KR20210126519A - Flavonoid glycosides comprising anti-corona activity - Google Patents

Abstract

The present invention relates to a use of a flavonoid glycoside compound for anti-corona virus. Particularly, the present invention relates to: an antiviral composition against corona virus; a pharmaceutical composition for preventing or treating corona virus infection diseases; a health-aid food composition for preventing or alleviating corona virus infection diseases; a feed composition; and a sanitary-aid composition, including a combination of three flavonoid glycoside compounds as an active ingredient. The composition including a compound represented by chemical formula 1, a compound represented by chemical formula 2 and a compound represented by chemical formula 3 as active ingredients according to the present invention shows an effect of inhibiting infection with corona virus, and thus can be used for developing an antiviral agent for preventing or treating the virus infection diseases.

Description

Flavonoid glycosides comprising anti-corona activity

The present invention relates to the anti-coronavirus use of a flavonoid glycoside compound, and specifically, the present invention relates to an antiviral composition for coronavirus comprising a combination of three flavonoid glycoside compounds as an active ingredient; A pharmaceutical composition for the prevention or treatment of a coronavirus infectious disease; It relates to a health functional food composition, feed composition and quasi-drug composition for preventing or improving coronavirus infectious disease.

Respiratory disease, which started in Wuhan, China in 2019, is a disease caused by coronavirus infection, showing severe respiratory symptoms, leading to death of a significant number of infected patients. Remdisivir ( GS-5734, remdesivir) and favipiravir are used for the treatment of coronavirus infection, but they are ineffective and have a high incidence of variant viruses due to the nature of the nucleic acid analog antivirals [Wang] M, Cao R, Koh Mun L, Yang X, Liu j, Xu M, Shi Z, Hu Z, Zhong W, Xiao G. 2020. Remdesvir and choloquinie effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Research 30: 269-271].

Most of the antiviral agents used for coronavirus infection, such as ribavirin and remdisivir, are substances with RNA polymerase inhibitory activity. However, a mouse hepatitis virus, a type of coronavirus with resistance to remdisivir, has been found, and the existing antiviral agents have no effect.

On the other hand, flavonoids are being studied on the inhibitory ability of the coronavirus polymerase enzyme protein, and it has been known that campferol acetyl 3-glucoside and quercetin acetyl 3-glucoside exhibit inhibitory activity on eukaryotic DNA polymerase (Mizushina Y, Ishdoh T, Takemura M, Sugawara F, Yoshida H, Sakaguchi K. 2003. Flavonoid glycoside: a new inhibitor of eukaryotic DNA polymerase alpha and a new carrier for inhibitor-affinity chromatography. Biochem Biophys. Res. Commun. 301(2):480-487 ), an antiviral composition for coronavirus using a plant extract is known (Korean Patent No. 10-0697309), but the anti-corona activity of the flavonoid glycoside mixture of the present invention has not yet been studied.

In addition, since the antivirals developed to date exhibit severe side effects, much attention is required in their application. These treatments are not effective and side effects are also present. Therefore, there is an increasing need for the development of excellent novel coronavirus agents with excellent anti-infection effects and low toxicity for preventing and treating coronavirus outbreaks.

Under this background, the present inventors, as a result of continuing efforts to develop novel coronavirus therapeutics, each isolated from a plant extract or a mixture of three flavonoid glycosides chemically synthesized by mimicking it has anti-coronavirus activity By confirming, the present invention was completed.

One object of the present invention is to provide an antiviral composition for coronavirus comprising a combination of three types of flavonoid glycoside compounds as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a coronavirus infection disease comprising a combination of three types of flavonoid glycoside compounds or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a health functional food composition for the prevention or improvement of coronavirus infection disease comprising a combination of three kinds of flavonoid glycoside compounds as an active ingredient.

Another object of the present invention is to provide a feed composition for the prevention or improvement of a coronavirus infection disease comprising a combination of three types of flavonoid glycoside compounds as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for the prevention or improvement of a coronavirus infection disease comprising a combination of three kinds of flavonoid glycoside compounds as an active ingredient.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object is a coronavirus comprising a compound represented by the following formula 1, a compound represented by formula 2, and a compound represented by formula 3 (three flavonoid glycoside compounds) as an active ingredient. It provides an antiviral composition for antiviral.

