WO2022084908A1

WO2022084908A1 - Compositions for inhibiting sars-cov-2 virus

Info

Publication number: WO2022084908A1
Application number: PCT/IB2021/059714
Authority: WO
Inventors: Upasana Arora; Alessandro MARCELLO
Original assignee: Sun Pharmaceutical Industries Limited
Priority date: 2020-10-21
Filing date: 2021-10-21
Publication date: 2022-04-28

Abstract

The present invention relates to an extract of Cocculus hirsutus, or one or more components thereof, for inhibiting a SARS-CoV-2 virus. The disclosure also discloses a method for inhibiting or reducing the proliferation of the SARS-CoV-2 virus by administering or treating a subject with the extract and/or marker compounds or with a pharmaceutical composition comprise of the extract and/or marker compounds. The present disclosure also describes in-vitro methods for inhibiting SARS-CoV-2 viral proliferations using an extract of Cocculus hirsutus or isolated compounds therefrom.

Description

COMPOSITIONS FOR INHIBITING SARS-CoV-2 VIRUS

FIELD OF THE INVENTION

The present disclosure relates to a composition comprising an extract of Cocculus hirsutus, for reducing or inhibiting the proliferation of a SARS-CoV-2 virus.

BACKGROUND OF THE INVENTION

Severe Acute Respiratory Syndrome Associated CoronaVirus-2 (SARS-CoV-2), also known as COVID-19, is a highly contagious and novel virus belonging to the Coronoviridae family, which has caused a global pandemic and raised worldwide health concerns. SARS-CoV-2 appears to spread easily in the human population. Many healthcare workers have been infected, and more clusters of cases are being detected with each passing day. According to the World Health Organization (WHO), COVID- 19 Situation dashboard data of March 29, 2020, there were 638,146 confirmed cases infected with SARS-CoV-2 with 30,105 deaths, By October 18, 2020, the pandemic had grown to 39,442,444 confirmed cases of SARS-CoV-2 infections, including 1,106,181 deaths globally. Current therapeutic strategies for SARS-CoV-2 infections are only supportive and not sufficient to control this pandemic. The unprecedented rapidity of spread of this outbreak presents a critical need for an effective measure against SARS-CoV-2 that can effectively inhibit viral proliferation and/or reduce the virus load in a subject infected with SARS-CoV-2 virus.

SUMMARY OF THE INVENTION

The present invention provides a method for inhibiting the proliferation of SARS- CoV-2 virus in a mammalian subject such as human being infected with the virus by administering a composition comprising an extract of Cocculus hirsutus, and/or one or more marker compounds. The composition reduces the rate of viral proliferation and viral load of SARS-CoV-2 virus in the infected subject.

In one aspect, the present invention also provides an oral composition of an extract of Cocculus hirsutus for use in the prevention and treatment of SARS-CoV-2 virus infection.

In another aspect, the present invention provides an oral composition of one or more marker compounds of Cocculus hirsutus for use in the prevention and treatment of SARS- CoV-2 virus infection. In some aspects, the present invention also provides methods for reducing copies of viral RNA in a tissue sample of a subject infected with SARS-CoV-2. Further, it provides for a stable pharmaceutical composition comprising a therapeutically effective amount of an extract and one or more marker compounds for use in the treatment of SARS-CoV-2 virus infection in a mammal. It also relates to processes for the preparation of these extracts. It further provides the activity of these extracts against SARS-CoV-2 virus in mammals. Further, these extracts and marker compounds were found to be safe for administration to humans and did not show any toxic effects even at relatively high doses.

DRAWINGS

Figure 1: Dose response graph of percent SARS-CoV-2 inhibition or activity (sphere curve; left Y axis) and cell viability (square curve; right Y axis) of a tablet of purified aqueous extract of Cocculus hirsutus obtained via the plaque assay and AlamarBlue cytotoxicity assay, respectively. Their corresponding ECso and CC50 values are shown at the top left comer of the graph. The concentrations of test samples are denoted on the X axis in pg/ml.

Figure 2: Dose response graph of percent SARS-CoV-2 inhibition or activity (sphere curve; left Y axis) and cell viability (square curve; right Y axis) of a placebo tablet obtained via the plaque assay and AlamarBlue cytotoxicity assay, respectively. The concentrations of test samples are denoted on the X axis in pg/ml.

Figure 3 : Dose response graph of percent SARS-CoV-2 inhibition (sphere curve; left Y axis) and cell viability (square curve ; right Y axis) of marker compound A obtained via the plaque assay and AlamarBlue cytotoxicity assay, respectively. Their corresponding EC50 and CC50 values are shown at the top left comer of the graph. The concentrations of Compound A are denoted on the X axis in pg/mL.

DESCRIPTION OF THE INVENTION

The terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” are understood to follow unless explicitly stated otherwise. As used herein, the term “about” is meant to account for variations due to any experimental errors which may be commonly accepted in the analytical chemistry field. All measurements reported herein are understood to be modified by the term “about,” whether or not the term is explicitly used, unless explicitly stated otherwise. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other suitable methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and not intended to be limiting by any means. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict, the present specification, including definitions, will control.

The present disclosure provides an extract of Cocculus hirsutus, or one or more components thereof, for inhibiting the SARS-CoV-2 virus. The present disclosure describes a purified extract and a pharmaceutical composition of such extract from Cocculus hirsutus which was found to be effective in inhibiting SARS-CoV-2 virus in-vitro.

In an embodiment, the extract of Cocculus hirsutus according to present disclosure was found to be effective in reducing the viral load of SARS-CoV-2 virus.

In another embodiment, the present disclosure provides a method of inhibiting proliferation of SARS-CoV-2 virus comprising treating said SARS-CoV-2 virus with an extract of Cocculus hirsutus, or one or more components thereof. In one aspect said treatment is performed in-vivo. In a preferred aspect, said treatment is performed in a mammalian subject. In a more preferred aspect, said mammalian subject is a human. In another aspect, said treatment is performed in-vitro.

In yet another embodiment, the present disclosure provides a method of inhibiting proliferation of SARS-CoV-2 virus comprising treating said SARS-CoV-2 virus with an extract of Cocculus hirsutus, or one or more components thereof, wherein inhibition is characterized by a reduced rate of proliferation of SARS-CoV-2 virus.

