JP6709030B2 - Anti-norovirus agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to an anti-norovirus agent using an extract of chestnut astringent skin, which has an excellent effect on norovirus, and an anti-norovirus composition containing the anti-norovirus agent and useful for disinfecting norovirus and preventing infection.

Norovirus is a single-stranded positive-stranded RNA virus with a non-envelope plus diameter of about 30 nm classified into the Caliciviridae genus Norovirus, and was discovered in the US in 1963 as a pathogen of food poisoning and acute gastroenteritis. Since then, it has been detected all over the world. Looking at the number of outbreaks of food poisoning due to norovirus in Japan over the past 6 years, the number of outbreaks was about 270 each year (about 18% of all food poisoning cases), and the number of patients was about 10,000 each year (about 30% of all food poisoning cases). Has become. As the source of infection, there are infections from foodstuffs such as oysters that cause clams and secondary infections from patients' vomit/excretion, and the latter is a major cause of the spread of infection. Moreover, the infectivity of norovirus is extremely strong, and it is said that 10 or more viruses are also infected.

Currently, there are no vaccines or remedies for Norovirus, and the only countermeasure is to prevent Norovirus infection by heating the causative food and disinfecting cookware and hands. However, benzalkonium chloride and ethanol, which are currently widely used as antibacterial agents in food processing facilities such as restaurants, lunch facilities, and factories, have not been found to have an effect on norovirus, and therefore chlorine bleach (next Sodium chlorite, etc.), iodine agents (povidone iodine, etc.), aldehyde agents (glutaral, etc.), etc. are considered to be effective to some extent. However, it is not appropriate to use these chemicals for the fingers and the cooking utensils of the worker from the viewpoint of safety for the human body, and it is impossible to directly contact the food with them.

Against this background, there is a strong demand for cleaning agents and disinfectants having excellent anti-norovirus properties that can keep workers' hands and cooking utensils clean in a safe manner. To date, few ingredients that are highly effective and highly safe for the human body have been found, and the development of such ideal cleaning agents has not progressed. As an example, as an antiseptic solution that promotes safety and gentleness to the hand skin as well as the inactivating effect of norovirus, it contains 0.05 to 0.5% by weight of polyhexamethylene biguanide compound and 40 to 40% of alcohol. A disinfecting solution containing 80% by weight and having a pH adjusted to a range of 9 to 12 has been proposed (Patent Document 1). Further, as another example, Patent Document 2 describes an anti-norovirus agent containing an extract of a plant of the genus Diospyros containing tannin as an active ingredient. A plant extract of the genus Diospyros is specifically an oyster extract (oyster tannin), and since this oyster extract is recognized as a food additive, the anti-norovirus agent described in Patent Document 2 is It does not matter even if it adheres to food and tableware and is eaten.

On the other hand, chestnut, which is a deciduous tree of the genus Chrysanthemum, is distributed in warm and humid regions such as Asia, Europe, and North America, and there are species such as Japanese currant, cinnabar, European currant and American currant. The Japanese ginseng distributed in Japan is classified into more than 200 varieties when classified in a lower rank. Chestnuts have a hard demon skin on the outside and a thin astringent skin on the inside (hereinafter sometimes referred to as "chestnut astringent skin"). Chestnut astringent skin contains 1.5 to 2.5 times more active ingredients such as condensed tannins and polyphenols that are effective for the human body than tea leaves and persimmon leaves. Development of products utilizing these active ingredients Cases are scattered.

For example, Patent Document 3 describes that the chestnut astringent skin extract has a body fat reduction promoting action, and specifically reduces fat accumulated in adipocytes, particularly fat accumulated in visceral cells. It is described that it has an action.

Further, for example, Patent Document 4 describes that an extract of chestnut astringent skin has a glycation inhibitory effect, and the extract of chestnut astringent skin prevents or prevents diabetic complications, arteriosclerosis, Alzheimer's disease, aging and inflammatory reaction or the like. It is stated that it can be used for therapeutic purposes.

In addition, for example, Patent Document 5 describes that an extract of chestnut astringent skin has an antioxidant effect, and an extract of chestnut astringent skin prevents the occurrence of adult diseases such as cancer, arteriosclerosis, ischemic disease, and diabetes. It is described that it is expected to prevent various diseases caused by the production of oxidized lipids, prevent aging, have a whitening effect, reduce hangover, and recover fatigue from sports.

Further, Patent Document 6 describes a norovirus-killing composition containing proanthocyanidins, and specifically, it is described that proanthocyanidins, which are grape seed-derived polyphenols, have norovirus-killing activity. Note that proanthocyanidins have a different chemical structure from condensed tannins. For example, proanthocyanidins produce anthocyanidins by hydrolysis, but condensed tannins are flavonoids having carbon-carbon bonds and are not hydrolyzed.

