (PDB ID: 2AJF) and the

different residues involved in their interaction. The phytochemicals with high affinity for

human ACE2 are, Withaferin A, Stigmasterol, Vicenin, Ursolic acid, Withanolide B, Oleanolic

acid, Withanone, Beta-sitosterol, Campesterol and Orientin. Their predicted binding energies

are -9.1, -8.8, -8.8, -8.7, -8.7, -8.5, -8.5, -8.3, -8.3 and -8.0 kcal/mole respectively. These

phytochemicals share same binding pocket in human ACE2 (Fig 2 b).

Furin is another protease found in the host cells which acts on the viral spike protein and

facilitates its interaction with the human ACE2. Table 5 shows the predicted binding of top 12

phytochemicals with human furin (PDB ID: 5MIM) and the different residues involved in their

interactions. These molecules are, Withanolide, Somniferine A, Apigenin, Withanolide B,

Ursolic acid, Hesperetin, Campesterol, Crategolic acid, Withanone, Chavicine, Rosmarinic acid

and Stigmasterol. Their predicted binding energies are -9.5, -8.7, -8.5, -8.3, -8.3, -8.2, -8.1, -

8.1, -8.1, -8.0, -8.0 and -8.0 kcal/mole respectively. Many of these molecules share same

binding pocket as its native ligand. However, our docking study also showed that Apigenin,

Withanolide, Hespertin, Campesterol, Chavicine, Rosmarinic acid, Stigmastereol bind to the

residues at a site adjacent to binding site of native ligand (Fig 3).

The molecular docking performed against other proteins from SARS-CoV-2 and SARS-CoV-

1proteins are shown in Table 6. Many of the phytochemicals present in the Kadha have

significant binding affinity with SARS-CoV-2 and SARS-CoV-1 proteins. Some of the

phytochemicals which have high binding affinity with NSP15 Endoribonuclease (PDB ID:

6vww) are Orientin, Withanolide, Withanolide B, Crategolic acid, Ursolic acid, Withaferin A,

Apigenin, Eriodictyol, Hesperetin, Oleanolic acid, Stigmasterol and Withanone. Their

predicted binding energies are -9.4, -9.3, -9.2, -9.1, -9.1, -9.1, -9.0, -9.0, -9.0, -9.0, -8.9 and -

8.9 kcal/mole respectively. A list of the phytochemicals which were found to exhibit high

affinity with SARS-CoV-2 ADP ribose phosphatase, NSP3 (PDB ID: 6w02) are Berberine,

Withanolide, Rosmarinic acid, Cyclocurcumin, Piperettine, Withaferin A, Withanolide B,

Bisdemethoxycurcumin, Chavicine, Cirsimaritin, Demethoxycurcumin and Apigenin. Their

predicted binding energies are -9.7, -9.6, -9.3, -9.2, -9.2, -9.2, -9.2, -8.8, -8.8, -8.8, -8.8, and -

8.7 kcal/mole respectively. Among the phytochemicals tested, the following molecules were

predicted to bind with Nsp9 RNA binding protein of SARS CoV-2 (PDB ID: 6w4b) protein,

Withanone, Withanolide B, Withaferin A, Withanolide, Beta-sitosterol, Campesterol,

Stigmasterol, Flavonol glucoside and Somniferine A. Their predicted binding energies are -9.1,

-9.0, -8.2, -8.1, -8.0, -8.0, -7.9, -7.5 and -7.5 kcal/mole respectively.

More than ten phytochemicals were predicted to interact with SARS-CoV-2 RNA binding

domain of nucleocapsid protein (PDB ID: 6vyo) viz, Withanolide, Withaferin A, Somniferine A,

Withanone, Withanolide B, Ursolic acid, Crategolic acid, Quercetin, Stigmasterol,

Tinocordioside, Tinosporaside and Oleanolic acid. Their predicted binding energies are -8.7, -

8.3, -8.2, -8.2, -8.0, -7.8, -7.7, -7.7, -7.7, -7.7, -7.7 and -7.6 kcal/mole respectively.

An array the phytochemicals (having binding energy ≤ 7.5 kcal/mole) were also predicted to

interact with SARS-CoV main protease (PDB ID: 4mds) viz, Somniferine A, Withanolide B,

Withanone, Withanolide, Oleanolic acid, Cordioside, Crategolic acid, Tinosporaside, Calebin

A, Ursolic acid, Stigmasterol, Orientin, Pentadienoylpiperidine, Campesterol, Cyclocurcumin,

Rosmarinic acid, Tinosporide, Withaferin A, Demethoxycurcumin, Jatrorrhizine, Piperettine,

Tinocordioside and Trichostachine. Their predicted binding energies are -9.3, -8.9, -8.5, -8.3,

-8.2, -8.1, -8.1, -8.1, -8.0, -8.0, -7.9, -7.8, -7.8, -7.7, -7.7, -7.7, -7.7, -7.7, -7.6, -7.5, -7.5, -7.5 and

-7.5 kcal/mole respectively. Similarly, the phytochemicals (having energy ≤7.5 kcal/mole)

which may interact with SARS-CoV-1 spike protein (PDB ID: 2ajf) include, Withanolide,

Stigmasterol, Withanolide B, Somniferine A, Ursolic acid, Beta-sitosterol, Oleanolic acid,

Pentadienoylpiperidine, Piperettine, Tinosporide, Withanone, Bisdemethoxycurcumin,

Calebin A, Campesterol, Cyclocurcumin, Piperine, Retrofractamide A, Trichostachine and

Quercetin. Their predicted binding energies are -8.7, -8.6, -8.6, -8.5, -8.2, -8.0, -7.9, -7.9, -7.9,

-7.8, -7.8, -7.6, -7.6, -7.6, -7.6, -7.6, -7.6, -7.6 and -7.5kcal/mole respectively. Many of the

phytochemicals (having energy ≤7.5 kcal/mole) were also predicted to interact with SARS-

CoV-1 nucleocapsid protein (PDB ID: 2cjr) viz, Ursolic acid, Somniferine A, Oleanolic acid,

Magnoflorine, Withanolide B, Withanolide, Withanone, Orientin, Withaferin A, Quercetin,

Rhamnetin and Isothymusina. Their predicted binding energies are -9.0, -8.6, -8.4, -8.2, -8.2,

-7.9, -7.8, -7.7, -7.7, -7.6, -7.6 and -7.5 kcal/moles respectively.

Table 7 shows the predicted binding energy of different phytochemicals with molecules

involved in the inflammatory processes such as COX2, PLA2, NIK and IRAK-4. From Table 7, it

is clear that Withaferin A, Withanolide B, Withanolide, Withanone, Campesterol,

Cyclocurcumin,Somniferine A, Stigmasterol, Eriodictyol, Isopiperine, Oleanolic acid,

Rhamnetin, Orientin, Quercetin, Piperine and Vicenin may possess high binding affinity with

most of the inflammatory molecules used in the study. Our study proposes that many of the

phytochemicals present in these herbs may directly inhibit COX-2, PLA2 and IRAK-4 which are

involved in the inflammation. Many of these phytochemicals may also bind to the active site

of the protein and at the same time few of them may have very high affinity for other regions

of the target protein which may affect its function. Our study also shows that several

phytochemicals present in the Kadha have significant predicted inhibitory activity towards

NIK. Inhibition of the NIK by phytochemicals may moderate the NF-κB dependent genes which

are involved in inflammation. Based on these docking studies, it may be predicted that the

phytochemicals present in the Kadha may exhibit anti-inflammatory property.

Discussion

Use of herbs and phytochemicals has a long history in the management of various respiratory

diseases (Santana et al., 2016; Alamgeer et al., 2018; Pinn et al., 2001). Currently, the demand

of complementary medicine, including herbal medicine has become more popular in

healthcare for both general maintenance of health and for treatment of minor illnesses

(Barnes, 2004). In European countries, several species of herbs have been used against flu

and common cold (Weiss and Fintelmann, 2000). Similarly, in Russia and Estonia various herbs

and medicinal plants have also been used for centuries for management of common cold and

flu (Raal et al., 2013). In India, use of spices and herbs for treatment of various diseases

including cough, cold is a common practice with recorded history of over 2000 years (Vasanthi

and Parameswari, 2010, Sachan et al., 2018).

It is well-known that SARS-CoV viral genome encodes more than 20 proteins, among which

two proteases i.e 3-chymotrypsin-like protease (3CLpro, main protease, Mpro) and papain-

like protease (PLpro) are vital for virus replication (Lindner et al. 2005). They cleave the two

translated polyproteins (PP1A and PP1AB) into individual functional components, resulting in

release of 16 non-structural proteins (NSPs) (Jo et al., 2020). Thus SARS-CoV-2 main protease

is considered as a promising druggable target. The viral NSPs play an important role in

replication and transcription. Our study predicts that many of the phytochemicals of Kadha

have significant binding affinity with the main protease. Twelve phytochemicals namely

Somniferine A, Tinosporide, Tinocordioside, Orientin, Flavonol glucoside, Withanolide,

Apigenin, Cyclocurcumin, Withanolide B, Kaempferol, Withanone and Withaferin A, have

predicted binding energy lower than the pharmacological inhibitor, N3 (Fig 1, Table 2). The

binding of these phytochemicals with main protease may slow down the cleavage of PPs to

releases NSPs and decrease the process of viral replication and transcription.

The SARS-CoV spike protein plays an important role in virus entry into the host (Li, 2016).

Initial interactions between the S1 domain and its host receptor (ACE2), and subsequent S2

segment mediated fusion of the host and viral membrane allows the viral RNA genome to

enter inside the host cells. Thus, these proteins represent as important targets for designing

drug (Li, 2016). Our study predicts that an array of the phytochemicals have significant binding

affinity with the SARS-CoV-2 spike proteins (Table 3, Figure 2a) as well as with host ACE2

https://cmjournal.biomedcentral.com/articles/10.1186/s13020-018-0204-y#auth-1

protein (Table 4, Figure 2b) and furin protein (which facilitate spike and ACE2 interaction)

(Table 5, Figure 3). Thus, phytochemicals may significantly inhibit viral interaction with the

host receptor and slow down or stop the entry of the viral genome inside the host. The spike

protein is also known to activate the immune response of the host cell towards CoVs (Li,

2016). The S1 domain of spike acts as a major antigen on the surface of the virus (Yuan et al.,

2016).

SARS-CoV nucleocapsid protein (SARS-CoV NP) is another vital structural protein which shows

intrinsic multimerization and interacts with M protein, suggesting that NP is both critical to

formation of the viral nucleocapsid core and is involved in virion assembly (He et al. 2004, He

et al. 2004). Our study predicts that the phytochemicals such as Withanone, Withanolide B,

Withanolide, Withaferin A, Ursolic acid, Tinosporaside, Tinocordioside, Stigmasterol,

Somniferine A, Quercetin, Oleanolic acid and Crategolic acid have significant binding energy

with nucleocapsid protein (Table 6). The N protein of SARS-CoV are also known to up-regulate

the expression of the proinflammatory protein COX2 and also interact with the proteasome

subunit p42, which affects a variety of basic cellular processes and inflammatory responses

(Hu et al. 2017; Wang et al. 2010).

Nsp3 protein is another key component for Coronavirus and is essential for

replication/transcription complex (RTC) formation. It plays various roles in Coronavirus

infection. It releases Nsp1, Nsp2, and itself from the polyproteins and interacts with other

viral Nsps as well as RNA to form the replication/ transcription complex (Leia et al. 2018).

Similarly, Nsp15 protein also acts as an endoribonuclease and preferentially cleaves 3’ of

uridylates through a ribonuclease A (RNase A)-like mechanism and also facilitates viral

replication and transcription (Alcantara et al. 2010; Sawicki and Sawicki, 1998). Our study

predicted that many of the phytochemicals of the Kadha have significant binding affinity with

Nsp3 and Nsp15. Thus, the phytochemicals identified in Table 6 may disrupt the formation of

RTC and stop the viral genome replication (Table 6).

During the SARS-CoV infection, human lung epithelial cells are among the first targets for viral

entry. In response to viral multiplication and host cell damage, lung epithelial cells secrete

inflammatory mediators to initiate and exacerbate host innate inflammatory responses,

causing detrimental immune-mediated pathology within the lungs. SARS-CoV-2 infects ACE-2

expressing epithelial cells in the air sacs (alveoli) in lower lungs. The damaged epithelium leads

and interlobular septal thickening lead to leaky cell junctions, accumulation of fluid which is

rich in proteins, inflammatory mediators and Pneumonia. The spread of virus from lung to

systemic circulation and excessive production of pro-inflammatory mediator can damage

different vital organs. Our study predicts that an array of the phytochemicals such as

Withaferin A, Withanolide B, Withanolide, Withanone, Campesterol,

Cyclocurcumin,Somniferine A,Stigmasterol, Eriodictyol, Isopiperine, Oleanolic acid,

Rhamnetin, Orientin, Quercetin, Piperine, Vicenin etc. found in the preparation of the Kadha,

have significant binding affinity with the many of these inflammatory mediators or the

molecules involved in this process (Table 7). It is well known that, NF-κB is the master

regulator for several genes such as COX-2, VEGF (vascular endothelial growth Factor), pro-

inflammatory cytokines (IL-1, IL-2, IL-6, and TNFα), chemokines (e.g., IL-8, MIP-1α, and MCP-

1), adhesion molecules, immunoreceptors, growth factors, and other agents involved in

proliferation and invasion. NF-κB activation is mediated by two distinctly different redox-

related signalling pathways. The first pathway involves NIK/IKK whereas second pathway

involve MAPKs and both causes the induction of transcriptional activation of NF-κB. NF-κB-

inducing kinase (NIK) is a key mediator of the non-canonical NF-κB signalling pathway. NF-κB

is also one of the main inducible transcription factors shown to respond directly to oxidative

stress. Oxidative stress activates nuclear factor-inducing kinase (NIK)/IκB kinase (IKK) and

mitogen-activated protein kinases (MAPKs). NIK/IKK and MAPK pathways activate NF-κB and

migrates to the nucleus and binds to κ elements on DNA in enhancers and promoter regions.

