TW202207960A - Composition and use of cocculus hirsutus in viral infections - Google Patents

Abstract

The present invention relates to a composite extract ofCocculus hirsutus, use in the prevention and treatment of infection caused by positive stranded RNA viruses and its pharmaceutical compositions. Further, it provides for a stable pharmaceutical composition comprising a therapeutically effective amount of the said extract for use in the prevention and/or treatment of virus infections in a mammal. It also provides for a method for reducing viral load, cytokine load and methods of improvement in signs and symptoms of virus infection by administering the composite extract or its pharmaceutical composition or particularly isolated compounds like Sinococuline, Magnoflorine, Makisterone A or 20-hydroxyecdysone or derivatives thereof to a mammal in need thereof. The present invention also provides various methods for reducing or inhibiting the proliferation of positive stranded RNA viruses in infected patients by administering a therapeutically effective amount of a composite extract ofCocculus hirsutus or compositions thereof.

Description

Hardwood tetragoni composition and its use in virus infection

The present invention relates to a composition and a pharmaceutical composition comprising a compound extract of Tetrachia sclera, for preventing and/or treating infection caused by positive single-stranded RNA virus in mammals. The present invention also provides various methods for reducing or inhibiting the proliferation of positive single-stranded RNA viruses in infected patients by administering a therapeutically effective amount of a complex extract of Tetrachia solani.

A forward single-stranded RNA virus is a virus that uses forward single-stranded RNA as its genetic material. Single-stranded RNA viruses are classified as positive or negative, depending on the orientation or polarity of the RNA. Forward viral RNA genomes act as messenger RNAs and can be translated into proteins in host cells. Positive single-stranded RNA viruses are classified into three orders—Reticuloviruses, Picornaviruses, and Turnip Mosaicviruses—and 33 families, 20 of which are not assigned to an order. A wide range of hosts can be infected with positive single-stranded RNA viruses, including bacteria, eukaryotic microorganisms, plants, invertebrates and vertebrates.

Positive-stranded RNA viruses cover one-third of all virus genera and include a large number of viral pathogens such as hepatitis C virus, dengue virus, Chikungunya virus, SARS and MERS virus, hCoV-OC43 virus; Japanese encephalitis virus (JEV) , Zika (Asian/Brazilian strains and African strains), Yellow Fever Virus (YFV), Usutu virus (Usutu, a growing concern in Europe), West Nile virus (WNV), tick-borne encephalitis Viruses (TBEV), coronaviruses and SARS-CoV-2, and other less clinically serious pathogens such as rhinoviruses, which cause the common cold.

The recent outbreak of coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 in December 2019, has caused global health concerns. In December 2019, some cases with unexplained lower respiratory tract infections were observed in Wuhan, China. The etiology of the disease is attributed to a novel virus belonging to the coronavirus (CoV) family, and the disease caused by this novel coronavirus is called COVID-19, short for "Coronavirus disease 2019" Character. This new virus has been found to be very contagious and has spread rapidly around the world. The WHO has declared the outbreak a Public Health Emergency of International Concern (PHEIC). Originally called 2019-CoV, the new virus was subsequently called SARS-CoV-2 (severe acute respiratory syndrome-associated coronavirus-2) by mission experts from the International Committee on Taxonomy of Viruses (ICTV). CoVs are large positive single-stranded RNA viruses of the family Coronaviridae, which can be isolated in different animal species. For reasons not yet explained, these viruses can cross species barriers and can cause diseases in humans ranging from the common cold to more severe conditions such as MERS and SARS. The elderly and those with primary disease are susceptible and prone to severe outcomes, which may be associated with acute respiratory distress syndrome (ARDS) and cytokine storm. (Guo et al., Origin, spread, and clinical treatment of the coronavirus disease-19 (Covid-19) outbreak—a status update, Mil Med Res, 2020 Mar 13;7(1):11; and Cascella et al., Coronavirus (COVID-19) Characteristics, Evaluation and Treatment, StatPearls, 20 May 2020). Based on the speed with which the COVID-19 outbreak has developed, SARS-CoV-2 appears to spread easily in the human population. Many health workers are already infected, and more clusters of cases are detected with each passing day. According to the World Health Organization's COVID-19 Situation Dashboard, as of 09 August 2020, there were 18,187,943 confirmed cases of SARS-CoV-2 infection and 716,075 deaths. World governments are committed to building countermeasures to stop potentially damaging effects. At the same time, scientists around the world are working tirelessly to understand transmission mechanisms, the clinical map of disease, and the development of new diagnostics and prevention and treatment strategies. Currently, treatment strategies to deal with infection are only supportive, and prevention aimed at reducing community transmission is considered the best weapon (Cacella et al., Coronavirus (COVID-19) Characterization, Evaluation and Treatment, StatPearls, May 2020 18th update).

Different antiviral drugs and systemic corticosteroid therapy have previously been commonly used in clinical trials, including neuraminidase inhibitors (oseltamivir, peramivir, zanamivir, etc.), ganciclovir, Ciclovir and ribavirin, as well as methyl penicillin cortisol for influenza virus, were found to be ineffective against COVID-19 and are not recommended. (Guo et al.). Some other antiviral drugs like remdesivir, lopinavir and ritonavir, and antimalarial drugs like chloroquine and hydroxychloroquine have also been studied for their effectiveness against COVID-19. This unprecedented rate of outbreak spread presents an important need for effective treatments to combat COVID-19 that can relieve symptoms, reduce the severity of common symptoms, prevent the development of serious complications, and be easy to prepare. In addition, there is an urgent need to develop a therapy against SARS-CoV-2 and all other similar positive single-stranded RNA viruses that can reduce viral load in the early stages and thus potentially prevent its infection and spread. There are also many pathogenic positive single-stranded RNA viruses that are arthropod-borne (called arboviruses)—that is, transmitted by a biting insect and capable of replicating in it, and then transferring the pathogen to an animal host. Recent environmental genomics studies have also identified a large number of RNA viruses whose host ranges are specific to insects that act as vectors for virus transmission.

Zika virus, also a mosquito-borne xanthovirus, was first identified in monkeys in Uganda in 1947. It was later identified in humans in 1952 in Uganda and the United Republic of Tanzania. The first documented outbreak of Zika virus disease was reported from Yap Island (Federated States of Micronesia) in 2007. A large-scale outbreak of Zika virus infection followed in 2013 in French Polynesia and other countries and territories in the Pacific. In October 2015, Brazil reported an intersection between a Zika virus infection and cerebellar disease. Evidence of outbreak and spread quickly emerged throughout the Americas, Africa, and other parts of the world. To date, a total of 86 countries and territories have reported evidence of mosquito-borne Zika infection. According to the World Health Organization's website, there are no specific treatments available for Zika virus infection or related diseases.

Therefore, there is a need for effective treatments or therapies that can be used to prevent and/or treat a wide range of viral infections.

The present invention provides a compound extract of Fangji hardwood or its medicinal composition for preventing and/or treating virus infection caused by positive single-stranded RNA virus in mammals.

In another aspect, the present invention also provides the treatment of positive single-stranded RNA virus ( Methods containing viral infections caused by the novel coronavirus (COVID-19).

In addition, there is also provided a stable pharmaceutical composition comprising a therapeutically effective amount of the extract for the prevention and treatment of infections caused by positive single-stranded RNA viruses (+ssRNA), such as hepatitis C, dengue virus ( All 4 serotypes DENV 1-4), Chikungunya virus (CHIKV), SARS and MERS, hCoV-OC43; Japanese encephalitis virus (JEV), Zika virus (Asian/Brazilian and African strains), yellow fever virus (YFV), Usutu virus (Usutu, a growing concern in Europe), West Nile virus (WNV), tick-borne encephalitis virus (TBEV), coronaviruses and, in mammals, SARS-CoV- 2 (COVID-19).

In yet another aspect, the present invention further provides a method for preventing and/or treating viral infection caused by positive single-stranded RNA virus in mammals using a complex extract of Tetrachia sclerata.

In yet another aspect, the present invention further provides for the prevention and/or treatment of mammals using individual chemical entities (eg, tetrandrine, sclerophylline, derived from a complex extract of tetrandrine sclera) A method for viral infection caused by a medium to positive single-stranded RNA virus.

The present invention also relates to processes for the preparation of these extracts and the isolation and enrichment of relevant therapeutic compounds. Furthermore, these extracts have been found to be safe for administration to humans and do not exhibit any toxic effects at the concentrations required to prevent and/or treat viral infection by positive single-stranded RNA viruses in mammals.

In some embodiments, the present invention provides the use of a composition for the treatment of viral infection caused by positive single-stranded RNA virus, the composition comprising: a therapeutically effective amount of a compound extract of Fangji sclera; and a medicine Scientifically acceptable excipients including diluents, binders, disintegrants, lubricants, slip agents, polymers, flavoring agents, surfactants, preservatives, antioxidants, buffers, tonicity modifiers , or a combination thereof.

In some embodiments, the present invention provides the use of a compound extract of Tetranychus serrata for preparing a medicament for viral infection caused by a positive single-stranded RNA virus.

In some embodiments, the present invention provides a composition comprising: a therapeutically effective amount of a complex extract of Tetrachia sclera; and a pharmaceutically acceptable excipient including a diluent, a binder, a disintegrant , lubricants, slip agents, polymers, flavoring agents, surfactants, preservatives, antioxidants, buffers, tonicity modifiers, or combinations thereof.

In some embodiments, the composite extract of Tetrachia serrata comprises about 50 wt % to about 80 wt % of the composition. In some embodiments, the composite extract of Tetrachia serrata comprises about 60 wt % to about 70 wt % of the composition.

In some embodiments, the complex extract of Tetrachia sclera includes tetrandrine, magnolidine, coniferyl alcohol, quercetin, rutin, syringaresinol diglucoside, marxosterone A, bristle hook Metaphylline, 20-hydroxyecdysone, derivatives thereof, or combinations thereof.

In some embodiments, the positive single-stranded RNA virus is hepatitis C virus, West Nile virus (WNV), Dengue virus, Chikungunya virus (CHIKV), Japanese encephalitis virus (JEV), Zika virus, yellow Fever virus (YFV), Usutu virus, tick-borne encephalitis virus (TBEV), coronavirus or a combination thereof. In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV or hCoV-OC43.

In some embodiments, the compound extract of Tetrachia serrata is an alcohol extract, a hydrated alcohol extract or an aqueous extract. In some embodiments, the complex extract of Tetrachia serrata includes water, ethanol, or a combination thereof. In some embodiments, the compound extract of Tetrachia serrata is an aqueous extract. In some embodiments, the extract includes alcohol to water in a ratio of about 1:99 to 99:1.

In some embodiments, the composite extract of Tetrachia serrata comprises from about 50 weight (wt) % to about 80 wt % of the composition. In some embodiments, the composite extract of Tetrachia serrata comprises about 60 wt % to about 70 wt % of the composition. In some embodiments, the complex extract of Tetrachia serrata includes from about 0.1 wt % to about 1 wt % magnolanine.

In some embodiments, the pharmaceutically acceptable excipients include magnesium aluminum trisilicate, lactose monohydrate, dicalcium phosphate, starch, calcium silicate, microcrystalline cellulose, colloidal silica, Croscarmellose, magnesium stearate, or a combination thereof. In some embodiments, the composition further includes a film coating, a solvent, or a combination thereof. In some embodiments, the composition or the medicament is an oral composition selected from powders, pellets, granules, spheres, mini-lozenges, caplets, lozenges, capsules, liquids, or combinations thereof.

In some embodiments, the composition or the medicament comprises at least three compounds selected from the group consisting of tetrandrine, magnolidine, marxosterone A, sclerophylline, and 20-hydroxyecdysone, wherein the at least three compounds About 0.1% to about 10% w/w of the composition or the agent. In some embodiments, the tetradine accounts for about 0.1 wt % to about 20 wt % of the composition or the agent; the magnolidine is about 0.01 wt % to about 5 wt %; the magsterone A is about 0.005 wt% to about 5 wt%; and the 20-hydroxyecdysone is about 0.01 wt% to about 5 wt%.

In some embodiments, the present invention provides a composition comprising: a composite extract of Tetranychus sclera; and an isolated compound selected from the group consisting of tetrandrine, magnolidine, macosterone A, bristle Unchristine, 20-hydroxyecdysone, a derivative thereof, or a combination thereof, wherein the ratio of the complex extract to the isolated compound is from about 99:1 to about 1:99.

In some embodiments, the isolated compound is tetrandine. In some embodiments, the isolated compound is magnolone. In some embodiments, the ratio of the composite extract to the isolated compound is from about 70:30 to about 30:70. In some embodiments, the ratio of the complex extract to the isolated compound is from about 40:60 to about 60:40. In some embodiments, the ratio of the complex extract to the isolated compound is about 1:1.

In some embodiments, the present invention provides a composition comprising: a composite extract of Fangji sclera; magnesium aluminum trisilicate; a diluent selected from lactose monohydrate, dicalcium phosphate, pregelatin starch, calcium silicate, or combinations thereof; microcrystalline cellulose; colloidal silica; croscarmellose; magnesium stearate; and an optional film coating.

In some embodiments, the composition includes from about 10 wt% to about 90 wt% of the composite extract of Tessyl sclera; from about 5 wt% to about 50 wt% of magnesium aluminum trisilicate; about 0.1 wt% to about 20 wt% diluent; about 0.1 wt% to about 10 wt% microcrystalline cellulose; about 0.1 wt% to about 10 wt% colloidal silica; about 0.1 wt% to about 10 wt% croscarmellose; and about 0.01 wt % to about 5 wt % magnesium stearate.

In some embodiments, the composition includes from about 30 wt% to about 85 wt% of a composite extract of Tessyl sclera; from about 8 wt% to about 40 wt% of magnesium aluminum trisilicate; about 0.5 wt% to about 15 wt% diluent; about 0.5 wt% to about 8 wt% microcrystalline cellulose; about 0.2 wt% to about 8 wt% colloidal silica; about 0.5 wt% to about 8 wt% croscarmellose; and about 0.02 wt % to about 2 wt % magnesium stearate.

