WO2023026867A1 - Aqueous catechin-containing solution composition having excellent storage stability, and use thereof - Google Patents

Aqueous catechin-containing solution composition having excellent storage stability, and use thereof Download PDF

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Publication number
WO2023026867A1
WO2023026867A1 PCT/JP2022/030616 JP2022030616W WO2023026867A1 WO 2023026867 A1 WO2023026867 A1 WO 2023026867A1 JP 2022030616 W JP2022030616 W JP 2022030616W WO 2023026867 A1 WO2023026867 A1 WO 2023026867A1
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catechins
cyclodextrin
egcg
composition
ppm
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French (fr)
Japanese (ja)
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正則 金山
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株式会社Hpg
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present invention provides various formulations containing catechins with excellent storage stability in aqueous solvents, particularly foods, beverages and cosmetics that have excellent storage stability and do not cause color tone changes such as browning over time. It belongs to the technical field of preparations related to aqueous compositions containing catechins, which are useful as compounding agents for their materials, pharmaceuticals, industrial products, and the like. Furthermore, the present invention uses formulations containing catechins, and relates to various products for various uses such as pharmaceuticals, foodstuffs, and cosmetics, which contain the formulations as active ingredients.
  • Catechins are a group of naturally-occurring organic compounds, and are one kind of flavonoids, which are plant secondary metabolites derived from chalcone, which is formed by polymerization of coumaric acid CoA and malonyl CoA. Below are the structures of the optical isomers (+)-catechin and ( ⁇ )-catechin.
  • the major catechin components in green tea include about four types, such as epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate (EGCg).
  • ECCg epigallocatechin gallate
  • viruses have protrusions (spikes) in their structures, through which they have joints that attach to cells, and the spikes are unique structures for each virus.
  • prevention by means of a vaccine is to create antibodies against the virus in the body, which then capture specific structures on the spikes and prevent the virus from attaching to cells. Because of this mechanism, for example, there are multiple influenza viruses with different spike structures, and if the antibody produced by the vaccine does not bind to the specific structure of the spike, influenza virus infection cannot be prevented. There is a problem.
  • the antiviral effect of catechins that is, the effect of preventing viral infection in the human body, etc., is thought to be due to the covering of the joints where the virus attaches to the cell, and the difference in the specific structure of the spikes of various viruses. You can do it regardless.
  • polyphenols including catechins
  • polyphenols exhibit different specific effects depending on their types. For example, anthocyanins have been reported to have an effect of improving eyesight (for example, Non-Patent Document 3), and isoflavones have been reported to have osteoporosis prevention and treatment effects (for example, see Non-Patent Document 4). Rutin has been reported to have a vasodilating effect (see, for example, Non-Patent Document 5), and is expected to be applied to many uses such as food and drink, cosmetics, and pharmaceuticals.
  • polyphenols including catechins
  • polyphenols are very unstable because they are easily oxidized by oxygen dissolved in water and oxygen in the air.
  • the expected useful action such as the action of removing active oxygen is reduced, and the storage stability is poor, such as discoloration due to oxidation.
  • Patent Document 1 discloses a polyphenol preparation containing a lower alcohol and a monoamine monocarboxylic acid.
  • Patent Document 2 discloses a polyphenol preparation containing polyoxyethylene alkyl sulfosuccinate.
  • Patent Document 3 discloses a technique for producing an s/o/w formulation in which an oil containing a polyhydric alcohol fatty acid ester in which polyphenols are dispersed is dispersed in water.
  • Patent Document 4 discloses a polyphenol preparation to which an organic reducing agent is added.
  • Patent Documents 1 to 3 all of the polyphenol preparations described in Patent Documents 1 to 3 have alcohol or surfactant dissolved in aqueous solvent.
  • the lower alcohols and surfactants are often known to have skin irritation and cytotoxicity to living organisms and induce skin inflammation (see, for example, Non-Patent Document 6). Therefore, it is not preferable from the viewpoint of safety to the living body to use it as it is for food, drink, cosmetics, pharmaceuticals, and the like. Therefore, in consideration of the safety to the living body, so-called “alcohol-free” and “surfactant-free” products that do not use alcohol or surfactants are desired, especially in the fields of food and drink, cosmetics, and oral care products. It is rare.
  • the object of the present invention is to stably exist catechins as an aqueous solution for a long period of time without causing color tone changes such as browning over time, and to be used in foods, cosmetics, pharmaceuticals, etc.
  • An object of the present invention is to provide a preparation containing catechins which is an aqueous liquid (aqueous agent) useful as a compounding agent and has excellent storage stability.
  • Another object of the present invention is to provide health foods and cosmetic adjuvants, which are often stored at room temperature on a daily basis, containing catechins as an active ingredient, since the water-soluble composition of the present invention has extremely good storage stability. and to provide additives to oral care products.
  • the present inventors have extensively studied the production of formulations containing catechins that have excellent storage stability in aqueous solvents. It was found that the decomposition of the genus can be greatly suppressed.
  • the effect of suppressing the decomposition of the catechins can be enhanced cooperatively, and the browning of the color tone can be enhanced. It was also found that the Moreover, the inventors have also found that the storage stability of the catechins in the resulting preparation containing catechins is improved by removing the dissolved components of the water used for dissolution as much as possible and increasing the purity thereof. Furthermore, as described above, it was found that preparations (stabilized compositions) containing catechins have antiviral activity, and that the antiviral activity can be exhibited by adding them to various materials, and the present invention has been completed. Arrived.
  • an aqueous solution composition containing catechins, cyclodextrin and an antioxidant, The catechins are dissolved at a concentration below the solubility,
  • the cyclodextrin is in a molar amount of 1/10 or more with respect to the catechins, 96% by weight or more of catechins in the aqueous solution composition remains at 40° C. for 14 days, Aqueous composition with excellent storage stability.
  • An aqueous solution composition containing catechins, cyclodextrin and an antioxidant The catechins are dissolved in the aqueous solution composition at a concentration of 1000 ppm or more, In the aqueous solution composition, the cyclodextrin is in a molar amount of 1/10 or more with respect to the catechins, When the aqueous composition is stored at 40° C. for 14 days, 50% by weight or more of catechins remain in the aqueous composition. Aqueous composition with excellent storage stability.
  • item [1] or [2] wherein after mixing cyclodextrin and catechins as an encapsulating agent, the mixture is added to water to dissolve, and then an antioxidant is added to dissolve.
  • Composition [5] The composition according to any one of items [1] to [4], wherein the catechin is epigallocatechin gallate.
  • a tablet containing catechins in which the composition according to any one of items [1] to [7] is impregnated in a solid matter and formed into a tablet form.
  • An antiviral agent which is an aqueous solution composition containing catechins or a pharmaceutically acceptable salt thereof, cyclodextrin and an antioxidant, which contains catechins as an active ingredient.
  • the antiviral agent according to item [10] wherein the virus is a coronavirus.
  • the antiviral agent according to item [10], wherein the virus is a novel coronavirus (COVID-19).
  • the catechins used in the aqueous composition of the present invention are not particularly limited. Furthermore, since the present invention aims to provide a water-soluble composition having excellent storage stability, specific examples include epigallocatechin gallate (EGCg), epicatechin gallate (ECg), epigallocatechin (EGC), and epicatechin (EC) are preferred.
  • any one of epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate (EGCg) may be used alone, or a mixture of any combination thereof may be used.
  • it has been reported that anti-aging action based on its active oxygen scavenging action and whitening action by inhibiting tyrosinase are also reported for the skin, and skin cancer suppressing action (Non-Patent Document 13). See also), etc.
  • catechins such as epigallocatechin gallate (EGCg), epicatechin gallate (ECg), epigallocatechin (EGC), and epicatechin (EC) are preferred.
  • the cyclodextrin used as an inclusion agent is not particularly limited.
  • ⁇ -cyclodextrin, ⁇ -cyclodextrin, or ⁇ -cyclodextrin, or alkylated cyclodextrins such as methylation and ethylation, hydroxyhydroxyethylation, hydroxypropylation, etc. for each of the above agents alkoxylated cyclodextrin, various cyclodextrin derivatives modified by acetylation, glucosylation, etc.
  • cyclodextrins and their derivatives show relatively excellent stabilizing ability by clathration to many catechins, and also has excellent heat stability, is colorless and odorless, and is used in foods, beverages, and cosmetics. Considering that it is permitted to be used for a wide range of applications such as food and pharmaceuticals, ⁇ -cyclodextrin, ⁇ -cyclodextrin, or ⁇ -cyclodextrin, or a mixture of any one or a combination thereof is preferable.
  • cyclodextrin clathrate epigallocatechin gallate (EGCg)
  • ⁇ -cyclodextrin ⁇ ⁇ -cyclodextrin ⁇ ⁇
  • the stability of EGCg tends to improve in the order of -cyclodextrin, and it was found that the combination of ⁇ -cyclodextrin and ⁇ -cyclodextrin further increases the stability of EGCg. Therefore, as the cyclodextrin, it is preferable to apply ⁇ -cyclodextrin, ⁇ -cyclodextrin, or a combination of ⁇ -cyclodextrin and ⁇ -cyclodextrin.
  • the antioxidant is not particularly limited.
  • ascorbic acid, derivatives thereof, or salts thereof can be used, and specific examples include L-ascorbic acid, L-ascorbic acid alkyl esters, L-ascorbic acid phosphates, and L-ascorbic acid sulfates. and derivatives thereof, or salts thereof such as alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, and the like.
  • Phytic acid or its salts, erythorbic acid or its salts, rosmarinic acid, chlorogenic acid, phytic acid, citric acid, ferulic acid, rutin, myricetin, myricitrin, quercetin, ubiquinol, melanoidin, caffeic acid, gallic acid Acids, other antioxidant carotenoids, flavone derivatives, flavonoids, tannins and their salts and esters, natural plant extracts such as rosemary extract, tea extract, apple extract, grape seed extract , sunflower seed extract, rice bran extract, sage extract, thyme extract, oregano extract, soybean extract, propolis extract and the like.
  • natural plant extracts such as rosemary extract, tea extract, apple extract, grape seed extract , sunflower seed extract, rice bran extract, sage extract, thyme extract, oregano extract, soybean extract, propolis extract and the like.
  • Purified water treated by a known method is usually used as the water used in the present invention, and the purification method for obtaining purified water is not particularly limited.
  • the water used in the present invention may be water obtained by purifying raw water before treatment by means of distillation, filtration, reverse osmosis, ion exchange, or the like.
  • water with extremely high purity generally water with a conductivity of 18.2 M ⁇ cm or less, is called ultrapure water.
  • the water used for dissolving these is highly pure and has an extremely high dissolved oxygen content. Due to limitations, ultrapure water is preferred.
  • the concentration of dissolved catechins in the aqueous composition of the present invention is preferably lower than the solubility of catechins under predetermined temperature conditions, although it varies depending on the type of catechins.
  • the solubility of EGCg in water is 4 g/100 mL at 20° C., that is, 40000 ppm
  • the solubility of catechins in the aqueous composition of the present invention is determined by the type, concentration, temperature, and other conditions of coexisting substances. It is.
  • aqueous solution for example, by containing 1000 ppm or more, 2000 ppm or more, 5000 ppm or more, or 10000 ppm or more of catechins in the aqueous solution, it can be used for various purposes. Specifically, in the case of a water-soluble composition containing 2000 ppm in an aqueous solution, 1000 ppm of catechins remain even if 50% of catechins do not remain due to decomposition or the like during storage. If 1000 ppm of catechins remain, antiviral effects can still be fully expected. Alternatively, even in the case of food to which 10% by volume of the total amount of the water-soluble composition containing 10000 ppm in the aqueous solution is added, 1000 ppm of catechins are contained.
  • the concentration at which the included catechins are dissolved may vary depending on the form of existence of the catechins, so the solubility should be measured as necessary. Good.
  • the mixing ratio of the above catechins and cyclodextrins is such that when the concentration of catechins dissolved in water for dissolving catechins is fixed, the higher the concentration of dissolved cyclodextrin, the more catechins are produced by clathration. Stabilization efficiency to the class is improved. Conversely, the lower the dissolved cyclodextrin concentration, the lower the efficiency of stabilization by clathration of catechins, and the lower the stability of catechins.
  • the molar concentration of catechins (the number of moles of catechins contained in 1 L of the aqueous solution composition at the above-mentioned dissolved concentration; the same applies hereinafter) of cyclodextrin is 1/ If it is less than 10, the stability of catechins may be remarkably lowered. Therefore, in the case of the present invention, the molar concentration of cyclodextrin to the molar concentration of catechins is preferably 1/10 or more, more preferably 1/10 to 1/1, particularly 1/6 to 1/3.
  • the above is 10 mol% or more, further 10 mol% to 66.6 mol%, particularly 16.6 mol% to 33.3 mol% when the molar ratio of the cyclodextrin to be added to the catechins is 10 mol% or more. preferable.
  • the higher the dissolved concentration of cyclodextrin the lower the stability of the dissolved system, which may result in precipitation over time.
  • each molecular species of cyclodextrin ( ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin, etc.) under predetermined conditions.
  • the solubility of each molecular species of cyclodextrin in water at 25° C. is 14.5 g/100 mL for ⁇ -cyclodextrin, 1.8 g/100 mL for ⁇ -cyclodextrin, and 23.2 g/100 mL for ⁇ -cyclodextrin. be.
  • the water-soluble composition of the present invention contains catechins, cyclodextrins and antioxidants, and as a result has high storage stability in products for various uses. As shown in the examples below, even after storage at 40°C for 14 days, the remaining amount of catechins is 96% by weight or more relative to the amount of catechins before storage at 40°C. Considering this data as an accelerated deterioration test, it can be expected that the storage period at room temperature (25° C.) in which foods and the like are normally stored can be set for a long period of time. For example, if it is possible to store food or functional food at 40°C for 14 days, it is thought that storage at 25°C for 3 months or more is possible for several months or more. It can be expected that the antiviral effect can be maintained.
  • the catechins contained therein are highly stable, and one of the reasons for this stability is that the catechins contained therein remain at an extremely high ratio without decomposition or the like.
  • the hue is stabilized, and coloring due to browning or the like is extremely small.
  • the amount of residual catechins was measured before and after storage of a water-soluble composition containing catechins at 40° C. for 14 days. In that case, it is important to maintain efficacy such as antiviral effect with respect to the residual amount of catechins after storage.
  • the water-soluble composition of the present invention preferably contains 50% by weight or more of catechins, preferably 80% by weight of the catechins before storage at 40°C. It is preferably at least 90% by weight, more preferably at least 96% by weight, and particularly preferably at least 96% by weight. Due to such a high residual rate, the water-soluble composition of the present invention can be used for various purposes.
  • the aqueous solution preferably contains 1000 ppm or more, preferably 2000 ppm or more, more preferably 5000 ppm or more, and particularly preferably 10000 ppm or more.
  • 1000 ppm or more preferably 2000 ppm or more, more preferably 5000 ppm or more, and particularly preferably 10000 ppm or more.
  • 50% of the catechins present at the time of preparation are decomposed after long-term storage or exposure to high temperatures. 1000 ppm of catechins still remain even if they do not remain. If 1000 ppm of catechins remain, antiviral effects can still be fully expected.
  • ⁇ Antiviral effect of aqueous composition containing catechins> As described above, it was found that the aqueous composition containing catechins of the present invention surprisingly has an antiviral effect. Therefore, when the present inventors further examined various dosage forms, it was found that the influenza virus can be effectively suppressed.
  • agents that can be applied to various uses based on the antiviral effect of the aqueous composition containing catechins of the present invention will be described.
  • catechins are said to exert an antiviral effect against viruses as well as an inhibitory effect on bacteria.