JP, 2007-045732, A WO2008/153077 Re-table 2007-111242 gazette JP 2012-171909 A JP, 2006-131916, A JP, 2013-047196, A

However, as described above, Patent Document 2 discloses an anti-norovirus agent containing tannin contained in an oyster extract as an active ingredient, but there is no description that the chestnut astringent skin is effectively used. In addition, as described above, there is a demand for a natural extract other than the oyster extract that is highly safe to the human body. And, although the above-mentioned Patent Documents 3 to 5 describe the predetermined efficacy of the chestnut astringent skin extract, the anti-norovirus effect is not described at all. In Patent Document 3, there is a line describing that the chestnut astringent skin extract can be expected to be applied to the suppression of microbial growth. However, in Patent Document 3, there is no description of an example of suppressing microbial growth, let alone in the environment. It has not even been suggested to describe the effect on norovirus which is durable and difficult to remove with most disinfectants and bactericides. Patent Document 6 describes a norovirus-killing composition consisting of proanthocyanidins contained in a grape extract, but Patent Document 6 does not describe chestnut astringent skin, but describes at all the anti-norovirus effect of the chestnut astringent skin extract. However, in the first place, the present invention uses components other than tannin contained in the chestnut astringent skin extract as active components having an anti-norovirus effect, as will be shown in Examples of the present application.

As described above, it has been earnestly desired to provide a composition having excellent anti-norovirus properties, but on the other hand, the development of a composition to which the active ingredient of chestnut astringent skin extract is applied is not sufficient. The present invention has been made in view of such problems, and an object thereof is to provide a safe and effective anti-norovirus agent and an anti-norovirus composition.

The anti-norovirus agent according to the present invention is characterized by containing a chestnut astringent skin extract as an active ingredient.

Further, the anti-norovirus composition according to the present invention comprises the anti-norovirus agent according to the present invention and at least one component selected from the group consisting of alcohols, surfactants, antibacterial agents, moisturizers and cosmetic fats and oils. It is characterized by containing.

According to the present invention, a safe and effective anti-norovirus agent and a composition for anti-norovirus can be obtained. The anti-norovirus properties of the chestnut astringent skin extract used as the anti-norovirus agent of the present invention are far superior to those of conventional fungicides and the like. The anti-norovirus agent of the present invention is not only a substance that can be used alone as it is for disinfection or infection prevention of norovirus, but also an alcohol preparation, a cleaning composition, a hand soap, a disinfecting composition, a lotion. It is also extremely useful as an active ingredient of a composition such as a milky lotion, a cream, or a drug against norovirus.

Further, since chestnut astringent skin extract is recognized as a food additive, all components of the composition for anti-norovirus of the present invention are composed of foods or food additives, and adhered to foods/tableware and taken in the mouth. Even if the composition does not matter, it becomes possible. By using the composition for anti-norovirus of the present invention containing chestnut astringent skin extract as an active ingredient, it becomes possible to efficiently disinfect and prevent infection of norovirus under conditions where food is handled or in medical institutions. The occurrence of diseases (food poisoning) caused by norovirus can be significantly suppressed.

It is a figure which shows the bacteriophage MS2 inactivating effect of the chestnut astringent skin extract concerning this Example. It is a figure which shows the norovirus inactivating effect of the chestnut astringent skin extract concerning this Example. FIG. 3 is a graph showing the bacteriophage MS2 inactivating effect of a decolorized chestnut astringent skin extract in the case of adding an organic acid. FIG. 6 is a view showing the bacteriophage MS2 inactivating effect of a decolorized chestnut astringent skin extract in the case of adding an organic acid. It is a figure which shows the norovirus inactivating effect of the chestnut astringent skin extract in the case of adding an organic acid. It is a figure which compares and shows each bacteriophage MS2 inactivating effect of a chestnut astringent skin extract and a persimmon astringent extract.

Hereinafter, an embodiment of the present invention will be specifically described with reference to the accompanying drawings, but the embodiment is for facilitating the understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

1. <Anti-Norovirus agent>
The anti-norovirus agent according to the present invention is characterized by containing a chestnut astringent skin extract as an active ingredient. In the present invention, "anti-norovirus (activity)" means at least either to kill the norovirus or to act on the surface protein of the norovirus even if it remains to lose the infectious ability (proliferative ability). Including one, it has the property of inactivating (ie, "disinfecting") norovirus.

The chestnut to be extracted is not particularly limited, and for example, any of Japanese chestnut, western chestnut, Chinese chestnut or American chestnut can be used, but Japanese chestnut is preferable. The variety of chestnuts is not particularly limited, and examples thereof include Morishaya, Tamatsukuri, Tanzawa, Izumo, Ginyose, Rihei, Tsukuba chestnut or Kunimi chestnut, but Tsukuba chestnut or Kunimi chestnut are preferable.

In the present invention, the astringent skin collected from the raw chestnut without the demon skin may be used as it is or the dried astringent skin may be used. Alternatively, astringent skin recovered from baked, heated, frozen, dried, etc. chestnuts or boiled ones may be used. For extraction with a solvent or the like, it is preferable to use finely crushed astringent skin.

The extract from astringent skin can be prepared using various extraction methods such as solvent extraction, enzymatic decomposition, compression extraction, centrifugation, and supercritical extraction. In particular, it is preferable to use solvent extraction because of the simplicity of the operation.

