Composition

Abstract

The objective is to provide a composition that has a more effective anti-influenza virus effect. [Solution] The above problem can be solved by a composition comprising rosmarinic acid or a salt thereof, and at least one flavonoid glycoside.

Description

Technical Field

[0001] The present invention relates to a composition.

Background Art

[0002] Influenza is a respiratory infection caused by the influenza virus. Patent Document 1 discloses an anti-influenza virus agent having, as an active ingredient, one or more selected from β-nicotinamide mononucleotide, a pharmacologically acceptable salt thereof, and a solvate thereof, and having an action of suppressing the growth of influenza virus in the lung.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a composition having an anti-influenza virus action.

Means for Solving the Problems

[0006] \]> Rosmarinic acid is a component contained in various plants and is known to inhibit influenza neuraminidase (Non-Patent Document 1). The inventors diligently researched compositions that have anti-influenza virus activity. As a result, they found that combining rosmarinic acid and flavonoid glycosides yields a more effective anti-influenza virus effect. This invention is based on these findings. Therefore, the present invention relates to the following invention. [1] Rosmarinic acid or its salts, and at least one flavonoid glycoside A composition containing the following: [2] The composition according to [1], wherein the ratio of the amount of substance of at least one flavonoid glycoside to rosmarinic acid or a salt thereof (flavonoid glycoside / rosmarinic acid or a salt thereof) is 0.0010 to 100.0. [3] The molar concentration of rosmarinic acid or its salt is 0.10 to 1000.0 μM based on the total amount of the composition. The composition according to [1] or [2], wherein the molar concentration of at least one flavonoid glycoside is 0.0010 to 1000.0 μM on a basis of the total amount of the composition. [4] The composition according to any one of [1] to [3], wherein the structure of the flavonoid in at least one flavonoid glycoside is a flavanone or a flavone, and the sugar is a monosaccharide or a disaccharide. [5] The composition according to [4], wherein at least one flavonoid glycoside is selected from the group consisting of eriocitrin, bisenin 2, and luteolin-7-rutinoside. [6] A composition according to any one of [1] to [5], comprising rosmarinic acid or a salt thereof, eriocitrin, luteolin-7-rutinoside, and bisenin 2. [7] The composition according to [6], comprising, on a total basis, 0.050 to 1000.0 μM of rosmarinic acid or a salt thereof, 0.0050 to 1000.0 μM of eriocitrin, 0.010 to 1000.0 μM of luteolin-7-rutinoside, and 0.0010 to 1000.0 μM of bisenin-2. [8] A composition according to any one of [1] to [7], which is for use against influenza viruses. [9] A composition comprising rosmarinic acid or a salt thereof, for ingestion or administration in combination with a flavonoid glycoside.

[10] A composition comprising at least one flavonoid glycoside, for ingestion or administration in combination with rosmarinic acid or a salt thereof.

[0007] Furthermore, from a different perspective, the present invention relates to the following inventions. [A] A composition for use against influenza viruses comprising at least one flavonoid glycoside, wherein the structure of the flavonoid in at least one flavonoid glycoside is a flavanone or a flavone, and the sugar is a monosaccharide or a disaccharide. [B] The composition for use against influenza virus comprising at least two flavonoid glycosides, wherein the structure of the flavonoids in at least two flavonoid glycosides is a flavanone or a flavone, and the sugar is a monosaccharide or a disaccharide, as described in [A]. [C] An anti-influenza virus composition comprising at least three flavonoid glycosides, wherein the structure of the flavonoids in at least three flavonoid glycosides is a flavanone or a flavone, and the sugar is a monosaccharide or a disaccharide, as described in [A]. [D] A composition according to any one of [A] to [C], wherein at least one, two, or three flavonoid glycosides are selected from the group consisting of eriocitrin, bisenin 2, and luteolin-7-rutinoside.

[0008] Furthermore, from a different perspective, the present invention relates to the following inventions. [E] A composition comprising at least two flavonoid glycosides. [F] The composition according to [E], comprising at least three flavonoid glycosides. [G] The composition according to [E] or [F], wherein the flavonoid structure in at least two or three flavonoid glycosides is flavanone or flavone, and the sugar is monosaccharide or disaccharide. [H] The composition according to [G], wherein at least two or three flavonoid glycosides are each selected from the group consisting of eriodictyol, vicenin 2, and luteolin-7-rutinoside. [I] The composition according to any one of [E] to [H], which is for anti-influenza virus use. [J] A composition comprising at least one flavonoid glycoside, which is for ingestion or administration in combination with a flavonoid glycoside different from the said flavonoid glycoside. [Advantages of the Invention]