In one embodiment, the inhibition of SARS-CoV-2 virus is characterized by a reduced viral load of SARS-CoV-2 virus in a subject infected with SARS-CoV-2 virus.

In another embodiment, the present disclosure provides a method for the treatment of a viral infection characterized by a presence of a SARS-CoV-2 virus in a mammalian subject, comprising administering to said subject an effective amount of an extract of Cocculus hirsutus, or one or more components thereof. In one aspect, said extract of Cocculus hirsutus, or one or more components thereof, is provided in the form of a pharmaceutical composition. In yet another embodiment, the present disclosure provides a method of determining the anti-viral activity of an extract of Cocculus hirsutus, or one or more components thereof, or a pharmaceutical composition comprising said extract or one or more components thereof, wherein said method comprises contacting one or more concentrations and/or volumes of said extract, or one or more components thereof, or said composition to the SARS-CoV-2 virus, and identifying the therapeutically effective concentration and/or volume inhibiting the proliferation of said virus. In an aspect, said inhibition is determined by in-vitro biochemical assays, cell-based assays evaluating the activity of inhibitors like, plaque reduction assays, flow-cytometry assays and other antiviral activity assays.

In another embodiment, the present disclosure provides a method of determining the anti-viral activity of an extract of Cocculus hirsutus, or one or more components thereof, or a pharmaceutical composition comprising said extract or one or more components thereof, wherein the method comprises administering one or more concentrations and/or volumes of the extract, or one or more components thereof, or the composition to a subject infected with the SARS-CoV-2 virus, and identifying the therapeutically effective concentration and/or volume causing inhibition of the proliferation of the said virus. In an aspect, said inhibition is determined by in-vitro biochemical assays, cell-based assays evaluating the activity of inhibitors like, plaque reduction assays, flow-cytometry assays and other antiviral activity assays.

In a further embodiment, the present disclosure provides an extract of Cocculus hirsutus for use in inhibition of the proliferation of the SARS-CoV-2 virus.

In an embodiment, the disclosure provides a pharmaceutical composition comprising an extract of present disclosure, wherein said extract reduces viral load of the SARS-CoV-2 virus in a subject infected with such virus.

In yet another embodiment, the disclosure provides a pharmaceutical composition comprising an extract of present disclosure, wherein said extract reduces the rate of proliferation of the SARS-CoV-2 virus.

Reduced viral load or reduced proliferation of the SARS-CoV-2 virus may be characterized by methods commonly known in the art to detect and/or quantify the presence and viral load of SARS-CoV-2 virus. Non-limiting examples of such characterization methods may be identifying the copies of viral RNA using PCR or RT-PCR method in a sample, in-vitro biochemical assays, cell-based assays evaluating the activity of inhibitors like, plaque reduction assays, flow-cytometry assays and other antiviral activity assays.

In a further embodiment, the present disclosure provides a pharmaceutical composition comprising an extract of Cocculus hirsutus or an extract as such for use in inhibiting the proliferation of the SARS-CoV-2 virus. In an aspect of the disclosure the extract of Cocculus hirsutus effectively reduces viral load of the SARS-CoV-2 virus in a subject infected with such virus.

In one embodiment, the present disclosure provides an anti-SARS-CoV-2 composition for inhibiting SARS-CoV-2 viral proliferation, wherein said composition comprises of a therapeutically effective amount of an extract of Cocculus hirsutus. In an aspect, the anti-SARS-CoV-2 composition is an oral dosage form. In another aspect, said composition further comprises a pharmaceutically acceptable excipient selected form a diluent, a binder, a disintegrant, lubricant, glidant, polymers, flavoring agents, surfactants, preservatives, buffers and tonicity modifying agents.

In another embodiment, the composition of present disclosure is used in a method of treating a SARS-CoV-2 virus infection wherein the composition effectively inhibits the proliferation of the SARS-CoV-2 virus. In another aspect, said method of treating or preventing viral infections caused by a SARS-CoV-2 virus comprises using said extract to reduce the viral load by inhibiting the proliferation of virus growth.

In one aspect, the present disclosure concerns a method for inhibiting proliferation of a SARS-CoV-2 virus comprising administering a therapeutically effective amount of an extract of Cocculus hirsutus or a composition thereof.

In another aspect, the disclosure concerns a method for the treatment of SARS-CoV- 2 infection in a mammalian subject, comprising administering to the subject an effective amount of an extract of Cocculus hirsutus, or one or more components thereof.

In yet another aspect, the disclosure concerns a method for identifying an anti-viral activity of an extract of Cocculus hirsutus, or one or more components thereof, wherein the method comprises treating or exposing one or more concentrations and/or volumes of said extract or one or more components thereof, to a SARS-CoV-2 virus, and identifying the concentration and/or volumes of the extract or one or more components thereof, that inhibits the proliferation of the virus. In an aspect, said inhibition of the virus is determined by in- vitro biochemical assays, cell-based assays evaluating the activity of inhibitors like, plaque reduction assays, flow-cytometry based assays and other antiviral activity assays.

The terms “extract” as used herein, refer to a product of an extraction of one or more components obtained from Cocculus hirsutus in any concentration comprising one or more of components such as marker compounds, either alone or in a combination. In some embodiments, one or more components of Cocculus hirsutus are removed during the extraction, e.g., the plant mass is removed, and other components in the extract are separated into and concentrated in the extract. Thus, the present disclosure provides for an extract which is a new composition of compounds and components in a concentration and ratio that is not found in nature. Additionally, the present disclosure provides for an extract which is free from one or more contaminants, which may, in some embodiments, cause side effects and/or reduce efficacy. In some embodiments, the extracts as described herein exhibit properties not found or appreciated in the Cocculus hirsutus plant, e.g., increased stability, increased inhibitory activity against SARS-CoV-2, increased bioavailability, increased solubility, reduced side-effects, etc. The extract may be a purified extract or a crude extract. The purified extract is an extract that is substantially free of pesticide residues, aflatoxins or any microbial impurities. The purified extract may be an extract enriched for any one of the Marker compounds up to 65%, preferably 70%, more preferably 85% and even more preferably up to 90-99% w/w of said extract. The enrichment may be carried out any time before, during or after purification by methods commonly known in the art for such enrichment, such as by way of repeated purifications and/or fractionations using a combination of solvents and other conditions. The extract may be present in the form of a liquid, semisolid, solid powder, solid cake, gel, paste, dispersion, solution or a distillate. In one embodiment, the extract is a dried extract.