The solvent used for solvent extraction may be any solvent that is acceptable under the Food Sanitation Law, and water, hydrophilic organic solvents, acetone, hexane and the like can be used. Examples of the hydrophilic organic solvent include lower alcohols such as methanol, ethanol, propanol, isopropanol and butanol, polyhydric alcohols such as propylene glycol and glycerin, a mixed solution thereof or a mixed solution thereof with water. .. It is preferable to use ethanol or a mixed solution of water and ethanol. The concentration of ethanol can be set in the range of 10% by volume to 100% by volume, preferably 30% by volume to 60% by volume.

The extraction treatment is carried out by adding 0.1 to 5 parts by weight of an extraction solvent to 1 part by weight of chestnut astringent skin and stirring at room temperature or under normal pressure or pressure, if necessary, for several minutes to several days. Preferably for about 5 minutes to 30 hours. The extraction may be either batch type or reflux type. Further, the temperature control at the time of extraction does not have to be particularly strict, but it is possible to set the temperature in the range of approximately 20 to 70°C.

In order to efficiently obtain the chestnut astringent skin extract according to the present invention, the insoluble matter is removed by filtration or centrifugation after extraction with hydrous ethanol or acetone, and the chestnut astringent skin extract according to the present invention is recovered as it is. Alternatively, a soluble fraction obtained by further fractionating the extract with methanol or the like is used. Further, the chestnut astringent skin extract according to the present invention can be obtained by removing the solvent from the extract by a known means such as vacuum distillation, spray drying and freeze drying.

The concentration of the chestnut astringent skin extract according to the present invention is not particularly limited as long as it has anti-norovirus properties, but is, for example, 5 to 30% (w/v), preferably 10 to 20% ( w/v). Here, the chestnut astringent skin extract concentration is obtained by adding chestnut astringent skin powder to 50% ethanol, stirring the mixture for 10 minutes, and then extracting by centrifugation and filtration, as will be described in Examples below. In, the extraction conditions for defining the concentration of chestnut astringent skin extract are defined by such conditions.

In the present invention, the chestnut astringent skin extract is preferably decolorized with a decolorizing agent. Chestnut astringent skin extract has an amber color due to the contained catechins, but the commercial value may be reduced with surfactants, antibacterial agents, etc. that use the chestnut astringent skin extract as it is colored. .. For example, when the colored chestnut astringent skin extract is used for disinfecting a cookware, the cookware is colored even if the norovirus attached to the cookware can be removed. Further, for example, when the colored chestnut astringent skin extract is used for hand disinfection, the hands are colored even if the norovirus attached to the hands can be removed.

Therefore, in order to lose the color of the chestnut astringent skin extract, the chestnut astringent skin extract is decolorized with a decolorizing agent. The decolorizing agent is not particularly limited, for example, silica gel, ion exchange resin, column chromatography using an adsorbent such as activated carbon, or contact treatment with polyvinyl polypyrrolidone (PVPP), etc. can be used, preferably Decolorization with activated carbon.

2. <Composition for anti-norovirus>
The composition for anti-norovirus according to the present embodiment is at least one component selected from the group consisting of the anti-norovirus agent according to the present invention described above, an alcohol, a surfactant, an antibacterial agent, a moisturizer, and a fat and oil for cosmetics. Contains and. The composition for anti-norovirus according to the present embodiment is a composition further containing an organic acid such as citric acid and/or a salt thereof or vitamin Cs, if necessary.

The aspect is not particularly limited, but typically includes the following.
-Alcohol preparation containing at least anti-norovirus agent and alcohol-Cleaning composition containing at least anti-norovirus agent and surfactant-Disinfecting composition containing at least anti-norovirus agent and antibacterial agent-At least anti-norovirus Lotion, emulsion or cream containing a moisturizer and a moisturizer and/or cosmetic oil and fat should be used to remove stains on food, tableware, cooking utensils, workers' hands and clothes, and disinfect norovirus. The composition of the present invention can be provided as, for example, a liquid or solid detergent. Embodiments in which the alcoholic preparation and the disinfecting composition are used to inactivate norovirus and bacteria attached to food, tableware, cooking utensils, workers' hands, or devices handling norovirus patient's waste, etc. And is provided as a propellant similar to a conventional ethanol preparation, for example. Lotions, creams and emulsions are compositions (basic cosmetics) that can be applied to the hands and fingers of workers who are liable to be roughened by water work or the like for skin care and disinfection of norovirus.

In the composition for anti-norovirus of the present invention, two or more kinds of alcohols, surfactants, bactericides, moisturizers, and cosmetic fats and oils may be used in combination. For example, for alcohol preparations, it is also preferable to further mix a surfactant such as a fatty acid ester in order to further improve antibacterial properties. Further, the cleaning composition can take a mode such as a hand soap containing a bactericide or alcohol in addition to the surfactant, and the cream keeps the hand skin clean together with the component for protecting the hand skin. It is possible to adopt a mode in which an antibacterial agent and alcohol are mixed.