Various herbs have potential to control inflammation-associated disease by decreasing the

production of the pro-inflammatory mediators by suppressing pro-inflammatory pathways

(Pan et al., 2011). Our study also predicted that phytochemicals found in the Kadha have

significant binding affinity with NIK (Table 7) which can stop NF-κB mediated downstream

events. In a very recent study, Huang et al (2020) have shown that the patients infected with

SARS-CoV-2 had high amounts of IL1, IFNγ, IP10, and MCP1, which can mediate cytokine

storm associated multi-organ damage. At the same time, SARS-CoV-2 infection also initiates

increased secretion of T-helper-2 (Th2) cytokines (eg, IL4 and IL10) which suppress

inflammation (Huang et al., 2020). The increased secretion of inflammatory mediators was

also associated with moderation of helper T cell responses in COVID19 patients.

Taken together, our current findings and the recent knowledge about SARS-CoV and SARS-

CoV-2 pathology, profess the use of Ayurvedic Kadha in the prevention and management of

COVID-19. The phytochemicals found in the Kadha have significant binding affinity with the

different CoVs proteins (Scheme 1), indicating that they may control viral infection and

multiplication in the host cells. Molecular docking study with human inflammatory mediators

predicts that many of the phytochemicals present in this preparation have significant anti-

inflammatory property. Most of the phytochemicals found in the herbs ashwagandha, giloy,

tulsi, clove and black pepper have potential to interact with most of the druggable proteins

selected in this study. In conclusion, regular consumption of ayurvedic Kadha in consultation

with ayurvedic practitioner may decrease the inflammatory response, boost the individual’s

immunity and reduce the risk of CoVs infection including SARS-CoV-2.

Acknowledgement: The authors would like to thank Dr S. Santosh Kumar, Radiation

Biology & Health Sciences Division, Bhabha Atomic Research Centre for his valuable

constructive suggestions and critical review of the article. The authors also acknowledge Mrs.

Sunitee Maurya (M.Sc, Chemistry) for her valuable constructive suggestions in formulation of

this work.

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of

interest with respect to the research, authorship, and/or publication of this article.

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Legends

Figure 1: SARS-CoV-2 main protease (PDB ID: 6LU7) showing top 12 phytochemicals

superimposed on it active site.

Figure 2: Selected phytochemicals superimposed on SARS-CoV-2 spike and human ACE2. a)

SARS-CoV-2 spike, b) Human ACE2.

Figure 3: Selected phytochemicals superimposed on human furin (PDB ID: 5MIM).

Scheme 1: Possible targets for the phytochemicals found in the Kadha against different SARS-

CoV-2 proteins.

Table 1: The predicted binding of different phytochemicals against SARS-CoV-2 main

protease, spike protein, human ACE2 and furin proteins. Only those phytochemicals name are

included in the table which have binding energy ≤ 7.5 kcal/mole.

Table 2: The predicted binding and 2D interaction of top 12 phytochemicals against SARS-

CoV-2 main protease (PDB ID: 6LU7) and their different protein residues which are involved

in the interaction.


CoV-2 spike protein (PDB ID: 6VXX) and their different protein residues which are involved in

the interaction.

Table 4: The predicted binding and 2D interaction of top 10 phytochemicals against human

ACE2 protein (PDB ID: 2AJF) and their different protein residues which are involved in the

interaction.


furin protein (PDB ID: 5MIM) and their different protein residues which are involved in the

interaction.

Table 6: The predicted binding of different phytochemicals against SARS-CoV-2 and SARS-CoV

target proteins. Only those phytochemicals name are included in the table which have binding

energy ≤ 7.5 kcal/mole.

Table 7: The predicted binding of different phytochemicals against different anti-

inflammatory macromolecules. Only those phytochemicals name are included in the table

which have binding energy ≤ 7.5 kcal/mole.



Somniferine A

Tinosporide

Tinocordioside

Orientin

Flavonol glucoside

Withanolide

Apigenin

Cyclocurcumin

Withanolide B

Kaempferol Withanone

Withaferin A



a.

b.

Withanolide

Somniferine A

Apigenin

Withanolide B

Ursolic acid

Hesperetin

Campesterol Crategolic acid

Withanone

Chavicine

Rosmarinic acid

Stigmasterol.


CoV-2 proteins.

Compounds

COVID-19 main

protease (PDB

ID: 6lu7)

SARS-CoV-2 spike

protein (PDB ID:

6vxx)

Human ACE2

(PDB ID: 2ajf)

Human Furin (PDB

ID: 5mim)

Native ligand (N3) -7.6 -9.3

Apigenin -7.8 -7.5 -8.5

Berberine -7.5 -7.8

Beta-sitosterol -7.6 -8.3 -7.6

Bisdemethoxycurcumin -8.1 -7.7

Brachyamide B -7.7 -7.7

Campesterol -8.3 -0.1

Chavicine -8

Cordifolioside A -7.7 -7.5

Cordioside -7.8

Crategolic acid -7.6 -7.5 -8.6 -8.1

Curcumin -7.8 -7.8

Cyclocurcumin -8.3

Demethoxycurcumin -7.6

Eriodictyol -7.7

Flavonol glucoside -8 -7.5

Hesperetin -7.7 -8.2

Isochavicine -7.8 -7.5

Isopiperine -7.6

Isothymonin -7.6

Jatrorrhizine -7.5

Kaempferol -7.7 -7.9

Oleanolic acid -8.5 -7.9

Orientin -8.1 -8 -7.9

Piperettine -8 -7.8

Pipericide -7.6 -7.5

Piperolein b -7.6

Quercetin -8

Retrofractamide A -7.5

Rhamnetin -7.5

Rosmarinic acid -8

Somniferine A -8.6 -7.8 -9 -8.7

Stigmasterol -8.8 -8.5

Tinocordifolioside -7.6

Tinocordioside -8.1 -8.5 -8

Tinosporaside -8.1 -8

Tinosporide -8.6 -8.3 -7.6

Target molecules; binding energy (kcal/mole) (Only those

phytochemcals are included in the table which have binding energy ≤ -

7.5)

Phytochemicals

Table 1

Trichostachine -7.8

Ursolic acid -7.7 -7.5 -8.7 -8.3

Vicenin -8.8 -7.7

Withaferin A -7.6 -8.2 -9.1 -7.9

Withanolide -8 -8.4 -9.2

Withanolide B -7.8 -8.7 -8.7 -8.4

Withanone -7.6 -7.9 -8.5 -8.1

Table 2: 2D interaction of top 12 phytochemicals with SARS-CoV-2 main protease (PDB ID:

6LU7)

S. No.

Name of the molecules 2D interaction between molecules and protein

Kinds of the binding

1 Native ligand N3 Binding energy= -7.6

Convention hydrogen bonding: THR45, SER46, LEU141, CYS145, GLN189 Van der vaals: THR25, THR26, LEU27, HIS41, PHE140, ASN142, GLY143, SER144, HIS163, MET165, GLU166, LEU167,ASP187, ARG188, THR190, GLN192 Pi-alkyl: MET49, PRO168 Pi-Sigma: HIS41 Pi-Sulphur: PRO168

2 Somniferine A Binding energy= -8.6

Convention hydrogen bonding: SER46, ASN142, GLU166 Van der vaals: THR24, THR25, THR26, THR45, GLY143, SER144, HIS163, HIS164, MET165, Pi-alkyl: HIS41, CYS145 Pi-sulphur: MET49 Carbon Hydrogen bond: PHE140, LEU141

3 Tinosporide Binding energy=-8.6

Convention hydrogen bonding: HIS163 Van der vaals: THR25, THR26, LEU27, MET49, PHE140, ASN142, GLY143, HIS164, GLU166, HIS172 Pi-alkyl: HIS41 Amide –Pi stacked: EU141, MET165 Pi-Sulphur: CYS145

4 Orientin Binding energy=-8.1

Convention hydrogen bonding: ASN142, GLU166, ARG188, THR190 Van der vaals: HIS41, MET49, PHE140, LEU142, GLY143, HIS163, HIS164,LEU167, PRO168, GLN189, ALA191, GLN192, HIS172,ALA191, GLN192 Pi-alkyl: CYS145, MET165

5 Tinocordioside Binding energy=-8.1

Convention hydrogen bonding: THR45, GLA143, SER144 Van der vaals: THR25, THR26, LEU27,SER46, PHE140, LEU141, ASN142, CYS145, MET165, GLU166, HIS172 Pi-alkyl: HIS41, MET49 Pi-amide: THR24

6 Flavonol glucoside Binding energy=-8.0

Convention hydrogen bonding: CYS145, GLU166 Van der vaals: LEU27, PHE140, LEU141, ASN142, GLY143, SER144, HIS163, HIS164, HIS172, ASP187, ARG188, GLN189 Pi-alkyl: MET49, MET165 Pi-Sulphur: CYS145

7 Withanolide Binding energy=-8.0

Convention hydrogen bonding: ARG40, PHE181, ARG188 Van der vaals: ASN53, TYR54, GLU55, ASN88, ARG105, ASN180, GLY183, PRO184, PHE185 Pi-alkyl: CYS85, PHE181, VAL186

8 Apigenin Binding energy=-7.8

Convention hydrogen bonding: LEU141, HIS163, ASP187 Pi donor hydrogen bond: GLU166 Van der vaals: HIS41, TYR154, PHE140, ASN142, SER144, HIS164, MET165, GLN89, ARG188, Pi-alkyl: MET49 Pi-Sulphur: CYS145

9 Cyclocurcumin Binding energy=-7.8

Convention hydrogen bonding: HIS163 Van der vaals: THR25, THR26, LEU27, VAL42, MET49, PHE140, LEU142, ASN142, GYL143, SER144, GLU166, LEU167, HIS172, ARG188, GLN189, THR190, ALA91, GLN192, Pi-cation:HIS41 Pi-sulphur: MET65 Pi-Donor hydrogen bond: CYS145

10 Withanolide B Binding energy=-7.8

Convention hydrogen bonding: THR45, ASN42, GLY43, SER144, CYS145 Van der vaals: THR24, THR25, THR26, SER46,PHE140, LEU142, HIS164, MET165, GLU166 Pi-alkyl: HIS41, MET49 Carbon-Hydrogen bond: GLN189

11 Cordifolioside A Binding energy=-7.7

Convention hydrogen bonding: LEU142, GLY143, SER144, HIS163, ARG188, THR190 Van der vaals: THR25, LEU27, MET49, PHE140, ASN142, CYS145, HIS164, GLU166, PRO168, LEU167, VAL186, GLN189, GLN192 Pi-alkyl: MET165 Carbon-Hydrogen bond: THR26

12 Kaempferol Binding energy=-7.7

Convention hydrogen bonding: ASP187 Van der vaals: PRO52, TYR54, PHE140, LEU141, ASN142, SER144, HIS164, ARG188, GLN189 Pi-alkyl: MET49 Pi-Sulphur: CYS145, MET165 Pi-Donor hydrogen bond: GLU166 Pi-Pi stacked: HIS41

13 Ursolic acid Binding energy=-7.7

Convention hydrogen bonding: SER144 Van der vaals: THR25, HIS41, LEU141, GLY143, HIS164, MET165, GLU166, PRO168, GLN189, HR190 Pi-alkyl: MET49, CYS145 Carbon-Hydrogen donor: ASN142

Table 3: 2D interaction of top 12 phytochemicals with SARS-CoV-2 Spike (PDB ID: 6VXX)

S. No. Name of the molecules

2D interaction between molecules and protein



Van der vaals: ILE418, ASP420, ARG454, ASP467, GLN493 Pi-alkyl: LYS417, TYR453, PRO491 Pi-Sigma:TYR421


Van der vaals: ILE418, ARG454, ASP467, TYR489, GLN493 Pi-alkyl: LYS417, TYR453, PRO491 Pi-Sigma: TYR421

3 Withaferin A Binding energy=-8.2

Van der vaals: ILE418, ARG454, ASP467, TYR489, GLN493 Pi-alkyl:CYS417, TYR453, PRO491 Pi-Sigma: TYR421

4 Withanone Binding energy=-7.9

Convention hydrogen bonding: SER530 Van der vaals: ASN331, THR333, LEU335, ASP364, GLY526, LYS528, GLN580 Pi-alkyl: VAL362, PRO527, LYS529

5 Somniferine A Binding energy=-7.8

Convention hydrogen bonding: ARG346 Van der vaals: GLU340,THR345, PHE347, ALA348, SER349, ASN354, LYS356, SER399, ASN448, ASN450, TYR451 Pi-alkyl: ALA344