In some embodiments, the present invention provides a composition comprising: about 50 wt % to about 80 wt % of a composite extract of Tessyl serrata; about 10 wt % to about 30 wt % magnesium aluminum trisilicic acid salt; about 1 wt% to about 10 wt% of a diluent selected from lactose monohydrate, dicalcium phosphate, pregelatinized starch, calcium silicate or combinations thereof; about 1 wt% to about 5 wt% of Microcrystalline cellulose; about 0.5 wt% to about 5 wt% colloidal silica; about 1 wt% to about 5 wt% croscarmellose; about 0.1 wt% to about 1 wt% hard Magnesium fatty acid; and optional film coating.

In some embodiments, the composition is an oral pharmaceutical composition. In some embodiments, the oral pharmaceutical composition is a powder, pellet, granule, sphere, mini-lozenge, caplet, lozenge, capsule, liquid, or a combination thereof. In some embodiments, the composite extract of Tetrachia serrata is a composite water extract, a composite primary alcohol extract, or a composite water/alcohol extract.

In some embodiments, the present invention provides a composition comprising at least three compounds selected from the group consisting of tetrandrine, magnolidine, maksterone A, sclerophylline, and 20-hydroxyecdysone, wherein the at least three compounds The compound comprises from about 0.1% to about 10% of the composition.

In some embodiments, the tetradine is about 0.1 wt% to about 20 wt% of the composition; the magnolin is about 0.01 wt% to about 5 wt%; the marquesterone A is about 0.005 wt% to about 5 wt%; and the 20-hydroxyecdysone is about 0.01 wt% to about 5 wt%.

In some embodiments, a composition of Tetrachia serrata complex extract is prepared, wherein the weight of the complex extract in the composition ranges from about 25 mg to 1500 mg. In some embodiments, a composition of the compound extract of Fangji hardwood is prepared, wherein the weight of the compound 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 single dosage form with the complex extract in any of the amounts listed above.

In some embodiments, the composite extract of Tetrachia serrata is substantially free of lignin, fiber, and tannins.

In some embodiments, the present invention provides a method for reducing or inhibiting the proliferation of a positive single-stranded RNA virus in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition as described herein. In some embodiments, the present invention provides a method for reducing the viral load of a positive single-stranded RNA virus in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition as described herein. In some embodiments, the composition is administered at a dose of about 1 mg/kg to about 500 mg/kg of the complex extract of Tetrachia solani by weight per subject.

In some embodiments, the present invention provides a method of treating a positive single-stranded RNA virus infection in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of a complex extract of Tetrachia serrata.

In some embodiments, the present invention provides a method of treating a positive single-stranded RNA virus infection in an individual in need thereof, the method comprising administering to the individual a complex extract comprising a therapeutically effective amount of Tetrachia solani and pharmaceutically acceptable excipients (including diluents, binders, disintegrants, lubricants, slip agents, polymers, flavoring agents, surfactants, preservatives, antioxidants, buffers, tonicity modifiers) agent, or a combination thereof).

In some embodiments, the present invention provides a method of treating a positive single-stranded RNA virus infection in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of tetrandrine, magnolidine , a composition of macosterone A, sclerophylline, 20-hydroxyecdysone, a derivative thereof, or a combination thereof. In some embodiments, the composition includes tetrandrine. In some embodiments, the composition includes magnolidine.

In some embodiments, the composition further includes pharmaceutically acceptable excipients including diluents, binders, disintegrants, lubricants, slip agents, polymers, flavoring agents, surfactants, Preservatives, antioxidants, buffers, tonicity modifiers, or combinations thereof. In some embodiments, the composite extract of Tetrachia serrata is substantially free of lignin, fiber, and tannins. In some embodiments, the composition is administered once daily, twice daily, three times daily, or four times daily. In some embodiments, the complex extract of Fangji sclera is administered once a day, twice a day, three times a day, or four times a day.

In some embodiments, the positive single-stranded RNA virus is hepatitis C virus, West Nile virus (WNV), Dengue virus, Chikungunya virus (CHIKV), Japanese encephalitis virus (JEV), Zika virus, yellow Fever virus (YFV), Usutu virus, tick-borne encephalitis virus (TBEV), coronavirus or a 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, Chikungunya virus, or Japanese encephalitis virus (JEV).

In some embodiments, the method further comprises co-administering to the individual an additional therapeutic agent selected from an antiviral agent, an antipyretic agent, an analgesic agent, or a combination thereof.

In some embodiments, the present invention provides a method of preparing a composite extract of T. sclera, comprising: a) using a solvent comprising ethanol, water, or both to extract the plant pieces of T. sclerosus, 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 at about 40°C to about Carry out at 95 DEG C, thereby obtain the compound extract of this Tetrachia serrata. In some embodiments, prior to (c), the method further comprises further extracting the concentrated extract of (b) with an additional solvent, wherein the additional solvent is ethanol, water, or both.

The present invention will be described in detail in conjunction with certain preferred and alternative embodiments so that its various aspects may be more fully understood and appreciated.

The inventors of the present invention have discovered that a complex extract of the plant known as Fangji hardwood has broad antiviral activity against positive single-stranded RNA viruses. In addition, the inventors also found that the composite extract interferes with the viral life cycle of normal-phase single-stranded RNA viruses. The inventors also found that the complex extract reduces the levels of TNF-α and IL-6 in mammals.

The complex extract can be used to prevent and/or treat different infectious diseases caused by positive single-stranded RNA viruses, such as hepatitis C virus, West Nile virus (WNV), dengue virus (all 4 serotypes DENV 1). -4), Chikungunya virus (CHIKV), SARS and MERS virus, hCoV-OC43 virus; Japanese encephalitis virus (JEV), Zika virus (Asian/Brazilian strain and African strain), yellow fever virus (YFV), Ukrainian Sutuvirus (Usutu, a growing concern in Europe), tick-borne encephalitis virus (TBEV), coronavirus and SARS-CoV-2 (COVID-19), or any other related positive virus in mammals Strand RNA virus.

The hardwood tree Linn (Teochondriaceae), commonly known as Jal-Jamni (Chopra et al., 1958) or Broom creeper, is found in tropical Asia and moderately cool and hot regions of Africa. It is a perennial climber and can reach 2 to 3 m above the ground. The present inventors have discovered extracts of Tetranychus serrata and isolated compounds of various tetrandrine (-) Syringaresinol diglucoside or 20-hydroxyecdysone and various combinations thereof can help effectively prevent and/or treat various infectious diseases caused by positive single-stranded RNA viruses including COVID-19, and reduce side effects . In one embodiment, the present invention provides a compound extract of Fangji sclera for preventing and/or treating infection caused by positive single-stranded RNA virus in mammals.

In another embodiment, the present invention provides a method of treating viral infection caused by a positive single-stranded RNA virus by administering a therapeutically effective amount of a complex extract of Tetranychus edulis.

In yet another embodiment, the present invention provides for reducing or inhibiting the proliferation of viral infections caused by positive single-stranded RNA viruses by administering to a mammal in need thereof a therapeutically effective amount of a complex extract of Tetrachia sclerata Methods.

In a further embodiment, the present invention provides the use of a complex extract of Tetranychus edulis for preventing and/or treating viral infection caused by positive single-stranded RNA virus in mammals.

In another embodiment, the present invention provides the use of a complex extract of Tetranychus edulis for reducing or inhibiting the proliferation of positive single-stranded RNA viruses in mammals.

In a further embodiment, the present invention also provides various synergistic composition compositions of compound extracts of Tetranychus serrata, tetrandrine, sclerophylline, quercetin, rutin, (-) syringin diglucoside, magnolidine, coniferyl alcohol, markasterone A or 20-hydroxyecdysone or derivatives thereof.

In a further embodiment, the present invention also provides methods of isolating various compounds from complex extracts of Tetranychus serrata and methods of treating or preventing viral infections in mammals caused by positive single-stranded RNA viruses use in.

中國木防己鹼              木蘭花鹼                   松柏醇

槲皮素                  芸香苷             丁香脂素雙葡萄糖苷

馬克甾酮A              20-羥基蛻皮酮                硬毛鈎藤鹼Compounds isolated from extracts of Tetranychus solani include tetrandrine, sclerophylline, magnolidine, coniferyl alcohol, quercetin, rutin, syringaresinol diglucoside, marxosterone A or 20 - Hydroxyecdysone or a derivative thereof. The structure of each of these compounds is provided below:

Chinese tetrandrine Magnolia alkaloid coniferyl alcohol

Quercetin Rutin Syringaresinol Diglucoside

Markosterone A 20-Hydroxyecdysone Sclerophylline

The term "composite extract" as used herein refers to an extract obtained at any concentration from T. hardwood, which includes a mixture of components and is available in the form of a liquid, semi-solid, solid, gel, paste, dispersion, solution , or in the form of distillate. Preferably, the complex extract is in solid form, such as a powder. The term "complex extract" is used interchangeably with "extract" in this specification.

In one aspect of the foregoing embodiments, the extract is an aqueous extract or an organic solvent extract, wherein the organic solvent is a polar or non-polar organic solvent.

In another aspect of the foregoing embodiments, the extract is an alcoholic or hydrated alcoholic extract from the trunk or other parts of the plant, such as shoots or roots. In one embodiment, the extract is an aqueous extract. By evaporation, the solvent in the extract can be completely removed to obtain a dry extract. The dried extract can be lyophilized to form a powder, which can then be filled into vials or capsules of suitable size or compressed into lozenges with or without pharmaceutically acceptable excipients. In related embodiments, the extract can be used in liquid or semi-solid form without further drying with a suitable pharmaceutically acceptable carrier for administration.

In another aspect of the foregoing embodiments, the extract is an alcoholic or hydrated alcoholic extract from the trunk or other parts of the plant, such as shoots or roots. In one of the preferred embodiments, the extract will be derived from the wet parts of the plant to form an aqueous extract. By evaporation, the solvent in the extract can be completely removed to obtain a dry extract. The dried extract can be lyophilized to form a powder, which can then be filled into vials or capsules of suitable size or compressed into lozenges with or without pharmaceutically acceptable excipients. In related embodiments, the extract can be used in liquid or semi-solid form without further drying with a suitable pharmaceutically acceptable carrier for administration.

The term "alcoholic extract" as used herein includes any alcohol-based extract, for example, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, or tert-butanol extraction of Tesco thing.

The term "hydrated alcohol extract" as used herein includes extracts prepared using a mixture of alcohol and pure water. It may also contain extracts prepared in denatured alcohol with other organic solvents. Examples of alcohols are methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol and tert-butanol. The ratio of alcohol to water in the hydrated alcohol extract may be the following ratio: 99:1 to 1:99, or 95:1 to 1:95, or 95:5 to 5:95, or 90:10 Alcohols and pure water mixture.

The term "aqueous extract" as used herein includes aqueous or pure water extract of Tessica serrata, also abbreviated as AQCH (Aqueous Extract of Tessica serrata).

The extract of Tessica serrata comprises (a) the extract obtained by extracting the plant pieces of Tessica serrata using one or more solvents, and (b) obtained by partitioning the extract of step (a) using one or more solvents part. In a preferred embodiment, the extract of T. sclera comprises (a) using pure water to extract a vegetative amount of T. sclerosus trunk to obtain the extract, and (b) using one or more solvents to pass through a partitioning step (a) Fraction obtained from the extract.

Solvents for extracts can be, for example, water; alcohols, such as methanol, ethanol, propanol, isopropanol, or butanol; ketones, such as acetone or methyl isobutyl ketone; esters, such as methyl acetate or ethyl acetate; halogenated hydrocarbons, such as chloroform, dichloromethane, or ethylene dichloride; petroleum fractions, such as hexane, petroleum ether, or heptane; or mixtures thereof.

Solvents used for distribution can be, for example, water; petroleum fractions, such as hexane, petroleum ether, or heptane; halogenated hydrocarbons, such as chloroform, dichloromethane, or ethylene dichloride; esters, such as ethyl acetate or Methyl acetate; ketones, such as acetone or methyl isobutyl ketone; alcohols, such as butanol; ethers, such as diethyl ether; or mixtures thereof.

As used herein, the term "plant pieces of Tessibuli" refers to a whole plant, which includes above-ground parts, eg, fruit, flowers, leaves, branches, trunk bark, trunk, seeds or heartwood, and roots. In a preferred embodiment, "the plant block of T. sclera" refers to the trunk of T. sclera. In one aspect, such plant pieces can be from wet or dry parts.

In yet another embodiment, the compound extract of Teochondrial sclera may include tetrandrine, magnolidine, coniferyl alcohol, marxosterone A or 20-hydroxyecdysone, uricine sclerophylline, quercetin rutin, (-) syringaresinol diglucoside or derivatives thereof. In a related embodiment, the complex extract of Tetrachia serrata can be used to isolate the compounds tetrandrine, magnolidine, maxosterone A or 20-hydroxyecdysone or derivatives thereof, as well as in the treatment or prevention of positive Use of a viral infection by a single-stranded RNA virus, or use thereof to inhibit viral proliferation in a mammal.

In a further preferred embodiment, the present invention provides an enriched fraction of a compound extract of Tetrachia sclerata for the treatment of viral infections caused by positive single-stranded RNA viruses in mammals. The extract can be enriched in any flavonoids, alkaloids, for example, tetrandrine or magnolidine; or other compounds such as sclerophylline, coniferyl alcohol, quercetin, rutin, (- ) syringaresinol diglucoside, markosterone A or 20-hydroxyecdysone or derivatives thereof. The extract can be enriched and normalized in terms of compounds such as tetrandrine, magnolone, macosterone A, sclerophylline, 20-hydroxyecdysone, or derivatives thereof. The enriched extract can be prepared using a solvent for the compound. The solvent can be any of the solvents previously discussed.

