Anti-rhinovirus agent

Abstract

To provide a novel antiviral agent against rhinovirus and a preventive agent or a therapeutic agent against rhinovirus.SOLUTION: An anti-rhinovirus agent contains at least one kind selected from the group consisting of laurel extract, tongkat ali extract, nemunoki bark extract, tulasi extract, chestnut astringent skin extract, coffee tree kernel extract, evening primrose extract, aronia extract, willow tree bark extract, Japanese mallotus extract, Terminalia bellirica extract, apricot kernel extract, ginger extract, angelica extract, Bixa orellana extract, leek extract, Japanese radish leaf extract, Citrus jabara fruit powder, apple fruit extract, green tea extract, grape seed extract, cassis extract, black soybean kernel extract, elderberry extract, and glycyrrhizinic acid as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an antiviral agent against rhinovirus.

Background Art

[0002] Rhinovirus belongs to the family Picornaviridae, is weak against acids, and infects through the upper respiratory tract such as the nose and throat, causing inflammation. It is considered that 80-90% of the causes of cold syndromes are viral infections, and approximately two-thirds of acute upper respiratory infections in adults in early autumn are caused by rhinovirus (Non-Patent Document 1). The incubation period of rhinovirus is about 1-2 days, and symptoms such as sore throat, runny nose, and nasal congestion may occur, and it is considered that health-related QOL such as activity and motivation may decline (Non-Patent Document 2). Furthermore, it is also involved in the exacerbation of respiratory diseases such as bronchial asthma and COPD (Non-Patent Document 3).

[0003] As therapeutic agents for the above symptoms, antipyretic analgesics, antitussive expectorants, anti-inflammatory agents, etc., which are symptomatic treatments, are used. Currently, there is no therapeutic agent on the market that has a direct effect on rhinovirus itself.

[0004] Therefore, there is a need for an antiviral agent against rhinovirus and a material that can be used for the prevention and treatment of rhinovirus infections.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0006] The object of the present invention is to provide a novel anti-rhinovirus agent. [Means for solving the problem]

[0007] The inventors diligently studied to find a new anti-rhinovirus agent and, unexpectedly, discovered that laurel extract, tongkat ali extract, mimosa bark extract, tulsi extract, chestnut peel extract, coffee seed extract, evening primrose extract, aronia extract, willow bark extract, red maple extract, Terminalia bellaria extract, apricot kernel extract, ginger extract, angelica extract, annatto extract, leek extract, radish leaf extract, jabara fruit powder, apple fruit extract, green tea extract, grape seed extract, blackcurrant extract, black bean seed coat extract, elderberry extract, and glycyrrhizic acid possess anti-rhinovirus activity, thus completing the present invention.

[0008] In other words, the present invention is (1) An antirhinovirus agent comprising at least one active ingredient from the group consisting of laurel extract, tongkat ali extract, mimosa bark extract, tulsi extract, chestnut peel extract, coffee seed extract, evening primrose extract, aronia extract, willow bark extract, red maple extract, Terminalia bellaria extract, apricot kernel extract, ginger extract, angelica extract, annatto extract, leek extract, radish leaf extract, jabara fruit powder, apple fruit extract, green tea extract, grape seed extract, blackcurrant extract, black bean seed coat extract, elderberry extract, and glycyrrhizic acid. (2) An anti-rhinovirus agent containing Tongkat Ali extract as an active ingredient, based on its inhibitory effect on the proliferation of rhinoviruses. (3) Food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetics that contain the anti-rhinovirus agent described in (1) or (2), for use against rhinoviruses. (4) An oral or nasal application preparation for anti-rhinovirus use, containing the anti-rhinovirus agent described in (1) or (2), (5) The indicated agent described in (4), which is a throat spray, nasal spray, candy, chewable tablet, granules, inhaler, or lozenge. (6) Food and beverages containing the anti-rhinovirus agent described in (1) or (2) and bearing a label indicating that they are used for the prevention or improvement of rhinovirus infections, That is the case. [Effects of the Invention]