  • the aqueous composition containing catechins of the present invention is a composition containing cyclodextrin and an antioxidant. and is useful as a compounding agent for foods, drinks, cosmetics, pharmaceuticals, and the like.
  • the antiviral effect can be exhibited without using organic solvents (excluding ethanol, etc.) and other materials that can be harmful to the human body.
  • oral care products such as mouth sprays
  • additives added to various foods such as chewing gum, candies, and pastries
  • various processed foods additives for cosmetics, and additives for alcoholic beverages.
  • raising dogs, cats, and other animals as pets raising livestock such as cattle, and raising pigs, chickens, etc.
  • virus infection to humans through animals, so an antiviral effect is required.
  • a form it is possible to soak a cloth in liquid to sterilize and remove viruses attached to shoes, as with general disinfectants.
  • it is desirable that the aqueous solution be in the form of a spray.
  • the aqueous composition containing catechins of the present invention is not particularly limited as a virus exhibiting the effect, as long as the effect of suppressing viruses is recognized.
  • viruses there are various viruses having double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, etc. (see Non-Patent Document 14).
  • influenza viruses such as influenza A virus, influenza B virus, and influenza C virus classified in the family Coronaviridae of the order Nidoviridae, and their mutant strains.
  • the aqueous solution composition containing catechins of the present invention has an inhibitory effect on influenza virus or its analogous viruses, has the same single-stranded RNA, and has the same single-stranded RNA, and from the viewpoint of cell infection via spikes, novel coronavirus (COVID-19) and its mutant strains can also be expected to have a sufficient suppressive effect. Furthermore, when an in vivo evaluation was performed, it was found that an antiviral effect was observed.
  • catechins-containing aqueous composition according to the present invention include, as described above, oral care products and additives. Examples include the form of shape.
  • a spray can include a container in which an aqueous solution composition containing catechins is introduced.
  • the container is not particularly limited as long as it does not affect the physical properties, stability, etc. of each main component in the aqueous solution composition containing catechins, such as glass, plastic, and metal.
  • tablets or tablets can be mentioned, which are obtained by impregnating a solid component with an aqueous solution composition containing catechins and making tablets or tablets by a conventional method. Before making tablets or tablets, they may be made into powder or granules having a smaller particle size and then tableted.
  • the form of the aqueous solution does not change. For this reason, it is produced in the form of an aqueous solution composition containing a predetermined amount of catechins and, if necessary, additives. Examples include antioxidants and pH adjusters.
  • the aqueous composition preferably contains 1000 ppm or more of catechins such as EGCg, preferably 2000 ppm or more, more preferably 5000 ppm or more, and particularly preferably 10000 ppm or more. Compositions containing the above catechins are included.
  • the formulation of the aqueous composition containing catechins as a spray is not particularly limited as long as the effects of the present invention can be obtained. and cyclodextrins to stabilize it, antioxidants, bulking agents, preservatives and other additives.
  • examples thereof include glycerin, D-sorbitol, propylene glycol, trehalose hydrate, fragrance, citric acid or its Na salt, l-menthol as a fragrance, and the like.
  • the aqueous composition containing catechins of the present invention has reduced bitterness and astringency while containing gallate-type catechins at a high concentration, and has excellent tableting aptitude.
  • this compressed tablet-form drug retains its antiviral effect, is excellent in persistence of the effect, and is easy to use.
  • the aqueous composition containing catechins of the present invention is in the form of a solution, it can be made into a tablet either by precipitation into a solid state, or by spraying on a powder or granular substance. and granulating it into a solid form. Specific solid forms may be those according to conventional methods.
  • the content of the catechins in the catechins-containing granules obtained from the aqueous solution composition containing the catechins of the present invention is not particularly limited as long as it is a blend that exhibits effects such as antiviral action.
  • the granules can be used as they are as granules, or can be further compressed and used as tablets.
  • the catechin-containing granules contain various ingredients such as the above-mentioned additives, stabilizers, and bulking agents in addition to catechins. Moreover, since it is to be drunk as granules, it is possible to use a high-intensity sweetener as an additive to make it easier to drink or to mask the specific taste of the additive. Examples of high-intensity sweeteners include acesulfame potassium, sucralose, stevia, aspartame, neotame, thaumatin, licorice, saccharin, etc. One or more of these can be used.
  • the content of the high-intensity sweetener in the catechins-containing granules containing the aqueous solution composition containing catechins of the present invention is not particularly limited as long as it is an amount capable of masking the specific taste of the additive. It is preferably 0.01% to 1.0% by weight, more preferably 0.1% to 0.5% by weight.
  • the particle size of the catechins-containing granules is not particularly limited, but may be relatively fine in order to make it smooth or easy to absorb when drinking or licking. For example, the particle size may be about 100 ⁇ m to 1 mm.
  • the bitter and astringent taste peculiar to catechins is less felt, and even though it has fluidity as a solid, it does not rise to powder when handled, and it is easy to eat. It is easy to drink and easy to lick without causing problems such as coughing or sticking in the mouth.
  • the catechin-containing granules are granular and easy to handle, they are also excellent in tableting aptitude.
  • the granules or chewable tablets become suitable for drinking or easy to lick, making them easy to use for children and the elderly.
  • Chewable Tablet A chewable tablet using the aqueous composition containing catechins of the present invention is obtained by compressing catechin-containing granules containing the above-mentioned aqueous composition containing catechins of the present invention. Therefore, the ingredients and composition used for the chewable tablet are the same as those for the catechins-containing granules.
  • catechin-containing granules containing various ingredients may be compressed to prepare a chewable tablet, but as another method, catechin-containing granules are prepared, sugar, Various ingredients such as acidulants, flavoring agents, coloring agents, dietary fibers, vitamins, minerals, amino acids, fats and oils, emulsifiers, polysaccharide thickeners, and lubricants are mixed, and the mixture is tableted to form chewable tablets. may be made. By appropriately selecting these various ingredients, the chewable tablet becomes suitable for drinking or easy to lick, and is easy to use for children and the elderly.
  • the chewable tablet is not particularly limited as long as it is a size that is easy for children, adults, and even the elderly to put in their mouths and eat.
  • the shape thereof may be cylindrical, triangular prismatic, quadrangular prismatic, or the like, but is not particularly limited.
  • catechins-containing granules (2) Method for producing catechins-containing granules and chewable tablets
  • the above-mentioned catechins-containing granules can be produced by mixing or spraying an aqueous solution containing catechins with powders or granules containing various ingredients, followed by granulation. .
  • catechins are dissolved in a solvent such as water to obtain an aqueous solution containing catechins.
  • a solvent such as water
  • the powder or granules containing various additives are mixed or sprayed with an aqueous solution containing catechins, and the powder or granules are mixed or sprayed, followed by fluid bed granulation, tumbling granulation, extrusion granulation, or stirring granulation. It can be granulated using a granulation method.
  • the fluid bed granulation method is preferably used because catechins can be uniformly coated on powders or granules containing various additives, and uniform particles with excellent tableting suitability can be easily obtained. can be done.
  • a granulation apparatus generally used for manufacturing pharmaceuticals, confectionery, etc. may be used.
  • a binder such as gum arabic for binding powder particles together can be used if necessary.
  • This granulation is preferably carried out at room temperature to about 90° C. so as not to deteriorate the catechins. It is also possible to dry the granules completely during the granulation process, but after taking them out of the granulator, the granules may be further dried by allowing them to stand at a predetermined temperature, for example around room temperature. Furthermore, for an aqueous solution containing catechins, granules with a desired particle size can be obtained by appropriately adjusting the granulation temperature, granulation time, etc., and the appropriate amount is used to make the particle sizes uniform. It is possible to use granules on the blocked side or currency side by passing them through a fine sieve.
  • the above chewable tablet is obtained by compressing the catechin-containing granules. Compression is preferably performed by dry compression.
  • the tableting machine used for tableting is not particularly limited, and a rotary tableting machine, a single shot tableting machine, or the like can be used.
  • the high-intensity sweetener that may be added to the above granules and chewable tablets can be added at any stage during the above process, but when dissolved in the catechins-containing aqueous solution, the catechins-containing granules or It is preferable because it can be uniformly dispersed in the chewable tablet.
  • Other optional components can also be added at any stage.
  • the water-soluble composition containing catechins obtained as described above and the preparation containing the same are aqueous preparations substantially composed of catechins, cyclodextrin, and antioxidants, and are water-soluble and easily oxidizable catechins. It has extremely high storage stability compared to the case of dissolving the same by a normal dissolving method. Therefore, oxidation of catechins during handling of a water-soluble composition containing catechins and preparations containing the same is prevented as much as possible, and as a result, the storage stability of catechins is remarkably improved.
  • the catechins are stably present in water for a long period of time. Moreover, since it is not accompanied by changes in color tone such as browning over time, it is extremely useful as a compounding agent for producing foods, beverages, cosmetics, pharmaceuticals, and the like. In particular, a sufficient inhibitory effect can be expected for influenza virus, novel coronavirus (COVID-19) having the same single-stranded RNA, and its mutants.
  • COVID-19 novel coronavirus
  • the water-soluble composition of the present invention which has excellent storage stability, as a compounding agent for food, beverages, cosmetics, pharmaceuticals, etc., it can be expected that the antiviral effect can be maintained for a long period of time. For this reason, a long consumption period or best-before period can be set as the storage period of food or the like.
  • At least one catechin, at least one or more cyclodextrins, and one or more antioxidants are dissolved in water such as purified water.
  • the catechins are dissolved at a concentration below the solubility of the catechins.
  • the cyclodextrin is dissolved at a molar concentration of 1/10 or more with respect to the molar concentration of the polyphenols and at a concentration lower than the solubility of the cyclodextrin.
  • at least one antioxidant is dissolved at a concentration equal to or lower than the solubility of the antioxidant.
  • cyclodextrin as a clathrate agent is dissolved in purified water using ultrapure water.
  • the catechins are dissolved.
  • the cyclodextrin and the catechins which are previously weighed and mixed in proportion, are simultaneously dissolved in purified water.
  • the water-soluble composition of the present invention can be used in the form of a spray or tablet, or as an additive to various foods.
  • it can be used as an antiviral agent for humans and animals, and as an antibacterial agent contained in a mat or the like for contact while wearing shoes or the like.
  • EMCg epigallocatechin gallate
  • Example 1 Preparation of aqueous solution composition (liquid sample A) containing catechins, cyclodextrin and antioxidant About 80 ml of ultrapure water as purified water is weighed into a 200 ml beaker, and the beaker is stirred with a magnetic stirrer. , and rotated and stirred at 200 rpm.
  • ⁇ -cyclodextrin and ⁇ -cyclodextrin are first added as clathrate agents to a molar concentration of 1/10 or more with respect to the molar concentration of catechins, and the cyclodextrin 0.1 g of .beta.-cyclodextrin and 0.4 g of .gamma.-cyclodextrin were slowly added little by little so as to obtain a concentration below the solubility of , and dissolved by mixing. As a result, the dissolved concentration of cyclodextrin was 0.5% with respect to the total weight (100 g) of the catechins-containing composition.
  • the molecular weight of catechins is 458 (Da)
  • the molecular weight of ⁇ -cyclodextrin is 1134 (Da)
  • the molecular weight of ⁇ -cyclodextrin is 1297 (Da). bottom.
  • epigallocatechin gallate (EGCg) powder manufactured by Taiyo Kagaku Co., Ltd., trade name: Sunphenon (registered trademark) EGCg; hereinafter referred to as powder sample a
  • EGCg epigallocatechin gallate
  • powder sample a which is one of catechins
  • Comparative Example 2 has a composition that does not contain an antioxidant.
  • Comparative Example 3 is a composition that does not contain cyclodextrin.
  • Example 2 Preparation of an aqueous solution composition containing catechins (liquid sample E) About 80 ml of ultrapure water as purified water was weighed into a beaker with a capacity of 200 ml, and the beaker was set in a magnetic stirrer and stirred at 200 rpm. to rotate and stir. In this ultrapure water, 1 g of ⁇ -cyclodextrin was slowly added little by little to 1% of the total weight of the final composition and dissolved by mixing. Further, 1 g of the above powder sample A (EGCg) was weighed so that its weight was 1% of the total weight of the composition, and slowly mixed little by little to dissolve.
  • the amount of cyclodextrin to catechins was set to 40.4 mol %.
  • 0.2 g of ascorbic acid is dissolved so as to be 0.2% with respect to the total weight of the composition, and ultrapure water is added so that the total weight of the composition is finally 100 g.
  • liquid sample E (catechin-containing composition) of Example 2 shown in "Table 1" below was prepared.
  • Example 3 Preparation of an aqueous solution composition containing catechins (liquid sample G) About 80 ml of ultrapure water as purified water was weighed into a beaker with a capacity of 200 ml, and the beaker was set in a magnetic stirrer and stirred at 200 rpm. to rotate and stir. In this ultrapure water, 1 g of ⁇ -cyclodextrin was slowly added little by little to 1% of the total weight of the final composition, and dissolved by mixing. Further, 1 g of the above powder sample A (EGCg) was slowly mixed little by little and dissolved so that the weight thereof was 1% with respect to the total weight of the composition.
  • the amount of cyclodextrin to catechins was set to 37.9 mol %. Further, 0.2 g of ascorbic acid is dissolved so as to be 0.2% with respect to the total weight of the composition, and ultrapure water is added so that the total weight of the composition is finally 100 g.
  • liquid sample G (catechin-containing composition) of Example 3 shown in "Table 1" below was prepared.
  • Comparative Example 5 Preparation of Aqueous Composition Containing Catechin (Liquid Sample H)
  • This Comparative Example 5 corresponds to Example 3 above, in which about 80 ml of ultrapure water is added as purified water in a beaker having a capacity of 200 ml. The beaker was weighed, set in a magnetic stirrer, and stirred by rotating at 200 rpm. In this ultrapure water, 1 g of ⁇ -cyclodextrin was slowly added little by little to 1% with respect to the total weight of the final composition, and dissolved by mixing.
  • Example 4 [Confirmation test 1 of storage stability] 30 mL of each of the liquid sample A (catechin-containing composition) obtained in Example 1 and the liquid samples B to D obtained in Comparative Examples 1-3 were placed in a sample bottle and placed at an ambient temperature of 40°C. Stored for 14 days. After that, EGCg concentration was measured for each sample by high performance liquid chromatography (HPLC). Furthermore, the change in the EGCg concentration over time was examined to determine the residual rate of EGCg with respect to the elapsed days.
  • HPLC high performance liquid chromatography
  • Example 1 of the present invention still maintains a residual EGCg rate of 100%, which is higher than the values of other Comparative Examples 1 to 3. , it was confirmed that the component stability was extremely high. In addition, the absorbance of Example 1 of the present invention was only 0.08, and it was confirmed that compared with the values of other Comparative Examples 1 to 3, browning did not occur by an order of magnitude.
  • Examples 2 and 3 of the present invention also show remarkably high component stability compared to Comparative Example 3 in which only ascorbic acid is blended. In addition, it was confirmed that even when compared with Comparative Examples 4 and 5 in which only cyclodextrin was blended, an incomparably higher color tone stability was exhibited.
  • Example 5 [Confirmation test of storage stability of aqueous solution for spray] ⁇ Spray containing EGCg-containing aqueous solution composition> The above aqueous solution composition was placed in a container with a nozzle, and the effect and stability of EGCg were evaluated according to the form of spray. With one push of the nozzle, 0.1 ml of the aqueous solution was atomized and could be sprayed. A single push dispensed a mist of the aqueous solution in a volume of approximately 10 liters (L). The evaluation was as follows. - The EGCg-containing aqueous solution composition was compared between a sample immediately after production and a sample stored at 40°C for 2 weeks. The evaluation items were as follows.
