TW202131918A - Compounds for treatment of dengue infection - Google Patents

Abstract

The present disclosure provides one or more compounds or their pharmaceutical acceptable salts or derivatives for prophylactic and/or curative treatment of an infection caused by Dengue virus. The disclosure also provides a stable pharmaceutical composition comprising one or more compounds or their pharmaceutical acceptable salts or derivatives and extracts for prophylactic and/or curative treatment of an infection caused by Dengue virus. The present disclosure also provides a method for reducing viral load and/or inhibiting growth or proliferation of Dengue virus serotypes in a mammal, the said method comprising administering one or more compounds or their pharmaceutical acceptable salts or derivatives or an extract comprising such compounds, as such or in a pharmaceutically acceptable composition, to a mammal in need thereof.

Description

Compound for the treatment of dengue fever infection

The present invention relates to the use of compounds for the treatment of dengue fever, their pharmaceutically acceptable salts and their derivatives, and their pharmaceutical compositions. These compounds can be separated from nature or can be synthesized by chemical synthesis. Methods of preparing or isolating or extracting these compounds and determining the activity of these isolated compounds against the serotype of dengue virus are also included in the present invention. The present invention also relates to an extract of Cocculus hirsutus used to prevent and treat dengue fever. Cross reference

This patent application claims the priority dates of the Indian Provisional Patent Application No. 201921044606 filed on November 4, 2019 and the Indian Provisional Patent Application No. 202021023699 filed on June 05, 2020.

Dengue fever is globally distributed, mainly distributed in the tropics and subtropics. As the main human arthropod-borne (arbo) viral disease, it is firmly established that the disease is its transmission vector, and the female mosquitoes of the genus Aedes provide native environment. In recent years, the worldwide incidence of dengue fever has increased significantly due to the combination of genetic diversity, geographic origin and distribution differences of different virus serotypes. The disease is currently endemic in more than one hundred countries in Central and South America, the Caribbean, the Mediterranean, Africa, Southeast Asia, and the Western Pacific, putting more than 2.5 billion people at risk of infection.

The pathogen of the infection is the enveloped positive single-stranded RNA virus dengue fever (DENV), which belongs to the genus Flavivirus. In contrast to other named species in the Flaviviridae (each of which is a single type), dengue virus has four clearly identified but very similar serotypes DENV 1-4, which are distinguished by virus plaque reduction neutralization analysis. The existence of multiple DENV serotypes can cause different onsets of mild to malignant diseases. Different dengue serotypes differ in their facilities that cause serious diseases. Today, in order to combat this international public health problem that has reappeared, there is neither a specific anti-dengue vaccine nor an effective approved treatment. The presence of several DENV serotypes in the same neighborhood poses a considerable risk to local residents (Taylor-Robinson, J Clin Diag Treat. 2018; 1 (2): 50-52). Therefore, there is a need for effective preventive and therapeutic treatments for dengue fever.

Commonly known as Jal-Jammi (Chopra et al., 1958) or Broom creeper, Cocculus hirsutus Linn (Menispermeaceae) is found in the cold and hot regions of India; especially Tamil Nadu ( Tamil nadu), Bihar (Bihar) and Punjab (Punjab). The leaves and roots of this plant are mainly used in traditional Indian medicines for various diseases, including liver obstruction, jaundice, bronchitis, diabetes, loss of appetite, gonorrhea and leprosy. Broom vine is documented for its anti-inflammatory, analgesic, anti-diabetic and spermatogenesis activities (Goodla et al., Egyptian Journal of Basic and Applied Sciences, Volume 4, Issue 4, December 2017, Pages 264-269) . Previous research on various parts of the plant led to the isolation of the following: d-tetrandrine, dl-aconitine, β-sitosterol, ginkgol, monomethyl ether of inositol, tetrandrine, aconitine, and magnolia Anthocyanine, camptothecin (hirsudiol), nonane-10-ol, shaheenine (shaheenine), cohirsinine (cohirsinine), sclerotin and cohirsutinine (Satyanarayana et al. , Indian Journal of Pharmaceutical Sciences, 2001, 63(1), 30-35).

The present invention provides certain compounds synthesized or isolated from plants and various extracts from plants to effectively prevent and treat dengue virus diseases.

The present invention relates to the use of compounds, pharmaceutically acceptable salts or derivatives thereof, and pharmaceutical compositions containing them to prevent and treat dengue fever. These compounds or their derivatives can be separated from nature or can be synthesized by chemical synthesis. The present invention also provides methods for preparing or isolating these compounds or their derivatives from plant species of the Menispermaceae family, such as the vine; and their use against dengue virus. It also provides a method for determining the activity of these isolated compounds against dengue virus of different serotypes, so as to identify these compounds as biomarkers in the treatment of dengue fever.

The present invention also provides methods for isolating or extracting these compounds from plants similar to S. sylvestris and determining the activity of these isolated compounds against different serotypes of dengue fever virus. The present invention also provides stable compositions containing one or more of these compounds. It has been found that administering these compounds to mammalian patients infected with dengue virus is highly effective, and is useful in the preventive and curative treatment of infections caused by viruses. It does not exhibit any toxic effects at the required therapeutically effective dose.

The present invention also provides extracts containing these compounds and compositions for the treatment and prevention of dengue fever virus infection. In addition, it provides a stable pharmaceutical composition containing a therapeutically effective amount of the extract, which is suitable for the prevention and/or treatment of dengue virus infection in mammals. The source of biological materials

The biological material disclosed in the present invention is the plant body of the broom vine obtained from Madhya Pradesh, India.

Unless otherwise explicitly stated the terms "such as" and "such as" and their grammatical equivalents, the phrase "and not limited to" should be understood to follow. As used herein, the term "about" means to account for changes due to any experimental error generally accepted in the field of analytical chemistry. Unless expressly stated otherwise, all measurements reported herein should be understood as modified by the term "about", regardless of whether the term is explicitly used. Unless otherwise expressly provided herein, as used herein, the singular forms "a/an" and "the" include plural indicators.

Unless otherwise defined, all technical and scientific terms used in this article have the same meaning as commonly understood by those who are familiar with the technology. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting by any means. All publications, patent applications, patents and other references mentioned in this article are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) will prevail.

式-I                                            式-II

式-III                      式-IV。The present invention relates to the use of compounds, pharmaceutically acceptable salts or derivatives thereof for preventing or treating dengue fever infection. The compound according to the present invention includes a compound of the structural formula provided below:

Formula-I Formula-II

Formula-III Formula-IV.

The compounds according to the present invention used for the prevention or treatment of dengue fever infection are synthesized synthetically, semi-synthetically prepared or isolated from various plant parts of plants through various extraction and isolation methods as disclosed in the present invention.

It was unexpectedly found that the compounds of structural formula I-IV, Sinococuline, magnolia, Makisterone A, and 20-hydroxyecdysone or pharmaceutically acceptable salts, derivatives or combinations thereof as disclosed in the present invention are effective against dengue fever. The virus is effective. Therefore, it has been found that the compounds according to the present invention are suitable for the prevention and treatment of dengue infections alone or in combination.

The effective inhibitory activity of these compounds or their derivatives against different serotypes of dengue virus has not been previously known. However, during the experimental test based on FACS neutralization test (FNT), the compound was found to be effective against dengue virus species. The present inventors found that each of the compound, copper octamide, Sinococuline, magnolia, Makisterone A and 20-hydroxyecdysone is extremely effective in inhibiting the DV4 (dengue fever virus) serotype.

In a specific example, the compound according to the present invention is isolated from an extract of a plant of the Broom vine family. In one aspect, the extract according to the present invention is an extract of a plant belonging to the genus Astragalus of the Broom vine family. In a preferred aspect, the extract according to the present invention is an extract of broom vine. It is a perennial climbing plant and is 2 to 3 m above the ground. The present invention describes an extract of Broom vine, a compound isolated therefrom, and its pharmaceutical composition. When tested in a test tube, it was found to be against all serums of dengue virus DENV-1, DENV-2, DENV-3 and DENV-4 The type is effective.

The present invention provides compounds and/or pharmaceutical compositions thereof isolated from extracts of S. sylvestris, which are used to prevent and treat infections caused by dengue fever virus. The compounds and compositions of the present invention reduce viral load during treatment and provide effective repair against dengue fever virus.

These compounds or their derivatives can be obtained from natural sources or can be synthesized chemically. In a specific example, the compound can be isolated from the plant species. In a preferred embodiment, the plant species is broom vine. Although the jointly-owned Indian application IN201821046412 discloses the use of the extract of Broom vine, the components or their combinations required for its activity against the dengue virus are not known. The inventors have identified and isolated these compounds from the extract of S. sphaerocarpa. Preferably, the extract is an aqueous, organic, and alcoholic or hydroalcoholic extract. More preferably, the extract is an aqueous extract.

In a specific example, the present invention provides a method for isolating compounds, such as Sinococuline, magnoliine, Makisterone A, and 20-hydroxyecdysone or derivatives from broom vine, for the treatment of dengue virus infection. Preferably, the present invention provides a method for separating Sinococuline, magnoliine, Makisterone A, and 20-hydroxyecdysone from extracts of Broom vine used to treat dengue virus infection.

The term "isolated compound" or "isolated compound" means a compound that is substantially separated or enhanced relative to other compounds produced from the compound in nature. The isolated compound is generally at least about 80% by weight, at least about 90% by weight, at least about 98% by weight, or at least about 99% by weight pure. The present invention is also intended to cover diastereomers and their racemic and resolved enantiomerically pure forms and their pharmaceutically acceptable salts.

In yet another specific example, the present invention provides an extract of Broomstick vine for the treatment of dengue virus infection.

As used herein, the term "extract" refers to an extract obtained from the Tentianaceae family, specifically a plant of the genus Tentiana, and more specifically a broom vine. In some specific examples, one or more components of the broom vine are removed during the extraction, for example, the plant body is removed, and other components in the extract are separated and concentrated in the extract. Therefore, the present invention provides an extract used as it is or for separating the compound according to the present invention and a new composition containing the extract or one or more compounds in a concentration and ratio not found in nature. In addition, the present invention provides an extract that does not contain one or more pollutants, which can be effectively used to treat dengue virus infection or can be used to isolate compounds, Sinococuline, magnolia, Makisterone A or 20-hydroxyecdysone or its pharmaceuticals The acceptable salt or derivative. In some specific examples, the extracts described herein also exhibit undiscovered or understood properties in the broom vine plant, such as increased stability, increased effectiveness against dengue virus, increased bioavailability, increased solubility, decreased side effects, etc. .

The extract may exist in one or more physical forms, for example in the form of liquid, semi-solid, solid powder, solid cake, gel, paste, dispersion, solution or distillate. In some specific examples, the extract used for separation according to the present invention may be a purified extract or a crude extract.

In some specific examples, the extract is a purified extract, that is, after the extract is obtained from the plant, the extract is purified by removing one or more contaminants. For example, in some specific examples, methods such as filtration, precipitation, crystallization, chromatography, additional solvent extraction, etc. may be used to purify the extract to remove one or more contaminants. In some specific examples, the extract is a purified and dried extract.

In some specific examples, the extract contains one or more of the following: Sinococuline, magnolia, Makisterone A, or 20-hydroxyecdysone or a combination thereof. In some embodiments, the isolate from the extract contains one or more compounds selected from the group consisting of Sinococuline, magnoliine, Makisterone A or 20-hydroxyecdysone or a combination thereof in any concentration. In some specific examples, the extract can be used to isolate a specific compound selected from the group consisting of Sinococuline, magnoliine, Makisterone A, or 20-hydroxyecdysone. In some specific examples, the extract may optionally be used to separate two compounds selected from the group consisting of Sinococuline, magnolinine, Makisterone A or 20-hydroxyecdysone. In some specific examples, the extract can be used to separate three compounds selected from the group consisting of Sinococuline, magnoliine, Makisterone A, or 20-hydroxyecdysone. In some specific examples, the extract may be used to separate all four compounds selected from the group consisting of Sinococuline, magnoliine, Makisterone A, or 20-hydroxyecdysone.

In some specific examples, the term "extraction" refers to the separation and removal of one or more components of the broom vine, for example, the extraction of plant solids (such as fiber, cellulose, etc.) from one or more fluids in the plant. In some specific examples, extraction is a solid/liquid separation operation: for example, plants are placed in contact with liquid (solvent). In some specific examples, the plant component of interest and the solvent are dissolved into a solution. The solution thus obtained is the desired extract. In some specific examples, the solvent will eventually be removed to obtain an extract. The separation operation may include mechanical methods, such as homogenization; chemical methods, such as acid, alcohol, or aqueous dissolution and heating methods. In some specific examples, extraction includes filtration, precipitation, crystallization, concentration, or centrifugation steps. In a preferred embodiment, the extraction can produce extracts that are very suitable for separating one or more of Sinococuline, magnolinine, 20-hydroxyecdysone, or Makisterone A in a higher yield.

In another specific example, the extract of the present invention is an aqueous extract or an organic solvent extract, wherein the organic solvent is a polar or non-polar organic solvent. In one aspect of the specific example, the extraction is alcoholic extraction, such as C ₁ -C ₄ alcohol extraction, hydroalcoholic extraction, or aqueous extraction.

In one aspect, the extract is an aqueous extract. The solvent in the extract can be completely removed by evaporation to obtain a dry extract, such as those with less than 5% water, less than 4% water, less than 3% water, less than 2% water, or less than 1% water Extracts. The dried extract can be lyophilized, for example to form a powder, which can then be used to isolate the desired compound according to the invention, which compound can then be formed in the presence or absence of pharmaceutically acceptable excipients It is a dosage form having a suitable size, such as a capsule, or compressed into a dosage form suitable for oral administration, such as a lozenge. In related specific examples, the extract can be used as it is in liquid or semi-solid form without further drying, or used to isolate the desired compound according to the present invention with reduced drying. In a specific example, the extract is a purified extract. In another specific example, the extract is a crude extract. In some specific examples, the extract is a concentrated extract, which can be suitably used to isolate a specific compound selected from Sinococuline, magnolinine, Makisterone A or 20-hydroxyecdysone with a significantly higher yield.

In another aspect of the above specific example, the extract is an alcohol extract or a hydroalcoholic extract from a stem or other part of a plant, such as an aerial part or root. In a specific example, the extract will be derived from the moist part of the plant to obtain an aqueous extract. The solvent in the extract can be completely removed, for example, by evaporation to obtain a dry extract. The dried extract can be lyophilized, for example, to form a powder. Such extracts can be used to isolate the desired compound according to the present invention.

The term "alcohol extract" as used herein includes any alcohol-based extract, for example, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutyric acid or tertiary butyric acid extracts .

The term "hydroalcoholic extract" as used herein includes extracts prepared by using a mixture of alcohol and purified water. Hydroalcoholic extracts can also include extracts prepared in denatured alcohol together with other organic solvents. The alcohol may include, for example, C ₁ -C ₄ alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and tert-butanol. The ratio of alcohol to water in the hydroalcoholic extract can be 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: A ratio of 30 to 30:70, or 60:40 to 40:60, or a 1:1 mixture of alcohol and purified water.

The term "aqueous extract" as used herein includes the purified water extract of S. sylvestris.

The extract of S. sylvestris may include, but is not limited to (a) extracts obtained by extracting the plant body of S. sylvestris with one or more solvents (for example, aqueous, alcohol or hydroalcohol), and (b) by using a Or the dissociated fraction obtained by dividing the extract with multiple solvents. In some specific examples, the extract of Broom vine includes (a) an extract obtained by extracting the stem of Broom vine with purified water, and (b) a dissociated fraction obtained by dividing the extract with one or more solvents . Such extracts can then be suitably used to isolate a compound according to the invention selected from the group consisting of Sinococuline, magnolia, Makisterone A or 20-hydroxyecdysone or a pharmaceutically acceptable salt or derivative thereof. In some embodiments, a solvent is used to dissolve the plant component/extract of the present invention in the solution. The solution thus obtained is the required reservoir for the separation of the compound according to the invention. In some specific examples, the solvent will eventually be removed to obtain isolated compounds. The separation operation may include mechanical methods, such as homogenization; chemical methods, such as acid, alcohol, or aqueous dissolution and heating methods. In some specific examples, extraction and separation include filtration, precipitation, crystallization, concentration, or centrifugation steps. In a preferred embodiment, the extraction step can separate one or more of Sinococuline, magnolinine, 20-hydroxyecdysone or Makisterone A in high yield.

The solvent used for extraction and separation can be, for example, water; alcohol, such as methanol, ethanol, propanol, isopropanol, or butanol; ketone, such as acetone or methyl isobutyl ketone; ester, such as methyl acetate or ethyl acetate Esters; halogenated hydrocarbons, such as chloroform, dichloromethane, or ethylene dichloride; petroleum isolates, such as hexane, petroleum ether, or heptane; or mixtures thereof.

The solvent used to separate the components in the extract can be, for example, water; petroleum fractions, such as hexane, petroleum ether, or heptane; halogenated hydrocarbons, such as chloroform, dichloromethane, or dichloroethylene; esters, such as acetic acid Ethyl or methyl acetate; ketones, such as acetone or methyl isobutyl ketone; alcohols, such as butanol; ethers, such as diethyl ether; or mixtures thereof.