The term “marker compound” as used in the present disclosure comprises Compound A, Compound B, Compound C, Compound D or Compound E which are isolated using various isolation techniques from an extract of Cocculus hirsutus.

The “marker compound” according to present disclosure are synthetic compounds or isolated from an extract of Cocculus hirsutus. The marker compounds are represented by following structures as follows:

Compound A, Compound B, Compound C,

Compound D, and Compound E.

The term “SARS-CoV-2” or “COVID-19” or “COVID” or “novel coronavirus” are used interchangeably throughout the specification, and refer to the novel coronavirus related to the Sever Acute Respiratory Syndrome (SARS) virus, and named by International Committee on Taxonomy of Viruses (ICTV) as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The term “Anti-SARS-CoV-2 composition” or “Anti- SARS-CoV-2 effect” as used in the specification refers to an inhibitory action of the extract or the composition of the present disclosure in inhibiting or reducing the proliferation of SARS-CoV-2 virus.

The present disclosure also provides a dosage form of a purified extract from Cocculus hirsutus which was found to be effective in inhibiting SARS-CoV-2 virus when tested in-vitro. The dosage form is useful in prevention and/or treatment of infections caused by SARS-CoV-2 virus.

The term “treatment” as used within the context of the present disclosure, is meant to include inhibition of viral proliferation and/or reducing the SARS-CoV-2 viral load.

In one aspect, the present disclosure concerns a method of inhibiting proliferation of SARS-CoV-2 virus comprising administering a therapeutically effective amount of an extract of Cocculus hirsutus, or one or more components thereof, or a composition thereof. In another aspect, the disclosure concerns a method for the treatment of SARS-CoV- 2 infection in a mammalian subject, comprising administering to the subject an effective amount of an extract of Cocculus hirsutus, or one or more components thereof, or a composition thereof.

In yet another aspect, the disclosure concerns a method for identifying an anti-viral activity of an extract of Cocculus hirsutus, or one or more components thereof, wherein the method comprises treating or exposing or contacting or incubating one or more concentrations and/or volumes of the said extract or one or more components thereof, with SARS-CoV-2 virus, and identifying the concentration and/or volumes of the said extract agent or one or more components thereof, that inhibits the proliferation of the virus. In one aspect, said inhibition is determined by in-vitro biochemical assays, cell-based assays evaluating the activity of inhibitors like, plaque reduction assays, flow-cytometry assays and other antiviral activity assays.

In one embodiment, the present disclosure provides a method of reducing or inhibiting proliferation of a SARS-CoV-2 virus, using a pharmaceutical composition comprising a therapeutically effective amount of an extract of Cocculus hirsutus, or one or more components thereof, or a composition thereof.

In one embodiment, the reduction of SARS-CoV-2 viral load is achieved by inhibition or reduction of the rate of proliferation of a SARS-CoV-2 virus using a therapeutically effective amount of an extract of Cocculus hirsutus, or one or more components thereof, or a composition thereof.

In one aspect, the pharmaceutical composition or dosage form according to the present disclosure is an oral dosage form.

In a preferred embodiment, the oral dosage form is selected from powder, pellets, granules, spheroids, mini-tablets, caplets, tablets, sachet or a capsule comprising such powder, pellets, granules, spheroids, min-tablets or caplets, or a liquids selected from solutions, suspensions, emulsions, syrups, linctuses, elixirs or drops.

In another aspect, the present disclosure provides a pharmaceutical composition of the purified extract of Cocculus hirsutus for non-oral administration.

In yet another embodiment, the present disclosure provides a pharmaceutical composition comprising an extract of Cocculus hirsutus, or one or more components thereof, or a composition thereof, for reducing the SARS-CoV-2 viral load. In another embodiment, the present disclosure provides a pharmaceutical composition comprising a Cocculus hirsutus extract comprising marker compounds or a combination thereof for reducing the SARS-CoV-2 viral infection wherein such reduction is characterized by an inhibition of SARS-CoV-2 virus proliferation.

In yet another embodiment, the extract of the present disclosure can be used for inhibition of SARS-CoV-2 viral proliferation.

In a further embodiment the present disclosure provides a pharmaceutical composition comprising an extract of Cocculus hirsutus for inhibiting the SARS-CoV-2 virus proliferation and/or reducing the SARS-CoV-2 viral load in a dose of about 0.02 mg / kg to about 150 mg/kg body weight. In an aspect, said extract is present in the composition in a concentration range of about 25 mg to about 1200 mg by weight of composition. The dose can be administered as a single dosage form or as multiple dosage forms equating to the required dose. In another aspect, the extract of present disclosure is administered at a dose of 0. 1 pg/mL to 1 mg/mL to a subject infected with SARS-CoV-2 virus.

In a further embodiment, the present disclosure provides a pharmaceutical composition comprising an extract of Cocculus hirsutus or one or more components thereof such as a marker compound, or a combination of other marker compounds, isolated form the extract of Cocculus hirsutus for inhibiting the SARS-CoV-2 virus proliferation and/or reducing the SARS-CoV-2 viral load in a dose of about 0.02 mg/kg to about 150 mg/kg body weight. In one aspect, the composition comprises a marker compound in concentration range of about 0.25 mg to about 100 mg by weight of the composition. The dose can be administered as a single dosage form or as multiple dosage forms equating to the required dose. In another aspect, a marker compound of present disclosure is administered at a dose of 0.1 pg/mL to 1 mg/mL to a subject infected with SARS-CoV-2 virus, for inhibition of proliferation of SARS-CoV-2 virus.

In another embodiment, the present disclosure provides a method for inhibition of SARS-CoV-2 virus proliferation.

In some embodiments, the term “extraction” refers to the separation and removal of one or more components of Cocculus hirsutus, e.g., plant solids (e.g., fibers, cellulose, etc.) extracted from one or more fluids in the plant. In some embodiments, the extraction is a solid/liquid separation operation: e.g., a plant is placed in contact with a fluid (a solvent). In some embodiments, the plant components of interest are solubilised into solution with the solvent. The solution thus obtained is the desired extract. In some embodiments, the solvent will eventually be eliminated to arrive at the extract.