6 Beta-sitosterol Binding energy=-7.6

Van der vaals:CYS336, GLY339, ASN343, ALA363, ASP364, SER371, SER373 Pi-alkyl: LEU335, PHE338, PHE342, VAL362, VAL367, LEU368, PHE374, PRO527

7 Pipericide Binding energy=-7.6

Convention hydrogen bonding: ASP364, TRP436 Van der vaals: CYS336, PHE338, GLY339, PHE342, ASN343, VAL362, ALA363, LEU368, SER371, SER373, PHE374, ASN437, SER438, ASN440, LEU441, ARG509 Pi-alkyl: LEU335, VAL367

8 Crategolic acid Binding energy=-7.5

Convention hydrogen bonding: THR376, VAL503 Van der vaals: TYR380, GLY404, ASP405,ILE410, GLUY504, TYR508 Pi-alkyl: LYS378, VAL407, ARG408, ALA411, VAL433

9 Retrofractamide A Binding energy=-7.5

Convention hydrogen bonding: ASP364 Van der vaals: LEU335, CYS336, PHE338, GLY339, ASN343, PHE342, VAL367, LEU368, SER373 Pi-Pi: TRP436


Convention hydrogen bonding: ASP364 Van der vaals: PHE329, PRO330, ASN331, ILE332, THR333, GLY526, LYS528, SER530, GLN580, Pi-alkyl: LEU335, VAL362, PRO527, LYS529

11 Piperolein B Binding energy=-7.4

Van der vaals: LEU335, PHE338, GLY339, PHE342, ASP364, PHE374,SER438, ARG509 Carbon hydrogen bond: CYS336, ASN343, SER373 Pi-alkyl: LEU368, LEU441 Pi-Sigma: VAL367, TRP436


Convention hydrogen bonding: PRO330, SER530 Van der vaals: PHE329, ASN331, ILE332, THR333, LEU335, GLY526, PRO527, LYS528, GLN580 Pi-alkyl: VAL362, LYS529

Table 4: 2D interaction of top 10 phytochemicals with human ACE2 (PDB ID: 2AJF)

S. No. Name of the molecules 2D interaction between molecules and protein


1 Withaferin A Binding Energy:-9.1

Convention hydrogen bonding: AL99, ASP350, ASN394 Van der vaals: PHE32, SER77, GLN102, ASP382, TYR385, ARG393 Pi-alkyl: PHE40, LEU73, LEU100, PH1390, PHE391

2 Stigmasterol Binding Energy:-8.8

Convention hydrogen bonding: SER77, GLN102 Van der vaals: PHE32, TRP69, ALA99, LEU100, ASP350, TYR385, LEU391 Pi-alkyl: PHE40, LEU73, ARG393 Pi-Sigma: PHE390

3 Vicenin Binding Energy:-8.8

Convention hydrogen bonding: ASP350, HIS378, TYR385, ARG393, ASN394, HIS401 Van der vaals: PHE40, THR347, ALA348, TRP349, GLU375, PHE390, LEU391, GLY395, GLU402 Pi-Pi stack: HIS401 Pi-cation: ASP382

4 Ursolic acid Binding Energy:-8.7

Convention hydrogen bonding: ASN394, HIS01 Van der vaals: SER43, SER44, SER47, ALA348, ASP350, ASP382, TYR385 Pi-alkyl: PHE40, TRP349, HIS378 Pi-Sigma: HIS378

5 Withanolide B Binding Energy:-8.7

Pi-alkyl: PHE40, ALA348, ARG393, HIS401 Van der vaals: THR347, ASP350, ASP382, TYR385, ASN394, GLU402 Pi-Sigma: HIS378, PHE390

6 Oleanolic acid Binding Energy:-8.5

Van der vaals: SER77, LEU100,GLN102,GLY104,LEU392, ASN394, LYS562 Pi-alkyl: PHE40, TRP69, LEU73, ALA99, PHE390, LEU391

7 Withanone Binding Energy:-8.5

Convention hydrogen bonding: ALA348 Van der vaals: SER44, SER47, LEU351, HIS378, HIS401, FLU402 Pi-alkyl: TRP349 Pi-Sigma: PHE40

8 Beta-sitosterol Binding Energy:-8.3

Convention hydrogen bonding: LEU100 Van der vaals: PHE32, TRP69, SER77, ALA99, GLN102, ASP350, TYR385, LEU391, ASN394 Pi-alkyl: PHE40, PHE73, PHE390, ARG393

9 Campesterol Binding Energy:-8.3

Convention hydrogen bonding: LEU100 Van der vaals: PHE32, TRP69, SER77, ALA99, ASP350, GLY352, ASN394 Pi-alkyl: PHE40, LEU73, TYR385, PHE390, LEU391, ARG393

10 Orientin Binding Energy:-8.0

Convention hydrogen bonding: PHE390, ARG393 Van der vaals: PHE40, SER43, SER44, SER47, ASP350, ASP382, TYR385, LEU391, ASN394, HIS401

Table 5: 2D interaction of top 12 phytochemicals with human furin (PDB ID: 5MIM)



1 Native ligand, 1N Binding energy= -9.3

Convention hydrogen bonding: LEU227, GLY255, PRO256, ASP258, THR308, THR365 Van der vaals: ARG185, ASN192, GLY229, SER253, TRP254, GLU257, GLY265, ASN295, HIS364,GLY366, SER368, Pi-alkyl: VAL231 Attractive charge: ASP154, ASP191, ASP228, ASP258, ASP264


Convention hydrogen bonding: PRO266, ASN29, ALA532 Van der vaals: VAL263, ASP264, GLY265, ALA267, GLU271, GLY307, ASN310, SER311, ARG498, ASP526, GLY527, PHE528 Pi-alkyl: TRP531


Convention hydrogen bonding: GLY255 Van der vaals:LEU227, SER253, TRP254, ASP258, ASP259, GLY294, ASN295, GLY296,ARG298, GLU299, TRP328, SER368 Carbon hydrogen bond: ALA292, ASP306


Convention hydrogen bonding: GLU271, ASN310, ALA532 Van der vaals: GLY265, PRO266, GLY307, SER311, ILE312, TYR313, GLN488, MET534 Pi-alkyl: ALA532 Pi-Pi: TRP531


Convention hydrogen bonding: SER330 Van der vaals: GLY297, HIS300, ASP301, ASN325, TRP328, GLU331, ALA332, ASN407, ALA408, ASN409, SER423 Pi-alkyl: VAL326


Convention hydrogen bonding: ASP154, ASN192, HIS194, GLY255 Van der vaals: GLU236, PRO256, GLU257 Pi-alkyl: LEU227, VAL231, TRP254

7 Hesperetin Binding energy=-8.2

Convention hydrogen bonding: PRO266, GLU271, ASN310, ALA532 Van der vaals: GLY265, ALA267, PHE274, GLY307, SER311, ILE312, TYR313, GLN488, MET534 Pi-alkyl: TRP531

8 Campesterol Binding energy=-8.1

Van der vaals: ASP264, GLY265, GLU271, GLY307, ASN310, SER311, GLN488, ARG498, GLY527, PHE528, ASN529, ASP530 Pi-alkyl:VAL263, PRO266, TRP531, ALA532


Convention hydrogen bonding: ASP154 Van der vaals: ASP153, ASP191, ASP192, GLU236, GLY255, PRO256, GLU257, ASP258 Pi-alkyl: HIS194, LEU227, VAL231, TRP254


Convention hydrogen bonding: ASP154, HIS194, ASN295 Van der vaals: ASP153, ASP191, ASN192, LEI227, ASP228, ASP258, SER368 Pi-alkyl:TRP328 Pi-Sigma: HIS194

11 Chavicine Binding energy=-8.0

Convention hydrogen bonding: GLN488 Van der vaals: GLY265, PRO266, GLU271, GLY307, ASN310, SER311, ILE312, TYR313, ASN529,KTRP531 Pi-alkyl: ALA532

12 Rosmarinic acid Binding energy=-8.0

Convention hydrogen bonding: VAL263, GLY307, GLY527 Van der vaals: ASP264, GLY265, PRO266, ASN310, SER311, ARG498,PHE528, ASN529 Pi-alkyl: ALA532 Pi-Pi: TRP531

13 Stigmasterol Binding energy=-8.0

Convention hydrogen bonding: SER311 Van der vaals: ASP264, GLY265, GLU271, GLY307, ILE312, TYR313, GLN488, ARG498, GLY527, PHE528, ASN529, ASP530 Pi-alkyl: VAL263, PRO266, TRP531, ALA532

Phytochemicals6vww (SARS-

CoV-2 NSP15

Endoribonucle

ase )

6w02 (SARS-CoV-2

ADP ribose

phosphatase of

NSP3)

6vyo (SARS-

CoV-2

nucleocapsid

RBD)

6w4b (SARS-

CoV-2 Nsp9

RBP)

4mds

(SARS-CoV

3CLpro)

2ajf (SARS-

CoV spike)

2cjr-SARS-CoV

Nucleocapsid

1-dehydrogingerdione -7.5 -7.5

6-gingerdione -7.5

6-gingerol -7.5

Apigenin -9 -8.7

Berberine -8.2 -9.7

Beta-sitosterol -8.6 -7.6 -8 -8



Calebin A -8.1 -8 -7.6

Campesterol -8.5 -7.9 -8 -7.7 -7.6

Chavicine -7.6 -8.8

Cirsilineol -7.6 -8.3

Cirsimaritin -7.6 -8.8

Cordifolioside A -7.7

Cordioside -8.1 -8.1


Curcumin -8.6 -8.2

Cyclocurcumin -8.3 -9.2 -7.7 -7.6

Demethoxycurcumin -8.8 -7.6

Eriodictyol -9 -8.4

Eugenitin -7.6

Flavonol glucoside -8.5 -8.6 -7.5

Guineensine -7.8

Hesperetin -9 -8.5

Isochavicine -7.5

Target molecules; binding energy (kcal/mole) (Only those phytochemcals are included in

the table which have binding energy <- 7.5)

Table 6

Isopiperine -8.1 -7.8

Isothymonin -7.6

Isothymusin -7.6 -8 -7.5

Jatrorrhizine -7.9 -7.5

Kaempferol -8 -8.6

Magnoflorine -7.9 -8.2

Oleanolic acid -9 -7.7 -7.6 -8.2 -7.9 -8.4

Orientin -9.4 -8.2 -7.8 -7.7

Pentadienoylpiperidine -8.7 -8.6 -7.8 -7.9

Piperamine -7.7

Piperettine -7.9 -9.2 -7.5 -7.9

Pipericide -7.6

Piperine -8.7 -7.6

Piperolein B -8.7

Quercetin -8.1 -8.3 -7.7 -7.5 -7.6

Retrofractamide A -7.9 -7.8 -7.6

Rhamnetin -7.9 -8.4 -7.6

Rosmarinic acid -7.5 -9.3 -7.7

Somniferine A -8.5 -8.3 -8.2 -7.5 -9.3 -8.5 -8.6

Stigmasterol -8.9 -7.9 -7.7 -7.9 -7.9 -8.6

Tinocordifolin -7.5 -7.6

Tinocordioside -7.7 -7.5

Tinosporaside -7.6 -7.7 -8.1

Tinosporide -8.1 -8.2 -7.7 -7.8

Tricholein -8.1

Trichostachine -8.4 -8.6 -7.5 -7.6

Umbelliferone -7.5

Ursolic acid -9.1 -7.6 -7.8 -8 -8.2 -9

Vicenin -7.8 -7.6

Withaferin A -9.1 -9.2 -8.3 -8.2 -7.7 -7.7

Withanolide -9.3 -9.6 -8.7 -8.1 -8.3 -8.7 -7.9

Withanolide B -9.2 -9.2 -8 -9 -8.9 -8.6 -8.2

Withanone -8.9 -8.3 -8.2 -9.1 -8.5 -7.8 -7.8

Compounds

COX2 (PDB ID:

5f1a)

PLA2 (PDB

ID:4uy1) NIK(PDB ID:4dn5)

IRAK-4 (PDB

ID:2nru)

Native ligand -6 -7.7 -6.9 -9.4

Apigenin -9.2 -9.5 -8.3

Berberine -9 -8.4 -9

Beta-sitosterol -8.7 -9.7

Bisdemethoxycurcumin -9.4 -8.5 -8.6


Calebin-A -9.1 -8.5

Campesterol -8.7 -8.1 -9.5 -9.9

Chavicine -8.2 -8.4 -8.3

Cirsilineol -8.8 -8.8 -8.4

Cirsimaritin -8.9 -9.1 -8.4


Cordioside -9.6

Crategolic acid -8.9 -8.3 -8.1

Curcumin -8.2 -8 -8.4

Cyclocurcumin -9.3 -8.4 -8.8 -9.4

Demethoxycurcumin -8.9 -8 -8.9

Eriodictyol -9.7 -8.1 -9.7 -8.7

Eugenitin -8

Flavonol glucoside -8.9 -8.7

Guineensine -8.7

Hesperetin -8.9 -9.4 -8.4

Isochavicine -9.3 -8.7 -8.9

Isopiperine -8.6 -8.1 -8.7 -8.9

Isothymonin -9 -8.8 -8.3

Isothymusin -8.7 -8.9 -8.4

Jatrorrhizine -9.1 -9

Kaempferol -8.9 -8.6 -8.1


Oleanolic acid -8.9 -8 -8.2 -9.2

Orientin -9.6 -8.9 -8.6 -9.5

Pentadienoylpiperidine -8.7 -8.3

Piperamine -8.1 -8.3

Piperettine -8.5 -9.1

Piperine -8.7 -8.5 -8.1

Piperolein b -8.6

Quercetin -9.6 -8 -9.1 -8.4

Retrofractamide A -8

Rhamnetin -9.1 -8 -9.3 -8.9


phytochemcals are includen in the table which have binding energy <-

8.0)