[0009] The anti-rhinovirus agent of the present invention has anti-rhinovirus activity. The anti-rhinovirus agent of the present invention can be used as a preventive or therapeutic agent for rhinovirus infections, such as upper respiratory tract-related diseases. Furthermore, the present invention is useful for preventing the spread of rhinovirus infections and alleviating symptoms in infected individuals. [Modes for carrying out the invention]

[0010] The laurel extract used in this invention is derived from the leaves of the laurel genus of the family Lauraceae, and can be extracted with water or a solvent usable in the production of food ingredients such as ethanol, or a mixture of the said solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used by processing such as heat treatment, freeze-drying, or vacuum drying.

[0011] The Tongkat Ali extract used in this invention is derived from the roots of Tongkat Ali, a plant belonging to the genus Tongkat Ali of the family Simaroubaceae. It can be extracted using water or a solvent such as ethanol that can be used in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available Tongkat Ali extract can also be used.

[0012] The Albizia julibrissin bark extract used in this invention is derived from the bark of Albizia julibrissin, a member of the Fabaceae family, subfamily Mimosoideae, and can be extracted with water or ethanol, or other solvents usable in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available Albizia julibrissin bark extract can also be used.

[0013] The tulsi extract used in this invention is derived from the leaves of Ocimum sanctum, a plant belonging to the Lamiaceae family. It can be extracted using water or a solvent such as ethanol that can be used in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, hygiene products, or cosmetic ingredients, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available tulsi extract can also be used.

[0014] The chestnut peel extract used in this invention is derived from the pericarp of the chestnut tree (Castanea genus) of the Fagaceae family, and can be extracted with water or ethanol, or other solvents usable in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available chestnut peel extract can also be used.

[0015] The coffee seed extract used in this invention is derived from the seeds of the coffee tree (Coffea japonica), a member of the Rubiaceae family. It can be extracted using water or a solvent such as ethanol that can be used in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available coffee seed extracts can also be used.

[0016] The evening primrose extract used in this invention is derived from the seeds of Oenothera biennis, a species of the Onagraceae family, and can be extracted with water or ethanol, or other solvents usable in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available evening primrose extract can also be used.

[0017] The aronia extract used in this invention is derived from the fruit of the Aronia genus of the Rosaceae family, and can be extracted with water or ethanol, or other solvents usable in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, hygiene products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used by processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available aronia extract can also be used.

[0018] The willow bark extract used in this invention is derived from the bark of the Salix genus of the Salicaceae family, and can be extracted with water or ethanol, or other solvents usable in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used by processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available willow bark extract can also be used.

[0019] The Rubus sieboldianus extract used in the present invention is derived from the bark of the genus Rubus of the Rosaceae family, and can be extracted with a solvent such as water or ethanol or a mixed solution of the solvent that can be used for the production of food and drink products, pharmaceuticals, quasi-drugs, sanitary products or cosmetic raw materials. Further, the form of the extract is not particularly limited, and soft extract, dried extract powder, extract powder, etc. can also be used by treatments such as heat treatment, freeze drying or vacuum drying. Furthermore, commercially available Rubus sieboldianus extract can also be used.

[0020] The Terminalia bellirica extract used in the present invention is derived from the fruit of the genus Terminalia of the Combretaceae family, and can be extracted with a solvent such as water or ethanol or a mixed solution of the solvent that can be used for the production of food and drink products, pharmaceuticals, quasi-drugs, sanitary products or cosmetic raw materials. Further, the form of the extract is not particularly limited, and soft extract, dried extract powder, extract powder, etc. can also be used by treatments such as heat treatment, freeze drying or vacuum drying. Furthermore, commercially available Terminalia bellirica extract can also be used.