In some specific examples, various parts of the broom vine can be used, for example, the extraction can be performed from the stem or other parts of the plant, such as the aerial parts or roots. The term "plant body of broom vine" as used herein refers to the entire plant, which includes above-ground parts such as fruits, flowers, leaves, branches, bark, stems, seeds or heartwood and roots. In a preferred embodiment, "the plant body of the broom vine" refers to the stem of the broom vine. In some specific examples, the compound according to the present invention can be directly separated from the plant body using a suitable solvent without drying the extract after extraction.

In one aspect, before separating a specific extract, the extract may be concentrated and standardized with respect to labeled compounds, such as Sinococuline, magnoliine, Makisterone A, 20-hydroxyecdysone, or a combination thereof. This type of concentrated extract can be used to separate each labeled compound with a higher yield using a separate solvent for the labeled compound. The solvent can be any of the solvents as disclosed above.

The extract according to the present invention is suitable for direct administration to mammals and for the preparation of pharmaceutical compositions, or can be used to isolate compounds according to the present invention. The dosage of the extract may be in the range of about 0.01 mg/kg to about 1500 mg/kg of body weight, specifically about 0.05 mg/kg to about 1200 mg/kg of body weight, more specifically about 0.1 mg/kg To about 500 mg/kg body weight, more specifically about 1 mg/kg to about 150 mg/kg body weight. Preferably, the dosage of the extract may be in the range of about 2 mg/kg to about 70 mg/kg of body weight. The compound extract or its composition can be administered once, twice, three times or four times a day.

In a specific example, the extract of S. chinensis contains one or more ingredients selected from the group consisting of flavonoids, resin brains, steroids and alkaloids or combinations thereof. Preferably, the extract of Broom vine contains Sinococuline, magnolia base, Makisterone or 20-hydroxyecdysone as one of the ingredients. More preferably, the extract of S. chinensis contains magnoliine in an amount of 0.1% to 1% of the total weight of the extract in the composition. In a preferred embodiment, the extract of S. chinensis contains magnolia in an amount of 0.45% of the total weight of the extract in the composition. In another aspect of the specific example, the compound extract of S. serrata contains quercetin as one of the flavonoids.

The term "mammal" as used herein refers to all mammals including humans. Mammals include, by way of example only, humans, non-human primates, cows, dogs, cats, goats, sheep, pigs, rats, mice, and rabbits.

Recovery from infection with a dengue serotype provides lifetime immunity against that specific serotype. However, the cross-immunity to other serotypes after recovery is only partial and temporary. Subsequent infections (secondary infections) of other serotypes increase the risk of severe dengue fever. Therefore, the compounds of the present invention provide advantages over treatments known in the art that are effective only for specific serotypes, because it was unexpectedly found that the compounds, their pharmaceutically acceptable salts, derivatives, and compositions are all against dengue virus. Serotypes, namely DENV-1, DENV-2, DENV-3 and DENV-4 are active.

In a specific example, the present invention provides a method for preventing and treating dengue fever infection by administering to a mammal a therapeutically effective amount of one or more selected from Sinococuline, magnolinine, Makisterone A or 20-hydroxyecdysone Or its combination or pharmaceutically acceptable salt or derivative compound. The therapeutically effective amount of the compound used for the treatment of dengue fever can be determined by those familiar with the technology, for example, by observation of the patient and the physician, including the patient's general health status, response to therapy, and the like. In one aspect of the specific example, the present invention provides a method for preventing and treating dengue fever infection by administering a therapeutically effective amount of Sinococuline. In another aspect of the specific example, the present invention provides a method for preventing and treating dengue fever infection by administering a therapeutically effective amount of magnoliine. In another aspect of the specific example, the present invention provides a method for preventing and treating dengue fever infection by administering a therapeutically effective amount of Makisterone A. In another aspect of the specific example, the present invention provides a method for preventing and treating dengue fever infection by administering an effective amount of 20-hydroxyecdysone.

The terms "therapeutically effective amount" and "effective amount" are used interchangeably herein and can be referred to as being sufficient for the intended use, including but not limited to the administration of compounds for the treatment of dengue virus infection or the treatment of diseases or conditions associated with dengue virus infection ( It can be called reagent, medicinal agent or drug and included in composition or medical composition). For example, an effective amount of the compound according to the present invention reduces one or more of the symptoms or the symptoms of the disease being treated, and/or the amount is related to the risk that the host being treated has or produces, such as prevention to a certain extent Fever, pain, headache, joint pain, or other symptoms of illness. The term also applies to the dose that triggers a specific response in the target cell. The specific dosage will depend on the following: the specific compound selected, the protocol to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the body delivery system that carries it.

In a specific example, the present invention provides a method for preventing and treating dengue fever infection by administering an effective amount of one or more compounds selected from the group consisting of formula I-IV or a combination or derivative thereof, wherein one of these compounds Co-administration of two or more of them produces a synergistic effect against dengue fever virus. In one aspect of the specific example, the co-administration of Sinococuline (Formula I) and Magnoline (Formula II) produces a synergistic effect against dengue fever virus. In another aspect of the specific example, co-administration of Sinococuline and Makisterone A (Formula III) produces a synergistic effect against dengue fever virus. In another aspect of the specific example, co-administration of Sinococuline and 20-hydroxyecdysone (Formula IV) produces a synergistic effect against dengue fever virus. In another aspect of the specific example, co-administration of magnoline and Makisterone A caused a synergistic effect against dengue fever virus. In another aspect of the specific example, co-administration of magnoline and 20-hydroxyecdysone produces a synergistic effect against dengue fever virus. In another aspect of the specific example, co-administration of Makisterone A and 20-hydroxyecdysone produces a synergistic effect against dengue fever virus. In another aspect of the specific example, the co-administration of Sinococuline, magnolinine and Makisterone A produces a synergistic effect against dengue fever virus. In another aspect of the specific example, the co-administration of Sinococuline, magnolin and 20-hydroxyecdysone produces a synergistic effect against dengue fever virus. In another aspect of the specific example, co-administration of Sinococuline, Makisterone A, and 20-hydroxyecdysone produces a synergistic effect against dengue fever virus. In another aspect of the specific example, co-administration of magnoline, Makisterone A, and 20-hydroxyecdysone produces a synergistic effect against dengue fever virus.

The term "derivative" herein refers to a chemically or biologically modified version of the compound described above, that is, structurally similar to and (actually or theoretically) derived from the parent compound. For example, hydrogen can be replaced by halogen, or hydroxyl (-OH) can be replaced by carboxylic acid moiety (-COOH) or ester and amide. The term "derivative" also includes conjugates and prodrugs of the parent compound (that is, chemically modified derivatives that are converted into the original compound under physiological conditions). The term "derivative" is also used to describe all solvates, such as hydrates or adducts of compounds (such as adducts with alcohols), active metabolites and salts.

The term "co-administration" herein refers to the administration of two or more compounds to a mammal. The two or more compounds may be in the form of a single pharmaceutical composition, or may be in the form of separate pharmaceutical compositions. Each of the two or more compounds can be administered via the same or different routes of administration. Co-voting covers simultaneous or sequential voting.

The term "synergistic" as used herein refers to a combination of two or more compounds of the present invention, which when combined are more effective than the additive effects of individual compounds.

In another specific example, the present invention provides a pharmaceutical composition for the treatment of dengue fever virus infection in mammals, which comprises Sinococuline, magnoline, Makisterone A or 20-hydroxyecdysone or a derivative or combination thereof. The pharmaceutical composition according to the present invention is a pharmaceutical composition suitable for intestinal, such as oral or rectal, transdermal, topical, and parenteral administration to mammals, including humans. In a preferred embodiment, the present invention provides an oral pharmaceutical composition for the treatment of dengue virus infection, which comprises Sinococuline, magnolia, Makisterone A or 20-hydroxyecdysone or a derivative or combination thereof and one or more medicines. Academically acceptable excipients. The oral pharmaceutical composition may be in the form of powder, pellets, granules, mini-tablets, lozenges, capsules or liquids, including but not limited to solutions, suspensions, emulsions or syrups. As used herein, the term "pharmaceutically acceptable excipients" includes diluents, binders, disintegrants, lubricants, slip agents, polymers, flavoring agents, surfactants, preservatives, antioxidants, Buffer and tension regulator. In one aspect of the specific example, the present invention is an oral pharmaceutical composition containing Sinococuline for the treatment of dengue fever virus infection. In another aspect of the specific example, the present invention provides an oral pharmaceutical composition containing magnoline for treating dengue fever virus infection. In another aspect of the specific example, the present invention provides an oral pharmaceutical composition containing Makisterone A for the treatment of dengue fever virus infection. In another aspect of the specific example, the present invention provides an oral pharmaceutical composition containing 20-hydroxyecdysone for the treatment of dengue fever virus infection.

In another specific example, the present invention provides a method for preventing and treating dengue fever infection by administering an effective amount of one or more pharmaceutical compositions selected from the group consisting of: Sinococuline, magnoliine, Makisterone A or 20-Hydroxyecdysone or its combination or pharmaceutically acceptable salt or derivative. The pharmaceutical composition is as described above. In one aspect of specific examples, the present invention provides a method for preventing and treating dengue fever infection by administering an effective amount of a pharmaceutical composition containing Sinococuline. In another aspect of the specific example, the present invention provides a method for preventing and treating dengue fever infection by administering an effective amount of a pharmaceutical composition containing magnoliine. In another aspect of the specific example, the present invention provides a method for preventing and treating dengue fever infection by administering an effective amount of a pharmaceutical composition containing Makisterone A. In another aspect of the specific example, the present invention provides a method for preventing and treating dengue fever infection by administering an effective amount of a pharmaceutical composition containing 20-hydroxyecdysone.

In another specific example, the present invention provides compounds for the treatment of dengue virus infection in mammals, such as Sinococuline, magnolin, Makisterone A or 20-hydroxyecdysone or a pharmaceutically acceptable salt or derivative thereof Or a combination, where the compounds exhibit platelet protection.

In another specific example, the present invention provides compounds such as Sinococuline, magnolinine, Makisterone A and 20-hydroxyecdysone or derivatives or combinations thereof to reduce the viral load in the early stage of treatment of dengue virus infection in mammals.

In another specific example, the present invention provides compounds such as Sinococuline, magnolinine, Makisterone A and 20-hydroxyecdysone or a pharmaceutically acceptable salt or derivative or combination thereof for the treatment of dengue fever virus in mammals. The initial stage of infection reduces viral load, where these compounds exhibit platelet protection.

In another specific example, the present invention provides a pharmaceutical composition comprising one or more compounds selected from the group consisting of Sinococuline, magnoliine, Makisterone A and 20-hydroxyecdysone for the treatment of dengue fever virus infection in mammals Or a pharmaceutically acceptable salt or derivative or combination thereof, and one or more pharmaceutically acceptable excipients, wherein the compounds exhibit platelet protection. Preferably, the pharmaceutical composition is an oral pharmaceutical composition.

In another specific example, the present invention provides a pharmaceutical composition comprising Sinococuline, magnolia, Makisterone A and 20-hydroxyecdysone or a derivative or combination thereof and one or more pharmaceutically acceptable excipients , To reduce the viral load in the early stage of treatment of dengue virus infection in mammals. Preferably, the pharmaceutical composition is an oral pharmaceutical composition. In one aspect of a specific example, the present invention provides a pharmaceutical composition comprising Sinococuline and one or more pharmaceutically acceptable excipients to reduce the viral load in the early stage of treatment of dengue virus infection in mammals. In another aspect of the specific example, the present invention provides a pharmaceutical composition comprising magnoline and one or more pharmaceutically acceptable excipients to reduce the virus in the early stage of treatment of dengue fever virus infection in mammals load. In another aspect of the specific example, the present invention provides a pharmaceutical composition comprising Makisterone A and one or more pharmaceutically acceptable excipients to reduce the viral load in the early stage of treatment of dengue virus infection in mammals . In another aspect of the specific example, the present invention provides a pharmaceutical composition comprising 20-hydroxyecdysone and one or more pharmaceutically acceptable excipients to treat dengue fever virus infection in mammals in the early stage Reduce virus load.

In another specific example, the present invention provides a pharmaceutical composition comprising Sinococuline, magnoliine, Makisterone A, 20-hydroxyecdysone or a derivative or combination thereof, and one or more pharmaceutically acceptable excipients , In order to reduce the viral load in the early stage of treatment of dengue virus infection in mammals, wherein these compounds exhibit platelet protection. Preferably, the pharmaceutical composition is an oral pharmaceutical composition. In one aspect of a specific example, the present invention provides a pharmaceutical composition comprising Sinococuline and one or more pharmaceutically acceptable excipients to reduce the viral load in the early stage of treatment of dengue virus infection in mammals, wherein These compounds exhibit platelet protection. In another aspect of the specific example, the present invention provides a pharmaceutical composition comprising magnoline and one or more pharmaceutically acceptable excipients to reduce the virus in the early stage of treatment of dengue fever virus infection in mammals Load, where these compounds exhibit platelet protection. In another aspect of the specific example, the present invention provides a pharmaceutical composition comprising Makisterone A and one or more pharmaceutically acceptable excipients to reduce the viral load in the early stage of treatment of dengue virus infection in mammals , Where these compounds exhibit platelet protection. In another aspect of the specific example, the present invention provides a pharmaceutical composition comprising 20-hydroxyecdysone and one or more pharmaceutically acceptable excipients to treat dengue fever virus infection in mammals in the early stage Reduce viral load, where these compounds exhibit platelet protection.

In another specific example, the present invention provides a pharmaceutical composition comprising Sinococuline, magnoliine, Makisterone A and 20-hydroxyecdysone or derivatives or combinations thereof, wherein the compounds are selected from DENV-1, DENV -2. One or all of the dengue virus serotypes of DENV-3 and DENV-4 exhibit inhibitory activity. Preferably, the compound exhibits inhibitory activity against all dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4. Preferably, the pharmaceutical composition is an oral pharmaceutical composition. In one aspect of specific examples, the present invention provides a pharmaceutical composition comprising Sinococuline, wherein the pharmaceutical composition is selected from one of the dengue virus serotypes selected from DENV-1, DENV-2, DENV-3, and DENV-4 Or all exhibit inhibitory activity. In another aspect of the specific example, the present invention provides a pharmaceutical composition comprising magnoliine, wherein the pharmaceutical composition is selected from the group of dengue virus serotypes DENV-1, DENV-2, DENV-3, and DENV-4 One or all exhibit inhibitory activity. In another aspect of the specific example, the present invention provides a pharmaceutical composition comprising Makisterone A, wherein the pharmaceutical composition is selected from DENV-1, DENV-2, DENV-3, and DENV-4. One or all exhibit inhibitory activity. In another aspect of the specific example, the present invention provides a medical composition comprising 20-hydroxyecdysone, wherein the medical composition is selected from DENV-1, DENV-2, DENV-3 and DENV-4. One or all of the serotypes exhibit inhibitory activity.

In a specific example, the present invention relates to the use of these compounds and their derivatives as biomarkers in the treatment of dengue fever. Currently, there are no routine laboratory biomarkers that can predict the severity of dengue fever infection or monitor the effectiveness of standard management (Soo, Kuan-Meng et al. "Meta-analysis of biomarkers for severe Dengue infections." PeerJ, 2017, No. 5 Volume e3589). Due to the discovery of the efficacy of the compounds of the present invention on the dengue virus, the content of the compounds of the present invention in a given treatment can therefore be used to predict the effectiveness of the treatment. Different pharmacokinetic parameters, such as the concentration, elimination rate, and half-life of the compound or derivative in the serum or plasma of dengue fever patients, can be used to optimize the pharmacodynamic response of these compounds or derivatives in a given patient. Therefore, these compounds or their derivatives serve as biomarkers to predict effectiveness in patients with dengue fever. In addition, the content of these compounds in a given plant extract can serve as an indicator of the efficacy of the extract.

式-I                       式-II

或

式-III                       式-IV 或其醫藥學上可接受之鹽，用於治療登革熱病毒感染。In a specific example, the present invention provides a compound selected from formula I-IV:

Formula-I Formula-II

or

Formula-III Formula-IV or a pharmaceutically acceptable salt thereof is used for the treatment of dengue fever virus infection.

In one aspect, the present invention provides a method of treating dengue virus infection, the method comprising administering to a mammal in need a therapeutically effective amount of one or more compounds selected from formula I-IV. In another aspect, the treatment method is selected from preventive treatment or curative treatment in mammals. In one aspect, the compound according to the present invention or the method for treating dengue virus infection is effective against one or more serotypes of dengue virus selected from DENV-1, DENV-2, DENV-3, and DENV-4. In yet another aspect, the treatment is characterized by a reduced titer of dengue virus in a tissue sample of the mammal. In one aspect, the compound used or the treatment method according to the present invention, wherein the compound is a synthetic compound, a semi-synthetic compound, or a compound isolated from an extract of the plant Broom vine.