[0009] According to the composition of the present invention, an anti-influenza virus effect can be obtained more effectively. According to the composition of the present invention, an enhanced anti-influenza virus effect and / or a composite anti-influenza virus effect can be obtained. [Brief Description of the Drawings]

[0010] [Figure 1] It is a diagram showing the neuraminidase inhibitory effect of eriodictyol alone on influenza virus. <00​​​​​​​This figure shows the inhibitory effect of a composition containing four components—rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside—on the expression of influenza virus matrix protein 1. [Figure 5] This figure shows the inhibitory effect of a composition containing four components—rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside—on the expression of influenza virus matrix protein 1. [Figure 6] This figure shows the neuraminidase inhibitory effect of luteolin-7-rutinoside alone on influenza virus. [Figure 7] This figure shows the anti-influenza virus effect of a combination of rosmarinic acid and at least one flavonoid glycoside. [Figure 8] This figure shows the inhibitory effect of a composition containing four components—rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside—on the expression of influenza virus matrix protein 1 at low concentrations. [Modes for carrying out the invention]

[0011] The present invention will be described in detail below. In this specification, unless otherwise specified, the notation "A~B" for numerical values ​​A and B shall be equivalent to "A or greater and B or less". That is, the notation "A~B" includes numerical values ​​A and B at both ends. If a unit is attached only to numerical value B in such notation, that unit shall also apply to numerical value A. The notation "including A or B" shall include embodiments that include both A and B. Furthermore, the following embodiments described herein also apply to compositions [A] to [J] and their sub-concepts described in the section on means for solving the problem.

[0012] (Rosmarinic acid or its salts) The composition of the present invention contains rosmarinic acid or a salt thereof as an active ingredient. Rosmarinic acid, also known as α-O-caffeoyl-3,4-dihydroxyphenyllactic acid, is an ester of caffeic acid (CA) and 3,4-dihydroxyphenyllactic acid. Rosmarinic acid has the following structural formula.

[0013] [ka]

[0014] Rosmarinic acid has several optical isomers, and any of them may be used. In the composition of the present invention, rosmarinic acid may be a commercially available product or may be chemically synthesized according to conventional methods. Alternatively, it may be obtained by extraction and purification from plant material such as plants containing rosmarinic acid.

[0015] The compositions of the present invention may contain salts of rosmarinic acid to the extent that the effects of the present invention are obtained. Examples of such salts include alkali metal and alkaline earth metal salts such as sodium salts, potassium salts, and calcium salts, or acid addition salts. The salts of rosmarinic acid are pharmaceutically acceptable salts of rosmarinic acid.

[0016] (Flavonoid glycosides) The composition of the present invention contains a flavonoid glycoside as an active ingredient. A flavonoid glycoside is a compound having a structure in which a flavonoid molecule and a sugar molecule are linked by a glycosidic bond.

[0017] Flavonoids are a general term for compounds having a basic C6-C3-C6 structure in which two benzene rings (A ring and B ring) are linked by three carbon atoms. Flavonoids can be classified into flavanones, flavanols, flavones, isoflavones, isoflavonoids, chalcones, anthocyanidins, etc., according to their chemical structure. The structure of the flavonoid in the flavonoid glycoside included in the composition of the present invention may be any structure as long as the effects of the present invention are obtained, but it is preferably a flavanone or a flavone. In the composition of the present invention, the flavonoid glycoside may be a commercially available product or may be chemically synthesized according to conventional methods. Alternatively, it may be obtained by extraction and purification from plant material such as plants containing flavonoid glycosides.

[0018] The sugar structure in the flavonoid glycoside contained in the composition of the present invention may be any structure, such as a monosaccharide, disaccharide, or polysaccharide, as long as the effects of the present invention are obtained, but a monosaccharide or disaccharide is preferred. In the flavonoid glycoside, the number of bonded sugar molecules may be one, two, or more.

[0019] The composition of the present invention preferably contains one or more flavonoid glycosides selected from the group consisting of eriocitrin, bisenin 2, and luteolin-7-rutinoside, and more preferably contains all of eriocitrin, bisenin 2, and luteolin-7-rutinoside. Eriocitrin, bisenin 2, and luteolin-7-rutinoside each have the following structural formulas: Eriocitrin has a structure in which rutinose is bonded to eriodicthiool, a type of flavanone. Bisenin 2 has a structure in which two molecules of glucose are bonded to apigenin, a type of flavone. Luteolin-7-rutinoside has a structure in which rutinose is bonded to luteolin, a type of flavone.