[0021] The Prunus mume extract used in the present invention is derived from the seeds of Prunus mume of the genus Prunus of the Rosaceae family, and can be extracted with a solvent such as water or ethanol or a mixed solution of the solvent that can be used for the production of food and drink products, pharmaceuticals, quasi-drugs, sanitary products or cosmetic raw materials. Further, the form of the extract is not particularly limited, and soft extract, dried extract powder, extract powder, etc. can also be used by treatments such as heat treatment, freeze drying or vacuum drying. Furthermore, commercially available Prunus mume extract can also be used.

[0022] The ginger extract used in this invention is derived from the rhizome of Zingiber officinale, a member of the Zingiberaceae family, and can be extracted with water or ethanol, or other solvents usable in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available ginger extract can also be used.

[0023] The Angelica extract used in this invention is derived from the rhizome of the Angelica genus of the Apiaceae family, and can be extracted with water or ethanol, or other solvents usable in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available Angelica extract can also be used.

[0024] The annatto extract used in this invention is derived from the seeds of the genus Annatto of the family Annattoaceae, and can be extracted with water or a solvent such as ethanol that can be used in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used by processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available annatto extract can also be used.

[0025] The onion extract used in this invention is derived from the leaves of the onion plant, genus Allium, of the Amaryllidaceae family, and is used in the form of a dried leaf extract. The dried leaf powder used in this invention may be, for example, a powdered dried product obtained by further grinding a dried chopped product. The onion extract used in this invention can be extracted with water or a solvent that can be used in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, hygiene products, or cosmetic raw materials, such as ethanol, or a mixture of the above solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used by processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available onion extracts can also be used.

[0026] The radish leaf extract used in this invention is derived from the leaves of the radish plant (Raphanus sativus), a member of the Brassicaceae family, and is used in the form of dried leaves or an extract. The dried leaf powder used in this invention may be, for example, a powdered dried product obtained by further grinding a dried chopped product. The radish leaf extract used in this invention can be obtained by extracting with water or a solvent usable in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, hygiene products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available radish leaf extract can also be used.

[0027] The jabara fruit powder used in this invention is derived from the fruit of the jabara plant, a member of the citrus family, and is used in the form of dried fruit. The fruit powder used in this invention may be, for example, a dried powder obtained by further grinding a dried chopped product.

[0028] The apple fruit extract used in this invention is derived from the fruit of the apple tree, a member of the genus Malus in the family Rosaceae. The type of apple used as the raw material for extraction in this invention may be any type of apple, such as red apples or yellow apples, for example, Fuji or Ohlin. The apple fruit extract used in this invention can be extracted with water or a solvent that can be used in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available apple fruit extracts can also be used.

[0029] The green tea extract used in this invention is derived from the leaves of Camellia sinensis O.KZE, a species of the Camellia genus in the Theaceae family, and can be extracted using water, ethanol, or other solvents usable in the production of food ingredients, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying.

[0030] The grape seed extract used in this invention is derived from the seeds of plants belonging to the Vitaceae family and can be extracted with water or a solvent such as ethanol that can be used in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used by processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available grape seed extracts can also be used.

[0031] The blackcurrant extract used in this invention is derived from the fruit of a species of currant in the genus Ribes of the family Saxifragaceae, known in Japanese as Kurofusasuguri (or Kurosuguri). It can be extracted using water or ethanol, or a solvent usable in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic ingredients, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used after processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available blackcurrant extract can also be used.

[0032] The black soybean seed coat extract used in this invention is derived from the seed coat of the black soybean, a member of the legume family. It can be extracted using water or a solvent such as ethanol that can be used in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, hygiene products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used by processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available black soybean seed coat extract can also be used.

[0033] The elderberry extract used in this invention is derived from the fruit of the elderberry genus of the Adoxaceae family, and can be extracted with water or ethanol, or other solvents usable in the manufacture of food and beverages, pharmaceuticals, quasi-drugs, sanitary products, or cosmetic raw materials, or a mixture of such solvents. Furthermore, the form of the extract is not particularly limited, and soft extracts, dried extract powders, and extract powders can also be used by processing such as heat treatment, freeze-drying, or vacuum drying. In addition, commercially available elderberry extract can also be used.

