Antiviral agent

Abstract

The present invention addresses the problem of providing an antiviral agent effective even against enveloped viruses, the antiviral agent being highly safe and having low manufacturing cost.　A protease is used as an active ingredient of the antiviral agent to be used against enveloped viruses. The protease is preferably a serine protease, and more preferably produced by a bacterium selected from the group consisting of a bacterium belonging to the genus Bacillus, a bacterium belonging to the genus Flavobacterium, a bacterium belonging to the genus Arthrobacter, a bacterium belonging to the genus Streptomyces, a bacterium belonging to the genus Geobacillus, a bacterium belonging to the genus Aspergillus, and a bacterium belonging to the genus Rhizopus. A cationic polymer is preferably used together with the protease as the active ingredient of the antiviral agent, and the cationic polymer is more preferably polylysine or polyethylenimine.

Description

antiviral agents

[0001] The present invention relates to antiviral agents for use against enveloped viruses, which contain proteases.

[0002] Disinfectants containing ethanol, hypochlorous acid, hypochlorites, etc. are commonly used to remove viruses and bacteria. However, they sometimes fail to provide sufficient inactivation effects, and sodium hypochlorite cannot be used on hands due to safety concerns. Furthermore, its corrosiveness to metals limits its use. To address these issues, a method using protease has been proposed for inactivating non-enveloped viruses, including norovirus (Patent Documents 1 and 2). Antiviral agents containing proteases as active ingredients are orally ingestible, highly safe, and have excellent antiviral activity against non-enveloped viruses.

[0003] International Publication No. 2021 / 230358 International Publication No. 2021 / 230359

[0004] However, no effective and safe means for inactivating enveloped viruses, including influenza, has been proposed to date. Furthermore, Patent Documents 1 and 2 do not disclose the action of proteases on enveloped viruses. In order to inactivate a wide range of influenza viruses, it is necessary for antiviral agents to have an inactivating effect not only on non-enveloped viruses but also on enveloped viruses. However, there is a concern that safety may not be ensured by using the antiviral agents disclosed in Patent Documents 1 and 2 in combination with other active ingredients that inactivate enveloped viruses. In view of the above circumstances, an object of the present invention is to provide an antiviral agent that is highly safe, inexpensive to produce, and effective against enveloped viruses.

[0005] As a result of extensive research aimed at solving the above problems, the present inventors have found that proteases, particularly serine proteases such as subtilisin, nattokinase, and trypsin, have the effect of inactivating enveloped viruses. Furthermore, they have also found that the inactivating effect of these proteases on enveloped viruses is further enhanced by combining them with a cationic polymer, leading to the completion of the present invention.

[0006] That is, this application provides the following inventions: [1] An antiviral agent containing a protease for use against enveloped viruses. [2] The antiviral agent according to [1], wherein the protease is a serine protease. [3] The antiviral agent according to [2], wherein the serine protease is produced by bacteria selected from the group consisting of bacteria belonging to the genus Bacillus, bacteria belonging to the genus Flavobacterium, bacteria belonging to the genus Arthrobacter, bacteria belonging to the genus Streptomyces, bacteria belonging to the genus Geobacillus, bacteria belonging to the genus Aspergillus, and bacteria belonging to the genus Rhizopus. [4] The antiviral agent according to [3], wherein the bacterium belonging to the genus Bacillus is selected from the group consisting of Bacillus licheniformis, Bacillus clausii, Bacillus polymyxa, Bacillus subtilis, Bacillus alcalophilus, and Bacillus amyloliquefaciens, and the bacterium belonging to the genus Geobacillus is Geobacillus stearothermophilus. [5] The antiviral agent according to any one of [2] to [4], wherein the serine protease is a protein having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, or a protein having an amino acid sequence 90% or more identical to an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7 and having antiviral activity. [6] The antiviral agent according to any one of [2] to [5], wherein the serine protease is subtilisin, nattokinase, or oryzen. [7] The antiviral agent according to any one of [2] to [5], wherein the serine protease is trypsin. [8] The antiviral agent according to any one of [1] to [7], further comprising a cationic polymer. [9] The antiviral agent according to [8], wherein the cationic polymer is polylysine or polyethyleneimine.

[10] The antiviral agent according to any one of [1] to [9], further comprising an alcohol.

[11] The antiviral agent according to any one of [1] to

[10] , wherein the enveloped virus is a mumps virus, a herpes virus, or an influenza virus.

[12] The antiviral agent according to any one of [1] to

[11] , which is in the form of a spray.

[13] A textile product to which the antiviral agent according to any one of [1] to

[11] is applied.

[14] A food or beverage composition comprising the antiviral agent according to any one of [1] to

[11] .

[15] A food cleaning treatment agent comprising the antiviral agent according to any one of [1] to

[11] .

[0007] The present invention provides a novel antiviral agent having virus inactivation ability that is highly safe and is composed of components that can be produced at low cost, and that has a virus inactivation ability that is further enhanced and stable compared to when serine protease is used alone.