In another embodiment, the present invention provides a method for inhibiting the growth and/or proliferation of dengue virus in mammals by administering a therapeutically effective amount of one or more compounds selected from formula I-IV. In one aspect, according to the treatment method of the present invention, the inhibition of the growth and/or proliferation of the dengue virus is determined by measuring the protective effect of platelets. In one aspect, the treatment method according to the present invention in which the growth and/or proliferation of dengue virus is inhibited occurs in a test tube or in vivo.

In some embodiments, the present invention provides a stable pharmaceutical composition comprising a therapeutically effective amount of one or more compounds selected from formula I-IV, wherein the composition exhibits a platelet protective effect. In another aspect, the present invention provides a stable pharmaceutical composition comprising a therapeutically effective amount of two or more compounds selected from formula I-IV.

In some specific examples, the present invention provides a compound selected from the structure of Formula I-IV or a pharmaceutically acceptable salt thereof for the treatment or prevention of dengue fever virus infection. In one aspect, the compounds according to the invention are used to treat dengue virus infections in mammals. In another aspect, the compounds of formula I-IV according to the present invention may be provided in the form of a composition containing one or more of these compounds.

In another specific example, the present invention provides a stable pharmaceutical composition for the treatment or prevention of dengue virus infection, which contains an extract of S. sylvestris. Compared with S. sylvestris, the extract contains at least one of the 400% increase in concentration or A variety of compounds of formula I to IV. In one aspect according to the present invention, about 95% of the fiber is removed from the extract. In another aspect according to the present invention, about 95% of lignin is removed from the extract. In another aspect according to the present invention, about 95% of tannin is removed from the extract. In one aspect according to the invention, the pharmaceutically acceptable extract contains a therapeutically effective amount of one or more of the compounds of formula I-IV. In one aspect, the pharmaceutically acceptable extract is formed by extraction with an aqueous solution. In another aspect, the pharmaceutically acceptable extract according to the present invention comprises compounds of formula I-IV, and these compounds are stable at 25° C. under 75% relative humidity for at least 3 months. In one aspect, the extract has less than 5% water. In one aspect, the extract is in the form of a solid oral dosage form.

In a specific example, the present invention provides a pharmaceutical composition comprising one or more compounds of formula I-IV, wherein the one or more compounds are stable at 25° C. and 75% relative humidity for at least 3 months. In another embodiment, the present invention provides a pharmaceutical composition comprising one or more compounds of formula I-IV and pharmaceutically acceptable excipients, wherein the one or more compounds are at 25° C. and 75% relative humidity Stable for at least 3 months. In one aspect, the composition has less than 5% water. In one aspect, the composition is a solid oral dosage form.

In another embodiment, the present invention provides a method of treating dengue virus infection, the method comprising administering to a mammal in need a therapeutically effective amount of one or more compounds of formula I to IV or the pharmaceutically acceptable compound of the present invention Accepted extracts, or pharmaceutical compositions containing compounds I-IV or extracts as currently disclosed. In one aspect, the treatment method is selected from preventive treatment and curative treatment. In another aspect, the treatment method for dengue virus infection, wherein the infection is caused by one or more serotypes of dengue virus selected from DENV-1, DENV-2, DENV-3, and DENV-4. In one aspect, the treatment method is characterized in that the titer of the dengue virus in the mammalian tissue sample is reduced.

In another specific example, the present invention provides a method for inhibiting the growth and/or proliferation of dengue virus in a mammal, the method comprising administering a therapeutically effective amount of one or more of the compounds of formula I-IV of the present invention, the present invention The pharmaceutically acceptable extract of the invention or a pharmaceutical composition comprising one or more compounds or extracts according to the invention.

In another embodiment, the present invention provides a method for providing platelet protection to an individual in need, the method comprising administering a therapeutically effective amount of one or more compounds of formula I-IV according to the present invention, and the pharmaceuticals of the present invention An acceptable extract or a pharmaceutical composition comprising one or more compounds or extracts according to the present invention.

In some specific examples, the isolated compounds of formula I-IV Sinococuline, magnoliine, Makisterone A, 20-hydroxyecdysone are present in the composition according to the present invention in the following weight percentages: WT% Sinococuline 0.01%-20% 0.05%-15% 0.5%-10% 1%-5% Magnolia 0.01%-10% 0.03%-3% 0.1%-2% 0.3%-1% Makisterone-A 0.005%-10% 0.01%-3% 0.05%-2% 0.2%-1% 20-hydroxyecdysone 0.01%-10% 0.05%-3% 0.2%-2% 0.3%-1%

In a specific example, the present invention provides a stable pharmaceutical composition comprising one or more compounds selected from the group consisting of Sinococuline, magnolinine, Makisterone-A and 20-hydroxyecdysone in one or more of the following ratios (by weight): ratio Sinococuline Magnolia Makisterone-A 20-hydroxyecdysone Ratio 1 1-15 1-15 1-15 1-15 Ratio 2 1 1-5 1-5 1-5 Ratio 3 1-5 1 1-5 1-5 Ratio 4 1-5 1-5 1 1-5 Ratio 5 1-5 1-5 1-5 1 Ratio 6 3-5 1-2 1-2 1-2

In some specific examples, the composition may include two labeled compounds in the following ratios (by weight), such as Sinococuline and magnoliine: ratio Sinococuline Magnolia Ratio 1 1-15 1-15 Ratio 2 1 1-5 Ratio 3 1-5 1 Ratio 4 3-5 1-2

In some specific examples, the extract may contain two labeling compounds, such as Sinococuline and Makisterone-A in the following ratios (by weight): ratio Sinococuline Makisterone-A Ratio 1 1-15 1-15 Ratio 2 1 1-5 Ratio 3 1-5 1 Ratio 4 3-5 1-2

In some specific examples, the extract may contain two labeled compounds in the following ratios (by weight), such as Sinococuline and 20-hydroxyecdysone: ratio Sinococuline 20-hydroxyecdysone Ratio 1 1-15 1-15 Ratio 2 1 1-5 Ratio 3 1-5 1 Ratio 4 3-5 1-2

In some specific examples, the extract may contain two labeled compounds in the following ratios (by weight), such as magnoline and Makisterone-A: ratio Magnolia Makisterone-A Ratio 1 1-15 1-15 Ratio 2 1 1-5 Ratio 3 1-5 1

In some specific examples, the extract may contain two labeled compounds in the following ratios (by weight), such as magnoline and 20-hydroxyecdysone: ratio Magnolia 20-hydroxyecdysone Ratio 1 1-15 1-15 Ratio 3 1 1-5 Ratio 4 1-5 1

In some specific examples, the extract may contain two labeled compounds in the following ratios (by weight), such as Makisterone-A and 20-hydroxyecdysone: ratio Makisterone-A 20-hydroxyecdysone Ratio 1 1-15 1-15 Ratio 3 1 1-5 Ratio 4 1-5 1

In some specific examples, the compounds of formulae I-IV according to the present invention, Sinococuline, orchidine, Makisterone-A or 20-hydroxyecdysone are present in the composition in a concentration range of 0.1-100% w/w, respectively, and in the combination In the composition, it exists in different ratios of (0.5 to 99):(0.05 to 99):(0.01 to 99):(0.05 to 99).

In some specific examples, one or more compounds according to the present invention can be administered in a therapeutically effective amount with or without a suitable vehicle to treat or prevent dengue virus infection.

In some embodiments, one or more compounds according to the present invention can be administered in a therapeutically effective amount in a suitable dosage form together with one or more pharmaceutically acceptable excipients for the treatment or prevention of dengue virus infection.

In another embodiment, the present invention provides a pharmaceutical composition comprising one or more compounds as disclosed for use in the preventive and curative treatment of infections caused by dengue fever virus.

In one aspect, the pharmaceutical composition according to the present invention is an oral dosage form selected from powder, pellets, granules, spheroids, mini-tablets, capsules, lozenges or capsules.

In another aspect, the present invention provides a pharmaceutical composition of the compound according to the present invention for parenteral administration.

In another specific example, the present invention provides a pharmaceutical composition for the prevention and treatment of dengue fever virus infection in mammals, which comprises an extract of broom vine and one or more pharmaceutically acceptable excipients. In one aspect of the above specific examples, the present invention provides a stable pharmaceutical composition for the prevention and treatment of dengue fever virus infection in mammals, the pharmaceutical composition comprising a therapeutically effective amount of extract of broom vine or one or more of An isolated compound in which the composition reduces the viral load when administered to a mammal in need.

The stable pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable excipients. The term "medical composition" as used herein includes any composition that can effectively deliver the compound according to the present invention to the desired site of action to treat or prevent dengue virus infection. The composition can be delivered by any suitable route of administration, such as oral, nasal, pulmonary, transdermal, or transrectal. The pharmaceutical composition includes one or more pharmaceutically acceptable excipients. The oral pharmaceutical composition may be in the form of powder, pellets, granules, spheroids, mini-tablets, capsules, lozenges or capsules. The powder may be in the form of a lyophilized powder filled with a pharmaceutically acceptable excipient into a capsule of a suitable size. Preferably, the pharmaceutical composition is in the form of a lozenge. Oral pharmaceutical compositions can exist in liquid form, including but not limited to solutions, suspensions, emulsions, or syrups.

In another specific example, the pharmaceutical composition containing the compound according to the present invention is an oral dosage form selected from powder, pellets, granules, spheroids, mini-tablets, capsules, lozenges or capsules. In one aspect, the pharmaceutical composition containing one or more compounds according to the present invention is under accelerated conditions of 40±2°C/75±5% RH, long-term storage conditions of 30±2°C/75±5% RH, or Stable storage at 25±2℃/75±5% RH for at least 3 months. The product can be stored at room temperature for a storage period of 6 months to 2 years. It is believed that this is unexpected, because the composition contains isolated compounds or extracts containing flavonoids, alkaloids, such as magnolia, resin brain, etc., as ingredients, and it is challenging to prepare a stable composition containing these ingredients . The stability of the composition can be evaluated by measuring some parameters, such as the analysis of one or more labeled substances during storage. In a preferred embodiment, the label is a compound of formula II. In addition, loss on drying (LOD) and disintegration time (in the tablet composition) after storage can serve as a measure of the stability of the composition. For stable compositions, the specification of LOD is determined to be no more than 2-5% w/w of NMT of the composition.

"Stable pharmaceutical composition" as used herein refers to a composition that is stable during storage for an extended period of time as assessed by the content of one or more impurities in the composition as described in standard textbooks. It is found that the stable pharmaceutical composition of the present invention is stable for at least 3 months under accelerated conditions of 40±2°C/75±5% RH; and under long-term storage conditions of 30±2°C/75±5% RH and at 25± Stable for at least 3 months under 2℃/75±5% RH. Alternatively, the product can be stored at room temperature for a storage period of 6 months to 2 years.

When the composition of the present invention was stored in an HDPE bottle under accelerated conditions of 40°C/75% RH for 6 months, no degradation of the content of the compound of formula II was shown, and it was found that the LOD was in the range of 2-5% w/w And there is no change in disintegration time. In one aspect of the present invention, the pharmaceutical composition comprising one or more compounds according to the present invention comprises selected from diluents, binders, disintegrants, lubricants, gliding agents, polymers, flavoring agents, and surfactants One or more of the pharmaceutically acceptable excipients of, preservatives, antioxidants, buffers and tonicity regulators.

As used herein, the term "pharmaceutically acceptable excipients" includes diluents, binders, disintegrants, lubricants, slip agents, polymers, flavoring agents, surfactants, preservatives, antioxidants, Buffer and tension regulator.

Non-limiting examples of diluents include microcrystalline cellulose, powdered cellulose, starch, pregelatinized starch, glucose binders, lactitol, fructose, compressible sugar, powdered sugar, dextrose, anhydrous lactose, two Primary calcium phosphate, ternary calcium phosphate, calcium sulfate and mixtures thereof.

Non-limiting examples of binding agents include water-soluble starch, such as pregelatinized starch; polysaccharides, such as agar, acacia, dextrin, sodium alginate, tragacanth, sanxian gum, hyaluronic acid, pectin or chondroitin sulfate Sodium; synthetic polymers, such as polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymers, polyacrylic acid series polymers, polylactic acid or polyethylene glycol; cellulose ethers, such as methyl cellulose, ethyl fiber Cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose or hydroxypropyl methyl cellulose; and mixtures thereof.

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

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

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

Non-limiting examples of surfactants include anionic surfactants, such as sulfonic acids or their salts, such as benzenesulfonic acid, dodecylbenzenesulfonic acid, or dodecylsulfonic acid; alkyl sulfates, such as dodecane Sodium sulfate or sodium lauryl sulfate; cationic surfactants, such as tetraalkylammonium salts, such as tetraalkylammonium halides, benzethonium chloride, benzalkonium chloride or cetylpyridinium chloride; nonionic surface activity Agents, such as (poly)oxyethylene sorbitan long-chain fatty acid esters, such as polyoxyethylene sorbitan monolaurate, such as polysorbate; amphoteric surfactants, such as glycine compounds, such as ten Dialkyl-di-(aminoethyl)glycine; betaine compounds, such as betaine or dimethyldodecylcarboxybetaine; and phospholipid derivatives, such as lecithin; polymeric surfactants , For example polyoxyethylene polyoxypropylene glycol, such as Pluronic® or poloxamer; 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, and mixtures thereof.

Non-limiting examples of solvents used in the preparation of pharmaceutical compositions include water; water-miscible organic solvents such as isopropanol or ethanol; dipolar aprotic solvents; methylene chloride; acetone; polyethylene glycol; polyethylene glycol Alcohol ethers; polyethylene glycol derivatives of monoglycerides or diglycerides; buffers; organic solvents; and combinations thereof.

The pharmaceutical excipients used in the present invention can be used interchangeably for various functions in the pharmaceutical composition, and are not limited by their applications as widely known. For example, a diluent can be used as a binding agent at a specific concentration.

In a preferred embodiment, the pharmaceutically acceptable excipients in the composition of the present invention include microcrystalline cellulose, anhydrous lactose, croscarmellose sodium, colloidal silica and stearic acid magnesium.

In another embodiment, the present invention provides a stable pharmaceutical composition comprising a therapeutically effective amount of one or more compounds of the present invention for the treatment of dengue virus infection in mammals, wherein the composition is administered to a mammal in need Time to reduce the virus load.

In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds according to the present invention and one or more pharmaceutically acceptable excipients, so as to treat dengue fever in mammals. Reduce the virus load in the early stage of virus infection. Preferably, the composition is a stable pharmaceutical composition. More preferably, the composition is a stable oral pharmaceutical composition.

In another embodiment, the present invention provides a stable pharmaceutical composition comprising a therapeutically effective amount of one or more of the compounds of the present invention for the prevention of dengue fever virus infection in mammals.

The term "treatment" as used in the context of the present invention is intended to include therapeutic treatments, including prophylactic or preventive and curative treatments. For example, the term treatment may include administering a therapeutically effective amount of one or more of the compounds or compositions according to the invention or in combination with any other standard care before or after the onset of symptoms, thereby preventing or removing a disease or condition or by Signs and symptoms caused by dengue virus infection. As another example, the administration of a compound or pharmaceutical composition before or after the clinical manifestation of dengue virus infection to prevent or combat the signs and symptoms and/or complications and conditions associated with dengue virus infection includes the "treatment" of the disease. In addition, in the case where symptoms affecting the disease or condition have appeared and may improve the disease, before or after the onset or when clinical symptoms and/or complications have appeared, one or more compounds according to the present invention or The composition or in combination with any standard of care includes the "treatment" of dengue virus infection. In a specific example, the treatment of the individual includes inducing and maintaining remission of the individual's dengue virus infection. In another specific example, treating a dengue virus infection in the individual includes maintaining remission of the dengue virus in the individual.

In one of the specific examples, the present invention provides a method for treating dengue virus infection in mammals, which comprises administering a compound of formula I, II, III or IV or a pharmaceutically acceptable salt or derivative thereof Or combination.

In another embodiment, the present invention provides a method for preventing dengue virus infection in mammals, which comprises administering a compound of formula I, II, III or IV or a pharmaceutically acceptable salt or derivative thereof or combination.

In another embodiment, the present invention provides a method for treating dengue virus infection in mammals, which comprises administering a compound of formula I, II, III or IV or a pharmaceutically acceptable salt or derivative or combination thereof , Where these compounds exhibit platelet protection.

In another specific example, the present invention provides a method for reducing the viral load in the early stage of treatment of dengue virus infection in mammals, which comprises administering a compound of formula I, II, III or IV or its pharmaceutically acceptable The salt or derivative or combination.

In another specific example, the present invention provides a method for reducing the viral load in the early stage of treatment of dengue virus infection in mammals, which comprises administering a compound of formula I, II, III or IV or a pharmaceutically acceptable compound thereof Salts or derivatives or combinations thereof, wherein these compounds exhibit platelet protection.

The appropriate dose of the compound or its derivative or combination or extract used for the treatment of dengue fever can be determined by those who are familiar with the technology, such as observations by the patient and physician, including the patient's overall health, response to therapy and the like .