In another embodiment, the extract of the present disclosure is an aqueous extract or an organic solvent extract, wherein the organic solvent is a polar or non-polar organic solvent. In an aspect of the embodiment, the extraction is an alcoholic extraction, e.g., a Ci- C4 alcohol extraction, a hydroalcoholic extraction, or an aqueous extraction. In some embodiments, various parts of Cocculus hirsutus can be used, e.g., the extraction can be performed from the stem or other parts of the plant, such as aerial parts or roots. In one aspect, the extract is an aqueous extract. Any solvents in the extract may be removed completely by evaporation to obtain a dried extract. A dried extract may be lyophilized to form a powder, which can then be filled into a capsule of suitable size or compressed into tablets with or without pharmaceutically acceptable excipients. In a related embodiment, the extract may be used as such in liquid or semisolid form without further drying along with a suitable pharmaceutically acceptable carrier for administration. In one embodiment, the extract is a purified extract. In another embodiment, the extract is a crude extract.

In another aspect of the above embodiment, the extract is an alcoholic extract, or a hydro-alcoholic extract from the stem or other parts of the plant, such as aerial parts or roots. In one embodiment, the extract will be derived from wet parts of the plant to arrive at an aqueous extract. Any solvents in the extract may be removed completely, e.g., by evaporation to obtain a dried extract. The dried extract may be lyophilized to form a powder, which can then be filled into a vial or a capsule of suitable size or compressed into tablets with or without pharmaceutically acceptable excipients. In a related embodiment, the extract may be used as such in liquid or semisolid form without further drying along with a suitable pharmaceutically acceptable carrier for administration. In one aspect, the extract may be derived from dry parts of the plant to arrive at an aqueous extract.

The term “alcoholic extract,” as used herein, includes any alcohol-based extract, for example, methanolic, ethanolic, n-propanolic, isopropanolic, n-butanolic, iso-butanolic or t-butanolic extract of Cocculus hirsutus.

The term “hydro-alcoholic extract,” as used herein, includes an extract prepared by using a mixture of alcohol and purified water. It may also include an extract prepared in denatured spirit with other organic solvents. Examples of alcohols are methanol, ethanol, n- propanol, isopropanol, n-butanol, iso-butanol, and t-butanol. The ratio of alcohol to water in the said hydroalcoholic extract may be in the ratio of 99: 1 to 1:99, or 95:5 to 5:95, or 90: 10 to 10:90, or 80:20 to 20:80, or 70:30 to 30:70, or 60:40 to 40:60, or a 1: 1 mixture of alcohol and purified water.

The term “aqueous extract,” as used herein, includes a water or purified water extract of Cocculus hirsutus.

The extracts of Cocculus hirsutus include (a) the extracts obtained by extraction of plant mass of Cocculus hirsutus with one or more solvents, and (b) the fractions obtained by partitioning of the extracts with one or more solvents. In a preferred embodiment, the extracts of Cocculus hirsutus include (a) the extracts obtained by extraction of stem of Cocculus hirsutus with purified water, and (b) the fractions obtained by partitioning of the extracts with one or more solvents.

The solvents for extraction may be, for example, water; alcohols, for example, methanol, ethanol, propanol, isopropanol or butanol; ketones, for example, acetone or methyl isobutyl ketone; esters, for example, methyl acetate or ethyl acetate; halogenated hydrocarbons, for example, chloroform, dichloromethane or ethylene dichloride; petroleum fractions, for example, hexane, petroleum ether or heptane; or mixture(s) thereof.

The solvents for partitioning may be, for example, water; petroleum fractions, for example, hexane, petroleum ether or heptane; halogenated hydrocarbons, for example, chloroform, dichloromethane or ethylene dichloride; esters, for example, ethyl acetate or methyl acetate; ketones, for example, acetone or methyl isobutyl ketone; alcohols, for example, butanol; ethers, for example, diethyl ether; or mixture(s) thereof.

The term “plant mass of Cocculus hirsutus." as used herein, refers to the whole plant, which includes aerial parts, for example, fruits, flowers, leaves, branches, stem bark, stems, seeds or heartwood, and roots. In a preferred embodiment, the “plant mass of Cocculus hirsutus" refers to stem of Cocculus hirsutus. In an aspect such plant mass may be from a wet part or a dry part.

In yet another embodiment, the extract of Cocculus hirsutus may be comprised of various marker compounds. In a related embodiment, the extract of Cocculus hirsutus may be used for isolating the marker compounds and there use in a method of treating or preventing viral infection caused by a SARS-CoV-2 viruses by inhibiting and/or reducing the proliferation of the virus. The pharmaceutical composition of the present disclosure is a stable dosage form that may further comprise one or more pharmaceutically acceptable excipient. The term “pharmaceutical composition,” as used herein, includes any composition that can effectively deliver the extracts of Cocculus hirsutus to the site of action for inhibition of SARS-CoV-2 virus.

A “stable dosage form” or “a stable pharmaceutical composition” as used herein refers to a composition which is stable over extended period of time on storage as assessed from the content of one or more impurities in the composition as described in standard textbooks. The stable pharmaceutical composition of the present disclosure was found to be stable for at least 3 months at accelerated conditions of stability testing, i.e., 40 ± 2°C temperature and 75 ± 5% Relative Humidity (RH), and at long term storage stability conditions of 30 ± 2°C/75 ± 5% RH and 25 ± 2°C/60 ± 5% RH. The product can be stored at room temperature for a shelf life of about 6 months to 2 years or more. The composition was found to be stable in spite of the presence of flavonoids, alkaloids, lignanas etc., as constituents in the aqueous extract which together may be difficult to formulate and may not be stable during storage.

A “therapeutically effective amount” as used herein refers to an amount of the extract of the disclosure sufficient to inhibit or reduce the viral proliferation of SARS-CoV- 2 virus.

The term “pharmaceutically acceptable excipients,” as used herein may include diluents, binders, disintegrants, lubricants, glidants, polymers, flavoring agents, surfactants, preservatives, antioxidants, buffers, and tonicity modifying agents.

Non-limiting examples of diluents include microcrystalline cellulose, powdered cellulose, starch, pre-gelatinized starch, dextrates, lactitol, fructose, sugar compressible, sugar confectioners, dextrose, anhydrous lactose, calcium phosphate-dibasic, calcium phosphate-tribasic, calcium sulfate, and mixtures thereof.