[Formula 1]

[Formula 2]

[Formula 3]

The compound having the structure of Chemical Formula 1 is a flavonoid glycoside compound _{having a molecular weight of 448.38 and Chemical Formula C 21} H ₂₀ O _{11 as Quercetin 3-rhamnoside (Quercitrin).}

In addition, the compound having the structure of Chemical Formula 2 is a flavonoid glycoside compound _{having a molecular weight of 464.38 and Chemical Formula C 21} H ₂₀ O _{12 as Quercetin 3-glucoside (Isoquercetin).}

In addition, the compound having the structure of Formula 3 is a flavonoid glycoside compound _{having a molecular weight of 464.38 and Formula C 21} H ₂₀ O _{12 as Quercetin 3-galactoside (Hyperoside).}

The compounds represented by Formulas 1 to 3 of the present invention, for example, the compound represented by Formula 1, the compound represented by Formula 2, and the compound represented by Formula 3 The composition comprising the compound represented by Formula 3 may be in the range expected by some skilled in the art from these compounds. Derivatives may be included, and are included without limitation as long as they have the same effect in the present invention. These compounds may be obtained from an extract containing them, but are not limited thereto, and may be synthesized and used by a known method. The extract may be, for example, an extract or a fraction of Eoseongcho, as long as it is a plant extract or fraction containing the compound, but is not limited thereto.

In the present specification, the compounds represented by the terms Formulas 1 to 3 may be used in combination with three types of flavonoid glycoside compounds, a mixture of flavonoid glycosides, a mixture of three types of flavonoid glycosides, and a mixture of three types.

The content ratio of each compound in the composition comprising the compound represented by Formula 1, the compound represented by Formula 2, and the compound represented by Formula 3 of the present invention may be 1 to 2: 1: 1 to 5, preferably may be 1:1:1, but is not limited thereto.

The compounds represented by Chemical Formula 1, Chemical Formula 2 and Chemical Formula 3 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is used. Can be used. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids, and organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, and fumaric acid. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol late, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving methylinderone of Formula 1 in an organic solvent, such as methanol, ethanol, acetone, methylene chloride, acetonitrile, or the like, and adding an organic or inorganic acid to the precipitate. It can be prepared by filtration and drying, or by drying or crystallization in an organic solvent after distilling the solvent and excess acid under reduced pressure.

In addition, a pharmaceutically acceptable metal salt may be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. Also, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate). In addition, the present invention includes all possible solvates, hydrates, stereoisomers, etc. that can be prepared therefrom, as well as methylinderone represented by Formula 1 and a pharmaceutically acceptable salt thereof.

As used herein, the term "coronavirus" is a virus belonging to the family Coronaviridae, and in one embodiment of the present invention, a composition comprising a combination of compounds represented by Chemical Formulas 1, 2 and 3 It was confirmed that there is a coronavirus inhibitory activity (Table 1 and Table 2).

In one embodiment of the present invention, the coronavirus is swine epidemic diarrhea virus (PEDV), swine infectious gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), Middle East respiratory syndrome coronavirus (MERS-CoV), severe It may be acute respiratory syndrome coronavirus (SARS-CoV) and novel coronavirus (CoVID-19), but is not limited thereto.

In one embodiment of the present invention, the inhibitory activity effect of the antiviral composition on porcine epidemic diarrhea virus (PEDV), porcine infectious gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) was confirmed (Example 2 and 3).

The above result shows that the homogeneity of the polymerase amino acid sequence between each coronavirus is 50% or more, and when the amino acid sequence similarity is included, the similarity is very high, over 75%. Considering that it reaches 100%, the antiviral activity against the coronavirus is applicable to other coronaviruses, suggesting that the antiviral composition of the present invention has anticorona activity.

In addition, in one embodiment of the present invention, it is confirmed that the antiviral composition comprising a combination of three types of compound 1, compound 2 and compound 3 has a more significant anti-corona activity than each of the compounds or a combination of two types of compounds. did. In addition, it was confirmed that it has a superior effect than the conventional anti-coronavirus agent as a positive control.

The above results were greater than the effect of combining three types of single compounds, and were more significant than the effects of combining the combinations of two types of compounds, confirming that they correspond to a synergistic effect (Table 1, FIGS. 1 to 4).

Another aspect of the present invention is a compound represented by Formula 1, a compound represented by Formula 2, and a compound represented by Formula 3; Or it provides a pharmaceutical composition for preventing or treating a coronavirus infection disease comprising a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term "coronavirus infectious disease" refers to a disease caused by infection with a coronavirus.