[0034] The above-mentioned extract and fruit powder of the present invention have anti-rhinovirus activity and are therefore useful as anti-rhinovirus agents.

[0035] The glycyrrhizic acid used in this invention has a molecular weight of C 42 H 62 O 16= 822.94, and it is a substance consisting of 99% or more by weight. Glycyrrhizic acid has anti-rhinovirus activity and is therefore useful as an anti-rhinovirus agent in this invention.

[0036] The extract, fruit powder, and glycyrrhizic acid of the present invention have anti-rhinovirus activity and can therefore be used as a preventive or therapeutic agent for rhinovirus infection. Rhinovirus infection refers to upper respiratory tract symptoms caused by rhinovirus infection, specifically, for example, sore throat, runny nose, nasal congestion, etc.

[0037] The anti-rhinovirus agent of the present invention can be used as is or mixed with other components as a food or beverage, pharmaceutical, quasi-drug, hygiene product, cosmetic, or reagent.

[0038] The amounts of the above-mentioned extract, fruit powder, or glycyrrhizic acid used in the present invention are not particularly limited, but when provided as cosmetics, quasi-drugs, pharmaceuticals, food and beverages, hygiene products, or reagents, each is 0.000001 to 10% by mass, preferably 0.0001 to 5% by mass, and more preferably 0.001 to 1% by mass, relative to the total composition.

[0039] The anti-rhinovirus agent of the present invention can be provided in various forms, such as food and beverages, pharmaceuticals, quasi-drugs, hygiene products, cosmetics, or reagents, and such products are suitable for anti-rhinovirus use. Preferably, the dosage form is a spray for the throat, nose, mouth, hands, masks, clothing, interior goods, etc., or a mouthwash, toothpaste, mouthwash, hand soap, throat lozenges, chewing gum, cough drops, or troches. Since rhinoviruses are easily transmitted through the upper respiratory tract, such as the nose and throat, agents applied to the throat, nose, and mouth are particularly preferred.

[0040] In this invention, "anti-rhinovirus use" includes treatment of rhinovirus infection or alleviation or improvement of symptoms of rhinovirus infection, prevention of rhinovirus infection, inactivation of rhinovirus, and suppression of rhinovirus proliferation. Furthermore, the fact that a product is for anti-rhinovirus use can be determined from the product name, the product itself, the container or packaging, or from advertisements used for promotion, including posters, television commercials, and the internet, as well as from in-store POP displays and explanations at informational meetings. In addition, the anti-rhinovirus agent of this invention, foods and beverages, pharmaceuticals, quasi-drugs, sanitary products, cosmetics, and their instruction manuals may bear indications that they are for anti-rhinovirus use, rhinovirus inactivation, or suppression of rhinovirus proliferation, or that they are for the prevention of rhinovirus infection.

[0041] While there are no particular limitations on the form of administration, food and beverages are preferred. When used as food or beverages, the dosage forms include capsules, powders, tablets, candies, syrups, granules, and inhalers. These can be manufactured by known methods. During manufacturing, various additives that can be contained in food and beverages can be incorporated, as long as they do not impair the effects of the present invention. [Examples]

[0042] The present invention will be further described below with reference to examples and test examples, but the present invention is not limited in any way by these examples.

[0043] Testing methods for evaluating antiviral activity include inactivation tests, which assess whether the virus is directly inactivated, and replication inhibition tests, which assess whether the virus's replication within cells is suppressed. In this study, to obtain a broader antiviral spectrum, antiviral activity was evaluated using a test system that combined inactivation and replication inhibition tests (Antiviral activity test in Test Example 1). The antiviral materials obtained from this test system are suggested to possess inactivation activity, replication inhibition activity, or both. By additionally conducting replication inhibition tests, it can be suggested that materials that showed a hit in this test system but did not exhibit replication inhibition activity exhibited activity due to virus inactivation.