[0020] [ka] (Eriocitrin)

[0021] [ka] (Bisenin 2)

[0022] [ka] (Luteolin-7-rutinoside)

[0023] The composition of the present invention may contain rosmarinic acid or a salt thereof in an amount of 0.050 to 1000.0 μM, 0.050 to 500.0 μM, 0.10 to 100.0 μM, or 0.14 to 50 μM based on the total amount of the composition.

[0024] The composition of the present invention may contain at least one flavonoid glycoside in an amount of 0.0010 to 1000.0 μM, 0.0010 to 500.0 μM, 0.010 to 100.0 μM, or 0.010 to 50.0 μM based on the total amount of the composition.

[0025] If at least one flavonoid glycoside is eriocitrin, the composition of the present invention may contain eriocitrin in amounts of 0.0050 to 1000.0 μM, 0.0050 to 500.0 μM, 0.010 to 100.0 μM, or 0.020 to 50.0 μM based on the total amount of the composition.

[0026] If at least one flavonoid glycoside is luteolin-7-rutinoside, the composition of the present invention may contain luteolin-7-rutinoside in amounts of 0.010 to 1000.0 μM, 0.010 to 100.0 μM, 0.020 to 50.0 μM, or 0.028 to 40.0 μM based on the total amount of the composition.

[0027] If at least one flavonoid glycoside is bisenin 2, the composition of the present invention may contain bisenin 2 in amounts of 0.0010 to 1000.0 μM, 0.0010 to 100.0 μM, 0.005 to 50.0 μM, or 0.010 to 25.0 μM based on the total amount of the composition.

[0028] The composition of the present invention may contain at least one flavonoid glycoside in an amount of 0.010 to 1000.0 μM, 0.020 to 500.0 μM, or 0.050 to 200.0 μM on a basis of the total amount of the composition, as the total amount of flavonoid glycosides (i.e., the total amount of eriocitrin, bisenin 2, and luteolin-7-rutinoside if they are included).

[0029] The above concentration ranges are applicable in combination even when rosmarinic acid or its salts and two or more flavonoid glycosides are included. Furthermore, the above concentration ranges are applicable to compositions [A] to [J], their sub-concepts, or their specific embodiments.

[0030] In the composition of the present invention, the ratio of the amount of substance of at least one flavonoid glycoside to rosmarinic acid or a salt thereof (flavonoid glycoside / rosmarinic acid or a salt thereof) can be 0.0010 to 100.0, 0.0050 to 50.0, 0.010 to 10.0, 0.025 to 5.0, or 0.050 to 2.0.

[0031] When the composition of the present invention contains four substances: rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside, a particular embodiment of the composition of the present invention may contain, on a basis of the total amount of the composition, 0.050 to 1000.0 μM of rosmarinic acid or a salt thereof, 0.0050 to 1000.0 μM of eriocitrin, 0.010 to 1000.0 μM of luteolin-7-rutinoside, and 0.0010 to 1000.0 μM of bisenin-2.

[0032] When the composition of the present invention contains four substances: rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside, a particular embodiment of the composition of the present invention may contain, on a basis of the total amount of the composition, 0.050 to 100.0 μM of rosmarinic acid or a salt thereof, 0.0050 to 100.0 μM of eriocitrin, 0.010 to 100.0 μM of luteolin-7-rutinoside, and 0.0010 to 100.0 μM of bisenin-2. When the composition of the present invention contains four substances: rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside, a particular embodiment of the composition of the present invention may contain, on a basis of the total amount of the composition, 0.10 to 50.0 μM of rosmarinic acid or a salt thereof, 0.010 to 50.0 μM of eriocitrin, 0.020 to 50.0 μM of luteolin-7-rutinoside, and 0.0050 to 50.0 μM of bisenin-2. When the composition of the present invention contains four substances: rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside, a particular embodiment of the composition of the present invention may contain, on a basis of the total amount of the composition, 0.14 to 50.0 μM of rosmarinic acid or a salt thereof, 0.020 to 50.0 μM of eriocitrin, 0.028 to 40.0 μM of luteolin-7-rutinoside, and 0.010 to 25.0 μM of bisenin-2.

[0033] The content of rosmarinic acid or a salt thereof in the composition of the present invention may be, for example, 0.01 ppm to 99.0% by mass, 0.1 ppm to 50.0% by mass, 1.0 ppm to 11.0% by mass, 1.5 ppm to 10.0% by mass, 2.0 ppm to 8.0% by mass, or 2.5 ppm to 6.0% by mass, based on the total amount of the composition. The upper limit may be 1% by mass, 0.1% by mass, or 100 ppm by mass.