In a specific example, the present invention provides a method for treating dengue fever virus infection in mammals, which comprises administering a medical composition to a mammal in need, the medical composition comprising administering a therapeutically effective amount of an extract of broom vine , Where the extract reduces the viral load.

In yet another embodiment, the present invention provides a method for treating dengue fever virus infection in mammals, which comprises administering to a mammal in need a pharmaceutical composition comprising a therapeutically effective amount of an extract of S. sylvestris, wherein the extract Shows platelet protection. It was found that when oral gavage was administered to Winstar rats at a dose of 600 or 1200 mg/kg/day for 14 days, higher platelet counts were observed.

In another specific example, the present invention provides a method for treating dengue fever virus infection in mammals, which comprises administering a complex extract of broom vine to mammals in need, wherein the extract exhibits platelet protection.

In another embodiment, the present invention provides a method for treating dengue fever virus infection in mammals, which comprises administering to a mammal in need a medical composition comprising a therapeutically effective amount of an extract of S. sylvestris, wherein the extract Reduce the viral load, and wherein the extract further exhibits platelet protection.

In another specific example, the present invention provides a method of treating dengue virus infection in mammals, which comprises administering a complex extract of broom vine to mammals in need, wherein the extract is reduced not only from serum but also from such as But it is not limited to the viral load of the intestinal tissues, and the extract further exhibits platelet protection.

In yet another specific example, the present invention provides a method for reducing viral load in the early stage of treatment of dengue fever virus infection in mammals, which comprises administering to mammals in need a medical composition containing a therapeutically effective amount of extracts of broom vine Where the extract exhibits platelet protection.

In another specific example, the present invention provides a method for reducing viral load in the early stage of treatment of dengue fever virus infection in mammals, which comprises administering a complex extract of broom vine to mammals in need, wherein the extract exhibits platelets Protective effects.

In another specific example, the present invention provides a method for preventing and/or treating primary and secondary dengue virus infections in mammals, which comprises administering a complex extract of broom vine to mammals in need.

In a specific example, the present invention provides a method for reducing the cytokine load in primary and secondary dengue virus infections in mammals, which comprises administering a complex extract of broom vine to mammals in need.

In another specific example, the present invention provides an oral composition comprising a composite extract of Broom vine, wherein the composition exhibits dengue fever viruses selected from DENV-1, DENV-2, DENV-3, and DENV-4 _{Inhibitory activity, wherein the IC 50} value is in the range of about 1 to 100 μg/ml as determined using FACS-based analysis. In one aspect of the above specific examples, the extract and composition are used to treat dengue virus infection caused by a serotype selected from DENV-1, DENV-2, DENV-3, and DENV-4 or a combination thereof. In another aspect of the above-mentioned specific example, the dengue virus infection is caused by a serotype selected from DENV-1 and/or DENV-2 or a combination thereof. In another aspect of the specific example, the dengue virus infection is caused by a serotype selected from DENV-1 and/or DENV-3 or a combination thereof. In another aspect of the specific example, the dengue virus infection is caused by a serotype selected from DENV-1 and/or DENV-4 or a combination thereof. In another aspect of the specific example, the dengue virus infection is caused by a serotype selected from DENV-2 and/or DENV-3 or a combination thereof. In another aspect of the specific example, the dengue virus infection is caused by a serotype selected from DENV-2 and/or DENV-4 or a combination thereof. In another aspect of the specific example, the dengue virus infection is caused by a serotype selected from DENV-3 and/or DENV-4 or a combination thereof.

The inhibitory activity of extracts of S. sylvestris against different serotypes of dengue virus is not known in the prior art. When tested in FACS-based neutralization test (FNT), which is a known method to test the efficacy of drugs against a given virus species, it was found that the complex extract of S. sphaerocarpa is very effective in inhibiting the DENV4 serotype. It is known that recovery from infection of a dengue serotype provides lifelong immunity against a specific serotype. However, the cross-immunity to other serotypes after recovery is only partial and temporary. Subsequent infections (secondary infections) of other serotypes increase the risk of severe dengue fever due to antibody-dependent enhancement (ADE). Therefore, the compounds of the present invention provide advantages over treatments known in the art that are only effective against specific serotypes, because it was unexpectedly found that the compound extract of S. sylvestris and the composition of the present invention are effective against all serotypes of dengue virus DENV- 1. DENV-2, DENV-3 and DENV-4 are active. The compound extract of Broomstick vine and the composition of the present invention exhibit inhibitory activity against all dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4, wherein the IC ₅₀ value is determined by FACS-based analysis In the range of about 1 to 20 μg/ml. Therefore, the extract and composition are effective against primary and secondary dengue virus infections. The inventors have discovered that when tested in animal models of primary and secondary dengue fever infections, they found that the extract, the isolated compounds from the extract, their synthetic equivalents, and all the components are in AG129. The murine model exhibits protection in primary and secondary dengue fever.

By obtaining the aerial part and stem part of the broom vine and using different solvents and different drying conditions, the inventors prepared a variety of extracts and tested the anti-dengue fever activity by virus inhibition analysis based on flow cytometry. It was found that the purified aqueous extract (AQCH) of the stem of the broom vine has the highest pan-DENV inhibitory activity. The purified extract proved its ability to reduce the secretion of NS1 and virus in the supernatant in the in vitro analysis, which was also dose-dependent. Through chemical fingerprint analysis, it was identified that five labeled compounds, Sinococuline, magnolia, 20-hydroxyecdysone, Makisterone-A and coniferyl alcohol, are always present in AQCH. When challenged with a lethal DENV dose, AQCH also protects AG129 mice, which further enhances its potential for development as a plant drug for the prevention and treatment of dengue fever.

The inventors of the present invention have also characterized the in vivo potential of AQCH in AG129 mice in primary and secondary DENV infection models. AG129 is a mouse model of immune insufficiency (deficiency of IFN α/β and γ receptors) in which DENV infection can be established to exhibit increased vascular leakage, viremia, and inflammatory cytokines. Therefore, this animal model is often used to evaluate the anti-dengue efficacy of various compounds and candidate vaccines. The antibody-dependent enhancer (ADE), which plays a major role in exacerbating secondary dengue infection, can also be studied in the AG129 mouse model. The inventors analyzed the effects of different doses, administration schedules, and AQCH delaying treatment in primary and secondary dengue infections in AG129 mice. The ability of AQCH was also assessed as a preventive model in secondary dengue fever AG129 mice. Serum viremia and small intestinal lesions, viremia, vascular leakage, and pro-inflammatory cytokines, which are regarded as signs of dengue infection, were also examined in the secondary dengue fever disease AG129 model.

In another aspect, the composition containing quercetin as a component exhibits an IC ₅₀ value ranging from about 2 to 10 μg/ml for DENV-1. In another aspect of the specific example, the composition containing quercetin exhibits an IC ₅₀ value in the range of about 1 to 10 μg/ml for DENV-2. In another aspect, the composition containing quercetin exhibits an IC50 value in the range of about 5 to 15 μg/ml for DENV-3. _{In another aspect, the composition containing quercetin exhibits an IC 50} value in the range of about 5 to 20 μg/ml for DENV-4.

In another specific example, the present invention provides a method for preventing vascular leakage in severe dengue infection caused by primary or secondary dengue infection in mammals, which comprises administering broom vine to mammals in need The compound extract.

In addition, it has been found that the extract and composition are effective in preventing vascular leakage in severe dengue fever infections caused by primary or secondary dengue fever infections. The results indicate that mice that were orally fed the extract or composition of the present invention exhibited lower vascular leakage (in a dose-dependent manner) compared to the untreated group, which confirmed that the extract inhibits DENV infection.

In another specific example, the present invention provides a method for inhibiting the secretion of cytokines in severe dengue fever infections in mammals, which comprises administering a complex extract of broom vine to mammals in need.

It has also been found that the extract and composition are effective in inhibiting the secretion of cytokines in severe dengue fever infections. Excessive production of pro-inflammatory cytokines similar to TNF-α and IFN-γ causes disease progression and vascular leakage. In in vivo studies, the extract or composition of the present invention inhibits the secretion of cytokines in the small intestine. The small intestine of AG129 mice attacked by the DENV-2 S221-4G2 immune complex (IC) and fed with the extract or composition of the present invention has a smaller amount of TNFα and IL-6.

In another embodiment, the present invention provides a method of treating dengue fever virus infection in mammals, which comprises administering a complex extract of broom vine to mammals in need, wherein the extract is effective in delayed initiation of treatment . When the extract and composition of the present invention were administered 6 to 12 hours after the establishment of primary dengue fever infection in the AG129 mouse model, it was found that the treatment delay did not affect the protective effect of the extract or composition.

In another embodiment, the present invention provides a method for preventing dengue fever virus infection in mammals, which comprises administering a complex extract of broom vine to mammals in need. When tested in an in vivo study in an AG129 mouse model, it was found that when the extract and composition of the present invention were administered, the pre-infection showed a protective effect against secondary dengue infection.

In another specific example, it was found that the extract and composition of the present invention are safe and do not show any toxic effects when administered to a mammal in need at a therapeutically effective dose. After repeated oral administration at a dose level of 100, 300, 600, or 1200 mg/kg/day in Weiss rats by oral gavage for 14 days, blood, overall, and histological changes were measured. At these doses, higher platelet counts were observed. Based on these results, the NOAEL in Weiss rats was established at 300 mg/kg/day. Therefore, the extract was found to be safe and non-toxic for human use at the maximum dose of 50 mg/kg/day.

In another specific example, the extract can be co-administered with one or more additional therapeutic agents. In another embodiment, the composition of the present invention may further include one or more additional therapeutic agents. The one or more additional therapeutic agents may be selected from antipyretic/analgesic compounds, such as NSAIDs, such as paracetamol, other antiviral drugs, and the like.

The term "co-administration" as used 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 in a single pharmaceutical composition or can be in separate pharmaceutical compositions. Each of the extract or additional therapeutic agent can be administered via the same or different routes of administration. Co-voting covers simultaneous or sequential voting.

Although the following examples are provided to some specific examples of the present invention, these examples are not intended to limit the scope of the present invention.

The extracts or isolated compounds and pharmaceutical compositions described herein can be administered via various delivery modes, such as oral delivery, enteral/gastrointestinal delivery, parenteral delivery, intravenous delivery, topical delivery, rectal delivery, vaginal delivery Delivery, transocular delivery, transmucosal delivery, etc. In some embodiments, the extracts and pharmaceutical compositions described herein can be administered via oral delivery.

Various doses can be delivered. For example, in some specific examples, the pharmaceutical composition contains 0.1 mg to 100 mg active substance (isolated compound or extract). In a specific example, an oral dose of 600 mg twice a day, 400 mg three times a day, 300 mg four times a day, or 200 mg six times a day is administered to a patient in need of the pharmaceutical composition according to the present invention for treatment Dengue virus infection. In an alternate specific example, the composition is administered in an oral dose of 800 mg twice a day, an oral dose of 400 mg four times a day, or an oral dose of 200 mg eight times a day. In yet another specific example, the composition is administered in an oral dose of 500 mg once, twice, three times, or four times a day. The dose for children can be selected from one-fourth, one-third, one-half, or two-thirds of the total daily dose for adults.

In a specific example, the present invention provides a medicinal composition of the extract of S. chinensis, wherein the composition contains about 10-80% of the extract based on the weight of the lozenge. In another specific example, the present invention provides a medical composition of S. chinensis extract, wherein the composition contains about 15%-70% of the extract based on the weight of the tablet.

In a specific example, the present invention provides a method for treating dengue virus infection by administering to a patient in need a stable composition comprising a therapeutically effective amount of an extract of S. sylvestris or an isolated compound selected from the group consisting of: Formula II Compound (magnoline) 0.1-200 µg/ml, compound of formula I (Sinococuline) 0.1-5 µg/ml, compound of formula IV (20-hydroxyecdysone)> 500 µg/ml and compound of formula III (Makisterone-A) 1-5 µg/ml.

In another specific example, the present invention provides the use of a pharmaceutical composition comprising one or more compounds according to the present invention selected from the group consisting of: Sinococuline, magnoliine, Makisterone-A, 20-hydroxy Ecdysone or a combination thereof by administering a composition to a patient in need, wherein the composition reduces the viral load.

In another embodiment, the present invention provides the use of a pharmaceutical composition comprising one or more compounds according to the present invention selected from the group consisting of: Sinococuline, magnoliine, Makisterone-A, 20 -Hydroxyecdysone or a combination thereof.

In another specific example, the present invention provides the use of a pharmaceutical composition comprising one or more compounds according to the present invention selected from the group consisting of: Sinococuline, magnoliine, Makisterone-A in a method for treating dengue virus infection in mammals , 20-hydroxyecdysone or a combination thereof, the method comprises administering a composition to a mammal in need, wherein the composition provides platelet protection.

In another specific example, the present invention provides the use of a pharmaceutical composition comprising one or more compounds according to the present invention selected from the group consisting of: Sinococuline, magnoliine, Makisterone-A, 20-Hydroxyecdysone or a combination thereof, the method comprises administering a complex extract of Broom vine to mammals in need.

In another embodiment, the present invention provides the use of one or more compounds according to the present invention for the manufacture of medicaments suitable for the treatment of dengue fever virus infection in patients.

In some embodiments, one or more compounds according to the invention are separated from the extract. The extract is prepared by the following method, which comprises extracting the plant body of S. sylvestris with one or more solvents, concentrating the extract, and drying the extract; or extracting the plant body of S. sylvestris with one or more solvents, concentrating the extract, and adding Divide the extract with water and use one or more solvents, and dry the extract; or use one or more solvents to extract the plant body of S. morifolia, concentrate the extract, extract the extract with one or more solvents, and dry the extract. In another aspect of the above specific example, the extraction of the plant body of the broom vine is performed at a temperature range of about 50°C to about 100°C. In another aspect of the above specific example, the extraction of the plant body of the broom vine is carried out at a temperature of about 80°C to about 85°C. In another aspect of the above specific example, the extraction of the plant body of the broom vine is carried out at a temperature of about 60°C to about 65°C. In another aspect of the above specific example, the drying of the extract of S. sylvestris is performed at a temperature ranging from about 40°C to about 95°C. In another aspect of the above specific example, the drying of the extract of S. morifolia is performed at a temperature ranging from about 40°C to about 45°C. In another aspect of the above specific example, the drying of the extract of S. chinensis is performed at a temperature ranging from about 45°C to about 50°C. In another aspect of the above specific example, the drying of the extract of S. sylvestris is performed at a temperature ranging from about 55°C to about 65°C. In another aspect of the above specific example, the drying of the extract of S. chinensis is performed at a temperature ranging from about 90°C to about 95°C. In another aspect, the plant body can be extracted from the dry part or the wet part of the plant.

In a specific example of the present invention, the isolated compound is obtained from the dry extract from S. sylvestris, wherein the dry extract is obtained by: a) performing C ₁ -C ₄ alcohol extraction or aqueous extraction, thereby alcohol extraction Or aqueous extraction uses heat, thereby forming a liquid phase and a solid phase; b) separating the liquid phase from the solid phase; c) drying the liquid phase to obtain a dry extract from Broom vine, whereby the extract contains less than 1% The solid body of the broom vine plant, wherein the dry extract is stable at 25°C for at least one week. In one aspect, the dry extract according to the present invention has a water concentration of not more than 5% w/w. In another aspect, the dry extract according to the present invention has a C ₁ -C ₄ alcohol concentration of not more than 10,000 ppm.

In another specific example, the present invention provides a method for preparing an extract of Broom vine, the method comprising: a) collecting dry or wet parts of the plant body of Broom vine; b) loading the plant body into an extractor and adding The solvent is used for extraction; c) heating the reaction mixture to obtain an extract; d) filtering the extract and collecting the filtrate; e) filtering the residue with a solvent at least once to obtain the filtrate if necessary; and f) concentrating the filtrate, and Dry as needed to obtain the extract.

In another embodiment, the present invention provides a method for preparing a tablet composition containing one or more compounds selected from the group consisting of: Sinococuline, Magnolin, Makisterone A, 20-Hydroxyecdysone for the treatment of dengue virus infection Or a combination thereof, the method includes the following steps: i. Screen one or more compounds and blend them with pharmaceutically acceptable excipients; ii. Lubricate the obtained blend and compress it into a lozenge, and iii. Film-coated tablets.

In another specific example, the present invention provides a method for preparing a tablet composition containing one or more compounds selected from the group consisting of formula I, II, III and IV or a combination thereof for the treatment of dengue virus infection, the method It includes the following steps: i. Blending one or more compounds with pharmaceutically acceptable excipients; ii. Granulate the blend with a solvent; iii. Lubricate and compress the blend into tablets, and iv. Film-coated tablets.

In another embodiment, the present invention provides a method for preparing a tablet composition containing one or more compounds selected from the group consisting of formula I, II, III and IV or a combination thereof for the treatment of dengue virus infection, the method It includes the following steps: i. Blend one or more compounds with pharmaceutically acceptable excipients and compact the mixture; ii. Grind compacts and blend them with extra-granular excipients; iii. Lubricate the blend and compress it into a lozenge, and iv. Film-coated tablets.