In the present invention, the inventors have found that the complex extract of Fangji hardwood possesses broad antiviral activity and has been found to interfere with the viral life cycle after it enters host cells. The complex extract was found to be effective against positive single-stranded RNA viruses. In addition, it was unexpectedly found that the complex extract also reduced TNF-α and IL-6 levels in mammals, thereby contributing to demonstrate its efficacy against severe cases of COVID-19.

In another embodiment, the present invention provides a complex extract of Tetranychus serrata for the treatment of positive single-stranded RNA viruses (selected from the group consisting of: hepatitis C, dengue virus (all) in mammals 4 serotypes (DENV 1-4), Chikungunya virus (CHIKV), SARS and MERS, hCoV-OC43) reduce viral load in the early stage of viral infection; Japanese encephalitis virus (JEV), Zika virus ( Asian/Brazilian strains and African strains), Yellow Fever Virus (YFV), Usutu virus (Usutu, a growing concern in Europe), West Nile virus (WNV), tick-borne encephalitis virus (TBEV), Coronavirus and SARS-CoV-2 (COVID-19).

In yet another embodiment, the present invention provides a pharmaceutical composition, which comprises the extract of Tetranychus sclera, or Tetrandrine, Magnoliaine, Markosterone A, Unchristine, 20-hydroxyl Ecdysone, or coniferyl alcohol, quercetin, rutin, (-) syringaresinol diglucoside, or a derivative or a combination thereof, wherein the composition exhibits resistance against a positive single-stranded RNA virus selected from the group consisting of Clusters: Hepatitis C, Yellow fever virus (YFV), Dengue virus (all 4 serotypes DENV 1-4), Chikungunya virus (CHIKV), SARS and MERS, Inhibitory activity of hCoV-OC43; Japanese encephalitis virus ( JEV), Zika virus (Asian/Brazilian strains and African strains), Usutu virus (Usutu, growing concerns in Europe), West Nile virus (WNV), tick-borne encephalitis virus (TBEV), Coronavirus and SARS-CoV-2 (COVID-19).

The complex extract obtained according to the present invention can be used for direct administration to mammals and for the preparation of pharmaceutical compositions in a dosage range of about 0.05 mg/kg to about 1500 mg/kg of body weight, especially in a range of about 0.1 mg/kg of body weight. kg to about 1200 mg/kg, a more specific range is 1 mg/kg to about 500 mg/kg body weight, a more specific range is 2 mg/kg to about 150 mg/kg body weight. The complex extract or composition thereof may be administered once, twice, three times, or four times daily.

In further related embodiments, the composite extracts of the present invention can be used to further isolate or enrich the aggregated compounds or derivatives for methods of preventing and/or treating viral infections caused by positive single-stranded RNA viruses.

In yet another related embodiment, the present invention provides methods for treating or reducing the proliferation of viral infections caused by positive single-stranded RNA viruses by administering to a mammal a suitable pharmaceutical dosage, including an effective amount of a or more compounds selected from the group consisting of tetrandrine, magnolidine, marxosterone A, sclerophylline, or 20-hydroxyecdysone, or combinations or derivatives thereof.

In another embodiment, the present invention provides a complex extract of Fangji hardwood to reduce viral load, exhibit platelet protection, inhibit viral proliferation in infected mammals, and other related mechanisms in the treatment of early stages of viral infection , thereby promoting the recovery of patients affected by infections caused by positive single-stranded RNA viruses.

In yet another preferred embodiment, the present invention provides use with or without one or more selected from the group consisting of tetrandrine, magnolanine, maxosterone A, sclerophylline, 20-hydroxyecdysone or derivatives thereof A method for treating and/or preventing a viral infection by a compound extract of the compound of T. solani. This may lead to a synergistic effect against viral infections caused by positive single-stranded RNA viruses, thereby enhancing the proliferative inhibitory power of the formulation (of the complex extract) or virotherapy.

In some embodiments, the present invention provides a composition comprising: a composite extract of Tetrachia sclera; and an isolated compound selected from the group consisting of tetrandrine, magnolidine, macosterone A, H. bristle Metaphylline, 20-hydroxyecdysone, a derivative thereof, or a combination thereof, wherein the ratio of the complex extract to the isolated compound is from about 99:1 to about 1:99.

As used herein, an "isolated compound" is essentially a purified compound isolated from a naturally-occurring source, eg, the Tetrachia serrata, or synthetic. In some embodiments, the isolated compound is substantially purified compound comprising less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% impurities.

In another embodiment, the present invention provides for the treatment and/or prevention of infection by a positive single-stranded RNA virus by administering a suitable composition dosage form of a complex extract of Tetranychus serrata and an isolated compound from a plant species. method of viral infection.

In yet another embodiment, the present invention provides a suitable composition dosage form of a complex extract of Tetrachia solani with an isolated compound from a plant species for the prevention and/or treatment of infection by a positive single-stranded RNA virus. Uses caused by viral infections.

In a related embodiment, when the composition is a composition of a composite extract of the isolated compound and Tetrachia serrata, 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 foregoing embodiments, the isolated compound is selected from the group consisting of tetrandrine, magnolidine, marxosterone A, sclerophylline, 20-hydroxyecdysone, or derivatives thereof. In some embodiments, the isolated compound is tetrandine. In some embodiments, the isolated compound is magnolone.

In some embodiments, the complex extract exhibits a therapeutic effect at a concentration of about 0.1 to about 500 μg/mL when administered. In some embodiments, the complex extract exhibits a therapeutic effect at a composition concentration of about 0.1 to about 200 μg/mL. In some embodiments, the complex extract exhibits a therapeutic effect at a composition concentration of about 1 to about 100 μg/mL. In some embodiments, the isolated compound exhibits a therapeutic effect at a composition concentration of from about 0.01 to about 100 μg/mL. In some embodiments, the isolated compound exhibits a therapeutic effect at a composition concentration of from about 0.01 to about 50 μg/mL. In some embodiments, the isolated compound is present at a concentration of about 0.01 to 20 μg/mL of the composition.

The term "combination" herein refers to a complex extract of Teochondrial sclera combined with a compound selected from the group consisting of tetrandrine, rhynchophylline, magnolin, coniferyl alcohol, quercetin, rutin, syringaresinol diglucoside, A mixture or combination of one or more isolated compounds of Markosterone A or 20-hydroxyecdysone or derivatives thereof. Furthermore, the term "combination" can also encompass administration as a single component or simultaneous, sequential or co-administration of different components.

The term "mammal" herein refers to all mammals including humans. Mammals include, by way of some non-limiting examples, humans, non-human primates, bovines, dogs, cats, goats, sheep, pigs, rats, mice, and rabbits.

In another embodiment, the present invention provides a pharmaceutical composition for preventing and/or treating viral infection caused by positive single-stranded RNA virus in mammals, comprising: a compound extract of Tetrachia sclera and one or Various pharmaceutically acceptable excipients. In one aspect of the foregoing embodiments, the present invention provides a stable pharmaceutical composition comprising: a therapeutically effective amount of a compound extract of Tetrachia sclerata, for preventing and/or treating viral infections in mammals , wherein the composition reduces viral load and/or inhibits viral proliferation when administered to a mammal in need thereof.

The stable pharmaceutical compositions of the present invention further comprise one or more pharmaceutically acceptable excipients. As used herein, the term "pharmaceutical composition" includes any composition effective to deliver an extract of Tetrachia serrata to the desired site of action, or an isolated compound or mixture thereof, for the treatment or prevention of infection caused by a positive single-stranded RNA virus Viral infection. The compositions can be delivered by any suitable route of administration, eg, oral, nasal, parenteral, pulmonary, transdermal, or rectal. The pharmaceutical composition includes one or more pharmaceutically acceptable excipients. The oral pharmaceutical compositions can be in the form of powders, pellets, granules, spheres, mini-lozenges, caplets, lozenges, or capsules. The powder can be filled into capsules of suitable size in the form of a freeze-dried powder together with a pharmaceutically acceptable excipient. Preferably, the pharmaceutical composition is in the form of a lozenge. The oral pharmaceutical composition may be presented in liquid form including, but not limited to, solutions, suspensions, emulsions or syrups. In accordance with the present invention, parenteral compositions may be suitable for administration routes such as, but not limited to, intravenous, subcutaneous, intramuscular, intraperitoneal, intrathecal, intravitreal, and the like.

The term "stable pharmaceutical composition" as used herein refers to a composition that is stable over long-term storage, as described in standard textbooks, as assessed from the content of one or more impurities in the composition. The stable pharmaceutical compositions of the present invention were found to be stable for at least 3 months under accelerated conditions of stability testing, i.e., 40 ± 2°C temperature and 75 ± 5% relative humidity (RH), and at 30 ± 2°C Long term storage stability at 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 despite the presence of flavonoids, alkaloids, resin brains, etc. as components in the aqueous extract, which together may be difficult to formulate and may not be stable during storage.

In some embodiments, the present invention provides a composition comprising: a therapeutically effective amount of a complex extract of Tetrachia sclera; and a pharmaceutically acceptable excipient including a diluent, a binder, a disintegrant , lubricants, slip agents, polymers, flavoring agents, surfactants, preservatives, antioxidants, buffers, tonicity modifiers, or combinations thereof.

In some embodiments, the composite extract of Tetrachia serrata comprises from about 20 wt % to about 95 wt % of the composition. In some embodiments, the composite extract of Tetrachia serrata comprises about 30 wt % to about 90 wt % of the composition. In some embodiments, the composite extract of Tetrachia serrata comprises about 40 wt % to about 85 wt % of the composition. In some embodiments, the composite extract of Tetrachia serrata comprises about 50 wt % to about 80 wt % of the composition. In some embodiments, the composite extract of Tetrachia serrata comprises about 60 wt % to about 70 wt % of the composition.

In some embodiments, the complex extract of Tetrachia sclera includes tetrandrine, magnolidine, coniferyl alcohol, quercetin, rutin, syringaresinol diglucoside, marxosterone A, bristle hook Metaphylline, 20-hydroxyecdysone, derivatives thereof, or combinations thereof.

In some embodiments, the complex extract of Teochondrial sclera includes from about 0.01 wt% to about 5 wt% of tetrandrine, magnolidine, coniferyl alcohol, quercetin, rutin, syringaresinol diglucose each of glycosides, maxosterone A, sclerophylline, and 20-hydroxyecdysone. In some embodiments, the complex extract of Tetrachia serrata includes from about 0.01 wt % to about 5 wt % magnolanine. In some embodiments, the complex extract of Tetrachia serrata includes from about 0.1 wt % to about 1 wt % magnolanine. In some embodiments, the complex extract of Tetrachia serrata includes about 0.29 wt % to about 0.43 wt % magnolanine.

The term "therapeutically effective amount" as used herein refers to an amount of the extract of the present invention sufficient to provide the benefit of treating or preventing viral infection caused by a positive single-stranded RNA virus, to delay or minimize symptoms associated with viral infection, or to cure or amelioration of infection or its etiology. In particular, a therapeutically effective amount refers to an amount sufficient to provide a therapeutic benefit in vivo . The term "pharmaceutically acceptable excipient" as used herein may include diluents, binders, disintegrants, lubricants, slip agents, polymers, flavoring agents, surfactants, preservatives, antioxidants, Buffers and tonicity modifiers.

Non-limiting examples of diluents include microcrystalline cellulose, powdered cellulose, starch, pregelatinized starch, glucose binders, lactitol, fructose, compressed sugar, powdered sugar, dextrose, anhydrous lactose, disalts Basic calcium phosphate, tribasic calcium phosphate, calcium sulfate and mixtures thereof.

Non-limiting examples of binders include water-soluble starches, such as pregelatinized starch; polysaccharides, such as agar, acacia, dextrin, sodium alginate, vetch gum, sanxian gum, hyaluronic acid, pectin, or sodium chondroitin sulfate ; Synthetic polymers such as polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymers, polyacrylic acid series polymers, polylactic acid, or polyethylene glycol; cellulose ethers such as methylcellulose, ethylcellulose, Carboxymethylcellulose, hydroxyethylcellulose, hypromellose, or hypromellose; and mixtures thereof.

Non-limiting examples of disintegrants include calcium carbonate, carboxymethyl cellulose, or salts thereof, such as croscarmellose sodium, crospovidone, low-substituted hydroxypropyl cellulose, and sodium starch glycolate .

Non-limiting examples of lubricants/slip agents include talc, magnesium stearate, hydrogenated vegetable oils, sodium fumarate, calcium stearate, colloidal silica, Aerosil®, stearic acid, sodium lauryl sulfate, Sodium benzoate, polyethylene glycol, hydrogenated castor oil, sucrose fatty acid esters, microcrystalline wax, yellow beeswax, white beeswax and mixtures thereof.

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

Non-limiting examples of surfactants include anionic surfactants, eg, sulfonic acids or salts thereof (eg, benzenesulfonic acid, dodecylbenzenesulfonic acid, or dodecylsulfonic acid); alkyl sulfates, For example, sodium dodecyl sulfate or sodium lauryl sulfate; cationic surfactants, such as quaternary ammonium salts (such as tetraalkylamine halides, benzalkonium chloride, benzalkonium chloride, or cetyl pyridine chloride) ; nonionic surfactants, eg, polyoxyethylene sorbitan long-chain fatty acid esters such as polyoxyethylene sorbitan laurate, eg, polysorbates; amphoteric surfactants, eg, glycine compounds For example dodecylbis(aminoethyl)glycine, betaine compounds such as betaine or dimethyldodecylcarboxybetaine and phosphatidic acid derivatives such as lecithin; polymeric surfactants such as Polyoxyethylene polyoxypropylene glycols such as Pluronic® or poloxamers; and mixtures thereof.