  • a sample to which cyclodextrin was not added (target sample 1) and a sample to which ascorbic acid was not added (target sample 2) were used.
  • the stability of EGCg, the presence or absence of precipitation, and the presence or absence of cloudiness were confirmed. (a. and b. below) Evaluation was by visual or optical turbidity measurement. a. transparent and dissolved b. white turbidity or precipitate 2) Hue of the aqueous solution composition The spectrum was measured with a spectrophotometer to determine whether the coloration increased from the transparency. 3) Odor of aqueous solution composition (sensory test, a., b., c.
  • the sample of the present invention has good stability, does not change color even under the severe conditions of 2 weeks at 40 ° C., has no odor, and when sprayed on the surface of the object. It can be seen that stable quality is maintained without any discomfort.
  • Control Sample 1 and Control Sample 2 which are comparative objects, coloration was observed, and changes in stability such as changes in odor and surface feel were observed. From this, it became clear that the aqueous composition of the present invention has excellent stability in the form of a spray.
  • Example 6 [Tablet storage stability confirmation test] ⁇ Tablet containing EGCg-containing aqueous solution composition> Solid tablets (diameter 1 cm, 0.5 cm (center), 0.3 cm (end)) were produced from the above aqueous composition by a conventional method. This tablet was evaluated for EGCg stability. 1) Stability of EGCg (solubility in water) 2) Color of tablet 3) Taste of aqueous solution composition (sensory test) The evaluation was as follows. - The EGCg-containing aqueous solution composition was compared between a sample immediately after production and a sample stored at 40°C for 2 weeks. The evaluation items were as follows.
  • the samples of the present invention have good stability, and even under the severe conditions of 2 weeks at 40°C, there is no change in hue and taste, and it can be seen that they maintain stable quality. . From this, it became clear that the stability of the tablets obtained based on the aqueous composition of the present invention in the form of tablets (tablets) is excellent.
  • Epigallocatechin gallate (EGCg) which is a kind of catechin, is known to inhibit the process of adsorption to virus-infected cells during the process of viral proliferation and exhibit an antiviral effect.
  • EGCg is highly lipid-soluble, it has been difficult to directly evaluate its antiviral effect using cultured cells. Therefore, the composition of the present invention in which the water solubility of EGCg is enhanced, and the formulation containing EGCg as a stabilized composition were used. That is, the anti-influenza virus activity was compared between a formulation containing EGCg and a solution (formulation) containing components other than EGCg.
  • the virus was diluted with an EGCg solution (EGCg/aqueous solution) or a solution of EGCg dissolved in white liquor (EGCg/white liquor solution), and the virus was adsorbed on MDCK cells. ⁇ Infected Similarly, the virus was diluted with a lysate (an aqueous solution in which EGCg was dissolved and containing additives, etc.) or white sake, and the virus was allowed to adsorb and infect MDCK cells. Regarding the inhibition after virus adsorption, EGCg treatment was performed on the infected MDCK cells at the above-mentioned dilution concentration after virus adsorption/infection to MDCK cells. In addition, the EGCg solution was diluted under the same conditions as in virus infection, and monolayer-cultured MDCK cells were treated (EGCg pretreatment), followed by virus adsorption and infection.
  • the method for adjusting the cells used is as follows. Thawed MDCK cells were cultured in 75 cm 2 flasks. Cells were confirmed to have proliferated to the corners of the 75 cm 2 flask, and were subcultured 2 to 3 times. After that, the cells were subcultured from the 75 cm 2 flask to a petri dish, and the cells in which the cells were evenly proliferating into a monolayer were used in the experiment.
  • plaque reduction method used in this example is as follows. The method and results will be shown below along with the exemplified conditions.
  • Example 8 When Influenza Virus Stock Solution was Diluted with EGCg Solution MDCK cells monolayer-cultured in a 60-mm petri dish were rinsed (washed) with 5 ml of PBS (phosphate-buffered saline).
  • PBS phosphate-buffered saline
  • Influenza virus (stock solution) was mixed with various concentrations of EGCg aqueous solution (stock solution: 10000 ppm) diluted with PBS to 5000 PFU / ml (control solution is PBS, indicated as control), and further, each of various concentrations 200 ⁇ l (100 PFU/200 ⁇ l/plate) of a 500 PFU/ml virus solution diluted 10-fold with PBS containing EGCg (0, 0.1, 0.3, 1, 3, 10, 30 ppm) was cultured in a monolayer and cultured in PBS. was adsorbed for 1 hour at room temperature to MDCK cells rinsed with At this time, the operation of tilting the petri dish every 15 minutes was repeated so that the cells were uniformly infected with the virus.
  • EGCg aqueous solution stock solution: 10000 ppm
  • control solution is PBS, indicated as control
  • the plaque assay medium (Table 4 above) and 2% agarose were mixed at a ratio of 3:2, and 5 ml of the mixed solution was added to each Petri dish. Three plates were prepared for the control and each concentration. After the agar medium solidified, the dishes were incubated at 37°C, 5% CO2 for 2 days. After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated. Table 5 shows the above results.
  • type A strains Bangkok (H1N1, labeled “Bangkok” in the table), PR8 (H1N1, labeled “PR8” in the table), and Aichi (H3N2, labeled “Aichi” in the table)
  • type B strain Singapore (indicated as “Singapore” in the table).
  • Example 9 In the case of diluting influenza virus with a lysate (an aqueous solution in which EGCg is dissolved, containing only additives, etc. that do not contain EGCg): MDCK cells monolayer-cultured in a 60 mm petri dish were rinsed with 5 ml of PBS. The lysate was diluted in the same manner as in the case of diluting the influenza virus stock solution with the EGCg solution to finally prepare a virus solution of 500 PFU/ml (100 PFU/200 ⁇ l/plate). 200 ⁇ l of the virus solution diluted variously with the lysate was adsorbed to MDCK cells which had been monolayer-cultured and rinsed with PBS for 1 hour at room temperature.
  • a lysate an aqueous solution in which EGCg is dissolved, containing only additives, etc. that do not contain EGCg
  • the operation of tilting the petri dish every 15 minutes was repeated so that the virus would uniformly infect the cells.
  • the plaque assay medium above, Table 2
  • 2% agarose were mixed at a ratio of 3:2, and 5 ml of the mixed solution was added to each Petri dish.
  • 3 petri dishes were prepared. After the agar medium had solidified, the dishes were incubated at 37° C., 5% CO 2 for 2 days. After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated. Table 6 shows the above results.
  • Example 10 When MDCK cells pretreated with EGCg were adsorbed and infected with influenza virus After rinsing MDCK cells monolayer-cultured in a 60 mm petri dish with 5 ml of PBS, EGCg aqueous solutions of various concentrations diluted with PBS (0, 0 1, 0.3, 1, 3, 10, 30 ppm) was added to each petri dish, and pretreatment was performed at 4°C for 1 hour while repeating the operation of tilting the petri dish every 15 minutes. After pretreatment with an EGCg aqueous solution, MDCK cells were rinsed twice with 5 ml of PBS, and PBS-diluted influenza virus was adsorbed and infected at 100 PFU/200 ⁇ l/plate for 1 hour at 4°C.
  • the operation of tilting the petri dish every 15 minutes was repeated so that the cells were uniformly infected with the virus.
  • the plate was rinsed once with 5 ml of PBS, and 5 ml of a solution obtained by mixing plaque assay medium (Table 2 above) and 2% agarose at a ratio of 3:2 was added to the petri dish.
  • Three plates were prepared for the control and each concentration. After the agar medium solidified, the dishes were incubated at 37°C, 5% CO 2 for 2 days. After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated. Table 7 shows the above results.
  • Example 11 EGCg Treatment After Influenza Virus Adsorption/Infection MDCK cells monolayer-cultured in a 60 mm petri dish were rinsed with 5 ml of PBS. Influenza virus (stock solution) was diluted with PBS and adsorbed and infected to MDCK cells at 100 PFU/200 ⁇ l/plate for 1 hour at room temperature. At this time, the operation of tilting the petri dish every 15 minutes was repeated so that the virus would uniformly infect the cells. After rinsing once with 5 ml of PBS, plaque assay medium containing various concentrations of EGCg (Table 2 above) and 2% agarose were mixed at a ratio of 3:2, and 5 mL of the mixed solution was added to each Petri dish. .
  • EGCg was added to each medium at final concentrations of 0, 0.3, 1, 3, 10, and 30 ppm, and 3 petri dishes were prepared for each concentration as a control. After the agar medium had solidified, the dishes were incubated at 37°C, 5% CO2 for 2 days. After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated. Table 8 shows the above results.
  • Example 12 When Influenza Virus was Diluted with EGCg Solution (Dissolved in Shirojiu) MDCK cells monolayer-cultured in a 60-mm petri dish were rinsed with 5 ml of PBS. Influenza virus (stock solution) was mixed with various concentrations of EGCg/white liquor solution (stock solution: 10000 ppm) diluted with PBS to 5000 PFU/ml (control solution was PBS), and each contained EGCg at various concentrations.
  • EGCg/white liquor solution stock solution: 10000 ppm
  • MDCK obtained by monolayer culture of 200 ⁇ l of virus solution of 500 PFU/ml (100 PFU/200 ⁇ l/plate) diluted 10-fold with PBS (0, 0.1, 0.3, 1, 3, 10, 30 ppm) and rinsed with PBS The cells were adsorbed for 1 hour at room temperature. At this time, the operation of tilting the petri dish every 15 minutes was repeated so that the virus would uniformly infect the cells.
  • the white liquor used was WULIANGYE Goryui (trade name) manufactured by WULIANGYE YIBIN CO, LTD.
  • the plaque assay medium (Table 2 above) and 2% agarose were mixed at a ratio of 3:2, and each 5 ml of the mixed solution was added to the petri dish.
  • 3 petri dishes were prepared. After the agar medium had solidified, the dishes were incubated at 37°C, 5% CO2 for 2 days. After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated. Table 9 shows the above results.
  • p ⁇ 0.05 is significant except for some, that is, the effect of suppressing virus growth due to contact between catechins and virus strains is statistically shown to be significant. Therefore, by diluting the influenza virus strain with a solution containing EGCg, that is, by contacting the virus with EGCg in white sake before adsorption to the cells, the adsorption or entry of the virus into the cells is inhibited, and the proliferation of the influenza virus strain is suppressed. I know you can. Furthermore, since the EC50 values in Table 9 are lower than the EC50 values in Table 5, it seems that EGCg has a higher anti-influenza virus activity in white liquor than in aqueous solution.
  • Example 13 When Influenza Virus was Diluted with Shirojiu Not Containing EGCg MDCK cells monolayer-cultured in a 60-mm petri dish were rinsed with 5 ml of PBS. Shirozake was diluted with PBS to finally prepare a virus solution of 500 PFU/ml (100 PFU/200 ⁇ l/plate) in the same manner as in the case of diluting influenza virus with EGCg solution (dissolved in white sake). 200 ⁇ l of virus solution diluted variously with PBS was adsorbed to MDCK cells which had been monolayer-cultured and rinsed with PBS for 1 hour at room temperature. At this time, the operation of tilting the petri dish every 15 minutes was repeated so that the virus would uniformly infect the cells.
  • the plaque assay medium (Table 4 above) and 2% agarose were mixed at a ratio of 3:2, and 5 ml of the mixed solution was added to each petri dish.
  • 3 petri dishes were prepared. After the agar medium had solidified, the dishes were incubated at 37° C., 5% CO 2 for 2 days. After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated. Table 10 shows the above results.
  • the EC 50 for post-virus adsorption inhibition of EGCg was 19.32 ⁇ 1.79 ppm in Bangkok, ⁇ 30 ppm for PR8, 22.89 ⁇ 1.44 ppm for Aichi, and 11.08 ⁇ 1.56 ppm for Singapore. This resulted in about 14-fold, more than 10-fold, about 8-fold, and about 12-fold higher pre-adsorption inhibition of the four viruses, respectively.
  • the EC50 for the four types of influenza viruses when EGCg pretreatment was performed was 30 ppm or more.
  • EGCg may have antiviral activity by exhibiting an inhibitory effect on the process of adsorption or entry of influenza virus particles into cells. did it.
  • influenza virus is an RNA virus like coronavirus, and coronavirus (Covid-19) and its mutant strains may also be protected by the mechanism by which the virus enters host cells such as humans and animals. Conceivable. Although this is not a specific action such as the effect of preventing viral infections by antibodies, it is conversely expected to have the effect of preventing infection with viruses in general.
  • ⁇ EGCg antiviral activity 2> In addition to the anti-influenza virus activity 1 of EGCg described above, the antiviral effect of EGCg against human coronavirus (HCoV)-229E, which is closely related to SARS-CoV-2 (COVID-19) and causes cold symptoms in humans, is reduced by 50%. Culture Infectious Dose (TCID 50 ) was used as an indicator. Furthermore, cytotoxicity against MRC-5 cells, which are host cells for HCoV-229E, Vero-E6/TMPRSS2 cells, which are host cells for SARS-CoV-2, and MDCK cells, which are host cells for influenza virus, were also evaluated. In tests to evaluate virus effects, we investigated conditions under which antiviral effects can be measured without cytotoxic effects or under conditions that can be evaluated.
  • MRC-5 cells which are host cells for HCoV-229E
  • Vero-E6/TMPRSS2 cells which are host cells for SARS-CoV-2
  • MDCK cells which are host cells for influenza virus
  • the virus solution was removed, medium was added, and the cells were cultured for 5 days.
  • the virus titer was calculated by the TCID 50 method using cytopathicity as an index.
  • the infectivity was calculated based on the EGCg titer of 0 ppm.
  • Each test was performed in duplicate with 4 wells. The results confirmed that mixing with 1 ppm and 10 ppm EGCg reduced the infectivity of HCoV-229E by an average of 45% and 55%, respectively. Also, the results of two tests revealed that the 50% effective concentration (EC 50 ) of EGCg against HCoV-229E in this experimental system was 0.54 ppm.
  • the above test protocol is as follows. 1) Water-soluble catechins were diluted with PBS to 0, 1, 10 and 100 ppm. 2) Dispense 10 ⁇ L of 0, 1, 10, 100 ppm water-soluble catechins into 1.5 mL tubes. 3) Add 90 ⁇ L of 1.0 ⁇ 10 3 TCID 50 /ml HCoV-229E. 4) Pipette and mix. 5) Immediately after mixing, dilute 2-fold with PBS 6) Then serially dilute 2-fold with PBS 7) Add virus solution to MRC-5 cells seeded at 1.0 ⁇ 10 4 cells/well the day before. 8) Adsorption for 1 hour at room temperature 9) Remove virus solution, wash with PBS, add medium, culture for 5 days 10) Measure TCID 50
  • EGCg 0.25, 0.5, 1, 2.5, 5 and 10 ppm of EGCg was mixed, and quickly diluted twice with PBS containing the same concentration of EGCg. After that, in order to confirm the inhibitory effect of EGCg on adsorption of HCoV-229E to MRC-5 cells, a 2-fold serial dilution was performed with PBS containing the same concentration of EGCg, and MRC-5 cells in a 96-well plate were added for 1 hour. adsorbed. After adsorption of HCoV-229E to the cells, the viral fluid was removed, the cells were washed with PBS, medium was added, and the cells were cultured for 5 days.
  • the virus titer was calculated by the TCID 50 method using cytopathicity as an index.
  • the infectivity was calculated based on the EGCg titer of 0 ppm.
  • Each test was performed in triplicate with 4 wells. As a result, an EGCg concentration-dependent reduction in HCoV-229E infectivity was confirmed. In particular, a significant reduction in infectivity was confirmed, 47% at 2.5 ppm and 82% at 5 ppm and 10 ppm.
