KR102762400B1 - Flavonoid glycosides comprising anti-corona activity - Google Patents

Abstract

The present invention relates to the use of flavonoid glycoside compounds for anti-coronaviruses, and specifically, the present invention relates to an anti-coronavirus composition comprising a combination of three flavonoid glycoside compounds as an active ingredient; a pharmaceutical composition for preventing or treating a coronavirus infectious disease; a health functional food composition for preventing or improving a coronavirus infectious disease; a feed composition; and a quasi-drug composition.
The composition containing the compound represented by chemical formula 1, the compound represented by chemical formula 2, and the compound represented by chemical formula 3 as active ingredients exhibits an infection-inhibiting effect against coronavirus, and therefore can be used to develop an antiviral agent for preventing or treating the viral disease.

Description

Flavonoid glycosides comprising anti-corona activity {Flavonoid glycosides comprising anti-corona activity}

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

The respiratory disease that started in Wuhan, China in 2019 is a disease caused by a coronavirus infection, and many infected patients are showing severe respiratory symptoms and leading to death. The approved treatments for coronavirus infection include ribavirin, penclovir, nitazoxanide, nafamostat, and chloroquine. In addition, remdesivir (GS-5734, remdesivir) and favipiravir are being used to treat coronavirus infection. However, the efficacy is low, and the incidence of mutant viruses is high due to the characteristics of nucleic acid analog-type antiviral drugs, so two or more antiviral drugs are being used together [Wang M, Cao R, Korm L, Yang X, Liu J, Xu M, Shi Z, Hu Z, Zhong W, Xiao G. 2020. Remdesvir and choloquine effectively inhibit the recently emerged novel coronavirus(2019-nCoV) in vitro. Cell Research 30: 269-271].

Most antiviral drugs used for coronavirus infections, including ribavirin and remdisivir, are substances that have RNA polymerase inhibitory properties. However, a mouse hepatitis virus, a type of coronavirus that is resistant to remdisivir, has been discovered, and existing antiviral drugs are ineffective.

On the other hand, the inhibitory ability of flavonoids to inhibit the coronavirus polymerase enzyme protein has been studied, and it has been known that kaempferol acetyl 3-glucoside and quercetin acetyl 3-glucoside exhibit inhibitory effects 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), and an antiviral composition for coronavirus using plant extracts has been 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 so far have shown severe side effects, great care is needed in their application. These treatments are ineffective and have side effects. Therefore, the need for the development of excellent new coronavirus agents with excellent infection suppression effects and low toxicity to prevent and treat the occurrence of coronaviruses is increasing.

Against this backdrop, the inventors of the present invention have continued their efforts to develop a novel coronavirus treatment agent, and as a result, they have confirmed that a mixture of three flavonoid glycosides, each isolated from a plant extract or chemically synthesized by imitating them, has anti-coronavirus activity, thereby completing the present invention.

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

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating coronavirus infectious disease, comprising a combination of three 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 preventing or improving coronavirus infectious disease, which comprises a combination of three flavonoid glycoside compounds as an effective ingredient.

Another object of the present invention is to provide a feed composition for preventing or improving coronavirus infectious disease, which comprises a combination of three flavonoid glycoside compounds as an effective ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or improving coronavirus infectious disease, which comprises a combination of three flavonoid glycoside compounds as an effective ingredient.

This is explained specifically as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention cannot be considered limited by the specific description described below.

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

[Chemical Formula 1]

[Chemical formula 2]

[Chemical Formula 3]

The compound having the structure of the above chemical formula 1 is quercetin 3-rhamnoside (Quercitrin), a flavonoid glycoside compound with a molecular weight of 448.38 and the chemical formula C ₂₁ H ₂₀ O ₁₁ .

In addition, the compound having the structure of the chemical formula 2 is quercetin 3-glucoside (Isoquercetin), a flavonoid glycoside compound with a molecular weight of 464.38 and a chemical formula of C ₂₁ H ₂₀ O ₁₂ .

In addition, the compound having the structure of the chemical formula 3 is Quercetin 3-galactoside (Hyperoside), a flavonoid glycoside compound with a molecular weight of 464.38 and a chemical formula of C ₂₁ H ₂₀ O ₁₂ .