[0008] <Antiviral Agent> The antiviral agent of the present invention contains a protease as an active ingredient. The protease is preferably a serine protease. Preferred examples of the serine protease include subtilisin-like serine proteases and chymotrypsin-like serine proteases. Preferred examples of the subtilisin-like serine proteases include subtilisin, oryzin, and nattokinase, and preferred examples of the chymotrypsin-like serine proteases include trypsin, chymotrypsin, acetyltrypsin, thrombin, elastase, and plasmin.

[0009] In the present invention, commercially available proteases may be used, or two or more types of proteases may be mixed depending on the application. Furthermore, the main component does not have to be protease as long as it contains protease.Examples of serine proteases include those commercially available under the following trade names: Alcalase (registered trademark) 2.4LFG, Protamex (registered trademark), alkaline protease produced using the JPBL001 strain, and protease produced using the JPFV001 strain (all manufactured by Novozyme Japan Co., Ltd.); Peptidase R, Protease P "Amano" 3SD, Protin SD-AY10, and Proteax (all manufactured by Amano Enzyme Inc.); Bioprase (registered trademark) OP, Bioprase (registered trademark) 30G, Bioprase (registered trademark) 30L, Bioprase (registered trademark) AL-15FG, Bioprase (registered trademark) APL-30, Bioprase (registered trademark) SP-20FG, Bioprase (registered trademark) XL-416F, Denateym (registered trademark) PMC SOFTER, and Denateym CPO. PEPRICH, Protease CL-15 (all manufactured by Nagase ChemteX Corporation); Aroase (registered trademark) XA-10, Aroase (registered trademark) AP-10, Aroase (registered trademark) NP-10 (all manufactured by Yakult Pharmaceutical Co., Ltd.); Orientase (registered trademark) 22BF (all manufactured by HIBI Corporation); Sumiteam (registered trademark) MP (all manufactured by Shin Nippon Chemical Industry Co., Ltd.); Maxipro BAP, Maxipro PSP, BakeZyme (registered trademark) B500BG, BakeZyme (registered trademark) PPU95,000 (all manufactured by DSM Nutrition Japan Co., Ltd.); Magnax MT103, Enthilon SA-100, Enthilon SA-150P, Enthilon NBS-100 (all manufactured by Rakuto Chemical Industry Co., Ltd.); EFFECTENZ (registered trademark) P 2020, EFFECTENZ (registered trademark) M 2020, EFFECTENZ (registered trademark) P 3020, EFFECTENZ (registered trademark) P 150, Optimase (registered trademark) PR, Multifect (registered trademark) PR 6L (all manufactured by Danisco Japan Co., Ltd.); ADMIL (manufactured by Godo Shusei Co., Ltd.); Coclase (registered trademark) P Granules (manufactured by Mitsubishi Chemical Foods Corporation).

[0010] Furthermore, in the present invention, the serine protease may be, but is not particularly limited to, bacteria belonging to the genus Bacillus, such as Bacillus licheniformis, Bacillus clausii, Bacillus polymyxa, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus alcalophilus; bacteria belonging to the genus Flavobacterium; bacteria belonging to the genus Arthrobacter; bacteria belonging to the genus Streptomyces; bacteria belonging to the genus Geobacillus, such as Geobacillus stearothermophilus; Aspergillus The serine protease may be produced by a bacterium belonging to the genus Aspergillus, such as Bacillus oryzae, Aspergillus niger, or Aspergillus melleus; or a bacterium belonging to the genus Rhizopus. In the present invention, the bacterium producing the serine protease is preferably Bacillus licheniformis, Bacillus clausii, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus alcalophilus, Geobacillus stearothermophilus, or Aspergillus melleus. More preferred are Bacillus licheniformis and Bacillus clausii, as the proteases produced by these bacteria exhibit strong and stable antiviral activity. An example of Bacillus subtilis is Bacillus subtilis var. natto.

[0011] Furthermore, although not particularly limited, in the present invention, the serine protease may be a protein having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, or may be a protein having an amino acid sequence that is 90% or more identical to an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, preferably 95% or more identical, and more preferably 98% or more identical, and having antiviral activity. The protein having the amino acid sequence represented by SEQ ID NO: 1 is a serine protease produced by Bacillus subtilis var. natto, and the protein having the amino acid sequence represented by SEQ ID NO: 2 is a serine protease produced by Bacillus subtilis var. the protein having the amino acid sequence represented by SEQ ID NO: 3 is a serine protease produced by Bacillus subtilis; the protein having the amino acid sequence represented by SEQ ID NO: 4 is a serine protease produced by Bacillus licheniformis; the protein having the amino acid sequence represented by SEQ ID NO: 5 is a serine protease produced by Bacillus clausii; the protein having the amino acid sequence represented by SEQ ID NO: 6 is a serine protease produced by Bacillus amyloliquefaciens; and the protein having the amino acid sequence represented by SEQ ID NO: 7 is a serine protease produced by Geobacillus stearothermophilus. The method for calculating the identity value of amino acid sequences can be a method known to those skilled in the art, for example, calculation can be performed using the specified parameters of blastp, which is used in amino acid homology searches provided by BLAST (registered trademark).

