FIELD OF THE INVENTION

[001] The present invention relates to a composition comprising composite extract of Cocculus hirsutus and pharmaceutical compositions for use in the prevention and/or treatment of infection in mammals caused by a positive sense single -stranded RNA virus. The present invention also provides various methods for reducing or inhibiting the proliferation of a positive stranded RNA virus in an infected patient by administering a therapeutically effective amount of a composite extract of Cocculus hirsutus.

BACKGROUND OF THE INVENTION

[002] A positive sense single-stranded RNA virus is a virus that uses positive sense single stranded RNA as its genetic material. Single stranded RNA viruses are classified as positive or negative depending on the sense or polarity of the RNA. The positive-sense viral RNA genome can serve as messenger RNA and can be translated into protein in the host cell. The positive sense single-stranded RNA viruses are classified into three orders — the Nidovirales, Picomavirales, and Tymovirales — and 33 families, of which 20 are not assigned to an order. A broad range of hosts can be infected by positive sense single-stranded RNA viruses, including bacteria, eukaryotic microorganisms, plants, invertebrates, and vertebrates.

[003] Positive-strand RNA viruses encompass over one-third of all virus genera and include numerous viral pathogens such as the Hepatitis C virus, Dengue virus, Chikunguniya virus, SARS and MERS virus, hCoV-OC43 virus; Japanese Encephalitis virus (JEV), Zika (Asian, Brazilian and African strains) virus, Yellow Fever Virus (YFV), Usutu Virus (Usutu, growing concern circulating in EU), West Nile Virus (WNV), Tick Borne Encephalitis Virus (TBEV), Coronaviruses and SARS-CoV-2 as well as other less clinically serious pathogens such as the Rhinoviruses that cause the common cold.

[004] The recent outbreak of Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 in December 2019 has raised global health concerns. In Dec 2019, a number of cases with unexplained low respiratory infections were detected in Wuhan, China. The etiology of the illness was attributed to a novel virus belonging to the coronavirus (CoV) family, and the disease caused by this novel virus was named as COVID- 19, which is acronym of “Coronavirus disease 2019”. This new virus has been found to be very contagious and has quickly spread globally. The disease outbreak has been declared by the WHO as a Public Health emergency of International Concern (PHEIC). Initially, the new vims was called 2019-CoV, subsequently the task of experts of International Committee on Taxonomy of Viruses (ICTV) termed it as SARS- CoV-2 (Sever Acute respiratory syndrome associated coronavirus-2). CoVs are large Coronoviridae family of positive sense single -stranded RNA viruses that can be isolated in different animal species. For reasons yet-to be explained, these viruses can cross species barriers and can cause in humans, illness ranging from the common cold to more severe cases such as MERS and SARS. The elderly and people with underlying diseases are susceptible to infection and prone to serious outcomes, which may be associated with acute respiratory distress syndrome (ARDS) and cytokine storm. (Guo et al, The origin, transmission and clinical therapies on coronavirus disease- 19 (Covid- 19) outbreak- an update on the status, Mil Med Res, 2020 Mar 13; 7(1): 11; also Cascella et al, Features, Evaluation and Treatment of Coronavirus (COVID-19), StatPearls, March 20, 2020). Based on the speed at which the outbreak of COVID- 19 has developed, 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. As per the World Health Organizations, COVID- 19 Situation dashboard, as on August 09, 2020, there were 18,187,943 confirmed cases infected with SARS-CoV-2 with 716,075 deaths. World governments are at work to establish counter measures to stem possible devastating effects. At the same time, scientists around the world are working tirelessly to understand the transmission mechanisms, the clinical spectrum of disease, new diagnostics, and development of preventive and therapeutic strategies. At the moment, the therapeutic strategies to deal with the infections are only supportive, and prevention aimed at reducing the transmission in the community is considered to be the best weapon (Cascella et al, Features, Evaluation and Treatment of Coronavirus (COVID-19), StatPearls, Updated May 18, 2020).

[005] Various antiviral drugs and systemic corticosteroid treatment commonly used in clinical practice previously, including neuraminidase inhibitors (Oseltamivir, Peramivir, Zanamivir, etc.), Ganciclovir, Acyclovir, and Ribavirin, as well as Methylprednisolone for influenza virus, were found to be invalid for COVID-19 and are not recommended. (Guo et al). Some other antiviral drugs like Remdesivir, Lopinavir and Ritonavir, antimalarial drugs like Chloroquine, Hydroxy cloroquine are also being investigated for their effects against COVID- 19. The unprecedented rapidity of spread of this out break presents a critical need for an effective treatment against CO VID-19 that can relieve the symptoms, reduce the severity of common symptoms, prevent the development of severe complications, and is easy to formulate. Furthermore, it is highly desirable to develop a treatment against SARS-CoV-2 and all other similar positive sense single-stranded RNA viruses that may reduce the viral load at an early stage, such that it may potentially prevent the infection and transmission thereof. Also many pathogenic positive sense single-stranded RNA viruses are arthropod-borne viruses (called arboviruses) — that is, transmitted by and capable of replicating in biting insects which then transfer the pathogen to animal hosts. Recent metagenomics studies have also identified large numbers of RNA viruses whose host range is specific to insects which acts as carrier for transmitting the viruses.

[006] Zika virus is also a mosquito-borne flavivirus that was first identified in Uganda in 1947 in monkeys. It was later identified in humans in 1952 in Uganda and the United Republic of Tanzania. The first recorded outbreak of Zika virus disease was reported from the Island of Yap (Federated States of Micronesia) in 2007. This was followed by a large outbreak of Zika virus infection in French Polynesia in 2013 and other countries and territories in the Pacific. In October 2015, Brazil reported an association between Zika virus infection and microcephaly. Outbreaks and evidence of transmission soon appeared throughout the Americas, Africa, and other regions of the world. To date, a total of 86 countries and territories have reported evidence of mosquito-transmitted Zika infection. As per Word Health Organization’s web information there is no particular treatment available for Zika virus infection or its associated diseases.

[007] Thus, there is a need for an effective treatment or therapy which could be used for prevention and/or treatment of a wide range of viral infections.

SUMMARY OF THE INVENTION

[008] The present invention provides a composite extract of Cocculus hirsutus or its pharmaceutical compositions for use in the prevention and/or treatment of viral infections caused by positive sense single -stranded RNA viruses in mammals.

[009] In another aspect, the present invention also provides methods for reducing viral load in the treatment of viral infections caused by positive sense single-stranded RNA viruses, including the novel coronavirus (COVID-19) by administering the composite extract or its pharmaceutical composition to a mammal in need thereof.

[010] Further, it provides for a stable pharmaceutical composition comprising a therapeutically effective amount of the said extract for use in prevention and treatment of infections caused by positive sense single -stranded RNA viruses (+ssRNA) liked Hepatitis C, Dengue Virus (all 4 serotypes DENV 1-4), Chikunguniya Virus (CHIKV), SARS and MERS, hCoV-OC43; Japanese Encephalitis Virus (JEV), Zika Virus (both Asian / Brazilian and African strain), Yellow Fever Virus (YFV), Usutu Virus (Usutu, growing concern circulating in EU), West Nile Virus (WNV), Tick Bome Encephalitis Virus (TBEV), Coronaviruses and SARS-CoV-2 (COVID- 19) in a mammal.

[OH] In yet another aspect, the present invention further provides method for use of an extract of Cocculus hirsutus in prevention and/or treatment of viral infections caused by positive sense single-stranded RNA viruses in mammals.

[012] In yet another aspect, the present invention further provides method for use of individual chemical entities, such as Sinococuline and Hirsutine, which is derived from the extract of Cocculus hirsutus in prevention and/or treatment of viral infections caused by positive sense single-stranded RNA viruses in mammals.

[013] The present invention also relates to processes for the preparation of these extracts and isolation of related therapeutic compounds and their enrichment. Further, these extracts were found to be safe for administration to humans and did not show any toxic effects at the concentration required for prevention and/or treatment of viral infections caused by positive sense single-stranded RNA viruses in mammals.

[014] In some embodiments, the present disclosure provides use of a composition comprising: a therapeutically effective amount of a composite extract of Cocculus hirsutus,' and a pharmaceutically acceptable excipient comprising a diluent, a binder, a disintegrant, a lubricant, a glidant, a polymer, a flavoring agent, a surfactant, a preservative, an antioxidant, a buffer, a tonicity modifying agent, or combination thereof, for treatment of a viral infection caused by a positive sense single-stranded RNA virus.

[015] In some embodiments, the present disclosure provides use of a composite extract of Cocculus hirsutus for preparation of medicament for treatment of a viral infection caused by a positive sense single-stranded RNA virus.

[016] In some embodiments, the present disclosure provides a composition comprising: a therapeutically effective amount of a composite extract of Cocculus hirsutus,' and a pharmaceutically acceptable excipient comprising a diluent, a binder, a disintegrant, a lubricant, a glidant, a polymer, a flavoring agent, a surfactant, a preservative, an antioxidant, a buffer, a tonicity modifying agent, or combination thereof.

[017] In some embodiments, the composite extract of Cocculus hirsutus is about 50 wt % to about 80 wt % of the composition. In some embodiments, the composite extract of Cocculus hirsutus is about 60 wt % to about 70 wt % of the composition. [018] In some embodiments, the composite extract of Cocculus hirsutus comprises Sinococuline, Magnoflorine, coniferyl alcohol, quercetin, rutin, syrinaresionl diglucoside, Makisterone A, Hirsutine, 20-hydroxyecdysone, a derivative thereof, or combination thereof.

[019] In some embodiments, the positive-stranded RNA virus is Hepatitis C virus, West Nile Virus (WNV), Dengue Virus, Chikunguniya Virus (CHIKV), Japanese Encephalitis Virus (JEV), Zika Virus, Yellow Fever Virus (YFV), Usutu Virus, Tick Borne Encephalitis Virus (TBEV), a coronavirus, or combination thereof. In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, or hCoV-OC43.

[020] In some embodiments, the composite extract of Cocculus hirsutus is an alcoholic extract, a hydro-alcoholic extract, or an aqueous extract. In some embodiments, the composite extract of Cocculus hirsutus comprises water, ethanol, or combination thereof. In some embodiments, the composite extract of Cocculus hirsutus is an aqueous extract. In some embodiments, the composite extract comprises alcohol and water at a ratio of about 1 : 99 to 99: 1.

[021] In some embodiments, the composite extract of Cocculus hirsutus is about 50 weight (wt) % to about 80 wt % of the composition. In some embodiments, the composite extract of Cocculus hirsutus is about 60 wt % to about 70 wt % of the composition. In some embodiments, the composite extract of Cocculus hirsutus comprises about 0. 1 wt% to about 1 wt% Magnoflorine.