The composition comprising the compounds represented by the chemical formulas 1 to 3 of the present invention, for example, the compound represented by the chemical formula 1, the compound represented by the chemical formula 2, and the compound represented by the chemical formula 3, may include derivatives of these compounds within a range that can be predicted by those skilled in the art, and are included without limitation as long as they have the same effect as in the present invention. These compounds can be obtained from extracts containing them, but are not limited thereto, and can also be synthesized and used by a known method. The extract may be, for example, an extract or fraction of Houttuynia cordata, but is sufficient as long as it is a plant extract or fraction containing the compound, and is not limited thereto.

In this specification, the compounds represented by the terms Chemical Formulas 1 to 3 may be used interchangeably with three flavonoid glycoside compounds, a mixture of flavonoid glycosides, a mixture of three flavonoid glycosides, and a mixture of three.

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

The compounds represented by the chemical formulas 1, 2 and 3 of the present invention can be used in the form of pharmaceutically acceptable salts, and as the salts, acid addition salts formed by pharmaceutically acceptable free acids can be used. Acid addition salts are obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanedioates, 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. These pharmaceutically non-toxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphate chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-1,4-dioate, hexane-1,6-dioate, benzoates, chlorobenzoates, methylbenzoate, dinitrobenzoate, hydroxybenzoates, methoxybenzoates, phthalates, Including but not limited to terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, 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, dissolving methylene dichloride of chemical formula 1 in an organic solvent, for example, methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., adding an organic acid or inorganic acid, and filtering and drying the resulting precipitate, or by distilling the solvent and an excess acid under reduced pressure and then drying or crystallizing the same in the presence of an organic solvent.

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. An 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 out the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate). In addition, the present invention includes not only methylene dichloride represented by Chemical Formula 1 and a pharmaceutically acceptable salt thereof, but also all possible solvates, hydrates, stereoisomers, etc. that can be prepared therefrom.

The term "coronavirus" of the present invention refers to a virus belonging to the Coronaviridae family, and in one embodiment of the present invention, it was confirmed that a composition including a combination of compounds represented by Chemical Formula 1, Chemical Formula 2, and Chemical Formula 3 has coronavirus inhibitory activity (Table 1 and Table 2).

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

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

The above results show that the homology of the polymerase amino acid sequences between each coronavirus is more than 50%, and the similarity is very high at more than 75% including the amino acid sequence similarity, and in particular, the 485th-545th amino acid sequence in the PEDV polymerase gene has a similarity of 100% with SARS coV2. Considering this, it is suggested that the antiviral activity against the above coronavirus can be applied to other coronaviruses, and that the antiviral composition of the present invention has anti-corona activity.

In addition, in one embodiment of the present invention, it was confirmed that an antiviral composition comprising a combination of three kinds of Compound 1, Compound 2, and Compound 3 had a remarkable anti-coronavirus activity compared to each compound or a combination of two kinds of compounds. In addition, it was confirmed that it had a superior effect than the existing anti-coronavirus agent, which was a positive control.

The above results were greater than the combined effects of the three single compounds, and were more remarkable than the combined effects of the two compounds, confirming that they correspond to a synergistic effect (Table 1, Figures 1 to 4).

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

The term "coronavirus infectious disease" in the present invention means a disease caused by infection with a coronavirus.

The above term, “infection,” refers to a state in which a pathogenic microorganism invades the body of a host organism and grows and proliferates.

In one specific example of the present invention, the coronavirus infectious disease may be, but is not limited to, a cold, cough, phlegm, bronchitis, or severe respiratory disease in humans, and hepatitis, encephalitis, neurological disease, severe respiratory disease, severe diarrhea or respiratory disease, peritonitis, or the like in animals.

The term “prevention” of the present invention means any act of inhibiting or delaying the onset of an infectious disease caused by a coronavirus by administering a pharmaceutical composition according to the present invention.

The term "treatment" of the present invention means any action whereby the symptoms of a subject suspected of or diagnosed with a coronavirus infectious disease are improved or beneficially changed by administration of the pharmaceutical composition.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions, and the carrier may comprise a non-naturally occurring carrier. Specific examples of the carrier, excipient and diluent include, but are not limited to, 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.

In addition, the pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, and suppositories, respectively, according to a conventional method, and may be various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Tablets, pills, powders, granules, capsules, etc. can be used as solid preparations for oral administration, and at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. can be used for the solid preparations. In addition, lubricants such as magnesium stearate and talc can be used in addition to simple excipients. Liquid preparations for oral administration can include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be used. Preparations for parenteral administration can include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, or suppositories. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include, but are not limited to, witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

The pharmaceutical composition of the present invention may contain, based on the total weight of the composition, 0.1 to 1000 ug/ml, specifically 0.1 to 200 ug/ml, 0.1 to 100 ug/ml of the compound represented by the chemical formula 1, the compound represented by the chemical formula 2, and the compound represented by the chemical formula 3, but is not limited thereto.