Phytochemicals

Table 7


Somniferine A -9.2 -8.5 -8.6 -8.4

Stigmasterol -9.6 -8 -8.2 -9.8

Tinocordifolioside -8.1 -9.6


Tinosporaside -8

Tinosporide -8

Trichostachine -8.5 -8.1

Ursolic acid -8.9

Vicenin -8.8 -8.5 -10.1

Withaferin A -8.8 -9.1 -8.7 -9.2

Withanolide -9.4 -9.7 -8.6 -8.2

Withanolide B -9.3 -9.4 -8.4 -11.5

Withanone -10 -8.1 -8.7

download fileview on ChemRxivComplete Manuscript 10042020.pdf (4.45 MiB)



Evaluation of traditional ayurvedic preparation for prevention and

management of the novel Coronavirus (SARS-CoV-2) using molecular docking

approach

Dharmendra Kumar Maurya 1, 2,* and Deepak Sharma1, 2

1Radiation Biology & Health Sciences Division, Bhabha Atomic Research Centre,

Mumbai 400 085, India

2Homi Bhabha National Institute, Anushaktinagar, Mumbai-400094, India

*Corresponding authors

Dharmendra Kumar Maurya, Ph.D

Scientific Officer/F

Free Radical Biology Section, Radiation Biology & Health sciences Division

Bhabha Atomic Research Centre, Mumbai 400085, Maharashtra, INDIA

Email: [email protected], [email protected]

Tel: +91-22-25590407



Abstract

Since the emergence of novel Coronavirus (SARS-CoV-2) infection in Wuhan, China in

December 2019, it has now spread to over 205 countries. The ever-growing list of globally

spread corona virus-19 disease (COVID19) patients has demonstrated the high transmission

rate among human population. Although 12 new drugs are being tried for management of

COVID19, currently there are no FDA approved drugs or vaccines to prevent and treat the

infection of the SARS-CoV-2. Considering the current state of affairs, there is an urgent unmet

medical need to identify novel and effective approaches for prevention and treatment of

COVID19 by re-evaluating the knowledge of traditional medicines and repurposing of drugs.

Here, we used molecular docking approach to explore the beneficial roles of an array of

phytochemicals and active pharmacological agents present in the Indian herbs (Tulsi, Haldi,

Giloy, Black pepper, Ginger, Clove, Cardamom, lemon, and Ashwagandha) which are widely

used in the preparation of Ayurvedic medicines in the form of Kadha to control various

respiratory disorders such as cough, cold and flu. The evaluation was made based on the

docking scores calculated by AutoDock Vina. Our study has identified an array of

phytochemicals present in these herbs which have significant docking scores and potential to

inhibit different stages of SARS-CoV-2 infection as well as other Coronavirus target proteins.

Molecular docking also indicated that, the phytochemicals present in these herbs possess

significant anti-inflammatory property. Apart from this, based on their pharmaceutical

characteristics, we have also performed in-silico drug-likeness and predicted

pharmacokinetics of the selected phytochemicals found in the Kadha. Overall our study

provides scientific justification in terms of binding of active ingredients present in different

plants used in Kadha preparation with viral proteins and target proteins for prevention and

treatment of the COVID19. This preparation can boost individual’s immunity and inhibit the

viral severity by interfering at different stages of virus multiplication in the infected person.

Introduction

Viral infections cause a wide spectrum of human diseases which appear with mild, severe or

life-threatening symptoms and underlie major clinical and socio-economic problems

worldwide. Human Coronaviruses (CoVs), including severe acute respiratory syndrome

Coronavirus (SARS-CoV), middle east respiratory syndrome Coronavirus (MERS-CoV) and

2019 novel Coronavirus (SARS-CoV-2) have caused three recent major global epidemics with

significant morbidity and mortality (Paules et al., 2020). Presently, there are no specific drugs

or vaccines approved for SARS-CoV-2. In light of the high infectivity rate of SARS-CoV-2 and

lack of treatment options, World Health Organization (WHO) has declared it as a global

emergency and researchers from all-over the world are trying to find possible cure for this

disease called Corona Virus Disease or COVID19. Phylogenetic analysis shows that SARS-CoV-

2 has very high nucleotide sequence identity with SARS-CoV-1 (79.7%) (Zhou et al., 2020b).

The envelope and nucleocapsid proteins of SARS-CoV-2 are two evolutionarily conserved

regions, with sequence identities of 96% and 89.6%, respectively. CoVs are enveloped viruses

with a positive-sense single-stranded RNA genome and contain at least four structural

proteins: Spike (S) protein (trimeric), envelope (E) protein, membrane (M) protein, and

nucleocapsid (N) protein. The Spike protein promotes host attachment and virion-cell

membrane fusion during infection. Therefore, Spike proteins play a crucial role in determining

the host range and tissue tropism. Zoonosis is common among CoVs and they can be

transmitted from one animal species to another, animals to humans, and humans to humans

(Salata et al., 2020; Ye et al., 2020).

Therapeutic interventions against CoVs can either activate the host defence machinery and

immune system or block viral life cycle events including transmission, cell binding, enzymes

involved in synthesis of the viral components, replication and assembly (Wu et al., 2020). In

search of therapeutics against CoVs, researchers are using following three broad strategies;

(i) Test existing broad-spectrum anti-viral drugs, (ii) in silico screening of molecular databases

to identify the lead molecules against viral or host proteins and (iii) rational drug design based

on the genomic information and pathological characteristics of COVID19 (Wu et al., 2020).

Among these, repurposing approach will shorten the time and reduce the cost as compared

to other strategies (Wu et al., 2020). Apart from the above strategies, alternative approaches

including traditional and herbal medicines may also have significant potential for

management of COVID19 both as prophylaxis and therapeutic purpose.

Ayurvedic medicines and their extracts are used for treatment of viral diseases for a very long

time (Alleva et al., 2010; Arora et al., 2011). Ayurvedic medicines are used in Indian

subcontinent since the Vedic period dating back to more than 2000 years (Saini, 2016). An

important Ayurvedic method for enrichment of active pharmacological agents from herbs

involves preparation of Kadha (decoction) for oral consumption. According to Panchvidh

Kashyapam described in the Charak Samhita (an ancient text on Ayurveda), there are five

prescribed ways to consume medicinal herbs and plants (Shingadiya et al., 2016). These

includes: i) Swaras (juicing), ii) Kwath (decoction), iii) Kalka (in paste form), iv) Hima (an herb

induced concoction), v) Phant (an herb-infused concoction). The decoction (Kadha) from a

mixture of spices and herbs is considered to be one of the oldest forms of medicine invented

by humans. Kadha is prepared from dry or less juicy ingredients like spices and herbs. The

Ministry of AYUSH (Ayurvedic, Yoga and Naturopathy, Unani, Siddha and Homeopathy),

Government of India, has recently recommended the use of Kadha for boosting the immunity

and reducing inflammation during COVID 19 crisis (AYUSH, 2020).

During viral infections, inflammation is part of the body’s immune response to reduce

infection, limit viral replication and transmission, reduce tissue injury and kill infected cells.

Acute inflammation is beneficial and it is followed by healing and regeneration. However,

CoVs are known to manipulate host machinery and subvert the immune system leading to

chronic inflammation (Chen et al., 2018; Takeuchi and Akira, 2007). The induction of pro-

inflammatory cytokines and chemokines in the host during SARS-CoV infection acts as a

double-edged sword which not only activates host immune response for viral clearance but

also aggravates tissue injury and organ toxicity during clinical evolution of the disease (Gu and

Korteweg, 2007; Tisoncik et al., 2012). CoVs infect cells using their spike proteins by making

interaction with cognate receptors present on the host cell surface. The spike proteins of

SARS-CoV and MERS-CoV attach to the cellular receptor angiotensin-converting enzyme 2

(ACE2) and dipeptidyl peptidase 4 (DPP4) receptor respectively for their entry inside the cells

(Gu and Korteweg, 2007; Wang et al., 2013). SARS-CoV-2 spike protein has higher affinity

towards human ACE2 as compared to that of SARS-CoV-1 (Zhou et al., 2020a). Upon infecting

the host cells, CoVs manipulate host machinery for new virus production and also elicit

inflammatory response in the host due to tissue injury (Gu and Korteweg, 2007; Wang et al.,

2013). Clinical investigation of critically ill patients infected with SARS-CoV-2 have shown high

concentration of cytokines and chemokines in human plasma, suggesting that cytokine storm

was associated with disease severity, multi-organ failure and mortality (Huang et al., 2020).

Cyclooxygenase-2 (COX-2), phospholipase A2 (PLA2), NF-κB-inducing kinase (NIK), and

interleukin-1 receptor associated kinase (IRAK) are important druggable targets involved in

SARS-CoV-2 induced inflammatory response and can be used to screen anti-inflammatory

molecules.

In the present study, the phytochemicals and active pharmacological ingredients found in

different herbs used in making Kadha were docked with different viral proteins (such as viral

capsid spike, proteases, NSP polymerase), host cell receptors & proteases (such as human

ACE2 and furin) as well as pro-inflammatory mediators (such as COX2, PLA2, IRAK-4 and NIK

proteins). Our study predicted that many of these phytochemicals possess significant affinity

towards functional region of viral proteins including spike, proteases, nucleoproteins and

polymerase as well as host surface receptors. These phytochemicals also showed significant

binding affinity for the functional region of different inflammatory mediators. Our findings

indicate that regular consumption of this ayurvedic Kadha in consultation with the ayurvedic

practitioner may significantly boost the host immunity and also help in the prevention of viral

infection and pathogenicity and reduce disease-severity in the infected individuals.

Materials and Methods

Phytochemicals name of commonly used herbs in making Kadha: Some of the commonly

used herbs in the preparations of Kadha includes, Tulsi (Ocimum sanctum), Haldi (Curcuma

longa), Giloy (Tinospora cordiofolia), Black pepper (Piper nigrum), Ginger (Zingiber officinale),

Clove (Syzygium aromaticum), Cardamom (Elettaria cardamomum), lemon (Citrus limon), and

Ashwagandha (Withania somnifera). A list of 108 phytochemicals present in herbs that are

used in preparation of Kadha or similar drink were collected from literature. Phytochemicals

found in Ocimum sanctum (Oleanolic acid, Ursolic acid, Rosmarinic acid, Eugenol, Carvacrol,

Linalool, Beta-caryophyllene, Estragole, Eugenic acid, Apigenin, Cirsimaritin, Isothymusin,

Isothymonin, Vicenin, Orientin and Cirsilineol) (Pattanayak et al., 2010), Curcuma longa

(Curcumin, Demethoxycurcumin, Bisdemethoxycurcumin, Ar-turmerone, Alpha-turmerone,

Beta-turmerone, Atlantone, Cyclocurcumin, Calebin A , Trans-Ferulic acid, Vanillin and Vanillic

acid) (Li et al., 2011), Tinospora cordiofolia (Magnoflorine, Berberine, Choline, Jatrorrhizine,

Beta-Sitosterol, Tinosporide, Tinosporaside, Cordifolioside A, Tinocordioside, Cordioside,

Tinocordifolioside and Tinocordifolin) (Sharma et al., 2019), Piper nigrum (Piperine,

Piperamide, Piperamine, Pipericide, Sarmentosine, Sarmentine, Brachyamide B,

Dihydropipericide, N-Formylpiperidine, Guineensine, Pentadienoylpiperidine, Tricholein,

Trichostachine, Piperettine, Piperolein B, Retrofractamide A, Chavicine, Isochavicine,

Isopiperine, Nerolidol, β-caryophyllene and Piperic acid) (Damanhouri and Ahmad, 2014),

Zingiber officinale (6-gingerol, 6-shogaol, 6-paradol, Zingiberene, Bisabolene, 1-

dehydrogingerdione, 6- gingerdione, 10-gingerdione, 4-gingerdiol, 6-gingerdiol, 10-

gingerdiol, Citral and Eucalyptol) (Bhattarai et al., 2018; Prasad and Tyagi, 2015), Syzygium

aromaticum (beta-caryophyllene, Vanillin, Eugenol, Acetyl eugenol, Crategolic acid, Eugenin,

Methyl salicylate, Kaempferol, Rhamnetin, Eugenitin, Oleanolic acid, Stigmasterol,

Campesterol, Gallic acid and Flavonol glucosides) (Cortes-Rojas et al., 2014), Elettaria

cardamomum (Protocatechualdehyde, Protocatechuic acid, Alpha-terpinyl acetate, 1,8-

cineole, Linalool, Linalyl acetate, Limonene, 4-terpineol andGeraniol) (Noumi et al., 2018),

Citrus limon (Eriodictyol, Quercetin, Hesperetin, Phloroglucinol, Umbelliferone, Vitamin C

(Rangel et al., 2011; Vandercook and Stephenson, 1966), and Withania somnifera (Withaferin

A, Somniferine, Choline, Anaferine, Withanolide A, Withanolide B, Withanone and

Withanolide) (Sangwan et al., 2004). 3D structures of these different phytochemicals were

downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov) in structure-data file (SDF).