[022] In some embodiments, the pharmaceutically acceptable excipient comprises magnesium aluminum trisilicate, lactose monohydrate, dicalcium phosphate, starch, calcium silicate, microcrystalline cellulose, colloidal silicon dioxide, croscarmellose, magnesium stearate, or combination thereof. In some embodiments, the composition further comprises a film coating, a solvent, or combination thereof. In some embodiments, the composition or the medicament is an oral composition selected from a powder, a pellet, a granule, a spheroid, a mini-tablet, a caplet, a tablet, a capsule, a liquid, or combination thereof.

[023] In some embodiments, the composition or the medicament comprises at least three compounds selected from Sinococuline, Magnoflorine, Makisterone A, Hirsutine, 20- hydroxyecdysone, wherein the at least three compounds are about 0.1% to about 10% w/w of the composition or the medicament. In some embodiments, the Sinococuline is about 0.1 wt% to about 20 wt%; the Magnoflorine is about 0.01 wt% to about 5 wt%; the Makisterone A is about 0.005 wt% to about 5 wt%; and the 20-hydroxyecdysone is about 0.01 wt% to about 5 wt% of the composition or the medicament. [024] In some embodiments, the present disclosure provides a composition comprising: a composite extract of Cocculus hirsutus,' and an isolated compound selected from Sinococuline, Magnoflorine, Makisterone A, Hirsutine, 20-hydroxyecdysone, a derivative thereof, or combination thereof, wherein the composite extract and the isolated compound are at a ratio of about 99: 1 to about 1:99.

[025] In some embodiments, the isolated compound is Sinococuline. In some embodiments, the isolated compound is Magnoflorine. In some embodiments, the ratio of the composite extract and the isolated compound is about 70:30 to about 30:70. In some embodiments, the ratio of the composite extract to the isolated compound is about 40:60 to about 60:40. In some embodiments, the ratio of the composite extract to the isolated compound is about 1: 1.

[026] In some embodiments, the present disclosure provides a composition comprising: a composite extract of Cocculus hirsutus,' magnesium aluminum trisilicate; a diluent selected from lactose monohydrate, dicalcium phosphate, pre-gelatinized starch, calcium silicate, or combination thereof; microcrystalline cellulose; colloidal silicon dioxide; croscarmellose; magnesium stearate; and optionally a film coating.

[027] In some embodiments, the composition comprises about 10 wt% to about 90 wt% of the composite extract of Cocculus hirsutus,' about 5 wt% to about 50 wt% of the magnesium aluminum trisilicate; about 0. 1 wt% to about 20 wt% of the diluent; about 0. 1 wt% to about 10 wt% of the microcrystalline cellulose; about 0.1 wt% to about 10 wt% of the colloidal silicon dioxide; about 0.1 wt% to about 10 wt% of the croscarmellose; and about 0.01 wt% to about 5 wt% of the magnesium stearate.

[028] In some embodiments, the composition comprises about 30 wt% to about 85 wt% of the composite extract of Cocculus hirsutus,' about 8 wt% to about 40 wt% of the magnesium aluminum trisilicate; about 0.5 wt% to about 15 wt% of the diluent; about 0.5 wt% to about 8 wt% of the microcrystalline cellulose; about 0.2 wt% to about 8 wt% of the colloidal silicon dioxide; about 0.5 wt% to about 8 wt% of the croscarmellose; and about 0.02 wt% to about 2 wt% of the magnesium stearate.

[029] In some embodiments, the present disclosure provides a composition comprising: about 50 wt% to about 80 wt% of a composite extract of Cocculus hirsutus,' about 10 wt% to about 30 wt% of magnesium aluminum trisilicate; about 1 wt% to about 10 wt% of a diluent selected from lactose monohydrate, dicalcium phosphate, pre-gelatinized starch, calcium silicate, or combination thereof; about 1 wt% to about 5 wt% of microcrystalline cellulose; about 0.5 wt% to about 5 wt% of colloidal silicon dioxide; about 1 wt% to about 5 wt% of croscarmellose; about 0. 1 wt% to about 1 wt% of magnesium stearate; and optionally a film coating.

[030] In some embodiments, the composition is an oral pharmaceutical composition. In some embodiments, the oral pharmaceutical composition is a powder, a pellet, a granule, a spheroid, a mini-tablet, a caplet, a tablet, a capsule, a liquid, or combination thereof. In some embodiments, the composite extract of Cocculus hirsutus is a composite water extract, a composite primary alcohol extract, or composite water/ethanol extract.

[031] In some embodiments, the present disclosure provides a composition comprising at least three compounds selected from Sinococuline, Magnoflorine, Makisterone A, Hirsutine, 20- hydroxyecdysone, wherein the at least three compounds are about 0.1% to about 10% of the composition.

[032] In some embodiments, the Sinococuline is about 0.1 wt% to about 20 wt%; the Magnoflorine is about 0.01 wt% to about 5 wt%; the Makisterone A is about 0.005 wt% to about 5 wt%; and the 20-hydroxyecdysone is about 0.01 wt% to about 5 wt% of the composition.

[033] In some embodiments, the composition of the composite extract of Cocculus hirsutus is prepared, wherein the weight of the composite extract in the composition is in a range of about 25 mg to 1500 mg. In some embodiments, the composition of the composite extract of Cocculus hirsutus is prepared, wherein the weight of the composite extract in the composition is selected from 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, or 1500 mg. In some embodiments, the composition is in a unitary dosage form having any of the above-listed amounts of composite extract.

[034] In some embodiments, the composite extract of Cocculus hirsutus is substantially free of lignin, fiber, and tannin.

[035] In some embodiments, the present disclosure provides a method for reducing or inhibiting proliferation of a positive sense single-stranded RNA virus in a subject, the method comprising administering to the subject, a therapeutically effective amount of the composition provided herein. In some embodiments, the present disclosure provides a method of reducing viral load of a positive sense single-stranded RNA virus in a subject, the method comprises administering to the subject, a therapeutically effective amount of the composition provided herein. In some embodiments, the composition is administered at a dose of about 1 mg/kg to about 500 mg/kg of the composite extract of Cocculus hirsutus per weight of the subject. [036] In some embodiments, the present disclosure provides a method of treating a positive sense single-stranded RNA virus infection in a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a composite extract of Cocculus hirsutus.

[037] In some embodiments, the present disclosure provides a method of treating a positive sense single-stranded RNA virus infection in a subject in need thereof, the method comprising administering to the subject, a composition comprising a therapeutically effective amount of a composite extract of Cocculus hirsutus and a pharmaceutically acceptable excipient comprising a diluent, a binder, a disintegrant, a lubricant, a glidant, a polymer, a flavoring agent, a surfactant, a preservative, an antioxidant, a buffer, a tonicity modifying agent, or combination thereof.

[038] In some embodiments, the present disclosure provides a method of treating a positive sense single-stranded RNA virus infection in a subject in need thereof, the method comprising administering to the subject, a composition comprising a therapeutically effective amount of Sinococuline, Magnoflorine, Makisterone A, Hirsutine, 20-hydroxyecdysone, a derivative thereof, or combination thereof. In some embodiments, the composition comprises Sinococuline. In some embodiments, the composition comprises Magnoflorine.

[039] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient comprising a diluent, a binder, a disintegrant, a lubricant, a glidant, a polymer, a flavoring agent, a surfactant, a preservative, an antioxidant, a buffer, a tonicity modifying agent, or combination thereof. In some embodiments, the composite extract of Cocculus hirsutus is substantially free of lignin, fiber, and tannin. In some embodiments, the composition is administered once daily, twice daily, three times daily, or four times daily. In some embodiments, the composite extract of Cocculus hirsutus. is administered once daily, twice daily, three times daily, or four times daily.

[040] In some embodiments, the positive sense single stranded RNA virus is Hepatitis C virus, West Nile Virus (WNV), Dengue Virus, Chikunguniya Virus (CHIKV), Japanese Encephalitis Virus (JEV), Zika Virus, Yellow Fever Virus (YFV), Usutu Virus, Tick Borne Encephalitis Virus (TBEV), a coronavirus, or combination thereof. In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, or hCoV-OC43. In some embodiments, the virus is SARS-CoV-2, Dengue virus, Chikunguniya virus or Japanese Encephalitis Virus (JEV). [041] In some embodiments, the method further comprises co-administering to the subject, an additional therapeutic agent selected from an antiviral agent, an antipyretic agent, an analgesic agent, or combination thereof.

[042] In some embodiments, the present disclosure provides a method of preparing a composite extract of Cocculus hirsutus, comprising: a) extracting a plant mass of Cocculus hirsutus with a solvent comprising ethanol, water, or both, wherein the extracting is performed at about 50 °C to about 100 °C; b) concentrating the extract of (a); and c) drying the concentrated extract of (b), wherein the drying is performed at about 40 °C to about 95 °C, thereby obtaining the composite extract of Cocculus hirsutus. In some embodiments, the method further comprises, prior to (c), further extracting the concentrated extract of (b) with a further solvent, wherein the further solvent is ethanol, water, or both.

BRIEF DESCRIPTION OF THE DRAWINGS

[043] The following drawings form part of the present specification and are included to further demonstrate exemplary embodiments of certain aspects of the present disclosure.

[044] Fig. 1(a): Graph of % inhibition of JEV on Y axis against tested concentrations of Sinococuline on X-axis in log.

[045] Fig. 1(b): Graph of % inhibition of JEV on Y axis against tested concentrations of AQCH tablet on X-axis in log.

DETAILED DESCRIPTION OF THE INVENTION

[046] The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

[047] The present inventors have found that a composite extract of a plant named Cocculus hirsutus has a broader anti-viral activity against the positive sense single-stranded RNA viruses. Further, it was also found by the inventors that the said composite extract interferes with the viral life cycle of the positive sense single-stranded RNA viruses. The inventors also found that the said composite extract lowers TNF-alpha and IL-6 levels in mammals.

[048] The said composite extract can be used for prevention and/or treatment of various infectious diseases caused by positive sense single-stranded RNA viruses like Hepatitis C virus, West Nile Virus (WNV), Dengue Virus (all 4 serotypes DENV 1-4), Chikunguniya Virus (CHIKV), SARS and MERS virus, hCoV-OC43 virus; Japanese Encephalitis Virus (JEV), Zika Virus (both Asian / Brazilian and African strains), Yellow Fever Virus (YFV), Usutu Virus (Usutu, growing concern circulating in EU), Tick Borne Encephalitis Virus (TBEV), Coronaviruses and SARS-CoV-2 (COVID-19), or any other related positive sense singlestranded RNA viruses in mammals.

[049] Cocculus hirsutus Linn (Menispermeaceae), commonly known as Jal-Jamni (Chopra et al., 1958) or Broom creeper is found in moderately cool and hot regions of Asia tropical and Africa. It is a perennial climber and reaches 2 to 3 m above ground. The present inventors have found that Cocculus hirsutus extracts as such and various isolated compounds from Cocculus hirsutus like Sinococuline, Magnoflorine, Makisterone A, Hirsutine, Quercetin, Rutin, (-) Syrinaresionl diglucoside or 20-hydroxyecdysone and their various combinations may help to effectively prevent and/or treat various infectious diseases caused by positive sense singlestranded RNA viruses including COVID- 19 effectively, with reduced side-effects. In an embodiment, the present invention provides a composite extract of Cocculus hirsutus for the prevention and/or treatment of infections caused by positive sense single-stranded RNA viruses in mammals.