[0012] Furthermore, although not particularly limited, the serine protease of the present invention is preferably a subtilisin-like serine protease, and specifically, subtilisin, nattokinase, oryzen, or trypsin. The amino acid sequence identity between subtilisin and nattokinase, which are produced by Bacillus subtilis var. natto and have the amino acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, is approximately 99%. Serine proteases such as subtilisin and nattokinase can be mass-produced industrially, which offers the advantage of lower production costs compared to natto extract peptides, which are not mass-produced industrially. Trypsin is a safe enzyme known for its use in food products, and because it can be mass-produced industrially, it is inexpensively available. Furthermore, the amino acid sequence of trypsin is not particularly limited, and any amino acid sequence may be used.

[0013] The serine protease "produced" by the aforementioned bacteria may mean not only a serine protease produced by the bacteria themselves, but also one "derived from" the aforementioned bacteria, and may include enzymes obtained by other methods as long as they have the same structure and properties as the serine protease. For example, a protein having the aforementioned amino acid sequence and having antiviral activity produced by genetic recombination technology using any host organism may also be included in the "serine protease produced by bacteria" herein.

[0014] The antiviral agent of the present invention preferably further contains a cationic polymer, from the viewpoint of enhancing the inactivating effect of the protease on enveloped viruses. The cationic polymer is not particularly limited as long as it enhances the virus inactivating effect, but preferred examples include polylysine, polyethyleneimine, protamine, and DEAE-dextran, and polylysine or polyethyleneimine is more preferred.

[0015] Although not particularly limited, when polylysine is used as the cationic polymer, it is particularly safe because polylysine is a naturally occurring polymer. The polylysine may be either α-polylysine or ε-polylysine, and although not particularly limited, ε-polylysine is preferred because of its low toxicity and easy availability. In addition, it is usually a polymer of L-lysine. ε-polylysine can be produced, for example, by the method described in Japanese Patent No. 1245361.

[0016] The weight-average molecular weight of polylysine is not particularly limited, but is preferably 3,000 or more, more preferably 4,000 or more, and is preferably 10,000 or less, more preferably 8,000 or less, even more preferably 6,000 or less, and is particularly preferably in the range of 3,000 to 6,000. The weight-average molecular weight of polyethyleneimine is not particularly limited, but is preferably 300 to 10,000,000. The weight-average molecular weight here is a value measured by the GPC-LALLS method.

[0017] Furthermore, the cationic polymer of the present invention is usually a homopolymer, but may contain other amino acids as monomers as long as the effects of the present invention are not impaired.

[0018] The cationic polymer of the present invention may be in a free form or in the form of a salt with at least one inorganic acid selected from hydrochloric acid, sulfuric acid, phosphoric acid, and hydrobromic acid, or at least one organic acid selected from acetic acid, propionic acid, fumaric acid, malic acid, and citric acid.

[0019] The content ratio of serine protease to cationic polymer in the antiviral agent of the present invention is not particularly limited as long as the antiviral agent exerts its antiviral effect; however, a mass ratio of 1:100 to 100:1 during use is preferred, and 1:50 to 1:1 is more preferred. Examples of the antiviral agent include powder, tablets, capsules, aqueous solutions, emulsions, and aqueous alcohol solutions. Components other than the serine protease and cationic polymer may also contain additives such as solvents, dispersion media, excipients, stabilizers, dispersants, pH adjusters, and bactericides / antibacterial agents. The amounts of serine protease and cationic polymer added are not particularly limited as long as they exert their antiviral effect, and may be determined appropriately depending on the intended use and the type of virus to be targeted. The amounts added may be increased for uses expected to involve a high level of viral contamination, or when a shorter period of time is required for the effect. Furthermore, the enhancement of antiviral activity by the cationic polymer is concentration-dependent, and the concentration and ratio may be selected depending on the intended use, the type of target virus, the desired level of effect, and the type of cationic polymer. Furthermore, if sufficient contact time with the object can be ensured, such as in soaking or pack cleaning, the added concentration can be reduced to still be effective, so the concentration can be selected according to the application.

[0020] The final pH of a formulation containing the antiviral agent of the present invention is not particularly limited as long as the antiviral agent exhibits its antiviral effect; however, it is preferably pH 6.0 or higher, more preferably pH 7.0 to 11.0, and even more preferably pH 7.3 to 10.3, and may be adjusted appropriately depending on the type and concentration of the target virus, or may be adjusted depending on the application.