The anti-norovirus composition of the present invention, various components for imparting desired performance and enhancing the quality of each composition, such as a thickener (xanthan gum, locust bean gum, sodium polyacrylate, etc.), antioxidant , Cosmetics such as fragrances and pigments, and lotions and the like can be appropriately blended with an anti-skin roughening agent, an anti-inflammatory agent and the like.

The content of the anti-norovirus agent in the composition for anti-norovirus of the present invention can be appropriately adjusted depending on aspects such as the component constitution and the method of use of the composition in the range where the anti-norovirus property is expressed. It is, for example, 5 to 30% (w/v), preferably 10 to 20% (w/v) based on the entire composition for norovirus.

The composition for anti-norovirus of the present invention preferably contains an organic acid and/or a salt thereof in addition to the above-mentioned anti-norovirus agent. The organic acid and/or salt thereof is preferably an organic acid having 2 to 10 carbon atoms and/or a salt thereof, more preferably a hydroxyl group-containing organic acid having 2 to 10 carbon atoms and/or a salt thereof, and more specifically Specific examples thereof include organic acids such as lactic acid, malic acid, citric acid, tartaric acid, salicylic acid, maleic acid, fumaric acid and succinic acid, and/or salts thereof. Among them, at least one organic acid selected from the group consisting of lactic acid, malic acid, citric acid, tartaric acid and salicylic acid and/or a salt thereof, particularly citric acid or a salt thereof is preferable. As the salt of the organic acid, sodium salts, potassium salts and the like of each of the above organic acids (for example, trisodium citrate) are preferably used. Organic acids such as citric acid and/or salts thereof do not show anti-norovirus activity by themselves, but their effects can be further enhanced by using them in combination with the chestnut astringent skin extract.

The amount of the above organic acid and/or salt thereof added is preferably 0.05 to 5.0% by weight, based on the entire composition for anti-norovirus (including solvent etc.), and 0.1 to 2. A ratio of 0% by weight is more preferable. Further, when the organic acid and/or its salt is used for the composition of the aqueous solution, it is preferable to use it in such an amount that the pH becomes 2 to 6.

In addition to the anti-norovirus agent described above, it is preferable to add vitamin Cs, which are known as antioxidants added to foods, to the composition for anti-norovirus of the present invention. Here, "vitamin Cs" is a general term that includes DL-ascorbic acid, ascorbic acid ester (palmitic acid ester, etc.), etc. in addition to L-ascorbic acid generally called vitamin C. The amount of vitamin C added is preferably 0.01 to 3.0% by weight, and preferably 0.05 to 1.0% by weight, based on the entire composition for anti-norovirus (including solvent etc.). Is more preferable.

As the alcohol, those similar to general alcohol preparations can be used, but ethanol and/or propanol, which has excellent antibacterial properties against bacteria and is recognized as a food additive, is preferable. The concentration of these alcohols may be the same as that of a general alcohol preparation and can be adjusted in consideration of antibacterial properties, but it is preferably about 20 to 80% with respect to the entire alcohol preparation. Alcohol may be used as a solvent in compositions other than alcoholic preparations, and may be added to cosmetics and the like as a component that brings astringency and antiseptic properties to the skin.

There are cationic, anionic, amphoteric, and nonionic surfactants, and in the present invention, it is preferable to use anionic and/or nonionic surfactants.

Examples of the anionic surfactant include soap (alkaline salt of higher fatty acid), monoalkyl sulfate, alkyl polyoxyethylene sulfate, alkylbenzene sulfonate, monoalkyl phosphate and the like.

Examples of nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, fatty acid partial esters of polyhydric alcohols (glycerin, sugar alcohols, etc.), and fatty acid diethanolamides.

Among these surfactants, those recognized as food additives such as glycerin fatty acid partial ester, sorbitan fatty acid partial ester, and sucrose fatty acid partial ester do not pose a problem even if they adhere to food, tableware, and cooking utensils. Is the preferred surfactant in the present invention.

The above-mentioned surfactants also have an action of destroying cell membranes of bacteria and envelopes of viruses. For example, a partial ester of glycerin with a fatty acid having 6 to 18 carbon atoms has antibacterial properties against Escherichia coli and Staphylococcus aureus. Since it is excellent, it is also preferable to add these surfactants to the above-mentioned alcohol preparation. Further, the surfactant is also used as a component for mixing the oil phase and the water phase in the cream or emulsion.