[0034] The flavonoid glycoside content in the composition of the present invention may be 0.001 ppm to 99.0% by mass, 0.01 ppm to 50.0% by mass, 0.1 ppm to 20.0% by mass, 0.5 ppm to 15.0% by mass, 1 ppm to 15.0% by mass, 5 ppm to 10.0% by mass, or 10 ppm to 8.0% by mass, based on the total amount of the composition. The above content represents the content of any one flavonoid glycoside contained in the composition of the present invention. The upper limit may be 1% by mass, 0.1% by mass, or 100 ppm by mass.

[0035] The total amount of flavonoid glycosides in the composition of the present invention can be 0.001 ppm to 99.0% by mass, 0.01 ppm to 50.0% by mass, 0.1 ppm to 20.0% by mass, 0.5 ppm to 15.0% by mass, 1 ppm to 15.0% by mass, 5 ppm to 10.0% by mass, or 10 ppm to 8.0% by mass, based on the total amount of the composition. The above content represents the content of any one flavonoid glycoside contained in the composition of the present invention. The upper limit may be 1% by mass or 0.1% by mass.

[0036] In certain embodiments, the composition of the present invention may contain 2.67 ppm to 5.00 ppm of rosmarinic acid or a salt thereof, 3.43 ppm to 5.39 ppm of eriocitrin, 0.59 ppm to 1.29 ppm of bisenin-2, and 2.31 ppm to 7.00 ppm of luteolin-7-rutinoside.

[0037] (Influenza virus) In this specification, influenza virus is a negative-sense single-stranded RNA virus belonging to the Orthomyxoviridae family. Human influenza viruses are further classified into influenza A, influenza B, and influenza C. Other influenza viruses include avian influenza virus, swine influenza virus, and equine influenza virus.

[0038] (Anti-influenza virus composition) In the present invention, "anti-influenza virus" includes an effect of suppressing influenza virus infection, an effect of inhibiting the adsorption of influenza virus (specifically, an effect of inhibiting the neuraminidase of influenza virus), and an effect of inhibiting the entry of influenza virus (specifically, an effect of suppressing the entry of influenza virus into host cells using endocytosis).

[0039] Influenza virus matrix protein 1 (M1) is a protein that plays a crucial role in viral replication and budding. If the M1 protein does not function properly, for example, viral particle formation and budding will be incomplete, leading to a decrease in the virus's replication ability. The degree of influenza virus infection can be assessed by comparing the expression levels of M1 protein in infected cells.

[0040] Neuraminidase is an enzyme present on the surface of the influenza virus. In influenza virus infection, neuraminidase is considered necessary for the degradation of sialic acid when the progeny viruses escape outside the cell after replication. However, recent studies have reported that neuraminidase also contributes to the degradation of sialic acid on false receptors (decoy receptors) that are expressed on the surface of host cells as a defense mechanism and do not contribute to the invasion process, thereby increasing the opportunity for interaction with receptors necessary for invasion. Therefore, if neuraminidase is inhibited in the pre-infection stage, the adsorption of the virus to cells is inhibited, and influenza virus infection is suppressed by depriving the virus of the opportunity to invade.

[0041] Endocytosis, the entry of influenza viruses into host cells, is an initial stage in the onset of infection. Endocytosis is broadly classified into clathrin-dependent and clathrin-independent types. Influenza viruses primarily enter endosomes through clathrin-dependent endocytosis. Inhibiting endocytosis can prevent influenza viruses from entering host cells and causing infection.

[0042] (composition) The dosage form of the composition of the present invention is not particularly limited, and can include oral and parenteral preparations, but oral preparations are preferred. Examples of oral preparations include solid or powdered preparations such as fine granules, granules, tablets, capsules, and pills, as well as liquid preparations such as suspensions, emulsions, syrups, and extracts. Examples of parenteral preparations include injectable preparations.

[0043] The composition of the present invention can exert anti-influenza virus activity upon contact with the influenza virus. Therefore, the composition of the present invention can be used in cosmetics, detergents for textile products, dish soaps, skin cleansers, hair cleansers, disinfectants, wet wipes, antibacterial sheets, mouthwashes, bath additives, anti-influenza virus coating agents, and the like.

[0044] The composition of the present invention may contain an active ingredient comprising rosmarinic acid or a salt thereof and at least one flavonoid glycoside. The composition of the present invention may contain an active ingredient comprising rosmarinic acid or a salt thereof, eriocitrin, bisenin 2, and luteolin-7-rutinoside.