Non-limiting examples of buffers include phosphate buffers such as sodium dihydrogen phosphate, citrate buffers such as sodium citrate, meglumine, tris(hydroxymethyl)aminomethane, and mixtures thereof.

Non-limiting examples of tonicity modifiers include sodium chloride, mannitol, dextrose, glucose, lactose, sucrose, calcium chloride, magnesium chloride, potassium chloride, other inorganic salts, urea, glycerol, glycerol, wood Sugar alcohol, fructose, mannose, maltitol, inositol or fucose or mixtures thereof.

Non-limiting examples of solvents used to prepare pharmaceutical compositions include water; water-miscible organic solvents such as isopropanol or ethanol; dipolar aprotic solvents; chlorinated methane; acetone; polyethylene glycol; Glycol ethers; polyethylene glycol derivatives of mono- or diglycerides; buffers; organic solvents; and combinations thereof.

In a preferred embodiment, the pharmaceutically acceptable excipients in the composition of the present invention comprise microcrystalline cellulose, anhydrous lactose, croscarmellose sodium salt, colloidal silica and stearic acid magnesium. In some embodiments, the pharmaceutically acceptable excipients include magnesium aluminum trisilicate, lactose monohydrate, dicalcium phosphate, starch, calcium silicate, microcrystalline cellulose, colloidal silica, Croscarmellose, magnesium stearate, or a combination thereof.

In some embodiments, the compositions provided herein further comprise a film coating, a solvent, or a combination thereof. In some embodiments, the film coating includes a polymer, a plasticizer, a colorant, an opacifying agent, a solvent, a vehicle, or a combination thereof. Non-limiting examples of film coatings include those commercially available under the trade names Opadry®, Opalux®, Acryl-Eze®, Sureteric®, Surelease® and Ethocel™. Film coatings are further described in, eg, Zaid, AN, Drug Des Devel Ther 14:4613-4623 (2020). In some embodiments, the solvent includes water, alcohol, glycerol, ketone, oil, cyclodextrin, or a combination thereof. In some embodiments, the composition includes an Opadry® film coat and water.

In some embodiments, the present invention provides a composition comprising: a composite extract of Fangji sclera; magnesium aluminum trisilicate; a diluent selected from lactose monohydrate, dicalcium phosphate, pregelatin starch, calcium silicate, or combinations thereof; microcrystalline cellulose; colloidal silica; croscarmellose; magnesium stearate; and an optional film coating.

In some embodiments, the composite extract of Tetrachia serrata comprises about 5 wt % to about 95 wt %, or about 10 wt % to about 90 wt %, or about 15 wt % to about 85 wt % of the composition , or about 20 wt% to about 90 wt%, or 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%. In some embodiments, the complex extract of Tetrachia serrata comprises about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 35%, about 35%, by weight of the composition. 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%.

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

In some embodiments, the diluent comprises 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% of the composition From about 15 wt%, or from about 1 wt% to about 10 wt%, or from about 2 wt% to about 8 wt%. 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% by weight of the composition %, about 1%, about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 18%, or about 20%.

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

In some embodiments, the colloidal silica comprises 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 % of the composition wt % to about 8 wt %, or about 1 wt % to about 5 wt %, or about 1.5 wt % to about 4 wt %. In some embodiments, the colloidal silica comprises 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 0.8%, about 0.8%, by weight of the composition. About 0.9%, about 1%, about 2%, about 5%, about 8%, about 10%, about 12%, about 15%, about 18%, or about 20%.

In some embodiments, the croscarmellose comprises 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%. In some embodiments, the croscarmellose comprises by weight 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%, about 20%.

In some embodiments, the magnesium stearate comprises 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% of the composition wt % to about 1 wt %, or about 0.1 wt % to about 1 wt %, or about 0.2 wt % to about 0.5 wt %. In some embodiments, the colloidal silica comprises by weight 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%.

In some embodiments, the composition includes from about 10 wt% to about 90 wt% of the composite extract of Tessyl sclera; from about 5 wt% to about 50 wt% of magnesium aluminum trisilicate; about 0.1 wt% to about 20 wt% diluent; about 0.1 wt% to about 10 wt% microcrystalline cellulose; about 0.1 wt% to about 10 wt% colloidal silica; about 0.1 wt% to about 10 wt% croscarmellose; and about 0.01 wt % to about 5 wt % magnesium stearate. In some embodiments, the composition further comprises a film coating.

In some embodiments, the composition includes from about 30 wt% to about 85 wt% of a composite extract of Tessyl sclera; from about 8 wt% to about 40 wt% of magnesium aluminum trisilicate; about 0.5 wt% to about 15 wt% diluent; about 0.5 wt% to about 8 wt% microcrystalline cellulose; about 0.2 wt% to about 8 wt% colloidal silica; about 0.5 wt% to about 8 wt% croscarmellose; and about 0.02 wt % to about 2 wt % magnesium stearate. In some embodiments, the composition further comprises a film coating.

In some embodiments, the present invention provides a composition comprising: about 50 wt % to about 80 wt % of a composite extract of Tessyl serrata; about 10 wt % to about 30 wt % magnesium aluminum trisilicic acid salt; about 1 wt% to about 10 wt% of a diluent selected from lactose monohydrate, dicalcium phosphate, pregelatinized starch, calcium silicate or combinations thereof; about 1 wt% to about 5 wt% of Microcrystalline cellulose; about 0.5 wt% to about 5 wt% colloidal silica; about 1 wt% to about 5 wt% croscarmellose; about 0.1 wt% to about 1 wt% hard Magnesium fatty acid; and optional film coating.

In another embodiment, the present invention provides a stable pharmaceutical composition comprising: a therapeutically effective amount of a compound extract of Tetrachia sclerata, for the treatment of rheumatoid arthritis caused by a positive single-stranded RNA virus in mammals Viral infections wherein viral load or viral proliferation is reduced when the composition is administered to a mammal in need thereof.

In a further embodiment, the present invention provides a pharmaceutical composition comprising: a complex extract of Tetrachia sclera and one or more pharmaceutically acceptable excipients, for use in mammals in the treatment of positive Single-stranded RNA viruses reduce viral load in the early stages of infection. Preferably, the composition is a stable pharmaceutical composition. More preferably, the composition is a stable oral pharmaceutical composition.

In a further embodiment, the present invention provides an oral composition comprising: a complex extract of Tetranychus edulis, wherein the composition exhibits inhibitory activity with _IC50 values ranging from about 1 to 100 μg/ml , as determined by viral inhibition assays based on plaque and flow cytometry.

In a further embodiment, the present invention provides an oral composition comprising: an aqueous co-extract of Tetrachia solani, wherein the composition exhibits an _IC50 value range of about 4-12 μg/ml, preferably about 5-11 μg/ml, more preferably about 6-10 μg/ml.

In a further embodiment, the present invention provides an oral composition comprising: an aqueous co-extract of Tetrachia solani, wherein the composition exhibits an _IC50 value range of about 40 against Chikungunya virus (CHIKV) -70 μg/ml, preferably about 45-65 μg/ml, more preferably about 50-60 μg/ml.

In a further embodiment, the present invention provides an oral composition comprising: tetrandrine, wherein the composition exhibits inhibitory activity with _IC50 values ranging from about 0.1-3 μg/ml. In one aspect, Chinese tetrandrine was found to have an _IC50 range of 0.1-2 μg/ml against dengue virus (DENV 1-4). In one aspect, Chinese tetrandrine was found to have an _IC50 range of 0.5-15 μg/ml, preferably 1-10 μg/ml, more preferably 1-5 μg/ml against Chikungunya virus (CHIKV). In another aspect, tetrandrine was found to have an _IC50 against Japanese encephalitis virus (JEV) in the range of about 0.1-3 μg/ml, preferably about 0.2-2 μg/ml, more preferably about 0.2-1 μg/ml.

The extracts and various compositions contemplated by the present invention have also been found to be effective in inhibiting the secretion of cytokines in many other viral infections. Pro-inflammatory cytokines like TNF-[alpha] and IFN-[gamma] are produced in large quantities, which contribute to disease progression. In in vivo studies, the extract and the composition of the present invention inhibited the secretion of cytokines in the small intestine. Mice induced with viral immune complexes (ICs) and fed 100 mg/kg/day of the extract were found to show lower amounts of TNFα and IL-6 compared to groups that did not receive any treatment. The extract-treated group secreted less cytokines confirming the ability of the extract to inhibit viral proliferation. Compared to virus-only controls, IC-induced mice without any extract treatment showed higher cytokine levels due to enhanced infection by ADE. Mechanistic studies have shown that the extract targets the initial stage, after the virus has entered the cell. Addition time and removal time study assays showed that the extract interfered as early as 3-4 hours before the virus entered the host cell. The present inventors also tested the antiviral activity of Chinese tetrandrine and found that Chinese tetrandrine showed antiviral activity by interfering with the post-entry step. Further time-elimination studies using Chinese tetrandrine also showed that it prevented the early steps of viral replication.

In yet another embodiment, the present invention provides a stable pharmaceutical composition, comprising: a therapeutically effective amount of a compound extract of Tetrachia sclerata, for the treatment of rheumatoid arthritis caused by a positive single-stranded RNA virus in mammals Viral infections in which the composition is effective for delayed onset of treatment when administered to a mammal in need thereof.

In a further embodiment, the present invention provides a stable pharmaceutical composition comprising: a therapeutically effective amount of a complex extract of Tetrachia sclerata, for the prevention and/or treatment of positive single-stranded RNA in mammals Viral infection. When tested in an in vivo study in an animal model of infection with dengue virus, the extracts and compositions of the present invention were found to show protective efficacy against secondary infection when administered prior to infection.

In one embodiment, the extract of T. similis includes one or more components selected from the group consisting of flavonoids, resinous alkaloids, and alkaloids or combinations thereof. In one aspect, the complex extract of Tetrachia serrata includes magnolanine as one of the alkaloids. In a further aspect, the complex extract of Tetrachia sclera includes magnolidin in an amount ranging from 0.1% to 1% by weight of the total extract in the composition. In yet another aspect, the complex extract of Tetrachia serrata includes magnolidin in an amount ranging from 0.29% to 0.43% by weight of the total extract in the composition.

In another aspect of this embodiment, the complex extract of Tetranychus serrata includes quercetin as one of the flavonoids. In another aspect of this embodiment, the complex extract of Tetranychus serrata includes tetrandrine as one of the flavonoids.

In some embodiments, the composite extracts of Tetrachia solani provided herein are substantially free of lignin, fibers, and tannins. In some embodiments, the composite extract of Fangji hardwood 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, fibers and tannins.

In some embodiments, the present invention provides a composition comprising at least three compounds selected from the group consisting of tetrandrine, magnolone, maksterone A, sclerophylline, and 20-hydroxyecdysone, wherein the at least The three compounds comprise from about 0.05% to about 10% of the composition, or from about 0.1% to about 5% of the composition. In some embodiments, the composition is substantially free of lignin, fibers and tannins.

In some embodiments, the tetradine comprises 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 0.5% by weight of the composition 15%, or about 1% to 10%. In some embodiments, the magnolidine comprises 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% by weight of the composition %, or from about 0.05% to about 3%, or from about 1% to about 2%.

In some embodiments, the Markosterone A comprises 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 0.02% by weight of the composition 2%. In some embodiments, the 2-hydroxyecdysone comprises 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 8% by weight of the composition About 5%, or about 0.05% to about 3%, or about 1% to about 2%.

In some embodiments, the composition includes about 0.1 wt% to about 20 wt% tetrandrine; about 0.01 wt% to about 5 wt% magnolidine; about 0.005 wt% to about 5 wt% Markus sterone A; and from about 0.01 wt % to about 5 wt % of 20-hydroxyecdysone. In some embodiments, the composition comprises about 0.5 wt% to about 15 wt% tetrandrine; about 0.05 wt% to about 3 wt% magnolidine; about 0.01 wt% to about 3 wt% Markus sterone A; and from about 0.05 wt% to about 3 wt% of 20-hydroxyecdysone.

In further examples, the extracts and compositions of the present invention were found to be safe and did not exhibit any toxic effects when administered in therapeutically effective doses to mammals in need thereof. NOAELs in Wistar rats were established at 300 mg/kg/day following oral gavage in Wistar rats at relevant oral doses of 100, 300, 600, and 1200 mg/kg/day. . Therefore, the extract was found to be safe for human use without any toxicity.

In one embodiment, the present invention provides a method of treating a positive single-stranded RNA virus infection in a mammal, comprising administering to a mammal in need thereof a complex extract comprising a therapeutically effective amount of Tetrachia solani The pharmaceutical composition, wherein the extract reduces viral load or viral proliferation or virus.

In yet another embodiment, the present invention provides a method for reducing viral load in an early stage of treatment of a viral infection in a mammal, comprising administering to a mammal in need thereof a complex extract of Tesco sclera .

In yet another embodiment, the present invention provides a method for preventing and/or treating a viral infection by positive single-stranded RNA in a mammal, comprising administering to a mammal in need thereof a hardwood tree A complex extract of fenugreek.

In a further embodiment, the present invention provides a method of inhibiting cytokine secretion in a mammal that is infected with a single-stranded RNA virus, comprising administering to a mammal in need thereof a complex extract of Tetranychus serrata.

In yet another embodiment, the present invention provides a method of preventing and/or treating a positive single-stranded RNA virus infection in a mammal, comprising administering to a mammal in need thereof a complex extract of Tesco sclera, wherein the The extract is effective for delayed onset of treatment.

In yet another embodiment, the present invention provides a method of preventing infection by a positive single-stranded RNA virus in a mammal comprising administering to a mammal in need thereof a complex extract of Tetrachia solani.

In a further embodiment, the invention provides the use of a complex extract of Tetranychus serrata for treating or preventing viral infection caused by positive single-stranded RNA virus or for reducing or inhibiting viral proliferation by administering A compound extract or a medicinal composition thereof with a therapeutically effective amount of Fangji sclera.