As used herein, the term “infection” refers to a state in which a pathogenic microorganism invades the body of a host organism and develops and proliferates.

In one embodiment of the present invention, the coronavirus infection disease may be a cold, cough, sputum, bronchitis or severe respiratory disease in humans, and hepatitis, encephalitis, neurological disease, severe respiratory disease, severe diarrhea or It may be a respiratory disease, peritonitis, etc., but is not limited thereto.

As used herein, the term "prevention" refers to any action that inhibits or delays the onset of an infectious disease caused by a coronavirus by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action in which the symptoms of a suspected and onset individual of a coronavirus infection are improved or beneficially changed by administration of the pharmaceutical composition.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition, and the carrier may include a non-naturally occurring carrier. Specific examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used, but is not limited thereto.

In addition, the pharmaceutical composition can be prepared as tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-drying agents and suppositories according to conventional methods, respectively. It may have any one formulation selected from the group consisting of, and may be oral or parenteral various formulations. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. A solid preparation for oral administration may include tablets, pills, powders, granules, capsules, and the like, and the solid preparation includes at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, Gelatin or the like may be used. In addition to simple excipients, lubricants such as magnesium stearate and talc may be used. Liquid formulations for oral administration may include suspensions, internal solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be used. can be used A sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, or a suppository may be used as a formulation for parenteral administration. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. may be used, but is not limited thereto.

The pharmaceutical composition of the present invention contains, with respect to the total weight of the composition, the compound represented by Formula 1, the compound represented by Formula 2, and the compound represented by Formula 3 at 0.1 to 1000ug/ml, specifically 0.1 to 200ug/ml, It may contain 0.1 ~ 100ug/ml, but is not limited thereto.

The pharmaceutical composition of the present invention may include the compound represented by Formula 1, the compound represented by Formula 2, and the compound represented by Formula 3 in an amount of 20-200 mg, specifically 70-100 mg, but is not limited thereto.

In an embodiment of the present invention, a composition (a mixture of three) comprising a compound represented by Formula 1, a compound represented by Formula 2, and a compound represented by Formula 3 is administered to a coronavirus-infected mouse model, and its weight loss inhibitory effect And by confirming the effect of reducing the increased blood cells at the location of the bubbles in the lung tissue due to the coronavirus infection, it was confirmed that it can be used for the prevention or treatment of coronavirus-infected diseases ( FIGS. 3 and 4 ).

Another aspect of the present invention provides a method for preventing or treating a coronavirus infection, comprising administering the pharmaceutical composition to an individual.

In this case, the definitions of "coronavirus infectious disease", "prevention" and "treatment" are the same as described above.

As used herein, the term “administration” refers to introducing the pharmaceutical composition to a subject by an appropriate method.

As used herein, the term "individual" refers to all animals, such as rats, mice, livestock, including humans, that have or can develop a coronavirus-infected disease. The animal may be a mammal, such as a cow, a horse, a sheep, a pig, a goat, a camel, an antelope, a dog, or a cat, in need of prevention or treatment of similar symptoms as well as humans, but is not limited thereto.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the subject, age, sex, activity of the drug, Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.

The pharmaceutical composition may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. In addition, single or multiple administration may be used. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

In addition, the pharmaceutical composition may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or locally applied) according to a desired method, and the dosage may vary depending on the condition and weight of the patient, and the disease. Although it varies depending on the degree, drug form, administration route and time, it may be appropriately selected by those skilled in the art. As a specific example, the pharmaceutical composition can generally be administered once to several times a day, but the preferred dosage is appropriate by those skilled in the art depending on the individual's condition and weight, the degree of disease, drug form, administration route and period. can be chosen

The pharmaceutical composition of the present invention may be administered within the range of 20-200mg per day for adults, the compound represented by the compound represented by Formula 1, the compound represented by Formula 2, and the compound represented by Formula 3, more specifically It may be administered within the range of 70-100 mg at a time, but is not limited thereto.

Another aspect of the present invention provides a health functional food composition for the prevention or improvement of a coronavirus infection disease comprising a compound represented by Formula 1, a compound represented by Formula 2, and a compound represented by Formula 3 as an active ingredient do.

At this time, the definition of "coronavirus infection disease" and "prevention" is the same as described above.

The food composition of the present invention can be ingested on a daily basis, and unlike general drugs, since compounds 1 to 3, which are active ingredients extracted from natural products, are used as raw materials, there is no side effect that may occur during long-term administration of the drug, It can be very usefully used for the purpose of preventing or improving coronavirus-infected diseases.