Antibacterial agents (including substances called bactericides and disinfectants) other than the above-mentioned ethanol, citric acid and the like that can be used in the present invention are not particularly limited, but for example, similar to Norovirus. Those which are effective against Escherichia coli, Staphylococcus aureus, MRSA, Salmonella, Vibrio parahaemolyticus, Pseudomonas aeruginosa, etc. which cause infections during food processing or nosocomial infections are preferable. Something like.
・Natural antibacterial agents: proteins (white protein, egg white lysozyme, etc.), peptides (polylysine, etc.), etc.;
・Antibiotics: Penicillin antibiotics, chloramphenicol, streptomycin, tetracycline antibiotics, cephalosporin antibiotics, etc.;
・Synthetic antibacterial agents...Chlorine compounds (Triclosan, etc.), Iodine compounds (Popidone iodine, etc.), Zinc compounds (Cetylpyridinium zinc, etc.), Benzenecarboxylic acids (benzoic acid, salicylic acid, isopropylmethylphenol, butyl parahydroxybenzoate (=butyl) Paraben), organic acid esters (glycerin ester, sucrose ester, etc.), aldehydes (glutaraldehyde, formaldehyde, etc.), biguanide compounds (chlorhexidine gluconate, etc.), quaternary ammonium salts (benzalkonium chloride, Cetyl ammonium bromide etc.) etc.

Of these antibacterial agents, isopropylmethylphenol, butylparahydroxybenzoate, triclosan, etc. are preferable because of their excellent antibacterial action.

The substances used as antibacterial agents as described above may be blended in the above-mentioned ethanol preparation, and may be used as preservatives in cosmetics and the like.

The moisturizers (wetting agents) that can be used in the present invention are the same as those used in cosmetics such as general lotions, emulsions and creams, and include, for example, glycerin, propylene glycol, sorbitol, polyethylene glycol, hyalurone. Acid, sodium chondroitin sulfate, ceramide, aloe extract and the like can be mentioned. Among these moisturizers, those recognized as foods or food additives such as aloe extract are preferred moisturizers in the present invention in that they do not pose a problem even if they adhere to foods, tableware and cooking utensils.

The oils and fats for cosmetics are components for forming a film on the skin surface to play a role of protecting the skin, imparting flexibility and lubricity, and imparting an appropriate feeling of use to cosmetics. In the present invention, the same cosmetic oils and fats as those used in general cosmetics such as emulsions and creams can be used, and examples thereof include the following.
・Fats and oils (esters of higher fatty acids and glycerin)... Vegetable fats and oils, animal fats and oils or hydrogenated products thereof (partially hydrogenated rapeseed oil, etc.), synthetic triglycerides (tri(capryl/capric acid)glyceryl, etc.), etc.;
Wax (ester of higher fatty acid and higher alcohol that is solid at room temperature)... Vegetable wax, animal wax (beeswax, lanolin, etc.), etc.;
・Hydrocarbons: mineral hydrocarbons (liquid paraffin, vaseline, paraffin, etc.), animal hydrocarbons (squalane, etc.), etc.;
・Higher fatty acid: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, etc.;
・Higher alcohol... Cetanol, stearyl alcohol, lanolin alcohol, etc.;
-Esters (esters of fatty acids and alcohols other than waxes)...myristyl myristate, propylene glycol dioleate, cetyl lactate, etc.

Among these cosmetic fats and oils, those recognized as foods or food additives such as beeswax are preferred cosmetic fats and oils in the present invention in that they do not pose a problem even if they adhere to foods, tableware and cooking utensils. ..

The method for producing an anti-norovirus composition of the present invention comprises an anti-norovirus agent (chestnut astringent skin extract), and optionally an organic acid such as citric acid and/or a salt thereof or vitamin C, The method is the same as the conventional method for producing alcohol preparations, cleaning agents, disinfectants, lotions, emulsions, creams, etc., except that appropriate adjustments are made in accordance with these matters. That is, by adding an anti-norovirus agent in addition to the general (or finely adjusted as necessary) production raw material of those conventional products, by the same (or finely adjusted as necessary) production process as conventional products, The anti-norovirus composition of the present invention can be produced. For example, in the case of a cream, an anti-norovirus agent and other components are added to purified water to prepare an aqueous phase part, while an oil phase part comprising cosmetic oils and fats is prepared, and these are prepared. It suffices to manufacture them by mixing them at a predetermined ratio.

The method of using the composition for anti-norovirus of the present invention is also the same as conventional alcohol preparations, detergents, disinfectants, lotions, emulsions, creams and the like. Further, for example, if it is a cleaning agent, it may be a concentrated type that is used by diluting it at the time of use, or if it is an alcohol preparation or a disinfectant, it may be a spray type, or a wipe type that is impregnated into a non-woven fabric, The composition for anti-norovirus of the present invention can be appropriately commercialized in a preferred embodiment depending on the method of use.

The anti-norovirus agent of the present invention can be used as an active ingredient of a therapeutic or preventive agent for infectious diseases caused by norovirus. The dosage form of such a pharmaceutical product can be appropriately selected from, for example, oral dosage forms such as liquids, syrups, tablets, capsules, powders, and granules, or injections, and if necessary, an excipient (Eg lactose and other sugars), binders (eg starch, methyl cellulose, polyvinyl alcohol), stabilizers (eg ascorbic acid), preservatives (eg paraoxybenzoate), sweeteners, solvents, and various other additives. It can be used in combination and can be produced according to a general formulation method. The effective dose of these drugs can be appropriately determined according to the age, weight, symptoms of the patient, drug administration route, administration schedule, formulation form, strength of inhibitory activity of the material, etc. The content of the anti-norovirus agent may be adjusted according to those conditions.