The pharmaceutical composition of the present invention may contain 20-200 mg, specifically 70-100 mg, of the compound represented by the chemical formula 1, the compound represented by the chemical formula 2, and the compound represented by the chemical formula 3, but is not limited thereto.

In one embodiment of the present invention, a composition (mixture of three types) comprising a compound represented by Chemical Formula 1, a compound represented by Chemical Formula 2, and a compound represented by Chemical Formula 3 was administered to a mouse model of coronavirus infection, and the effect of suppressing weight loss and reducing increased blood cells in the location of lung tissue bubbles due to coronavirus infection was confirmed, thereby confirming that the composition can be used for the prevention or treatment of coronavirus infectious diseases (Figs. 3 and 4).

Another aspect of the present invention provides a method for preventing or treating a coronavirus infectious disease, comprising administering the pharmaceutical composition to a subject.

At this time, the definitions of “coronavirus infectious disease,” “prevention,” and “treatment” are as described above.

The term "administration" in the present invention means introducing the pharmaceutical composition to a subject by an appropriate method.

The term "subject" of the present invention refers to all animals, including rats, mice, and livestock, including humans, that have developed or may develop a coronavirus infectious disease. The animal may be, but is not limited to, mammals such as cows, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require prevention or treatment of symptoms similar to humans.

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

The above term, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined based on factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

The above pharmaceutical composition can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. In addition, it can be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art.

In addition, the pharmaceutical composition may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may vary depending on the patient's condition and weight, the extent of the disease, the drug form, the administration route, and the time, but may be appropriately selected by those skilled in the art. As a specific example, the pharmaceutical composition may generally be administered once or several times a day, but the preferred dosage may be appropriately selected by those skilled in the art depending on the patient's condition and weight, the extent of the disease, the drug form, the administration route, and the time.

The pharmaceutical composition of the present invention can be administered to adults within the range of 20-200 mg per day of the compound represented by the chemical formula 1, the compound represented by the chemical formula 2, and the compound represented by the chemical formula 3, more specifically, 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 preventing or improving coronavirus infectious disease, comprising 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.

At this time, the definitions of “coronavirus infectious disease” and “prevention” are as described above.

The food composition of the present invention can be consumed on a daily basis, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs, as it uses compounds 1 to 3, which are active ingredients extracted from natural products, as raw materials, and therefore can be very usefully used for the purpose of preventing or improving coronavirus infectious diseases.

The term "improvement" of the present invention means any action that reduces a parameter related to a condition being treated by ingestion of the food composition, for example, the degree of a symptom.

The term "food" of the present invention includes meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, 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 conventional sense.

The term "health functional food" of the present invention is the same as food for special health use (FoSHU), and means a food with high medical and healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition.

Here, 'function' means regulating nutrients for the structure and function of the human body or obtaining a useful effect for health purposes such as physiological functions. The above health food refers to a food that has a more active health maintenance or promotion effect than general food, and health supplement food refers to a food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and health supplement food may be used interchangeably.

Specifically, the health functional food means a food product manufactured by adding a compound represented by the chemical formula 1, a compound represented by the chemical formula 2, and a compound represented by the chemical formula 3 to food materials such as beverages, teas, spices, gum, and confectionery, or by manufacturing the same in the form of encapsulation, powder, suspension, etc., and which has a specific health effect when consumed; however, unlike general drugs, it has the advantage of not having side effects that can occur with long-term use of drugs made from food.

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

In addition, the above health functional food composition can be manufactured without limitation in various forms of formulations as long as it is a formulation recognized as a food.

In addition, the health functional food composition may additionally include a physiologically acceptable carrier. The type of the carrier is not particularly limited, and any carrier commonly used in the relevant technical field may be used.

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

In addition, the health functional food composition may include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar color, etc.), colorants (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (glutinating agents), film agents, gum bases, foam suppressants, solvents, and improvers. The above additives can 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 coronavirus infectious disease, comprising 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.

At this time, the definitions of “coronavirus infectious disease,” “prevention,” and “improvement” are as described above.

In the present invention, the term "feed additive" collectively refers to substances added in small amounts to feed for nutritional or specific purposes, and in the present invention, refers to substances added for the purpose of preventing or improving coronavirus infectious diseases. Here, the term "animal" is a concept that includes livestock and pets.