In another specific example, the present invention provides a method for preparing a tablet composition containing broom vine extract for the treatment of dengue virus infection, the method comprising the following steps: i. Blend one or more compounds with pharmaceutically acceptable excipients and compact the mixture; ii. Grind compacts and blend them with extra-granular excipients; iii. Lubricate the blend and compress it into a lozenge, and iv. Film-coated tablets.

Although the above specific examples are related to lozenge composition, one or more compounds of the present invention can also be formulated into any other suitable oral dosage forms, such as powders, pellets, granules, spheroids, mini-tablets, capsules, Medicine pouch and so on.

In another related embodiment, one or more compounds can be co-administered simultaneously or sequentially with one or more additional therapeutic agents. In another embodiment, the composition of the present invention may further include one or more additional therapeutic agents. The one or more additional therapeutic agents can be selected from related antiviral therapies or compounds that can provide symptomatic relief, such as antibiotics, antipyretics, and analgesics. Example

The following embodiments only include illustrative specific examples to illustrate the practice of the present invention. It will be obvious to those skilled in the art that the present invention is not limited to the details of the following illustrative embodiments, and the present invention can be implemented in other specific forms without departing from its basic properties, and therefore it is necessary to combine specific examples and embodiments of the present invention in All aspects are to be regarded as illustrative and not restrictive.

Example 1 : Preparation of extract:

Plant raw materials (BRM), that is, the aboveground parts or stem parts of tin vines and broom vines are collected by botanists, and tin vines (deposit number RRLH-23148) and broom vines (deposit number RRLH-23152) The properly identified herbarium samples are submitted to Janaki Ammal Herbarium (RRLH) under CSIR-IIIM, Jammu. In addition, for some studies, the BRM of the broom vine collected from the same area was obtained from a local supplier, and the broom vine samples were properly identified, that is, the above-ground part (deposit numbers CDR-4037 and CDR-4038) and the stem part ( Deposit numbers CDR-4061, CDR-4064, CDR-4065 and CDR-4078) are submitted to the Crude Drug Repository (CDR) under CSIR-IIIM, Jammu. BRM is dried and crushed in a cool place. At ambient temperature, the ground plant bodies of tin vine and/or broom vine are separately loaded into the extractor. The extraction solvent (water-alcohol, 50:50/denatured alcohol/water) is added, and the mixture is heated to reflux temperature for about 3 hours. The extracted body is filtered, collected and stored in a container. Repeat the extraction method from the residue plant body more than two times. All three filtered extracts are combined, concentrated and further dried using rotating steam, tray under vacuum or spray drying. Depending on the part of the plant used and the extraction solvent, the yield obtained is 7-14%.

Example 1-A : preparation 95:5 Ethanol : Purified water extract of broom vine

Put the plant body (1 kg) of the broom vine into the extractor at ambient temperature*. A mixture of ethanol and purified water (95:5; 6 L) was added and the reaction mixture was heated at a temperature of 60-65°C for about 3 hours. The extracted body is filtered, collected and stored in a container. Repeat the extraction and filtration steps twice with a mixture of ethanol and purified water (95:5; 3 L). The three filtered extracts were combined and concentrated to the greatest possible extent under reduced pressure at low temperature. The resulting extract was decanted into a stainless steel pan, and then dried under vacuum at 45-50°C until the ethanol content did not exceed 10,000 ppm and the moisture content did not exceed 5%. The dried extract is cooled to about 20-25°C and unloaded under controlled humidity (RH no more than 40%). Yield obtained = 90 g to 120 g

Example 1-B : preparation 1:1 Ethanol : Purified water extract of broom vine

Put the plant body (1 kg) of the broom vine into the extractor at ambient temperature*. A mixture of ethanol and purified water (1:1; 6 L) was added and the reaction mixture was heated at a temperature of 60-65°C for about 3 hours. The extracted body is filtered, collected and stored in a container. Repeat the extraction and filtration steps twice with ethanol and purified water (1:1, 3 L). The three filtered extracts were combined and concentrated to the greatest possible extent under reduced pressure at low temperature. The resulting extract was decanted into a stainless steel pan, and then dried under vacuum at 45-50°C until the ethanol content did not exceed 10,000 ppm and the moisture content did not exceed 5%. The dried extract is cooled to about 20-25°C and unloaded under controlled humidity (RH no more than 40%). Yield obtained = 80 g to 120 g

Example 1-C : Preparation of the aqueous extract of broom vine

Put the plant body (1 kg) of the broom vine into the extractor at ambient temperature*. Purified water (6 L) was added and the reaction mixture was heated at a temperature of 60-65°C for about 3 hours. The extracted body is filtered, collected and stored in a container. Repeat the extraction and filtration steps twice with purified water (3 L). The three filtered extracts were combined and concentrated to the greatest possible extent under reduced pressure at low temperature. The resulting extract was decanted into a stainless steel pan, and then dried under vacuum at 45-50°C until the ethanol content did not exceed 10,000 ppm and the moisture content did not exceed 5%. The dried extract is cooled to about 20-25°C and unloaded under controlled humidity (RH no more than 40%). The yield obtained = 80 g to 120 g.

*The term "ambient temperature" as used herein includes temperatures in the range of about 18°C to about 25°C.

Example 2 : Isolated compound from broom vine:

The dried aqueous extract of vine broom (powder, 500 gm) (as described in Example 1C) was dissolved in distilled water and filtered. The filtrate was partitioned between CHCl ₃ (A) and H ₂ O (B). _{Next, the CHCl 3} soluble fraction (A) was divided with an aqueous HCl solution to _{obtain a CHCl 3} layer (C) and an acidic H ₂ O layer (D). The chloroform layer (C) was subjected to column chromatography, eluted with a _{gradient of CHCl 3} -MeOH and concentrated to obtain 30 eluted fractions. -Based on the TLC profile, the coniferous base (SP-A-01) is obtained in the pure form of the dissociation fraction. The _{acidic H 2} O layer (D) was then basified with an aqueous _{NH 4} OH solution to produce the free base, and then extracted with CHCl _{3 to} produce the alkaloid-containing chloroform layer (E). -The CHCl ₃ fraction (E) was dried and purified by column chromatography and eluted with a _{gradient of CHCl 3} -MeOH to isolate the compound of formula II (magnoliine). -In addition, the aqueous layer (B) _{is alkalized with ammonium hydroxide (NH 4} OH) solution, and then extracted with CHCl _{3 to} produce the alkaloid-rich dissociated fraction (E1) and the aqueous layer (F1). The CHCl ₃ layer (E1) is further processed to separate the compound of formula I (Sinococuline) _{eluted with CHCl 3 -MeOH via column chromatography.} -Use lyophilization to turn the aqueous layer (F1) into a powder and dissolve it in methanol. The methanol-soluble fraction was further purified by column chromatography, eluted with a _{gradient of CHCl 3} -MeOH and concentrated to obtain fifty fractions. Observe two UV active spots on TLC. This mixture containing two UV active compounds was further subjected to RP-HPLC (using a _{gradient solvent system of MeOH/H 2} O) to obtain a compound of formula IV (20-hydroxyecdysone) and a compound of formula III (Makisterone A).

Example 3 : In test tube and in vivo DENV Inhibition analysis

Materials and methods : Vero cell line was purchased from American Type Cell Culture (ATCC), Virginia, USA. The monkey kidney cell line was maintained at 37°C in a 10% CO ₂ humidified incubator with a modified Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% ΔFBS. WHO references DENV strains DENV-1 (WP 74), DENV-2 (S16803), DENV-3 (CH53489) and DENV-4 (TVP-360) from Dr. Aravinda de Silva's lab, University of North Carolina (UNC) , USA. These viruses were cultured in C6/36 cells obtained from ATCC, Virginia, USA. The mouse-adapted DENV-2 S221 used in animal experiments was obtained from Global Vaccines Inc., North Carolina, USA and cultured in Vero cells. Dengue cross-reactive monoclonal antibodies (mAb) 4G2 and 2H2 that recognize the fusion loop of DENV and the epitope in the prM protein, respectively, were produced inside their respective fusion tumors obtained from ATCC, Virginia, USA. 2H2 mAb was internally labeled with Alexa fluor-488 via a commercial labeling kit (Thermo Fischer Scientific, Eugene, USA).

In the case of the methanol extract of the aerial part of C. pareira (another plant known for its anti-dengue fever activity), in the virus inhibition analysis based on in vitro flow cytometry instead of In the previously used bioassay based on conventional plaques, each of three batches of methanol extracts of the aerial parts of tinned vine and broom vine was prepared, and the anti-dengue fever activities of all six methanol extracts were evaluated. Virus suppression based on flow cytometry and plaque-based biological analysis are generally similar. However, the evaluation of virus inhibition analysis based on flow cytometry is advantageous because of its high throughput and stricter, because it uses higher doses of DENV to evaluate anti-dengue activity. In the virus inhibition analysis based on flow cytometry used in the current study, Vero cells were infected with DECV, and after infection, the cells were incubated for 46 hours in a medium containing various concentrations of extracts. The cells were fixed and incubated, permeated and stained with Alexa fluor-labeled anti-dengue mAb, 2H2, which is reactive to all four DENV serotypes, and these serotypes were read in a flow cytometer to determine the percentage of DENV-infected cells. _{This is used to calculate the concentration (IC 50} ) of the extract that inhibits 50% of DENV infection.

Virus inhibition analysis based on flow cytometry : inoculate Vero cells in 200 μl DMEM+10% ΔFBS in a 96-well plate (20,000-25,000 cells/well), and at 37°C and 10% CO ₂ Incubate for 24 hours in a down-regulated incubator. The next day, with 100 μl of DENV-1, DENV-2, DENV-3 and DENV-4 diluent cells, 10% infection occurred in DMEM + 0.5% ΔFBS (diluted medium). After incubating the Vero cells and the virus _{for 2 hours at 37°C and 10% CO 2} , the virus is aspirated and 200 μl of the test substance (AQCH extract/isolated compound) in the appropriate range prepared in the diluted medium is prepared in duplicate Add ground to the hole. The cells were incubated in an incubator at 37°C and 10% CO ₂ for another 46 hours. Wells infected with virus but without any subsequent extract/drug treatment served as virus controls, while wells without infection and untreated served as cell controls. These experimental controls were used for relative virus infection calculation and antibody background signal adjustment. After completing the incubation period, the cells were stained with Alexa-488 labeled 2H2 mAb to observe the presence of cytoplasmic DENV. For staining, the medium is aspirated from the top of the cells and washed with PBS. The cells were trypsinized and transferred to a 96-well U-shaped tray. After transfer, the cells were centrifuged at 1500 rpm for 5 min and the supernatant was aspirated. The cells were washed again with PBS, and then fixed with 4% paraformaldehyde for 20 minutes. The cells were centrifuged at 2500 rpm for 5 minutes and the supernatant was aspirated. The cells were washed twice with permeation or permeation buffer and blocked with 1% normal mouse serum (prepared in permeation buffer) for 30 minutes. Without removing the blocking solution, Alexa-488 labeled 2H2 mAb was added to stain the cells for DENV, and the cells were incubated for 1 hour at 37°C under gentle shaking. After incubation, the cells were centrifuged at 2500 rpm for 5 minutes and the supernatant was aspirated. The cells were washed twice with permeation buffer and resuspended in 100 μl PBS. The above processed cells were analyzed by BD FACS Verse flow cytometer and 5000 cells were counted per well. The data was analyzed by FlowJo software to determine the relative percentage of each test substance concentration relative to the infected cells of the virus-only control group. GraphPad Prism software using non-linear regression analysis and calculation of 50% inhibitory concentration of the test substance IC ₅₀₎ was determined with respect to the virus control by 50% inhibitory concentration of dengue virus infection.

Results : After evaluating all six extracts in parallel in the virus suppression analysis based on flow cytometry, it was observed that all three batches of methanol extracts of S. sylvestris were specific to all four extracts compared with methanol extracts of S. sylvestris. The DENV serotype has significantly stronger anti-dengue fever activity (Figure 1).

Example 4 : Select the aqueous extract of the stem of the broom vine for further evaluation

When choosing broom vine instead of tin raw vine due to its more effective anti-dengue fever activity, we explored the use of various solvents such as denatured olein, water-alcohol (50:50) and the aboveground part of the extract of broom vine in water (Figure 2a; dashed curve) and stem part (Figure 2a; solid curve) are prepared separately. These extracts were evaluated against all four DENV serotypes by flow cytometry-based virus inhibition analysis and their IC ₅₀ values were compared (Figure 2b). It was observed that, regardless of the solvent used, the stem part of the broom vine was significantly more effective than the above-ground part. Therefore, the aqueous extract of the stem of the broom vine, hereinafter referred to as AQCH, was further studied.

Example 5 : NS1 ELISA Analysis and estimation of extracellular viruses

NS1 analysis : Inoculate Vero cells in 500 μl DMEM + 10% ΔFBS in a 48-well plate (40,000 cells/well). The next day, cells were infected with DENV-1, DENV-2, DENV-3 and DENV-4 at 0.1 MOI. After 2 hours of infection, the medium was aspirated and the cell monolayer was superimposed with different concentrations of extracts (100, 50, 25, and 12.5 μg/ml) in the virus infection medium (DMEM+0.5% ΔFBS). Wells that did not receive any infection but only received virus-infected media were used as negative controls. After infection, a commercial dengue fever NS1 Ag Microlisa kit (J. Mitra and Co. Pvt. Co., Ltd.) was used to extract 10 μl of supernatant culture supernatant from each well every day for 6 days for the detection of NS1 antigen.

Extracellular virus estimation : In this analysis, the culture supernatant of DENV-infected cells was collected and virus was titrated in AQCH-treated and untreated wells. Briefly, Vero cells were seeded and infected in six 96-well plates, as detailed in the analysis of viral suppression based on flow cytometry. After 2 hours of infection, the medium was aspirated and the monolayer overlapped the virus infection medium with 200 μl extracts of different concentrations (100, 50, 25, and 12.5 μg/ml). After 24 hours of infection, one culture plate was harvested every day for the next 6 days, and the supernatant was transferred to a U-shaped bottom 96-well plate. The samples were stored at 4°C until the 6th day. In the analysis based on flow cytometry, the titer of DENV-1 in the collected supernatant was evaluated on Vero cells [31] to obtain FACS infection unit/ml (FIU/ml).

result : AQCH inhibition DENV And its antigen secretion dose-dependently

Until now, the anti-DENV activity of AQCH was measured by virus inhibition analysis based on flow cytometry, which quantified cytosolic virus 46 hours after infection. In order to determine DENV inhibition and its kinetics, the effect of AQCH on the secreted virus and its secreted antigen NS1 was evaluated for up to 6 days after infection (Figure 3). An aliquot of the culture supernatant from Vero cells infected with DENV1-4 was collected from day 1 to day 6 after infection, and analyzed by flow cytometry and commercial ELISA kits respectively Analyze the titration of secreted DENV and NS1. AQCH of 100 and 50 µg/ml was observed to be extremely effective in completely inhibiting DENV secretion up to 6 days in the experiment, and it was observed that this inhibition was dose-dependent (Figure 3a; only data with DENV-1 is shown). The analysis of the NS1 content in the collected supernatants of all four types of DENV on day 6 confirmed that the results were 100 and 50 µg/ml AQCH exhibited 100% inhibition of the release of NS1, and this inhibition increased with the concentration of AQCH Decrease and decrease (Figure 3b).

Example 6 : Based on FACS Neutralization test ( FNT ) In vitro evaluation of the anti-dengue fever activity of the compound ( Sinococuline , Magnolia, Makisterone A and 20- Hydroxyecdysone )

FACS-based neutralization analysis was used to evaluate all compounds to screen for their DENV inhibitory activity. In FNT analysis, Vero cells were seeded in each well and incubated overnight. The next day, the cells were infected with individual DENV-4 and incubated at 37°C. After incubation, the medium was aspirated and a solution of Sinococuline, Magnolin, Makisterone A, and 20-hydroxyecdysone was added; analysis was performed in duplicate for each inhibitor concentration. The cells were incubated at 37°C. After incubation, the cells were stained with fluorescently labeled antibodies to observe the presence of cytoplasmic DENV. The cells were trypsinized and transferred to a 96-well U-shaped tray. Then centrifuge and aspirate the supernatant. The cells were washed again with PBS, and then fixed with 4% paraformaldehyde, and then centrifuged at 2500 rpm for 5 minutes, and the supernatant was aspirated. The cells were washed twice with permeation or permeation buffer and blocked with 1% normal mouse serum. Without removing the blocking solution, the 2H2-Alexa488 antibody was added to stain the cells for DENV, and the cells were incubated at 37°C with gentle shaking. A After 1 hour incubation, the cells were centrifuged and the supernatant was aspirated. The cells were washed twice and resuspended in PBS. The above processed cells were analyzed by flow cytometry and the cells were counted. Analyze the obtained data (via FlowJo software) to determine the relative percentage of each inhibitor concentration relative to the virus-only (without any inhibitor treatment) control group infected cells. Using GraphPad Prism software to calculate, the IC ₅₀ of all compounds was determined to be the concentration that inhibits 50% of dengue virus infection. _{For the DENV-4 serotype, the IC 50} values of Sinococuline, magnolin, Makisterone A and 20-hydroxyecdysone are provided in Table 1 below.