In a further embodiment, the present invention provides compounds such as tetrandrine, magnolidine, marxosterone A, sclerophylline, 20-hydroxyecdysone, or its Derivatives or combinations thereof) in the method for treating or preventing viral infection caused by positive single-stranded RNA viruses or for reducing or inhibiting viral proliferation, by administering a therapeutically effective amount of T. sclera of this compound or its pharmaceutical composition.

In yet another embodiment, the complex extracts of the present invention were found to be better and superior to other plant-based extracts in treating viral infections, especially the use of cinnabar vine extracts to treat dengue virus infections.

In some embodiments, the extract can be prepared by procedures known in the art. In some embodiments, the present invention provides a method of preparing a composite extract of T. sclera, comprising: a) using a solvent comprising ethanol, water, or both to extract the plant pieces of T. sclerosus, 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 at about 40°C to about Carry out at 95 DEG C, thereby obtain the compound extract of this Tetrachia serrata. In some embodiments, prior to (c), the method further comprises further extracting the concentrated extract of (b) with an additional solvent, wherein the additional solvent is ethanol, water, or both. In some embodiments, the additional solvent is ethanol. In some embodiments, the additional solvent is water. In some embodiments, the additional solvent comprises ethanol and water in 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 A combination of 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.

In some embodiments, the process includes extracting the plant pieces of T. sclera with one or more solvents and drying the extract, or extracting the plant pieces of T. sclera with one or more solvents, concentrating the extract, Water is added and the extract is partitioned with one or more solvents, and the extract is dried, or the plant pieces of T. sclera are extracted with one or more solvents, the extract is concentrated, the extract is extracted with one or more solvents, and The extract was dried. In another aspect of the foregoing embodiments, the extraction of the plant pieces of Tetrachia serrata is performed at a temperature ranging from about 50 ºC to about 100 ºC. In another aspect of the foregoing embodiments, the extraction of the plant pieces of Tetrachia serrata is performed at a temperature ranging from about 80°C to about 85°C. In another aspect of the foregoing embodiments, the extraction of the plant pieces of Tetrachia serrata is performed at a temperature ranging from about 60°C to about 65°C. In another aspect of the foregoing embodiment, the drying of the extract of Tetrachia serrata is performed at a temperature ranging from about 40°C to about 95°C. In another aspect of the foregoing embodiments, the drying of the extract of Tetrachia serrata is performed at a temperature ranging from about 40°C to about 45°C. In another aspect of the foregoing embodiments, the drying of the Tetrachia sclera extract is carried out at a temperature ranging from about 45°C to about 50°C. In another aspect of the foregoing embodiments, the drying of the Tetrachia serrata extract is carried out at a temperature ranging from about 55°C to about 65°C. In another aspect of the foregoing embodiments, the drying of the Tetrachia sclera extract is carried out at a temperature ranging from about 90°C to about 95°C. In yet another aspect, the plant pieces can be extracted from dry or wet parts of the plant.

In yet another embodiment, the present invention provides a process for preparing a lozenge composition according to the composite extract of the present invention for the treatment of viral infection caused by a positive single-stranded RNA virus, the process comprising the following steps: (i) sieving the extract and blending with a pharmaceutically acceptable excipient; (ii) lubricating the blend obtained from step (i) and compressing it into a lozenge, and (iii) coating the tablet of step (ii).

In yet another embodiment, the present invention provides a process for preparing a lozenge composition according to the composite extract of the present invention for the treatment of viral infection caused by a positive single-stranded RNA virus, the process comprising the following steps: (i) blending the extract with a pharmaceutically acceptable excipient; (ii) granulating the blend of step (i) with a solvent; (iii) lubricating and compressing the blend into a lozenge, and (iv) coating the tablet of step (iii).

In a further embodiment, the present invention provides a process for preparing a lozenge composition from a compound extract for the treatment of viral infection caused by a positive single-stranded RNA virus, the process comprising the following steps: (i) blending the extract with a pharmaceutically acceptable excipient and compacting the mixture; (ii) grinding the compacted body and mixing using a superstructure excipient; (iii) lubricating the blend of step (ii) and compressing it into a lozenge, and (iv) coating the tablet of step (iii).

Although the above examples relate to lozenge compositions, the compound extract of Tetrachia serrata can also be formulated into any other suitable oral dosage form, such as powders, pellets, granules, spheres, mini lozenges, caplets agent.

In further related embodiments, the extract may be co-administered simultaneously or sequentially with one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent includes an antiviral agent, an antipyretic agent, an analgesic agent, or a combination thereof. In another embodiment, the compositions of the present 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, eg, which may provide symptomatic relief from conditions, such as antipyretics and analgesics.

The term "co-administration" herein refers to the administration of one or more additional therapeutic agents and extracts to a mammal. The extract and additional therapeutic agent can be a single pharmaceutical composition, or can be separate pharmaceutical compositions. Each of the extract or additional therapeutic agents can be administered simultaneously or sequentially by the same or different routes of administration. Example

The following examples include exemplary embodiments only, and will serve to illustrate the practice of the invention. It will be apparent to those skilled in the art that the present 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 its essential attributes, and it is therefore intended that the present embodiments and examples be used in all Aspects are considered to be illustrative and non-limiting. Example 1: Preparation of 95:5 Ethanol:Pure Water Tetrachia serrata extract

This hardwood tetrakis plant piece (1 kg) was loaded into the 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 extract mass was filtered, collected, and stored in a container. The extraction and filtration steps were repeated twice with a mixture of ethanol and pure water (95:5; 3 L). The three filtered extracts were combined and concentrated to the maximum possible extent under reduced pressure at low temperature. The extract was poured into stainless steel trays and dried under vacuum at 45-50 ºC until the ethanol content did not exceed 10,000 ppm and the water content did not exceed 5%. Cool the dry extract to about 20-25 ºC and unload under controlled humidity (RH NMT 40%).

Get Yield = 90 g to 120 g Example 2: Preparation of 1 : 1 Ethanol:Pure Water Tetrachia serrata extract

This hardwood tetrakis plant piece (1 kg) was loaded into the 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 ºC for about 3 hours. The extract mass was filtered, collected, and stored in a container. The extraction and filtration steps were repeated twice with a mixture of ethanol and pure water (1:1, 3 L). The three filtered extracts were combined and concentrated to the maximum possible extent under reduced pressure at low temperature. The extract was poured into stainless steel trays and dried under vacuum at 45-50 ºC until the ethanol content did not exceed 10,000 ppm and the water content did not exceed 5%. Cool the dry extract to about 20-25 ºC and unload under controlled humidity (RH NMT 40%).

Get Yield = 80 g to 120 g Example 3: Preparation of Aqueous Extracts of Tessica serrata

This hardwood tetrakis plant piece (1 kg) was loaded into the extractor at ambient temperature ^* . Pure water (6 L) was added and the reaction mixture was heated at a temperature of 60-65 ºC for about 3 hours. The extract mass was filtered, collected, and stored in a container. The extraction and filtration steps were repeated twice with pure water (3 L). The three filtered extracts were combined and concentrated to the maximum possible extent under reduced pressure at low temperature. The extract was poured into stainless steel trays and dried under vacuum at 45-50 ºC until the ethanol content did not exceed 10,000 ppm and the water content did not exceed 5%. Cool the dry extract to about 20-25 ºC and unload under controlled humidity (RH NMT 40%).

Get Yield = 80 g to 120 g

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

Similarly, the extract can also be prepared by spray drying using an inlet temperature of 160 ^° C-190 ^° C and an outlet temperature of 105 ^° C to 120 ^° C. Example 4: Isolation of various compounds from Tesco sclera:

A dry aqueous extract of T. serrata (powder, 500 gm) was dissolved in distilled water and filtered. The filtrate was partitioned between _CHCl3 (A) and _H2O (B). The _CHCl3 soluble fraction (A) was then separated with aqueous HCl to yield the _CHCl3 layer (C) and the acidic _H2O layer (D). The chloroform layer (C) was subjected to column chromatography, eluted with a gradient of _CHCl3 -MeOH and concentrated to obtain 30 fractions. A fraction of coniferyl alcohol (SP-A-01) was obtained in pure form based on the TLC spectrum. The acidic _H2O layer (D) was then basified with aqueous _NH4OH to make the free base, followed by extraction with _CHCl3 , yielding the alkaloid-containing chloroform layer (E). The _CHCl3 fraction (E) was dried and purified by column chromatography and eluted with a _CHCl3 -MeOH gradient to isolate magnolidine. In addition, the aqueous layer (B) was basified with ammonium hydroxide ( _NH4OH ) solution, followed by extraction with _CHCl3 , yielding the alkaloid rich fraction (E1) and the aqueous layer (F1). The _CHCl3 layer (E1) was further processed by column chromatography using _CHCl3 -MeOH elution to isolate tetrandine. The aqueous layer (F1) was lyophilized powdered and dissolved in methanol. The methanol soluble fraction was further purified by column chromatography, eluted with a _CHCl3 -MeOH gradient, and concentrated to give 50 fractions. Two UV active spots were visible on TLC. The mixture containing the two UV active compounds was further subjected to RP-HPLC (using a gradient solvent system of (MeOH/H ₂ O) to obtain 20-hydroxyecdysone and maxosterone A. Example 5: Preparation from bristles using compression technique lozenge of fenugreek extract S/N Element Quantity (mg/ tablet ) (1) (2) (3) (4) 1 Extract (from Example 1, 2 or 3) 1000.0 1000.0 1000.0 1000.0 2 Magnesium Aluminum Trisilicate 300.0 300.0 300.0 300.0 3 Lactose monohydrate 90.0 - - - 4 Dicalcium Phosphate - 90.0 - - 5 pregelatinized starch - - 90.0 - 6 calcium silicate - - - 90.0 7 microcrystalline cellulose 40.0 40.0 40.0 40.0 8 Colloidal silica 20.0 20.0 20.0 20.0 9 Croscarmellose 40.0 40.0 40.0 40.0 10 Magnesium stearate 10.0 10.0 10.0 10.0 Core Lozenge Weight 1500.0 1500.0 1500.0 1500.0 11 Opadry Green 45.0 45.0 45.0 45.0 12 pure water Qs. Qs. Qs. Qs. The total weight of the coated tablet 1545.0 1545.0 1545.0 1545.0

Manufacturing procedure: 1. Pass the extract through a No. 10 mesh screen (2 mm); 2. Pass the step 1 material and magnesium aluminum trisilicate through a No. 14 mesh screen (1.4 mm); 3. Lactose monohydrate , dicalcium phosphate, pregelatinized starch and calcium silicate were screened through No. 36 mesh (420 µ); 4. Microcrystalline cellulose, colloidal silica and croscarmellose were screened through No. 25 mesh (600 μ). µ); 5. Blend the materials from Steps 3 and 4 with the materials from Step 2 in a blender; 6. Lubricate the blend from Step 5 with magnesium stearate and compress into a lozenge; 7. Disperse Opadry Green in pure water to prepare a dispersion; and 8. Apply the dispersion from step 7 to the compressed tablet from step 6. Example 5.1: Preparation of lozenges from extracts of Tessica serrata using direct compression technique: S/N Element Quantity (mg/ tablet ) (1) (2) (3) (4) (5) (6) 1 Extract (from Example 1, 2 or 3) 100.0 200.0 300.0 400.0 500.0 800.0 2 Magnesium Aluminum Trisilicate 30.0 60.0 90.0 120.0 150.0 240.0 5 Lactose monohydrate 9.0 - - - - - 6 Dicalcium Phosphate - 18.0 - - - - 7 pregelatinized starch - - 27.0 - - - 8 calcium silicate - - - 36.0 45.0 72.0 9 microcrystalline cellulose 4.0 8.0 12.0 16.0 20.0 32.0 10 Colloidal silica 2.0 4.0 6.0 8.0 10.0 16.0 11 Croscarmellose 4.0 8.0 12.0 16.0 20.0 32.0 12 Magnesium stearate 1.0 2.0 3.0 4.0 5.0 8.0 Core Lozenge Weight 150.0 300.0 450.0 600.0 750.0 1200.0 13 Opadry Green 4.47 8.94 13.41 17.88 22.35 35.76 14 pure water Qs. Qs. Qs. Qs. Qs. Qs. The total weight of the coated tablet 154.47 308.94 463.41 617.88 772.35 1235.76

The manufacturing procedure is as in Example 5. Example 6: Preparation of lozenges from extracts of Tessica serrata using wet granulation techniques (via a quick-mix granulator) S/N Element Quantity ( mg/tablet) (1) (2) (3) (4) Part (a) : Intragranular part 1 Extract (from Example 1, 2 or 3) 1000.0 1000.0 1000.0 1000.0 2 Magnesium Aluminum Trisilicate 250.0 250.0 250.0 250.0 3 methanol Qs. Qs. Qs. Qs. total 1250.0 1250.0 1250.0 1250.0 Part (b) : Extragranular part 4 Magnesium Aluminum Trisilicate 40.0 40.0 40.0 40.0 5 Lactose monohydrate 90.0 - - - 6 Dicalcium Phosphate - 90.0 - - 7 pregelatinized starch - - 90.0 - 8 calcium silicate - - - 90.0 9 microcrystalline cellulose 40.0 40.0 40.0 40.0 10 Colloidal silica 20.0 20.0 20.0 20.0 11 Croscarmellose 40.0 40.0 40.0 40.0 12 Magnesium stearate 10.0 10.0 10.0 10.0 Core Lozenge Weight 1490.0 1490.0 1490.0 1490.0 13 Opadry Green 44.7.0 44.7 44.7 44.7 14 pure water Qs. Qs. Qs. Qs. The total weight of the coated tablet 1534.7 1534.7 1534.7 1534.7