1. <Example 1-Bacteriophage MS2 inactivating effect>
Chestnut astringent skin powder was added to 50% ethanol, stirred for 10 minutes, and then centrifuged and filtered to obtain a 5,10% chestnut astringent skin extract (w/v). A solution obtained by passing 1 g of activated carbon through this was decolorized and used as a sample. A sample is shown below.

Sample 1・・・10% chestnut astringent skin extract before decolorization Sample 2・・・10% chestnut astringent skin extract after decolorization Sample 3・・・5% chestnut astringent skin extract before decolorization Sample 4・・・5% chestnut astringent skin after decolorization Extract Solution The chestnut astringent skin extract solutions of Samples 1, 2, 3 and 4 and bacteriophage MS2 were reacted at a ratio of 9:1 for 2 minutes and diluted with LB medium to stop the reaction. Further, it was serially diluted with LB medium, added to LB soft agar medium (0.5% agar) supplemented with Escherichia coli C3000, and overlaid on the LB agar medium. After static culture at 37° C. overnight, the number of plaques formed was counted. Bacteriophage MS2 is an icosahedral single-stranded (+) RNA virus and infects E. coli. The LB medium was one of the nutrient rich medium for bacteria, and was composed of tryptone 1% (w/v), yeast extract 0.5% (w/v) and sodium chloride 1% (w/v).

FIG. 1 is a diagram showing the bacteriophage MS2 inactivating effect of the chestnut astringent skin extract according to this example. As shown in Fig. 1, the anti-viral effect of the 5% chestnut astringent skin extract (w/v) after decolorization was slightly lower than that of the 5% chestnut astringent skin extract (w/v) before decolorization. The subsequent 5% chestnut astringent skin extract (w/v) and the decolorized 10% chestnut astringent skin extract (w/v) showed sufficient bacteriophage MS2 inactivating effect. The detection limit value of bacteriophage MS2 is log4.32 to log4.39, but the 10% chestnut astringent skin extract (w/v) after decolorization is below the detection limit value. The effect was also a good numerical value. The experiment was performed 3 times and the average value is shown in the figure. PFU means plaque forming uint, and 1 PFU is the number of viruses required to form one plaque. Specifically, plaques (dead cells) caused by virus infection by infecting cultured cells Is the unit of infectious titer of the virus. Further, PBS (phosphate-buffered saline) is phosphate-buffered saline.

2. <Example 2-Norovirus (NV) inactivating effect>
The above chestnut astringent skin extract samples 1 to 4 and Norovirus (NV) were reacted at a ratio of 9:1 for 2 minutes and diluted with LB medium to stop the reaction. RNA was extracted using the QIAamp (registered trademark) Viral RNA Mini Kit (Qiagen (registered trademark)), and the genome was quantified by real-time PCR through DNase treatment and cDNA synthesis. The QIAamp Viral RNA Mini Kit is a purification kit for extracting viral RNA by centrifugation using a spin column.

FIG. 2 is a view showing the norovirus inactivating effect of the chestnut astringent skin extract according to this example. As shown in FIG. 2, the 5% chestnut astringent skin extract (w/v) after decolorization had a slightly lower norovirus inactivating effect than the 5% chestnut astringent skin extract (w/v) before decolorization. The decolorized 5% chestnut astringent skin extract (w/v) and the decolorized 10% chestnut astringent skin extract (w/v) both showed sufficient norovirus inactivating effect. In particular, the 10% chestnut astringent skin extract (w/v) after decolorization showed a good value for the norovirus inactivating effect while increasing the commercial value by decolorization. The experiment was performed 3 times and the average value is shown in the figure. In FIG. 2, total control is PBS. The genomic residual rate (%) was determined by counting the number of viral genomic RNAs remaining in the diluted solution by real-time PCR.

3. <Example 3 -Bacteriophage MS2 inactivating effect of decolorized pear chestnut astringent skin extract in the case of adding an organic acid>
A 10% chestnut astringent skin extract (w/v) before bleaching was diluted 20 times with an organic acid adjusted to 0.01% with 50% ethanol to prepare an organic acid-added pre-bleaching chestnut astringent skin extract sample. The organic acids used were malic acid (MaA), succinic acid (SuA), and citric acid (CiA). A 10% chestnut astringent skin extract (w/v) before decolorization was diluted 20 times with 50% ethanol to prepare a diluted decolorized chestnut astringent skin extract sample. A sample is shown below.

Sample 5: 10% chestnut astringent skin extract with MaA added Decolorization Sample 6: 10% chestnut astringent skin extract with SuA added Decolorization Sample 7: CiA added 10% chestnut astringent skin extract before decolorization Sample 8 Diluted 10% chestnut astringent skin extract before decolorization In the same manner as in Example 1, the chestnut astringent skin extracts of Samples 5, 6, 7 and 8 were reacted with bacteriophage MS2 at a ratio of 9:1 for 2 minutes, The reaction was stopped by diluting with LB medium. Further, it was serially diluted with LB medium, added to LB soft agar medium (0.5% agar) supplemented with Escherichia coli C3000, and overlaid on the LB agar medium. After static culture at 37° C. overnight, the number of plaques formed was counted.