[050] In another embodiment, the present invention provides method of treating a viral infection caused by positive sense single-stranded RNA viruses, by administering a therapeutically effective amount of a composite extract of Cocculus hirsutus.

[051] In yet another embodiment, the present invention provides methods for reducing or inhibiting the proliferation of viral infections caused by positive sense single-stranded RNA viruses, by administering a therapeutically effective amount of a composite extract of Cocculus hirsutus to a mammal in need thereof.

[052] In a further embodiment, the present invention provides use of a composite extract of Cocculus hirsutus for prevention and/or treatment of viral infections caused by positive sense single-stranded RNA viruses in mammals.

[053] In another embodiment, the present invention provides use of a composite extract of Cocculus hirsutus for reducing or inhibiting the proliferation of positive sense single-stranded RNA viruses in mammals.

[054] In further embodiment, the present invention also provides various synergistic combination compositions of a composite extract of Cocculus hirsutus, Sinococuline, Hirsutine, Quercetin, Rutin, (-) Syrinaresionl diglucoside, Magnoflorine, Coniferyl alcohol, Makisterone A or 20-hydroxyecdysone or derivatives thereof.

[055] In a further embodiment, the present invention also provides methods for isolating various compounds from a composite extract of Cocculus hirsutus and use thereof in methods for treating or preventing viral infection in mammals caused by positive sense single-stranded RNA viruses.

[056] The compounds isolated from extract of Cocculus hirsutus comprise Sinococuline, Hirsutine, Magnoflorine, Coniferyl alcohol, Quercetin, Rutin, Syrinaresionl diglucoside, Makisterone A or 20-hydroxyecdysone or derivatives thereof. The structures of each of these compounds are as provided below:

Magnoflorine Coniferyl alcohol

Quercetin Rutin Syrinaresionl diglucoside

Makisterone A 20-hydroxyecdysone Hirsutine

[057] The term “composite extract” as used herein, refers to the extract obtained from

Cocculus hirsutus in any concentration comprising a mixture of constituents and is present in the form of a liquid, semisolid, solid, gel, paste, dispersion, solution, or distillate. Preferably, the composite extract is in the form of a solid, for example, in the form of a powder. The use of the term “composite extract” is used interchangeable with “extract” in the present specification. [058] In one aspect of the above embodiments, the extract is an aqueous extract or an organic solvent extract, wherein the said organic solvent is a polar or non-polar organic solvent.

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

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

[061] 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.

[062] 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: 1 to 1 :95, 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 1: 1 to 95:5, or a 1: 1 mixture of alcohol and purified water.

[063] The term “aqueous extract,” as used herein, includes a water or purified water extract of Cocculus hirsutus, also abbreviated as AQCH (Aqueous extract of Cocculus hirsutus).

[064] 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 of step (a) 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 of step (a) with one or more solvents.

[065] 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.

[066] 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.

[067] 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.

[068] In yet another embodiment, the composite extract of Cocculus hirsutus may comprise of Sinococuline, Magnoflorine, Coniferyl alcohol, Makisterone A or 20-hydroxyedysone, Hirsutine, Quercetin, Rutin, (-) Syrinaresionl diglucoside or their derivatives. In a related embodiment, the composite extract of Cocculus hirsutus may be used for isolating the compounds Sinococuline, Magnoflorine, Makisterone A or 20-hydroxyedysone or their derivatives and there use in method of treating or preventing viral infection caused by positive sense single-stranded RNA viruses or their use in inhibiting the proliferation of virus in mammals.

[069] In a further preferred embodiment, the present invention provides an enriched fraction of the composite extract of Cocculus hirsutus for use in treatment of viral infection in mammals caused by positive sense single-stranded RNA viruses. The extract may be enriched with respect to any of the flavonoid, alkaloids, for e.g., Sinococuline or Magnoflorine; or other compounds like Hirsutine, Coniferyl alcohol, Quercetin, Rutin, (-) Syrinaresionl diglucoside, Makisterone A or 20-hydroxyecdysone or derivative compounds thereof. The extract may be enriched and standardized with respect to compounds, for e.g., Sinococuline, Magnoflorine, Makisterone A, Hirsutine, 20-hydroxyecdyson, or their derivative compounds. The enriched extract may be prepared by using the solvent for the said compound(s). The solvent may be any of the solvent as discussed above.

[070] In the present invention, the inventors found that the composite extract of Cocculus hirsutus possesses a broader anti-viral activity and has been found to interfere with the viral life cycle post its entry into the host cell. The composite extract was found to be effective against positive sense single -stranded RNA viruses. Additionally, it was unexpectedly found that the composite extract also lowers TNF-alpha and IL-6 levels in mammals, thereby contributing in exhibiting its efficacy against severe cases of COVID-19.

[071] In another embodiment, the present invention provides a composite extract of Cocculus hirsutus to reduce the viral load at an early stage in the treatment of viral infections in mammals caused by positive sense single-stranded RNA viruses selected from a group comprising of Hepatitis C, Dengue Virus (all 4 serotypes DENV 1-4), Chikunguniya Virus (CHIKV), SARS and MERS, hCoV-OC43; Japanese Encephalitis Virus (JEV), Zika Virus (both Asian / Brazilian and African strain), Yellow Fever Virus (YFV), Usutu Virus (Usutu, growing concern circulating in EU), West Nile Virus (WNV), Tick Borne Encephalitis Virus (TBEV), Coronaviruses and SARS-CoV-2 (COVID-19).

[072] In yet another embodiment, the present invention provides a pharmaceutical composition comprising composite extract of Cocculus hirsutus, or Sinococuline, Magnoflorine, Makisterone A, Hirsutine, 20-hydroxyecdysone, or Coniferyl alcohol, Quercetin, Rutin, (-) Syrinaresionl diglucoside or derivatives or combinations thereof, wherein the composition exhibits an inhibitory activity against positive sense single-stranded RNA viruses selected from a group comprising of Hepatitis C, Yellow Fever Virus (YFW), Dengue Virus (all 4 serotypes DENV 1- 4), Chikunguniya Virus (CHIKV), SARS and MERS, hCoV-OC43; Japanese Encephalitis Virus (JEV), Zika Virus (both Asian / Brazilian and African strain), Usutu Virus (Usutu, growing concern circulating in EU), West Nile Virus (WNV), Tick Borne Encephalitis Virus (TBEV), Coronaviruses and SARS-CoV-2 (COVID-19).

[073] The composite extract obtained according to this invention is useful both to be directly administrated to a mammal and to be used in the preparation of a pharmaceutical composition, with the dose in the range of approximately 0.05 mg/kg to approximately 1500 mg/kg body weight, particularly in the range of approximately 0.1 mg/kg to approximately 1200 mg/kg body weight, more particularly in the range of approximately Img/kg to approximately 500 mg/kg body weight, more particularly in the range of approximately 2 mg/kg to approximately 150 mg/kg body weight. The composite extract or its composition may be administered once, twice, thrice or four times a day.

[074] In a further related embodiment, the composite extract of the present invention may be used to further isolate or enrich suitable compounds or derivatives for use in method for prevention and/or treatment of a viral infection caused by positive sense single-stranded RNA viruses.

[075] In yet another related embodiment, the present invention provides methods for treatment or reducing the proliferation of viral infection caused by positive sense single-stranded RNA viruses, by administering a suitable pharmaceutical dosage from comprising an effective amount of one or more compounds selected from Sinococuline, Magnoflorine, Makisterone A, Hirsutine or 20-hydroxyecdysone or combinations or derivatives thereof to the mammal.

[076] In another embodiment, the present invention provides a composite extract of Cocculus hirsutus to reduce the viral load at an early stage in the treatment of viral infection, exhibit a platelet protective effect, inhibits proliferation of virus in the infected mammals and other related mechanisms, thereby promoting the recovery of the patient affected by infections caused by positive sense single-stranded RNA viruses.

[077] In yet another preferred embodiment, the present invention provides method of treating and/or preventing viral infections using the composite extract of Cocculus hirsutus with or without one or more compounds selected from Sinococuline, Magnoflorine, Makisterone A, Hirsutine, 20-hydroxyecdysone or their derivatives. This may result in a synergistic effect against the viral infection caused by positive sense single-stranded RNA viruses, thereby enhancing the proliferation inhibiting capability of the formulation (of composite extract) or viral therapy.

[078] In some embodiments, the present disclosure provides a composition comprising : a composite extract of Cocculus hirsutus,' and an isolated compound selected from Sinococuline, Magnoflorine, Makisterone A, Hirsutine, 20-hydroxyecdysone, a derivative thereof, or combination thereof, wherein the composite extract and the isolated compound are at a ratio of about 99: 1 to about 1:99.

[079] As used herein, an “isolated compound” is a substantially purified compound that is isolated from a naturally-occurring source, e.g., the Cocculus hirsutus plant, or that is synthesized. In some embodiments, the isolated compound is a substantially purified compound that comprises less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0. 1% of impurities. [080] In another embodiment, the present invention provides methods for treatment and/or prevention of viral infection caused by positive sense single-stranded RNA viruses by administering a suitable combination dosage form of composite extract of Cocculus hirsutus and isolated compounds from the plant species.

[081] In yet another embodiment, the present invention provides use of suitable combination dosage form of composite extract of Cocculus hirsutus and isolated compounds from the plant species for prevention and/or treatment of viral infection caused by a positive sense singlestranded RNA virus.

[082] In a related embodiment, when the composition is a combination of the isolated compounds and the composite extract of Cocculus Hirsutus, the ratio is 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 1: 1. In the above embodiment, the isolated compound is selected from Sinococuline, Magnoflorine, Makisterone A, Hirsutine, 20-hydroxyecdysone or their derivatives. In some embodiments, the isolated compound is Sinococuline. In some embodiments, the isolated compound is Magnoflorine.

[083] In some embodiments, the composite extract when administered shows therapeutic effect at a concentration of about 0.1 to about 500 pg/mL. In some embodiments, the composite extract shows therapeutic effect at a concentration of about 0. 1 to about 200 pg/mL of the composition. In some embodiments, the composite extract shows therapeutic effect at a concentration of about 1 to 100 pg/mL of the composition. In some embodiments, the isolated compound shows therapeutic effect at a concentration of about 0.01 to about 100 pg/mL of the composition. In some embodiments, the isolated compound shows therapeutic effect at a concentration of about 0.01 to about 50 pg/mL of the composition. In some embodiments, the isolated compound is present at a concentration of about 0.01 to 20 pg/mL of the composition.