Manufacturing procedure: 1. Pass the extract through a No. 10 mesh screen (2 mm); 2. Pass the magnesium aluminum trisilicate through a No. 36 mesh screen (420 µ); Material from step 2 was granulated with methanol and dried; 4. Pass dried material from step 3 through No. 16 mesh (1 mm); 5. Magnesium aluminum trisilicate, lactose monohydrate, dicalcium phosphate, pregelatinized Starch and calcium silicate were screened through No. 36 mesh (420 µ); 6. Microcrystalline cellulose, colloidal silica and croscarmellose were passed through No. 25 mesh (600 µ); 7. 5. Mix the materials from step 6 with the materials from step 4 in a blender; 8. Lubricate and compress the blend from the above step with magnesium stearate into a lozenge; 9. Disperse Opadry Green in purified water to prepare and 10. Apply the dispersion from Step 9 to the compressed tablet from Step 8. Example 7: Preparation of lozenges from extract of Tessica serrata using dry granulation technique (by compactor) S/N Element Quantity (mg/ tablet ) (1) (2) (3) (4) Part (a) : Intragranular part 1 Extract (from Example 1, 2 or 3) 1000.0 1000.0 1000.0 1000.0 2 Magnesium Aluminum Trisilicate 300.0 300.0 300.0 300.0 3 Magnesium stearate 15.0 15.0 15.0 15.0 total 1315.0 1315.0 1315.0 1315.0 (b) Part of the extragranular part 4 Lactose monohydrate 30.0 - - - 5 Dicalcium Phosphate - 30.0 - - 6 pregelatinized starch - 30.0 - 7 calcium silicate - - 30.0 8 microcrystalline cellulose 100.0 100.0 100.0 100.0 9 Colloidal silica 10.0 10.0 10.0 10.0 10 Croscarmellose 30.0 30.0 30.0 30.0 11 Magnesium stearate 15.0 15.0 15.0 15.0 Core Lozenge Weight 1500.0 1500.0 1500.0 1500.0 12 Opadry Green 45.0 45.0 45.0 45.0 13 pure water Qs. Qs. Qs. Qs. The total weight of the coated tablet 1545.0 1545.0 1545.0 1545.0

Manufacturing Procedure: 1. Screen the extract through No. 10 mesh (2 mm); 2. Screen the Step 1 material with magnesium aluminum trisilicate through No. 14 mesh (1.4 mm); 3. Screen magnesium stearate through 36 mesh (420 µ) and mix with the material from step 2 in a blender; 4. Use a compactor to compact the blended material; 5. Grind the compacted body from step 4; 6. Shape the extra-granular 7. Compress the lubricating blend of Step 6 into a lozenge; 8. Disperse Opadry Green in purified water to prepare a dispersion; and 9. Apply the dispersion from Step 8 to the compressed tablet from Step 7. Example 8: Evaluation of Biological Activity in Treating Viral Infections Caused by Various Positive Single-Stranded RNA Viruses: A. Evaluation of the efficacy of aqueous extracts of Fangji hardwood (AQCH) in the treatment of patients with COVID-19 .

Patients selected according to the following inclusion and exclusion criteria were administered lozenges of 400-800 mg of aqueous extract of Tetrachia serrata three times daily (every 8±1 hour) before meals, preferably at the same time for 10 days. - Inclusion criteria : Individuals were included in the study if they met all of the following criteria: 1. Provided written informed consent. 2. Men or non-pregnant, non-breastfeeding women aged ≥18 years and ≤65 years old. 3. Patients with acute respiratory disease (fever and at least one of the following cough or shortness of breath) with: a) Travel history to high-risk COVID-19 infection countries within the last 14 days OR. b) close contact with a laboratory confirmed case of COVID-19 within 14 days or. c) When health care workers managing respiratory distress/severe acute respiratory illness cases are symptomatic. 4. Laboratory confirmed COVID-19. 5. Able to take medication by mouth and adhere to study procedures. 6. Females of reproductive potential must have a negative urine pregnancy test before entering the study, and agree to use a highly effective method of contraception to avoid pregnancy from study entry to the last administration of the study drug (such contraceptives may include hormonal contraception, such as , containing [oral, intravaginal, or transdermal] combined estrogens and progestins or progesterone only associated with ovulation inhibition [oral, injectable, or implantable] hormonal contraception, intrauterine devices, intrauterine hormone release Systemic or bilateral ureteral occlusion, vasectomy partner or sexual abstinence). -. Exclusion criteria : Patients were considered ineligible to participate in the study if they fell into any of the following criteria: 1. Patients with persistent vomiting (more than 3 times in 12 hours to prevent proper oral hydration), making it difficult to swallow drug. 2. Patients with altered mental status. 3. Laboratory screening abnormalities, including any of the following: a. AST or ALT > 5 ULN b. Oxygen saturation < 90% without oxygen therapy c. Absolute neutrophil count < 1500/μL d . Serum creatinine > ULN e. Total bilirubin > ULN 4. Patients with active hepatitis, tuberculosis, and established bacterial or fungal infections. 5. Patients with combined organ failure who require ICU monitoring and treatment. 6. Respiratory failure and patients requiring mechanical ventilation. 7. Shock patients. 8. Patients with any concurrent medical condition or uncontrolled clinically significant systemic disease (eg, renal failure, heart failure, hypertension, liver disease, diabetes, anemia, etc.), according to the investigator's opinion, were excluded from the study. Participation in the study or interference with the interpretation of study results. 9. Patients with a seropositive history of Hepatitis B, Hepatitis C, or Human Immunodeficiency Virus. 10. Patients receiving specific antiviral drugs ritonavir/lopinavir, chloroquine, hydroxychloroquine, monoclonal antibodies within one week prior to hospitalization. 11. Participate in other clinical studies thirty (30) days prior to screening. 12. Patients who participated in other research studies 3 months earlier were enrolled in this study. 13. Investigators, researchers, sponsor representatives and their first-degree relatives.

Efficacy assessment : Based on clinical as well as laboratory assessments. Perform the following assessments: ˙ At screening, daily until discharge, discharge day, day 17 and physical examination as clinically indicated. ˙ At screening, within 10 minutes before the first dose on Day 1, then every 24 hours (± 10 minutes) until discharge (on or after Day 11), at the FU visit on Day 17 and as clinically indicated Indicated vital signs (pulse rate, respiratory rate, systolic and diastolic blood pressure). ˙ FU will be administered at screening, within 30 minutes before the first dose on Day 1, then every 24 hours (± 10 minutes) until discharge day (on or after Day 11), on (or after) Day 17 Measure body temperature (oral) at the time of visit and as clinically indicated. ˙ Blood and kidney function tests for evaluation of liver hematology and biochemistry will be withdrawn at screening and subsequently on days 3, 5, 7, and 10. At the investigator's discretion, additional hematologic and biochemical evaluations may be performed until recovery. ˙ PCR for coronavirus school pricing: at screening, within 30 minutes before the first dose on Day 1, then every 24 hours (± 10 minutes) until Day 10 and at discharge. ˙ Chest X-rays – at screening and at discharge on days 3, 5, 7, 10.

Recorded by adverse events, vital signs (pulse rate, systolic and diastolic blood pressure (sitting), body temperature and respiratory rate), ECG, physical examination and clinical laboratory investigations (hematology, biochemistry and urinalysis) assessment to assess safety. Adverse events were classified according to their severity based on CTCAE v 5.0 criteria. Any abnormal change from baseline to any clinically significant abnormality should be recorded as an AE under concurrent medical conditions, physical examination, and/or laboratory data. A 12-lead ECG was performed at screening, 1 hour after dosing on Day 1 and repeated 24 hours later (± 10 minutes) until Day 10, on discharge day, and as clinically indicated. Any patient determined by the treating physician/PI to be at risk of developing serious disease will be managed in accordance with the standard of care.

The primary outcome measure was the reduction in viral load in nasopharyngeal swabs and the percentage of subjects reaching clinical treatment (clinical treatment was defined as negative viral load in two consecutive respiratory samples when measurement frequency was greater than or equal to one day, lung images improvement, normothermia for more than 3 days, and improvement in clinical features). The second evaluation indicators are: viral nucleic acid conversion rate and days from positive to negative (time frame: within 10 days after admission), time to clinical improvement as time to normalize fever, respiratory rate, oxygen saturation, and cough attenuation , Clinically defined time as time to death, mechanical ventilation or ICU introduction, drug safety and tolerability assessed by treatment-emergent adverse events. The medicinal composition of the extract is effective in treating COVID-19 patients without significant side effects. B. Evaluation of the efficacy of an aqueous extract of Fangji hardwood (AQCH) in the treatment of infections caused by Chikungunya virus (CHIKV) .

Assay protocol for testing AQCH inhibition of CHIKV using a viral plaque reduction assay: i. 200,000 green monkey kidney cells per ml seeded in DMEM + 10% ΔFBS in each well of a 12-well plate; ii. Incubate the plate in an incubator at 37 ºC and 10% CO2 setting for 24 hours; iii. After the incubation period, infect green monkey kidney cells with CHIKV; iv. After infection, incubate at 37 ºC with 10 The plate was incubated under % CO for ₂ hours; v. After two hours, the infection medium was removed and then different concentrations of the drug to be tested ( ^AQCH ) and methylcellulose overlays were placed; vi. Incubate the plate for more than 3 days under carbon dioxide and perform a double or triple test for each drug concentration; vii. At the end of the incubation period, remove the plate from the incubator; viii. Wash the plate with 1X PBS, dispensing 500 µl per well Three times; ix. Tap the plate on a stack of paper towels and add 500 μl of 10% formaldehyde (prepared in IX PBS) to fix the cells. x. Incubate the cells for 30 minutes at room temperature, then wash the plate three more times with 1X PBS, dispensing 500 µl per well; xi. Tap the plate on a stack of paper towels. Add 200 µl of 0.25% crystal violet stain (prepared in 30% methanol and filtered before use) and incubate the dish at room temperature for 5-10 minutes; and xii. Fill the jar with tap water and submerge the dish to remove excess of dyes. - Infection in step iii) is done by preparing the required virus stock dilutions in DMEM + 0.5 % ΔFBS to yield 80-100 virus control wells per 250 µl of infection medium (DMEM + 0.5 % ΔFBS) plaque. Cell control wells included in the experimental plate design did not receive any viral infection, but received only 250 µl DMEM + 0.5 % ΔFBS. - Different concentrations of drug were prepared in step v) by preparing 2% methylcellulose (MC) in hot water and autoclaving. Prepare appropriate test drug dilutions in 2X DMEM + 1.0% ΔFBS. 500 µL of 2% MC and 500 µL of the drug dilution were mixed in each well and added over the cells after removal of the viral infection medium (1 ml total).

Results: AQCH was screened using the plaque assay for CHIKV. In a total of four independent experiments using laboratory-scale and plant-scale _AQCH extracts, IC50s were in the range of 10-20 µg/ml. Therefore, the results show the potential inhibitory activity of the complex extracts of the present invention against the virus (CHIKV). C. To evaluate the efficacy of the extracts or isolated compounds of Tetranychus solani, magnolidine, 20-hydroxyecdysone in the treatment of infections caused by Dengue virus (DENV) , using a FACS -based neutralization assay:

This assay was used to detect the number of cells infected with DENV in the total cell population. Green monkey kidney cells (sterile, tissue culture) were seeded in 96-well plates in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum ΔFBS (20,000-25,000 cells/200 μl/well). treatment) and incubated at 37°C in a humidified incubator with 10% _CO for 24-26 hours (green monkey kidney cell doubling time). In this method, cells should be no less than 20,000/well. Media was aspirated and cells were infected with 0.1 MOI of DENV-1, 2, 3, 4 in DMEM supplemented with 0.5% ΔFBS media (100 μl/well). The plate was incubated for ₂ hours at 37°C in a humidified incubator with 10% CO. Preparation of the active stock solution and its different concentrations of the aqueous extract of hardwood repellent, i.e. 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml and 3.125 μg/ml to 96-well plates in each hole. The plates were allowed to incubate at 37°C in a humidified chamber with 10% carbon dioxide for 42-46 hours after infection. After the incubation period, cells were stained with fluorescently labeled antibodies to visualize cytosolic DENV. For staining, aspirate the medium from the top of the cells and wash with PBS. Cells were trypsinized and transferred to a 96-well U-bottom. Then centrifuge and aspirate the supernatant. Cells were washed twice with permeabilization buffer and blocked with 1% normal mouse serum (prepared in permeabilization buffer) for 30 min. 2H2-Alexa488 antibody was added to stain cells with DENV and incubated with gentle shaking. After incubation, the cells were centrifuged and the supernatant was aspirated. Cells were washed and resuspended in PBS. The above-treated cells were analyzed by flow cytometry and 5000 cells per well were counted. The data obtained were analyzed (by FlowJo software) to determine the relative percentage of infected cells for each extract concentration compared to the virus-only control group (without any extract treatment). The IC50 of the extract was determined as the concentration of the extract that inhibited 50% dengue virus infection and was calculated using GraphPad Prism software.

As assessed by FNT, the extract was found to be effective against all four DENV serotypes. IC50 values for each DENV serotype generally ranged from 5-10 μg/ml (Table 1), indicating that 5-10 μg/ml of extract was required to inhibit 50% of all four DENV infections in this assay. Table 1 : Tabular representation of _IC50 (μg/ml) of extracts examined by FNT against all four dengue virus serotypes: test candidates _IC50 (μg/ml) DENV-1 DENV-2 DENV-3 DENV-4 Hardwood water repellent extract 7.3 8.2 5.5 6.9

A similar evaluation study was also performed on other isolated compounds according to the invention against Dengue virus serotype 4 (DENV4 or DV4) by FNT examination, and the test results are provided in Table 2 below: Table 2 : Isolated compounds examined by FNT against Dengue virus Tabular representation of IC ₅₀ (μg/ml) for serotype 4 (DV4): compound DV4 (µg/ml) Chinese tetrandrine 0.34 - 0.80 Magnoliaine >100 20-Hydroxyecdysone ＞500

Therefore, based on the above Table 1 and Table 2, it is clear that the compound extracts or isolated compounds of Tetranychus denguetus (Tendrogine chinensis, magnolidine, 20-hydroxyecdysone) can effectively treat dengue fever virus (DENV) caused by Infect. Example 9: Evaluation of antiviral effect of Chinese tetrandrine against positive-stranded RNA viruses: 1. Chinese tetrandrine in vitro Antiviral effect: A. Against Dengue virus (DENV) : flow cytometry-based viral inhibition assay Law.