As used herein, the term “improvement” refers to any action that reduces a parameter related to a condition to be treated by ingestion of the food composition, for example, the severity of symptoms.

As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , vitamin complexes, health functional foods, and health foods, and includes all foods in the ordinary sense.

As used herein, the term "health functional food" is the same term as food for special health use (FoSHU), and in addition to nutrition, it is processed to efficiently exhibit bioregulatory functions. Foods that are highly effective.

Here, 'function (sex)' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological actions with respect to the structure and function of the human body. The health food means a food having an active health maintenance or promotion effect compared to general food, and the health supplement food means a food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and health supplement may be used interchangeably.

Specifically, the health functional food is a compound represented by Formula 1, a compound represented by Formula 2, and a compound represented by Formula 3 added to food materials, such as beverages, teas, spices, gums, and confectionery, or encapsulated or powdered. , suspension, etc., which means that certain health effects are obtained when ingested.

The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art.

In addition, the health functional food composition may be prepared without limitation in various types of formulations as long as the formulations are recognized as food.

In addition, the health functional food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.

In addition, the health functional food composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the health functional food composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), Butylhydroxytoluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclmate) , saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (flavors), film agents, gum base agents, foam inhibitors, solvents, improving agents, etc. of food additives. The additive may be selected according to the type of food and used in an appropriate amount.

Another aspect of the present invention provides a feed composition for preventing or improving a coronavirus infection disease comprising a compound represented by Formula 1, a compound represented by Formula 2, and a compound represented by Formula 3 as an active ingredient.

In this case, the definitions of "coronavirus infectious disease", "prevention", and "improvement" are the same as described above.

As used herein, the term "feed additive" refers to a substance added in a trace amount to a feed for nutritional or specific purposes. Here, an animal is a concept including livestock and pets.

As a component that can be included in the feed additive of the present invention, there is no particular limitation on other components other than containing an active ingredient as an essential component. The feed additive of the present invention may further include a binder, an emulsifier, a preservative, etc. added to prevent quality deterioration, and an amino acid agent, a vitamin agent, an enzyme agent, a probiotic agent, a flavor agent, and a non-protein nitrogen added to increase the utility. It may further include a compound, a silicate agent, a buffer, a colorant, an extractant, an oligosaccharide, and the like, and may further include a feed mixture, etc., but is not limited thereto.

Another aspect for the purpose of the present invention is to provide a quasi-drug for the prevention or improvement of a coronavirus infection disease comprising a compound represented by Formula 1, a compound represented by Formula 2, and a compound represented by Formula 3 as an active ingredient do.

In this case, the definitions of "coronavirus infectious disease", "prevention", and "improvement" are the same as described above.

The term "quasi-drug" used in the present invention refers to articles that are not instruments, machines, or devices, among articles used for the purpose of diagnosing, treating, alleviating, treating or preventing diseases of humans or animals, and those that are not pharmacologically related to the structure and function of humans or animals. Articles used for the purpose of influencing, other than those that are not instruments, machines or devices.

In the present invention, the quasi-drug composition may have an effect of preventing or improving cardiovascular disease, but is not limited thereto.

The quasi-drug composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent if necessary in addition to the above components. The pharmaceutically acceptable carrier, excipient or diluent is not limited as long as it does not impair the effects of the present invention, for example, a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, a lubricant, a sweetener, a fragrance, a preservative, etc. may include

Another aspect for the purpose of the present invention relates to the antiviral use of a composition comprising a compound represented by Formula 1, a compound represented by Formula 2, and a compound represented by Formula 3 as an active ingredient against coronavirus. .

Another aspect for the purpose of the present invention is for the prevention or treatment of a coronavirus-infected disease of a composition comprising a compound represented by Formula 1, a compound represented by Formula 2, and a compound represented by Formula 3 as an active ingredient It's about use.