As for the components that can be included in the feed additive of the present invention, other than those containing effective ingredients as essential components, there are no particular restrictions on the other components. The feed additive of the present invention may further include a binder, an emulsifier, a preservative, etc. added to prevent quality deterioration, and may further include an amino acid agent, a vitamin agent, an enzyme agent, a probiotic agent, a flavoring agent, a non-protein nitrogen compound, a silicate agent, a buffering agent, a coloring agent, an extractant, an oligosaccharide, etc. added to increase utility, and may further include a feed mixer, etc., but is not limited thereto.

Another embodiment for the purpose of the present invention provides a pharmaceutical product for preventing or improving coronavirus infectious disease, comprising 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.

At this time, the definitions of “coronavirus infectious disease,” “prevention,” and “improvement” are as described above.

The term "quasi-drug" used in the present invention means an article other than an instrument, machine or device used for the purpose of diagnosing, treating, alleviating, managing or preventing a disease in humans or animals, and an article other than an instrument, machine or device used for the purpose of exerting a pharmacological effect on the structure and function of humans or animals.

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

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, in addition to the above components, as needed. The pharmaceutically acceptable carrier, excipient or diluent is not limited as long as it does not impair the effects of the present invention, and may include, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc.

Another embodiment for the purpose of the present invention relates to the antiviral use of a composition comprising 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 against coronavirus.

Another embodiment for the purpose of the present invention relates to the use of a composition comprising 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 for preventing or treating coronavirus infectious disease.

The composition containing the compound represented by chemical formula 1, the compound represented by chemical formula 2, and the compound represented by chemical formula 3 as active ingredients exhibits an infection-inhibiting effect against coronavirus, and therefore can be used to develop an antiviral agent for preventing or treating the viral disease.

Figure 1 shows the results of confirming the anti-coronavirus activity of a mixture of three flavonoid glycosides.
Figure 2 shows the results comparing the anti-coronavirus activity of a mixture of three flavonoid glycosides and ribavirin.
Figure 3 shows the results of confirming the antiviral activity of a mixture of three flavonoid glycosides in a coronavirus infection model. (Square solid line marking: mixture of three, cross-shaped dotted line marking: quercetin 3-glucoside, X-shaped dotted line marking: quercetin 3-rhamnoside, open diamond-shaped dotted line marking: quercetin 3-galactoside, closed triangle dotted line marking: infected group that did not receive drug, circle solid line marking: non-infected group)
Figure 4 shows the results of confirming the antiviral effect of a mixture of three flavonoid glycosides in lung tissue of a coronavirus infection model.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of a mixture of three flavonoid glycosides

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

Each single compound of quercetin 3-galactoside, quercetin 3-glucoside, and quercetin 3-rhamnoside was dissolved in a dimethylsulfoxide solution at a concentration of 10 mg/ml, and the mixture of the three was prepared by mixing each flavonoid compound in an equal amount in a ratio to prepare a final mixture concentration of 10 mg/ml.

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

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

The method for measuring the inhibition of coronavirus proliferation was based on the method patented by Kwon Du-han et al. (Korean Patent Registration No. 0682069), and the virus activity inhibition effect against PEDV (strain CV777), a type of coronavirus, was measured.

Vero cells were cultured in each well of a 96-well microplate until the bottom was completely covered with cells. The existing culture medium was removed, and each well was washed twice with phosphate buffer. Then, a virus solution prepared at a concentration of TCID50 was added to each well, and a compound for which antiviral activity was to be confirmed 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, and combinations of two 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)) were each used as comparative groups 4 to 6. In addition, the three 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.

The above compounds were each dissolved in dimethyl sulfoxide, and each solution was administered to each well at a concentration of 0.1 to 100 μg/ml, cultured for 48 hours, and the morphology of MDCK cells was observed under a microscope to initially determine the antiviral activity. 100 μl of 70% acetone was added to each well, left at -20 °C for 1 hour, dried in a desiccator, and then 100 μl of 0.4% (w/v) SRB (sulforhodamine B) solution dissolved in 1% (v/v) acetic acid was added, stained for 30 minutes, and the SRB staining solution that was not bound to the cells was washed four times with 1% (v/v) acetic acid and then dried again.

