INTRODUCTION:

Human beings have been affected by many diseases in the past and this will continue to happen in the future as well. Multiple bacteria, fungi and viruses have attacked on the human population and have resulted in severe diseases^1,2.

Bacterial disease such as- cholera, tuberculosis, plague and syphilis, fungal diseases such as- aspergillosis, coccidioidomycosis, blastomycosis and viral diseases such as rubella, chickenpox, chikugunya have severely impacted the human lives².

The most recent pandemic caused by Corona virus has resulted in a great loss to lives throughout the world. Corona Virus Disease 2019 (COVID-19) is a contagious respiratory illness which has affected many people in different ways³. This disease is easily transmitted from animals to animals and animals to human beings which is the reason for its spread with such a high pace³. Some of the common symptoms of COVID-19 disease are dry cough, fever, loss of taste, loss of smell, headache, pain where as some of the serious symptoms include chest pain or pressure, loss of speech, shortness of breath.

Actually, the corona virus is not new to this world. It belongs to the corona virus family⁴. The following members of this family have already affected humans in the past for instance, SARS-COV-2, MERS-COV, HKU1 beta, 229E alpha⁵.

All the viruses present in the world – including SARS-CoV-2, the virus that causes COVID-19–show evolution during the course of time. When a virus replicates or makes copies of itself, certain changes occur in the genome of the virus called as ‘mutations’ which is normal for a virus. A virus with one or more new mutations is referred to as a “variant” of the original virus. There have been multiple mutants that have been seen in the corona virus like British variant seen in the patients in England⁶. Furthermore, the double mutant and most recently a triple mutant variety seen in the India patients are a cause of concern in the health care industry⁷.

Whatever little is known about the management of this disease relies mostly on the use of the allopathic medicines These medications not only cannot provide 100% cure to the disease but are also associated with significant side effects like allergy, constipation, skin rash or dermatitis. Some serious effects of allopathic medicine can be cancer, internal bleeding and suicidal thoughts etc⁸.

Due to the persistent issues of side effects associated with the allopathic medicines, an alternate to the same should be employed at the earliest. In this context, the Indian ayurvedic medicines are prepared naturally (plant based)⁹, therefore are mostly free from side effects. Apart from helping in the eradication of the diseases these medicines, if employed, would also function in increasing the strength and immunity of the body¹⁰.

With this concept in mind, we have tested the efficacy of certain ayurvedic compounds against the COVID-19 virus in a computational study.

Dragon fruit (Selenicereus undatus) whose active agent is alpha amyrin, (Pubchem CID- 73170) has antioxidant and antiviral properties. Furthermore, vitamin C present in this fruit can aid in improving immunity and preventing the viral disease¹¹. Ashwagandha (Withania somnifera) whose active agent is anaferine (Pubchem CID- 443143) is known to have medicinal properties against virus, bacteria and fungi¹². Punarnava (Boerhavia diffusa) whose active agent is beta-sitosterol (Pubchem CID- 222284), which is known to have medicinal properties and it also increases strength and immunity of the body¹¹. Broccoli (Brassica oleracea) has two active agents- glucoraphanin (Pubchem CID- 6602383) and sulforaphane (Pubchem CID- 5350), which are known to bind with the targets of COVID-19. Apart from the antiviral activity antioxidant and antibacterial properties of broccoli have also been reported¹². Lemon (Citrus limon) whose active agent is hydroxycinnamic acid (Pubchem CID- 637542) binds with the targets of COVID-19, citrus fruit are amongst those traditional food items which can increase strength of the body along with its antiviral properties¹³. Hydroxycinnamic acid has multiple important functions in aiding the plant to adapt to stress such as antioxidant, viruses, cold stress etc¹⁴. Triphala (Emblica officinalis) whose active agent is syringic acid (Pubchem CID- 10742), is also antiviral operator that acts against swine flu, HSV-1, HIV-1 and other viruses¹⁵. Moringa (Moringa oleifera) whose active agent is pantothenic acid (Pubchem CID- 6613) and niacin or nicotinic acid (Pubchem CID- 938) is known to bind with the targets of COVID-19¹⁶. Angilica (Angilica archangelica) whose active agent is apigenin, which is known to have antiviral activity¹⁷.