[0045] When the composition of the present invention is an oral preparation, other additives may include excipients, binders, disintegrants, emulsifiers, lubricants, flow enhancers, diluents, preservatives, colorants, flavorings, stabilizers, antiseptics, antioxidants, or suspending agents. Specifically, these may include, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, anhydrous silicic acid, or synthetic aluminum silicate.

[0046] When the composition of the present invention is a parenteral agent, other additives may include water-soluble solvents such as physiological saline or Ringer's solution, water-insoluble solvents such as vegetable oil or fatty acid esters, isotonic agents such as glucose or sodium chloride, solubilizers, stabilizers, preservatives, suspending agents, or emulsifiers.

[0047] The dosage or intake of the composition of the present invention can be appropriately adjusted depending on the formulation, the age, sex, and weight of the person using it.

[0048] The compositions of the present invention can be administered to humans, but the recipients may also be animals other than humans, such as pets like dogs, cats, rabbits, hamsters, guinea pigs, and squirrels; livestock like cattle and pigs; and laboratory animals like mice and rats.

[0049] The aforementioned pharmaceutical compositions include pharmaceuticals and quasi-drugs. Examples of pharmaceuticals include herbal medicine preparations and traditional Chinese medicine preparations. Examples of quasi-drugs include nutritional drinks and health supplements containing herbal medicines.

[0050] (Food composition) When the composition of the present invention is in the form of a food composition, examples of food compositions include: fresh prepared foods such as salads; cooked foods such as steaks, pizzas, and hamburgers; stir-fried foods such as stir-fried vegetables; vegetables such as tomatoes, bell peppers, celery, bitter melon, carrots, potatoes, and asparagus, and processed foods made from these vegetables; confectionery such as cookies, bread, biscuits, hardtack, cakes, rice crackers, yokan, puddings, jellies, ice creams, chewing gum, gummies, tablets, crackers, chips, chocolates, and candies; noodles such as udon, pasta, and soba; fish paste products such as kamaboko, ham, and fish sausage; dairy products such as cheese, cream, and butter; seasonings such as miso, soy sauce, dressings, ketchup, mayonnaise, soup bases, noodle soup bases, curry powder, mirin, and roux; soy products such as tofu; and konjac. The food products are preferably curry, stew sauce, pasta sauce, roux (for example, for curry or stew), seasoning spices, or supplements.

[0051] Food compositions include beverages. Examples of beverages include coffee beverages; cocoa beverages; vegetable juices obtained from the aforementioned vegetables; fruit juices such as grapefruit juice, orange juice, grape juice, and lemon juice; tea beverages such as green tea, black tea, sencha, and oolong tea; alcoholic beverages such as beer, wine (red wine, white wine, or sparkling wine, etc.), sake, plum wine, sparkling wine, whiskey, brandy, shochu, rum, gin, and liqueurs; dairy beverages; soy milk beverages; liquid foods; spiced teas, and sports drinks.

[0052] Food compositions include animal feed. Target animals include, for example, primates such as humans, cattle, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, or mice.

[0053] These food compositions may optionally contain food additives and food ingredients such as antioxidants, flavorings, acidulants, colorings, emulsifiers, preservatives, seasonings, sweeteners, spices, pH adjusters, stabilizers, vegetable oils, animal oils, sugars and sugar alcohols, vitamins, organic acids, fruit juice extracts, vegetable extracts, grains, legumes, vegetables, meats, and seafood, either individually or in combination of two or more. The amounts of these food ingredients and food additives can be appropriately determined within a range that does not impair the objectives of the present invention.

[0054] These food compositions can be subjected to common sterilization treatments such as heat and pressure sterilization in retorts and autoclaves, batch sterilization, plate sterilization, electrostatic sterilization, microwave sterilization, and steam sterilization such as injection and infusion. These food compositions can be packaged using appropriate containers, such as paper cartons, metal cans, or pouches.

[0055] Food compositions include functional foods (beverages) and health foods (beverages). In this specification, "health foods (beverages)" means foods or beverages that have some effect on health, or that can be expected to have some effect, and "functional foods (beverages)" means foods or beverages among the "health foods (beverages)" that are designed and processed to fully express biological regulatory functions. Functional foods and health foods may be in the form of granules, solids, liquids, capsules, gels, or tablets.

[0056] The food composition of the present invention may be provided and sold as a food or beverage labeled with uses such as suppressing influenza virus infection, inhibiting the adsorption of influenza virus, inhibiting influenza virus neuraminidase, inhibiting influenza virus entry, and suppressing entry into host cells by influenza virus using endocytosis.