Preparation of Ayurvedic Kadha: There are specific Ayurvedic methods to prepare the Kadha.

In India, variants of standard Kadha are also prepared using different combinations of herbs

depending on the severity of disease / ailment and availability of ingredients. The most

common ingredients are Tulsi leaves (10-15 leaves or ¼ teaspoon powder), Ginger (2-5g

rhizome or ¼ teaspoon powder), Clove (4-5 pieces), Black pepper (4-5 pieces), Cardamom (4-

5 pieces), Ashwagandha (2-5g raw or ¼ teaspoon powder) and Giloy (2-5g raw or ¼ teaspoon

powder). To make the Kadha, these herbs are boiled in 200 ml water for 5-10 minutes, and

jaggery or honey is added to make it sweet. The preparation is filtered and mixed with ¼

teaspoon of lemon juice. In case, if all ingredients are not available, it can be prepared using

locally available ingredients.

Protein structures: In order to study the mode of interaction of different phytochemicals with

various proteins including SARS-CoV-2, SARS-CoV-1, other important proteins and receptors

found on virus and host cells, molecular docking was performed. We have used following PDB

ID’s 6LU7 (SARS-CoV-2 main protease), 6m3m (SARS-CoV-2 nucleocapsid), 6vww (SARS-CoV-

2-NSP15 Endoribonuclease), 6vyo (SARS-CoV-2 RNA binding domain), 6w02 (SARS CoV-2-

NSP3), 6w4b(SARS-CoV-2-NSP9 replicase), 2ajf (ACE2 and SARS-CoV spike), 4mds (SARS-CoV

3CLpro) and 5mim (proprotein convertase; furin). In order to study the interaction of different

phytochemicals with different pro-inflammatory mediators like COX2, PLA2, NIK and IRAK-4,

we have used following PDB ID’s, 5f1a, 4uy1, 4dn5 and 2nrurespectively. All the protein

structures were retrieved from protein data bank (www.rcsb.org) and cleaned using UCSF

Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the

University of California.

Molecular docking: PyRx virtual screening tools was used for preparation of the input files

and performing molecular docking using Vina wizard (Dallakyan and Olson, 2015). For

preparation of protein input files, all water molecules, ligands and ions were removed from

*.pdb files. The polar hydrogens were added to protein structure and prepared files were

saved in *.pdbqt format. The molecule’s energy was minimized using energy minimization

tools of PyRx virtual screening tools and ligands were saved in *.pdbqt format after adding

polar hydrogens for further docking process. All docking results were sorted by the binding

energy. 2D interaction of the ligand and protein was visualized using Discovery Studio

Visualizer. Region-specific docking was performed against SARS-CoV-2 main protease and

spike protein as well as for human ACE2 & Furin protease. Following AutoDock Vina docking

parameters such as (center_x = -16.69, center_y = 27.23, center_z = 68.46, size_x = 36.65,

size_y = 42.12, size_z = 50.40), (center_x = 190.45, center_y = 197.88, center_z = 260.72,

size_x = 61.32, size_y = 41.03, size_z = 43.79), (center_x = 6.68, center_y = -2.42, center_z =

48.2, size_x = 37.03, size_y = 70.29, size_z = 55.72) and (center_x = 32.41, center_y = -37.97,

center_z = -11.64, size_x = 71.93, size_y = 55.05, size_z = 47.46) were used for SARS-CoV-2

main protease (PDB ID: 6LU7), SARS-CoV-2 spike (PDB ID: 6VXX), human ACE2 (PDB ID: 2AJF)

and furin (PDB ID: 5MIM) respectively. Other proteins from SARS-CoV-2, SARS-CoV and host

anti-inflammatory mediators were blindly docked using complete protein structure.

In-silico drug-likeness, pharmaceutical characterisation and pharmacokinetics: We sought

to predict in-silico drug-likeness and important pharmaceutical properties including

pharmacokinetics of the selected phytochemicals which exhibited high affinity for different

SARS-CoV-2 viral proteins and human ACE2 and Furin proteins. For this purpose pkCSM online

prediction platform was used (Pires et al., 2015). This online server calculates

pharmaceutically applicable properties such as molecular weight, octanol-water partition

coefficient (LogP), number of H-bond donor, number of H-bond acceptor and number of

rotatable bonds. This platform was also used to estimate additional parameters like ligand

Absorption, Distribution, Metabolism and Excretion (ADME), water solubility, human

intestinal absorption, total clearance, AMES test, human maximum tolerated dose, and

hepatotoxicity .

Results

Our molecular docking study revealed that different phytochemicals found in the traditional

Ayurvedic Kadha, may have high binding affinity (the lowest binding energy) with various viral

and host macromolecular targets and other human pro-inflammatory mediators and

proteins. Table 1 shows the list of the phytochemicals which showed significant binding

affinity (≤-7.5 kcal/mole) with SARS-CoV-2 main protease, spike protein, human ACE2 and

furin proteins.

Table 2 shows the predicted binding of top 12 phytochemicals and native ligand N3, against

SARS-CoV-2 main protease and their different protein residues which are involved in the

interaction. These molecules are Somniferine A, Tinosporide, Tinocordioside, Orientin,

Flavonol glucoside, Withanolide, Apigenin, Cyclocurcumin, Withanolide B, Kaempferol,

Withanone and Withaferin A. Their predicted binding energies are -8.6, -8.6, -8.1, -8.1, -8.0, -

8.0, -7.8, -7.8, -7.8, -7.7, -7.6 and -7.6 kcal/mol respectively. All these molecules share same

binding site with the well-known inhibitor, N3 and their binding energy is also comparable

(Fig 1).

SARS-CoV-2 spike protein interacts with the host ACE2 receptor present on the surface of the

host cells for their entry. Table 3 shows the binding of top 12 phytochemicals with SARS-CoV-

2 spike (PDB ID: 6VXX) and the different residues involved in their interaction. These

molecules are, Withanolide B, Withanolide, Withaferin A, Withanone, Somniferine A, Beta-

sitosterol, Pipericide, Crategolic acid, Retrofractamide A, Ursolic acid, Piperolein B and

Tinosporide. Their predicted binding energies are -8.7, -8.4, -8.2, -8.2, -7.9, -7.8, -7.6, -7.6, -

7.5, -7.5, -7.5, -7.5, -7.4 and -7.4 kcal/mol respectively. These phytochemicals were found to

share same binding pocket in the target proteins (Fig 2 a).

Table 4 shows the binding of top 10 phytochemicals with human ACE2 (PDB ID: 2AJF) and the

different residues involved in their interaction. The phytochemicals with high affinity for

human ACE2 are, Withaferin A, Stigmasterol, Vicenin, Ursolic acid, Withanolide B, Oleanolic

acid, Withanone, Beta-sitosterol, Campesterol and Orientin. Their predicted binding energies

are -9.1, -8.8, -8.8, -8.7, -8.7, -8.5, -8.5, -8.3, -8.3 and -8.0 kcal/mol respectively. These

phytochemicals share same binding pocket in human ACE2 (Fig 2 b).

Furin is another protease found in the host cells which acts on the viral spike protein and

facilitates its interaction with the human ACE2. Table 5 shows the predicted binding of top 12

phytochemicals with human furin (PDB ID: 5MIM) and the different residues involved in their

interactions. These molecules are, Withanolide, Somniferine A, Apigenin, Withanolide B,

Ursolic acid, Hesperetin, Campesterol, Crategolic acid, Withanone, Chavicine, Rosmarinic acid

and Stigmasterol. Their predicted binding energies are -9.5, -8.7, -8.5, -8.3, -8.3, -8.2, -8.1, -

8.1, -8.1, -8.0, -8.0 and -8.0 kcal/mol respectively. Many of these molecules share same

binding pocket as its native ligand. However, our docking study also showed that Apigenin,

Withanolide, Hespertin, Campesterol, Chavicine, Rosmarinic acid, Stigmastereol bind to the

residues at a site adjacent to binding site of native ligand (Fig 3).

The molecular docking performed against other proteins from SARS-CoV-2 and SARS-CoV-

1proteins are shown in Table 6. Many of the phytochemicals present in the Kadha have

significant binding affinity with SARS-CoV-2 and SARS-CoV-1 proteins. Some of the

phytochemicals which have high binding affinity with NSP15 Endoribonuclease (PDB ID:

6vww) are Orientin, Withanolide, Withanolide B, Crategolic acid, Ursolic acid, Withaferin A,

Apigenin, Eriodictyol, Hesperetin, Oleanolic acid, Stigmasterol and Withanone. Their

predicted binding energies are -9.4, -9.3, -9.2, -9.1, -9.1, -9.1, -9.0, -9.0, -9.0, -9.0, -8.9 and -

8.9 kcal/mol respectively. A list of the phytochemicals which were found to exhibit high

affinity with SARS-CoV-2 ADP ribose phosphatase, NSP3 (PDB ID: 6w02) are Berberine,

Withanolide, Rosmarinic acid, Cyclocurcumin, Piperettine, Withaferin A, Withanolide B,

Bisdemethoxycurcumin, Chavicine, Cirsimaritin, Demethoxycurcumin and Apigenin. Their

predicted binding energies are -9.7, -9.6, -9.3, -9.2, -9.2, -9.2, -9.2, -8.8, -8.8, -8.8, -8.8, and -

8.7 kcal/mol respectively. Among the phytochemicals tested, the following molecules were

predicted to bind with Nsp9 RNA binding protein of SARS CoV-2 (PDB ID: 6w4b) protein,

Withanone, Withanolide B, Withaferin A, Withanolide, Beta-sitosterol, Campesterol,

Stigmasterol, Flavonol glucoside and Somniferine A. Their predicted binding energies are -9.1,

-9.0, -8.2, -8.1, -8.0, -8.0, -7.9, -7.5 and -7.5 kcal/mol respectively.

More than ten phytochemicals were predicted to interact with SARS-CoV-2 RNA binding

domain of nucleocapsid protein (PDB ID: 6vyo) viz, Withanolide, Withaferin A, Somniferine A,

Withanone, Withanolide B, Ursolic acid, Crategolic acid, Quercetin, Stigmasterol,

Tinocordioside, Tinosporaside and Oleanolic acid. Their predicted binding energies are -8.7, -

8.3, -8.2, -8.2, -8.0, -7.8, -7.7, -7.7, -7.7, -7.7, -7.7 and -7.6 kcal/mol respectively.

An array the phytochemicals (having binding energy ≤ 7.5 kcal/mol) were also predicted to

interact with SARS-CoV main protease (PDB ID: 4mds) viz, Somniferine A, Withanolide B,

Withanone, Withanolide, Oleanolic acid, Cordioside, Crategolic acid, Tinosporaside, Calebin

A, Ursolic acid, Stigmasterol, Orientin, Pentadienoylpiperidine, Campesterol, Cyclocurcumin,

Rosmarinic acid, Tinosporide, Withaferin A, Demethoxycurcumin, Jatrorrhizine, Piperettine,

Tinocordioside and Trichostachine. Their predicted binding energies are -9.3, -8.9, -8.5, -8.3,

-8.2, -8.1, -8.1, -8.1, -8.0, -8.0, -7.9, -7.8, -7.8, -7.7, -7.7, -7.7, -7.7, -7.7, -7.6, -7.5, -7.5, -7.5 and

-7.5 kcal/mol respectively. Similarly, the phytochemicals (having energy ≤7.5 kcal/mol) which

may interact with SARS-CoV-1 spike protein (PDB ID: 2ajf) include, Withanolide, Stigmasterol,

Withanolide B, Somniferine A, Ursolic acid, Beta-sitosterol, Oleanolic acid,

Pentadienoylpiperidine, Piperettine, Tinosporide, Withanone, Bisdemethoxycurcumin,

Calebin A, Campesterol, Cyclocurcumin, Piperine, Retrofractamide A, Trichostachine and

Quercetin. Their predicted binding energies are -8.7, -8.6, -8.6, -8.5, -8.2, -8.0, -7.9, -7.9, -7.9,

-7.8, -7.8, -7.6, -7.6, -7.6, -7.6, -7.6, -7.6, -7.6 and -7.5kcal/mol respectively. Many of the

phytochemicals (having energy ≤7.5 kcal/mole) were also predicted to interact with SARS-

CoV-1 nucleocapsid protein (PDB ID: 2cjr) viz, Ursolic acid, Somniferine A, Oleanolic acid,

Magnoflorine, Withanolide B, Withanolide, Withanone, Orientin, Withaferin A, Quercetin,

Rhamnetin and Isothymusina. Their predicted binding energies are -9.0, -8.6, -8.4, -8.2, -8.2,

-7.9, -7.8, -7.7, -7.7, -7.6, -7.6 and -7.5 kcal/mol respectively.

Table 7 shows the predicted binding energy of different phytochemicals with molecules

involved in the inflammatory processes such as COX2, PLA2, NIK and IRAK-4. From Table 7, it

is clear that Withaferin A, Withanolide B, Withanolide, Withanone, Campesterol,

Cyclocurcumin,Somniferine A, Stigmasterol, Eriodictyol, Isopiperine, Oleanolic acid,

Rhamnetin, Orientin, Quercetin, Piperine and Vicenin may possess high binding affinity with

most of the inflammatory molecules used in the study. Our study proposes that many of the

phytochemicals present in these herbs may directly inhibit COX-2, PLA2 and IRAK-4 which are

involved in the inflammation. Many of these phytochemicals may also bind to the active site

of the protein and at the same time few of them may have very high affinity for other regions

of the target protein which may affect its function. Our study also shows that several

phytochemicals present in the Kadha have significant predicted inhibitory activity towards

NIK. Inhibition of the NIK by phytochemicals may moderate the NF-κB dependent genes which

are involved in inflammation. Based on these docking studies, it may be predicted that the

phytochemicals present in the Kadha may exhibit anti-inflammatory property.