[0021] The antiviral agent of the present invention is intended to inactivate enveloped viruses. As a result of virus inactivation, the infectivity of the virus can be attenuated or viral infection can be prevented. The antiviral composition of the present invention takes advantage of the advantages of disinfectants containing proteases, which are approved as food additives, i.e., a significantly wider range of targets, conditions, environments, and forms of application compared to hypochlorous acid or sodium hypochlorite, while expanding the scope of its antiviral effect to include enveloped viruses. Envelopeed viruses on which the antiviral agent of the present invention acts include, but are not limited to, influenza virus, mumps virus, and herpes virus, with influenza virus being preferred. The mechanism by which the antiviral agent of the present invention inactivates enveloped viruses is unknown, but it is presumed that the protease degrades a transmembrane protein present in the envelope structure, which is the outermost shell of the virus, triggering the breakdown of the lipid bilayer of the envelope or the degradation of the capsid protein, thereby inactivating the virus. Therefore, it is expected that the antiviral agent of the present invention will be effective against a wide range of enveloped viruses in addition to the viruses shown in the Examples below.

[0022] In this specification, the "antiviral activity" may be evaluated by, for example, a plaque assay method, which is a method known to those skilled in the art.

[0023] In the plaque assay, a sample is judged to have antiviral activity when the number of plaque-forming units (PFU) when the sample is added is smaller, more preferably 10 times smaller, and even more preferably 100 times smaller, than when no sample is added.

[0024] Another aspect of the present invention is the use of a protease for inactivating enveloped viruses. Another aspect is the use of a protease in the production of an antiviral agent for inactivating enveloped viruses. Another aspect is a protease used for inactivating enveloped viruses. Another aspect is a method for inactivating enveloped viruses in a subject, comprising applying a protease to the subject. Examples of applying a protease to a subject include orally ingesting the protease as a food or beverage composition to a mammal such as a human, contacting or mixing the protease with a food or beverage, or applying the protease to a textile product.

[0025] <Food and drink composition> The antiviral agent of the present invention can be orally ingested, and therefore can be incorporated into a food and drink composition.

[0026] The food and drink composition of the present invention may be, but is not limited to, a normal food, a beverage, a food additive, a functional food such as a functional food or a food for specified health uses, a supplement, etc. The supplement may be, for example, a solid supplement such as a powder, granules, tablets, or capsules; or a liquid supplement such as a solution, syrup, suspension, or emulsion.

[0027] The food and beverage composition may be in any form, such as liquid, paste, solid, or powder, and may also be in the form of tablet confectionery, liquid food, or feed. When preparing such a form, in addition to the antiviral agent of the present invention, components typically added during the production of food and beverages, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavorings, can be used. Examples of carbohydrates include common sugars such as monosaccharides, e.g., glucose and fructose; disaccharides, e.g., maltose, sucrose, and oligosaccharides; and polysaccharides, e.g., dextrin and cyclodextrin, as well as sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of flavorings that can be used include natural flavors (e.g., thaumatin, stevia extract, etc.) and synthetic flavors (e.g., saccharin and aspartame, etc.).

[0028] The food and beverage composition may further contain additives that are typically added to foods and beverages, such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, odorants, pH adjusters, and colorants.

[0029] Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and carboxymethyl cellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.

[0030] Examples of binders include gelatin, polyvinylpyrrolidone, macrogol, and the like, in addition to the above-mentioned excipients.

[0031] Examples of disintegrants include the above-mentioned excipients as well as chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and cross-linked polyvinylpyrrolidone.

[0032] Examples of lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and gaelt; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; and starch derivatives.

[0033] Examples of stabilizers include parahydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid; and CaCl.２ and divalent inorganic salts such as:

[0034] Examples of flavoring agents and odorants include sweeteners, acidulants, fragrances, etc. In the case of liquid preparations, the carrier used is not particularly limited, but examples thereof include solvents such as water.

[0035] Furthermore, as a form of food and drink composition, the composition may be incorporated into other general foods and drinks, for example, wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, and breadcrumbs; instant foods such as instant noodles, cup noodles, retort / prepared foods, canned foods, microwave foods, instant soup / stew, instant miso soup / cleaning soup, canned soup, and freeze-dried foods; processed agricultural products such as canned agricultural products, canned fruit, jams / marmalades, pickles, boiled beans, dried agricultural products, and cereals (processed grain products); processed marine products such as canned seafood, fish ham / sausage, fish paste products, marine delicacies, and tsukudani (simmered fish paste); processed livestock products such as canned livestock products / pastes, and livestock ham / sausage; milk and dairy products such as processed milk, milk drinks, yogurt, lactic acid bacteria drinks, cheese, ice cream, modified milk powder, cream, and other dairy products. basic seasonings such as soy sauce, miso, sauces, processed tomato seasonings, mirin, vinegars, and other complex seasonings and foods; frozen foods such as frozen ingredient foods, semi-cooked frozen foods, and cooked frozen foods; confectioneries such as caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice snacks, bean snacks, and dessert sweets; beverages such as carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks containing fruit juice, fruit pulp drinks, fruit drinks containing fruit particles, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, and alcoholic drinks; the antiviral agent of the present invention may be added to foods and beverages other than those listed above.

[0036] Another example of a food and beverage composition is a food cleaning treatment agent, which is used to perform antiviral treatment on food and beverages, including fresh foods, to prevent the spread of infection through food.