[084] The term “combination” herein refers to a mixture or combination of composite extract of Cocculus hirsutus and one or more of the isolated compounds selected from Sinococuline, Hirsutine, Magnoflorine, Coniferyl alcohol, Quercetin, Rutin, Syrinaresionl diglucoside, Makisterone A or 20-hydroxyecdysone or derivatives thereof. Further the term “combination” may also include the administration in the form of a single composition or different compositions for simultaneous, sequential or co-administration.

[085] The term "mammal" herein refers to all mammals including humans. Mammals include, by way of some non-limiting examples, humans, non-human primates, cows, dogs, cats, goats, sheep pigs, rats, mice, and rabbits. [086] In another embodiment, the present invention provides a pharmaceutical composition for use in the prevention and/or treatment of virus infection in a mammal caused by positive sense single-stranded RNA viruses, comprising a composite extract of Cocculus hirsutus and one or more pharmaceutically acceptable excipients. In one aspect of the above embodiment, the present invention provides a stable pharmaceutical composition comprising a therapeutically effective amount of a composite extract of Cocculus hirsutus for use in the prevention and/or treatment of a virus infection in a mammal, wherein the composition when administered to a mammal in need thereof reduces the viral load and/or inhibits the proliferation of virus.

[087] The stable pharmaceutical composition of the present invention further comprises 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 desired site of action or an isolated compound or their mixture to treat or prevent the viral infections caused by positive sense single-stranded RNA viruses. The composition can be delivered by any suitable route of administration, such as oral, nasal, parenteral, pulmonary, transdermal, or rectal. The pharmaceutical composition includes one or more pharmaceutically acceptable excipients. The oral pharmaceutical composition can be in the form of powder, pellets, granules, spheroids, mini-tablets, caplets, tablets, or capsules. The powder can be in the form of a lyophilized powder fdled, with pharmaceutically acceptable excipients, into a capsule of suitable size. Preferably, the pharmaceutical composition is in the form of a tablet. The oral pharmaceutical composition can be present in the form of liquid, including but not limited to solutions, suspensions, emulsions, or syrups. Parenteral compositions according to the invention may be suitable for administration routes such as but not limited to intravenous, subcutaneous, intra-muscular, intra-peritoneal, intrathecal, intravitreal, etc.

[088] 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 invention 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. [089] In some embodiments, the present disclosure provides a composition comprising: a therapeutically effective amount of a composite extract of Cocculus hirsutus,' and a pharmaceutically acceptable excipient comprising a diluent, a binder, a disintegrant, a lubricant, a glidant, a polymer, a flavoring agent, a surfactant, a preservative, an antioxidant, a buffer, a tonicity modifying agent, or combination thereof.

[090] In some embodiments, the composite extract of Cocculus hirsutus is about 20 wt % to about 95 wt % of the composition. In some embodiments, the composite extract of Cocculus hirsutus is about 30 wt % to about 90 wt % of the composition. In some embodiments, the composite extract of Cocculus hirsutus is about 40 wt % to about 85 wt % of the composition. In some embodiments, the composite extract of Cocculus hirsutus is about 50 wt % to about 80 wt % of the composition. In some embodiments, the composite extract of Cocculus hirsutus is about 60 wt % to about 70 wt % of the composition.

[091] In some embodiments, the composite extract of Cocculus hirsutus comprises Sinococuline, Magnoflorine, coniferyl alcohol, quercetin, rutin, syrinaresionl diglucoside, Makisterone A, Hirsutine, 20-hydroxyecdysone, a derivative thereof, or combination thereof.

[092] In some embodiments, the composite extract of Cocculus hirsutus comprises about 0.01 wt% to about 5 wt% of each of Sinococuline, Magnoflorine, coniferyl alcohol, quercetin, rutin, syrinaresionl diglucoside, Makisterone A, Hirsutine, and 20-hydroxyecdysone. In some embodiments, the composite extract of Cocculus hirsutus comprises about 0.01 wt% to about 5 wt% Magnoflorine. In some embodiments, the composite extract of Cocculus hirsutus comprises about 0. 1 wt% to about 1 wt% Magnoflorine. In some embodiments, the composite extract of Cocculus hirsutus comprises about 0.29 wt% to about 0.43 wt% of Magnoflorine.

[093] A “therapeutically effective amount” as used herein refers to an amount of the extract of the invention sufficient to provide a benefit in the treatment or prevention of viral infection caused by a positive sense single-stranded RNA virus, to delay or minimize symptoms associated with the viral infection, or to cure or ameliorate the infection or cause thereof. In particular, a therapeutically effective amount means an amount sufficient to provide a therapeutic benefit in-vivo. 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.

[094] 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.

[095] 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, paolyvinyl alcohol, carboxyvinyl 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.

[096] 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.

[097] 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.

[098] 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.

[099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] In a preferred embodiment, the pharmaceutically acceptable excipients in the composition of the invention include microcrystalline cellulose, anhydrous lactose, croscarmellose sodium, colloidal silicon dioxide and magnesium stearate. In some embodiments, the pharmaceutically acceptable excipient comprises magnesium aluminum trisilicate, lactose monohydrate, dicalcium phosphate, starch, calcium silicate, microcrystalline cellulose, colloidal silicon dioxide, croscarmellose, magnesium stearate, or combination thereof.

[0104] In some embodiments, the compositions provided herein further comprise a fdm coating, a solvent, or combination thereof. In some embodiments, the film coating comprises a polymer, a plasticizer, a colorant, an opacifier, a solvent, a vehicle, or combination thereof. Non-limiting examples of film coatings include commercial film coatings marketed under the tradenames Opadry®, Opalux®, Acryl-Eze®, Sureteric®, Surelease®, and Ethocel™. Film coatings are further described in, e.g., Zaid, AN, Drug Des Devel Ther 14:4613-4623 (2020). In some embodiments, the solvent comprises water, alcohol, glycol, ketone, oil, cyclodextrin, or combination thereof. In some embodiments, the composition comprises an Opadry® film coating and water.

[0105] In some embodiments, the present disclosure provides a composition comprising: a composite extract of Cocculus hirsutus,' magnesium aluminum trisilicate; a diluent selected from lactose monohydrate, dicalcium phosphate, pre-gelatinized starch, calcium silicate, or combination thereof; microcrystalline cellulose; colloidal silicon dioxide; croscarmellose; magnesium stearate; and optionally a film coating.

[0106] In some embodiments, the composite extract of Cocculus hirsutus is about 5 wt% to about 95 wt%, or about 10 wt% to about 90 wt%, or about 15 wt% to about 85 wt%, or about 20 wt% to about 90 wt%, or about 25% to about 85 wt%, or about 30 wt% to about 80 wt%, or about 35 wt% to about 75 wt%, or about 40 wt% to about 75 wt%, or about 45 wt% to about 70 wt%, or about 50 wt% to about 75 wt%, or about 55 wt% to about 70 wt%, or about 50 wt% to about 60 wt% of the composition. In some embodiments, the composite extract of Cocculus hirsutus is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% by weight of the composition.

[0107] In some embodiments, the magnesium aluminum trisilicate is about 1 wt% to about 70 wt%, or about 2 wt% to about 60 wt%, or about 5 wt% to about 50 wt%, or about 8 wt% to about 40 wt%, or about 10 wt% to about 30 wt%, or about 12 wt% to about 25 wt%, or about 15 wt% to about 20 wt% of the composition. In some embodiments, the magnesium aluminum trisilicate is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% by weight of the composition.

[0108] In some embodiments, the diluent is about 0.05 wt% to about 30 wt%, or about 0. 1 wt% to about 25 wt%, or about 0. 1 wt% to about 20 wt%, or about 0.5 wt% to about 15 wt%, or about 1 wt% to about 10 wt%, or about 2 wt% to about 8 wt% of the composition. In some embodiments, the diluent is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 18%, or about 20% by weight of the composition.

[0109] In some embodiments, the microcrystalline cellulose is about 0.05 wt% to about 20 wt%, or about 0.1 wt% to about 15 wt%, or about 0.1 wt% to about 10 wt%, or about 0.5 wt% to about 8 wt%, or about 1 wt% to about 5 wt%, or about 1.5 wt% to about 4 wt% of the composition. In some embodiments, the microcrystalline cellulose is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 18%, or about 20% by weight of the composition.

[0110] In some embodiments, the colloidal silicon dioxide is about 0.05 wt% to about 20 wt%, or about 0.1 wt% to about 15 wt%, or about 0.1 wt% to about 10 wt%, or about 0.5 wt% to about 8 wt%, or about 1 wt% to about 5 wt%, or about 1.5 wt% to about 4 wt% of the composition. In some embodiments, the colloidal silicon dioxide is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 18%, or about 20% by weight of the composition.

[oni] In some embodiments, the croscarmellose is about 0.05 wt% to about 20 wt%, or about 0.1 wt% to about 15 wt%, or about 0.1 wt% to about 10 wt%, or about 0.5 wt% to about 8 wt%, or about 1 wt% to about 5 wt%, or about 1.5 wt% to about 4 wt% of the composition. In some embodiments, the croscarmellose is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 18%, or about 20% by weight of the composition.

[0112] In some embodiments, the magnesium stearate is about 0.005 wt% to about 10 wt%, or about 0.01 wt% to about 5 wt%, or about 0.02 wt% to about 2 wt%, or about 0.05 wt% to about 1 wt%, or about 0.1 wt% to about 1 wt%, or about 0.2 wt% to about 0.5 wt% of the composition. In some embodiments, the colloidal silicon dioxide is about 0.001%, about 0.005%, about 0.01%, about 0.02%, about 0.05%, about 0.08%, about 0.1%, about 0.12%, about 0.15%, about 0.18%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, or about 5% by weight of the composition.

[0113] In some embodiments, the composition comprises about 10 wt% to about 90 wt% of the composite extract of Cocculus hirsutus,' about 5 wt% to about 50 wt% of the magnesium aluminum trisilicate; about 0.1 wt% to about 20 wt% of the diluent; about 0. 1 wt% to about 10 wt% of the microcrystalline cellulose; about 0.1 wt% to about 10 wt% of the colloidal silicon dioxide; about 0.1 wt% to about 10 wt% of the croscarmellose; and about 0.01 wt% to about 5 wt% of the magnesium stearate. In some embodiments, the composition further comprises a fdm coating.

[0114] In some embodiments, the composition comprises about 30 wt% to about 85 wt% of the composite extract of Cocculus hirsutus,' about 8 wt% to about 40 wt% of the magnesium aluminum trisilicate; about 0.5 wt% to about 15 wt% of the diluent; about 0.5 wt% to about 8 wt% of the microcrystalline cellulose; about 0.2 wt% to about 8 wt% of the colloidal silicon dioxide; about 0.5 wt% to about 8 wt% of the croscarmellose; and about 0.02 wt% to about 2 wt% of the magnesium stearate. In some embodiments, the composition further comprises a fdm coating. [0115] In some embodiments, the present disclosure provides a composition comprising: about 50 wt% to about 80 wt% of a composite extract of Cocculus hirsutus,' about 10 wt% to about 30 wt% of magnesium aluminum trisilicate; about 1 wt% to about 10 wt% of a diluent selected from lactose monohydrate, dicalcium phosphate, pre-gelatinized starch, calcium silicate, or combination thereof; about 1 wt% to about 5 wt% of microcrystalline cellulose; about 0.5 wt% to about 5 wt% of colloidal silicon dioxide; about 1 wt% to about 5 wt% of croscarmellose; about 0. 1 wt% to about 1 wt% of magnesium stearate; and optionally a film coating.