Non-limiting examples of binders include a water-soluble starch, for example, pregelatinized starch; a polysaccharide, for example, agar, gum acacia, dextrin, sodium alginate, tragacanth gum, xanthan gum, hyaluronic acid, pectin, or sodium chondroitin sulfate; a synthetic polymer, for example, polyvinylpyrrolidone, polyvinyl alcohol, carboxy vinyl polymer, polyacrylic acid-series polymer, polylactic acid, or polyethylene glycol; a cellulose ether, for example, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, or hydroxypropyl methyl cellulose; and mixtures thereof.

Non-limiting examples of disintegrants include calcium carbonate, carboxymethyl cellulose or a salt thereof, for example, croscarmellose sodium, crosslinked povidone, low- substituted hydroxypropyl cellulose, and sodium starch glycolate.

Non-limiting examples of lubricants / glidants include talc, magnesium stearate, hydrogenated vegetable oils, sodium stearyl fumarate, calcium stearate, colloidal silicon dioxide, Aerosil®, stearic acid, sodium lauryl sulphate, sodium benzoate, polyethylene glycol, hydrogenated castor oil, sucrose esters of fatty acids, microcrystalline wax, yellow beeswax, white beeswax, and mixtures thereof.

Non-limiting examples of flavoring agents include synthetic flavor oils and flavoring aromatics; natural oils or extracts from plants, leaves, flowers, and fruits; and combinations thereof. These may include cinnamon oil, oil of wintergreen, peppermint oils, bay oil, anise oil, eucalyptus, thyme oil, vanilla, citrus oil, including lemon, orange, lime, and grapefruit, and fruit essences including apple, banana, grape, pear, peach, strawberry, raspberry, cherry, plum, pineapple, and apricot.

Non-limiting examples of surfactants include anionic surfactants, for example, a sulfonic acid or a salt thereof such as benzenesulfonic acid, dodecylbenzenesulfonic acid, or dodecanesulfonic acid; an alkyl sulfate, for example, sodium dodecyl sulfate or sodium lauryl sulfate; cationic surfactants, for example, a tetraalkylammonium salt such as a tetraalkylammonium halide, benzethonium chloride, benzalkonium chloride, or cetylpyridinium chloride; a nonionic surfactant, for example, a (poly) oxyethylene sorbitan long-chain fatty acid ester such as a polyoxyethylene sorbitan monolaurate, for example, a polysorbate; amphoteric surfactants, for example, a glycine compound such as dodecyl-di- (aminoethyl)glycine, a betaine compound such as betaine or dimethyldodecylcarboxybetaine, and a phosphatidic acid derivative such as lecithin; polymeric surfactants, for example, a polyoxyethylene polyoxypropylene glycol such as Pluronic® or poloxamer; and mixtures thereof.

Non-limiting examples of buffers include phosphate buffers such as di-hydrogen sodium phosphate, citrate buffers such as sodium citrate, meglumine, tri(hydroxymethyl) aminomethane, and mixtures thereof. Non-limiting examples of tonicity modifying agents include sodium chloride, mannitol, dextrose, glucose, lactose, sucrose, calcium chloride, magnesium chloride, potassium chloride, other inorganic salts, urea, glycerin, glycerol, xylitol, fructose, mannose, maltitol, inositol or trehalose or mixture thereof.

Non-limiting examples of solvents for the preparation of the pharmaceutical composition include water; water miscible organic solvents, for example, isopropyl alcohol or ethanol; dipolar aprotic solvents; methylene chloride; acetone; polyethylene glycol; polyethylene glycol ether; polyethylene glycol derivatives of a mono- or di-glyceride; buffers; organic solvents; and combinations thereof.

In a preferred embodiment, the pharmaceutically acceptable excipients in a composition of the present disclosure include microcrystalline cellulose, anhydrous lactose, croscarmellose sodium, colloidal silicon dioxide and magnesium stearate.

In another embodiment, the present disclosure provides a stable pharmaceutical composition comprising a therapeutically effective amount of an extract of Cocculus hirsutus for inhibiting the SARS-CoV-2 virus. In an aspect the composition reduces the viral load or proliferation of the SARS-CoV-2 virus.

In a further embodiment, the present disclosure provides a pharmaceutical composition comprising an extract of Cocculus hirsutus and one or more pharmaceutically acceptable excipients to inhibit the proliferation of SARS-CoV-2 viruses. Preferably, the composition is a stable pharmaceutical composition. More preferably, the composition is a stable oral pharmaceutical composition.

In a further embodiment, the present disclosure provides an oral composition comprising an extract of Cocculus hirsutus, wherein said composition exhibits an inhibitory activity with an IC50 (or Effective Concentration EC50) value ranging from about 0.1 to about 100 pg/ml as determined by a plaque based virus inhibition assay.

In a further embodiment, the present disclosure provides an oral composition comprising an extract, wherein the said composition exhibits an inhibitory activity with an IC50 value ranging from about 0.1 to about 50 pg/ml.

In a further embodiment, the extracts or compositions of the present disclosure were found to be safe and didn’t show any toxic effect. A CC50 (Cytotoxic concentration) value was found to be 70 pg/ml, which indicates a high selectivity index for use as a safe drug (See Figure 1).

In a further embodiment, the present disclosure provides an oral composition comprising marker compound A from an extract of Cocculus hirsutus, wherein said composition exhibits an inhibitory activity with an ICso (or Effective Concentration ECso) value ranging from about 0.01 to about 10 pg/ml as determined by a plaque based virus inhibition assay.

In a further embodiment, the present disclosure provides an oral composition comprising marker compound A from an extract of Cocculus hirsutus, wherein the composition exhibits an inhibitory activity with an ICso value ranging from 0.1-5 pg/ml.

In a further embodiment, the marker compounds or compositions of the present disclosure were found to be safe and didn’t show any toxic effect. A CCso (Cytotoxic concentration) value was found to be 9 pg/ml, which indicates a high selectivity index for use as a safe drug (See Figure 3).

In one embodiment, the present disclosure provides a method of inhibiting a SARS- CoV-2 virus infection using an extract of Cocculus hirsutus, wherein said extract reduces the viral load, or proliferation of the virus or viruses.