[0057] The aforementioned "disclosure" includes all actions that inform consumers of the aforementioned uses, effects, and / or properties. The disclosure can take place on any medium, including packaging, containers, catalogs, brochures, and websites. [Examples]

[0058] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the invention. Unless otherwise specified, "%" indicates mass%.

[0059] [Reference Example 1] Anti-influenza virus effect of eriocitrin alone The anti-influenza virus effect of eriocitrin alone was tested.

[0060] (Inhibitory effect on the adsorption of influenza virus) The inhibitory effect of eriocitrin alone on influenza virus adsorption was tested using a neuraminidase activity quantification kit (EnzyChrom Neuraminidase Assay Kit; BioAssay Systems). The test procedure followed the instructions provided with the kit.

[0061] As a result, as shown in Figure 1, eriocitrin alone was found to have an inhibitory effect on influenza virus neuraminidase.

[0062] (Inhibitory effect on influenza virus entry) Cells were cultured in 10 mL of Eagle's minimum essential medium (EMEM) containing 10% fetal bovine serum (FBS) in a dish in a 37°C, 5% CO2 incubator until confluence. Once confluent, all of the medium was aspirated, the cells were washed once with 5 mL of phosphate-buffered saline (PBS), and then trypsinized. After detaching the cells from the dish using trypsin, they were centrifuged at 4°C, 10,000 rpm for 5 minutes. The supernatant was removed, the cells were suspended in EMEM medium, and then re-implanted in 10 mL for subculturing. After subculturing and reaching confluence, the same treatment as above was performed, and the number of cells in 10 μL of EMEM medium was counted to determine the total number of cells.

[0063] The cells are 1.0 × 10 5Cells were planted in 24-well plates at a concentration of cells / mL and incubated for 24 hours in a 37°C, 5% CO2 incubator. After washing three times with Live Cell Imaging Solution (Thermo Fisher Scientific), aqueous solutions of each component were added, and the cells were treated at 37°C for 1 hour. The control group was given the same amount of water. Subsequently, pHrodo Red-labeled transferrin (Thermo Fisher Scientific) was added at 10 μg / mL, and the cells were cultured for a further 15 minutes. After treatment, the cells were washed with PBS, detached using TrypLE Express, and collected. The cells were centrifuged at 10,000 rpm for 3 minutes using a benchtop centrifuge, the supernatant was removed, and the cells were centrifuged again with PBS before being resuspended in PBS. For measurement using a flow cytometer FACSCelesta (Becton Dickinson), the cells were transferred to a measurement tube while passing through a 40 μm mesh cell strainer, and data was acquired for 10,000 cells under excitation light irradiation at 560 nm. The obtained results were expanded into a histogram using FACSDiva software (Becton Dickinson), and the percentage of positive cells was calculated.

[0064] As a result, as shown in Figure 2, eriocitrin alone was found to be effective in suppressing the entry of influenza viruses into host cells via endocytosis.

[0065] [Reference Example 2] Anti-influenza virus effects of bisenin-2 and luteolin-7-rutinoside individually. The anti-influenza virus effects of bisenin 2 and luteolin-7-rutinoside were tested individually.

[0066] (Inhibitory effect on the expression of matrix protein 1 of the influenza virus) The following tests were conducted using MDCK cells derived from canine renal tubular epithelial cells.

[0067] (Cell culture and treatment with each component) Cells were cultured in 10 mL of EMEM medium containing 10% fetal bovine serum (FBS) in a dish in a 37°C, 5% CO2 incubator until confluence. Once confluent, all the medium was aspirated, the cells were washed once with 5 mL of phosphate-buffered saline (PBS), and then treated with TrypLE Express (Thermo Fisher Scientific). The TrypLE Express treatment detached the cells from the dish, and they were centrifuged at 4°C, 10,000 rpm for 5 minutes. The supernatant was removed, the cells were suspended in EMEM medium, and then re-implanted in 10 mL for subculturing. After subculturing and reaching confluence, the same treatment as above was performed, and the number of cells in 10 μL of EMEM medium was counted to determine the total number of cells. The cell count was 2.0 × 10⁶. 5 Cells were inoculated into 24-well plates at a concentration of cells / mL, 2 μL of aqueous solution of each component was added, and the mixture was incubated at 37°C in a 5% CO2 incubator for 1 hour. The same amount of water was added to the control group. An additional 2 μL of influenza virus solution diluted with PBS was added to each well, and the mixture was incubated at 37°C in a 5% CO2 incubator for 1 hour. Influenza virus strain A / Puerto Rico / 8 / 34 (H1N1 subtype) was used. After incubation, the cells were washed with 1 mL of PBS, and EMEM medium containing 10% FBS was added. The mixture was incubated at 37°C in a 5% CO2 incubator for 8 hours. After incubation, the culture medium was aspirated, the cells were washed with PBS, 1 mL of TRI Reagent was added, and the cells were detached and collected by pipetting.