  • the results of three tests also confirmed that the EC50 of EGCg against HCoV-229E in this experimental system was 1.66 ppm.
  • the above test protocol is as follows. 1) Water-soluble catechins were diluted with PBS to 0, 1, 2.5, 5, 10, 25, 50 and 100 ppm. 2) Dispense 40 ⁇ L of 0, 1, 2.5, 5, 10, 25, 50, 100 ppm water-soluble catechin into a 1.5 ml tube. 3) Add 360 ⁇ L of 2.5 ⁇ 10 2 TCID 50 /ml HCoV-229E. 4) Pipette and mix. 5) After mixing, immediately dilute 2-fold with PBS containing catechin at the same concentration 6) Then serially dilute 2-fold with PBS containing catechin at the same concentration 7) MRC- 5 Add the virus solution to the cells. 8) Adsorption for 1 hour at room temperature 9) Remove virus solution, wash with PBS, add medium, culture for 5 days 10) Measure TCID 50
  • the above test protocol is as follows. 1) Water-soluble catechins were diluted with PBS to 0, 1, 2.5, 5, 10, 25, 50 and 100 ppm. 2) Dispense 40 ⁇ L of 0, 1, 2.5, 5, 10, 25, 50, 100 ppm water-soluble catechin into a 1.5 ml tube. 3) Add 360 ⁇ L of 2.5 ⁇ 10 2 TCID 50 /ml HCoV-229E. 4) Pipette and mix. 5) After mixing, let stand at room temperature for 1 hour 6) Then serially dilute 2-fold with PBS containing catechin at the same concentration 7) Add the virus solution to MRC-5 cells seeded at 1.0 ⁇ 10 4 cells/well the day before. Add. 8) Adsorption for 1 hour at room temperature 9) Remove virus solution, wash with PBS, add medium, culture for 5 days 10) Measure TCID 50
  • EGCg Cytotoxicity To determine the cytotoxicity of EGCg, Vero-E6/TMPRSS2, MRC-5, MDCK cells in 24-well plates were added to final concentrations of 0, 0.1, EGCg was added dropwise to 1, 10, 100 and 300 ppm, and cultured for 2 days. Cytotoxicity was evaluated by adding WST-8 reagent, allowing to stand at 37°C for 1.5 hours, and measuring the number of viable cells based on absorbance. The 50% cytotoxic concentration (CC 50 ) was calculated based on the viable cell count of cells with an EGCg of 0 ppm. As a result, a decrease in the number of viable cells was confirmed from 10 ppm to 100 ppm in all cells tested.
  • CC 50 of Vero-E6/TMPRSS2 cells, MRC-5 cells and MDCK cells were found to be 67.53 ppm, 65.34 ppm and 85.61 ppm, respectively.
  • the above test protocol is as follows. Toxicity Test of Solubilized Catechin (EGCg) 1) Vero-E6-TMPRSS2 cells or MRC-5 cells were seeded in a well plate at 1.0 ⁇ 10 5 cells/ml (500 ⁇ L medium). 2) Incubate for 17 hours 3) Add 50 ⁇ L of catechin and its solvent so that the concentration is 0 to 1000 ppm. 4) Culture for 48 hours 5) Add 30 ⁇ L of WST-8 reagent and heat at 37° C. for 1.5 hours 6) Transfer 100 ⁇ L of the supernatant to a 96-well plate and measure OD450
  • EGCg reduces the infectivity of HCoV-229E through two actions: a direct inactivation effect and an adsorption inhibition effect.
  • 10 ppm EGCg decreased the HCoV-229E titer by 55% to 85%, demonstrating a certain antiviral effect.
  • its EC 50 was 0.54 ppm to 1.11 ppm, suggesting that it exhibits antiviral effects at very low concentrations.
  • the cytotoxicity results of test [4] revealed that EGCg has cytotoxicity to Vero-E6/TMPRSS2 cells, MRC-5 cells and MDCK cells.
  • Example 14 ⁇ In vivo evaluation using mice (anti-influenza virus activity)> After dropping 3 ⁇ l of catechin solution (0, 1000, 10000 ppm) from both nostrils of four mice in each group, 2 ⁇ l of influenza virus solution (2 ⁇ 10 7 PFU ml) was dropped into both nostrils within 5 minutes. Lungs were removed 3 days after infection, weighed, and homogenized with 2 ml of PBS. The homogenized suspension was centrifuged and the amount of virus in the supernatant was determined by the plaque method. As a result, as shown in Table 13 below, addition of catechin was found to have a statistically significant virus suppressing effect. Table 13-1 shows basic data for in vivo evaluation, and Table 13-2 shows the results of evaluation by T-test (T-test).
  • an aqueous composition containing catechin was added per 100 g of the above formulation.
  • the aqueous composition was charged as a binder liquid for granulation shown below.
  • the raw material for granulation and the raw material for mixing were separately weighed.
  • an aqueous solution composition containing catechin was added to the following materials and stirred. Lactose starch Grape peel pigment Sweetener ⁇ Then, dried at 55°C for 3 hours.
  • the tablets were sized through a sieve so that the diameter of each tablet was 1000 ⁇ m or less. ⁇ The rectified granules and the remaining raw materials were added and mixed. • The mixture was compressed using a tablet press.
  • Example 16 ⁇ Influenza virus inhibition test with tablets (tablets) containing EGCg-containing aqueous solution composition> [Example 16]
  • an in vitro test using MDCK cells was performed. In the in vitro experiment, equal amounts of 10000 PFU virus solution and EGCg solution dissolved in PBS were mixed, and 5000 PFU virus amount was treated at each concentration of EGCg. MDCK cells were infected with a mixture of virus and EGCg, and the plaque method was performed. From the number of plaques formed, the EC50 value was calculated as the concentration that inhibits virus growth by 50%. In vitro experiments were performed with tablets containing no EGCg as a control. Controls had no virus-suppressing effect.
  • compositions and formulations containing catechins according to the present invention have particularly excellent storage stability, are not accompanied by changes in color tone such as browning over time, and are substantially free of lower alcohols and surfactants. Therefore, it has extremely high safety to the living body and excellent storage stability. Therefore, when blended in foods, cosmetics, pharmaceuticals, etc., the beneficial health effects and antioxidant effects of catechins can be expected, and this is an extremely useful technology in industry.

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Abstract

[Problem] To provide a polyphenol preparation in which catechins are present in water stably over a long period of time, and which does not undergo change in color tone such as browning over time, and therefore, is useful for many applications such as beverage and food, cosmetics, medicines, and the like, and useful as a raw material for production thereof; and a production method for the polyphenol preparation. [Solution] This catechin preparation comprises purified water, at least one catechin component, at least one cyclodextrin, and at least one anti-oxidizing agent, and has excellent storage stability.

Description

保存安定性に優れたカテキン類含有水溶液組成物及びその用途Aqueous composition containing catechins with excellent storage stability and use thereof
　本発明は、水性溶剤中における保存安定性に優れたカテキン類を含む各種製剤、特に、優れた保存安定性を有し、しかも経時的な褐変化等の色調変化を伴わず、飲食品、化粧品やその材料、医薬品、工業製品等への配合剤として有用なカテキン類含有水溶液組成物に係る製剤の技術分野に属する。
　さらに本発明は、カテキン類を含む製剤を用い、これを有効成分とする医薬、食料品、化粧料などの各種用途に係る各種製品に係る。 The present invention provides various formulations containing catechins with excellent storage stability in aqueous solvents, particularly foods, beverages and cosmetics that have excellent storage stability and do not cause color tone changes such as browning over time. It belongs to the technical field of preparations related to aqueous compositions containing catechins, which are useful as compounding agents for their materials, pharmaceuticals, industrial products, and the like.
Furthermore, the present invention uses formulations containing catechins, and relates to various products for various uses such as pharmaceuticals, foodstuffs, and cosmetics, which contain the formulations as active ingredients.
　カテキン類は、天然に存在する有機化合物群で、クマル酸ＣｏＡとマロニルＣｏＡが重合してできるカルコンから派生する植物二次代謝物であるフラボノイドの１種である。下記は光学異性体である（＋）－カテキンと（－）－カテキンの構造である。
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000002
 Catechins are a group of naturally-occurring organic compounds, and are one kind of flavonoids, which are plant secondary metabolites derived from chalcone, which is formed by polymerization of coumaric acid CoA and malonyl CoA. Below are the structures of the optical isomers (+)-catechin and (−)-catechin.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000002
　さらに、例えば緑茶中の主なカテキンの成分はエピカテキン、エピガロカテキン、エピカテキンガレート、エピガロカテキンガレート（ＥＧＣｇ）などの約４種類が含まれる。以下はその化学構造を示す。
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000006
 Furthermore, for example, the major catechin components in green tea include about four types, such as epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate (EGCg). The following shows its chemical structure.
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000006
　このカテキン類には、細菌への抑制効果と共にウイルスに対して抗ウイルス効果を発揮するとされる。特にウイルスは、その構造において突起（スパイク）を持っており、これを介して細胞に取りつくジョイント部分があり、スパイクはウイルスによってそれぞれ固有の構造である。このため、ワクチンによる予防は、体の中にウイルスの抗体を作り、その抗体がスパイクの特定構造をとらえてウイルスが細胞へ取りつくのを防止するものである。
　このようなメカニズムであるため、例えばインフルエンザウイルスではスパイクの構造が異なるインフルエンザウイルスが複数あり、ワクチンで作られた抗体とスパイクの特定構造とが結合しなければ、インフルエンザウイルスの感染を防ぐことができないという課題がある。
　一方、カテキンの抗ウイルス効果、つまりウイルスの人体等への感染を予防する効果は、ウイルスが細胞に取りつくジョイント部分を覆ってしまうものと思われ、種々のウイルスのスパイクの特定構造の違いによらず発揮することができる。 These catechins are said to exert an antiviral effect against viruses as well as an inhibitory effect against bacteria. In particular, viruses have protrusions (spikes) in their structures, through which they have joints that attach to cells, and the spikes are unique structures for each virus. For this reason, prevention by means of a vaccine is to create antibodies against the virus in the body, which then capture specific structures on the spikes and prevent the virus from attaching to cells.
Because of this mechanism, for example, there are multiple influenza viruses with different spike structures, and if the antibody produced by the vaccine does not bind to the specific structure of the spike, influenza virus infection cannot be prevented. There is a problem.
On the other hand, the antiviral effect of catechins, that is, the effect of preventing viral infection in the human body, etc., is thought to be due to the covering of the joints where the virus attaches to the cell, and the difference in the specific structure of the spikes of various viruses. You can do it regardless.
　一般に、カテキン類を含むポリフェノール類については、生体内及び生体外での優れた抗酸化作用を持ち、体内の活性酸素を捕捉して除去する（例えば非特許文献１参照）ことにより、活性酸素が引き起こす種々の疾患、例えば、癌、心疾患、炎症といった酸化ストレス疾患に対する予防効果が認められている（例えば非特許文献２参照）。
　また、ポリフェノール類は、その種類に応じて、それぞれ異なる特有の効果が奏される。例えば、アントシアニンには視力の向上効果（例えば非特許文献３）が報告され、イソフラボンには骨粗しょう症予防および治療効果（例えば非特許文献４参照）が報告されている。ルチンには血管拡張効果（例えば非特許文献５参照）が報告されており、飲食品や化粧料、医薬品等々、多くの用途への応用が期待されている。 In general, polyphenols, including catechins, have excellent antioxidant activity in vivo and in vitro, and capture and remove active oxygen in the body (see, for example, Non-Patent Document 1), thereby reducing active oxygen. It has been found to have a preventive effect against various diseases caused by oxidative stress, such as cancer, heart disease, and inflammation (see, for example, Non-Patent Document 2).
In addition, polyphenols exhibit different specific effects depending on their types. For example, anthocyanins have been reported to have an effect of improving eyesight (for example, Non-Patent Document 3), and isoflavones have been reported to have osteoporosis prevention and treatment effects (for example, see Non-Patent Document 4). Rutin has been reported to have a vasodilating effect (see, for example, Non-Patent Document 5), and is expected to be applied to many uses such as food and drink, cosmetics, and pharmaceuticals.
　しかしながら、カテキン類を含むポリフェノール類は、水中に溶存する酸素や空気中の酸素により容易に酸化されるため非常に不安定である。水性溶剤に溶解した際には、期待される活性酸素除去作用等の有用な作用が低下するほか、酸化に伴って変色する等、その保存安定性に欠ける。その結果として、水性溶剤に溶解して液体状態で取り扱うことが困難であり、飲食品、化粧料、医薬品等へ応用する上で大きな障害になっている。 However, polyphenols, including catechins, are very unstable because they are easily oxidized by oxygen dissolved in water and oxygen in the air. When dissolved in an aqueous solvent, the expected useful action such as the action of removing active oxygen is reduced, and the storage stability is poor, such as discoloration due to oxidation. As a result, it is difficult to dissolve it in an aqueous solvent and handle it in a liquid state, which is a major obstacle in applying it to foods, beverages, cosmetics, pharmaceuticals, and the like.
　上記のようなポリフェノール類、特にカテキン類を含む物質を、水性溶剤中で安定化させる技術に関しては、従来既に幾つかの提案がなされている。
　例えば下記の特許文献１には、低級アルコールとモノアミンモノカルボン酸を含有させたポリフェノール製剤が開示されている。
　特許文献２には、ポリオキシエチレンアルキルスルホコハク酸塩を含有させたポリフェノール製剤が開示されている。
　特許文献３には、ポリフェノールを分散させた多価アルコール脂肪酸エステルを含有する油を水中に分散させたs/o/w製剤を製造する技術が開示されている。
　更に、特許文献４には、有機還元剤を添加したポリフェノール製剤が開示されている。 Several proposals have already been made with respect to techniques for stabilizing substances containing polyphenols, particularly catechins, in aqueous solvents.
For example, Patent Document 1 below discloses a polyphenol preparation containing a lower alcohol and a monoamine monocarboxylic acid.
Patent Document 2 discloses a polyphenol preparation containing polyoxyethylene alkyl sulfosuccinate.
Patent Document 3 discloses a technique for producing an s/o/w formulation in which an oil containing a polyhydric alcohol fatty acid ester in which polyphenols are dispersed is dispersed in water.
Furthermore, Patent Document 4 discloses a polyphenol preparation to which an organic reducing agent is added.
　しかしながら、特許文献１～３に記載されたポリフェノール製剤は、その何れも水性溶剤中にアルコールまたは界面活性剤が溶解している。前記低級アルコールや界面活性剤は、生体に対して皮膚刺激性や細胞毒性を有し、皮膚の炎症を誘発させることが多々知られている（例えば非特許文献６参照）。そのため、これをそのまま飲食品、化粧料、医薬品等へ使用するには、生体への安全性という点から好ましくない。
　そこで生体への安全性を考慮して、特に飲食品や化粧品、口腔ケア製品などの分野では、アルコール、界面活性剤を用いない、いわゆる「アルコールフリー」、「界面活性剤フリー」の商品が望まれている。 However, all of the polyphenol preparations described in Patent Documents 1 to 3 have alcohol or surfactant dissolved in aqueous solvent. The lower alcohols and surfactants are often known to have skin irritation and cytotoxicity to living organisms and induce skin inflammation (see, for example, Non-Patent Document 6). Therefore, it is not preferable from the viewpoint of safety to the living body to use it as it is for food, drink, cosmetics, pharmaceuticals, and the like.
Therefore, in consideration of the safety to the living body, so-called "alcohol-free" and "surfactant-free" products that do not use alcohol or surfactants are desired, especially in the fields of food and drink, cosmetics, and oral care products. It is rare.