[0044] (Preparation of test samples) The test samples used are listed below. Bay leaf extract: Product name "Lauresh" (manufactured by Tokiwa Botanical Chemical Research Institute). Contains 1.0% or more of deacetyllaurenobiolede as the main indicator component. Tongkat Ali Extract: Aqueous extract of Tongkat Ali root. Contains 0.8% or more of eurycomanone as the main indicator component. Albizia julibrissin bark extract: An extract obtained by extracting the bark of the Albizia julibrissin tree until the moisture content is 10% or less. Tulsi extract: Extract from tulsi leaves. Contains 2.0-5.0% ursolic acid as the main indicator component. Chestnut skin extract: An extract derived from the inner skin of chestnuts. It contains 20% or more total polyphenols as its main indicator component. Coffee tree seed extract: Aqueous ethanol extract of coffee tree seeds. It contains 24.0% or more chlorogenic acid and 45.0% or more chlorogenic acid derivatives as the main indicator component. Evening primrose extract: Aqueous ethanol extract of evening primrose seeds. It contains 60% or more polyphenols with a gallic acid skeleton as the main indicator component. Aronia extract: A hydrated ethanol extract of aronia fruit. Contains 35% or more anthocyanins as the main indicator component. Willow bark extract: Aqueous ethanol extract of willow bark or branches. Contains 15% or more salicin as the main indicator component. Japanese Red Maple Extract: Aqueous extract of the bark of the Japanese red maple tree. It contains approximately 10% bergenin as the main indicator component. Terminalia bellerica extract: An extract of Terminalia bellerica fruit. It contains 35% or more polyphenols as its main indicator component. Apricot kernel extract: A 30% ethanol extract of apricot seeds. Ginger extract: A aqueous extract of the rhizome of ginger. Angelica extract: Aqueous extract of the rhizome of Angelica. Contains 0.07% or more of ostor as the main indicator component. Annatina extract: Aqueous ethanol-containing extract of annatto leaves. Green onion extract: A water-based extract of green onion leaf powder. Radish leaf extract: A water-based extract of radish leaf powder. Jabara fruit powder: Contains naringin as the main indicator component. Apple fruit extract: Aqueous extract of apple fruit. Contains 5% or more of phloridine, a polyphenol, as the main indicator component. Green tea extract: Product name "Tiacaron 90S" (Tokiwa Botanical Chemical Research Institute). Extracted from tea leaves with aqueous alcohol. Contains 40% or more EGCG as the main indicator component. Grape seed extract: An extract of grape seeds. It contains more than 80% proanthocyanidins as its main indicator component. Blackcurrant extract: An extract derived from blackcurrant fruit. It contains 35% or more anthocyanins as its main indicator component. Black soybean seed coat extract: An extract derived from the seed coat of black soybeans. It contains 10% or more anthocyanins as the main indicator component. Elderberry extract: An extract obtained by osmotic membrane filtration of elderberry fruit. It contains 10% or more anthocyanins as the main indicator component. Glycyrrhizic acid: Manufactured by Fujifilm Wako Pure Chemical Corporation.

[0045] The above test product was dissolved in distilled water or DMSO to a concentration of 100 mg / mL, and then diluted 20-fold with distilled water to prepare the stock solution for the cytotoxicity test. This solution was then serially diluted 2-fold with distilled water to prepare the test samples.

[0046] (Cytotoxicity confirmation test) HeLa cells, intended for virus infection, were pre-seed in 96-well plates and cultured in a 37°C CO2 incubator for 1 day. After culture, the supernatant was removed and replaced with 0.1 mL of cell maintenance medium. The stock solution and 2-fold serially diluted test samples for cytotoxicity confirmation were added, and the cells were cultured in a CO2 incubator. After culture, 0.1 mL of 5% crystal violet-containing methanol was added per well, and the cells were stained by standing at room temperature for 15 minutes. After staining, each well was washed with tap water and air-dried. Then, 0.05 mL of ethanol was added to each well to elute the crystal violet, and the absorbance at 585 nm was measured using a microplate reader with a reference wavelength of 700 nm. The test was performed with n=3 (wells), and the viability of cells cultured with each test sample was calculated, with the viability of cells in PBS set to 100%. The cytotoxicity of the test samples was determined using the following formula (1), with the viable cell rate serving as the baseline value. Samples above this baseline value were judged to have no cytotoxicity. Formula (1) Reference value = 100 - (Standard deviation of viable cell rate in PBS × 2)