Table 1 : _{The IC 50} (μg/ml) of labeled compounds against dengue virus serotype 4 (DENV-4), if checked by FNT. Compound DV4 (µg/ml) Makisterone A 2.0- 2.38 Magnolia 0.34-0.80 Magnoflorine 146.7 20-hydroxyecdysone ＞500 Therefore, the above data indicate the effective inhibitory activity of the compounds of the present invention against dengue fever virus.

Example 7 : AQCH Chemical fingerprint leaving mark instrument and characterization method

Method : All NMR spectral data were recorded on a Bruker 400 MHz spectrometer. The chemical shift ( δ ) internally refers to the residual solvent peak (CD ₃ OD: ¹ H δ 3.30, ¹³ C δ 49.0 ppm; CDCl ₃ : ¹ H 7.26, ¹³ C 77.0 ppm) and the reference point is TMS ( δ _H and δ _C : 0.00 ppm). HR-ESIMS spectra were recorded on Agilent 1100 LC-Q-TOF mass spectrometer and HRMS-6540-UHD machine. HPLC purification was performed on a Thermo Scientific Dionex UltiMate 3000 HPLC system with a UV detector. Use silica gel (60-120 and 230-400 mesh) for column chromatography; monitor the dissociated fraction by TLC, pre-coated silica gel plate 60 F254 (Merck). Observe the spots by UV light or by _{spraying with H 2} SO ₄ -MeOH, anisaldehyde-H ₂ SO _{4 reagents.}

For HPLC fingerprint retention, transfer 100 mg of AQCH to a 20 ml volumetric flask and add about 10 ml of diluent for 5-10 minutes to dissolve under sonic processing/shaking/stirring. The volume is made up with diluent, mixed and filtered through a 0.45 µm filter for HPLC fingerprint retention. HPLC fingerprint trace was performed on the RP18e Purospher-STAR (Hibar) (250 × 4.6 mm; 5 µm) column. Use a mobile phase containing buffer (0.1% formic acid in water) and acetonitrile at a flow rate of 0.65 ml/min at a wavelength of 254 nm at a column temperature of 30°C. The injection volume is 5 μl and the total running time of the analysis is 75 minutes. The gradient program is used as follows: 0-15 min, 00-05% B; 15-40 min, 05-20% B; 40-55 min, 20-30% B; 55-65 min, 30-60% B; 65 -68 min, 60-00% B and 68-75 min, 00% B. For separation (Figure S1), 500 g of AQCH was suspended in distilled water and partitioned between ethyl acetate (A) and H2O (B). The aqueous layer (B) was basified with NH4OH solution (pH 9) and then extracted with chloroform, CHCl3. This separated the chloroform layer (4.0 g, C) from the aqueous layer (D). The CHCl3 layer (C) was further purified by repeated column chromatography in neutral alumina and eluted with a gradient of CHCl3-MeOH (100:0 to 0:100) to obtain compound 1 and compound 2 as main components. The aqueous layer (D) was lyophilized (480.5 g) and suspended in methanol. The methanol soluble fraction (400.0 g) was purified by column chromatography (silica gel, 100-200 mesh) with a gradient of CHCl3-MeOH (100:0 to 0:100, 500 ml, the volume of each fraction collected) It was eluted and concentrated to obtain fifty eluted fractions (Fr.1-Fr.50), and its composition was monitored by TLC. These fractions showing similar TLC curves will be composed of six main dissolution fractions (Fr-1a to Fr-5a). The dissociated fraction Fr-2a yielded two UV active compounds as crystals. These crystals containing two UV active compounds were further subjected to semi-preparative C18 reversed-phase HPLC {Eclipse 5 μm; 9.4 × 250 mm; 3 ml/min; water (B)/acetonitrile (A) gradient for 32 minutes; 100 -80% B (5 min), 80-60% B (5 min), 60-50% B (5 min), 50-40% B (5 min), 40-20% B (5 min), 20 -45% B (3 min), 45-70% B (2 min) and 70-100% B (2 min); column oven temperature 25℃} to obtain compounds 3 and 4. Perform column chromatography (silica gel, 100-200 mesh) on the ethyl acetate soluble fraction (A), using a gradient of CHCl3-MeOH (100:0 to 0:100, 250 ml, collect the volume of each fraction) It is eluted and concentrated to obtain 30 eluted fractions (Fr.51-Fr.80). Compound 5 was obtained from the purified fractions Fr.66-Fr.70. Through specific spectral analysis of 1D and 2D NMR and HRESI-MS data and comparison with reported spectral data, all isolated compounds 1-5 were identified.

Results : After confirming the pan anti-dengue activity of the batch by virus inhibition analysis based on flow cytometry (Figure 4a), the AQCH batch was subjected to HPLC chromatography for its chemical analysis. The obtained HPLC chromatogram is shown in Figure 4b. After that, the five labeled compounds were separated using repeated chromatography methods, which were characterized using advanced 1D and 2D NMR spectroscopy and mass spectrometry. The marker compounds were identified as Sinococuline (Compound 1), Magnolialine (Compound 2), 20-Hydroxyecdysone (Compound 3), Makisterone A (Compound 4) and Coniferyl Alcohol (Compound 5) (Figure 4c). The physicochemical data of all five identified labeled compounds are given in Table 1.

Table 2 : Physicochemical data of markers 1-5 identified in AQCH Sinococuline : pink amorphous powder; ¹ H NMR (CD ₃ OD, 400 MHz): δ 6.74 (1H, d, J= 8.4 Hz, H-2), 6.53 (1H, d, J= 8.4 Hz, H- 1), 4.37 (1H, d, J = 5.6 Hz, H-9), 4.28 (1H, d, J= 2.8 Hz, H-7), 3.85 (1H, ddd, J= 13.3, 3.7 and 3.5 Hz, C-6), 3.82 (3H, s, 3-OCH ₃ ), 3.69 (3H, s, 8-OCH ₃ ), 3.15 (1H, dd, J = 17.6 and 6.0 Hz, H-10), 2.92 (1H , dd, J= 13.3, 3.7 Hz, H-5), 2.88 (1H, d, J= 17.7 Hz, H-10), 2.63-2.71 (2H, ddd, J= 13.1, 12.5, 3.4 Hz, H- 16), 2.16 (1H, t, J= 13.2 Hz, H-5), 2.00 (1H, dd, J= 12.7, 3.4 Hz, H-15), 1.86 (1H, ddd, J= 12.7, 12.5 and 4.7 Hz, H-15); ¹³ C NMR (CD ₃ OD, 100 MHz): δ 147.2 (C-4), 145.9 (C-3), 145.4 (C-8), 131.2 (C-11), 130.2 ( C-14), 124.7 (C-12), 119.2 (C-1), 110.5 (C-2), 68.5 (C-6), 66.8 (C-7), 57.4 (3-OCH ₃ ), 56.7 ( 8-OCH ₃ ), 46.7 (C-9), 41.0 (C-16), 39.8 (C-13), 38.4 (C-15), 37.2 (C-5), 36.8 (C-10); ESI- MS m/z 334.25 [M+H] ⁺ (calculated as C ₁₈ H ₂₄ NO ₅ , 334.25). Magnolia base : yellow-brown powder; ¹ H NMR (400 MHz, CD ₃ OD): δ _H 6.72 (1H, d, J = 8.0 Hz, H-9), 6.56 (2H, t, J = 4.0 Hz, H -3, H-8), 3.84 (3H, s, OCH ₃ -10), 3.80 (3H, s, OCH ₃ -2), 3.56 (1H, dd, J = 18.0, 5.6 Hz, H-6a), 3.45 (1H, dd, J = 18.0, 5.2 Hz, H-5), 3.31 (3H, s, N-CH ₃ ), 3.22 (1H, m, H-4), 3.04 (1H, dd, J = 18.0 , 5.2 Hz, H-5), 2.89 (3H, s, N-CH ₃ ), 2.74 (1H, dd, J = 20.0, 4.0 Hz, H-4), 2.60 (1H, t, J = 13.2 Hz, H-5); ¹³ C NMR (100MHz, CD ₃ OD): δ _C 151.9 (C-2), 150.5 (C-10), 149.5 (C-1), 148.6 (C-11), 124.7 (C- 7a), 122.4 (C-11b), 122.3 (C-11a), 119.7 (C-6b), 115.6 (C-8), 114.4 (C-3a), 109.5 (C-9), 108.3 (C-3 ), 70.0 (C-6a), 61.2 (C-5), 55.0 (OCH ₃ ), 54.7 (OCH ₃ ), 52.6 (N-CH ₃ ), 42.2 (N-CH ₃ ), 30.5 (C-7) , 23.4 (C-4); ESIMS m/z 343.20 [M+H] ⁺ (calculated as C ₂₀ H ₂₄ NO ₄ ⁺ , m/z 342.20). 20 -Hydroxyecdysone : white soft crystals; ¹ H NMR (CD ₃ OD, 400 MHz): δ 5.81 (1H, d, J= 2.6 Hz, H-7), 3.95 (1H, q, H-3α), 3.84 (1H, ddd, J = 12.0, 4.0, 3.2 Hz, H-2α), 3.33 (1H, dd, J= 11.0, 1.7 Hz, H-22), 3.15 (1H, ddd, J= 11.2, 7.0, 2.6 Hz, H-9), 2.39 (1H, dd, J= 9.5, 8.0 Hz, H-17), 2.38 (1H, dd, J= 13.0, 4.5 Hz, H-5), 2.13 (1H, dt, J= 13.0, 13.0, 4.8 Hz, H-12α), 1.99 (1H, H-15α), 1.95 (1H, H-16α), 1.88 (1H, ddd, J= 12.8, 4.6, 2.3 Hz, H-12β ), 1.81 (1H, H-11β), 1.79 (2H, H-1α/24a), 1.75 (1H, H-4α), 1.73 (1H, H-16β), 1.70 (1H, H-4β), 1.69 (1H, H-11α), 1.66 (1H, H-23a), 1.60 (1H, H-15β), 1.43 (2H, dd, J= 13.3, 12.3 Hz, H-1β/24b) 1.28 (1H, dddd , J= 13.0, 11.5, 11.0, 4.6 Hz, H-23b) 1.20 (3H, s, 21-CH ₃ ), 1.20 (3H, s, 26-CH ₃ ), 1.19 (3H, s, 27-CH ₃ ), 0.97 (3H, s, 19-CH ₃ ), 0.89 (3H, s, 18-CH ₃ ); ¹³ C NMR (CD ₃ OD, 100 MHz): δ 206.4 (C-6), 167.9 (C- 8), 122.1 (C-7), 85.2 (C-14), 78.4 (C-22), 77.9 (C-20), 71.3 (C-25), 68.7 (C-2), 68.5 (C-3 ), 51.8 (C-5), 50.5 (C-17), 48.6 (C-13), 42.4 (C-24), 39.3 (C-10), 37.4 (C -1), 35.1 (C-9), 32.8 (C-4), 32.5 (C-12) 31.8 (C-15), 29.7 (C-26), 28.9 (C-27), 27.4 (C-23 ), 24.4 (C-19), 21.5 (C-16), 21.5 (C-11), 21.0 (C-21), 18.0 (C-18); HR-MS m/z 481.3161 [M+H] ⁺ (Calculated based on C ₂₇ H ₄₅ O ₇ , 481.3165). Makisterone A : white crystal; ¹ H NMR (CD ₃ OD, 400 MHz): δ 5.81 (1H, d, J= 2.6 Hz, H-7), 3.95 (1H, q, H-3α), 3.84 (1H, ddd, J = 12.0, 4.0, 3.2 Hz, H-2α), 3.46 (1H, dd, J= 11.0, 1.7 Hz, H-22), 3.15 (1H, ddd, J= 11.2, 7.0, 2.6 Hz, H -9), 2.39 (1H, dd, J= 9.5, 8.0 Hz, H-17), 2.38 (1H, dd, J= 13.0, 4.5 Hz, H-5), 2.13 (1H, dt, J= 13.0, 13.0, 4.8 Hz, H-12α), 1.99 (1H, H-15α), 1.95 (1H, H-16α), 1.88 (1H, ddd, J= 12.8, 4.6, 2.3 Hz, H-12β), 1.81 ( 1H, H-11β), 1.79 (2H, H-1α/24a), 1.75 (1H, H-4α), 1.73 (1H, H-16β), 1.70 (1H, H-4β), 1.69 (1H, H -11α), 1.60 (1H, H-23a), 1.57 (1H, H-15β), 1.43 (1H, dd, J= 13.3, 12.3 Hz, H-1β) 1.29 (1H, dddd, J= 13.0, 11.5 , 11.0, 4.6 Hz, H-23b), 1.19 (3H, s, 21-CH ₃ ), 1.17 (3H, s, 27-CH ₃ ), 1.14 (3H, s, 28-CH ₃ ), 0.97 (3H , s, 25-CH ₃ ), 0.94 (3H, s, 19-CH ₃ ), 0.90 (3H, s, 18-CH ₃ ); ¹³ C NMR (CD ₃ OD, 100 MHz): δ 204.4 (C- 6), 165.9 (C-8), 120.1 (C-7), 83.1 (C-14), 75.9 (C-20), 73.3 (C-22), 71.8 (C-26), 66.6 (C-2 ), 66.4 (C-3), 49.6 (C-5), 48.2 (C-17), 39.7 (C-10), 37.2 (C-1 ), 35.3 (C-9), 33.0 (C-12), 32.3 (C-4), 30.9 (C-15), 30.4 (C-13), 29.7 (C-15), 28.7 (C-23) , 28.6 (C-24), 25.8 (C-27), 23.8 (C-28), 22.5 (C-19), 19.5 (C-16), 19.3 (C-11), 19.0 (C-21), 16.1 (C-18), 13.2 (C-25); HR-MS m/z 495.3318 [M+H] ⁺ (calculated as C ₂₈ H ₄₇ O ₇ , 495.3322). Coniferyl alcohol : white solid; ¹ H NMR (CD ₃ OD, 400 MHz): δ 7.01 (1H, d, J = 1.8 Hz, H-3), 6.85 (1H, dd, J = 7.9 and 1.8 Hz, H- 5), 6.74 (1H, d, J = 7.9 Hz, H-6), 6.50 (1H, d, J = 15.9 Hz, H-7), 6.20 (1H, dt, J = 15.9 and 5.4 Hz, H- 8), 4.21 (2H, d, J = 6.1 Hz, H-9), 3.87 (3H, s, 2-OCH ₃ ); ¹³ C NMR (CD ₃ OD, 100 MHz): δ 149.2 (C-2) , 147.6 (C-1), 132.2 (C-7), 130.7 (C-4), 127.2 (C-8), 121.1 (C-5), 116.3 (C-6), 110.8 (C-3), 64.0 (C-9), 56.5 (C-2-OCH ₃ )

Example 8 : AG129 Primary Dengue Fever Lethal Mouse Model AQCH In vivo evaluation of its efficacy

Method : AG129 mice lacking IFN-α/β and IFN-γ were purchased from B&K Universal, United Kingdom, and were kept and raised in Genetic Engineering and Biotechnology (ICGEB), International Centre in New Delhi. The 6 to 8 week old experimental mice (six in each group) are housed in a BSL-2 containment facility. It was injected intravenously with a lethal dose (1.0×10 ⁵ FIU) of mouse-adapted DENV-2 strain S221. Half an hour after DENV-2 infection, mice were orally fed with 8.25 or 25 mg/kg/dose AQCH four times a day (QID) for a period of five days. Three groups of mice served as experimental controls. The first group, "uninfected", was neither infected with DENV-2 S221 nor treated with AQCH. The second group, "AQCH only", was not infected with DENV-2 S221, but was treated with AQCH. The third group, "V", was infected with DENV-2 S221, but was not treated with AQCH. The survival rate, weight change and morbidity score of all mouse groups were monitored for a period of 15 days after infection. The survival rate score was statistically evaluated by Log Rank (Mantel Cox) test, and the p value <0.05 was regarded as statistically significant. The morbidity score is based on a 5-point system: 0.5, slightly wrinkled fur; 1.0, wrinkled fur; 1.5, eye damage; 2, eye damage and hunchback; 2.5, soft stools; 3.0, limited exercise; 3.5, no exercise / Hind leg paralysis; 4.0, if the cumulative score is 5, euthanasia. All AQCH doses for feeding were immediately prepared in water with 0.1% methylcellulose (v/v) and stored at 4°C. Pre-incubate an appropriate volume of the dose at room temperature, and then feed the animal. For four doses per day, mice were fed AQCH in a 4/4/4/12 hour cycle (7 AM, 11 AM, 3 PM, and 7 PM per day for 5 days).