Since the composition comprising the compound represented by Formula 1, the compound represented by Formula 2, and the compound represented by Formula 3 as an active ingredient of the present invention exhibits an infection inhibitory effect on coronavirus, the prevention or treatment of the viral disease It can be used to develop antiviral drugs for

1 is a result confirming the anti-coronavirus activity of a mixture of three types of flavonoid glycosides.
2 is a result of comparing the anti-coronavirus activity of a mixture of three types of flavonoid glycosides and ribavirin.
3 is a result confirming the antiviral activity of a mixture of three types of flavonoid glycosides in a coronavirus infection model. (Square solid line marker: 3 mixtures, cross-dotted line marker: quercetin 3-glucoside, X-dotted line marker: quercetin 3-rhamnoside, open diamond-shaped dotted line marker: quercetin 3-galactoside, closed triangle dashed line marker: drug Non-infected group, solid circle mark: non-infected group)
4 is a result confirming the antiviral effect of a mixture of three types of flavonoid glycosides in lung tissue of a coronavirus infection model.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Preparation of a mixture of three types of flavonoid glycosides

In order to search for compounds capable of inhibiting virus proliferation, the present inventors from Sigma-Aldrich, quercetin 3-galactoside (Catalog No. 83388), quercetin 3-glucoside (Catalog No. 16654), quercetin 3-rhamnoside (Catalog No. 00740580-25MG) was purchased and used. In addition, as a positive control, ribavirin (R9644), ramivudin (L1295), and zidovudine (Catalog No. PHR1292-1G) used as antiviral agents were purchased and used in the antiviral activity test against coronavirus.

Each single compound of quercetin 3-galactoside, quercetin 3-glucoside, and quercetin 3-rhamnoside was dissolved in a dimethylsulfuroxide solution at a concentration of 10 mg/ml, respectively, and the mixture of the three types contained the flavonoid compound in the same amount was mixed to obtain a concentration of 10 mg/ml of the final mixture.

Example 2: Confirmation of anti-coronavirus activity of a mixture of three types of flavonoid glycosides

In order to measure the antiviral activity of the mixture against coronavirus, the present inventors performed the following experiment using Vero cells, a monkey kidney cell line.

As for the method of measuring coronavirus proliferation inhibitory ability, the virus activity inhibitory effect on PEDV (strain CV777), a kind of coronavirus, was measured based on the method applied for a patent by Kwon Doo-han et al. (Korean Patent Registration No.: 0682069).

Vero cells were cultured in each well of a 96-well microplate until the bottom was completely covered with cells. After removing the existing culture medium and washing each well twice with phosphate buffer, a virus solution prepared at a concentration of TCID50 was added to each well, and a compound to check antiviral activity was added.

At this time, quercetin 3-rhamnoside (A), quercetin 3-glucoside (B), and quercetin 3-galactoside (C) were each used as Comparative Groups 1 to 3 for three types of compounds, and a combination of these two types of compounds (quercetin 3-rhamnoside and quercetin 3-glucoside (A + B); quercetin 3-rhamnoside and quercetin 3-galactoside (A + C); and quercetin 3-glucoside and quercetin 3-galactoside (B + C)) was used as Comparative Groups 4 to 6, respectively. In addition, the three types of mixtures of the present invention were used as an experimental group, and ribavirin (R9644), known as an antiviral agent purchased from Sigma, was used as a positive control group.

After each of the compounds was dissolved in dimethyl sulfoxide, each solution was administered to each well at a concentration of 0.1 to 100 μg/ml, and cultured for 48 hours. judged. After adding 100 μl of 70% acetone to each well, it was left at -20 ° C for 1 hour, dried in a dryer, and a 0.4% (w/v) SRB (sulforhodamine B) solution dissolved in 1% (v/v) acetic acid. After adding 100 μl of staining for 30 minutes, the SRB stain solution not bound to cells was washed 4 times with 1% (v/v) acetic acid and dried again.

After dissolving the stain by adding 100 μl of a 10 mM Tris solution (pH 10.5) to each well of the cell-bound stain at the bottom of each well, the absorbance was measured at 562 nm to compare the virus inhibitory effect. At this time, after comparing cells treated with only DMSO and cells administered with DMSO and coronavirus infection solution together as a negative control group, the inhibitory effect on the proliferation of coronavirus shown by the comparison group, the experimental group and the control group was determined, and the following Table 1 and FIG. 1 shown in CC50 (cytotoxic concentration) refers to a concentration showing 50% cytotoxicity, and EC50 (effective concentration) refers to a concentration showing 50% medicinal efficacy. In addition, SI is an index that shows the safety and efficacy of a drug, and the higher the value, the better the drug is evaluated. In addition, the remark column shows the percentage comparison value expressed based on the SI value of quercetin 3-galactoside among the three types of flavonoids.