[0037] The preferred intake amount of the food and drink composition of the present invention is not particularly limited as long as it can exert an antiviral effect. For example, in the case of an average human weighing approximately 60 kg, the amount of serine protease per day is 0.01 mg to 10,000 mg, preferably 0.1 mg to 10,000 mg, and more preferably 1 mg to 5,000 mg, and the amount of cationic polymer is 0.01 mg to 100,000 mg, preferably 0.1 mg to 100,000 mg, and more preferably 1 mg to 50,000 mg.

[0038] The preferred timing of ingestion of the food and drink composition of the present invention is not particularly limited, and may be, for example, before a meal, after a meal, or between meals. The interval between ingestion is also not particularly limited.

[0039] The content of the antiviral agent of the present invention in the food and drink composition of the present invention is not particularly limited and may be any content as long as it has antiviral activity.

[0040] Another aspect of the present invention is the use of a serine protease, or a serine protease and a cationic polymer, in the production of a food or beverage composition. Another aspect is a method for ameliorating and / or preventing a viral infection, comprising ingesting a serine protease, a serine protease and a cationic polymer, or a food or beverage composition containing them as active ingredients.

[0041] <Other Product Forms> The antiviral agent of the present invention can also be formulated into various other forms, such as daily necessities and hygiene products. The form in which it is formulated is not particularly limited, and it can be in the form of a spray. The spray is used, for example, in the form of a spray, aerosol, or dry spray. The spray is processed into a dosage form suitable for spraying and / or ejection using a compressed gas or a pump. For example, the antiviral agent of the present invention can be dissolved in water, a buffer solution having a pH of, but not limited to, 6.0 or higher, preferably about pH 7.0 to 11.0, and more preferably about pH 7.3 to 10.3, or physiological saline, or can be suspended in a volatile solvent to form a formulation. Sprays, such as oral sprays and nasal sprays, can be administered directly to a living body, or can be applied to textile products such as face masks and clothing, or can be sprayed indoors using a humidifier or the like.

[0042] The present invention also encompasses textile products coated with the antiviral agent of the present invention. Examples of textile products include clothing, sanitary products, bedding, films, and interior goods made from synthetic fibers, regenerated fibers, natural fibers, or blends thereof. Examples of textile products include, but are not limited to, shirts, coats, trousers, skirts, nightwear, underwear, socks, gloves, hats, white coats, nursing wear, care wear, work clothes, face masks, sheets, pillowcases, futons, cushions, wallpaper, various filters, disinfecting paper, kitchen paper, cleaning cloths such as mops, curtains, and carpets. The textile product of the present invention may be prepared by applying the antiviral agent of the present invention to the textile product or fibers before processing by spraying, dipping, impregnation, coating, or other methods.

[0043] When the antiviral agent of the present invention is formulated into various products (including food and beverage compositions), it can be used in combination with any suitable ingredient as long as the effects of the present invention are not impaired. Such combined ingredients may be appropriately selected depending on the dosage form of the product, and examples thereof include water; lower alcohols having 5 or less carbon atoms, such as ethanol and isopropanol; polyhydric alcohols, such as glycerin, polyethylene glycol, butylene glycol, propylene glycol, dipropylene glycol, and sorbitol; glycine, organic acids, glycerin fatty acid esters, sucrose fatty acid esters, sodium benzoate, sodium sorbate, sodium propionate, sodium dehydrogenase, parahydroxybenzoate esters, and sodium sulfite. Examples of antibacterial agents that can be used in combination include, but are not limited to, thorium, EDTA, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, alkyldiaminoethylglycine chloride, iodine tincture, povidone-iodine, cetyl benzalkonium oxide, triclosan, chloroxylenol, isopropylmethylphenol, lactoferrin, nisin, bacteriocin, Aralia udo extract, Styrax japonica extract, Artemisia capillaris extract, enzymatically hydrolyzed Job's tears extract, Shirako protein extract, thujaplicin, and pectin hydrolysate. Among these, at least one selected from the group consisting of water, lower alcohols, and polyhydric alcohols is preferred as a co-component, and from the viewpoint of being able to also impart antibacterial properties, at least one selected from the group consisting of lower alcohols and polyhydric alcohols is more preferred.

[0044] Other surfactants include anionic surfactants such as sodium fatty acid, sodium alpha sulfo fatty acid ester, sodium linear alkylbenzene sulfonate, and sodium alkyl sulfate; cationic surfactants such as alkyltrimethylammonium salts and dialkyldimethylammonium salts; nonionic surfactants such as polyoxyethylene alkyl ethers and polyoxyethylene alkylphenyl ethers; amphoteric surfactants such as sodium alkylamino fatty acid, alkyl betaine, and alkylamine oxide; and various surfactants added for the purposes of solubilization, emulsification, dispersion stabilization, cleaning, and sterilization, as well as citric acid, sodium citrate, sodium hydroxide, and triethanolamine. Other preferred additives that can be used in combination include pH adjusters such as dimethylamine, antioxidants such as tocopherol acetate, thickeners such as carboxyvinyl polymers and hydroxyethyl cellulose, aminocarboxylic acid chelating agents such as ethylenediaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, hydroxyethylethylenediaminetriacetic acid, triethylenetetraminehexaacetic acid, and cyclohexanediaminetetraacetic acid, phosphonic acid chelating agents such as hydroxyethylidene diphosphonic acid, nitrilotris(methylenephosphonic acid), phosphonobutanetricarboxylic acid, and ethylenediaminetetramethylenephosphonic acid, and amine chelating agents such as triethanolamine and polyamines.