In a further embodiment, the present disclosure provides the use of an extract of Cocculus hirsutus in a method of inhibiting a SARS-CoV-2 virus by reducing or inhibiting the virus proliferation. In an aspect, the extract of the disclosure is used in an anti-SARS- CoV-2 composition.

The extract may be prepared by a process comprising extracting the plant mass of Cocculus hirsutus with one or more solvents, concentrating the extract, and drying the extract, or extracting the plant mass of Cocculus hirsutus with one or more solvents, concentrating the extract, adding water and partitioning the extract with one or more solvents, and drying the extract, or extracting the plant mass of Cocculus hirsutus with one or more solvents, concentrating the extract, extracting the extract with one or more solvents, and drying the extract. In another aspect of the above embodiment, the extraction of the plant mass of Cocculus hirsutus is done at a temperature in the range of about 50 °C to about 100 °C. In another aspect of the above embodiment, the extraction of the plant mass of Cocculus hirsutus is done at a temperature of about 80 °C to about 85 °C. In another aspect of the above embodiment, the extraction of the plant mass of Cocculus hirsutus is done at a temperature of about 60 C to 65 C. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 40 °C to about 95 °C. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 40 °C to about 45 °C. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 45 °C to about 50 °C. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 55 °C to about 65 °C. In another aspect of the above embodiment, the drying of extract of Cocculus hirsutus is done at a temperature in the range of about 90 °C to about 95 °C. In yet another aspect, the plant mass can be extracted from a dry part or a wet part of the plant.

In yet another embodiment, the present disclosure provides a process of preparation of a tablet composition of an extract according to the present invention for inhibiting the SARS-CoV-2 virus, the process comprising the steps of: sifting the extract and blending the extract with pharmaceutically acceptable excipients; lubricating the blend obtained and compressing into tablets, and film coating the tablets.

In yet another embodiment, the present disclosure provides a process of preparation of a tablet composition of an extract according to the present invention for inhibiting the SARS-CoV-2 virus, the process comprising the steps of: blending the extract with pharmaceutically acceptable excipients; granulating the blend with a solvent; lubricating and compressing the blend into tablets, and film coating the tablets.

In a further embodiment, the present disclosure provides a process of preparation of a tablet composition of an extract for reducing the SARS-CoV-2 viral proliferation, the process comprising the steps of: blending the extract with pharmaceutically acceptable excipients and compacting the mixture; milling the compacts and blending with extragranular excipients; lubricating the blend and compressing into tablets, and film coating the tablets.

Although the above embodiments are related to a tablet composition, the extract of Cocculus hirsutus may also be formulated into any other suitable oral dosage forms like powder, pellets, granules, spheroids, mini-tablets, caplets.

In a further related embodiment, the extract may be co-administered simultaneously or sequentially with one or more additional therapeutic agents. EXAMPLES

The following examples which include only exemplary embodiments will serve to illustrate the practice of the present invention.

Example 1: Preparation of a 95:5 Ethanol: purified water extract of Cocculus hirsutus

The plant mass of Cocculus hirsutus (1 kg) was charged into an extractor at ambient temperature*. A mixture of ethanol and purified water (95:5; 6L) was added and the reaction mixture was heated at a temperature of 60-65 °C for about 3 hours. The extracted mass was filtered, collected and stored in a container. The extraction and filtration steps were repeated with a mixture of ethanol and purified water (95:5; 3L) twice. The three filtered extracts were combined and concentrated to the maximum possible extent under reduced pressure at a low temperature. The extract was decanted into stainless steel trays, and then dried under vacuum at 45-50 °C until the ethanol content was not more than 10,000 ppm and the moisture content was not more than 5%. The dried extract was cooled to about 20-25 °C and unloaded at controlled humidity (RH NMT 40%).

Yield obtained = 90 g to 120 g

Example 2: Preparation of a 1 : 1 Ethanol : purified water extract of Cocculus hirsutus

The plant mass of Cocculus hirsutus (1kg) was charged into an extractor at ambient temperature* . A mixture of ethanol and purified water (1: 1; 6L) was added and the reaction mixture was heated at a temperature of 60-65 °C for about 3 hours. The extracted mass was filtered, collected and stored in a container. The extraction and filtration steps were repeated with a mixture of ethanol and purified water (1: 1, 3L) twice. The three filtered extracts were combined and concentrated to the maximum possible extent under reduced pressure at a low temperature. The extract was decanted into stainless steel trays, and then dried under vacuum at 45-50 °C until the ethanol content was not more than 10,000 ppm and the moisture content was not more than 5%. The dried extract was cooled to about 20-25 °C and unloaded at controlled humidity (RH NMT 40%).

Yield obtained = 80 g to 120 g Example 3: Preparation of an aqueous extract of Cocculus hirsutus

The plant mass of Cocculus hirsutus (1kg) was charged into an extractor at ambient temperature*. Purified water (6L) was added and the reaction mixture was heated at a temperature of 60-65 °C for about 3 hours. The extracted mass was filtered, collected and stored in a container. The extraction and filtration steps were repeated with purified water (3L) twice. The three filtered extracts were combined and concentrated to the maximum possible extent under reduced pressure at a low temperature. The extract was decanted into stainless steel trays, and then dried under vacuum at 45-50 °C until the ethanol content was not more than 10,000 ppm and the moisture content was not more than 5%. The dried extract was cooled to about 20-25 °C and unloaded at controlled humidity (RH NMT 40%).

Yield obtained = 80 g to 120g

*The term "ambient temperature" as used herein, includes a temperature ranging from about 18 °C to about 25 °C.

Similarly the extract was also prepared by spray drying using an inlet temperature of 160°C- 190°C and outlet temperature of 105°C to 120°C.