Result : AG129 is an immunologically insufficient mouse lacking interferon α/β and γ receptor signaling, which makes the mice adapt to the development of diseases caused by the reproduction of the DENV-2 S221 strain. Therefore, this mouse model is used to evaluate the efficacy of AQCH in vivo. The design of the analysis is depicted in Figure 6a and allowed AG129 mice to be infected with a lethal dose of DENV-2 S221 (1.0×105 FIU) via the intravenous route. After that, use 25 mg/kg ("V+AQCH 25 mg/kg four times a day" group, blue curve) or 8.25 mg/kg ("V+AQCH 8.25 mg/kg four times a day" group, pink curve) AQCH Oral feeding was given four times a day for 5 days and survival rate, morbidity score, and weight change were monitored for up to 15 days after infection. Uninfected and unfed AQCH ("uninfected" group, black curve) and uninfected but fed AQCH ("AQCH only" group, orange curve) AG129 mice served as negative controls, and virus-infected but unfed AQCH AG129 mice ("V" group, gray curve) served as a positive control. The AG129 mice in the "V" group did not survive more than six days and showed the highest incidence score and weight change% (Figure 6b-d, gray curve). However, the infected AG129 mice fed with 25 and 8.25 mg/kg AQCH four times a day were significantly protected (p<0.05), showing 100% and 50% survival rates, respectively (Figure 6b, blue and pink curves, respectively) ); its incidence score and weight change% gradually increase after reaching the peak on about 4-6 days (Figure 6c-d). In the negative control group, "Uninfected" and "AQCH only" did not show any mortality (Figure 6b, black and orange curves respectively), and no significant morbidity and weight loss were observed (Figure 6c-d, black respectively) And orange curve).

Example 9 : Paracetamol and AQCH In-vitro evaluation of the interaction

The interaction between paracetamol and AQCH was determined in vitro via a virus inhibition analysis based on flow cytometry. Infect Vero cells with DENV-1 for 2 hours (as described in virus inhibition analysis based on flow cytometry). After 2 hours, the virus infection medium was aspirated and the cells were treated with AQCH with a concentration in the range of 0 to 25 μg/ml together with 1, 10, and 100 μg/ml paracetamol in the different channels of the 96-well plate. Duplicate assess the treatment and analyzed via non-linear regression using GraphPad Prism calculate IC _50.

Result : As AQCH exhibits anti-dengue efficacy in vitro and in vivo , it becomes obvious that it has a strong potential to be developed as a drug. However, its clinical suitability needs to be evaluated. Our first study in that area was to evaluate its interaction with paracetamol, which is a standard care drug for the treatment of dengue fever. In this study, in the absence (0 µg/ml) and the presence of 1, 10, and 100 µg/ml paracetamol, a certain range of DENV-1 was evaluated by virus inhibition analysis based on flow cytometry. The concentration of AQCH. Importantly, it was found that the anti-dengue fever activity of AQCH was not affected by paracetamol in this experiment (Figure 7).

Example 10 : Primary and secondary dengue fever diseases AG129 In vivo evaluation of mouse models

Statistical analysis : The data set is subjected to two-way analysis of variance (multiple comparisons using Bonferroni's correction) to determine the statistical significance of the differences. The significance of Kaplan-Meier survival curve was analyzed by log-level test. The survival rate (p) level is less than or equal to 0.05 and less than 0.001 as significant and extremely significant respectively. All statistical calculations are performed using GraphPad Prism (v8.0) software.

Fatal primary and secondary dengue disease AG129 Establishment of mouse model

The DENV-2 S221 strain was used to attack AG129 mice with immune insufficiency and DENV sensitivity, in which primary and secondary dengue fever were determined. In the primary model, the lethal dose (1×105 FIU) of S221 was used, and in the secondary model, the second lethal dose (2×104 FIU) of S221 was used intracutaneously and the neutralization concentration of mAb 4G2 was used. (10 µg). In both cases, if left untreated, the mouse died of dengue infection within 4-7 days.

Five days after the establishment of the infection, we established the protective efficacy of AQCH in the two models by feeding the mice orally with the selected AQCH dosage and dosing schedule. After the challenge, after monitoring the mouse group for 15 days, the level of protection brought by the survival curve, morbidity score, and weight change was scored. Three to four groups of AG129 mice were kept as experimental controls parallel to the test group. In the "uninfected" group, they were neither infected with S221 nor treated with AQCH. In the "V" group, it received a lethal dose of S221 in the primary model, and a sublethal dose in the secondary model, but without AQCH treatment. In the "AQCH only" group, they were not infected with S221 and also fed with AQCH (25 mg/kg four times a day). The control "IC" group was included in the secondary model, in which mice were inoculated intracutaneously with S221 and 4G2.

The mice in the "uninfected" group and the "AQCH only" group are expected to survive the entire study period. Because the infection is lethal, the mice in the "V" group died of the infection within 4-7 days in the primary model, and in the secondary model, because the infection is sublethal, the mice Survive. The 4G2-mediated ADE increased the secondary lethal dose to the lethal dose, so that the intradermal inoculated AG129 mice died of infection within 4-7 days. Various studies have been conducted on these two models, such as the effect of AQCH dose and dosing regimen, the effect of delaying the initial AQCH treatment, and the effect of AQCH treatment on the pathology of the small intestine. For the in-depth characterization of the protective efficacy of AQCH, Figure 9 provides a schematic overview of the experiments conducted in the primary and secondary dengue AG129 mouse models in this study.

Evaluation in the AG129 mouse model of primary dengue fever disease : Primary dengue fever was determined by attacking it with a lethal dose (1×105 FIU) of the DENV-2 S221 strain in immunocompromised AG129 mice. Starting from 30 minutes after infection, mice were gavaged with AQCH by oral gavage at 33 and 100 mg/kg/day twice a day, three times a day, and four times a day for 5 days. After the lethal challenge, after monitoring the mouse group for 15 days, the protection level brought by AQCH was scored by the survival curve. The effect of delaying the initial AQCH treatment was also evaluated. Mice infected with a lethal dose of DENV-2 S221 were treated with 25 mg/kg/dose of AQCH four times a day 6 and 12 hours later for another 5 days. 15 days after the lethal attack, the protection level was scored via the survival curve. Monitor the weight change, disease symptoms and survival rate of mice.

Evaluation in the AG129 mouse model of secondary dengue fever disease : the secondary lethal dose of the immune complex (IC) of DENV-2 S221 (2x104 FIU) and the neutralization of the 4G2 mAb (10 µg) concentration were established to establish secondary Dengue fever AG129 mouse model. After intradermal inoculation, mice challenged intradermally with oral gavage with different doses of AQCH at 33 and 100 mg/kg/day twice a day, three times a day, and four times a day (n=6) 5 days. In a similar way to the AG129 model of primary dengue fever, the level of protection conferred by AQCH and the delayed treatment effect were checked. In order to test the preventive potential, in mice with secondary diseases, AQCH was orally fed at a dose of 50 mg/kg/dose twice a day for 3 days before and after intradermal infection. Monitor the weight change, disease symptoms and survival rate of mice.

Evaluation of serum viremia and small intestinal pathologist viremia, vascular leakage, and proinflammatory cytokine levels in the secondary dengue fever model of AG129 mice: two groups of AG129 mice injected intracutaneously (n=6 ) AQCH was orally fed four times a day at 8.25 and 25 mg/kg/dose for 4 days. Serum was collected from each group (n=3) on day 4 for evaluation of viral RNA equivalents via real-time quantitative PCR (RT-qPCR), and Evans blue dye was used to evaluate serum collection in these mice Vascular leakage, as detailed in the supplementary information. On the 4th day, the remaining three mice from each group were euthanized, and the small intestines were collected to check the levels of enteroviremia and TNFα and IL-6 cytokines.

result : Primary dengue fever disease AG129 In vivo evaluation of the aqueous extract of broomsticks against dengue fever activity in a mouse model

In this study, the inventors used twice a day (orange curve) and three times a day (purple curve) schemes (Figure 2a) to evaluate two different doses of 100 mg/kg/day (solid curve) and 33 mg/kg /Day (dashed curve) under the protection of AQCH. The twice a day and three times a day regimens also confer protection similar to four times a day (100 and approximately 33% for two doses). Therefore, there was no significant difference in the survival rate score of the mice between the dosing regimens under either of the two AQCH doses. However, for each of the two doses, the morbidity and weight loss scores of mice under the twice-daily regimen were appropriately higher than three times a day and four times a day (Figure 10c, d). The mice in the control group behaved as expected. Of the two doses, significantly lower protection and higher morbidity were observed in the twice-daily and three-times daily dosing regimens under 33 mg/kg/day AQCH treatment (p=0.01). An additional dose of 15 mg/kg four times a day was also evaluated, resulting in an efficacy of approximately 80% survival rate (data not shown).

Secondary dengue fever AG129 In vivo evaluation of the aqueous extract of broomsticks against dengue fever activity in a mouse model

ADE has been implicated in causing severe dengue fever. The inventors here study the therapeutic potential of AQCH for severe dengue fever. For this reason, AQCH was evaluated in a secondary model of dengue disease in AG129 mice. On 5 days after vaccination, in the twice daily (orange curve), three times a day (purple curve) and four times a day (blue curve) dosing regimens, 33 mg/kg/day (dashed curve) and 100 mg/ Different doses of Kg/day (solid curve) were fed AQCH to mice that were challenged intracutaneously (Figure 11). Including the control group "Uninfected" (magenta curve), "V" (green curve), "AQCH only" (black curve) and "IC" (gray curve) that produced the expected results. Unlike the primary dengue fever disease model, the efficacy of AQCH is not similar in the three evaluated dosing regimens. Four times a day is the most effective dosing regimen, followed by three times a day and twice a day, because 100%, 50%, and 0% survival rates were observed at 100 mg/kg/day AQCH (Figure 11b). An additional dose of 15 mg/kg four times a day was also evaluated, and the efficacy of about 80% survival rate (data not shown) was evaluated.

Because the 100 mg/kg/day AQCH dose did not provide protection in secondary dengue AG129 mice in the twice-daily regimen, it was only evaluated in the three-times-a-day and four-times-a-day regimens exhibiting 33% and 50% protection, respectively. The lower AQCH dose of 33 mg/kg/day (Figure 11b). Morbidity and weight change scores are extremely correlated with survival scores (Figure 11c and d). Under the result that the twice-daily regimen was unprotected in secondary disease AG129 mice, the inventors wanted to evaluate whether pre-feeding AQCH to these mice in the twice-daily regimen before intradermal vaccination was protective effect. Therefore, AG129 mice were fed AQCH at 50 mg/kg twice daily for three days before and after intradermal vaccination (Figure 12a). Interestingly, all mice survived (Figure 12b) and showed only mild morbidity (Figure 12c) and rapid recovery with insignificant weight loss (Figure 12d).

After the start of treatment is delayed , Determination of the aqueous extract of broom cane AG129 Protective efficacy of primary and secondary dengue fever infections in mice

After the injection, the efficacy of AQCH at the most effective dose of 25 mg/kg four times a day was also evaluated by delaying the initial treatment of primary and secondary dengue fever disease AG129 mice for 6 and 12 hours (Figure 13a). In the primary and secondary dengue fever disease models, it was observed that almost all mice can tolerate the treatment delay for 6 hours (orange solid curve), providing 100% and 83% in the primary and secondary models, respectively Survival rate. In both models, after a 12-hour delay in the start of AQCH treatment (orange dashed curve), the survival rate dropped slightly to about 70% (Figure 13b, c). Therefore, even after a 6-12 hour delay in the initiation of treatment, AQCH can substantially protect immunocompromised AG129 mice.

In secondary AG129 Mouse model , Determination of the water-based extract of broom cane to prevent the pathogenesis of dengue fever - Serum viremia and small enteric viremia, interleukin storm and vascular leakage

AG129 mice with secondary dengue fever disease were orally fed with AQCH (8.25 mg/kg and 25 mg/kg four times a day) for 4 days. The mice in the "IC" group were visibly lethargic, and the mice in the 25 mg/kg AQCH treatment group four times a day were as active as the "AQCH only" group. The level of viremia was determined in the serum and a small intestine sample was collected on the 4th day. It was observed that AQCH significantly reduced both serum (Figure 14a) and small intestine (Figure 14b) viremia levels. However, unlike enteroviremia, the reduction in serum viremia is not dose-dependent, because treatment with 8.25 mg/kg (blue hollow bars) AQCH four times a day produces serum viruses compared to 25 mg/kg A higher degree of reduction in hememia (blue solid bars, Figure 14a).

Evans blue dye is used to detect vascular leakage, which acts as a marker for albumin extravasation. Quantitative leakage is estimated by colorimetry of dye absorption. Compared with the "uninfected" control, the leakage is expressed as a multiple increase of OD620/gram of tissue wet weight. It was found that vascular leakage was significantly reduced after AQCH treatment at 25 mg/kg four times a day (Figure 15a, solid blue bars) and 8.25 mg/kg four times a day (Figure 15a, open blue bars). It is expected that the highest level of vascular leakage was observed in the intradermal group of mice (Figure 15a, gray bars), which is due to the enhanced persistence of infection mediated by 4G2 mAb. The visible examination of the small intestine of mice in these groups on day 4 also reflected similar findings of the unique fluid accumulation in the intradermal group; and groups "V", "IC+25 mg/kg AQCH four times a day" And "IC+8.25 mg/kg AQCH four times a day" showed normal small intestine, as in the "AQCH only" group (Figure 15b).

We also measured the expression levels of pro-inflammatory cytokines TNFα (Figure 15c) and IL-6 (Figure 15c) in the small intestine to gauge the degree of cytokine storm. The pattern is similar to that observed for vascular leakage, where AQCH treatment inhibits the secretion of intestinal cytokines in a dose-dependent manner, in which mice in the intradermal group showed the highest levels of cytokines due to enhanced infection by ADE Expression (Figure 15b, c).

Example 11 : Stability analysis

Use approved excipients to formulate AQCH into tablets of different strengths (100 mg, 300 mg, and 500 mg). By exposing AQCH and AQCH lozenges to different conditions (30±2°C/65±5% RH and 40±2°C/75±5% RH) to evaluate their acceleration and long-term stability. The anti-dengue activity and the content of the selected labeled compound in the test tube were evaluated on the stored samples at different time points (1, 2, 3, and 6 months) through virus inhibition analysis based on flow cytometry and HPLC chromatography. .

Result : The next aspect of AQCH used for its clinical utility is to evaluate its feasibility to be formulated into tablets. Therefore, AQCH tablets with concentrations of 100, 300, and 500 mg were formulated and subjected to accelerated and long-term stability studies together with the batch of AQCH extract from which the tablets were formulated. By flow cytometry-based virus inhibition analysis, samples from the stability study were analyzed for the content of magnoliine and the anti-DENV-2 activity in the test tube to evaluate any degradation or biological activity during storage under the tested conditions. loss. It has been found that under the tested conditions for up to 6 months, there is neither a significant change in the content of magnolin (S2 table), nor the anti-DENV-2 activity of AQCH (Table 1) and all three concentrations (100, 300). And 500 mg) any loss of AQCH tablets. Long-term stability studies are ongoing and samples will be evaluated for storage for up to 3 years. This data is encouraging because it ensures the feasibility of formulating AQCH into a stable tablet dosage form, which facilitates its clinical evaluation.

Table 2 : Anti- DENV-2 activity of AQCH and AQCH tablets under the stated storage conditions AQCH extract / tablet (batch ID ) Storage conditions DENV-2 IC ₅₀ ( μg/ml ) ^a 1 month 2 months 3 months 6 months Extracts 40±2℃, 75±5% RH 4.2 4.0 5.4 7.2 30±2℃, 65±5% RH nd* nd* 6.1 5.7 Tablet 100 mg 40±2℃, 75±5% RH 4.7 4.6 7.3 5.4 30±2℃, 65±5% RH nd* nd* 7.8 4.7 Tablet 300 mg 40±2℃, 75±5% RH 5.2 6.2 6.2 5.3 30±2℃, 65±5% RH nd* nd* 6.1 4.0 Tablet 500 mg 40±2℃, 75±5% RH 4.6 2.7 5.1 5.1 30±2℃, 65±5% RH nd* nd* 6.8 4.7 The anti-DENV-2 activity is measured as IC ₅₀ , which corresponds to the concentration at which the virus control inhibits 50% of the virus* nd: not determined; these specific storage conditions are used for long-term stability studies and are therefore the first in storage Month and second month without sampling

Table 3 : Stability data of AQCH and AQCH lozenges relative to the content of magnolin as an analytical marker AQCH extract / tablet (batch ID ) Storage conditions Magnolia content ( w/w% ) 1 month 2 months 3 months 6 months Extracts 40±2℃, 75±5% RH 0.39 0.43 0.41 nd* 30±2℃, 65±5% RH nd* nd* 0.41 0.34 Tablet 100 mg 40±2℃, 75±5% RH 0.37 0.37 0.38 0.43 30±2℃, 65±5% RH nd* nd* 0.38 0.42 Tablet 300 mg 40±2℃, 75±5% RH 0.41 0.4 0.41 0.45 30±2℃, 65±5% RH nd* nd* 0.41 0.46 Tablet 500 mg 40±2℃, 75±5% RH 0.4 0.4 0.41 0.45 30±2℃, 65±5% RH nd* nd* 0.41 0.46 * nd: Not determined; these specific storage conditions are used for long-term stability studies and therefore have not been sampled in the first and second months of storage.