CC50
(μg/ml) EC50
(μg/ml) SI
(CC50/EC50) note(%) Comparative group 1 (A) 1000> 69.18 ± 19.21 14.5< 9.01 Comparative group 2 (B) 875.35 ND ND ND Comparative group 3 (C) 466.03 2.88 ± 0.50 161 100 Control group 4 (A + B) 1000> 142.42 ± 60.97 7< 4.35 Control group 5 (A + C) 535.30 2.44 ± 0.80 219.4 136.27 Control group 6 (B + C) 326.02 5.98± 0.65 54.5 33.85 Experimental group (A+B+C) 229.86. 0.041 7,143 4,436.65 Positive control (ribavirin) 271 4.57 ± 0.28 59.30 36.83

As can be seen in Table 1, it was confirmed that the experimental group of the three mixtures had a significantly higher SI value compared to Comparative Groups 1 to 3 of a single compound and Comparative Groups 4 to 6, which is a combination of two compounds. That is, when the comparative group 3 (quercetin 3-galactoside), which has the highest anti-corona activity among single compounds, is 100, in Comparative Example 5, which is a mixture of two compounds, it is only about 136%, but a three-type mixture In the case of 4,436%, it was confirmed that the effect was superior.

In addition, it was confirmed that the positive control group had a more significant effect than the existing anti-coronavirus drug.

This is greater than the combined value of each of the three single compounds, and as showing a more significant effect than the combined value of the combination of two compounds, it cannot be seen as an effect on a simple combination, and it can be confirmed that it is a synergistic effect.

In addition, as can be seen in FIG. 1, in the case of the experimental group of the three mixtures, compared to the compounds of Comparative Groups 1, 2, and 3 of a single compound, at concentrations of 0.01, 0.1, and 1 μg/ml, the antiviral effect was very superior. was able to confirm

In the same manner as the above method, the antiviral activity of the three types of flavonoid glycoside compounds against coronavirus was compared with ribavirin, which is another anti-coronavirus, and the efficacy by concentration of 0.01, 0.1, 1, 10, 100 μg/ml was compared three times. and the average value and error are presented in FIG. 2 .

As shown in FIG. 2, the three types of flavonoid glycoside compounds exhibited anti-coronavirus activity of 50% or more in all of the tested concentration ranges of 0.01, 0.1, 1, 10, and 100 μg/ml, and as the concentration increased, the anti-coronavirus activity also increased. did. In contrast, it was confirmed that ribavirin did not exhibit antiviral activity of 50% or less except for 100 μg/ml.

As a result of the above, it was confirmed that the mixture of three types of flavonoid glycosides of the present application has excellent effects compared to the existing therapeutic agent, ribavirin, and exhibits a significant effect compared to a single flavonoid glycoside.

Example 3. Confirmation of antiviral activity against other coronaviruses of a mixture of three types of flavonoid glycosides

To measure the antiviral activity of the three mixtures of the present invention, quercetin 3-galactoside, quercetin 3-rhamnoside and quercetin 3-glucoside against PEDV and other coronaviruses PRCV and TGEV, the pig seminal vesicle cell line ST An experiment was carried out in the same manner as in Example 2 using cells.

In addition, as a positive control group, the anti-corona activity was compared with the conventional antiviral agents acycloguanine, azidothymidine, lamivudine and ribavirin, and the results are shown in Table 2.

As can be seen in Table 2, the existing antiviral agents acyclovir, zidovudine, and ramivudine for PRCV or TGEV coronavirus were confirmed to be ineffective against two types of coronavirus, but the mixture of three types of flavonoid glycosides of the present invention In the case of the virus, it was possible to confirm the anti-corona activity.

Example 4. Confirmation of antiviral activity of a mixture of three types of flavonoid glycosides in a coronavirus infection model

4-1. Construction of a coronavirus-infected mouse model

In order to confirm the anti-coronavirus activity in animals of the three types of mixtures of the present invention, the following experiment was performed. Rat coronavirus (ATCC®VR-1410) purchased from the US ATCC was cultured in Vero cells and then injected into the nose of mice in an amount of 100TCID50 to induce infection of the rat coronavirus.

According to Funk et al. (Rat respiratory coronavirus infection: replication in airway and alveolar epithelial cells and the innate immune response. J. Gen. Virol. 2005. 90: 2956-2964), rat coronavirus is a severe respiratory disease in mice. is a virus that causes

The experiment consisted of 3 mice in each group, and was divided into an uninfected group, an infected group but not administered with a drug, a group administered with a single flavonoid glycoside compound to the infected group, and a group administered with a mixture of three flavonoid glycosides.