Example 4: Preparation of Tablets from the extract of Cocculus hirsutus using Direct compression technique

MANUFACTURING PROCEDURE:

1. The extract was passed through sieve #10 mesh (2 mm);

2. The extract along with Magnesium aluminium trisilicate was sifted through #14 mesh (1.4 mm);

3. Lactose monohydrate, Dicalcium Phosphate, Starch Pre-gelatinised and Calcium silicate were sifted through #36 mesh (420 p);

4. Microcrystalline cellulose, Colloidal silicon dioxide and Croscarmellose were passed through #25 mesh (600 p);

5. The excipients and extract with magnesium aluminium silicate were mixed in a blender;

6. The blend was then lubricated with magnesium stearate and compressed into tablets;

7. Opadry green was dispersed in purified water to prepare a dispersion; and

8. The compressed tablets were coated with the dispersion.

Example 5: Preparation of Tablets from the extract of Cocculus hirsutus with different strengths:

1. Sifted Cocculus hirsutus aqueous extract, micro-crystalline cellulose, anhydrous lactose, croscarmellose sodium and colloidal silicon dioxide through a suitable sieve in the range of ASTM # 10 sieve to # 60 sieve;

2. Blended the material in a blender for 5-20 minutes;

3. Sifted lubricant through a suitable sieve and blended with the material for 2-10 minutes;

4. Compacted the material and passed the compacts through an upper granulation screen followed by a lower granulation screen followed by sifting over a suitable sieve until the desired percentage of granules were obtained;

5. Blended the material above the sieve and below the sieve to get a intragranular part;

6. Sifted a second set of excipients (extragranular part) through a suitable sieve, and then blended them with intragranular part;

7. Sifted lubricant through a suitable sieve and blended with the blended intragranular- extragranular material followed by compression of the blend to get sufficient strength; 8. Dispersed the coating material in a solvent and stirred well to get uniform dispersion followed by coating of the tablets;

The Extract used in below examples is an aqueous extract of Cocculus hirsutus.

Preparation of tablets of strength 25 mg, 50 mg, 100 mg, 300 mg, 400 mg, 500 mg, 600 mg & 800 mg:

Example 6: Preparation of Tablets from the extract of Cocculus hirsutus using Wet Granulation technique (by Rapid mixer granulator)

MANUFACTURING PROCEDURE:

1. The extract was passed through sieve #10 mesh (2 mm);

2. Magnesium aluminium trisilicate was sifted through sieve #36 mesh(420 p); 3. The extract and sifter material were granulated with methanol and dried;

4. The dried material was then passed through #16 mesh (1 mm);

5. Magnesium aluminium trisilicate, Lactose monohydrate, Dicalcium Phosphate, Starch Pre-gelatinised and Calcium silicate were sifted through #36 mesh (420 p);

6. Microcrystalline cellulose, Colloidal silicon dioxide and Croscarmellose were passed through #25 mesh (600 p);

7. Then the excipients were mixed in a blender along with the granulated extract;

8. The blend obtained was then lubricated with magnesium stearate and compressed into tablets;

9. Opadry green was dispersed in purified water to prepare a dispersion; and 10. The compressed tablets were coated with dispersion.

Example 7: In-vitro activity of tablet of purified aqueous extract of Cocculus hirsutus and its marker compound A against SARS-CoV-2:

A. Procedure: Virus plaque reduction assays were performed for assessing the anti-viral activity and cytotoxicity of these samples was evaluated using an AlamarBlue method, which are explained below in brief.

- Cells and Virus Used: Vero E6 cells (African green monkey kidney cells) were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum, FBS (Gibco). SARS-CoV isolate ICGEB FVG S5 (Licastro D et al. Journal of Virology 2020) was grown and quantified using Vero E6 cells.

- Virus Plaque Reduction Assay: The Vero cells were seeded in a 48 well plate, at 6xl0⁴ cells/well density and incubated at 37 °C overnight. Next day, test compound dilutions were prepared. For this, the tablet (150 mg total weight, 25 mg active principle) was used fresh for the assay. The tablet was ground and 12.5 mg (equivalent to 2 mg of active principle) of powder was dissolved in 1 mb of DMEM (2 mg/mL final concentration of active principle) and kept at room temperature on agitation for 2 hours. Similarly, 2 mg/mL stock of marker compound A was prepared. Cells were infected with 10-20 viral PFU per well, and incubated at 37 °C for 1 hour. Post-infection, the virus was removed and the plate was washed with IX PBS. The infected cells were covered with 800 pL of overlay medium containing 1% carboxymethylcellulose (CMC) with DMEM + 2% FBS, and test compound dilutions. Test concentrations used in plaque assay were 16, 8, 4, 2, 1, 0.5 and 0.25 pg/mL for tablet and 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.19 pg/mL for Compound A. Cells were then incubated at 37 °C for 3 days. Finally, cells were fixed with 3.7% PFA and stained with crystal violet. Plaques were counted and values were normalized to controls (vehicle-treated infected cells and non-infected cells). The plaque reduction assays were conducted in 2X replicates in two independent experiments. The half of maximal effective concentration (EC50) was calculated using GraphPad Prism (Version 7).

- AlamarBlue Cytotoxicity Assay: The cytotoxicity assay was conducted using AlamarBlue (Invitrogen), as recommended by the manufacturer’s protocol. The Vero cells were seeded at IxlO⁴ cells per well in a 96 well plate, and incubated at 37 °C overnight. Next day, 50 pL of compounds at the desired concentrations were added to 150 pL of medium (final 200 pL). Test concentrations used in cytotoxicity assay were 160, 80, 40, 20, 10, 5 and 2.5 pg/mL for tablets and 40, 20, 10, 5, 2.5, 1.25 and 0.625 pg/mL for Compound A. Plates were incubated at 37 °C for 3 days and then the colorimetric reagent was added (20 pL for 4 hours). Measurements from sample-treated or vehicle-treated cells were normalized against those from untreated cells. The cytotoxicity assays were conducted in 3X replicates in two independent experiments. The half of maximum cytotoxic concentration (CC50) was calculated using GraphPad Prism (Version 7).

- Data analysis: Cytotoxicity assay fluorescence readings were normalized for vehicle and percent plotted against dilutions expressed as antilog. Anti-viral activity is calculated with the formula:

[1 -(average plaques with test sample)/(average plaques with vehicle)]xl00 and plotted against dilutions expressed as antilog.

B. RESULTS:

Plaque and cytotoxicity assays of test samples were performed as described above.

Tablet of purified aqueous extract of Cocculus hirsutus was observed to inhibit SARS-CoV-2 virus and ECso was found to be 9 pg/ml.

- A low ECso was indicative of strong inhibitory action of the drug against virus. Its CC50 value was found to be 70 pg/ml, which indicates a high selectivity index for use as a safe drug (See Figure 1).

Placebo tablet showed no inhibitory activity against SARS-CoV-2 virus and did not exhibit any significant cytotoxicity either. So, EC50 and CC50 values could not be established (See Figure 2).