　また、特許文献１および４に記載されたポリフェノール製剤は、経時的な色調変化のみ評価している。しかし、ポリフェノール製剤の安定化を図るには、成分残存率と色調変化の両方を評価して、双方の安定性を保持させる必要があると考えられている。 In addition, the polyphenol preparations described in Patent Documents 1 and 4 are evaluated only for changes in color tone over time. However, in order to stabilize polyphenol preparations, it is considered necessary to evaluate both the component retention rate and the color tone change and maintain the stability of both.
特開２００１－１１４６５１号公報Japanese Patent Application Laid-Open No. 2001-114651 特開平１１－１９３２２１号公報JP-A-11-193221 特開２００１－３１６２５９号公報JP-A-2001-316259 特開平０６－２３９７１６号公報JP-A-06-239716
　本発明の目的は、上記の背景技術に鑑み、カテキン類が水溶液として長期間安定的に存在し、しかも経時的な褐変化等の色調変化を伴わず、飲食品や化粧料、医薬品等への配合剤として有用な水性液体（水性の剤）で保存安定性に優れたカテキン類を含む製剤を提供することにある。
　本発明の別の目的は、本発明の水溶性組成物の保存安定性が極めて良いことから、カテキン類を有効成分とする、日常的に常温で存置されることが多い健康食品、化粧品補助剤や、口腔ケア製品への添加剤を提供することにある。 In view of the above-mentioned background art, the object of the present invention is to stably exist catechins as an aqueous solution for a long period of time without causing color tone changes such as browning over time, and to be used in foods, cosmetics, pharmaceuticals, etc. An object of the present invention is to provide a preparation containing catechins which is an aqueous liquid (aqueous agent) useful as a compounding agent and has excellent storage stability.
Another object of the present invention is to provide health foods and cosmetic adjuvants, which are often stored at room temperature on a daily basis, containing catechins as an active ingredient, since the water-soluble composition of the present invention has extremely good storage stability. and to provide additives to oral care products.
　本発明者らは、水性溶剤中における保存安定性に優れたカテキン類を含む製剤を製造することについて鋭意検討した結果、意外なことに、カテキン類をサイクロデキストリンにより包接化することで、カテキン類の分解を大幅に抑制できることを見出した。 The present inventors have extensively studied the production of formulations containing catechins that have excellent storage stability in aqueous solvents. It was found that the decomposition of the genus can be greatly suppressed.
　さらに、上記の通りカテキン類をサイクロデキストリンにより包接化すると共に、抗酸化剤を添加することで、カテキン類の分解を抑制する効果を協働的に高めることができ、さらに、色調の褐変化を抑制できることも見出した。
　しかも溶解する際に用いる水の溶存成分を可能な限り除去し、その純度を高めるほどに、得られるカテキン類を含む製剤中のカテキン類の保存安定性が向上することをも見出した。
　さらに上記の通りカテキン類を含む製剤（安定化した組成物）について抗ウイルス活性を有すると共に、これを種々の材料に加えることで当該抗ウイルス活性を発揮させることも見出し、本発明を完成するに至った。 Furthermore, by clathrating the catechins with cyclodextrin as described above and adding an antioxidant, the effect of suppressing the decomposition of the catechins can be enhanced cooperatively, and the browning of the color tone can be enhanced. It was also found that the
Moreover, the inventors have also found that the storage stability of the catechins in the resulting preparation containing catechins is improved by removing the dissolved components of the water used for dissolution as much as possible and increasing the purity thereof.
Furthermore, as described above, it was found that preparations (stabilized compositions) containing catechins have antiviral activity, and that the antiviral activity can be exhibited by adding them to various materials, and the present invention has been completed. Arrived.
　すなわち本発明は以下の発明に係る。
［１］　カテキン類、サイクロデキストリン及び抗酸化剤を含む水溶液組成物であって、
　前記カテキン類は溶解度以下の濃度に溶解され、
　水溶液組成物中、前記サイクロデキストリンは、前記カテキン類に対して１／１０以上のモル量であり、
　４０℃、１４日間で、水溶液組成物中のカテキン類が９６重量％以上残存している、
　保存安定性に優れた水溶液組成物。
［２］　カテキン類、サイクロデキストリン及び抗酸化剤を含む水溶液組成物であって、
　前記カテキン類は前記水溶液組成物中に１０００ｐｐｍ以上の濃度で溶解しており、
　前記水溶液組成物中、前記サイクロデキストリンは、前記カテキン類に対して１／１０以上のモル量であり、
　前記水溶液組成物を４０℃、１４日間保存したときに、水溶液組成物中のカテキン類が５０重量％以上残存している、
　保存安定性に優れた水溶液組成物。
［３］　水に包接化剤としてのサイクロデキストリンを溶解させた後、カテキン類を溶解し、さらに抗酸化剤を加えて溶解させてなる、項［１］又は［２］に記載の組成物。
［４］　包接化剤としてのサイクロデキストリンとカテキン類とを混合した後、水に加えて溶解させ、さらに抗酸化剤を加えて溶解させてなる、項［１］又は［２］に記載の組成物。
［５］　カテキン類が、エピガロカテキンガレートである、項［１］～［４］のいずれかに記載の組成物。
［６］　サイクロデキストリンが、α-サイクロデキストリン、β-サイクロデキストリン及びγ-サイクロデキストリンからなる群より選ばれる１種または２種以上の混合物である、項［１］～［５］のいずれかに記載の組成物。
［７］　抗酸化剤が、アスコルビン酸もしくはその誘導体、またはフィトン酸もしくはその誘導体である、項［１］～［６］のいずれかに記載の組成物。
［８］　項［１］～［７］のいずれかに記載の組成物を、内容物を噴射可能な容器に充填してなる、カテキン類を含むスプレー。
［９］　項［１］～［７］のいずれかに記載の組成物が固形物に含侵されており、錠剤の形態とされてなる、カテキン類を含む錠剤。
［１０］　カテキン類もしくはこれの薬学的に許容可能な塩、サイクロデキストリン及び抗酸化剤を含む水溶液組成物であって、カテキン類を有効成分として含む抗ウイルス剤。
［１１］　ウイルスがコロナウイルスである、項［１０］に記載の抗ウイルス剤。
［１２］　ウイルスが新型コロナウイルス（ＣＯＶＩＤ－１９）である、項［１０］に記載の抗ウイルス剤。 That is, the present invention relates to the following inventions.
[1] An aqueous solution composition containing catechins, cyclodextrin and an antioxidant,
The catechins are dissolved at a concentration below the solubility,
In the aqueous solution composition, the cyclodextrin is in a molar amount of 1/10 or more with respect to the catechins,
96% by weight or more of catechins in the aqueous solution composition remains at 40° C. for 14 days,
Aqueous composition with excellent storage stability.
[2] An aqueous solution composition containing catechins, cyclodextrin and an antioxidant,
The catechins are dissolved in the aqueous solution composition at a concentration of 1000 ppm or more,
In the aqueous solution composition, the cyclodextrin is in a molar amount of 1/10 or more with respect to the catechins,
When the aqueous composition is stored at 40° C. for 14 days, 50% by weight or more of catechins remain in the aqueous composition.
Aqueous composition with excellent storage stability.
[3] The composition according to item [1] or [2], which is obtained by dissolving cyclodextrin as an encapsulating agent in water, dissolving catechins, and then adding and dissolving an antioxidant. .
[4] Item [1] or [2], wherein after mixing cyclodextrin and catechins as an encapsulating agent, the mixture is added to water to dissolve, and then an antioxidant is added to dissolve. Composition.
[5] The composition according to any one of items [1] to [4], wherein the catechin is epigallocatechin gallate.
[6] Any one of items [1] to [5], wherein the cyclodextrin is one or a mixture of two or more selected from the group consisting of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin The described composition.
[7] The composition according to any one of items [1] to [6], wherein the antioxidant is ascorbic acid or a derivative thereof, or phytonic acid or a derivative thereof.
[8] A spray containing catechins, obtained by filling the composition according to any one of items [1] to [7] into a container capable of being sprayed.
[9] A tablet containing catechins, in which the composition according to any one of items [1] to [7] is impregnated in a solid matter and formed into a tablet form.
[10] An antiviral agent, which is an aqueous solution composition containing catechins or a pharmaceutically acceptable salt thereof, cyclodextrin and an antioxidant, which contains catechins as an active ingredient.
[11] The antiviral agent according to item [10], wherein the virus is a coronavirus.
[12] The antiviral agent according to item [10], wherein the virus is a novel coronavirus (COVID-19).
＜カテキン類、サイクロデキストリン及び抗酸化剤を含む水溶液組成物＞
　本発明の水溶液組成物において用いられるカテキン類については、特に制限されない。さらに本発明が保存安定性に優れる水溶性組成物の提供を目的としていることから、具体的には例えば、エピガロカテキンガレート（EGCg）、エピカテキンガレート（ECg）、エピガロカテキン（EGC）、及びエピカテキン（EC）等のカテキン類が好適である。 <Aqueous composition containing catechins, cyclodextrin and antioxidant>
The catechins used in the aqueous composition of the present invention are not particularly limited. Furthermore, since the present invention aims to provide a water-soluble composition having excellent storage stability, specific examples include epigallocatechin gallate (EGCg), epicatechin gallate (ECg), epigallocatechin (EGC), and epicatechin (EC) are preferred.
　上記のカテキン類について、例えばエピカテキン、エピガロカテキン、エピカテキンガレート、エピガロカテキンガレート（ＥＧＣｇ）のいずれか一つを単独で用いても、これを任意に組み合わせた混合物を用いてもよい。
　殊に、皮膚に対しても、その活性酸素除去作用に基づく抗老化作用や、チロシナーゼを阻害することによる美白作用（非特許文献１２参照）が報告され、皮膚癌の抑制作用（非特許文献１３参照）等も報告されていることから、エピガロカテキンガレート（EGCg）、エピカテキンガレート（ECg）、エピガロカテキン（EGC）、及びエピカテキン（EC）等のカテキン類が好ましい。 As for the above catechins, for example, any one of epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate (EGCg) may be used alone, or a mixture of any combination thereof may be used.
In particular, it has been reported that anti-aging action based on its active oxygen scavenging action and whitening action by inhibiting tyrosinase (see Non-Patent Document 12) are also reported for the skin, and skin cancer suppressing action (Non-Patent Document 13). See also), etc., and catechins such as epigallocatechin gallate (EGCg), epicatechin gallate (ECg), epigallocatechin (EGC), and epicatechin (EC) are preferred.
　次に、包接化剤として用いるサイクロデキストリンについて、特に制限されるものではない。例えば、α-サイクロデキストリン、β-サイクロデキストリン、あるいはγ-サイクロデキストリン、又は前記それぞれの剤に対してメチル化、エチル化等のアルキル化サイクロデキストリン、ヒドロキシヒドロキシエチル化、ヒドロキシプロピル化等に装飾化されたアルコキシル化サイクロデキストリン、アセチル化、グルコシル化等に修飾化された様々なサイクロデキストリン誘導体等の適用例を挙げることができる。
　これらのサイクロデキストリンやその誘導体の内でも、多くのカテキン類に対して比較的に優れた包接化による安定化能を示し、また、熱安定性に優れ、無色無臭であって飲食品、化粧料、医薬品等の幅広い用途に使用が許可されていることを考慮すると、α-サイクロデキストリン、β-サイクロデキストリン、もしくはγ-サイクロデキストリン、又はこれらのいずれかあるいは組み合わせた成分とする混合物が好ましい。 Next, the cyclodextrin used as an inclusion agent is not particularly limited. For example, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or alkylated cyclodextrins such as methylation and ethylation, hydroxyhydroxyethylation, hydroxypropylation, etc. for each of the above agents alkoxylated cyclodextrin, various cyclodextrin derivatives modified by acetylation, glucosylation, etc.
Among these cyclodextrins and their derivatives, it shows relatively excellent stabilizing ability by clathration to many catechins, and also has excellent heat stability, is colorless and odorless, and is used in foods, beverages, and cosmetics. Considering that it is permitted to be used for a wide range of applications such as food and pharmaceuticals, α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a mixture of any one or a combination thereof is preferable.
　なお、エピガロカテキンガレート（ＥＧＣｇ）に対するサイクロデキストリンの包接化能に関する本発明者らの検討によれば、サイクロデキストリンの構成糖数が大きいほど、つまりα-サイクロデキストリン＜β-サイクロデキストリン＜γ-サイクロデキストリンの順に、ＥＧＣgの安定性が向上する傾向にあり、β-サイクロデキストリンとγ-サイクロデキストリンを組み合わせることで一層ＥＧＣｇの安定性が高くなることが判明した。よって、サイクロデキストリンとしては、β-サイクロデキストリン、γ-サイクロデキストリン、又はβ-サイクロデキストリンとγ-サイクロデキストリンの組み合わせを適用することが好ましい。 According to the present inventors' study on the ability of cyclodextrin to clathrate epigallocatechin gallate (EGCg), the greater the number of constituent sugars of cyclodextrin, that is, α-cyclodextrin < β-cyclodextrin < γ The stability of EGCg tends to improve in the order of -cyclodextrin, and it was found that the combination of β-cyclodextrin and γ-cyclodextrin further increases the stability of EGCg. Therefore, as the cyclodextrin, it is preferable to apply β-cyclodextrin, γ-cyclodextrin, or a combination of β-cyclodextrin and γ-cyclodextrin.
　本発明において、抗酸化剤について、特に制限されない。例えば、アスコルビン酸及びその誘導体、又はそれらの塩を用いることができ、具体的には、Ｌ－アスコルビン酸や、Ｌ－アスコルビン酸アルキルエステル、Ｌ－アスコルビン酸リン酸エステル、Ｌ－アスコルビン酸硫酸エステル等の誘導体等、又はこれらの塩であるナトリウム塩、カリウム塩等のアルカリ金属塩、カルシウム塩、マグネシウム塩等のアルカリ土類金属塩等を挙げることができる。また、フィトン酸（フィチン酸）またはその塩、エリソルビン酸またはその塩、ロズマリン酸、クロロゲン酸、フィチン酸、クエン酸、フェルラ酸、ルチン、ミリセチン、ミリシトリン、クエルセチン、ユビキノール、メラノイジン、コーヒー酸、没食子酸、その他酸化防止効果を持つカロテノイド類、フラボン誘導体、フラボノイド類、タンニン類及びその塩並びにエステル類、天然植物抽出物としては、ローズマリー抽出物、茶抽出物、りんご抽出物、ブドウ種子抽出物、ひまわり種子抽出物、米ぬか抽出物、セージ抽出物、タイム抽出物、オレガノ抽出物、大豆抽出物、プロポリス抽出物等の適用例を挙げることができる。
　これらの中でも本発明者らの研究によれば、多くのカテキン類に対して比較的優れた抗酸化能を示し、また、熱安定性に優れ、無色無臭であって、飲食品、化粧料、医薬品等々の幅広い用途に使用が許可されていることから、抗酸化剤については、アスコルビン酸、もしくはその誘導体あるいはこれらを主成分とする混合物が好ましい。 In the present invention, the antioxidant is not particularly limited. For example, ascorbic acid, derivatives thereof, or salts thereof can be used, and specific examples include L-ascorbic acid, L-ascorbic acid alkyl esters, L-ascorbic acid phosphates, and L-ascorbic acid sulfates. and derivatives thereof, or salts thereof such as alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, and the like. Phytic acid (phytic acid) or its salts, erythorbic acid or its salts, rosmarinic acid, chlorogenic acid, phytic acid, citric acid, ferulic acid, rutin, myricetin, myricitrin, quercetin, ubiquinol, melanoidin, caffeic acid, gallic acid Acids, other antioxidant carotenoids, flavone derivatives, flavonoids, tannins and their salts and esters, natural plant extracts such as rosemary extract, tea extract, apple extract, grape seed extract , sunflower seed extract, rice bran extract, sage extract, thyme extract, oregano extract, soybean extract, propolis extract and the like.