[0068] (RNA preparation from cultured cells) The TRI Reagent reagent containing the lysed cells was left at room temperature for 5 minutes, 200 μL of chloroform was added, and the mixture was vigorously shaken by hand for 15 seconds. Afterward, it was left at room temperature for 3 minutes, centrifuged at 15,000 rpm at 4°C for 15 minutes, and 400-500 μL of the supernatant was collected. 500 μL of isopropyl alcohol was added to the collected supernatant and vigorously shaken by hand for 15 seconds. The mixture was left to stand at room temperature for 10 minutes, and centrifuged at 15,000 rpm at 4°C for 15 minutes. After removing the isopropyl alcohol, 1 mL of 75% EtOH / RNase-free water was added, and the mixture was centrifuged at 15,000 rpm at 4°C for 5 minutes. The supernatant was completely removed using a pipette. The cells were lysed in 30 μL of RNase-free water until no precipitate was visible. RNA levels were measured using 5 μL of the 30 μL cell suspension, and cDNA synthesis was performed using the remainder. RNA levels were measured by absorbance using a spectrophotometer (GE healthcare Ultrospec 3300 pro). 5 μL of cell suspension was mixed with 95 μL of RNase-free water to prepare a 20-fold diluted sample of 100 μL. Absorbance was measured at 260 nm and 280 nm to calculate the RNA concentration in the suspension. Based on the calculated concentration, DPEC water was added to the remaining cell suspension used for absorbance measurement to prepare an RNA sample of 1 μg.

[0069] (cDNA synthesis from RNA) 5.5 μL of RNA sample + RNase-free water and 0.5 μL of 10 μM random dT18 primer were dispensed into PCR tubes, mixed by centrifugation, and then placed in a thermal cycler and heated at 65°C for 5 minutes. The samples were removed, rapidly cooled on ice, and gently centrifuged. In a separate Eppendorf tube, 2.0 μL of 5× First strand buffer, 1.0 μL of 0.1 M DTT, and 0.5 μL of 10 mM dNTP Mix were added per sample while cooling on ice, and mixed by centrifugation. Then, 0.5 μL (200 U / L) of reverse transcriptase (enough for the number of samples + 1 tube) was added, and 4 μL was dispensed into tubes warmed in the thermal cycler, and mixed by centrifugation. The tubes were placed back in the thermal cycler and heated at 42°C for 50 minutes and then at 75°C for 15 minutes for the RT reaction. After the program was completed, the samples were removed while kept at 15°C.

[0070] (RT-PCR analysis) The expression of the M1 protein gene was evaluated by PCR analysis using the Eco Real Time PCR system. A cDNA template prepared by RT reaction was diluted 20-fold with purified water for PCR. A PCR master mix was prepared by adding 2.2 μL of sterile distilled water, 0.4 μL of PCR forward primer (10 μL), 0.4 μL of PCR reverse primer (10 μL), and 5.0 μL of TB Green Premix Ex Taq II to an Eppendorf tube per reaction. The layout was determined on a 48-well reaction plate, and 8.0 μL of master mix and 2.0 μL of cDNA template were dispensed into three compartments. The plate was completely sealed with a sealer, and the plate was centrifuged on a benchtop plate to allow the samples to settle at the bottom of the wells. The 48-well reaction plate was placed in the chamber and the reaction was started. The expression level of the M1 protein gene was expressed as the relative expression level relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0071] As a result, as shown in Figure 3, both bisenin 2 and luteolin-7-rutinoside were found to have neuraminidase inhibitory effects on the influenza virus.

[0072] Examples 1-2, 11-13, 21-23: Anti-influenza virus effect of a composition containing four components: rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside. In this example, the inhibitory effect of a composition containing rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside on the expression of influenza virus matrix protein 1 was tested. The procedure was the same as in Reference Example 2.

[0073] The following shows the molar concentrations (in μM) of each component contained in the compositions of each example, and the ratios of the components to each other. RA: Rosmarinic acid ·EC:Eriocitrin ·L7R :Luteolin-7-O-rutinoside ·V2 : Vicenin 2 [Table 1]

[0074] The results are shown in Figures 4 and 5. As a result, compositions containing rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside showed an inhibitory effect on the expression of influenza virus matrix protein 1, as shown in Figures 4 and 5.