These ayurvedic products were tested for their efficacy against the different targets identified in the corona virus like-

Spike glycoprotein (6vxx): Spike glycoprotein is used by the covid-19 virus to attach to the host cell and henceforth cause infection to host¹⁸.

Nucleocapsid protein (6m3m): The covid-19 nucleocapsid protein is essential for viral RNA replication and packaging into new virions¹⁹.

Hemagglutinin esterases (3cl5): The hemagglutinin-esterases (HEs) comprise a family of viral envelope glycoproteins that facilitate reversible attachment to O-acetylated sialic acids by acting both as lectins and as receptor-destroying enzymes (RDEs)²⁰.

MATERIALS AND METHODS:

Protein targets retrieval:

Structure of the corona virus protein targets (Spike glycoprotein, Nucleocapsid, Hemagglutinin) were retrieved from protein data bank.

Ligands structure retrieval:

Pubchem database was used to obtain the 3D structures of the plant products viz. alpha-amyrin, anaferine, beta-sitosterol, glucoraphanin, sulforaphane, hydroxycinnamic acid, syringic acid, pantothenic acid, nicotinic acid and apigenin.

Open babel²¹: This is a downloadable software, which is mainly used to interconvert different file formats. In the current study SDF format obtained from the Pubchem database of compounds- alpha-amyrin, anaferine, beta-sitosterol, glucoraphanin, hydroxycinnamic acid, sulforaphane, syringic acid, pantothenic acid, nicotinic acid and apigenin were converted to the PDB format using this software.

UCSF chimera²²: This software is an extensible program for interactive 3D visualization of molecular structure data, density maps, supramolecules assemblies, sequence alignment, docking etc. In the current study, it was used as a visualization software for setting up the calculations and analyzing the results obtained from PatchDock and FireDock.

Patchdock and Firedock²³: PatchDock and FireDock are online web servers which have been used in this study for the purpose of molecular docking. With the aid of these softwares, the best suited ligands were identified against the corona virus.

RESULTS:

The 3D structure of the proteins (Spike glycoprotein, Nucleocapsid and Hemagglutinin) were obtained from the protein data bank (Fig 1).

Figure 1: Structure of the target proteins. a) Spike glycoprotein b) Nucleocapsid c) Hemagglutinin

The structures of the ligands (alpha-amyrin, anaferine, beta-sitosterol, glucoraphanin, sulforaphane, hydroxycinnamic acid, syringic acid, pantothenic acid, nicotinic acid and apigenin) were obtained from Pubchem (Figure 2)

Figure 2: Structure of the ligands. a) alpha amyrin b) anaferine c) apigenin d) beta-sitosterol e) glucoraphanin f) hydroxycinnamic acid g) nicotinic acid h) pantothenic acid i) sulforaphane j) syringic acid

The binding energies of different ligands were determined against the 3 protein targets using PatchDock and FireDock softwares. The results of the binding energies have been mentioned in table 1.

Table 1: Binding energies of ligands against the 3 protein targets (Binding energies are in KJ/mol).

	Protein Targets
Ligands	Spike	Hemagglutinin Esterase	Nucleocapsid
1. Alpha-amyrin	-48.9	-31.76	-59.42
2. Anaferine	-32.8	-30.34	-39.68
3. Beta-sitosterol	-49.4	-25.41	-55.44
4. Glucoraphanin	-51.2	-26.32	-53.79
5. Hydroxycinnamic	-36.0	-20.24	-25.57
6. Nicotinic acid	-27.2	-15.26	-17.18
7. Pantothenic acid	-24.5	-17.26	-33.83
8. Sulforaphane	-35.8	-23.79	-33.54
9. Syringic acid	-34.8	-2235	-28.36
10. Apigenin	-35.2	-27.2	-40.26

Amidst all the ligands one ligand was identified against each protein target having the best binding energy as depicted in table 2. It was found that glucoraphanin had the best binding energy of -51.44 KJ/mol against the spike protein, alpha-amyrin against hemagglutinin (-31.76 KJ/mol), and beta-sitosterol against nucleocapsid (-55.44 KJ/mol).