Among these, according to the research of the present inventors, it exhibits relatively excellent antioxidant ability against many catechins, has excellent heat stability, is colorless and odorless, and is used in food and drink, cosmetics, Ascorbic acid, derivatives thereof, or mixtures containing these as main ingredients are preferred as antioxidants because they are permitted to be used in a wide range of applications such as pharmaceuticals.
　本発明で用いられる水は、通常、公知の方法により処理された精製水が用いられ、精製水を得る精製方法については特に限定されない。本発明で用いられる水は、処理前の原水を、蒸留やろ過、逆浸透、イオン交換などの手法で精製された水で良い。このような精製水の中でも極めて純度の高い水、一般的には伝導率が１８．２ＭΩｃｍ以下の水を超純水と言う。本発明者らの検討によれば、カテキン類、サイクロデキストリン、及び抗酸化剤を含む組成物からなる製剤の製造において、これらの溶解に用いられる水は、高純度で、溶存酸素量が極度に制限されることから、超純水が好ましい。 Purified water treated by a known method is usually used as the water used in the present invention, and the purification method for obtaining purified water is not particularly limited. The water used in the present invention may be water obtained by purifying raw water before treatment by means of distillation, filtration, reverse osmosis, ion exchange, or the like. Among such purified water, water with extremely high purity, generally water with a conductivity of 18.2 MΩcm or less, is called ultrapure water. According to the studies of the present inventors, in the production of a formulation comprising a composition containing catechins, cyclodextrins, and antioxidants, the water used for dissolving these is highly pure and has an extremely high dissolved oxygen content. Due to limitations, ultrapure water is preferred.
　本発明の水溶液組成物やそれを用いた製剤中のカテキン類が溶解している濃度は、その濃度が高いほど有益な効果を高められるため、溶解している濃度は高いほどよい。ただし、溶解している濃度が所定温度条件下でのカテキン類の水に対する溶解度を超えてしまうと、溶解系の安定性が低下し、経時的に沈殿を生じる場合がある。
　したがって、カテキン類の種類によっても異なるが、本発明の水溶液組成物中のカテキン類の溶解している濃度は、所定温度条件下でのカテキン類の溶解度以下であることが好ましい。例えばＥＧＣｇの水への溶解度は２０℃で４ｇ／１００ｍＬ、すなわち４００００ｐｐｍであり、本発明の水溶液組成物中のカテキン類の溶解度は共存する物質の種類、濃度、温度、その他の条件の下で定まるものである。 The higher the concentration of catechins dissolved in the aqueous composition of the present invention and the formulation using the same, the higher the beneficial effect, so the higher the dissolved concentration, the better. However, if the concentration of dissolved catechins exceeds the solubility of catechins in water under predetermined temperature conditions, the stability of the dissolved system decreases, and precipitation may occur over time.
Therefore, the concentration of dissolved catechins in the aqueous composition of the present invention is preferably lower than the solubility of catechins under predetermined temperature conditions, although it varies depending on the type of catechins. For example, the solubility of EGCg in water is 4 g/100 mL at 20° C., that is, 40000 ppm, and the solubility of catechins in the aqueous composition of the present invention is determined by the type, concentration, temperature, and other conditions of coexisting substances. It is.
　例えば、水溶液中に１０００ｐｐｍまたはそれ以上、２０００ｐｐｍまたはそれ以上、５０００ｐｐｍまたはそれ以上、１００００ｐｐｍまたはそれ以上のカテキン類を含有させることで、各種用途に用いることができる。
　具体的には水溶液中に２０００ｐｐｍ含有した水溶性組成物の場合、保存により５０％のカテキン類が分解等して残存しなくなっても１０００ｐｐｍのカテキン類が残存する。１０００ｐｐｍのカテキン類が残存しておれば、抗ウイルス効果などもまだ十分に期待できる。
　あるいは、水溶液中に１００００ｐｐｍ含有した水溶性組成物を全量の１０容量％の量を添加した食品等の場合でも、１０００ｐｐｍのカテキン類を含有することになる。このような濃度であれば、抗ウイルス効果などもまだ十分に期待できる。
　なお、包接化合物であるサイクロデキストリン共存下の場合、包接されるカテキン類の溶解する濃度は、カテキン類の存在形態により変動することがあるため、必要に応じて、溶解度を測定しておくとよい。 For example, by containing 1000 ppm or more, 2000 ppm or more, 5000 ppm or more, or 10000 ppm or more of catechins in the aqueous solution, it can be used for various purposes.
Specifically, in the case of a water-soluble composition containing 2000 ppm in an aqueous solution, 1000 ppm of catechins remain even if 50% of catechins do not remain due to decomposition or the like during storage. If 1000 ppm of catechins remain, antiviral effects can still be fully expected.
Alternatively, even in the case of food to which 10% by volume of the total amount of the water-soluble composition containing 10000 ppm in the aqueous solution is added, 1000 ppm of catechins are contained. At such a concentration, antiviral effects and the like can still be fully expected.
In addition, in the presence of cyclodextrin, which is an inclusion compound, the concentration at which the included catechins are dissolved may vary depending on the form of existence of the catechins, so the solubility should be measured as necessary. Good.
　上記カテキン類とサイクロデキストリンとの混合比率は、カテキン類を溶解するための水における溶解する濃度を固定した場合には、サイクロデキストリンの溶解している濃度が高くなればなるほど、包接化によるカテキン類への安定化効率が向上する。逆に、サイクロデキストリンの溶解している濃度が低くなればなるほど、カテキン類の包接化による安定化効率が低下して、カテキン類の安定性が低下する。
　カテキン類およびサイクロデキストリンの種類よっても異なるが、カテキン類のモル濃度（前記溶解濃度において水溶液組成物１Ｌ中に含まれるカテキン類のモル数。以下同じ。）に対する、サイクロデキストリンのモル濃度が１／１０未満になると、カテキン類の安定性が著しく低下することがある。そのため本発明の場合は、カテキン類のモル濃度に対するサイクロデキストリンのモル濃度は１／１０以上、さらに１／１０～１／１、特に１／６～１／３であることが好ましい。
　なお上記は、カテキン類に対する加えるサイクロデキストリンのモル比としたときは、１０モル％以上、さらに１０モル％～６６．６モル％、特に１６．６モル％～３３．３モル％であることが好ましい。
　ただし、サイクロデキストリンの溶解している濃度が高くなればなるほど、溶解系の安定性が低下し、経時的に沈殿を生じる場合がある。
　したがって、サイクロデキストリンの溶解している濃度の上限は、共存する物質の種類、濃度、温度、その他の条件の下で定まるものの、サイクロデキストリンの各分子種（α－サイクロデキストリン、β－サイクロデキストリン、γ－サイクロデキストリンなど）の所定条件下での溶解度以下であることが好ましい。
　なお、サイクロデキストリン各分子種の水への溶解度は、２５℃でα－サイクロデキストリンが １４．５ｇ／１００ｍＬ、β－サイクロデキストリンは１．８ｇ／１００ｍＬ、γ－サイクロデキストリンは２３．２ｇ／１００ｍＬである。 The mixing ratio of the above catechins and cyclodextrins is such that when the concentration of catechins dissolved in water for dissolving catechins is fixed, the higher the concentration of dissolved cyclodextrin, the more catechins are produced by clathration. Stabilization efficiency to the class is improved. Conversely, the lower the dissolved cyclodextrin concentration, the lower the efficiency of stabilization by clathration of catechins, and the lower the stability of catechins.
Although it varies depending on the types of catechins and cyclodextrins, the molar concentration of catechins (the number of moles of catechins contained in 1 L of the aqueous solution composition at the above-mentioned dissolved concentration; the same applies hereinafter) of cyclodextrin is 1/ If it is less than 10, the stability of catechins may be remarkably lowered. Therefore, in the case of the present invention, the molar concentration of cyclodextrin to the molar concentration of catechins is preferably 1/10 or more, more preferably 1/10 to 1/1, particularly 1/6 to 1/3.
In addition, the above is 10 mol% or more, further 10 mol% to 66.6 mol%, particularly 16.6 mol% to 33.3 mol% when the molar ratio of the cyclodextrin to be added to the catechins is 10 mol% or more. preferable.
However, the higher the dissolved concentration of cyclodextrin, the lower the stability of the dissolved system, which may result in precipitation over time.
Therefore, although the upper limit of the dissolved cyclodextrin concentration is determined by the type, concentration, temperature, and other conditions of coexisting substances, each molecular species of cyclodextrin (α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, etc.) under predetermined conditions.
The solubility of each molecular species of cyclodextrin in water at 25° C. is 14.5 g/100 mL for α-cyclodextrin, 1.8 g/100 mL for β-cyclodextrin, and 23.2 g/100 mL for γ-cyclodextrin. be.
　本発明の水溶性組成物はカテキン類、サイクロデキストリン及び抗酸化剤を含み、その結果種々の用途製品において、高い保存安定性を有する。
　以下の実施例に示すように、４０℃で１４日間の保存であっても、４０℃の保存前のカテキン類の量に対し、カテキン類の残存している量が９６重量％以上ある。このデータを加速劣化試験として鑑みると、食品等が通常保管される常温（２５℃）での保管期間を長期間に設定することが期待できる。例えば、４０℃で１４日間の保存が可能であれば、食品あるいは機能性食品などとすれば２５℃で３か月以上など数か月以上の保管が可能と考えられ、その期間、カテキン類による抗ウイルス効果を維持できることが期待できる。 The water-soluble composition of the present invention contains catechins, cyclodextrins and antioxidants, and as a result has high storage stability in products for various uses.
As shown in the examples below, even after storage at 40°C for 14 days, the remaining amount of catechins is 96% by weight or more relative to the amount of catechins before storage at 40°C. Considering this data as an accelerated deterioration test, it can be expected that the storage period at room temperature (25° C.) in which foods and the like are normally stored can be set for a long period of time. For example, if it is possible to store food or functional food at 40°C for 14 days, it is thought that storage at 25°C for 3 months or more is possible for several months or more. It can be expected that the antiviral effect can be maintained.
　本発明の水溶性組成物は、含有するカテキン類の安定性が高く、その安定性とは一つには含有するカテキン類が分解等せず残存する比率が極めて高い。またそのことにより、二つ目に色相が安定し、褐変等などによる着色が極めて少ない。
　以下で示す実施例ではカテキン類を含む水溶性組成物を４０℃で１４日間の保存の前後でのカテキン類の残存量を測定している。
　その場合、保存後のカテキン類の残存量について、抗ウイルス効果などの効能を維持できているかが重要である。本発明の水溶性組成物は、当該水溶液組成物の使用目的にもよるが、４０℃の保存前のカテキン類の量に対し、カテキン類の残存が５０重量％以上あるとよく、好ましくは８０重量％以上あるとよく、さらに好ましくは９０重量％以上あるとよく、特に好ましくは９６重量％以上あるとよい。このように残存率が高いため、本発明の水溶性組成物は各種用途に用いることができる。 In the water-soluble composition of the present invention, the catechins contained therein are highly stable, and one of the reasons for this stability is that the catechins contained therein remain at an extremely high ratio without decomposition or the like. In addition, secondly, the hue is stabilized, and coloring due to browning or the like is extremely small.
In the examples shown below, the amount of residual catechins was measured before and after storage of a water-soluble composition containing catechins at 40° C. for 14 days.
In that case, it is important to maintain efficacy such as antiviral effect with respect to the residual amount of catechins after storage. Depending on the purpose of use of the aqueous composition, the water-soluble composition of the present invention preferably contains 50% by weight or more of catechins, preferably 80% by weight of the catechins before storage at 40°C. It is preferably at least 90% by weight, more preferably at least 96% by weight, and particularly preferably at least 96% by weight. Due to such a high residual rate, the water-soluble composition of the present invention can be used for various purposes.
　上記したように、水溶液中に１０００ｐｐｍまたはそれ以上含まれるとよく、好ましくは２０００ｐｐｍまたはそれ以上、さらに好ましくは５０００ｐｐｍまたはそれ以上、特に好ましくは１００００ｐｐｍまたはそれ以上のカテキン類を含有させることで、本発明の水溶性組成物は各種用途に用いることができる。
　具体的には水溶液中に２０００ｐｐｍ含有した水溶性組成物の場合、長期の保存や、高温に晒されるなどした後に仕込み時に存在していたカテキン類の量に対して５０％のカテキン類が分解等して残存しなくなっても、まだ１０００ｐｐｍのカテキン類が残存する。１０００ｐｐｍのカテキン類が残存しておれば、抗ウイルス効果などもまだ十分に期待できる。
　また、水溶液中に１００００ｐｐｍ含有した水溶性組成物を全量の１０％容量分添加した食品等の場合、あるいは水溶液中に５０００ｐｐｍ含有した水溶性組成物を全量の２０％容量分添加した食品等の場合でも、１０００ｐｐｍのカテキン類を含有することになる。このような濃度であれば、抗ウイルス効果などもまだ十分に期待できる。 As described above, the aqueous solution preferably contains 1000 ppm or more, preferably 2000 ppm or more, more preferably 5000 ppm or more, and particularly preferably 10000 ppm or more. can be used for various purposes.
Specifically, in the case of a water-soluble composition containing 2000 ppm in an aqueous solution, 50% of the catechins present at the time of preparation are decomposed after long-term storage or exposure to high temperatures. 1000 ppm of catechins still remain even if they do not remain. If 1000 ppm of catechins remain, antiviral effects can still be fully expected.
In addition, in the case of foods to which 10% volume of the total amount of water-soluble composition containing 10000 ppm in the aqueous solution is added, or in the case of food etc. to which 20% volume of the total amount of the water-soluble composition containing 5000 ppm in the aqueous solution is added. However, it will contain 1000 ppm of catechins. At such a concentration, antiviral effects and the like can still be fully expected.
　＜カテキン類を含む水溶液組成物の抗ウイルス効果＞
　上記の通り、本発明のカテキン類を含む水溶液組成物は、驚くべきことに抗ウイルス効果を有することが分かった。そこで、本発明者らがさらに各種剤型にて検討したところ、インフルエンザウイルスに効果的に抑制できることが分かった。以下では、本発明のカテキン類を含む水溶液組成物の抗ウイルス効果に基づいた、各種用途に適用可能できる剤について説明する。 <Antiviral effect of aqueous composition containing catechins>
As described above, it was found that the aqueous composition containing catechins of the present invention surprisingly has an antiviral effect. Therefore, when the present inventors further examined various dosage forms, it was found that the influenza virus can be effectively suppressed. Hereinafter, agents that can be applied to various uses based on the antiviral effect of the aqueous composition containing catechins of the present invention will be described.
　上記の通り、カテキン類には細菌への抑制効果と共にウイルスに対して抗ウイルス効果を発揮するとされるが、本発明のカテキン類を含む水溶液組成物は、サイクロデキストリンと抗酸化剤を含む組成物であり、飲食品や化粧料、医薬品等への配合剤として有用である。特に有機溶剤（エタノールなどは除く）その他の人体に有害となりうる材料を用いることなく、抗ウイルス効果を発揮できる。 As described above, catechins are said to exert an antiviral effect against viruses as well as an inhibitory effect on bacteria. The aqueous composition containing catechins of the present invention is a composition containing cyclodextrin and an antioxidant. and is useful as a compounding agent for foods, drinks, cosmetics, pharmaceuticals, and the like. In particular, the antiviral effect can be exhibited without using organic solvents (excluding ethanol, etc.) and other materials that can be harmful to the human body.