The phytochemicals which were predicted to bind with different SARS-CoV-2 proteins and

human ACE2 & Furin proteins were analysed for their in-silico drug-likeness and

pharmacokinetics using pkCSM server (Pires et al., 2015) (Table 8). Some of these

phytochemicals did not follow the Lipinski’s rule of five for all the parameters. The ADME

properties derived from pkCSM server indicated reasonable intestinal absorption and

bioavailability of the phytochemicals such as Apigenin, Berberine, Beta-sitosterol,

Bisdemethoxycurcumin, Brachyamide B, Campesterol, Chavicine, Crategolic acid, Curcumin,

Cyclocurcumin, Demethoxycurcumin, Eriodictyol, Isochavicine, Isopiperine, Isothymonin,

Jatrorrhizine, Kaempferol, Oleanolic acid, Piperettine, Pipericide, Piperolein B, Quercetin,

Retrofractamide A, Rhamnetin, Somniferine A, Stigmasterol, Tinocordioside, Tinosporide,

Trichostachine, Ursolic acid, Withaferin A, Withanolide, Withanolide B and Withanone are

significantly very high (>70%). Among these phytochemicals, only Berberine was shown to be

negative for AMES test (Table 8).

Discussion

Use of herbs and phytochemicals has a long history in the management of various respiratory

diseases (Alamgeer. et al., 2018; Pinn, 2001; Santana et al., 2016). Currently, the demand of

complementary medicine, including herbal medicine has become more popular in healthcare

for both general maintenance of health and for treatment of minor illnesses (Barnes, 2004).

In European countries, several species of herbs have been used against flu and common cold

(Weiss and Fintelmann, 2000). Similarly, in Russia and Estonia various herbs and medicinal

plants have also been used for centuries for management of common cold and flu (Raal et al.,

2013). In India, use of spices and herbs for treatment of various diseases including cough, cold

is a common practice with recorded history of over 2000 years (Sachan et al., 2018; Vasanthi

and Parameswari, 2010).

It is well-known that SARS-CoV viral genome encodes more than 20 proteins, among which

two proteases i.e 3-chymotrypsin-like protease (3CLpro, main protease, Mpro) and papain-

like protease (PLpro) are vital for virus replication (Lindner et al., 2005). They cleave the two

translated polyproteins (PP1A and PP1AB) into individual functional components, resulting in

release of 16 non-structural proteins (NSPs) (Jo et al., 2020). Thus SARS-CoV-2 main protease

is considered as a promising druggable target. The viral NSPs play an important role in

replication and transcription. Our study predicts that many of the phytochemicals of Kadha

have significant binding affinity with the main protease. Twelve phytochemicals namely

Somniferine A, Tinosporide, Tinocordioside, Orientin, Flavonol glucoside, Withanolide,

Apigenin, Cyclocurcumin, Withanolide B, Kaempferol, Withanone and Withaferin A, have

predicted binding energy lower than the pharmacological inhibitor, N3 (Fig 1, Table 2). The

binding of these phytochemicals with main protease may slow down the cleavage of PPs to

releases NSPs and decrease the process of viral replication and transcription.

The SARS-CoV spike protein plays an important role in virus entry into the host (Li, 2016).

Initial interactions between the S1 domain and its host receptor (ACE2), and subsequent S2

segment mediated fusion of the host and viral membrane allows the viral RNA genome to

enter inside the host cells. Thus, these proteins represent as important targets for designing

drug (Li, 2016). Our study predicts that an array of the phytochemicals have significant binding

affinity with the SARS-CoV-2 spike proteins (Table 3, Figure 2a) as well as with host ACE2

protein (Table 4, Figure 2b) and furin protein (which facilitate spike and ACE2 interaction)

(Table 5, Figure 3). Thus, phytochemicals may significantly inhibit viral interaction with the

host receptor and slow down or stop the entry of the viral genome inside the host. The spike

protein is also known to activate the immune response of the host cell towards CoVs (Li,

2016). The S1 domain of spike acts as a major antigen on the surface of the virus (Yuan et al.,

2017).

SARS-CoV nucleocapsid protein (SARS-CoV NP) is another vital structural protein which shows

intrinsic multimerization and interacts with M protein, suggesting that NP is both critical to

formation of the viral nucleocapsid core and is involved in virion assembly (He et al., 2004a;

He et al., 2004b). Our study predicts that the phytochemicals such as Withanone, Withanolide

B, Withanolide, Withaferin A, Ursolic acid, Tinosporaside, Tinocordioside, Stigmasterol,

Somniferine A, Quercetin, Oleanolic acid and Crategolic acid have significant binding energy

with nucleocapsid protein (Table 6). The N protein of SARS-CoV are also known to up-regulate

the expression of the proinflammatory protein COX2 and also interact with the proteasome

subunit p42, which affects a variety of basic cellular processes and inflammatory responses

(Hu et al., 2017; Wang et al., 2010).

Nsp3 protein is another key component for Coronavirus and is essential for

replication/transcription complex (RTC) formation. It plays various roles in Coronavirus

infection. It releases Nsp1, Nsp2, and itself from the polyproteins and interacts with other

viral nsps as well as RNA to form the replication/ transcription complex (Lei et al., 2018).

Similarly, Nsp15 protein also acts as an endoribonuclease and preferentially cleaves 3’ of

uridylates through a ribonuclease A (RNase A)-like mechanism and also facilitates viral

replication and transcription (Alcantara et al., 2010; Sawicki and Sawicki, 1998). Our study

predicted that many of the phytochemicals of the Kadha have significant binding affinity with

Nsp3 and Nsp15. Thus, the phytochemicals identified in Table 6 may disrupt the formation of

RTC and stop the viral genome replication (Table 6).

During the SARS-CoV infection, human lung epithelial cells are among the first targets for viral

entry. In response to viral multiplication and host cell damage, lung epithelial cells secrete

inflammatory mediators to initiate and exacerbate host innate inflammatory responses,

causing detrimental immune-mediated pathology within the lungs. SARS-CoV-2 infects ACE-2

expressing epithelial cells in the air sacs (alveoli) in lower lungs. The damaged epithelium leads

and interlobular septal thickening lead to leaky cell junctions, accumulation of fluid which is

rich in proteins, inflammatory mediators and Pneumonia. The spread of virus from lung to

systemic circulation and excessive production of pro-inflammatory mediator can damage

different vital organs. Our study predicts that an array of the phytochemicals such as

Withaferin A, Withanolide B, Withanolide, Withanone, Campesterol,

Cyclocurcumin,Somniferine A,Stigmasterol, Eriodictyol, Isopiperine, Oleanolic acid,

Rhamnetin, Orientin, Quercetin, Piperine, Vicenin etc. found in the preparation of the Kadha,

have significant binding affinity with the many of these inflammatory mediators or the

molecules involved in this process (Table 7). It is well known that, NF-κB is the master

regulator for several genes such as COX-2, VEGF (vascular endothelial growth Factor), pro-

inflammatory cytokines (IL-1, IL-2, IL-6, and TNFα), chemokines (e.g., IL-8, MIP-1α, and MCP-

1), adhesion molecules, immunoreceptors, growth factors, and other agents involved in

proliferation and invasion. NF-κB activation is mediated by two distinctly different redox-

related signalling pathways. The first pathway involves NIK/IKK whereas second pathway

involve MAPKs and both causes the induction of transcriptional activation of NF-κB. NF-κB-

inducing kinase (NIK) is a key mediator of the non-canonical NF-κB signalling pathway. NF-κB

is also one of the main inducible transcription factors shown to respond directly to oxidative

stress. Oxidative stress activates nuclear factor-inducing kinase (NIK)/IκB kinase (IKK) and

mitogen-activated protein kinases (MAPKs). NIK/IKK and MAPK pathways activate NF-κB and

migrates to the nucleus and binds to κ elements on DNA in enhancers and promoter regions.

Various herbs have potential to control inflammation-associated disease by decreasing the

production of the pro-inflammatory mediators by suppressing pro-inflammatory pathways

(Pan et al., 2011). Our study also predicted that phytochemicals found in the Kadha have

significant binding affinity with NIK (Table 7) which can stop NF-κB mediated downstream

events. In a very recent study, Huang et al (2020) have shown that the patients infected with

SARS-CoV-2 had high amounts of IL1, IFNγ, IP10, and MCP1, which can mediate cytokine

storm associated multi-organ damage (Huang et al., 2020). At the same time, SARS-CoV-2

infection also initiates increased secretion of T-helper-2 (Th2) cytokines (eg, IL4 and IL10)

which suppress inflammation (Huang et al., 2020). The increased secretion of inflammatory

mediators was also associated with moderation of helper T cell responses in COVID19

patients.

Our in-silico drug-likeness and pharmacokinetic prediction study shows that many of the

active phytochemicals found in the Ayurvedic Kadha have reasonably high intestinal

absorption (Table 8). Except berberine, others phytochemicals are negative for AMES test.

Some of the phytochemicals such as Berberine, Brachyamide B, Chavicine, Crategolic acid,

Jatrorrhizine, Oleanolic acid, Piperettine, Pipericide, Piperolein B, Retrofractamide A,

Tinosporide and Ursolic acid were also predicted to be hepatotoxic at higher doses. Thus,

consumption of Ayurvedic Kadha would allow absorption of active phytochemicals which can

interact with the key viral proteins including spike and main protease and also human proteins

needed for viral life cycle. This interaction will be help in the inhibiting virus entry and

multiplication in the host cells.

Taken together, our current findings and the recent knowledge about SARS-CoV and SARS-

CoV-2 pathology, profess the use of Ayurvedic Kadha in the prevention and management of

COVID-19. The phytochemicals found in the Kadha have significant binding affinity with the

different CoVs proteins (Scheme 1), indicating that they may control viral infection and

multiplication in the host cells. Molecular docking study with human inflammatory mediators

predicts that many of the phytochemicals present in this preparation have significant anti-

inflammatory property. Most of the phytochemicals found in the herbs ashwagandha, giloy,

tulsi, clove and black pepper have potential to interact with most of the druggable proteins

selected in this study. In conclusion, regular consumption of ayurvedic Kadha in consultation

with ayurvedic practitioner may decrease the inflammatory response, boost the individual’s

immunity and reduce the risk of CoVs infection including SARS-CoV-2.

Acknowledgement: The authors would like to thank Dr S. Santosh Kumar, Radiation

Biology & Health Sciences Division, Bhabha Atomic Research Centre for his valuable

constructive suggestions and critical review of the article. The authors also acknowledge Mrs.

Sunitee Maurya (M.Sc, Chemistry) for her valuable constructive suggestions in formulation of

this work.

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of

interest with respect to the research, authorship, and/or publication of this article.

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Legends







CoV-2 proteins.

Table 1: The predicted binding of different phytochemicals against SARS-CoV-2 main

protease, spike protein, human ACE2 and furin proteins. Only those phytochemicals name are

included in the table which have binding energy ≤ 7.5 kcal/mol.


CoV-2 main protease (PDB ID: 6LU7) and their different protein residues which are involved

in the interaction.


CoV-2 spike protein (PDB ID: 6VXX) and their different protein residues which are involved in

the interaction.


ACE2 protein (PDB ID: 2AJF) and their different protein residues which are involved in the

interaction.


furin protein (PDB ID: 5MIM) and their different protein residues which are involved in the

interaction.

Table 6: The predicted binding of different phytochemicals against SARS-CoV-2 and SARS-CoV

target proteins. Only those phytochemicals name are included in the table which have binding

energy ≤ 7.5 kcal/mol.

Table 7: The predicted binding of different phytochemicals against different anti-

inflammatory macromolecules. Only those phytochemicals name are included in the table

which have binding energy ≤ 7.5 kcal/mol.

Table 8: In-silico drug-likeness and ADME prediction of the selected phytochemicals found in

the Ayurvedic Kadha.



Somniferine A

Tinosporide

Tinocordioside

Orientin

Flavonol glucoside

Withanolide

Apigenin

Cyclocurcumin

Withanolide B

Kaempferol Withanone

Withaferin A



a.

b.

Withanolide

Somniferine A

Apigenin

Withanolide B

Ursolic acid

Hesperetin

Campesterol Crategolic acid

Withanone

Chavicine

Rosmarinic acid

Stigmasterol.


CoV-2 proteins.