[0116] In another embodiment, the present invention provides a stable pharmaceutical composition comprising a therapeutically effective amount of a composite extract of Cocculus hirsutus for use in the treatment of viral infection in a mammal caused by positive sense singlestranded RNA viruses, wherein the composition when administered to a mammal in need thereof reduces the viral load or proliferation of virus.

[0117] In a further embodiment, the present invention provides a pharmaceutical composition comprising a composite extract of Cocculus hirsutus and one or more pharmaceutically acceptable excipients to reduce the viral load at an early stage in the treatment of infection in mammals caused by positive sense single -stranded RNA viruses. Preferably, the composition is a stable pharmaceutical composition. More preferably, the composition is a stable oral pharmaceutical composition.

[0118] In a further embodiment, the present invention provides an oral composition comprising a composite extract of Cocculus hirsutus, wherein the said composition exhibits an inhibitory activity with ICso value ranging from about 1 to 100 pg/ml as determined by plaque and flowcytometry based virus inhibition assays.

[0119] In a further embodiment, the present invention provides an oral composition comprising an aqueous composite extract of Cocculus hirsutus, wherein the said composition exhibits an ICso value against Japanese Encephalitis Virus (JEV) ranging from about 4-12 pg/ml, preferably about 5-11 pg/ml, more preferably about 6-10 pg/ml.

[0120] In a further embodiment, the present invention provides an oral composition comprising an aqueous composite extract of Cocculus hirsutus, wherein the said composition exhibits an IC50 value against Chikungunya (CHIKV) ranging from about 40-70 pg/ml, preferably about 45- 65 pg/ml, more preferably about 50-60 pg/ml.

[0121] In a further embodiment, the present invention provides oral composition comprising Sinococuline, wherein the said composition exhibits an inhibitory activity with IC50 value ranging from about 0.1-3 pg/ml. In an aspect, the IC50 of Sinococuline against Dengue virus (DENV 1-4) was found to be in the range of 0.1-2 pg/ml. In an aspect, the ICso of Sinococuline against Chikungunya virus (CHIKV) was found to be in the range of 0.5-15 pg/ml, preferably 1- 10 pg/ml, more preferably 1-5 pg/ml. In another aspect, the ICso of Sinococuline against Japanese Encephalitis Virus (JEV) was found to be in the range of about 0.1-3 pg/ml, preferably about 0.2-2 pg/ml, more preferably about 0.2-1 pg/ml.

[0122] The extract and the various compositions as contemplated in the present invention were also found to be effective in inhibiting the secretion of cytokines in many other virus infections. Pro-inflammatory cytokines like TNF-a and IFN-y are produced in excessive amounts which lead to disease progression. In an In-vivo study, the extract and the composition of the invention inhibited the secretion of cytokines in small intestine. It was found that mice challenged with Viral Immune complex (ICs) and fed with 100 mg/kg/day of the extract showed lesser amounts of TNFa and IL-6 in comparison to the group which did not receive any treatment. Lesser cytokine secretion by the extract treated group is in confirmation with the ability of the extract to inhibit virus proliferation. IC challenged mice without any extract treatment displayed higher cytokine levels due to enhanced infection through ADE, in comparison to virus alone control. The mechanistic studies have shown that the extract targets the initial stages i.e., post entry of virus into the cell. The time-of-addition and time of removal studies assay showed that the extract interfered as early as 3-4 hours of viral entry into the host cells. The inventors also tested Sinococuline for the antiviral activity, and it was found that Sinococuline shows anti-viral activity by interfering the post entry steps. Further time of elimination study with Sinococuline also showed that it arrests the early steps in viral replication.

[0123] In yet another embodiment, the present invention provides a stable pharmaceutical composition comprising a therapeutically effective amount of a composite extract of Cocculus hirsutus for use in the treatment of viral infection in a mammal caused by a positive sense single-stranded RNA virus, wherein the composition when administered to a mammal in need thereof is effective in a delayed treatment onset.

[0124] In a further embodiment, the present invention provides a stable pharmaceutical composition comprising a therapeutically effective amount of a composite extract of Cocculus hirsutus for use in the prevention and/or treatment of positive sense single-stranded RNA virus infection in a mammal. The extract and the composition of the present invention when administered pre-infection is found to show protective efficacy against secondary infection when tested in an in-vivo study in an animal model infected with Dengue virus. [0125] In one embodiment, the extract of Cocculus hirsutus comprises one or more constituents selected from the group consisting of flavonoids, lignans and alkaloids or combinations thereof. In an aspect, the composite extract of Cocculus hirsutus comprises Magnoflorine as one of the alkaloids. In a further aspect, the composite extract of Cocculus hirsutus comprises Magnoflorine in an amount ranging from 0.1% to 1% of the total weight of extract in the composition. In yet another aspect, the composite extract of Cocculus hirsutus comprises Magnoflorine in an amount ranging from 0.29% to 0.43% of the total weight of extract in the composition.

[0126] In another aspect of the embodiment, the composite extract of Cocculus hirsutus comprises quercetin as one of the flavonoids. In another aspect of the embodiment, the composite extract of Cocculus hirsutus comprises Sinococuline as one of the flavonoids.

[0127] In some embodiments, the composite extract of Cocculus hirsutus provided herein is substantially free of lignin, fiber, and tannin. In some embodiments, the composite extract of Cocculus hirsutus comprises less than 10 wt%, less than 8 wt%, less than 5 wt%, less than 2 wt%, less than 1 wt%, less than 0.5 wt%, or less than 0.1 wt% of lignin, fiber, and tannin.

[0128] In some embodiments, the present disclosure provides a composition comprising at least three compounds selected from Sinococuline, Magnoflorine, Makisterone A, Hirsutine, 20- hydroxyecdysone, wherein the at least three compounds are about 0.05% to about 10% of the composition, or about 0. 1% to about 5% of the composition. In some embodiments, the composition is substantially free of lignin, fiber, and tannin.

[0129] In some embodiments, the Sinococuline is about 0.01% to about 30%, or about 0.05% to about 25%, or about 0.1% to about 20%, or about 0.5% to about 15%, or about 1% to about 10% by weight of the composition. In some embodiments, the Magnoflorine is about 0.001% to about 20%, or about 0.005% to about 8%, or about 0.01% to about 10%, or about 0.08% to about 5%, or about 0.05% to about 3%, or about 1% to about 2% by weight of the composition.

[0130] In some embodiments, the Makisterone A is about 0.0001% to about 10%, or about 0.005% to about 5%, or about 0.01% to about 3%, or about 0.02% to about 2%, by weight of the composition. In some embodiments, the 2-hydroxyecdysone is about 0.001% to about 20%, or about 0.005% to about 8%, or about 0.01% to about 10%, or about 0.08% to about 5%, or about 0.05% to about 3%, or about 1% to about 2% by weight of the composition.

[0131] In some embodiments, the composition comprises about 0.1 wt% to about 20 wt% Sinococuline; about 0.01 wt% to about 5 wt% Magnoflorine; about 0.005 wt% to about 5 wt% Makisterone A; and about 0.01 wt% to about 5 wt% 20-hydroxyecdysone. In some embodiments, the composition comprises about 0.5 wt% to about 15 wt% Sinococuline; about 0.05 wt% to about 3 wt% Magnoflorine; about 0.01 wt% to about 3 wt% Makisterone A; and about 0.05 wt% to about 3 wt% 20-hydroxyecdysone.

[0132] In a further embodiment, the extract and composition of the present invention were found to be safe and didn’t show any toxic effect when administered in a therapeutically effective dose to the mammal in need thereof. After related oral administration at dose levels of 100, 300, 600 and 1200 mg/kg/day by oral gavage in Wistar rats for 14 days the NOAEL in Wistar rats, was established at 300 mg/kg/day. Thus, the extract was found to be safe and nontoxic for human use.

[0133] In one embodiment, the present invention provides a method of treating a positive sense single -stranded RNA virus infection in a mammal comprising administering a pharmaceutical composition comprising a therapeutic effective amount of a composite extract of Cocculus hirsutus to a mammal in need thereof, wherein the extract reduces the viral load, or proliferation of virus or viruses.

[0134] In yet another embodiment, the present invention provides a method for reducing the viral load at an early stage in the treatment of virus infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.

[0135] In yet another embodiment, the present invention provides a method of preventing and/or treating virus infection in mammals caused by positive sense single -stranded RNA comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.

[0136] In a further embodiment, the present invention provides a method of inhibiting the secretion of cytokines in positive sense single-stranded RNA virus infection in mammals, comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.

[0137] In yet another embodiment, the present invention provides a method of prevention and/or treatment of a positive sense single-stranded RNA virus infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof, wherein the extract is effective in a delayed treatment onset.

[0138] In yet another embodiment, the present invention provides a method of prevention of positive sense single-stranded RNA virus infection in mammals comprising administering a composite extract of Cocculus hirsutus to the mammals in need thereof.

[0139] In a further embodiment, the present invention provides use of a composite extract of Cocculus hirsutus in method for treatment or prevention of viral infection caused by positive sense single-stranded RNA virus or for reducing or inhibiting the virus proliferation, by administering a therapeutically effective amount of composite extract of Cocculus hirsutus or a pharmaceutical composition thereof.

[0140] In a further embodiment, the present invention provides use of compounds such as Sinococuline, Magnoflorine, Makisterone A, Hirsutine, 20-hydroxyecdysone or their derivatives or combinations thereof from an extract of Cocculus hirsutus in method for treatment or prevention of viral infection caused by a positive sense single-stranded RNA virus or for reducing or inhibiting the virus proliferation, by administering a therapeutically effective amount of such compound or a pharmaceutical composition thereof.

[0141] In yet another embodiment, the present invention composite extract was found to be better and superior to other plant based extracts in treating viral infections, more particularly in treating Dengue virus infection using Cissampelos pareirci extract.

[0142] In some embodiments, the extract may be prepared by process known in the art. In some embodiments, the present disclosure provides a method of preparing a composite extract of Cocculus hirsutus, comprising: a) extracting a plant mass of Cocculus hirsutus with a solvent comprising ethanol, water, or both, wherein the extracting is performed at about 50 °C to about 100 °C; b) concentrating the extract of (a); and c) drying the concentrated extract of (b), wherein the drying is performed at about 40 °C to about 95 °C, thereby obtaining the composite extract of Cocculus hirsutus. In some embodiments, the method further comprises, prior to (c), further extracting the concentrated extract of (b) with a further solvent, wherein the further solvent is ethanol, water, or both. In some embodiments, the further solvent is ethanol. In some embodiments, the further solvent is water. In some embodiments, the further solvent comprises a combination of ethanol and water at a ratio of about 1:99 to about 99: 1, or about 5:95 to about 95:5, or about 10:90 to about 90: 10, or about 20:80 to about 80:20, or about 30:70 to about 70:30, or about 40:60 to about 60:40, or about 1: 1.