[0047] Test Example 1: Antiviral Efficacy Test (Preparation of virus solution) When HeLa cells were infected with the virus and more than 90% of the cell culture area showed a cytopathic effect (CPE), the cells and culture medium were collected and frozen at -30°C. Subsequently, the virus solution was frozen and thawed, and the supernatant obtained by centrifugation at 3000×g for 10 minutes was concentrated in a Kvic Lab Packet 100 KD and stored at -80°C as the stored virus solution. In the tests, the virus was used after dilution with PBS and EMEM. (Plaque formation inhibition test) Based on the results of cytotoxicity confirmation tests, the test product was dissolved to a concentration 110 times higher than the highest concentration that did not show toxicity to virus-infected cells, and then diluted five times with distilled water to prepare a solution. 1.0 mL of test sample contains 1.0 × 10 4 0.1 mL of virus solution, prepared to a PFU / mL concentration, was added and mixed with a test tube mixer, then left to stand in a 25°C incubator for 1 hour. After 1 hour, the sample was diluted 20-fold with EMEM to prepare the plaque formation inhibition test sample. Pirodavir was used as a positive control. Various solvents (water, DMSO) were prepared to the same concentration as the plaque formation inhibition test sample and used as negative controls. HeLa cells were pre-seed in 6-well plates and cultured in a 37°C CO2 incubator for 1-4 days. Before inoculation with the virus, the culture supernatant was removed and replaced with fresh cell maintenance medium. Next, 0.2 mL of the plaque formation inhibition test sample was inoculated into each well and the cells were infected at 37°C for 1 hour. After virus inoculation, the plates were tilted every 15 minutes to prevent drying and ensure even contact of the virus with the cells. After 1 hour, 4 mL of plaque formation medium was added per well. After solidifying the agarose, it was cultured in a CO2 incubator. After culturing, 2 mL of 4% formalin-added PBS (formalin / PBS) was added to each well and left to stand at room temperature for 1 hour. After 1 hour, the formalin / PBS and agarose were removed from each well, and 1 mL of 1% crystal violet and 5% methanol-added PBS was added and left to stand at room temperature for about 10 minutes to stain the cells. After washing with tap water and air drying, the number of plaques formed by viral proliferation was counted. (judgement) Plaque formation inhibitory activity was calculated by determining the inhibition rate from the average number of plaques with each solvent (water, DMSO) of the test product (A) and the number of plaques formed when each test product was added (B). Equation (2) is shown below. For convenience, test products with zero plaque formation were expressed as having a plaque formation inhibition rate of 100%. In this study, test products with a plaque formation inhibition rate of 50% or higher were judged to have anti-rhinovirus activity. Equation (2) Plaque formation inhibition rate (%) = (1 - (A ÷ B)) × 100 The results are shown in Table 1.

[0048] [Table 1]

[0049] Table 1 shows that laurel extract, tongkat ali extract, mimosa bark extract, tulsi extract, chestnut peel extract, coffee seed extract, evening primrose extract, aronia extract, willow bark extract, red maple extract, Terminalia bellaria extract, apricot kernel extract, ginger extract, angelica extract, annatto extract, leek extract, radish leaf extract, jabara fruit powder, apple fruit extract, green tea extract, grape seed extract, blackcurrant extract, black bean seed coat extract, elderberry extract, and glycyrrhizic acid have anti-rhinovirus activity.