Compounds

COVID-19 main

protease (PDB

ID: 6lu7)

SARS-CoV-2 spike

protein (PDB ID:

6vxx)

Human ACE2

(PDB ID: 2ajf)

Human Furin (PDB

ID: 5mim)

Native ligand (N3) -7.6 -9.3

Apigenin -7.8 -7.5 -8.5

Berberine -7.5 -7.8

Beta-sitosterol -7.6 -8.3 -7.6



Campesterol -8.3 -0.1

Chavicine -8

Cordifolioside A -7.7 -7.5

Cordioside -7.8


Curcumin -7.8 -7.8

Cyclocurcumin -8.3

Demethoxycurcumin -7.6

Eriodictyol -7.7

Flavonol glucoside -8 -7.5

Hesperetin -7.7 -8.2

Isochavicine -7.8 -7.5

Isopiperine -7.6

Isothymonin -7.6

Jatrorrhizine -7.5

Kaempferol -7.7 -7.9

Oleanolic acid -8.5 -7.9

Orientin -8.1 -8 -7.9

Piperettine -8 -7.8

Pipericide -7.6 -7.5

Piperolein b -7.6

Quercetin -8

Retrofractamide A -7.5

Rhamnetin -7.5

Rosmarinic acid -8

Somniferine A -8.6 -7.8 -9 -8.7

Stigmasterol -8.8 -8.5

Tinocordifolioside -7.6

Tinocordioside -8.1 -8.5 -8

Tinosporaside -8.1 -8

Tinosporide -8.6 -8.3 -7.6


phytochemcals are included in the table which have binding energy ≤ -

7.5)

Phytochemicals

Table 1

Trichostachine -7.8

Ursolic acid -7.7 -7.5 -8.7 -8.3

Vicenin -8.8 -7.7

Withaferin A -7.6 -8.2 -9.1 -7.9

Withanolide -8 -8.4 -9.2

Withanolide B -7.8 -8.7 -8.7 -8.4

Withanone -7.6 -7.9 -8.5 -8.1

Table 2: 2D interaction of top 12 phytochemicals with SARS-CoV-2 main protease (PDB ID:

6LU7)

S. No.

Name of the molecules 2D interaction between molecules and protein


1 Native ligand N3 Binding energy= -7.6

Convention hydrogen bonding: THR45, SER46, LEU141, CYS145, GLN189 Van der vaals: THR25, THR26, LEU27, HIS41, PHE140, ASN142, GLY143, SER144, HIS163, MET165, GLU166, LEU167,ASP187, ARG188, THR190, GLN192 Pi-alkyl: MET49, PRO168 Pi-Sigma: HIS41 Pi-Sulphur: PRO168

2 Somniferine A Binding energy= -8.6

Convention hydrogen bonding: SER46, ASN142, GLU166 Van der vaals: THR24, THR25, THR26, THR45, GLY143, SER144, HIS163, HIS164, MET165, Pi-alkyl: HIS41, CYS145 Pi-sulphur: MET49 Carbon Hydrogen bond: PHE140, LEU141


Convention hydrogen bonding: HIS163 Van der vaals: THR25, THR26, LEU27, MET49, PHE140, ASN142, GLY143, HIS164, GLU166, HIS172 Pi-alkyl: HIS41 Amide –Pi stacked: EU141, MET165 Pi-Sulphur: CYS145

4 Orientin Binding energy=-8.1

Convention hydrogen bonding: ASN142, GLU166, ARG188, THR190 Van der vaals: HIS41, MET49, PHE140, LEU142, GLY143, HIS163, HIS164,LEU167, PRO168, GLN189, ALA191, GLN192, HIS172,ALA191, GLN192 Pi-alkyl: CYS145, MET165

5 Tinocordioside Binding energy=-8.1

Convention hydrogen bonding: THR45, GLA143, SER144 Van der vaals: THR25, THR26, LEU27,SER46, PHE140, LEU141, ASN142, CYS145, MET165, GLU166, HIS172 Pi-alkyl: HIS41, MET49 Pi-amide: THR24

6 Flavonol glucoside Binding energy=-8.0

Convention hydrogen bonding: CYS145, GLU166 Van der vaals: LEU27, PHE140, LEU141, ASN142, GLY143, SER144, HIS163, HIS164, HIS172, ASP187, ARG188, GLN189 Pi-alkyl: MET49, MET165 Pi-Sulphur: CYS145


Convention hydrogen bonding: ARG40, PHE181, ARG188 Van der vaals: ASN53, TYR54, GLU55, ASN88, ARG105, ASN180, GLY183, PRO184, PHE185 Pi-alkyl: CYS85, PHE181, VAL186


Convention hydrogen bonding: LEU141, HIS163, ASP187 Pi donor hydrogen bond: GLU166 Van der vaals: HIS41, TYR154, PHE140, ASN142, SER144, HIS164, MET165, GLN89, ARG188, Pi-alkyl: MET49 Pi-Sulphur: CYS145

9 Cyclocurcumin Binding energy=-7.8

Convention hydrogen bonding: HIS163 Van der vaals: THR25, THR26, LEU27, VAL42, MET49, PHE140, LEU142, ASN142, GYL143, SER144, GLU166, LEU167, HIS172, ARG188, GLN189, THR190, ALA91, GLN192, Pi-cation:HIS41 Pi-sulphur: MET65 Pi-Donor hydrogen bond: CYS145


Convention hydrogen bonding: THR45, ASN42, GLY43, SER144, CYS145 Van der vaals: THR24, THR25, THR26, SER46,PHE140, LEU142, HIS164, MET165, GLU166 Pi-alkyl: HIS41, MET49 Carbon-Hydrogen bond: GLN189

11 Cordifolioside A Binding energy=-7.7

Convention hydrogen bonding: LEU142, GLY143, SER144, HIS163, ARG188, THR190 Van der vaals: THR25, LEU27, MET49, PHE140, ASN142, CYS145, HIS164, GLU166, PRO168, LEU167, VAL186, GLN189, GLN192 Pi-alkyl: MET165 Carbon-Hydrogen bond: THR26

12 Kaempferol Binding energy=-7.7

Convention hydrogen bonding: ASP187 Van der vaals: PRO52, TYR54, PHE140, LEU141, ASN142, SER144, HIS164, ARG188, GLN189 Pi-alkyl: MET49 Pi-Sulphur: CYS145, MET165 Pi-Donor hydrogen bond: GLU166 Pi-Pi stacked: HIS41


Convention hydrogen bonding: SER144 Van der vaals: THR25, HIS41, LEU141, GLY143, HIS164, MET165, GLU166, PRO168, GLN189, HR190 Pi-alkyl: MET49, CYS145 Carbon-Hydrogen donor: ASN142

Table 3: 2D interaction of top 12 phytochemicals with SARS-CoV-2 Spike (PDB ID: 6VXX)

S. No. Name of the molecules

2D interaction between molecules and protein



Van der vaals: ILE418, ASP420, ARG454, ASP467, GLN493 Pi-alkyl: LYS417, TYR453, PRO491 Pi-Sigma:TYR421


Van der vaals: ILE418, ARG454, ASP467, TYR489, GLN493 Pi-alkyl: LYS417, TYR453, PRO491 Pi-Sigma: TYR421

3 Withaferin A Binding energy=-8.2

Van der vaals: ILE418, ARG454, ASP467, TYR489, GLN493 Pi-alkyl:CYS417, TYR453, PRO491 Pi-Sigma: TYR421


Convention hydrogen bonding: SER530 Van der vaals: ASN331, THR333, LEU335, ASP364, GLY526, LYS528, GLN580 Pi-alkyl: VAL362, PRO527, LYS529


Convention hydrogen bonding: ARG346 Van der vaals: GLU340,THR345, PHE347, ALA348, SER349, ASN354, LYS356, SER399, ASN448, ASN450, TYR451 Pi-alkyl: ALA344

6 Beta-sitosterol Binding energy=-7.6

Van der vaals:CYS336, GLY339, ASN343, ALA363, ASP364, SER371, SER373 Pi-alkyl: LEU335, PHE338, PHE342, VAL362, VAL367, LEU368, PHE374, PRO527

7 Pipericide Binding energy=-7.6

Convention hydrogen bonding: ASP364, TRP436 Van der vaals: CYS336, PHE338, GLY339, PHE342, ASN343, VAL362, ALA363, LEU368, SER371, SER373, PHE374, ASN437, SER438, ASN440, LEU441, ARG509 Pi-alkyl: LEU335, VAL367


Convention hydrogen bonding: THR376, VAL503 Van der vaals: TYR380, GLY404, ASP405,ILE410, GLUY504, TYR508 Pi-alkyl: LYS378, VAL407, ARG408, ALA411, VAL433

9 Retrofractamide A Binding energy=-7.5

Convention hydrogen bonding: ASP364 Van der vaals: LEU335, CYS336, PHE338, GLY339, ASN343, PHE342, VAL367, LEU368, SER373 Pi-Pi: TRP436


Convention hydrogen bonding: ASP364 Van der vaals: PHE329, PRO330, ASN331, ILE332, THR333, GLY526, LYS528, SER530, GLN580, Pi-alkyl: LEU335, VAL362, PRO527, LYS529

11 Piperolein B Binding energy=-7.4

Van der vaals: LEU335, PHE338, GLY339, PHE342, ASP364, PHE374,SER438, ARG509 Carbon hydrogen bond: CYS336, ASN343, SER373 Pi-alkyl: LEU368, LEU441 Pi-Sigma: VAL367, TRP436


Convention hydrogen bonding: PRO330, SER530 Van der vaals: PHE329, ASN331, ILE332, THR333, LEU335, GLY526, PRO527, LYS528, GLN580 Pi-alkyl: VAL362, LYS529

Table 4: 2D interaction of top 10 phytochemicals with human ACE2 (PDB ID: 2AJF)



1 Withaferin A Binding Energy:-9.1

Convention hydrogen bonding: AL99, ASP350, ASN394 Van der vaals: PHE32, SER77, GLN102, ASP382, TYR385, ARG393 Pi-alkyl: PHE40, LEU73, LEU100, PH1390, PHE391

2 Stigmasterol Binding Energy:-8.8

Convention hydrogen bonding: SER77, GLN102 Van der vaals: PHE32, TRP69, ALA99, LEU100, ASP350, TYR385, LEU391 Pi-alkyl: PHE40, LEU73, ARG393 Pi-Sigma: PHE390

3 Vicenin Binding Energy:-8.8

Convention hydrogen bonding: ASP350, HIS378, TYR385, ARG393, ASN394, HIS401 Van der vaals: PHE40, THR347, ALA348, TRP349, GLU375, PHE390, LEU391, GLY395, GLU402 Pi-Pi stack: HIS401 Pi-cation: ASP382

4 Ursolic acid Binding Energy:-8.7

Convention hydrogen bonding: ASN394, HIS01 Van der vaals: SER43, SER44, SER47, ALA348, ASP350, ASP382, TYR385 Pi-alkyl: PHE40, TRP349, HIS378 Pi-Sigma: HIS378

5 Withanolide B Binding Energy:-8.7

Pi-alkyl: PHE40, ALA348, ARG393, HIS401 Van der vaals: THR347, ASP350, ASP382, TYR385, ASN394, GLU402 Pi-Sigma: HIS378, PHE390

6 Oleanolic acid Binding Energy:-8.5

Van der vaals: SER77, LEU100,GLN102,GLY104,LEU392, ASN394, LYS562 Pi-alkyl: PHE40, TRP69, LEU73, ALA99, PHE390, LEU391

7 Withanone Binding Energy:-8.5

Convention hydrogen bonding: ALA348 Van der vaals: SER44, SER47, LEU351, HIS378, HIS401, FLU402 Pi-alkyl: TRP349 Pi-Sigma: PHE40

8 Beta-sitosterol Binding Energy:-8.3

Convention hydrogen bonding: LEU100 Van der vaals: PHE32, TRP69, SER77, ALA99, GLN102, ASP350, TYR385, LEU391, ASN394 Pi-alkyl: PHE40, PHE73, PHE390, ARG393

9 Campesterol Binding Energy:-8.3

Convention hydrogen bonding: LEU100 Van der vaals: PHE32, TRP69, SER77, ALA99, ASP350, GLY352, ASN394 Pi-alkyl: PHE40, LEU73, TYR385, PHE390, LEU391, ARG393

10 Orientin Binding Energy:-8.0

Convention hydrogen bonding: PHE390, ARG393 Van der vaals: PHE40, SER43, SER44, SER47, ASP350, ASP382, TYR385, LEU391, ASN394, HIS401

Table 5: 2D interaction of top 12 phytochemicals with human furin (PDB ID: 5MIM)



1 Native ligand, 1N Binding energy= -9.3

Convention hydrogen bonding: LEU227, GLY255, PRO256, ASP258, THR308, THR365 Van der vaals: ARG185, ASN192, GLY229, SER253, TRP254, GLU257, GLY265, ASN295, HIS364,GLY366, SER368, Pi-alkyl: VAL231 Attractive charge: ASP154, ASP191, ASP228, ASP258, ASP264


Convention hydrogen bonding: PRO266, ASN29, ALA532 Van der vaals: VAL263, ASP264, GLY265, ALA267, GLU271, GLY307, ASN310, SER311, ARG498, ASP526, GLY527, PHE528 Pi-alkyl: TRP531


Convention hydrogen bonding: GLY255 Van der vaals:LEU227, SER253, TRP254, ASP258, ASP259, GLY294, ASN295, GLY296,ARG298, GLU299, TRP328, SER368 Carbon hydrogen bond: ALA292, ASP306


Convention hydrogen bonding: GLU271, ASN310, ALA532 Van der vaals: GLY265, PRO266, GLY307, SER311, ILE312, TYR313, GLN488, MET534 Pi-alkyl: ALA532 Pi-Pi: TRP531


Convention hydrogen bonding: SER330 Van der vaals: GLY297, HIS300, ASP301, ASN325, TRP328, GLU331, ALA332, ASN407, ALA408, ASN409, SER423 Pi-alkyl: VAL326