Green monkey kidney cells in 200 μl DMEM + 10% ΔFBS were seeded in 96-well dishes (20,000-25,000 cells/well) and cultured for 24 hours in an incubator adjusted to 37 ^° C and 10% _CO2 . The next day, cells were infected with 100 μl of DENV-1, DENV-2, DENV-3 and DENV-4 dilutions to yield 10% infection in DMEM + 0.5% ΔFBS (dilution medium). After culturing the green monkey kidney cells for 2 hours at 37 ^° C, 10% CO ₂ , the virus was aspirated and 200 μl of the appropriate range of test substance (tetrandine Chinese) prepared in dilution medium was added to the wells in duplicate. Cells were further cultured in an incubator at 37 ^o C, 10% CO ₂ for an additional 46 h. Wells infected with virus but without any subsequent extract/drug treatment served as virus controls, while wells uninfected and untreated served as cell controls. These experimental control groups were used for relative viral infection calculations and antibody background signal adjustments, respectively. After the incubation period, cells were stained with Alexa-488-labeled 2H2 mAb to visualize cytosolic DENV. For staining, aspirate the medium from the top of the cells and wash with PBS. Cells were trypsinized and transferred to a 96-well U-bottom. After transfer, cells were centrifuged at 1500 rpm for 5 minutes and the supernatant was aspirated. Cells were washed again with PBS and then fixed with 4% paraformaldehyde for 20 min. The cells were centrifuged at 2500 rpm for 5 minutes and the supernatant was aspirated. Cells were washed twice with permeabilization buffer and blocked with 1% normal mouse serum (prepared in permeabilization buffer) for 30 min. Without removing blocking solution, Alexa-488-labeled 2H2 mAb was added to stain cells with DENV and incubated for 1 hr at 37 °C with gentle shaking. After incubation, the cells were centrifuged at 2500 rpm for 5 minutes and the supernatant was aspirated. Cells were washed twice with permeabilization buffer and resuspended in 100 μl of PBS. The above-treated cells were analyzed by a BD FACS Verse flow cytometer and 5000 cells were counted per well. The data were analyzed by FlowJo software to determine the relative percentage of infected cells for each analyte concentration to the virus-only control group. The 50% inhibitory concentration ( _IC50 ) of the analyte was determined as the concentration that inhibited 50% of dengue virus infection relative to the virus control group and was calculated using nonlinear regression analysis of GraphPad Prism software. Table 3 : shows the _IC50 of Chinese tetrandrine against DENV (1-4): IC 50 of Chinese tetrandrine against DENV ₍ μg/ml) DENV-1 DENV-2 DENV-3 DENV-4 0.62 0.73 0.74 0.92

Results : The _IC50 of Chinese tetrandrine against DENV 1-4 was found to be 0.62-0.91 μg/ml. B. Against Chikungunya Virus (CHIKV) : Viral Plaque Reduction Assay .

Green monkey kidney cells were seeded at 200,000/well/ml in DMEM + 10% ΔFBS in a 12-well dish and cultured overnight at 37 ^° C, 10% CO ₂ . After 24 hours, thaw the virus on ice to set up viral infection. Viruses were diluted in DMEM + 2% ΔFBS (diluent) in virus control wells to generate ~80 plaques per 250 μl/well. A control group of cells was kept free of virus infection during the experiment and received only the dilution. Shake the plate every 15 minutes after setting the infection. After 2 hours, the viral infection medium was removed and the wells were washed with DMEM. 1 ml of a dilution of the desired test substance (tetrandine) containing 1% methylcellulose was added to each well and incubated for ₂ days at 37 ^° C, 10% CO2. Cell and virus control wells will receive analyte-free methylcellulose covers prepared in diluent. At the end of the incubation period, the dish was removed from the incubator. Dispense 500 µl of 1X PBS into each well and wash the plate three times with 1X PBS. Cells were fixed by adding 500 μl of 4% paraformaldehyde (prepared in IX PBS). Cells were incubated for 30 minutes at room temperature. Dispense 500 µl of 1X PBS into each well and wash the plate three more times with 1X PBS. 100 μl of 0.25% crystal violet stain (prepared in 30% methanol and filtered before use) was added and the plate was incubated at RT for 10 minutes. Wash the dish with water to remove excess stain and count plaques.

Results : The _IC50 of Chinese tetrandrine against CHIKV was found to be ~2 μg/ml. Table 4 : Summary of _IC50s of Chinese tetrandrine against different viruses tested Forward single-stranded RNA virus _IC50 ( μg/ml) In vitro assay DENV 1-4 0.62-0.91 Flow Cytometry-Based Viral Inhibition Assay CHIKV 2 Plaque Reduction Assay 2. Antiviral effect of Chinese tetrandrine in vivo in AG129 mice

A second dengue AG129 mouse model was established by IV inoculation with a sublethal dose of immune complex (IC) of DENV- ² S221 (2x104 FIU) and neutralization of the 4G2 mAb (10 μg) concentration. Tetrandrine was dissolved in water and a 1 mg/ml solution was prepared in 0.1% methylcellulose. IC-induced mice (n=4-6) were orally fed and injected with 10 mg/kg/day of tetrandrine with QID and BID dosing, respectively, within 5 days after IC inoculation. Mice were monitored for weight changes, disease symptoms, and survival. Body weight was monitored twice a day in the morning and in the evening, and averaged on a graph. Incidence score on a 5-point scale: 0.5, mildly uneven fur; 1.0, uneven fur; 1.5, damaged eyes; 2, damaged eyes and hunchback; 2.5, loose stools; 3.0, Movement limitation; 3.5, no movement/hind leg paralysis; 4.0, euthanasia at a cumulative score of 5. A p value of < 0.05 was considered significant (*p < 0.05).

Results : Chinese tetrandrine provided protection against second dengue infection in AG129 mice. Following oral administration of QID, a 70% survival rate was observed. Complete protection was observed when BID was administered intraperitoneally.

While considerable emphasis is placed herein on specific extracts and specific isolated compounds of the Tetrachia solani components in various preferred formulations and bioactivity evaluation studies, it should be understood that many additional components can be added and the preferred Many changes are made to the formula or extract. These and other variations in the preferred extracts and formulations of the present invention will be apparent to those skilled in the art in light of the present invention, from which it should be clearly understood that the foregoing recited matters are to be construed as illustrative of the present invention, and Not restricted content. Example 10: Evaluation of the efficacy of AQCH lozenges and Chinese tetrandrine against Chikungunya virus (CHIKV):

An additional plaque reduction neutralization test (PRNT) was performed according to the assay procedure in Example 8B. The in vitro activity of AQCH and Chinese tetrandrine was scored against the African CHIKV strain against Chikungunya virus (CHIKV) by the viral plaque reduction neutralization test (PRNT), and the drug concentration that caused 50% viral inhibition was calculated as 50% inhibition concentration or _IC50 .

For efficacy evaluation, samples of Chinese tetradine, AQCH lozenges, and placebo lozenges were prepared at the following concentration ranges: ˙ 100 to 3.12 µg/ml for AQCH and placebo lozenges; ˙ Chinese tetrandrine is 10 to 0.31 µg/ml.

The test results are summarized in Table-5 below: Table - 5 : _IC50 values obtained from in vitro anti- CHIKV PRNT experiments Sample Details _IC50 value against CHIKV ( µg/ml) Chinese tetrandrine 3.95 AQCH lozenges 58.2 placebo lozenges NA *NA: No activity observed at concentrations assessed from 100 to 3.12 µg/ml

Conclusion: Chinese tetrandrine and AQCH inhibit CHIKV in PRNT experiments. Example 11: Evaluation of the efficacy of AQCH lozenge and Chinese tetrandrine against Japanese encephalitis virus (JEV):

The 50% inhibitory concentration or IC50 was calculated based on the in vitro activity of AQCH and Chinese tetrandrine against Japanese encephalitis virus (JEV) based on flow cytometric neutralization assay (FNT) and the drug concentration that caused ₅₀ % viral inhibition .

For efficacy evaluation, samples of Chinese tetradine, AQCH lozenges, and placebo lozenges were prepared at the following concentration ranges: ˙ 50 to 1.56252 µg/ml for AQCH and placebo lozenges; ˙ Chinese tetrandrine is 10 to 0.31 µg/ml.

Experimental details: 1. Seeding of green monkey kidney cells: Confluent T-75 flasks of green monkey kidney cells were trypsinized with 1x TE. For this, the mulch medium was aspirated from the flask and rinsed with 4 mL of DMEM. Add 4 mL of 1x TE and incubate the flask at 37˚C for 5-10 min in a 10% _CO incubator to remove attached green monkey kidney cells. Collect the cell suspension into tubes containing 0.4 mL of DMEM with 10% ΔFBS. Centrifuge at 1500 rpm for 5 minutes at room temperature. The supernatant was discarded and the pellet was resuspended in the desired volume of 10% ΔFBS-DMEM to yield 20,000 - 25,000 green monkey kidney cells per 200 μl of suspension. Seed 200 µl of green monkey kidney cell suspension per well in a 96-well plate. Incubate at 37˚C for 24 hours in a 10% _CO2 incubator. 2. Prepare test item stock solutions: 2 hours before use, dissolve tetrandrine at 1 mg/mL, AQCH at 5 mg/mL, and placebo at 5 mg/mL in 1x DMEM and keep at RT on a flip-flop for even mixing. 3. JEV infection: Dilute JEV stock to desired volume in 1x DMEM + 0.5% ΔFBS to produce 10% infection. Remove the medium on top of the seeded green monkey kidney cells and add 80 µl of JEV's reaction solution. The assay was set up in double replicates for the concentrations of all tested items. Incubate the plate at 37˚C for 2 hours in a 10% _CO2 incubator. 4. Test item dilution: Prepare a dilution of the desired test item from the stock solution prepared in step 2 in sterile 1x DMEM + 0.5% ΔFBS; sterile filter the first dilution using a syringe filter with a 0.45u filter, It is used to prepare successive dilutions. 5. Treatment with test items: 2 hours after JEV infection ends, aspirate virus and add 200 µl of vehicle containing the drug dilution from step 4 to the cells. Incubate the plate at 37˚C for 22-24 hours in a 10% _CO2 incubator. 6. Staining: Staining can be performed 22-24 hours after adding the virus inoculum. In this regard, cells were trypsinized, fixed, permeabilized and blocked prior to staining with Alexa488-labeled 4G2 mAb. The specific steps are as follows: 6.1. Aspirate the medium from the top of the cells after completing the 22-24 hour incubation. Wash cells with 1X-PBS (100 µl/well). Add 1x Trypsin EDTA (25 µl/well) by aspiration and incubate at 37˚C until cells detach (1-2 min), and add 150 µl of 10% ΔFBS in PBS to each well. Mix appropriately and transfer to a 96-well round dish. 6.2. Centrifuge the contents of the dish at 1500 rpm for 5 minutes at room temperature (RT); aspirate the supernatant. 6.3. Add 1x PBS (100 µl/well) to wash the cell pellet. Do not mix. 6.4. Centrifuge the contents of the dish at 1500 rpm for 5 minutes at RT. Aspirate the supernatant. 6.5. Add 4% paraformaldehyde (50 µl/well) to the cell pellet. Mix appropriately and incubate at RT for 15-30 minutes. 6.6. Centrifuge the plate at 2500 rpm for 5 minutes at RT. Aspirate the supernatant. 6.7. Wash the cell pellet twice with 1x PBS (100 µl/well). Plates can be stored at 2-8°C for 2-3 days at this stage. 6.8. Prepare 1x permeabilization buffer in PBS using 10x permeabilization buffer. 6.9. Centrifuge the plate at 2500 rpm for 5 minutes at RT. Aspirate the supernatant. 6.10. Add 1x permeabilization buffer (100 µl/well) to the cell pellet. Do not mix. 6.11. Centrifuge the plate at 2500 rpm for 5 minutes at RT. Aspirate the supernatant. 6.12. Add 50 µl of blocking solution (1% normal mouse serum in 1x permeabilization buffer) to each well. Mix properly. 6.13. Incubate the plate for 30 minutes at room temperature. 6.14. After blocking, add 25 µl of the pre-optimized dilution of 4G2-alexa 488 mAb prepared in blocking solution to each well on top of the blocking solution. The optimal amount of 4G2-Alexa 488 mAb was predetermined to identify the desired saturation concentration of mAb [diluted 1:400 in blocking buffer, which was used with this batch of 4G2-Alexa 488]. From here, the disc is processed in dark or dim light. 6.15. Incubate the plate for 1 hour at 37°C in the dark with gentle shaking. 6.16. Centrifuge the plate at 2500 rpm for 5 minutes at room temperature; aspirate the supernatant. 6.17. Add 1x permeabilization buffer (100 µl/well) to the cell pellet. Do not mix. 6.18. Centrifuge the plate at 2500 rpm for 5 minutes at room temperature; aspirate the supernatant. 6.19. Add 1x permeabilization buffer (100 µl/well) to the cell pellet. Do not mix. 6.20. Centrifuge the plate at 2500 rpm for 5 minutes at room temperature; aspirate the supernatant. 6.21. Suspend cells in 1x PBS. 7. Read the plate by flow cytometer (within 7 days of staining; the plate can be covered with aluminum foil and stored at 2-8°C until then) and count 5000 cells. Data were analyzed using Flowjo with Graphpad prism software and _IC50 calculated, which corresponds to the concentration of drug at which 50% viral inhibition (or 50% infection) was observed.