[0050] Test Example 2: Measurement of the effect of inhibiting viral replication (Preparation of test samples) Test Example 1: Based on the results of the cytotoxicity test, the test sample was prepared in solvents (water, DMSO) to a concentration 100 times higher than the test concentration. Then, samples were prepared by diluting the test sample 5-fold and 25-fold with the solvent in which it was dissolved. The prepared solutions were added to plaque-forming culture medium at a ratio of 1 / 100 and used for the test. The test samples used were Tongkat Ali extract, Albizia julibrissin bark extract, Chestnut peel extract, Coffee tree seed extract, and Terminalia bellerica extract. (Plaque formation inhibition test) HeLa cells were pre-seed in 6-well plates and cultured in a 37°C CO2 incubator for 1-4 days. Before virus inoculation, the culture supernatant was removed and replaced with fresh cell maintenance medium. 0.2 mL of virus solution, adjusted to approximately 500 PFU / mL, was inoculated into each well, and the cells were infected at 37°C for 1 hour. After virus inoculation, the plates were tilted every 15 minutes to prevent drying and ensure even contact of the virus with the cells. After 1 hour, the virus solution was removed, the cells in each well were washed with 2 mL of EMEM, and 4 mL of plaque-forming medium containing the test sample was added per well. After agarose solidification, the cells were cultured in a CO2 incubator. After culturing, 2 mL of 4% formalin-added PBS (formalin / PBS) was added to each well and allowed to stand at room temperature for 1 hour. After 1 hour, the formalin / PBS and agarose were removed from each well, and 1 mL of PBS with 1% crystal violet and 5% methanol was added. The cells were allowed to stand at room temperature for about 10 minutes to stain them. After washing with tap water and air drying, the number of plaques formed by viral proliferation was counted. Pirodavir was used as a positive control. As a negative control, various solvents (water, DMSO) were prepared to the same concentration as the plaque formation inhibition test samples and used. (judgement) In this study, antiviral activity was determined from plaque size. The results are shown in Table 2.

[0051] [Table 2]

[0052] Tongkat Ali extract, which showed a reduction in plaque size, was found to have an inhibitory effect on rhinovirus proliferation. Therefore, Tongkat Ali extract is suitable for use in throat lozenges, chewing gum, cough drops, troches, and as an application agent for the throat, nose, and mouth.

[0053] Albizia julibrissin bark extract, chestnut husk extract, coffee tree seed extract, and Terminalia bellerica extract did not show any inhibitory effect on rhinovirus proliferation. Therefore, these four extracts were suggested to have rhinovirus inactivating properties. Accordingly, these are suitable for external use in sprays for the throat, nose, mouth, hands, masks, clothing, and interior items, as well as in mouthwashes, toothpastes, and hand soaps. [Industrial applicability]

[0054] The antiviral agent against rhinovirus of the present invention can be used in the fields of food and beverages, quasi-drugs, pharmaceuticals, and hygiene products. Furthermore, the anti-rhinovirus agent of the present invention can be used as a positive control drug when performing material screening and the like.

Claims

1. An anti-rhinovirus agent containing at least one active ingredient selected from the group consisting of ginger extract, glycyrrhizinic acid, green tea extract, apricot kernel extract, bay laurel extract, tongkat ali extract, silk tree bark extract, tulsi extract, chestnut astringent skin extract, coffee seed extract, evening primrose extract, aronia extract, willow bark extract, Mallotus japonicus extract, Terminalia belerica extract, angelica tree extract, annatto extract, green onion extract, radish leaf extract, jabara fruit powder, apple fruit extract, grape seed extract, black currant extract, black bean seed coat extract, and elderberry extract.

2. 10. An anti-rhinovirus food or drink, medicine, quasi-drug, sanitary product, or cosmetic, which contains the anti-rhinovirus agent according to claim 1.

3. An antirhinovirus agent for oral or nasal application, comprising the antirhinovirus agent according to claim 1.

4. 4. The application agent according to claim 3, which is a throat spray, a nasal spray, a lozenge, a chewable tablet, a granule, an inhaler or a troche.

5. A food or drink containing the antirhinovirus agent according to claim 1 and labeled as being used for the prevention or amelioration of rhinovirus infections.