[0045] The present invention will be described in more detail below with reference to examples, but it goes without saying that the scope of the present invention is not limited to the examples.

[0046] Example 1: Anti-influenza virus activity test using protease (evaluation by plaque assay) Influenza A virus (Influenza virus / A / NWS / 33 (H1N1 subtype); Denka Seiken Co., Ltd.) was diluted to a predetermined concentration relative to cells. The diluted virus solution was added to MDCK cells whose surfaces had been washed with phosphate-buffered saline (PBS(-) (pH 7.4)). Five mL of cell growth medium (Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum, Nakarai Techno) was added, and the cells were incubated with CO until cytopathic effect (CPE) was confirmed. ２The incubator was maintained at 37°C and 5% CO ２ The cells were cultured for 2 to 3 days under the conditions listed above. The suspension was collected in a 50 mL conical tube and centrifuged (3,000 rpm, 10 minutes) to pellet the cells. The supernatant obtained by centrifugation was dispensed into vials as virus stock solutions and stored at -85°C.

[0047] Subtilisin (Amano Enzyme "Protin SD-AY10", composition (weight percent): enzyme approximately 15%; dextrin approximately 85%) was prepared in phosphate buffered saline (PBS(-) (pH 7.4)) to the specified final concentration (volume percent concentration) for each test group in the table. The cationic polymers used were ε-polylysine (JNC Corporation, Lot No. 2221101, weight-average molecular weight 4700, weight percent concentration 25%), polyethyleneimine (Junsei Chemical Co., Ltd., Product No. 2018I1645, molecular weight approximately 1200), protamine sulfate (Fujifilm Wako Pure Chemical Corporation, Product No. 168-05192, salmon-derived), and DEAE-dextran hydrochloride (Sigma-Aldrich, Product No. 29100346). Each was prepared in purified water, adjusting the pH to 8.5 or higher with hydrochloric acid or sodium hydroxide to the specified final concentration (volume percent concentration) for each test group in the table. The prepared protease solution or a mixture of the protease solution and cationic polymer solution was used as a sample for the following tests. Specifically, test groups were prepared as examples using a protease solution alone or a mixture of a protease solution and a cationic polymer solution, and as a comparative example using a cationic polymer solution alone. In particular, in the test group designed for soaking and washing, the contact time between the virus and the prepared protease solution or the mixture of the protease solution and the cationic polymer solution was set to a long time of 60 minutes.

[0048] 0.5 mL of the virus stock solution was added to 0.5 mL of the sample, and the mixture was left to stand at room temperature for the time indicated in the table. After that, the mixture was diluted 10 times with cell growth medium (Eagle medium supplemented with 10% fetal bovine serum, Nakarai Techno), and then diluted 10 times with the same 10% ２ From 10 ３The solution was serially diluted with phosphate buffered saline (PBS(-) (pH 7.4)) up to 1:1.

[0049] Using a cell growth medium, host cells (MDCK cells) were cultured in a 35 x 10 mm cell culture dish (FALCON 353001) at approximately 4 x 10 cells per dish. ５ 100 cells were seeded onto the 100 cells / well plate. ２ The incubator was maintained at 38°C and 5% CO ２ The cells were cultured for 1 to 2 days under the conditions of 0.5% agarose solution (Agarose-ME Classic Type, Nacalai Tesque) and 0.01 mg / mL acetyltrypsin in Eagle's minimum essential medium. 0.1 mL of each serial dilution was inoculated onto one dish of cells, cultured at room temperature of 25°C for 1 hour, and then layered with the previously prepared culture medium. ２ The incubator was maintained at 38°C and 5% CO ２ After culturing for two days under the conditions, the plates were fixed with 10% neutral buffered formaldehyde and stained with gentian purple (crystal violet) solution, and the number of plaques was measured. The measured number of plaques was applied to the formula ((number of plaques per dish / volume of measurement sample per dish (mL)) x dilution factor = PFU / mL) to calculate the virus titer (PFU / mL), and the antiviral activity value (LogΔPFU / mL) was calculated from the difference from the untreated control. This test was performed twice for each condition, and the average value was recorded.