[0143] In some embodiments, the process comprises 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.

[0144] In yet another embodiment, the present invention provides a process of preparation of a tablet composition of the composite extract according to the invention for use in the treatment of viral infection caused by positive sense single-stranded RNA viruses, the process comprising the steps of:

(i) sifting the extract and blending with pharmaceutically acceptable excipients;

(ii) lubricating the blend obtained from step (i) and compressing into tablets, and

(iii) film coating the tablets of step (ii).

[0145] In yet another embodiment, the present invention provides a process of preparation of tablet composition of the composite extract according to the invention for use in the treatment of viral infection caused by positive sense single-stranded RNA viruses, the process comprising the steps of:

(i) blending the extract with pharmaceutically acceptable excipients;

(ii) granulating the blend of step (i) with a solvent;

(iii) lubricating and compressing the blend into tablets, and

(iv) film coating the tablets of step (iii).

[0146] In a further embodiment, the present invention provides a process of preparation of tablet composition of the composite extract for use in the treatment of viral infection caused by positive sense single -stranded RNA viruses, the process comprising the steps of:

(i) blending the extract with pharmaceutically acceptable excipients and compacting the mixture;

(ii) milling the compacts and blending with extragranular excipients; (iii) lubricating the blend of step (ii) and compressing into tablets, and

(iv) film coating the tablets of step (iii).

[0147] Although the above embodiments are related to tablet composition, however the said composite extract of Cocculus hirsutus may also be formulated into any other suitable oral dosage forms like powder, pellets, granules, spheroids, mini-tablets, caplets.

[0148] In further related embodiment, the extract may be co-administered simultaneously or sequentially with one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent comprises an antiviral agent, an antipyretic agent, an analgesic agent, or combination thereof. In another embodiment, the composition of the invention may further comprise one or more additional therapeutic agents. The one or more additional therapeutic agents may be selected from related antiviral therapies or compounds such as which may provide symptomatic relief from the conditions, for examples antipyretic and analgesic drugs.

[0149] The term "co-administration" herein refers to administration of one or more additional therapeutic agents with the extract to a mammal. The extract and additional therapeutic agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the extract or additional therapeutic agents may be administered through the same or different routes of administration simultaneously of sequentially.

EXAMPLES

[0150] The following examples, which include only exemplary embodiments, will serve to illustrate the practice of this invention. It will be evident to those skilled in the art that the invention is not limited to the details of the following illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive.

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

[0151] 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 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 till ethanol content is not more than 10,000 ppm and moisture content is not more than 5%. The dried extract was cooled to about 20-25 °C and unloaded at controlled humidity (RH NMT 40%).

[0152] Yield obtained = 90 g to 120 g

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

[0153] 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 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 ethanol content is not more than 10,000 ppm and moisture content is not more than 5%. The dried extract was cooled to about 20-25 °C and unloaded at controlled humidity (RH NMT 40%).

[0154] Yield obtained = 80 g to 120 g

Example 3: Preparation of an aqueous extract of Cocculus hirsutus

[0155] 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 till ethanol content is not more than 10,000 ppm and moisture content is not more than 5%. The dried extract was cooled to about 20-25 °C and unloaded at controlled humidity (RH NMT 40%).

[0156] Yield obtained = 80 g to 120g

[0157] * The term "ambient temperature" as used herein, includes a temperature ranging from about 18 °C to about 25 °C. [0158] 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: Isolation of various compounds from Cocculus hirsutus:

[0159] The dried aqueous extract (powder, 500 gm) of Cocculus hirsutus was dissolved in distilled water and fdtered. The fdtrate was partitioned between CHCh (A) and H2O (B). The CHCh soluble fraction (A) was then partitioned using aq. HC1 to yield CHCh layer (C) and acidic H2O layers (D). Chloroform layer (C) was subjected to column chromatography, eluted with a gradient of CHCh-MeOH, and concentrated, giving thirty fractions. Coniferol_(SP-A-01) was obtained in pure form fractions based on TLC profile. The acidic H2O layer (D) was then basified with aq. NH4OH to produce free bases, and then extracted with CHCh, resulting in alkaloid-containing chloroform layer (E). The CHCh fraction (E) was dried and purified by column chromatography and eluted with a gradient of CHCh-MeOH, for separation of Magnoflorine. Further, the aqueous layer (B) was basified with ammonium hydroxide (NH4OH) solution, and subsequently extracted with CHCh, resulted in alkaloid rich fraction (El) and aqueous layer (Fl). The CHCh layer (El) was further processed for separation of Sinococuline through column chromatography eluted with CHCh-MeOH. The Aqueous layer (Fl) was powdered using lyophilization and dissolved in methanol. The methanol dissolved portion was further purified by column chromatography, eluted with a gradient of CHCh-MeOH, and concentrated, giving fifty fractions. Two UV active spots were visualized on TLC. This mixture containing two UV active compounds further subjected to RP-HPLC (using a gradient solvent system of (McOH/FhO) to obtain 20-hydroxyecdysone and Makisterone A.

Example 5: Preparation of Tablets from the extract of Cocculus Hirsutus using Direct compression technique

[0160] MANUFACTURING PROCEDURE:

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

2. Step 1 material 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 material from Steps 3 and 4 was mixed in a blender with step 2 material;

6. The blend obtained from step 5 was 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 from step 6 were coated with dispersion of step 7.

Example 5.1: Preparation of Tablets from the extract of Cocculus hirsutus using direct compression technique:

[0161] Manufacturing procedure same as Example 5.

Example 6: Preparation of Tablets from the extract of Cocculus hirsutus using Wet

Granulation technique (by Rapid mixer granulator)

[0162] 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 material from Step 1 and step 2 was granulated with methanol and dried;

4. The dried material from step 3 was 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. The material from steps 5 and 6 were mixed in a blender along with step 4 material;

8. The blend obtained from above step was 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 from step 8 were coated with dispersion of step 9.

Example 7: Preparation of Tablets from the extract of Cocculus Hirsutus using Dry granulation technique (by Roller compactor)

[0163] MANUFACTURING PROCEDURE:

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

2. The step 1 material was sifted along with Magnesium aluminium trisilicate through #14 mesh (1.4 mm);

3. Magnesium stearate was sifted through #36 mesh (420 p) and mixed with step 2 material in a blender;

4. The blended material was compacted using roll compactor;

5. The compacts obtained from step 4 were milled;

6. The extra granular excipients were sifted and blended with magnesium stearate to obtain a lubricated blend;

7. The lubricated blend of step 6 was compressed into tablets;

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

9. The compressed tablets from step 7 were coated with dispersion of step 8.

Example 8: Biological activity evaluation in treatment of various viral infections caused by positive sense single-stranded RNA viruses: A. To evaluate the efficacy of the aqueous extract of Cocculus hirsutus (AQCH) in treatment of COVID- 19 patients.

[0164] The selected patients as per the inclusion and exclusion criteria below are administered tablets of aqueous extract of Cocculus hirsutus 400-800 mg thrice daily (every 8±1 hours) before meal, preferably at same time, for 10 days.

- Inclusion Criteria: Subjects are included in the study if they meet all of the following criteria:

1. Provides written informed consent.

2. Male or non-pregnant, non-lactating female aged > 18 and < 65 years.

3. Patients with acute respiratory illness (fever with at least one of the following cough or shortness of breath) with: a) History of travel to high risk COVID-19 affected countries within last 14 days OR. b) Close contact with a laboratory confirmed case of COVID- 19 in 14 days OR. c) Healthcare personnel managing respiratory distress / severe acute respiratory illness cases when they are symptomatic.

4. Laboratory confirmed diagnosis of COVID-19.

5. Able to take the drug orally and comply with study procedures.

6. Women of childbearing potential must have a negative urine pregnancy test prior to study entry and agree to use highly effective methods of contraception to prevent pregnancy from study entry till the last dose of the study medication (such contraception may include hormonal birth control e.g., combined estrogen and progestogen containing [oral, intravaginal, or transdermal] or progesterone only [oral, injectable, or implantable] hormonal contraception associated with inhibition of ovulation, intrauterine devices, intrauterine hormone releasing system OR bilateral tubal occlusion, vasectomized partner, or sexual abstinence).

- Exclusion Criteria: Patient are deemed ineligible to participate in the study if they fall under any of the following criteria:

1. Patients with persistent vomiting (more than three episodes of vomiting in 12 hours, preventing adequate oral hydration) that make it difficult to swallow the drug.

2. Patients with altered mental state.

3. Laboratory abnormalities at Screening, including any of the following: a. AST or ALT > 5 times ULN b. Room air oxygen saturation < 90% c. Absolute neutrophil count < 1500/pL d. Serum Creatinine > ULN e. Total bilirubin > ULN

4. Patients with active hepatitis, tuberculosis and definite bacterial or fungal infections.

5. Patients with Combined organ failure requiring ICU monitoring and treatment.

6. Patients of respiratory failure and requiring mechanical ventilation.

7. Patients with shock.

8. Patients with any concurrent medical condition or uncontrolled, clinically significant systemic disease (e.g., renal failure, heart failure, hypertension, liver disease, diabetes, anaemia etc.) that, in the opinion of the Investigator precludes the subject’s participation in the study or interferes with the interpretation of the study results.

9. Patients with history of serology tests positive for hepatitis B, hepatitis C, or human immunodeficiency virus.

10. Patients who received specific antiviral drugs Ritonavir/Ropinavir, Chloroquine, Hydroxychloroquine, monoclonal antibodies within 1 week before admission.

11. Participation in another clinical study within thirty (30) days prior to screening.

12. Patient who has participated in another investigational study within 3 months prior to enrolment in this study.

13. Investigators, study personnel, sponsor’s representatives, and their first-degree relatives.

[0165] Efficacy evaluation: It is based on both clinical and laboratory assessments. Following assessments are conducted:

• Physical examination at screening, everyday till day of discharge, Day of Discharge, Day 17 and as clinically indicated.

• Vital signs (pulse rate, respiratory rate, systolic and diastolic pressure) at screening, within 10 minutes before first dosing on day 1 and then every 24 hours (± 10 minutes) until discharge (on day 11 or later), at the FU visit on Day 17 and as clinically indicated.

• Body temperature (oral) will be measured at screening, within 30 minutes before first dosing on day 1 and then every 24 hours (± 10 minutes) until day of discharge (on day 11 or later), at the FU visit on day 17 (or later) and as clinically indicated.

• Blood for evaluation of hematology and biochemistry for liver and renal function test will be withdrawn at screening, and subsequently on days 3, 5, 7, 10. Additional hematological and biochemistry estimations could be performed until recovery as per the discretion of the investigator.