　このため、例えばマウススプレーなどの口腔ケア製品や、チューインガム、キャンディー類、菓子パンなど各種食品へ添加する添加剤、種々の加工食品、化粧品への添加剤、酒類への添加剤など、各種用途に有用である。
　また犬、猫、その他動物のペット飼育や、牛などの牧畜、豚、鶏等の飼育において、動物を介しての人へのウイルス感染もありうるため、その抗ウイルス効果が求められる。またその形態として、一般の殺菌剤と同様に靴などに付着したウイルスを殺菌、除去するために布に液体を浸した措置も考えられるが、カテキン類という自然に存在する茶葉の成分を水溶液とし、当該水溶液を噴射するスプレーの形態とするが望ましい。 Therefore, it is useful for various applications such as oral care products such as mouth sprays, additives added to various foods such as chewing gum, candies, and pastries, various processed foods, additives for cosmetics, and additives for alcoholic beverages. is.
In addition, in raising dogs, cats, and other animals as pets, raising livestock such as cattle, and raising pigs, chickens, etc., there is a possibility of virus infection to humans through animals, so an antiviral effect is required. In addition, as a form, it is possible to soak a cloth in liquid to sterilize and remove viruses attached to shoes, as with general disinfectants. , it is desirable that the aqueous solution be in the form of a spray.
　本発明のカテキン類を含む水溶液組成物は、特にウイルス類の抑制効果が認められるところ、その効果を示すウイルス類としては特に制限されない。
　例えばウイルスの分類によれば、２本鎖ＤＮＡ、１本鎖ＤＮＡ、２本鎖ＲＮＡ、１本鎖ＲＮＡなどを有した各種ウイルスがあり（非特許文献１４参照）、これらに分類されるウイルスの中でも、ニドウイルス目コロナウイルス科に分類されるＡ型インフルエンザウイルス、Ｂ型インフルエンザウイルス、Ｃ型インフルエンザウイルスなどのインフルエンザウイルスやその変異株に抑制効果が認められる。また近年その爆発的な感染が問題となっている新型コロナウイルス（ＣＯＶＩＤ－１９）やその変異株についての抑制効果も気がかりとなる。
　本発明のカテキン類を含む水溶液組成物は、インフルエンザウイルス又はその類縁ウイルスに抑制効果が認められ、同じ１本鎖ＲＮＡを有し、突起（スパイク）を介した細胞感染という観点で、新型コロナウイルス（ＣＯＶＩＤ－１９）やその変異株についても抑制効果を十分に期待できる。
　さらにｉｎ　ｖｉｖｏによる評価を実施すると、抗ウイルス効果が認められることが分かった。 The aqueous composition containing catechins of the present invention is not particularly limited as a virus exhibiting the effect, as long as the effect of suppressing viruses is recognized.
For example, according to the classification of viruses, there are various viruses having double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, etc. (see Non-Patent Document 14). Among them, the inhibitory effect is observed in influenza viruses such as influenza A virus, influenza B virus, and influenza C virus classified in the family Coronaviridae of the order Nidoviridae, and their mutant strains. In addition, there is concern about the effect of suppressing the new coronavirus (COVID-19) and its mutant strains, whose explosive infections have become a problem in recent years.
The aqueous solution composition containing catechins of the present invention has an inhibitory effect on influenza virus or its analogous viruses, has the same single-stranded RNA, and has the same single-stranded RNA, and from the viewpoint of cell infection via spikes, novel coronavirus (COVID-19) and its mutant strains can also be expected to have a sufficient suppressive effect.
Furthermore, when an in vivo evaluation was performed, it was found that an antiviral effect was observed.
　本発明に係るカテキン類含有水溶液組成物の用途としては、上記の通り、口腔ケア製品や添加剤があるが、その形態としては、スプレーなどに用いられる水溶液の形態、タブレットあるいは錠剤のような固形状の形態が挙げられる。 Applications of the catechins-containing aqueous composition according to the present invention include, as described above, oral care products and additives. Examples include the form of shape.
　水溶液の形態として端的に示せばスプレーとして、容器にカテキン類を含む水溶液組成物を導入したものを挙げることができる。容器としては、ガラス、プラスチック、金属等のカテキン類を含む水溶液組成物中の各主成分の物性、安定性などに影響を与えないものであれば特に制限されない。 As an example of the form of an aqueous solution, a spray can include a container in which an aqueous solution composition containing catechins is introduced. The container is not particularly limited as long as it does not affect the physical properties, stability, etc. of each main component in the aqueous solution composition containing catechins, such as glass, plastic, and metal.
　また固体状の形態として端的に示せば錠剤あるいはタブレットとして、カテキン類を含む水溶液組成物を固体状の成分へ含浸させるなどし、常法により錠剤あるいはタブレットとしたものを挙げることができる。錠剤あるいはタブレットとする前に、より粒径の小さな粉末あるいは顆粒とした後に錠剤化することもできる。 In addition, as a solid form, tablets or tablets can be mentioned, which are obtained by impregnating a solid component with an aqueous solution composition containing catechins and making tablets or tablets by a conventional method. Before making tablets or tablets, they may be made into powder or granules having a smaller particle size and then tableted.
　以下、カテキン類を含む水溶液組成物の用途として、スプレー、タブレットの形態を例として説明する 　Hereinafter, as examples of applications of the aqueous composition containing catechins, the forms of sprays and tablets will be explained.
＜スプレー＞
　容器にカテキン類を含む水溶液組成物を導入したスプレーの形態では、水溶液形態としては変わらない。このため、その製造は所定量のカテキン類を含む水溶液組成物に、さらに必要に応じて添加物を加えた形態とする。
　例えば、酸化防止剤、ｐＨ調整剤などを挙げることができる。
　実際にスプレーとして使用する場合、当該水溶液組成物中にＥＧＣｇなどのカテキン類を１０００ｐｐｍまたはそれ以上含まれるとよく、好ましくは２０００ｐｐｍまたはそれ以上、さらに好ましくは５０００ｐｐｍまたはそれ以上、特に好ましくは１００００ｐｐｍまたはそれ以上のカテキン類を含有する組成物が挙げられる。 <Spray>
In the form of a spray in which an aqueous solution composition containing catechins is introduced into a container, the form of the aqueous solution does not change. For this reason, it is produced in the form of an aqueous solution composition containing a predetermined amount of catechins and, if necessary, additives.
Examples include antioxidants and pH adjusters.
When actually used as a spray, the aqueous composition preferably contains 1000 ppm or more of catechins such as EGCg, preferably 2000 ppm or more, more preferably 5000 ppm or more, and particularly preferably 10000 ppm or more. Compositions containing the above catechins are included.
　スプレーの一押しで０．１ｍｌの水溶液が噴霧される場合、その噴霧された水溶液が１０リットルの空間に噴霧されれば、当該空間中のカテキン類は空間中の濃度としては、１０リットル÷０．１ｍｌ＝１００００倍の希釈となる。しかしながら、水溶液が小さな液滴として１０リットルの空間に噴霧されただけであるため、液体が揮発してガスにならない限り、液滴中のカテキン類濃度は当初通りであり、当該液滴がウイルスと接触すれば十分な抗ウイルス効果を発揮することを期待できる。 When 0.1 ml of aqueous solution is sprayed with one push of the spray, if the sprayed aqueous solution is sprayed in a 10 liter space, the concentration of catechins in the space is 10 liters/0 .1 ml = 10000 fold dilution. However, since the aqueous solution was only sprayed as small droplets into a 10-liter space, the concentration of catechins in the droplets remained the same as before, unless the liquid volatilized into gas, and the droplets were considered to be viruses. It can be expected that sufficient antiviral effects will be exerted by contact.
　スプレーとしてのカテキン類を含む水溶液組成物の配合としては、本発明の効果が得られる範囲であれば特に制限はないが、通常、カテキン類を１０００ｐｐｍ含有する水溶液組成物の場合であれば、カテキン類とそれを安定化するためのサイクロデキストリン、抗酸化剤や、増量剤、防腐剤、その他の添加剤が挙げられる。例えば、グリセリン、Ｄ－ソルビトール、プロピレングリコール、トレハロース水和物、香料、クエン酸もしくはそのＮａ塩、香料としてのｌ－メントールなどが挙げられる。 The formulation of the aqueous composition containing catechins as a spray is not particularly limited as long as the effects of the present invention can be obtained. and cyclodextrins to stabilize it, antioxidants, bulking agents, preservatives and other additives. Examples thereof include glycerin, D-sorbitol, propylene glycol, trehalose hydrate, fragrance, citric acid or its Na salt, l-menthol as a fragrance, and the like.
＜タブレット＞
　本発明のカテキン類を含む水溶液組成物は、ガレート型カテキンを高濃度で含有しながら苦渋味が低減され、かつ優れた打錠適性を有します。また、この打錠されたタブレット形態の剤は、抗ウイルス効果を保持したままであり、その効果の持続性についても優れており、使いやすいという特徴がある。 <Tablet>
The aqueous composition containing catechins of the present invention has reduced bitterness and astringency while containing gallate-type catechins at a high concentration, and has excellent tableting aptitude. In addition, this compressed tablet-form drug retains its antiviral effect, is excellent in persistence of the effect, and is easy to use.
（１）ガレート型カテキン含有顆粒
　本発明のカテキン類を含む水溶液組成物は溶液の形態であるため、タブレットにするには沈殿にして固体状にするか、粉末あるいは顆粒状の物質に噴霧等して含浸させ、それを造粒して固体形態にすることでよい。具体的な固体形態は常法によるものとでよい。
　上記の通り、本発明のカテキン類を含む水溶液組成物より得られたカテキン類含有顆粒におけるカテキン類の含有量は、抗ウイルス作用などの効果を奏する配合であれば特に制限はない。顆粒の用途としては、そのまま顆粒剤として用いる場合や、さらに顆粒を打錠して錠剤、タブレットとして用いることもできる。 (1) Gallated catechin-containing granules Since the aqueous composition containing catechins of the present invention is in the form of a solution, it can be made into a tablet either by precipitation into a solid state, or by spraying on a powder or granular substance. and granulating it into a solid form. Specific solid forms may be those according to conventional methods.
As described above, the content of the catechins in the catechins-containing granules obtained from the aqueous solution composition containing the catechins of the present invention is not particularly limited as long as it is a blend that exhibits effects such as antiviral action. The granules can be used as they are as granules, or can be further compressed and used as tablets.
　カテキン類含有顆粒には、カテキン類の他、上記した添加剤、安定化剤、増量剤等の各種成分が含まれる。また顆粒として飲用するため、飲みやすくするための添加剤とししたり、添加物の特異的な呈味をマスキングするために、高甘味度甘味料を使用することもできる。例えば高甘味度甘味料としては、例えば、アセスルファムカリウム、スクラロース、ステビア、アスパルテーム、ネオテーム、ソーマチン、カンゾウ、サッカリン等が挙げられ、これらの１種または２種以上を使用することができる。 The catechin-containing granules contain various ingredients such as the above-mentioned additives, stabilizers, and bulking agents in addition to catechins. Moreover, since it is to be drunk as granules, it is possible to use a high-intensity sweetener as an additive to make it easier to drink or to mask the specific taste of the additive. Examples of high-intensity sweeteners include acesulfame potassium, sucralose, stevia, aspartame, neotame, thaumatin, licorice, saccharin, etc. One or more of these can be used.
　本発明のカテキン類を含む水溶液組成物を含むカテキン類含有顆粒における高甘味度甘味料の含有量は添加物の特異的な呈味をマスキングすることができる量であれば特に制限されないが、例えば０．０１重量％～１．０重量％、さらに０．１重量％～０．５重量％であることが好ましい。
　またカテキン類含有顆粒の粒径としては特に制限されるものではないが、飲用時あるいは舐める際、円滑あるいは馴染みやすくするため、比較的細かな粒径としてもよい。例えば１００μｍ～１ｍｍ程度の粒径とすることが挙げられる。このような粒径とすることで、カテキン類特有の苦渋味を感じることが少なくなり、固形物としての流動性がありながらも、取り扱いの際に粉状に舞い上がったりせず、食べる際にも咳き込んだり、さらには口内に付着したりするなどの支障がなく、飲用し易く、また舐めやすくなる。また、カテキン類含有顆粒は、顆粒状で取り扱い易いことから打錠適性にも優れる。 The content of the high-intensity sweetener in the catechins-containing granules containing the aqueous solution composition containing catechins of the present invention is not particularly limited as long as it is an amount capable of masking the specific taste of the additive. It is preferably 0.01% to 1.0% by weight, more preferably 0.1% to 0.5% by weight.
The particle size of the catechins-containing granules is not particularly limited, but may be relatively fine in order to make it smooth or easy to absorb when drinking or licking. For example, the particle size may be about 100 μm to 1 mm. By making it into such a particle size, the bitter and astringent taste peculiar to catechins is less felt, and even though it has fluidity as a solid, it does not rise to powder when handled, and it is easy to eat. It is easy to drink and easy to lick without causing problems such as coughing or sticking in the mouth. In addition, since the catechin-containing granules are granular and easy to handle, they are also excellent in tableting aptitude.
　上記カテキン類含有顆粒中には、食感を阻害しない程度に、糖質、酸味料、香料、着色料、食物繊維、ビタミン類、ミネラル類、アミノ酸、油脂、乳化剤、増粘多糖類、滑沢剤等の各種成分を含有してもよい。これらの各種成分を適宜選択することで、顆粒またはチュアブルタブレットを飲用に適し、あるいは舐めやすくなり、小児や高齢者にも使用しやすくなる。 Carbohydrates, acidulants, flavors, coloring agents, dietary fibers, vitamins, minerals, amino acids, fats and oils, emulsifiers, polysaccharide thickeners, lubricants, and It may contain various components such as agents. By appropriately selecting these various ingredients, the granules or chewable tablets become suitable for drinking or easy to lick, making them easy to use for children and the elderly.
（２）チュアブルタブレット
　本発明のカテキン類を含む水溶液組成物を用いたチュアブルタブレットは、上記の本発明のカテキン類を含む水溶液組成物を含むカテキン類含有顆粒を打錠して得られる。このため、チュアブルタブレットに使用される成分やその組成は、カテキン類含有顆粒と同じである。
　また、上記の通り各種成分を含有しているカテキン類含有顆粒を打錠してチュアブルタブレットを作製してもよいが、その他の手法として、カテキン類含有顆粒を作製し、これに、糖質、酸味料、香料、着色料、食物繊維、ビタミン類、ミネラル類、アミノ酸、油脂、乳化剤、増粘多糖類、滑沢剤等の各種成分を混合し、この混合物を打錠することでチュアブルタブレットを作製してもよい。これらの各種成分を適宜選択することで、チュアブルタブレットが飲用に適し、あるいは舐めやすくなり、小児や高齢者にも使用しやすくなる。 (2) Chewable Tablet A chewable tablet using the aqueous composition containing catechins of the present invention is obtained by compressing catechin-containing granules containing the above-mentioned aqueous composition containing catechins of the present invention. Therefore, the ingredients and composition used for the chewable tablet are the same as those for the catechins-containing granules.
As described above, catechin-containing granules containing various ingredients may be compressed to prepare a chewable tablet, but as another method, catechin-containing granules are prepared, sugar, Various ingredients such as acidulants, flavoring agents, coloring agents, dietary fibers, vitamins, minerals, amino acids, fats and oils, emulsifiers, polysaccharide thickeners, and lubricants are mixed, and the mixture is tableted to form chewable tablets. may be made. By appropriately selecting these various ingredients, the chewable tablet becomes suitable for drinking or easy to lick, and is easy to use for children and the elderly.