The test results are summarized in Table-6 below: Table - 6 : _IC50 values and percentage inhibition graphs obtained from in vitro anti- JEV FNT experiments : Sample Details _IC50 value against JEV (µg/ml) Chinese tetrandrine 0.4022 AQCH lozenges 6.239 placebo lozenges NA *NA: No activity observed at concentrations assessed from 50 to 1.5625 µg/ml

Conclusion: Chinese tetrandrine and AQCH inhibit CHIKV in PRNT experiments. Example 12: Evaluation of in vitro anti-dengue activity of AQCH extracts and pharmaceutical formulations under stability:

AQCH extracts and drug formulations were scored for in vitro activity against all four dengue virus serotypes (DENV-1-4) based on flow cytometric neutralization assays (FNT). The drug concentration that caused 50% viral inhibition was calculated as the 50% inhibitory concentration or _IC50 .

For efficacy evaluation, test samples of AQCH extract and AQCH lozenge were used to evaluate the activity of the test samples against dengue virus serotypes (DENV-1, -2, -3 and -4).

The concentration range of the test samples was 50 to 1.5625 µg/ml.

Test results are provided in Table-7 and Table-8 as follows: Table 7: _IC50 values obtained from in vitro anti-dengue FNT experiments of AQCH extracts under stability (at 30°C ± 2°C/60% ± 5% RH for 24 months): Sample Details Storage conditions IC ₅₀ value for resistance (µg/ml) DENV-1 DENV-2 DENV-3 DENV-4 AQCH extract 30°C ± 2°C/ 60% ± 5% RH for 24 months 3.827 5.874 2.981 4.366 Table 8: _IC50 values obtained from in vitro anti-dengue FNT experiments of AQCH lozenges under stability (18 months at 30°C/65% RH): Sample Details Storage conditions IC ₅₀ value for resistance (µg/ml) DENV-1 DENV-2 DENV-3 DENV-4 AQCH lozenge (25 mg) 30°C ± 2°C/ 60% ± 5% RH for 24 months 6.434 6.228 5.398 7.945 AQCH lozenge (100 mg) 6.750 6.635 5.710 7.930 AQCH lozenge (300 mg) 5.610 5.167 5.049 7.589 AQCH Lozenges (500 mg) 6.520 6.296 4.694 7.405

CONCLUSION: In in vitro FNT experiments, AQCH extracts of different strengths and AQCH lozenges inhibited all four dengue serotypes (DENV-1, -2, -3 and -4).

While considerable emphasis is placed herein on specific extracts and specific isolated compounds of the Tetrachia solani components in various preferred formulations and bioactivity evaluation studies, it should be understood that many additional components can be added and the preferred Many changes are made to the formula or extract. These and other variations in the preferred extracts and formulations of the present invention will be apparent to those skilled in the art in light of the present invention, from which it should be clearly understood that the foregoing recited matters are to be construed as illustrative of the present invention, and Not restricted content.

without

The following drawings form a part of this specification and are included to further demonstrate illustrative embodiments of certain aspects of the invention.

[FIG. 1(a)]: Plot of the logarithmic relationship between the percent inhibition of JEV on the Y-axis and the test concentration of tetrandrine on the X-axis.

[FIG. 1(b)]: Plot of the logarithmic relationship between percent inhibition of JEV on the Y-axis and AQCH assay concentration on the X-axis.

Claims (49)

Use of a composition for treating viral infection caused by a positive single-stranded RNA virus, the composition comprising: a therapeutically effective amount of a compound extract of Fangji sclera; and a pharmaceutically acceptable excipient, It includes diluents, binders, disintegrants, lubricants, slip agents, polymers, flavoring agents, surfactants, preservatives, antioxidants, buffers, tonicity modifiers, or combinations thereof. The invention relates to the use of a compound extract of Fangji hardwood for preparing a medicament for viral infection caused by a forward single-stranded RNA virus. The use as claimed in claim 1 or 2, wherein the complex extract of Tetranychus serrata includes tetrandrine, magnolidine, coniferyl alcohol, quercetin, rutin, syringaresinol diglucoside, macosterol Ketone A, sclerophylline, 20-hydroxyecdysone, derivatives thereof, or combinations thereof. The use according to any one of claims 1 to 3, wherein the positive single-stranded RNA virus is hepatitis C virus, West Nile virus (WNV), Dengue virus, Chikungunya virus (CHIKV), Japanese encephalitis Virus (JEV), Zika virus, Yellow fever virus (YFV), Ussutu virus, tick-borne encephalitis virus (TBEV), coronavirus or a combination thereof; as the case may be, the coronavirus is SARS-CoV-2, SARS -CoV, MERS-CoV or hCoV-OC43. The use according to any one of claims 1 to 4, wherein the composite extract of Tetrachia serrata is an alcohol extract, a hydrated alcohol extract or an aqueous extract. The use according to any one of claims 1 to 4, wherein the composite extract of Tetrachia serrata comprises water, ethanol or a combination thereof. The use according to claim 5, wherein the composite extract of Tetrachia serrata is an aqueous extract. The use of claim 5, wherein the complex extract comprises alcohol and water in a ratio of about 1:99 to 99:1. The use according to any one of claims 1 to 8, wherein the composite extract of Tetrachia serrata accounts for about 50 weight (wt) % to about 80 wt % of the composition. The use as claimed in claim 9, wherein the composite extract of Tetrachia serrata accounts for about 60 wt % to about 70 wt % of the composition. The use as claimed in claim 3, wherein the composite extract of Tetrachia serrata comprises about 0.1 wt % to about 1 wt % magnolanine. The use according to any one of claims 1 or 3 to 11, wherein the pharmaceutically acceptable excipient comprises magnesium aluminum trisilicate, lactose monohydrate, dicalcium phosphate, starch, calcium silicate , microcrystalline cellulose, colloidal silica, croscarmellose, magnesium stearate, or a combination thereof. The use as claimed in any one of claims 1 or 3 to 12, wherein the composition further comprises a film coating, a solvent or a combination thereof. The use as claimed in any one of claims 1 to 13, wherein the composition or the medicament is an oral composition selected from the group consisting of powders, pellets, granules, spheres, mini-lozenges, caplets, lozenges, Capsules, liquids or combinations thereof. The use as described in any one of claims 1 to 14, wherein the composition or the medicament comprises a compound selected from the group consisting of tetrandrine, magnolidine, maxosterone A, sclerophylline, 20-hydroxyecdysone of at least three compounds, wherein the at least three compounds comprise from about 0.1% to about 10% w/w of the composition or the agent. The use as claimed in claim 15, wherein the tetrandrine accounts for about 0.1 wt % to about 20 wt % of the composition or the medicament; the magnolanine is about 0.01 wt % to about 5 wt %; Ketone A is about 0.005 wt% to about 5 wt%; and the 20-hydroxyecdysone is about 0.01 wt% to about 5 wt%. A composition, which comprises: a composite extract of Tetranychus serrata; and an isolated compound selected from the group consisting of tetrandrine, magnolidine, macosterone A, sclerophylline, 20-hydroxyecdysone, A derivative or combination thereof, wherein the ratio of the complex extract to the isolated compound is from about 99:1 to about 1:99. The composition of claim 17, wherein the isolated compound is tetrandrine. The composition of claim 17, wherein the isolated compound is magnolanine. The composition of any one of claims 17 to 19, wherein the ratio of the composite extract to the isolated compound is from about 70:30 to about 30:70. The composition of any one of claims 17 to 20, wherein the ratio of the composite extract to the isolated compound is from about 40:60 to about 60:40. The composition of any one of claims 17 to 20, wherein the ratio of the composite extract to the isolated compound is about 1:1. A composition comprising: Complex extract of tetragoni hardwood; magnesium aluminum trisilicate; a diluent selected from lactose monohydrate, dicalcium phosphate, pregelatinized starch, calcium silicate or a combination thereof; Microcrystalline cellulose; Colloidal silica; Croscarmellose; Magnesium stearate; and Optional film coat. The composition of claim 23, comprising: From about 10 wt% to about 90 wt% of a complex extract of Tetrachia serrata; from about 5 wt% to about 50 wt% of magnesium aluminum trisilicate; from about 0.1 wt% to about 20 wt% of a diluent; from about 0.1 wt% to about 10 wt% of microcrystalline cellulose; about 0.1 wt% to about 10 wt% colloidal silica; from about 0.1 wt% to about 10 wt% of croscarmellose; and From about 0.01 wt% to about 5 wt% of magnesium stearate. The composition of claim 23 or 24, comprising: From about 30 wt% to about 85 wt% of a complex extract of Tetrachia serrata; about 8 wt% to about 40 wt% magnesium aluminum trisilicate; from about 0.5 wt% to about 15 wt% of a diluent; from about 0.5 wt% to about 8 wt% of microcrystalline cellulose; about 0.2 wt% to about 8 wt% colloidal silica; from about 0.5 wt% to about 8 wt% of croscarmellose; and From about 0.02 wt% to about 2 wt% of magnesium stearate. A composition comprising: From about 50 wt% to about 80 wt% of a complex extract of Tetrachia serrata; from about 10 wt% to about 30 wt% of magnesium aluminum trisilicate; from about 1 wt% to about 10 wt% of a diluent selected from lactose monohydrate, dicalcium phosphate, pregelatinized starch, calcium silicate, or combinations thereof; from about 1 wt% to about 5 wt% of microcrystalline cellulose; about 0.5 wt% to about 5 wt% colloidal silica; from about 1 wt% to about 5 wt% of croscarmellose; about 0.1 wt% to about 1 wt% of magnesium stearate; and Optional film coat. The composition according to any one of claims 17 to 26, wherein the composition is an oral pharmaceutical composition. The composition of claim 27, wherein the oral pharmaceutical composition is a powder, pellet, granule, sphere, mini-lozenge, capsule-type lozenge, lozenge, capsule, liquid, or a combination thereof. The composition according to any one of claims 17 to 28, wherein the composite extract of Tetrachia serrata is a composite water extract, a composite primary alcohol extract, or a composite water/alcohol extract. A composition comprising at least three compounds selected from the group consisting of tetrandrine, magnolidine, maksterone A, sclerophylline, 20-hydroxyecdysone, wherein the at least three compounds account for about 0.1% of the composition to about 10%. The composition of claim 28, wherein the tetrandrine accounts for about 0.1 wt % to about 20 wt % of the composition; the magnolanine is about 0.01 wt % to about 5 wt %; the magsterone A is about 0.005 wt% to about 5 wt%; and the 20-hydroxyecdysone is about 0.01 wt% to about 5 wt%. The use as claimed in any one of Claims 1 to 17 or the composition as claimed in any one of Claims 17 to 31, wherein the composite extract of Tetrachia serrata is substantially free of lignin, fiber and Tannins. A method for reducing or inhibiting the proliferation of positive single-stranded RNA viruses in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition of any one of claims 17-32. A method for reducing the viral load of a positive single-stranded RNA virus in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition of any one of claims 17-32. The method of claim 33 or 34, wherein the composition is administered in a dose of about 1 mg/kg to about 500 mg/kg of the complex extract of Tetrachia serrata by weight per individual. A method of treating a positive single-stranded RNA virus infection in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of a complex extract of Fangji sclera. A method of treating a positive single-stranded RNA virus infection in an individual in need, the method comprising administering to the individual a complex extract comprising a therapeutically effective amount of Fangji sclera and a pharmaceutically acceptable excipient (including diluents, binders, disintegrants, lubricants, slip agents, polymers, flavoring agents, surfactants, preservatives, antioxidants, buffers, tonicity modifiers, or combinations thereof). A method of treating a positive single-stranded RNA virus infection in an individual in need, the method comprising administering to the individual a therapeutically effective amount comprising tetrandrine, magnolidine, macosterone A, Uncaria hirsutes Compositions of base, 20-hydroxyecdysone, derivatives thereof, or combinations thereof. The method of claim 38, wherein the composition comprises tetrandrine. The method of claim 38, wherein the composition comprises magnolanine. The method of any one of claims 38 to 40, wherein the composition further comprises pharmaceutically acceptable excipients including diluents, binders, disintegrants, lubricants, slip agents, Polymers, flavors, surfactants, preservatives, antioxidants, buffers, tonicity modifiers, or combinations thereof. The method of any one of claims 36 to 41, wherein the composite extract of Tetrachia serrata is substantially free of lignin, fibers and tannins. The method of any one of claims 33 to 35 or 37 to 42, wherein the composition is administered once a day, twice a day, three times a day, or four times a day . The method of claim 36, wherein the complex extract of Fangji sclera is administered once a day, twice a day, three times a day, or four times a day. The method of any one of claims 33 to 44, wherein the positive single-stranded RNA virus is hepatitis C virus, West Nile virus (WNV), Dengue virus, Chikungunya virus (CHIKV), Japanese encephalitis Virus (JEV), Zika virus, Yellow fever virus (YFV), Ussutu virus, tick-borne encephalitis virus (TBEV), coronavirus or a combination thereof; as the case may be, the coronavirus is SARS-CoV-2, SARS -CoV, MERS-CoV or hCoV-OC43. The method of any one of claims 33 to 45, wherein the virus is SARS-CoV-2, Dengue virus, Chikungunya virus or Japanese encephalitis virus (JEV). The method of any one of claims 33 to 46, further comprising co-administering to the individual an additional therapeutic agent selected from an antiviral agent, an antipyretic agent, an analgesic agent, or a combination thereof. A method for preparing the compound extract of Tetrachia serrata, comprising: a) using a solvent comprising ethanol, water, or both to extract the plant pieces of Tesco sclera, wherein the extraction is performed at about 50°C to about 100°C; b) Concentrate 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, the compound extract of Tessica serrata is obtained. The method of claim 48, further comprising, prior to (c), extracting the concentrated extract of (b) with an additional solvent, wherein the additional solvent is ethanol, water, or both.

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