The dye bound to the cells at the bottom of each well was added to 100 ㎕ of 10 mM Tris solution (pH 10.5) to each well to dissolve the dye, and the absorbance was measured at 562 nm to compare the virus inhibition effect. At this time, the cells treated with only DMSO and the cells administered with DMSO and coronavirus infection solution together were compared as a negative control group, and the coronavirus proliferation inhibition effect exhibited by the comparison group, experimental group, and control group was determined and shown in Table 1 and Figure 1 below. CC50 (cytotoxic concentration) indicates the concentration that exhibits 50% cytotoxicity, and EC50 (effective concentration) indicates the concentration that exhibits 50% medicinal efficacy. In addition, SI is an index that indicates both safety and efficacy of a drug, and a higher SI is evaluated as a better drug, and ND (not determined) means that the value of 50% inhibition cannot be calculated even at the maximum concentration due to a low value. In addition, the note column shows the percentage comparison value based on the SI value of quercetin 3-galactoside among the three flavonoids.

CC50
(μg/ml) EC50
(μg/ml) SI
(CC50/EC50) note(%) Comparison group 1(A) 1000> 69.18 ± 19.21 14.5< 9.01 Comparison group 2(B) 875.35 ND ND ND Comparison group 3(C) 466.03 2.88 ± 0.50 161 100 Comparison group 4 (A + B) 1000> 142.42 ± 60.97 7< 4.35 Comparison group 5 (A + C) 535.30 2.44 ± 0.80 219.4 136.27 Comparison group 6 (B + C) 326.02 5.98±0.65 54.5 33.85 Experimental group (A+B+C) 292.86. 0.041 7,143 4,436.65 Positive control group (ribavirin) 271 4.57 ± 0.28 59.30 36.83

As can be seen in Table 1 above, it was confirmed that the experimental group of the three mixtures had significantly higher SI values than the comparative groups 1 to 3 of single compounds and the comparative groups 4 to 6 of the two-compound combination. That is, when the comparative group 3 (quercetin 3-galactoside) with the highest anti-corona activity among the single compounds is considered to be 100, the comparative example 5, which is a mixture of two compounds, only corresponds to about 136%, but the effect of the three mixtures was confirmed to be superior at 4,436%.

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

This is greater than the combined value of the three single compounds, and shows a more remarkable effect than the combined value of the two compounds, so it cannot be considered an effect of a simple combination, but rather a synergistic effect.

In addition, as can be seen in Figure 1, it was confirmed that the experimental group of the three mixtures exhibited a significantly superior antiviral effect at concentrations of 0.01, 0.1, and 1 ㎍/ml compared to the single compound comparison groups 1, 2, and 3.

In the same manner as the above method, the antiviral activity of three flavonoid glycoside compounds against coronavirus was compared with that of ribavirin, another anti-coronavirus antiviral agent, at concentrations of 0.01, 0.1, 1, 10, and 100 ㎍/ml three times, and the average value and error are presented in Figure 2.

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

As a result of the above, it was confirmed that the mixture of three flavonoid glycosides of this invention is more effective than the existing treatment, ribavirin, and shows a remarkable effect compared to a single flavonoid glycoside.

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

In order to measure the antiviral activity of the three mixtures of the present invention, quercetin 3-galactosidase, quercetin 3-rhamnoside, and quercetin 3-glucoside, against PRCV and TGEV, which are other coronaviruses than the PEDV, experiments were conducted using ST cells, a porcine seminal vesicle cell line, using the same method as in Example 2.

In addition, the anti-coronavirus activity was compared with that of existing antiviral agents such as acyclovir, zidovudine, and lamivudine as positive controls, and the results are shown in Table 2.

As can be confirmed in Table 2 above, existing antiviral agents such as acyclovir, zidovudine, and lamivudine were confirmed to be ineffective against two types of coronaviruses, PRCV or TGEV, but the mixture of three flavonoid glycosides of the present invention was confirmed to have anti-coronavirus activity against the above viruses as well.

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

4-1. Production of a mouse model of coronavirus infection

In order to confirm the anti-coronavirus activity of the three types of mixture of the present invention in animals, the following experiment was performed. Murine coronavirus (ATCC®VR-1410) purchased from ATCC in the United States was cultured in Vero cells, and then injected into the nose of mice at a dose of 100 TCID50 to induce infection with the murine 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), murine coronavirus is a virus that causes severe respiratory disease in mice.

The experiment involved three mice per group, and the mice were divided into a non-infected group, an infected group that was not administered drugs, a group administered a single flavonoid glycoside compound to the infected group, and a group administered a mixture of three flavonoid glycosides.