Marker compound A was observed to inhibit SARS-CoV-2 virus and EC50 was found to be 1 pg/mL. A low EC50 was indicative of a strong inhibitory action of the drug against virus. The CCso value of compound A was found to be 9 pg/mL, which indicates a high selectivity index for its use as a safe drug.

Marker compound A showed a lower ICso than a tablet of purified aqueous extract of Cocculus hirsutus against SARS-CoV-2, which indicates the strong inhibitory potential of Compound A (See Figure 3).

The results of in-vitro plaque assay and cytotoxicity assays of test samples are summarized in table- 1 below:

Table- 1 : In-vitro Plaque and cytotoxic assay data:

Placebo and tablet sample was prepared according to Example 5.

While considerable emphasis has been placed herein on the extract of the ingredients of the Cocculus hirsutus, marker compound A and its preferred formulation for anti-viral activity against SARS-CoV-2 viruses, it will be appreciated that many further ingredients can be added and that many changes can be made in the preferred formulation or extract without departing form the principle of the present invention. These and other changes in the preferred extract and formulations of the present invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the present invention and not as a limitation.

Claims

Claims:

1. A method of inhibiting proliferation of a SARS-CoV-2 virus comprising treating said SARS-CoV-2 virus with an extract of Cocculus hirsutus, and/or one or more marker compounds.

2. The method of claim 1 wherein said treatment is performed in vivo.

3. The method of claim 1 wherein said treatment is performed in a mammalian subject.

4. The method of claim 3 wherein said mammalian subject is human.

5. The method of claim 1 wherein said treatment is performed in vitro.

6. The method of claim 1 wherein inhibition is characterized by reduced rate of proliferation of the SARS-CoV-2 virus.

7. The method of claim 1 wherein the inhibition is characterized by a reduced viral load of the SARS-CoV-2 virus.

8. A method for the treatment of a viral infection characterized by the presence of a SARS-CoV-2 virus in a mammalian subject, comprising administering to said subject an effective amount of an extract of Cocculus hirsutus, and/or one or more marked compounds.

9. The method as claimed in any of the preceding claims, wherein the marker compound is selected from compound A, compound B, compound C, compound D, compound E or a combination thereof.

10. The method of claim 1 or 5, wherein the extract of Cocculus hirsutus is provided in the form of a pharmaceutical composition.

11. A method of determining the anti-viral activity of an extract of Cocculus hirsutus, or one or more components thereof, wherein the method comprises contacting one or more concentrations and/or volumes of said extract, or one or more components thereof, to a SARS-CoV-2 virus, and identifying the concentration and/or volume of said extract, or one or more components thereof, that inhibits the proliferation of the virus.

12. An extract of Cocculus hirsutus for use in inhibition of the proliferation of a SARS- CoV-2 virus.

13. A pharmaceutical composition comprising the extract according to claim 12, wherein said extract reduces viral load of a SARS-CoV-2 virus in a subject infected with such virus.

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14. The method of claim 7, wherein the reduced viral load is characterized by reduced copies of viral RNA in a tissue sample of the subject infected with such virus.

15. The extract according to claim 12, wherein aid extract reduced the rate of proliferation of a SARS-CoV-2 virus.

16. The method of claim 6, wherein the reduced rate of proliferation of the SARS-CoV- 2 virus is characterized by reduced copies of viral RNA in a tissue sample of the subject infected with such virus.

17. The extract according to claim 12, wherein said extract is effective in reducing viral proliferation at a dose of 1 to 100 pg/mL.

18. An anti-SARS-CoV-2 composition for inhibiting SARS-CoV-2 viral proliferation, wherein said composition comprises of a therapeutically effective amount of an extract of Cocculus hirsutus.

19. The composition according to claim 13 or 18, wherein said composition is an oral dosage form.

20. The composition according to claim 13 or 18, wherein said composition further comprises a pharmaceutically acceptable excipient selected form a diluent, a binder, a disintegrant, a lubricant, a glidant, polymer, a flavoring agent, a surfactant, a preservative, a buffer and a tonicity modifying agent.

21. The extract according to any of the preceding claims wherein the extract is an aqueous or hydroalcoholic extract.

22. The composition as claimed in claims 13 or 18, for use in a method of treating a SARS-CoV-2 virus infection wherein said composition effectively inhibits the proliferation of the SARS-CoV-2 virus.

23. The extract according to any of the preceding claims, for use in a method of treating or preventing viral infections caused by a SARS-CoV-2 virus, wherein said extract reduces the viral load by inhibiting the proliferation of virus growth.

24. The composition as claimed in claim 19, wherein said composition exhibits inhibitory activity with an IC50 (or Effective Concentration EC50) value ranging from about 0.1 to about 100 pg/ml.

25. The composition as claimed in claim 19, wherein the inhibitory activity with IC50 (or Effective Concentration EC50) is determined by a plaque based virus inhibition assay.

26. The composition as claimed in claim 19, wherein said composition exhibits an inhibitory activity with an IC50 (or Effective Concentration EC50) value ranging from about 0.1 to about 50 pg/ml.

27. The composition as claimed in claim 19, wherein the composition is safe with a high selectivity index for use as a safe drug, and a CC50 (Cytotoxic concentration) value of 70 pg/ml.

28. The composition as claimed in claim 19, wherein the composition is an oral composition comprising an extract of Cocculus hirsutus, wherein the said composition exhibits an inhibitory activity with an IC50 (or Effective Concentration EC50) value ranging from about 0.01 to about 10 pg/ml as determined by a plaque based virus inhibition assay.

29. The composition as claimed in claim 19, wherein the composition further comprises a marker compound, wherein the composition exhibits an inhibitory activity with an IC50 (or Effective Concentration EC50) value ranging from about 0.1 to about 5 pg/ml as determined by a plaque based virus inhibition assay.

30. The composition as claimed in claim 19, wherein the composition further comprises a marker compound, wherein the composition is safe with a high selectivity index for use as a safe drug, and a CC50 (Cytotoxic concentration) value of 9 pg/ml.

31. An oral anti-SARS-CoV-2 composition for inhibiting SARS-CoV-2 viral proliferation, wherein the composition comprises a therapeutically effective amount of a marker compound.

32. The composition as claimed in claim 31, wherein the marker compound is selected from a compound A, compound B, compound C, compound D, compound E or a combination thereof.