• PCR for Corona virus titer: At screening, within 30 minutes before first dosing on day 1 and then every 24 hours (± 10 minutes) till day 10 and on Discharge. • X-Ray Chest - At screening on Day 3, 5, 7, 10 and on Discharge.

[0166] Safety is assessed by assessments of recording of Adverse events, vital signs (pulse rate, systolic and diastolic blood pressure (seated), body temperature and respiratory rate), ECG, physical examination, and clinical laboratory investigations (hematology, biochemistry and urinalysis). Adverse events will be classified according to their severity based on CTCAE v 5.0 criteria. Any clinically significant abnormal change from baseline in the concurrent medical condition(s), physical examination and/or laboratory data shall be recorded as an AE. 12 lead ECG is conducted at screening, 1-hour post-dose on day 1 and repeated after 24 hours (± 10 minutes) until day 10, on day of discharge and as clinically indicated. Any subject who is judged by the treating physician/ PI to be at risk for developing severe disease will be managed as per standard of care.

[0167] The Primary end points are reduction in viral load in nasopharyngeal swab and percentage of subject achieving Clinical Cure (clinical Cure is defined as negative viral load of the respiratory specimen for two consecutive times when measured on frequency of greater than or equal to one day, improvement in lung image, normal body temperature for more than 3 days, and improvement in clinical manifestation). Secondary end points are: Viral nucleic acid conversion rate and days from positive to negative (Time frame: within 10 days of admission), Time to clinical improvement as the time to normalization of fever, respiratory rate, oxygen saturation and alleviation of cough, Time to clinically defined as time to death, mechanical ventilation or ICU admission, Safety and tolerability of the drug as assessed by treatment emergent adverse events. The pharmaceutical composition for the extract is effective in the treatment of patients with COVID-19 with no significant side-effects.

B. To evaluate the efficacy of the aqueous extract of Cocculus hirsutus (AQCH) in treatment of infection caused by Chikunguniya Virus (CHIKV),

[0168] Assay protocol for testing the AQCH for CHIKV inhibition using virus plaque reduction assay: i. Seed 0.2 million Vero cells per ml per well in a 12 well plate in DMEM + 10% AFBS; ii. Incubate the plate for 24 hrs in an incubator set at 37 °C, and 10% CO2; iii. After the incubation period is over, infect the Vero cells with CHIKV; iv. After setting up the infection, incubate the plate in an incubator for 2 hr at 37 °C and

10% CO _2; v. After two hours, remove the infection media and put drug to be tested (AQCH) in different concentrations, along with the methylcellulose overlay; vi. Each drug concentration tested in duplicates or triplicates by incubating the plate for 3 more days in an incubator set at 37 °C and 10% CO2; vii. After the incubation period is over, remove the plate from the incubator; viii. Wash the plate 3 times with IX PBS by dispensing 500 pl in each well; ix. Gently tap the plate on a wad of paper towels and fix the cells by adding 500 pl of 10% formaldehyde (prepared in IX PBS); x. Incubate the cells at RT for 30 mins followed by wash the plate again 3 times with IX PBS by dispensing 500 pl in each well; xi. Gently tap the plate on a wad of paper towels. Add 200 pl of 0.25% crystal violet stain (prepared in 30% methanol and filtered before use) and incubate the plates 5-10 mins at RT; and xii. Fill a tub with tap water and immerse the plate to remove excess stain.

Infection in step iii) is done by preparing required dilution of the virus stock in DMEM + 0.5 %A FBS such that virus control wells yield 80-100 plaques per 250 pl of infection media (DMEM + 0.5 %AFBS). Cell control wells included in the experimental plate design, which do not receive any virus infection but only 250 pl of DMEM + 0.5 %AFBS.

In step v) Different cone, of drug prepared by, preparing 2% methylcellulose (MC) in hot water and autoclave it. Prepare appropriate dilutions of the drug to be tested in 2X DMEM + 1.0 %A FBS. Mix 500 pL of 2% MC and 500pL of drug dilution for each well and added (total 1 ml) on top of the cells after removing the virus infection media.

[0169] Results: AQCH was screened using the plaque assay against CHIKV. In total of four independent experiments using lab scale and plant scale AQCH extract, the IC50 was in the range of 10-20 pg/ml. Thus, the results demonstrate the potent inhibitory activity of the composite extract of the invention against the virus (CHIKV).

C. To evaluate the efficacy of the extract of Cocculus hirsutus or the isolated compounds Sinococuline. Magnoflorine. 20-hvdroxyecdvsone in treatment of infections caused by Dengue Virus (DENY), FACS based neutralization assay was used:

[0170] This assay was used to detect the number of DENV-infected cells in total cell population. Vero cells in a 96-well flat bottom plate (sterile, tissue culture treated) Dulbecco modified Eagle medium supplemented with 10% heat inactivated Fetal Bovine Serum AFBS (20,000-25,000 cells/200pl/well) were seeded and incubated at 37°C in a humidified incubator with 10% CO2 for 24-26 h (doubling time for Vero cells). In this method, the cells should not be less than 20,000/well. The media was aspirated and cells were infected with DENV-1, 2, 3, 4 at 0.1 MOI in DMEM supplemented with 0.5% AFBS media (lOOpl/well). The plates were incubated for 2 h at 37°C in a humidified incubator with 10% CO2. The working stocks of Cocculus hirsutus aqueous extract were prepared and its different concentrations viz. lOOpg/ml, 50 pg/ml, 25 pg/ml, 12.5 pg/ml, 6.25 pg/ml, and 3.125 pg/ml to the respective wells of the 96-well plate. The plates were allowed to incubate at 37°C in a humidified chamber with 10% CO2 for 42-46 h post infection. After completion of the incubation period, cells were stained for the presence of cytosolic DENVs with a fluorescent labelled antibody. For staining, media was aspirated from the top of the cells, washed with PBS. Cells trypsinised and transferred to a 96 well U bottom plate. Then centrifuged and supernatant was aspirated. Cells were washed twice with permeabilization or perm buffer and blocked with 1% normal mouse sera (prepared in perm buffer) for 30 mins. 2H2-Alexa488 antibody was added to stain the cells for DENVs and incubated with gentle shaking. After incubation, cells were centrifuged and supernatant aspirated. Cells were washed and re-suspended in PBS. The above processed cells were analyzed through flow cytometer and 5000 cells were counted per well. Data obtained was analyzed (through FlowJo software) to determine the relative percentage of infected cells for each extract concentration with respect to virus only (without any extract treatment) control group. The IC50 of extract was determined as the extract concentration that inhibited 50% of dengue virus infection, calculated using GraphPad Prism software.

[0171] Upon evaluation through FNT, the extract was found to be efficacious in inhibiting all four DENV serotypes. The IC50 values for each DENV serotypes typically ranged between 5-10 pg/ml (Table 1), indicating that 5-10 pg/ml of extract is required to inhibit all four DENV infection by 50% in this assay.

Table 1: Tabular representation of ICso (pg/ml) of the extract against all the four dengue virus serotypes as examined through FNT: [0172] A similar evaluation study was also done with respect to other isolated compounds according to the invention against Dengue virus serotype 4 (DENV4 or DV4) as examined through FNT, the test results are provided in below Table 2:

Table 2: Tabular representation of ICso (pg/ml) of isolated compounds against dengue virus serotype 4 (DV4) as examined through FNT:

[0173] Thus, based on above table 1 and 2, it is clear that the composite extract of Cocculus hirsutus or the isolated compounds (Sinococuline, Magnoflorine and 20-hydroxyecdysone) are effective in treatment of infections caused by Dengue Virus (DENV).

Example 9: Evaluation of anti-viral effects of Sinococuline against positive strand RNA viruses:

1. In-vitro anti-viral effects of Sinococuline:

A. Against Dengue Virus (DENV): Flow cytometry based virus inhibition assay

[0174] Vero cells were seeded in a 96-well plate (20,000-25,000 cells/well) in 200 pl DMEM + 10% AFBS and incubated for 24 hr in an incubator adjusted at 37 °C and 10% CO2. Next day, cells were infected with 100 pl of DENV-1, DENV-2, DENV-3, and DENV-4 dilutions to yield 10% infection in DMEM + 0.5% AFBS (dilution media). After a 2 hr incubation of Vero cells with the virus at 37 °C, 10% CO2, virus was aspirated and 200 pl of a suitable range of test substance (Sinococuline) prepared in dilution media was added to the wells in duplicates. Cells were incubated further for another 46 hr in an incubator at 37 °C, 10% CO2. Wells infected with the virus but without any subsequent extract/drug treatment served as virus controls whereas wells with no infection and no treatment served as cell controls. These experimental controls were utilized for relative virus infection calculations and antibody background signal adjustments, respectively. After completion of the incubation period, cells were stained for the presence of cytosolic DENVs with Alexa-488 labelled 2H2 mAb. For staining, media was aspirated from the top of the cells and washed with PBS. Cells were trypsinised and transferred to a 96 well U bottom plate. After transfer, cells were centrifuged at 1500 rpm for 5 mins and supernatant was aspirated. Cells were washed with PBS again and then fixed with 4% paraformaldehyde for 20 mins. Cells were centrifuged at 2500 rpm for 5 mins and supernatant was aspirated. Cells were washed twice with permeabilization or perm buffer and blocked with 1% normal mouse sera (prepared in perm buffer) for 30 mins. Without removing the blocking solution, Alexa-488 labelled 2H2 mAb was added to stain the cells for DENVs and incubated for 1 hr at 37 °C with gentle shaking. Post-incubation, cells were centrifuged at 2500 rpm for 5 mins and supernatant aspirated. Cells were washed twice with perm buffer and re-suspended in 100 pl of PBS. The above processed cells were analysed through a BD FACS Verse flow cytometer and 5000 cells were counted per well. Data was analyzed through FlowJo software to determine the relative percentage of infected cells for each test substance concentration with respect to virus only control group. The 50% inhibitory concentration (ICso) of the test substance was determined as the concentration that inhibited 50% of dengue virus infection with respect to virus control, calculated using non-linear regression analysis of GraphPad Prism software.

Table 3: Shows ICso of Sinococuline against DENV (1-4):

[0175] Result: ICso of Sinococuline against DENV 1-4 was found to be 0.62-0.91 pg/ml.