[0050] As shown in Table 1, it was found that the application of protease significantly reduced the virus titer. This indicates that protease alone has the effect of inactivating influenza viruses. Furthermore, as shown in Table 2, when protease was used in combination with a cationic polymer, the virus titer tended to be further reduced. This indicates that cationic polymers enhance the influenza virus inactivation effect of proteases. The enhancing effect of cationic polymers is concentration-dependent, and the concentration and ratio can be selected depending on the application, type of target virus, the required degree of effect, and the type of cationic polymer. Furthermore, as shown in Table 3, when sufficient contact time with the target object is ensured, such as in soaking or pack washing, sufficient effect can be obtained even at a lower added concentration, so the concentration can be selected depending on the application.

[0051]

[0052]

[0053]

[0054] Example 2: Anti-mumps virus activity test using protease (evaluation by plaque assay) Mumps virus (EY strain; Toyama Prefectural Institute of Public Health) was diluted to a predetermined concentration relative to the cells. The diluted virus solution was added to Vero cells whose surfaces had been washed with phosphate-buffered saline (PBS(-) (pH 7.4)). Five mL of cell growth medium (Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum, Nakarai Techno) was added, and the cells were incubated with CO until cytopathic effect (CPE) was confirmed. ２ The incubator was maintained at 38°C and 5% CO ２ The cells were cultured for 2 to 3 days under the conditions listed above. The suspension was collected in a 50 mL conical tube and centrifuged (3,000 rpm, 10 minutes) to pellet the cells. The supernatant obtained by centrifugation was dispensed into vials as virus stock solutions and stored at -85°C.

[0055] The proteases used were subtilisin (Amano Enzyme "Protin SD-AY10," composition (weight percent): approximately 15% enzyme; approximately 85% dextrin, and Nagase ChemteX "Bioprase® OP," composition (weight percent): 10-25% enzyme; 75-90% dextrin), nattokinase (Fujifilm Wako Pure Chemical Industries "Nattokinase," composition (weight percent): 100% nattokinase), and Origin (Amano Enzyme "Protease P 'Amano' 3SD," composition (weight percent): approximately 30% enzyme). Each protease solution and each cationic polymer solution were prepared in the same manner as in Example 1. Experimental samples included the protease solution alone and a mixture of the protease solution and the cationic polymer solution, while comparative experiments included the cationic polymer solution alone.

[0056] 1 mL of virus stock solution was added to 1 mL of sample, and the mixture was left to stand at room temperature for the specified time in the table. After that, the mixture was diluted 10 times with cell growth medium (Eagle medium supplemented with 10% fetal bovine serum, Nakarai Techno), and then diluted 10 times with the same 10% ２ From 10 ３ The solution was serially diluted with phosphate buffered saline (PBS(-) (pH 7.4)) up to 1:1.

[0057] Using a cell growth medium, host cells (Vero cells) were cultured in a 35 x 10 mm cell culture dish (FALCON 353001) at approximately 4 x 10 cells per dish. ５ 100 cells were seeded onto the 100 cells / well plate. ２ The incubator was maintained at 37°C and 5% CO ２ The cells were cultured for 1 to 2 days under the conditions of 0.8% methylcellulose in Eagle's minimum essential medium. 0.1 mL of each serial dilution was inoculated onto one dish of cells, cultured at room temperature of 25°C for 1 hour, and then layered with the previously prepared medium. ２ The incubator was maintained at 38°C and 5% CO ２After culturing for one day under the conditions, the plates were fixed with 10% neutral buffered formaldehyde and stained with gentian purple (crystal violet) solution, and the number of plaques was measured. The measured number of plaques was applied to the formula ((number of plaques per dish / volume of measurement sample per dish (mL)) x dilution factor = PFU / mL) to calculate the virus titer (PFU / mL), and the antiviral activity value (LogΔPFU / mL) was calculated from the difference with the untreated control. This test was performed twice for each condition, and the average value was recorded.

[0058] As shown in Table 4, it was found that the application of protease significantly reduced the virus titer. This indicates that protease alone has the effect of inactivating the mumps virus. Furthermore, as shown in Table 5, when protease was used in combination with a cationic polymer, the virus titer tended to be further reduced. This indicates that the cationic polymer enhances the mumps virus inactivation effect of protease.

[0059]

[0060]

[0061] Example 3: Anti-herpes simplex virus activity test using protease (evaluation by plaque assay) Herpes simplex virus (Herpes simplex virus type 1 (KOS strain); Toyama Prefectural Institute of Public Health) was diluted to a predetermined concentration relative to the cells. The diluted virus solution was added to Vero cells whose surfaces had been washed with phosphate-buffered saline (PBS (-) (pH 7.4)). Five mL of cell growth medium (Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum, Nakarai Techno) was added, and the cells were incubated with CO until cytopathic effect (CPE) was confirmed. ２ The incubator was maintained at 38°C and 5% CO ２ The cells were cultured for 2 to 3 days under the conditions listed above. The suspension was collected in a 50 mL conical tube and centrifuged (3,000 rpm, 10 minutes) to pellet the cells. The supernatant obtained by centrifugation was dispensed into vials as virus stock solutions and stored at -85°C.