FIG. 3 is a diagram showing the bacteriophage MS2 inactivating effect of an organic acid-added decolorized chestnut astringent skin extract. As shown in FIG. 3, the chestnut astringent skin extract with addition of MaA, SuA and CiA also showed a good anti-virus effect as compared with the diluted chestnut astringent skin extract.

4. <Example 4 -Bacteriophage MS2 inactivating effect of decolorized chestnut astringent skin extract in the case of adding organic acid>
The decolorized 5% chestnut astringent skin extract (w/v) and the decolorized 10% chestnut astringent skin extract (w/v) were diluted with an organic acid dissolved in 50% ethanol. Further, the decolorized 5% chestnut astringent skin extract (w/v) and the decolorized 10% chestnut astringent skin extract (w/v) were diluted with 50% ethanol. The organic acid was diluted with 50% ethanol. The organic acids used were malic acid (MaA) and citric acid (CiA). The final concentration was adjusted so that the extract was 1% and the organic acid was 0.01%. A sample is shown below.

Sample 9 ... 10% chestnut astringent skin extract after decolorization with CiA added Sample 10 ... 10% chestnut astringent skin extract after decolorized with MaA sample 11 ... 5% chestnut astringent skin extract after decolorized with CiA added Sample 12 ... MaA addition 5% chestnut astringent skin extract after decolorization Sample 13: diluted 10% chestnut astringent skin extract after decolorization Sample 14: diluted 5% chestnut astringent skin extract after decolorization Sample 15: diluted CiA
Sample 16...diluted MaA
In the same manner as in Example 1, the above samples 9 to 16 were allowed to react with bacteriophage MS2 at a ratio of 9:1 for 2 minutes and diluted with LB medium to stop the reaction. Furthermore, it was serially diluted with LB medium, added to LB soft agar medium (0.5% agar) supplemented with Escherichia coli C3000, and layered on LB agar medium. After static culture at 37° C. overnight, the number of plaques formed was counted.

FIG. 4 is a diagram showing the bacteriophage MS2 inactivating effect of the chestnut astringent skin extract after decolorization with the addition of an organic acid. As shown in FIG. 4, the chestnut astringent skin extract with both MaA and CiA addition exhibited a good anti-virus effect as compared with the diluted chestnut astringent skin extract.

5. <Example 5: Norovirus (NV) inactivating effect of decolorized pear chestnut astringent skin extract in the case of adding an organic acid>
A 10% chestnut astringent skin extract (w/v) before bleaching was diluted 20 times with an organic acid adjusted to 0.01% with 50% ethanol to prepare an organic acid-added pre-bleaching chestnut astringent skin extract sample. The organic acids used were malic acid (MaA) and citric acid (CiA). A sample is shown below.

Sample 5... 10% chestnut astringent skin extract before decolorization with MaA added Sample 7... 10% chestnut astringent skin extract before decolorized with CiA Similar to Example 2, the above chestnut astringent skin extract samples 5 and 7, Norovirus (NV) was reacted at a ratio of 9:1 for 2 minutes and diluted with LB medium to stop the reaction. RNA was extracted using the QIAamp (registered trademark) Viral RNA Mini Kit (Qiagen (registered trademark)), and the genome was quantified by real-time PCR through DNase treatment and cDNA synthesis.

FIG. 5: is a figure which shows the norovirus inactivation effect of the chestnut astringent skin extract in the case of adding an organic acid. The experiment was performed 3 times and the average value is shown in the figure. Total control is PBS. As shown in FIG. 5, the norovirus (NV) inactivating effect of the chestnut astringent skin extract was very high when an organic acid was added.

6. <Example 6-Comparison between bacteriophage MS2 inactivating effect of chestnut astringent skin extract and bacteriophage MS2 inactivating effect of persimmon astringent extract>
Chestnut astringent skin powder was added to 50% ethanol, stirred for 10 minutes, and then centrifuged and filtered to obtain a 5,10% chestnut astringent skin extract (w/v). A solution obtained by passing 1 g of activated carbon through this was decolorized and used as a sample.

Next, a crushed product of an immature fruit of Japanese persimmon (Shibaki persimmon: PT) was placed in a cloth bag with fine mesh and squeezed, and the squeezed juice was freeze-dried to obtain a powder. The yield was not constant because it was a natural product, but was about 5 to 7%. This persimmon astringent powder was dissolved in 50% ethanol, stirred for 10 minutes and then centrifuged and filtered to obtain a 5% persimmon astringent skin extract (w/v).

A sample is shown below.

Sample 1 ... 10% chestnut astringent skin extract before decolorization Sample 2 ... 10% chestnut astringent skin extract after decolorization Sample 3 ... 5% chestnut astringent skin extract before decolorization Sample 4 ... 5% chestnut astringent skin after decolorization Extract sample 17: Persimmon astringent extract solution The tannin content of 5,10% chestnut astringent skin extract (w/v) before and after decolorization and the persimmon astringent extract solution was measured by the vanillin hydrochloride method with 50% ethanol. It was diluted and adjusted to 0.2 to 0.05 mg/ml.