[0075] [Reference Example 2] Anti-influenza virus effect of luteolin-7-rutinoside alone The anti-influenza virus effect of luteolin-7-rutinoside alone was evaluated using a neuraminidase activity quantification kit (EnzyChrom Neuraminidase Assay Kit; BioAssay Systems), similar to the method used in Reference Example 1.

[0076] As a result, as shown in Figure 6, luteolin-7-rutinoside alone was found to have an inhibitory effect on influenza virus neuraminidase.

[0077] Example 4: Anti-influenza virus effect of a combination of rosmarinic acid and at least one flavonoid glycoside. The inhibitory effect of a combination of rosmarinic acid and at least one flavonoid glycoside on the expression of influenza virus matrix protein 1 was tested using the same procedure as in Reference Example 2. For example, RA+EC is a composition that combines rosmarinic acid and eriocitrin. In each test group, the tests were conducted to achieve the following molar concentrations, as shown in the table below Figure 7. RA = 7.41 μM EC = 9.04 μM L7R = 3.88 μM V2 = 2.18 μM

[0078] The results are shown in Figure 7. As a result, the following test groups showed a higher inhibitory effect on the expression of influenza virus matrix protein 1 compared to the test group to which only rosmarinic acid was added. Rosmarinic acid + eriocitrin Rosmarinic acid + Bisenin 2 Rosmarinic acid + luteolin-7-rutinoside Rosmarinic acid + eriocitrin + bisenin 2 Rosmarinic acid + Bisenin 2 + Luteolin-7-rutinoside

[0079] Furthermore, in the following test groups, a higher inhibitory effect on the expression of influenza virus matrix protein 1 was observed compared to the test group to which only rosmarinic acid was added. • Visenin 2 alone Luteolin-7-rutinoside alone • Bisenin 2 + Luteolin-7-rutinoside • Eriocitrin + Bisenin 2 + Luteolin-7-rutinoside

[0080] Example 3: Anti-influenza virus effect in the low-concentration range of a composition containing four components: rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside. In this example, a composition containing rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside was used to test the inhibitory effect on the expression of influenza virus matrix protein 1 in the low-concentration range. The procedure was the same as in Reference Example 2.

[0081] The molar concentrations (in μM) of each component contained in the composition of Example 3 are shown below. • RA: 0.07 μM EC: 0.09μM L7R: 0.04μM V2: 0.02 μM

[0082] The results are shown in Figure 8. As a result, the composition containing rosmarinic acid, eriocitrin, bisenin-2, and luteolin-7-rutinoside showed an inhibitory effect on the expression of influenza virus matrix protein 1 even at low concentrations, as shown in Figure 8. [Industrial applicability]

[0083] The composition of the present invention provides a composition that has a more effective anti-influenza virus activity.

Claims

1. Rosmarinic acid or its salts, and at least one flavonoid glycoside A composition containing the following:

2. The composition according to claim 1, wherein the ratio of the amount of substance of at least one flavonoid glycoside to rosmarinic acid or a salt thereof (flavonoid glycoside / rosmarinic acid or salt thereof) is 0.0010 to 100.

0.

3. The molar concentration of rosmarinic acid or its salt is 0.10 to 1000.0 μM based on the total amount of the composition. The composition according to claim 1 or 2, wherein the molar concentration of at least one flavonoid glycoside is 0.0010 to 1000.0 μM on a basis of the total amount of the composition.

4. The composition according to claim 1 or 2, wherein the structure of the flavonoid in at least one flavonoid glycoside is a flavanone or a flavone, and the sugar is a monosaccharide or a disaccharide.

5. The composition according to claim 4, wherein at least one flavonoid glycoside is selected from the group consisting of eriocitrin, bisenin 2, and luteolin-7-rutinoside.

6. The composition according to claim 5, comprising rosmarinic acid or a salt thereof, eriocitrin, luteolin-7-rutinoside, and bisenin 2.

7. The composition according to claim 6, comprising, on a basis of the total amount of the composition, 0.050 to 1000.0 μM of rosmarinic acid or a salt thereof, 0.0050 to 1000.0 μM of eriocitrin, 0.010 to 1000.0 μM of luteolin-7-rutinoside, and 0.0010 to 1000.0 μM of bisenin-2.

8. The composition according to claim 1 or 2, which is for use against influenza viruses.

9. A composition comprising rosmarinic acid or a salt thereof, for ingestion or administration in combination with a flavonoid glycoside.

10. A composition comprising at least one flavonoid glycoside, for ingestion or administration in combination with rosmarinic acid or a salt thereof.

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