B. Against Chikungunya Virus (CHIKV): Viral plaque reduction assay

[0176] Seed 0.2 million/well/ml of Vero cells in DMEM + 10% AFBS in 12 well plate and incubate overnight at 37° C, 10% CO2. After 24 hrs, thaw the virus on ice for setting up virus infection. Dilute the virus using DMEM + 2% AFBS (diluent) to yield ~80 plaques per 250 pl/well in virus control well. Keep cell controls in the experiment which receive no virus infection but only diluent. Rock the plate every 15 min after setting up the infection. After 2 hrs, remove the virus infection media and wash the wells with DMEM. Add 1 ml of required dilutions of test substance (Sinococuline) with 1% methylcellulose in respective wells and incubate for 2 days at 37 °C, 10% CO2. Cell and Virus control wells will receive methylcellulose overlay prepared in diluent with no test substance. After the completion of incubation period, remove the plate from the incubator. Wash the plate 3 times with IX PBS by dispensing 500 pl of IX PBS into each well every time. Fix the cells by adding 500 pl of 4% para-formaldehyde (prepared in IX PBS). Incubate the cells at RT for 30 mins. Wash the plate again 3 times with IX PBS by dispensing 500 pl of IX PBS into each well every time. Add 100 pl of 0.25% crystal violet stain (prepared in 30% methanol and fdtered before use) and incubate the plate for 10 mins at RT. Wash the plate with water to remove excess stain and count the plaques.

[0177] Result: ICso of Sinococuline against CHIKV was found to be ~2 pg/ml.

Table 4: Summarising the IC50 of Sinococuline against different viruses tested

2. In-vivo anti-viral effects of Sinococuline in AG129 mice

[0178] Secondary dengue AG 129 mouse model was established through IV inoculation of immune complex (IC) of sub-lethal dose of DENV-2 S221 (2xl0 ⁴ FIU) and neutralizing concentration of 4G2 mAb (10 pg). Sinococuline was dissolved in water and a solution of 1 mg/ml was prepared in 0.1% methylcellulose. IC challenged mice (n=4-6) were orally fed and injected intraperitoneally with Sinococuline, 10 mg/kg/day, in a QID and BID dosing, respectively, for 5 days post IC inoculation. Mice were monitored for body weight change, disease symptoms and survival. Body weight was monitored twice a day in morning and evening, and the mean taken for plotting graphs. Morbidity score was based on 5 point system: 0.5, mild ruffled fur; 1.0, ruffled fur; 1.5, compromised eyes; 2, compromised eyes with hunched back; 2.5, loose stools; 3.0, limited movement; 3.5, no movement/hind leg paralysis; 4.0, euthanized if cumulative score was 5. The p values of <0.05 were considered significant (*p < 0.05).

[0179] Results: Sinococuline could provide protection against the secondary dengue infection in AG129 mice. Upon oral QID administration, 70% survival was observed. While under intraperitoneal administration BID, complete protection was observed.

[0180] While considerable emphasis has been placed herein on the specific extract of the ingredients of the Cocculus hirsutus and specifically isolated compounds in various preferred formulation and biological activity evaluation studies, 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 invention. These and other changes in the preferred extract and formulations of the 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 invention and not as a limitation.

Example 10: Evaluation of efficacy of AQCH Tablet and Sinococuline against Chikungunya Virus (CHIKV):

[0181] Additional Plaque Reduction Neutralization Test (PRNT) was done as per assay procedure in example 8.B. The in vitro activity of AQCH and Sinococuline was scored against African CHIKV strain of Chikungunya Virus (CHIKV) by Plaque Reduction Neutralization Test (PRNT) and concentration of drug that caused 50% inhibition of the virus was calculated as 50% Inhibitory Concentration or ICso.

[0182] For evaluation of efficacy, samples of Sinococuline, AQCH tablet and placebo tablet were prepared in concentration range of:

• 100 to 3. 12 pg/ml for AQCH and Placebo tablet;

• 10 to 0.31 pg/ml for Sinococuline.

[0183] The test results are summarized in Table-5 below:

Table-5: ICso values obtained from in vitro anti-CHIKV PRNT experiment

*NA: No activity observed within the evaluated concentrations of 100 to 3.12 pg/ml

[0184] Conclusion: Sinococuline and AQCH inhibited CHIKV in the PRNT experiment.

Example 11: To evaluate the efficacy of AQCH Tablet and Sinococuline against Japanese Encephalitis Virus (JEV):

[0185] The in vitro activity of AQCH and Sinococuline was scored against Japanese Encephalitis Virus (JEV) by Flow-cytometry based Neutralisation Test (FNT) and concentration of drug that caused 50% inhibition of the virus was calculated as 50% Inhibitory Concentration or ICso.

[0186] For evaluation of efficacy, samples of Sinococuline, AQCH tablet and placebo tablet were prepared in concentration range of:

• 50 to 1.56252 pg/ml for AQCH and Placebo tablets;

• 10 to 0.31 pg/ml for Sinococuline.

[0187] Experiment details:

1. Seeding of Vero cells: Typsinize confluent T-75 flask of Vero cells using lx TE. For this, aspirate the overlay media from the flask and rinse it with 4 mb DMEM. Add 4 mL of lx TE and incubate the flask 5-10 min at 37° C in 10% CO2 incubator to dislodge the adhered Vero cells. Collect the cell suspension in tube containing 0.4 mL of DMEM with 10% AFBS. Centrifuge at RT at 1500 rpm for 5 min. Discard the supernatant and re-suspend the pellet in desired volume of 10% AFBS-DMEM to yield 20,000 - 25,000 Vero cells/200 pl of suspension. Seed 200 pl of Vero cell suspension per well in 96 well flat bottom plate. Incubate for 24 h at 37° C in 10% CO2 incubator.

2. Preparation of test item stock: Dissolve Sinococuline at Img/mL, AQCH at 5mg/mL and Placebo at 5mg/mL concentrations in lx DMEM 2 h prior to usage and keep on flip flop for equal mixing at RT.

3. JEV Infection: Dilute the desired volume of stock of JEV in lx DMEM + 0.5% AFBS to yield 10% infection. Remove the media on top of the seeded Vero cells and add 80pl of working solution of JEV. The assay is set up in duplicates for all the test item concentrations. Incubate the plates for 2 h at 37°C in 10% CO2 incubator.

4. Test item dilution: Prepare the required dilutions of test item from the stock prepared in step 2 in sterile lx DMEM + 0.5 % AFBS; first dilution is filter sterilized using a syringe filter with 0.45u filter, which is used to prepare the subsequent dilutions.

5. Treatment with test item: At the end of 2 h of JEV infection, aspirate the virus and add 200 pl media containing the drug dilutions from step 4 on to the cells. Incubate the plates for 22- 24 h at 37°C in 10% CO2 incubator.

6. Staining: Staining can be performed between 22-24 h post-addition of virus inoculum. For this, the cells are trypsinized, fixed, permeabilized and blocked before staining with Alexa488 labelled 4G2 mAb. The steps are detailed below:

6.1. Aspirate media from top of cells post completion of 22-24 h incubation. Wash cells with 1X-PBS (100 pl/ well). Aspirate. Add Ix Typsin EDTA (25 pl/well). Incubate at 37°C till cells detach (1-2 min) and add to each well 150 pl of 10% AFBS in PBS. Mix properly and transfer cells to 96 well round bottom plates. Centrifuge the contents of the plate at 1500 rpm for 5 min at Room Temperature (RT); Aspirate supernatant. Wash the cell pellet by adding lx PBS (100 pl/well). Do not mix. Centrifuge the contents of the plate at 1500 rpm for 5 minutes at RT. Aspirate supernatant. To the cell pellet add 4% Para-Formaldehyde (50 pl/well). Mix properly and incubate for 15-30 min at RT. Centrifuge the plates at 2500 rpm for 5 min at RT. Aspirate supernatant. Wash the cell pellet twice with lx PBS ( 100 pl/well). Plates can be stored at this stage for 2-3 days at 2-8°C. Using lOx Perm buffer, prepare lx Perm buffer in PBS. Centrifuge the plates at 2500 rpm for 5 min at RT. Aspirate supernatant. To the cell pellet add lx Perm Buffer (100 pl/ well). Do not mix. Centrifuge the plates at 2500 rpm for 5 min at RT. Aspirate supernatant. To each well add 50 pl blocking solution (1% Normal Mouse Serum in lx Perm buffer). Mix properly. Incubate the plate for 30 min at RT. Post-blocking, to each well add 25 pl of 4G2-alexa 488 mAb prepared in blocking buffer on top of the blocking solution at pre-optimized dilution. The optimum amount of 4G2-alexa 488 mAb was pre -determined to identify the saturating concentration of mAb required [1:400 diluted in blocking buffer used with the present batch of 4G2- Alexa 488], From here on the plates are processed in dark or in dim light. Incubate the plate in dark for 1 h at 37°C under gentle shaking. Centrifuge the plate at 2500 rpm for 5 min at RT; Aspirate supernatant. To the cell pellet add lx Perm buffer (100 pl/ well). Do not mix. Centrifuge the plate at 2500 rpm for 5 min at RT; Aspirate supernatant. To the cell pellet add lx Perm buffer (100 pl/ well). Do not mix. Centrifuge the plate at 2500rpm for 5 min at RT; Aspirate supernatant. Re-suspend the cells in lx PBS. 7. Read the plate (within 7 days of staining; plates can be covered with aluminum foil and stored at 2-8°C till then) using flow cytometer and count 5000 cells. Analyze the data using Flowjo and Graphpad prism softwares and calculate ICso which corresponds to the concentration of drug at which 50% inhibition (or 50% infection) of virus is observed.

[0188] The test results are summarized in Table-6 below:

Table-6: ICso values and graph of % inhibition obtained from in vitro anti-JEV FNT experiment:

*NA: No activity observed within the evaluated concentrations of 50 to 1.5625 pg/ml

[0189] Conclusion: Sinococuline and AQCH inhibited CHIKV in the PRNT experiment.

Example 12: Evaluation of the In vitro anti-Dengue activity of AQCH extract and drug formulations under stability:

[0190] The in vitro activity of AQCH extract and drug formulations was scored against all four serotypes of Dengue Virus (DENV-1-4) by Flow-cytometry based Neutralisation Test (FNT). Concentration of drug that caused 50% inhibition of the virus was calculated as 50% Inhibitory Concentration or ICso.

[0191] For evaluation of efficacy, test samples of AQCH Extract and AQCH tablet were used to evaluate activity of the test samples against Dengue Virus serotypes (DENV-1, -2, -3 and -4).

[0192] Concentration range of test samples was in the range of 50 to 1.5625 pg/ml.

[0193] Test results are provided in Table-7 and 8 as below:

Table 7: ICso values obtained from in vitro anti-Dengue FNT experiment with AQCH Extract under stability (24 Months at 30°C ± 2°C/ 60% ± 5% RH):

Table 8: ICso values obtained from in vitro anti-Dengue FNT experiment with AQCH Tablets under stability (18 Months at 30°C/ 65% RH):

[0194] Conclusion: AQCH extract and AQCH tablets of different strengths inhibited all four Dengue serotypes (DENV-1, -2, -3 and -4) post-storage at 30°C for 24 months and 18 Months, respectively in the in vitro FNT experiment.

[0195] While considerable emphasis has been placed herein on the specific extract of the ingredients of the Cocculus hirsutus and specifically isolated compounds in various preferred formulation and biological activity evaluation studies, 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 invention. These and other changes in the preferred extract and formulations of the 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 invention and not as a limitation.