Convention hydrogen bonding: ASP154, ASN192, HIS194, GLY255 Van der vaals: GLU236, PRO256, GLU257 Pi-alkyl: LEU227, VAL231, TRP254

7 Hesperetin Binding energy=-8.2

Convention hydrogen bonding: PRO266, GLU271, ASN310, ALA532 Van der vaals: GLY265, ALA267, PHE274, GLY307, SER311, ILE312, TYR313, GLN488, MET534 Pi-alkyl: TRP531

8 Campesterol Binding energy=-8.1

Van der vaals: ASP264, GLY265, GLU271, GLY307, ASN310, SER311, GLN488, ARG498, GLY527, PHE528, ASN529, ASP530 Pi-alkyl:VAL263, PRO266, TRP531, ALA532


Convention hydrogen bonding: ASP154 Van der vaals: ASP153, ASP191, ASP192, GLU236, GLY255, PRO256, GLU257, ASP258 Pi-alkyl: HIS194, LEU227, VAL231, TRP254


Convention hydrogen bonding: ASP154, HIS194, ASN295 Van der vaals: ASP153, ASP191, ASN192, LEI227, ASP228, ASP258, SER368 Pi-alkyl:TRP328 Pi-Sigma: HIS194

11 Chavicine Binding energy=-8.0

Convention hydrogen bonding: GLN488 Van der vaals: GLY265, PRO266, GLU271, GLY307, ASN310, SER311, ILE312, TYR313, ASN529,KTRP531 Pi-alkyl: ALA532

12 Rosmarinic acid Binding energy=-8.0

Convention hydrogen bonding: VAL263, GLY307, GLY527 Van der vaals: ASP264, GLY265, PRO266, ASN310, SER311, ARG498,PHE528, ASN529 Pi-alkyl: ALA532 Pi-Pi: TRP531

13 Stigmasterol Binding energy=-8.0

Convention hydrogen bonding: SER311 Van der vaals: ASP264, GLY265, GLU271, GLY307, ILE312, TYR313, GLN488, ARG498, GLY527, PHE528, ASN529, ASP530 Pi-alkyl: VAL263, PRO266, TRP531, ALA532

Phytochemicals6vww (SARS-

CoV-2 NSP15

Endoribonucle

ase )

6w02 (SARS-CoV-2

ADP ribose

phosphatase of

NSP3)

6vyo (SARS-

CoV-2

nucleocapsid

RBD)

6w4b (SARS-

CoV-2 Nsp9

RBP)

4mds

(SARS-CoV

3CLpro)

2ajf (SARS-

CoV spike)

2cjr-SARS-CoV

Nucleocapsid

1-dehydrogingerdione -7.5 -7.5

6-gingerdione -7.5

6-gingerol -7.5

Apigenin -9 -8.7

Berberine -8.2 -9.7

Beta-sitosterol -8.6 -7.6 -8 -8



Calebin A -8.1 -8 -7.6

Campesterol -8.5 -7.9 -8 -7.7 -7.6

Chavicine -7.6 -8.8

Cirsilineol -7.6 -8.3

Cirsimaritin -7.6 -8.8


Cordioside -8.1 -8.1


Curcumin -8.6 -8.2

Cyclocurcumin -8.3 -9.2 -7.7 -7.6

Demethoxycurcumin -8.8 -7.6

Eriodictyol -9 -8.4

Eugenitin -7.6

Flavonol glucoside -8.5 -8.6 -7.5

Guineensine -7.8

Hesperetin -9 -8.5

Isochavicine -7.5

Target molecules; binding energy (kcal/mole) (Only those phytochemcals are included in

the table which have binding energy <- 7.5)

Table 6

Isopiperine -8.1 -7.8

Isothymonin -7.6

Isothymusin -7.6 -8 -7.5

Jatrorrhizine -7.9 -7.5

Kaempferol -8 -8.6


Oleanolic acid -9 -7.7 -7.6 -8.2 -7.9 -8.4

Orientin -9.4 -8.2 -7.8 -7.7

Pentadienoylpiperidine -8.7 -8.6 -7.8 -7.9

Piperamine -7.7

Piperettine -7.9 -9.2 -7.5 -7.9

Pipericide -7.6

Piperine -8.7 -7.6

Piperolein B -8.7

Quercetin -8.1 -8.3 -7.7 -7.5 -7.6

Retrofractamide A -7.9 -7.8 -7.6

Rhamnetin -7.9 -8.4 -7.6


Somniferine A -8.5 -8.3 -8.2 -7.5 -9.3 -8.5 -8.6

Stigmasterol -8.9 -7.9 -7.7 -7.9 -7.9 -8.6

Tinocordifolin -7.5 -7.6


Tinosporaside -7.6 -7.7 -8.1

Tinosporide -8.1 -8.2 -7.7 -7.8

Tricholein -8.1

Trichostachine -8.4 -8.6 -7.5 -7.6

Umbelliferone -7.5

Ursolic acid -9.1 -7.6 -7.8 -8 -8.2 -9

Vicenin -7.8 -7.6

Withaferin A -9.1 -9.2 -8.3 -8.2 -7.7 -7.7

Withanolide -9.3 -9.6 -8.7 -8.1 -8.3 -8.7 -7.9

Withanolide B -9.2 -9.2 -8 -9 -8.9 -8.6 -8.2

Withanone -8.9 -8.3 -8.2 -9.1 -8.5 -7.8 -7.8

Compounds

COX2 (PDB ID:

5f1a)

PLA2 (PDB

ID:4uy1) NIK(PDB ID:4dn5)

IRAK-4 (PDB

ID:2nru)

Native ligand -6 -7.7 -6.9 -9.4

Apigenin -9.2 -9.5 -8.3

Berberine -9 -8.4 -9

Beta-sitosterol -8.7 -9.7

Bisdemethoxycurcumin -9.4 -8.5 -8.6


Calebin-A -9.1 -8.5

Campesterol -8.7 -8.1 -9.5 -9.9

Chavicine -8.2 -8.4 -8.3

Cirsilineol -8.8 -8.8 -8.4

Cirsimaritin -8.9 -9.1 -8.4


Cordioside -9.6

Crategolic acid -8.9 -8.3 -8.1

Curcumin -8.2 -8 -8.4

Cyclocurcumin -9.3 -8.4 -8.8 -9.4

Demethoxycurcumin -8.9 -8 -8.9

Eriodictyol -9.7 -8.1 -9.7 -8.7

Eugenitin -8

Flavonol glucoside -8.9 -8.7

Guineensine -8.7

Hesperetin -8.9 -9.4 -8.4

Isochavicine -9.3 -8.7 -8.9

Isopiperine -8.6 -8.1 -8.7 -8.9

Isothymonin -9 -8.8 -8.3

Isothymusin -8.7 -8.9 -8.4

Jatrorrhizine -9.1 -9

Kaempferol -8.9 -8.6 -8.1


Oleanolic acid -8.9 -8 -8.2 -9.2

Orientin -9.6 -8.9 -8.6 -9.5

Pentadienoylpiperidine -8.7 -8.3

Piperamine -8.1 -8.3

Piperettine -8.5 -9.1

Piperine -8.7 -8.5 -8.1

Piperolein b -8.6

Quercetin -9.6 -8 -9.1 -8.4

Retrofractamide A -8

Rhamnetin -9.1 -8 -9.3 -8.9


phytochemcals are includen in the table which have binding energy <-

8.0)

Phytochemicals

Table 7


Somniferine A -9.2 -8.5 -8.6 -8.4

Stigmasterol -9.6 -8 -8.2 -9.8

Tinocordifolioside -8.1 -9.6


Tinosporaside -8

Tinosporide -8

Trichostachine -8.5 -8.1

Ursolic acid -8.9

Vicenin -8.8 -8.5 -10.1

Withaferin A -8.8 -9.1 -8.7 -9.2

Withanolide -9.4 -9.7 -8.6 -8.2

Withanolide B -9.3 -9.4 -8.4 -11.5

Withanone -10 -8.1 -8.7

Phytochemicals MW LogP

No. of

rotata

ble

bonds

No. of

HBA

No.

of

HBD

Surface

Area

Water

solubility

(log mol

/L)

Intestinal

absorption

(human) (%

Absorbed)

Total

Clearance

(log ml/

min/kg)

AMES

toxicity

Max.

tolerated

dose

(human)

(log

mg/kg/da

y)

Hepato-

toxicityApigenin 270.24 2.577 1 5 3 112.519 -3.329 93.25 0.566 No 0.328 No

Berberine 336.367 3.096 2 4 0 144.867 -3.973 97.147 1.27 Yes 0.144 Yes

Beta-sitosterol 414.718 8.025 6 1 1 187.039 -6.773 94.464 0.628 No -0.621 No

Bisdemethoxycurcumin 308.333 3.353 6 4 2 133.575 -3.38 91.159 -0.008 No -0.081 No

Brachyamide B 327.424 4.168 7 3 0 143.298 -4.631 93.346 0.237 No -0.418 Yes

Campesterol 400.691 7.635 5 1 1 180.674 -7.068 94.543 0.572 No -0.458 No

Chavicine 285.343 2.997 3 3 0 124.203 -3.464 94.444 0.232 No -0.38 Yes

Cordifolioside A 504.485 -2.65 10 13 7 200.81 -2.121 25.173 0.751 No 0.356 No

Cordioside 538.546 -0.14 5 12 5 218.29 -3.324 42.456 0.844 No -0.018 No

Crategolic acid 472.71 6.204 1 3 3 206.148 -3.042 100 -0.071 No 0.127 Yes

Curcumin 368.385 3.37 8 6 2 156.532 -4.01 82.19 -0.002 No 0.081 No

Cyclocurcumin 368.385 3.743 5 6 2 156.479 -3.978 90.24 0.072 No 0.046 No

Demethoxycurcumin 338.359 3.361 7 5 2 145.054 -3.566 91.393 0.026 No -0.12 No

Eriodictyol 288.255 2.216 1 5 4 119.029 -3.253 74.687 -0.013 No 0.014 No

Flavonol glucoside 400.383 0.639 4 8 4 164.724 -2.89 66.122 0.178 No 0.377 No

Hesperetin 302.282 2.519 2 6 3 125.714 -3.047 70.277 0.044 No 0.25 No

Isochavicine 285.343 2.997 3 3 0 124.203 -3.464 94.444 0.232 No -0.38 No

Isopiperine 285.343 2.997 3 3 0 124.203 -3.464 94.444 0.232 No -0.38 No

Isothymonin 360.318 2.603 4 8 3 146.955 -3.241 96.094 0.546 No 0.414 No

Jatrorrhizine 338.383 3.082 3 4 1 145.554 -3.871 94.465 1.222 No 0.175 Yes

Kaempferol 286.239 2.282 1 6 4 117.313 -3.04 74.29 0.477 No 0.531 No

Oleanolic acid 456.711 7.234 1 2 2 201.354 -3.074 99.931 -0.081 No 0.203 Yes

Orientin 448.38 -0.2 3 11 8 178.788 -2.905 43.733 0.374 No 0.567 No

Piperettine 311.381 3.553 4 3 0 136.244 -4.117 94.151 0.266 No -0.492 Yes

Pipericide 341.451 4.626 9 3 1 149.769 -5.605 93.876 1.364 No -0.213 Yes

Piperolein b 343.467 4.782 8 3 0 150.353 -5.029 92.451 1.202 No -0.382 Yes

Table 8- In-silico ADME prediction of the selected phytochemicals

Quercetin 302.238 1.988 1 7 5 122.108 -2.925 77.207 0.407 No 0.499 No

Retrofractamide A 327.424 327.4 8 3 1 143.404 -4.965 93.741 0.306 No -0.259 Yes

Rhamnetin 316.265 2.291 2 7 4 128.792 -3.212 80.214 0.473 No 0.56 No

Rosmarinic acid 360.318 1.761 6 7 5 147.396 -3.059 32.516 0.25 No 0.152 No

Somniferine A 608.691 2.698 3 9 2 260.855 -3.12 94.052 1.051 No -0.873 No

Stigmasterol 412.702 7.801 5 1 1 186.349 -6.682 94.97 0.618 No -0.664 No

Tinocordifolioside 412.479 -0.09 4 8 4 170.004 -3.584 59.166 0.93 No 0.116 No

Tinocordioside 478.538 1.451 4 9 4 197.856 -3.739 73.253 0.729 No -0.533 No

Tinosporaside 492.521 0.63 4 10 4 202.017 -3.648 67.679 0.665 No -0.507 No

Tinosporide 374.389 1.744 1 7 1 155.494 -3.393 100 0.414 No -0.373 Yes

Trichostachine 271.316 2.607 3 3 0 117.838 -3.118 94.833 0.23 No -0.327 Yes

Ursolic acid 456.711 7.09 1 2 2 201.354 -3.072 100 0.083 No 0.199 Yes

Vicenin 594.522 -2.39 5 15 11 235.468 -2.844 14.664 -0.108 No 0.46 No

Withaferin A 470.606 3.353 3 6 2 201.317 -5.063 85.345 0.435 No -0.695 No

Withanolide 470.606 3.495 2 6 2 201.317 -5.127 99.2 0.347 No -0.867 No

Withanolide B 454.607 4.381 2 5 1 196.523 -5.355 97.465 0.354 No -0.593 No

Withanone 470.606 470.6 2 6 2 201.317 -5.127 100 0.385 No -1.093 No

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