Table 2: Protein targets with their respective ligands showing the highest binding energy

Protein:	Spike protein	Hemagglutinin	Nucleocapsid
Alpha-amyrin		-31.76
Beta-sitosterol			-55.44
Glucoraphanin	-51.44

The best docked structures of alpha- amyrin against hemagglutinin, beta sitosterol against nucleocapsid and glucoraphanin against spike protein have been provided in Figure 3.

Figure 3: Best docked structures a)Hemagglutinin vs alpha-amyrin b) Nucleocapsid vs beta- sitosterol c) Spike glycoprotein and glucoraphenin

DISCUSSION:

Covid-19 is fast spreading disease and has shown its effect worldwide. Severely hit countries of the world include USA, India, Brazil, Russia, Italy and the United Kingdom²⁴. As this paper is being written, India and certain other countries are facing the second wave of this virus²⁵. The medicines and the hospital facilities are proving to be insufficient for these developing countries. Therefore, keeping this problem in mind, this manuscript was focused on finding suitable remedies against this virus. Products from Indian traditional medicine were used in this study and their effect was tested against 3 important proteins targets (Hemagglutinin, Nucleocapsid, Spike glycoprotein) of the corona virus. The aim behind choosing the ayurvedic compounds was that these are plant based, free from side effects, easily accessible, cheap, can be used as home remedies and would also result in boosting the immune system. A combination of these compounds would ensure that the patients do not reach a critical condition. These compounds can be orally administered in the patient’s body in the form of an ayurvedic drink called ‘Karha’ or ‘Kadha’ (commonly used in India) which comprises of number of herbs and spices. This could alleviate the immune response and prove wonders against the corona virus.

There are numerous studies in which the plant products have been used against the corona virus for instance Puttaswamy and coworkers virtually screened 4704 plant secondary metabolites (PSM) against 4 targets of corona virus. The results showed that amongst all the triterpenoids tested 50% of them showed good interactions with spike protein²⁶. In a similar study 10 Mexican natural products (emodin anthrone, kaempferol, quercetin, aesculin, cichoriin, luteolin, matricin, riolozatrione, monocaffeoyl tartaric acid, aucubin) were screened against the corona virus and it was found that only three compounds target the important proteins of SARS-CoV-2 viz. quercetin, riolozatrione and cichoriin, but only one was found to be safe (cichoriin)²⁷. In a separate study 14,011 phytochemicals produced by Indian medicinal plants were tested for their inhibitory activity against two human proteases, TMPRSS2 and cathepsin L. It was found that 96 compounds could inhibit TMPRSS2 and 9 compounds could inhibit cathepsin L²⁸. Additionally, Alrasheid and co-workers identified Gallic acid (− 17.45), Quercetin (− 15.81), Naringin (− 14.50), Capsaicin (− 13.90), and Psychotrine (− 13.5) are important sources for novel antiviral drugs targeting COVID-19²⁹.

In our study also we got descent binding energies of -51.44 KJ/mol obtained by docking glucoraphanin against the spike protein, -31.76 KJ/mol obtained by docking alpha-amyrin against hemagglutinin, -55,44 KJ/mol obtained by docking beta-sitosterol against nucleocapsid (-55.44 KJ/mol) and thus these are prospective compounds against the corona virus however their in-vitro and in-vivo effect still remains to be seen.

CONCLUSION:

The COVID-19 pandemic has caused some serious issues worldwide and developing countries like India, Brazil and developed countries like United States have suffered severely due to it. The limited hospital resources have worsened the situation in India. In this paper we have proposed that alpha-amyrin, beta-sitosterol and glucoraphanin present in Dragon fruit (Selenicereus undatus) broccoli (Brassica oleracea) and Punarnava (Boerhavia diffusa) respectively if administered to the covid positive patients it can reduce the infection load and help the patients to recover early.

CONFLICT OF INTEREST:

The authors state that there is no conflict of interest.

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Received on 07.05.2021 Modified on 22.05.2021

Asian J. Pharm. Res. 2021; 11(3):167-172.

DOI: 10.52711/2231-5691.2021.00031