In this study, it was found that AQCH inhibited the secretion of both DENV and NS1 in a dose-dependent manner, with complete inhibition observed at 100 and 50 µg/ml extracts. This is related because DENV load and NS1 have been involved in the pathogenesis of dengue fever in humans. Based on the HPC profile, five labeled compounds in the AQCH batch were also identified-Sinococuline, magnolia, 20-hydroxyecdysone, Makisterone A and coniferol (Figure 4). The in vivo protective efficacy of AQCH was also evaluated in the AG129 mouse model (Figure 6), and when fed at 25 mg/kg/dose four times a day, it was found to provide 100 %protect. It was also found that paracetamol, a standard care drug for the treatment of dengue fever, had no effect on the anti-dengue fever activity of AQCH (Figure 7). In addition, the spray-dried AQCH was formulated into tablets of various concentrations, and it was found that the tablets were stable during storage (Table 1). It was also tested whether AQCH could protect AG129 mice at 100 mg/kg/day four times a day in the two infection models. Unexpectedly, AQCH can survive the delayed treatment in both infection models. Nearly complete protection was observed when treatment was delayed by 6 hours (100% in the primary model and approximately 85% in the secondary model), and approximately 70% when treatment was delayed by 12 hours in both infection models. % Survival rate (Figure 13). Considering that AG129 mice are immunologically insufficient and prone to death from infection, these results indicate sufficient therapeutic efficacy of AQCH in the two infection models after the delay in the initiation of treatment, without any interferon immunity. The increased frequency of dosing produces a significantly higher survival rate effect. The effect of the dosing regimen is not obvious in the primary AG129 mice, however, it becomes more discernible in the secondary dengue infection model, where four times a day of 100 mg/kg/day makes 100% Animals survived, but no animals survived twice daily feeding (Figures 10 and 11). However, after the preventive treatment of AQCH, the twice-daily regimen became extremely effective in keeping 100% of the mice alive, reflecting the preventive ability of AQCH (Figure 12). In conclusion, as assessed by in vivo studies in the AG129 mouse model, AQCH exhibits strong protection.

without

[ Figure 1 ] : Anti-dengue fever activity of broom vine and C. pareira (in test tube): (a) As reported in the table, separately calculate each of the three extracts prepared from the two plants The IC _{50 value of the patient and its geometric mean IC 50} value for each of the four DENV (Dengue fever virus) serotypes. (B) The DENV-2 infection% chart observed with each of the six extracts, in which the gray and blue curves represent the methanol extracts from the ground of the three types of broom vines and cinnamon vines. The dashed horizontal line represents 50% DENV-2 infection value.

[ Figure 2 ] : By flow cytometry-based virus inhibition analysis, compared to DENV-1 (magenta curve), DENV-2 (green curve), DENV-3 (blue curve) and DENV-4 (Black curve) Evaluate the anti-dengue fever activity of extracts at various concentrations. (A) The percentage of DENV infection relative to the virus control is represented graphically: denatured alcohol (left picture), water-alcohol, 50:50 (middle picture) and water (right picture) above ground (dashed curve) and stem (solid curve) )Extracts. (B) As shown in the table for all extracts, the extract concentration (µg/ml) that produces 50% inhibition of virus infection compared with the virus control (indicated by the horizontal dotted line in group "a") uses Graphpad Prism Calculated as IC ₅₀ .

[ Figure 3 ] : AQCH (aqueous extract of Broom vine) inhibits the secretion of DENV and its antigen NS1 in a dose-dependent manner: (a) DENV secreted by virus titration analysis based on FACS from day 1-6 -1 amount to produce FIU/ml, and (b) on the 6th day, the% inhibition of viral antigen NS1 secretion against all four DENV serotypes was evaluated by a commercial ELISA kit.

[ Figure 4 ] : Chemical fingerprint traces of AQCH: (a) Prepare AQCH batch materials and target DENV-1 (magenta curve), DENV-2 (green curve), DENV-3 (blue curve) and DENV The anti-dengue fever activity of -4 (black curve) was confirmed by virus inhibition analysis based on flow cytometry, as shown in the graph of DENV infection and extract concentration %. Compared with the virus control (denoted by horizontal dashed line), it has been reported IC ₅₀ values corresponding to a concentration of 50% DENV AQCH infected suppressed in the table in the inset. (B) AQCH HPLC chemical fingerprint retention curve, in which the peaks correspond to the five identified labeled compounds. (C) The chemical structures of five labeled compounds (1-5).

[ Figure 5 ] : AQCH preparation method is consistent and stable: various batches of AQCH are prepared through three different methods, namely rotary steam drying (RD), vacuum tray drying (VTD) or spray drying (SD). One is dry. The effect of the drying method was evaluated by evaluating the following: (a) Anti-dengue fever activity based on virus inhibition analysis based on flow cytometry to generate IC ₅₀ value (compared to virus control, it is necessary to reduce DENV infection by 50% The extract concentration), and (b, c) chemical fingerprint retention profile; the stacked HPLC chromatograms of the three batches corresponding to the three drying conditions and the retention time table of the five labeled compounds are shown in Figure 『b "And "c".

[ Figure 6 ] : AQCH protects AG129 mice from lethal DENV-2 S221 infection: (a) Schematic diagram of the experimental design using five groups of AG129 mice (n=6). A group of "uninfected persons" represented by the black curve was neither infected with DENV-2 S221 nor administered AQCH. Another group called "AQCH only" and represented by the orange curve was not infected with DENV-2 S221 but received AQCH dose (25 mg/kg four times a day). The third group called "V" and represented by the gray curve was infected with DENV-2 S221 but not administered with AQCH. Mice in the remaining two groups were infected with DENV-2 S221 and were dosed with 25 mg/kg four times a day (blue curve) or 8.25 mg/kg four times a day (pink curve). DENV-2 S221 infection was given intravenously at a lethal dose of 1.0×105 FIU, while AQCH was given orally after infection. The (b) survival rate, (c) morbidity score and (d) weight change of all groups were monitored 15 days after infection. The log-rank (Mantel-Cox) test was used to analyze the survival rate data (group "b") in order to statistically evaluate the significance level of the difference in survival rate. The survival rates of the mice in the groups "V+AQCH 25 mg/Kg four times a day" and "V+AQCH 8.25 mg/Kg four times a day" were not significantly different from each other (p=0.14), but compared with the group "V The mice in "are significantly different (p=0.006 and p=0.016, respectively). The p value<0.05 is considered significant. The incidence in group "c" is based on a 5-point system: 0.5, slightly wrinkled fur; 1.0, wrinkled fur; 1.5, eye damage; 2, eye damage and hunchback; 2.5, soft stool; 3.0, limited movement ; 3.5, no movement/paralysis of hind legs; 4.0, if the cumulative score is 5, euthanasia. The body weight in the group "d" was monitored twice a day in the morning and at night, and the average value used to draw the curve was obtained.

[ Figure 7 ] : Paracetamol does not inhibit the anti-dengue fever activity of AQCH. Vero cells infected with DENV-1 are used in the absence (black curve) and 1 (orange curve), 10 (magenta curve) and 100 (blue curve) µg/mL paracetamol. Various concentrations of AQCH extract (0-25 µg/ml) are processed. The percentage of DENV-1 infection achieved under these conditions was evaluated in the virus inhibition analysis based on flow cytometry, which is depicted in the graph on the left. Compared with the virus control, the AQCH concentration that reduces DENV-1 infection by 50% is calculated for each condition as its corresponding IC50, and is depicted in the table on the right.

[ Figure 8 ] : Separation procedure of labeled compounds 1-5 from AQCH.

[ Figure 9 ] : Schematic diagram of the total in vivo protective efficacy study conducted with AQCH.

[ Figure 10 ] : The effect of AQCH dose and dosing schedule for fatal primary DENV infection. (A) Schematic diagram of the research design. A lethal dose of DENV-2 S221 virus (105 FIU/mouse) was used to inoculate the AG129 mouse group (n=6), followed by three different dosing schedules, namely twice a day (BID; orange Curve), three times a day (TID; purple curve) and four times a day (QID; blue curve) with two doses of AQCH, namely 100 mg/kg/day (solid curve) and 33 mg/kg/day ( Dotted curve) oral gavage for a period of 5 days. Then monitor the mice for 10 days (b) survival period, (c) morbidity and (d) body weight changes. Uninfected (neither infected with virus nor fed AQCH; indicated by magenta), virus-infected V (infected with virus but not fed AQCH; indicated by the green curve) and AQCH only (not infected with virus but at 25 mg/ The group of AG129 mice fed with AQCH (black curve) four times a day served as the experimental control. The insignificant (ns) and single star (*) in group "b" indicate the statistical difference between the same administration time course of different doses.

[ Figure 11 ] : The effect of AQCH dose and dosing schedule on secondary DENV infection: (a) Explain that AG129 mice were injected with sub-lethal doses of DENV-2 S221 (marked as V; green curve) or as DENV- 2 Schematic diagram of S221-4G2 IC (IC). IC injection of AG129 mice to indicate higher (solid curve) and lower (dashed curve) doses orally fed AQCH, or only twice a day (orange), three times a day (purple) and four times a day (blue) ) Dosing schedule Methyl cellulose (marked as IC; gray curve) is administered for 5 days. A group of AG129 mice that were not infected (magenta curve) and only AQCH (black curve) were used as experimental controls. (B), (c) and (d) respectively represent the survival curve, morbidity score and weight change observed during the entire 15-day study period. The statistical difference (ns; not significant and *; less significant) of the survival rate between different administration time courses is shown on the right side of the figure.

[ Figure 12 ] : Preventive effect of AQCH in AG129 mice with secondary dengue fever disease. (A) Show a schematic representation of the experimental method, in which mice were pre-fed with 50 mg/kg AQCH twice a day (orange curve) for three days, followed by intradermal inoculation with DENV-2 S221-4G2, where in the skin After vaccination, AQCH was fed for three days. Then monitor the mice's (b) survival rate, (c) morbidity score and (d) weight change for 12 days. Uninfected (neither infected with virus nor fed AQCH; indicated by magenta), virus-infected V (infected with sub-lethal dose of virus but not fed AQCH; indicated by the green curve) and intradermal (intradermal but inoculated but not fed AQCH) A group of AG129 mice not fed with AQCH; gray curve) served as experimental control.

[ Figure 13 ] : Delayed therapeutic effects of AQCH on primary and secondary DENV infections: (a) Schematic diagram of the study design, in which mice were injected with a lethal dose of DENV-2 S221 strain (primary Dengue fever disease model) or intradermal injection of lethal doses of DENV-2 S221 and 4G2 mAb (secondary dengue fever disease model). After the inoculation, the mice were orally fed with AQCH at 25 mg/kg four times a day for 5 days, in which the initial treatment was delayed. For one group, the treatment was delayed for 6 hours (solid blue curve) and another 12 hours (dashed blue curve). The survival of the mice was monitored for another 10 days. The survival curves were shown in (b) and (c) of studies conducted in AG129 mice with primary and secondary dengue fever disease, respectively. Only the AQCH (black curve), virus alone, V (green curve) and intradermal (gray curve; in group c) mouse groups were used as experimental controls. The statistical difference is shown on the right side of each survival rate graph, ns=not significant (p>0.05), **=significant (p=0.0024).

[ Figure 14 ] : Serum and enterovirus inhibition by AQCH in secondary DENV infection: AG129 mice that were inoculated with DENV-2 S221-4G2 IC to establish secondary dengue fever were orally fed 25 mg/kg AQCH four times a day (blue solid bars) or 8.25 mg/kg four times a day (blue hollow bars). Serum samples were drawn on the 4th day and the mice were sacrificed to collect small intestine tissue. SYBR Green-based real-time PCR was used to determine the serum viremia level (a) and intestinal virus load (b). The "AQCH only", "V" and "IC" control groups are displayed in the black, green and gray bars respectively. The statistical differences between the groups are presented at the top of the bar graph, ns=not significant (p=>0.05), ***=very significant difference (p<0.0001).

[ Figure 15 ] : AQCH in secondary DENV infection inhibits intestinal vascular leakage and reduces cytokine storm. The secondary dengue fever AG129 mice (n=6) were treated with AQCH at 25 mg/kg (solid blue bars) or 8.25 mg/kg (open blue bars) four times a day for 4 days. (A) On the 4th day, the vascular leakage of the small intestine of the three AG129 mice of each group was tested by Evans blue staining assay. Refer to the untreated control that has not received any AQCH treatment, and the data is depicted as the fold change of dye staining. The multiple change of 1 corresponds to the basal vascular leakage, which is equivalent to the untreated group. On the 4th day, the remaining three mice of each group were euthanized. The mice were perfused with sterile PBS, and the luminal contents were flushed again with PBS to identify visual intestinal vascular leakage; representative images of the small intestine of each group Shown in (b). Prepare 50 mg of the small intestine from "b" for the determination of TNF-α(c) and IL-6(d) by ELISA. The control group used in this study is "AQCH only" group (black bar), virus only group (V; green bar); IC group (gray bar). The statistical difference between the two groups was calculated by two ANOVAs with Bonferroni correction (Bonferroni correction) and displayed at the top of the bar graph; ns=not significant, *p=0.01, **p=0.001 and * **p<0.0001.

Claims (26)

A compound selected from formula I-IV,

Formula-I Formula-II

or

Formula-III Formula-IV or a pharmaceutically acceptable salt thereof, which is used for the treatment of dengue fever virus infection in mammals in need. A method for treating dengue virus infection, which comprises administering to a mammal in need a therapeutically effective amount of one or more compounds selected from formula I-IV:

Formula-I Formula-II

or

Formula-III Formula-IV. The method of claim 2, wherein the treatment is a preventive treatment or a curative treatment. The compound of claim 1 or the method of claim 2, wherein the dengue virus infection is caused by one or more serotypes of dengue virus. The compound or method of claim 4, wherein the dengue virus serotype is selected from DENV-1, DENV-2, DENV-3 and DENV-4. The method of claim 2, wherein the treatment is characterized by a reduction in the titer of dengue virus in a tissue sample of the mammal. A method for inhibiting the growth and/or proliferation of dengue fever virus in mammals in need, which comprises administering a therapeutically effective amount of one or more compounds selected from formula I-IV:

Formula-I Formula-II

or

Formula-III Formula-IV. The compound of claim 1, wherein the compound is a synthetic compound, a semi-synthetic compound, or a compound isolated from the extract of the plant Cocculus hirsutus. A stable pharmaceutical composition comprising a therapeutically effective amount of one or more of the compounds according to claim 1, wherein the composition exhibits a platelet protective effect. The method of claim 7, wherein the inhibition of the growth and/or proliferation of the dengue virus is determined by measuring platelet protection. A stable pharmaceutical composition comprising a therapeutically effective amount of one or more of the compounds according to claim 1, wherein the composition inhibits the growth and/or proliferation of dengue fever virus. Such as the composition of claim 11, wherein in an in vitro condition, the composition inhibits the growth and/or proliferation of the dengue virus. The composition of claim 11, wherein the composition inhibits the growth and/or proliferation of the dengue virus under in vivo conditions. A pharmaceutical composition comprising a therapeutically effective amount of two or more compounds selected from formula I-IV:

Formula-I Formula II

or

Formula-III Formula-IV, wherein the two or more compounds are stable at 25°C and 75% relative humidity for at least 1 month. The composition of claim 14, wherein the composition further comprises a pharmaceutically acceptable excipient, and wherein the composition is stable at 25° C. and 75% relative humidity for at least 3 months. A stable pharmaceutical composition for the treatment or prevention of dengue fever virus infection, which comprises an extract of S. sylvestris, wherein the extract contains one or more compounds as claimed in claim 1 whose concentration is increased by at least 400% relative to that of S. sylvestris. Such as the composition of claim 16, wherein 95% of the fiber has been removed from the extract. Such as the composition of claim 16 or 17, in which 95% of the lignin has been removed from the extract. Such as the composition of any one of claims 16 to 18, wherein 95% of tannin has been removed from the extract. The composition of any one of claims 16 to 19, wherein the one or more compounds of claim 1 present in the extract comprise the one or more compounds in a therapeutically effective concentration. The composition according to any one of claims 16 to 20, wherein the extract is formed by extraction of an aqueous solution. The composition according to any one of claims 16 to 21, wherein the composition is stable at 25°C under 75% relative humidity for at least 3 months. The composition of any one of claims 14 to 22, wherein the composition has less than 5% water. The composition according to any one of claims 14 to 22, wherein the composition is an oral dosage form. A method for treating dengue virus infection, which comprises administering a therapeutically effective amount of the composition according to any one of claims 14 or 16 to a mammal in need. A method for inhibiting the growth and/or proliferation of dengue virus in mammals, which comprises administering a therapeutically effective amount of the composition according to any one of claims 14 or 16.

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