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

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

The body weight of all mice used in the above experiment was measured daily, and the body weight of each individual in the non-infected group, the infected group that was not administered the drug, the infected group that was administered a single flavonoid glycoside drug, and the group administered a mixture of three flavonoid glycoside drugs was measured daily, and the change in daily body weight of each group is presented in Figure 3.

As shown in Fig. 3, the drug administration group was administered 100 μg orally every day. The non-infected group continued to increase in body weight during the experimental period, with a final increase of 6 g, and among the infected groups, the non-drug administration group decreased in body weight, with a decrease of 5.77 g on the 9th day. Among the single flavonoid glycoside drug administration groups, the quercetin 3-glucoside administration group decreased by 1.74 g, the quercetin 3-rhamnoside administration group decreased by 0.85 g, and the quercetin 3-galactoside administration group decreased by 1.19 g.

On the other hand, the group administered the three-flavonoid mixture drug showed an increase of 3.65 g, confirming an excellent antiviral effect by confirming the ability to suppress weight loss caused by mouse coronavirus infection compared to the group administered the single flavonoid drug.

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

After the above mouse infection experiment was completed, lung tissue was isolated from the mice in each group, and lung tissue sections were made. The results of comparing lung tissue sections of the non-infected group, the infected group that was not administered drugs, the infected group that was administered a single flavonoid glycoside drug, and the group administered a mixture of three flavonoid glycoside drugs are presented in Figure 4.

As shown in Figure 4, the non-infected group had activated bubbles in their lung cells, but the lung tissue sections of mice infected with the coronavirus showed bubble locations filled with blood cells. In the quercetin 3-glucoside administration group, the quercetin 3-rhamnoside administration group, and the quercetin 3-galactoside administration group, the number of blood cells in the bubble locations was confirmed to be smaller than in the non-administered infected group, but it was confirmed that some lung cells were filled with blood cells.

However, in the lung tissue sections of the three-type mixture administration group, it was confirmed that the number of cells filled with blood cells was significantly less than that of the single flavonoid administration group, and that the overall distribution of blood cells within the lung tissue was low.

In this way, the antiviral activity of the mixture of three flavonoid glycosides was confirmed in a coronavirus infection model mouse, confirming that it can be used for the treatment of coronavirus infectious diseases. In addition, it was confirmed that the antiviral effect was better in the group administered the three mixtures than in the group administered a single flavonoid glycoside, confirming that the three mixtures showed a synergistic effect.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical idea or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present invention should be interpreted as including all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts rather than the above detailed description.

Claims (7)

An antiviral composition for coronavirus comprising a combination of three compounds represented by the following chemical formula 1, a compound represented by the following chemical formula 2, and a compound represented by the following chemical formula 3 as an active ingredient.
A composition wherein the coronavirus is at least one selected from porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV).
[Chemical Formula 1]

[Chemical formula 2]

[Chemical Formula 3]

delete A pharmaceutical composition for the prevention or treatment of coronavirus infectious disease, comprising a combination of three compounds represented by the following chemical formula 1, a compound represented by the following chemical formula 2, and a compound represented by the following chemical formula 3; or a pharmaceutically acceptable salt thereof, as an active ingredient.
A pharmaceutical composition, wherein the coronavirus is at least one selected from porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV).
[Chemical Formula 1]

[Chemical formula 2]

[Chemical Formula 3]


delete A health functional food composition for preventing or improving coronavirus infectious disease, comprising a combination of three compounds represented by the following chemical formula 1, a compound represented by the following chemical formula 2, and a compound represented by the following chemical formula 3 as an effective ingredient.
A health functional food composition, wherein the coronavirus is at least one selected from porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV).
[Chemical Formula 1]

[Chemical formula 2]

[Chemical Formula 3]

A feed composition for preventing or improving coronavirus infectious disease, comprising a combination of three compounds represented by the following chemical formula 1, a compound represented by the following chemical formula 2, and a compound represented by the following chemical formula 3 as an effective ingredient.
A feed composition, wherein the coronavirus is at least one selected from porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV).
[Chemical Formula 1]

[Chemical formula 2]

[Chemical Formula 3]

A pharmaceutical composition for preventing or improving coronavirus infectious disease, comprising a combination of three compounds represented by the following chemical formula 1, a compound represented by the following chemical formula 2, and a compound represented by the following chemical formula 3 as an active ingredient,
A pharmaceutical composition, wherein the coronavirus is at least one selected from porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV).
[Chemical Formula 1]

[Chemical formula 2]

[Chemical Formula 3]