The vanillin hydrochloride method was performed in the steps shown below.
(1) A dry powder sample (2.0 g) was placed in a 500 mL flask, and 100 mL of hydrochloric acid-70% methanol solution (a mixed solution of methanol:distilled water:concentrated hydrochloric acid=70:29:1) was added.
(2) It was attached to a cooling tube, heated in a water bath at 80° C., and extracted for 30 minutes after the inside of the flask began to boil.
(3) The extract was collected by filtering with filter paper. The powder sample on the filter paper was collected and again extracted with 100 mL of hydrochloric acid-70% methanol solution.
(4) It was filtered in the same manner and the extract was recovered. It was combined with the first extract to give a tannin quantitative sample solution (preserved from light).
(5) 200 μL of the above tannin quantitative sample solution diluted 5-fold with methanol was placed in a light-shielding test tube, 4% vanillin-methanol solution (1.2 mL) and concentrated hydrochloric acid (600 μL) were added, mixed well, and left for 15 minutes.
(6) The absorbance of the sample solution was measured at an optical path wavelength of 500 nm.
(7)(+)-Catechin (1,000 μg/mL diluted in 2-fold with methanol in five-step dilutions) was performed in the same manner, and a calibration curve was created to determine the amount of condensed tannin in the sample solution as the catechin conversion amount. As shown.
(8) Each sample solution was tested three times and the average value was calculated.

As in Example 1, the above sample and bacteriophage MS2 were reacted at a ratio of 9:1 for 2 minutes and diluted with LB medium to stop the reaction. Further, it was serially diluted with LB medium, added to LB soft agar medium (0.5% agar) supplemented with Escherichia coli C3000, and overlaid on the LB agar medium. After static culture at 37° C. overnight, the number of plaques formed was counted.

FIG. 6 is a view showing comparison of the bacteriophage MS2 inactivating effect of each of the chestnut astringent skin extract and the persimmon astringent extract. As shown in FIG. 6, in the persimmon astringent extract, the bacteriophage MS2 inactivating effect decreased as the amount of tannin decreased to 0.2 mg/ml, 0.1 mg/ml, 0.05 mg/ml. The persimmon astringent extract has an excellent anti-norovirus effect, because tannin is contained in the extract of the plant of the genus Diospyros. Since the amount of tannin is reduced to about 1/5 by passing the extract through the activated carbon, the effect of inactivating bacteriophage MS2 is reduced as the amount of tannin is reduced in the persimmon astringent extract.

Next, in the case of the chestnut astringent skin extract, both the 10% chestnut astringent skin extract before decolorization and the 5% chestnut astringent skin extract before decolorization showed a bacteriophage MS2 inactivating effect with a tendency similar to that of the persimmon astringent extract. It is considered that the tannin contained also in the chestnut astringent skin extract exhibits the bacteriophage MS2 inactivating effect, and the bacteriophage MS2 inactivating effect decreases as the amount of tannin decreases.

However, the decolorized 10% chestnut astringent skin extract and the decolorized 5% chestnut astringent skin extract showed almost constant bacteriophage MS2 inactivating effect even when the amount of tannin was reduced. Also, when the tannin amount was 0.2 mg/ml and 0.1 mg/ml, no significant difference was observed between the chestnut astringent skin extract samples, but when the tannin amount was 0.05 mg/ml, after decolorization The sample had a greater effect of inactivating cteriophage MS2 than the pre-bleached sample. This experimental result means that the chestnut astringent skin extract has a virus inactivating ability independent of the amount of tannin. That is, it was revealed that the chestnut astringent skin extract contains an active ingredient other than tannin, which is not contained in the persimmon astringent extract, and the inactivating effect is exhibited by the active ingredient. In addition, Example 6 shows the bacteriophage MS2 inactivating effect of the chestnut astringent skin extract, whereas Example 2 described above shows the norovirus inactivating effect of the chestnut astringent skin extract. Therefore, the facts of Example 6 and Example 2 revealed that the active ingredients other than tannin contained in the chestnut astringent skin extract contributed to the inactivation of norovirus.

It can be used to inactivate the black bean.

Claims (5)

An anti-norovirus composition comprising a chestnut astringent skin extract and malic acid and/or a salt thereof. The composition for anti-norovirus according to claim 1, comprising a component other than tannin as an anti-norovirus component. The composition for anti-norovirus according to claim 1 or 2, wherein the chestnut astringent skin extract has a concentration of 5% (w/v) or more. Further, at least one component selected from the group consisting of alcohols, surfactants, antibacterial agents, moisturizers, and cosmetic fats and oils is contained, and any one of claims 1 to 3 is included. Composition for anti-norovirus. A therapeutic or prophylactic agent for infectious diseases caused by norovirus, comprising the composition for anti-norovirus according to any one of claims 1 to 6 .

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