[0062] Each protease solution and each cationic polymer solution was prepared in the same manner as in Example 2, and test plots were prepared using the protease solution alone and a mixture of the protease solution and the cationic polymer solution as samples for the example, and a test plot for the comparative example using the cationic polymer solution alone as sample. In particular, in the test plots designed for soaking and washing, the contact time between the virus and the prepared protease solution or the mixture of the protease solution and the cationic polymer solution was set to a long time of 30 minutes.

[0063] 1 mL of virus stock solution was added to 1 mL of sample, and the mixture was left to stand at room temperature for the specified time in the table. After that, the mixture was diluted 10 times with cell growth medium (Eagle medium supplemented with 10% fetal bovine serum, Nakarai Techno), and then diluted 10 times with the same 10% ２ From 10 ３ The cells were serially diluted with phosphate buffered saline (PBS(-) (pH 7.4)) up to 1:1. The host cells (Vero cells) were cultured in advance in a cell growth medium in a 35 x 10 mm cell culture dish (FALCON 353001) at a concentration of approximately 4 x 10 per dish. ５ 100 cells were seeded onto the 100 cells / well plate. ２ The incubator was maintained at 37°C and 5% CO ２ The cells were cultured for 1 to 2 days under the conditions of 0.8% methylcellulose in Eagle's minimum essential medium. 0.1 mL of each serial dilution was inoculated onto one dish of cells, cultured at room temperature of 25°C for 1 hour, and then layered with the previously prepared medium. ２ The incubator was maintained at 35°C and 5% CO ２ After culturing for 1 or 2 days under the culture conditions, the plates were fixed with 10% neutral buffered formaldehyde and stained with gentian purple (crystal violet) solution, and the number of plaques was measured. The measured number of plaques was applied to the formula ((number of plaques per dish / volume of measurement sample per dish (mL)) x dilution factor = PFU / mL) to calculate the virus titer (PFU / mL), and the antiviral activity value (LogΔPFU / mL) was calculated from the difference from the untreated control. This test was performed twice for each condition, and the average value was recorded.

[0064] As shown in Table 6, the application of protease significantly reduced virus titer. This indicates that protease alone has the effect of inactivating herpes viruses. Furthermore, as shown in Table 7, the combined use of protease and cationic polymer tended to further reduce virus titer. This indicates that cationic polymer enhances the herpes virus inactivation effect of protease. The enhancing effect of the cationic polymer is concentration-dependent, and the concentration and ratio can be selected depending on the application, type of target virus, desired level of effect, and type of cationic polymer. Furthermore, as shown in Table 8, when sufficient contact time with the target object is ensured, such as in soaking or pack washing, sufficient effect can be obtained even at a lower concentration, so the concentration can be selected depending on the application.

[0065]

[0066]

[0067]

[0068] The present invention provides a novel antiviral agent that exhibits an inactivating effect against enveloped viruses, is highly safe, can be applied to hands or can be taken orally, and is therefore extremely useful industrially.

Claims

1. An antiviral agent containing a protease for use against enveloped viruses.

2. The antiviral agent of claim 1, wherein the protease is a serine protease.

3. The antiviral agent according to claim 2, wherein the serine protease is produced by bacteria selected from the group consisting of bacteria belonging to the genus Bacillus, bacteria belonging to the genus Flavobacterium, bacteria belonging to the genus Arthrobacter, bacteria belonging to the genus Streptomyces, bacteria belonging to the genus Geobacillus, bacteria belonging to the genus Aspergillus, and bacteria belonging to the genus Rhizopus.

4. The antiviral agent according to claim 3, wherein the bacterium belonging to the genus Bacillus is selected from the group consisting of Bacillus licheniformis, Bacillus clausii, Bacillus polymyxa, Bacillus subtilis, Bacillus alcalophilus, and Bacillus amyloliquefaciens, and the bacterium belonging to the genus Geobacillus is Geobacillus stearothermophilus.

5. The antiviral agent according to claim 2, wherein the serine protease is a protein having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, or a protein having an amino acid sequence that is 90% or more identical to an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7 and that has antiviral activity.

6. The antiviral agent according to claim 2, wherein the serine protease is subtilisin, nattokinase, or oryzen.

7. The antiviral agent according to claim 2, wherein the serine protease is trypsin.

8. The antiviral agent of claim 1, further comprising a cationic polymer.

9. The antiviral agent according to claim 8, wherein the cationic polymer is polylysine or polyethyleneimine.

10. The antiviral agent of claim 1, further comprising alcohol.

11. The antiviral agent according to any one of claims 1 to 10, wherein the enveloped virus is a mumps virus, a herpes virus, or an influenza virus.

12. The antiviral agent according to any one of claims 1 to 10, which is in the form of a spray.

13. A textile product coated with the antiviral agent according to any one of claims 1 to 10.

14. A food or beverage composition containing the antiviral agent according to any one of claims 1 to 10.

15. A food cleaning treatment agent containing the antiviral agent according to any one of claims 1 to 10.

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