After culturing the mouse coronavirus in Vero cells, an amount of 100TCID50 was injected into the nose of each mouse (Balb/C 4 weeks old) to infect the mouse coronavirus, and the experimental results are as shown in FIGS. 3 and 4 .

4-2. Confirmation of weight loss inhibition by a mixture of three types of flavonoid glycosides in a coronavirus infection model

The weights of all mice used in the above experiment were measured daily, and the weight of each individual in the uninfected group, the infected group but not administered with the drug, the group administered with a single drug flavonoid glycoside to the infected group, and the group administered with a three-type flavonoid glycoside mixture drug was measured every day, and the change in the daily body weight of each group is presented in FIG. 3 .

As can be seen in FIG. 3 , the drug administration group was orally administered with 100 μg daily. The non-infected group continued to gain weight during the experiment, resulting in a final increase of 6 g. In the single flavonoid glycoside drug administration group, the quercetin 3-glucoside administration group decreased 1.74 g, the quercetin 3-rhamnoside administration group decreased 0.85 g, and the quercetin 3-galactoside administration group decreased 1.19 g.

On the other hand, the three flavonoid mixture drug administration group increased 3.65 g, confirming the ability to inhibit weight loss due to rat coronavirus infection compared to the single flavonoid drug administration group, confirming the excellent antiviral effect.

4-3. Confirmation of lung tissue section of coronavirus infection model by administration of a mixture of three types of flavonoid glycosides

After the rat infection experiment was completed, lung tissue was isolated from the mice in each group to make a lung tissue section, and the non-infected group, the infected group but not administered with the drug, and the group in which a single agent of flavonoid glycoside was administered to the infected group and 3 types The results of comparing the lung tissue sections of the flavonoid glycoside mixture drug administration group are presented in FIG. 4 . .

Referring to FIG. 4 , it was confirmed that the non-infected group had bubbles in the lung cells, but the lung tissue sections of the coronavirus-infected mice were filled with blood cells at the location of the bubbles. Although it can be seen that the number of blood cells in the bubble location is lower in all of the quercetin 3-glucoside administration group, quercetin 3-rhamnoside administration group, and quercetin 3-galactoside administration group than in the non-medicated infected group, some lung cells are filled with blood cells. was confirmed.

However, in the lung tissue section of the three-type mixture administered group, it was confirmed that the total number of cells filled with blood cells was significantly less than that of the single flavonoid administration group, and it was confirmed that the distribution of blood cells was small in the lung tissue as a whole.

As a result, the antiviral activity of the three-type flavonoid glycoside mixture was confirmed in the coronavirus-infected model mouse, and it was confirmed that it could be used for the treatment of coronavirus-infected diseases. It was confirmed that the synergistic effect was confirmed in the case of the three types of mixture by confirming that it appeared better.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (7)

An antiviral composition for coronavirus comprising the compound represented by the following formula (1), the compound represented by the formula (2), and the compound represented by the formula (3) as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

According to claim 1, wherein the coronavirus is porcine epidemic diarrhea virus (PEDV), swine infectious gastroenteritis virus (TGEV), swine respiratory coronavirus (PRCV), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus Virus (SARS-CoV) and novel coronavirus (CoVID-19) will any one or more selected from, the composition.
a compound represented by the following formula (1), a compound represented by formula (2), and a compound represented by formula (3); Or a pharmaceutical composition for preventing or treating a coronavirus infection disease comprising a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

According to claim 3, wherein the coronavirus is swine epidemic diarrhea virus (PEDV), swine infectious gastroenteritis virus (TGEV), swine respiratory coronavirus (PRCV), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus Virus (SARS-CoV) and novel coronavirus (CoVID-19) will any one or more selected from, the pharmaceutical composition.
A health functional food composition for the prevention or improvement of a coronavirus infection disease comprising a compound represented by the following formula (1), a compound represented by formula (2), and a compound represented by formula (3) as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

A feed composition for preventing or improving a coronavirus infection disease comprising the compound represented by the following formula (1), the compound represented by formula (2), and the compound represented by formula (3) as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

A quasi-drug composition for the prevention or improvement of a coronavirus infection disease comprising the compound represented by the following formula (1), the compound represented by the formula (2), and the compound represented by the formula (3) as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

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