　チュアブルタブレットは、子供から大人、さらには高齢者まで口に入れて食べ易いサイズであればよく、特に限定されない。その形状としては、円柱状、三角柱状、四角柱状等が挙げられるが、特に限定されない。 The chewable tablet is not particularly limited as long as it is a size that is easy for children, adults, and even the elderly to put in their mouths and eat. The shape thereof may be cylindrical, triangular prismatic, quadrangular prismatic, or the like, but is not particularly limited.
（３）カテキン類含有顆粒およびチュアブルタブレットの製造方法
　上記のカテキン類含有顆粒は、各種成分を含む粉末あるいは顆粒に、カテキン類を含有する水溶液を混合または噴霧し、次いで造粒することで製造できる。 (3) Method for producing catechins-containing granules and chewable tablets The above-mentioned catechins-containing granules can be produced by mixing or spraying an aqueous solution containing catechins with powders or granules containing various ingredients, followed by granulation. .
　具体的には、まず、カテキン類を水などの溶媒に溶解させてカテキン類を含む水溶液を得る。
　次に、各種添加物を含む粉末または顆粒に、カテキン類を含有する水溶液を混合または噴霧し、流動層造粒法、転動造粒法、押出造粒法、撹拌造粒法のいずれかの造粒法を用いて造粒できる。
　これらの造粒法の中でも、流動層造粒法が、カテキン類を、各種添加物を含む粉末または顆粒に均一に被覆でき、均一で打錠適性の優れた粒子が得られやすいため好ましく用いることができる。用いる造粒法には、製薬、菓子製造等に使用する一般に使用されている造粒装置を用いればよい。また粉体粒子どうしを結着させるためのアラビアガムなどのバインダーは必要であれば用いることができる。 Specifically, first, catechins are dissolved in a solvent such as water to obtain an aqueous solution containing catechins.
Next, the powder or granules containing various additives are mixed or sprayed with an aqueous solution containing catechins, and the powder or granules are mixed or sprayed, followed by fluid bed granulation, tumbling granulation, extrusion granulation, or stirring granulation. It can be granulated using a granulation method.
Among these granulation methods, the fluid bed granulation method is preferably used because catechins can be uniformly coated on powders or granules containing various additives, and uniform particles with excellent tableting suitability can be easily obtained. can be done. As for the granulation method used, a granulation apparatus generally used for manufacturing pharmaceuticals, confectionery, etc. may be used. A binder such as gum arabic for binding powder particles together can be used if necessary.
　この造粒は、カテキン類を劣化させないために室温～９０℃くらいで行うことが好ましい。
　また、顆粒を造粒工程中に完全に乾燥させることも可能であるが、造粒装置から取り出してから所定の温度、例えば常温付近で静置するなどしてさらに乾燥を行なってもよい。
　さらに、カテキン類を含む水溶液について、造粒温度、造粒時間などを適宜調整して造粒を行うことで、所望の粒径の顆粒を得ることができ、さらにその粒径を揃えるために適度な篩を通過させて、阻止された側あるいは通貨側の顆粒を用いることができる。 This granulation is preferably carried out at room temperature to about 90° C. so as not to deteriorate the catechins.
It is also possible to dry the granules completely during the granulation process, but after taking them out of the granulator, the granules may be further dried by allowing them to stand at a predetermined temperature, for example around room temperature.
Furthermore, for an aqueous solution containing catechins, granules with a desired particle size can be obtained by appropriately adjusting the granulation temperature, granulation time, etc., and the appropriate amount is used to make the particle sizes uniform. It is possible to use granules on the blocked side or currency side by passing them through a fine sieve.
　次に、上記のチュアブルタブレットは、カテキン類含有顆粒を打錠することで得られる。打錠は乾式打錠により行うことが好ましい。打錠に使用する打錠機は特に限定されず、ロータリー打錠機、単発打錠機などを用いることができる。 Next, the above chewable tablet is obtained by compressing the catechin-containing granules. Compression is preferably performed by dry compression. The tableting machine used for tableting is not particularly limited, and a rotary tableting machine, a single shot tableting machine, or the like can be used.
　上記の顆粒やチュアブルタブレットに添加することがある高甘味度甘味料は、以上の工程中の任意の段階で添加することができるが、カテキン類含有水溶液中に溶解させると、カテキン類含有顆粒またはチュアブルタブレット中に均一に分散させることができるため好ましい。その他任意成分も任意の段階で添加することができる。 The high-intensity sweetener that may be added to the above granules and chewable tablets can be added at any stage during the above process, but when dissolved in the catechins-containing aqueous solution, the catechins-containing granules or It is preferable because it can be uniformly dispersed in the chewable tablet. Other optional components can also be added at any stage.
　以上のようにして得られるカテキン類を含有する水溶性組成物、及びこれを含む製剤は、実質的にカテキン類とサイクロデキストリンと抗酸化物からなる水性製剤であり、水溶性で酸化され易いカテキン類を通常の溶解方法で溶解させた場合と比較して極めて保存安定性が高い。よって、カテキン類を含有する水溶性組成物、及びこれを含む製剤の取扱中におけるカテキン類の酸化は可及的に防止され、結果としてカテキン類の保存安定性が顕著に向上する。 The water-soluble composition containing catechins obtained as described above and the preparation containing the same are aqueous preparations substantially composed of catechins, cyclodextrin, and antioxidants, and are water-soluble and easily oxidizable catechins. It has extremely high storage stability compared to the case of dissolving the same by a normal dissolving method. Therefore, oxidation of catechins during handling of a water-soluble composition containing catechins and preparations containing the same is prevented as much as possible, and as a result, the storage stability of catechins is remarkably improved.
　本発明のカテキン類を含有する水溶性組成物、及びこれを含む製剤は、カテキン類が水中で長期間安定的に存在する。しかも経時的な褐変化等の色調変化を伴わないので、飲食品や化粧料、医薬品等を製造する配合剤として極めて有用である。特に、インフルエンザウイルスや同じ１本鎖ＲＮＡを有する新型コロナウイルス（ＣＯＶＩＤ－１９）やその変異株についても抑制効果を十分に期待できる。 In the water-soluble composition containing the catechins of the present invention and the formulation containing the same, the catechins are stably present in water for a long period of time. Moreover, since it is not accompanied by changes in color tone such as browning over time, it is extremely useful as a compounding agent for producing foods, beverages, cosmetics, pharmaceuticals, and the like. In particular, a sufficient inhibitory effect can be expected for influenza virus, novel coronavirus (COVID-19) having the same single-stranded RNA, and its mutants.
　さらに、優れた保存安定性を有する本発明の水溶性組成物を飲食品や化粧料、医薬品等への配合剤として用いることで、抗ウイルス効果を長期間維持できることが期待できる。このため、食品等の保存期間として、長期の消費期間、あるいは賞味期間を設定することができる。 Furthermore, by using the water-soluble composition of the present invention, which has excellent storage stability, as a compounding agent for food, beverages, cosmetics, pharmaceuticals, etc., it can be expected that the antiviral effect can be maintained for a long period of time. For this reason, a long consumption period or best-before period can be set as the storage period of food or the like.
　精製水等の水中に少なくとも１種のカテキン類、および少なくとも１種以上のサイクロデキストリン、並びに１種以上の抗酸化剤を溶解させて成る。
　前記カテキン類は、カテキン類の溶解度以下の濃度に溶解させる。
　前記サイクロデキストリンは、前記ポリフェノール類のモル濃度に対して１／１０以上のモル濃度で、且つサイクロデキストリンの溶解度以下の濃度に溶解させている。
　更に少なくとも１種の抗酸化剤を、同抗酸化剤の溶解度以下の濃度で溶解させて成る。
　超純水を用いた精製水中に、先ず包接化剤としてのサイクロデキストリンを溶解させる。続いてカテキン類を溶解させる。又は予め配合割合を計量して混合したサイクロデキストリンとカテキン類を同時に精製水中へ溶解させる。 At least one catechin, at least one or more cyclodextrins, and one or more antioxidants are dissolved in water such as purified water.
The catechins are dissolved at a concentration below the solubility of the catechins.
The cyclodextrin is dissolved at a molar concentration of 1/10 or more with respect to the molar concentration of the polyphenols and at a concentration lower than the solubility of the cyclodextrin.
Furthermore, at least one antioxidant is dissolved at a concentration equal to or lower than the solubility of the antioxidant.
First, cyclodextrin as a clathrate agent is dissolved in purified water using ultrapure water. Subsequently, the catechins are dissolved. Alternatively, the cyclodextrin and the catechins, which are previously weighed and mixed in proportion, are simultaneously dissolved in purified water.
　本発明の水溶性組成物は、上記に説明した通り、スプレー、タブレットの形態で、あるいは各種食品等に添加剤として用いることができる。
　また、ヒトや動物向けの抗ウイルス剤としての用途、靴等を履いたまま接触させるためのマットなどに含ませた抗菌剤などの使用ができる。 As described above, the water-soluble composition of the present invention can be used in the form of a spray or tablet, or as an additive to various foods.
In addition, it can be used as an antiviral agent for humans and animals, and as an antibacterial agent contained in a mat or the like for contact while wearing shoes or the like.
　以下に、本発明によるポリフェノール類製剤及びその製造方法の実施例を具体的に説明し、合わせて比較例、及び試験例と共にその特性を説明する。但し、本発明は実施例に示す態様に限定されるものではない。 Examples of the polyphenols formulation and the method for producing the same according to the present invention will be specifically described below, along with comparative examples and test examples to describe their characteristics. However, the present invention is not limited to the embodiments shown in the examples.
　エピガロカテキンガレート（ＥＧＣｇ）濃度の分析は以下の通り行なった。
１．抽出操作
　分析対象の試料０．２ｇを、ヘキサン２ｍｌ、メタノール：水：酢酸＝８０：２０：０．５の混合溶媒２ｍｌを加えて、抽出１０分行なう。次いで、３０００ｒｐｍ×１０ｍｉｎの遠心分離を行ない、メタノール：水：酢酸＝８０：２０：０．５の層分取を行なった後、分析に供した。
２．高速液体クロマトグラフィー（ＨＰＬＣ）操作条件
　抽出された試料を以下の条件でＨＰＬＣ分析した。
機種：ＰＵ２０８０ＰＬＵＳ　ｐｕｍｐ（ＪＡＳＣＯ）
検出器：ＵＶ２０７５ＰＬＵＳ　ＵＶ／ＶＩＳｄｉｔｅｃｔｏｒ（ＪＡＳＣＯ）
カラム：ＹＭＣ－Ｐａｃｋ　ＯＤＳ－Ａ　ＡＡ１２ＳＯ５－１５０ＷＴ　Φ ６．０ｍｍ×１５０ｍｍ
移動相： 水：メタノール：０．２ｍｏｌ／ｌリン酸緩衝液(ｐＨ３．０)の混液（３３：１２：５）
吸光測定波長：２７０ｎｍ
流速：１．０ｍｌ／ｍｉｎ
カラム温度：４０℃ Analysis of epigallocatechin gallate (EGCg) concentration was performed as follows.
1. Extraction Operation To 0.2 g of a sample to be analyzed, 2 ml of hexane and 2 ml of a mixed solvent of methanol:water:acetic acid=80:20:0.5 are added, and extraction is performed for 10 minutes. Then, it was subjected to centrifugation at 3000 rpm×10 min, and separated into layers of methanol:water:acetic acid=80:20:0.5, and subjected to analysis.
2. High Performance Liquid Chromatography (HPLC) Operating Conditions The extracted sample was analyzed by HPLC under the following conditions.
Model: PU2080PLUS pump (JASCO)
Detector: UV2075PLUS UV/VIS detector (JASCO)
Column: YMC-Pack ODS-A AA12SO5-150WT Φ 6.0mm x 150mm
Mobile phase: water: methanol: 0.2 mol/l phosphate buffer (pH 3.0) mixture (33:12:5)
Absorbance measurement wavelength: 270 nm
Flow rate: 1.0ml/min
Column temperature: 40°C
　［実施例１］　カテキン類、サイクロデキストリン及び抗酸化剤を含む水溶液組成物（液体試料Ａ）の調製
　容量２００ｍｌのビーカー中に精製水として超純水約８０ｍｌを量り取り、そのビーカーをマグネチックスターラーにセットし、２００ｒｐｍの条件で回転し撹拌させた。
　この超純水中に、先ずは包接化剤としてのβ-サイクロデキストリン及びγ-サイクロデキストリンを、最終的にカテキン類のモル濃度に対して１／１０以上のモル濃度で、且つ同サイクロデキストリンの溶解度以下の濃度となるように、β－サイクロデキストリンは０．１ｇを、γ－サイクロデキストリンは０．４ｇを、各々を少しずつゆっくりと加えながら混合して溶解させた。その結果、サイクロデキストリンの溶解濃度は、カテキン類含有組成物の全重量（１００ｇ）に対して０．５％となった。すなわち、カテキン類（ＥＧＣｇ）の分子量４５８（Ｄａ）、β-サイクロデキストリンの分子量１１３４（Ｄａ）、γ-サイクロデキストリンの分子量１２９７（Ｄａ）として、カテキン類に対するサイクロデキストリン量を１８．２モル％とした。
　次に、カテキン類としては、カテキン類の１種であるエピガロカテキンガレート（ＥＧＣｇ）粉末（太陽化学株式会社製商品名：サンフェノン（登録商標）ＥＧＣｇ；以下、粉末試料イという。）を、ＥＧＣｇとしての重量が組成物の全重量に対して１重量％（１０ｍｇ／ｇであり、１０，０００ｐｐｍ相当）となるように１ｇを量り、少しずつゆっくりと混合して溶解させた。
　さらに、抗酸化剤としてアスコルビン酸を、組成物全重量に対して０．２％となるように０．２ｇ溶解して、最終的に組成物全量が１００ｇ（１００ｍＬ）となるように超純水を追加して十分に混合させ、下記「表１」に示す実施例１の液体試料Ａ（カテキン類含有組成物）を調製した。 [Example 1] Preparation of aqueous solution composition (liquid sample A) containing catechins, cyclodextrin and antioxidant About 80 ml of ultrapure water as purified water is weighed into a 200 ml beaker, and the beaker is stirred with a magnetic stirrer. , and rotated and stirred at 200 rpm.
In this ultrapure water, β-cyclodextrin and γ-cyclodextrin are first added as clathrate agents to a molar concentration of 1/10 or more with respect to the molar concentration of catechins, and the cyclodextrin 0.1 g of .beta.-cyclodextrin and 0.4 g of .gamma.-cyclodextrin were slowly added little by little so as to obtain a concentration below the solubility of , and dissolved by mixing. As a result, the dissolved concentration of cyclodextrin was 0.5% with respect to the total weight (100 g) of the catechins-containing composition. That is, the molecular weight of catechins (EGCg) is 458 (Da), the molecular weight of β-cyclodextrin is 1134 (Da), and the molecular weight of γ-cyclodextrin is 1297 (Da). bottom.
Next, as catechins, epigallocatechin gallate (EGCg) powder (manufactured by Taiyo Kagaku Co., Ltd., trade name: Sunphenon (registered trademark) EGCg; hereinafter referred to as powder sample a), which is one of catechins, was added to EGCg. (10 mg/g, corresponding to 10,000 ppm) with respect to the total weight of the composition, 1 g was weighed and slowly mixed little by little to dissolve.
Furthermore, 0.2 g of ascorbic acid as an antioxidant is dissolved so as to be 0.2% with respect to the total weight of the composition, and ultrapure water is added so that the total composition is finally 100 g (100 mL). was added and thoroughly mixed to prepare a liquid sample A (catechin-containing composition) of Example 1 shown in "Table 1" below.
　［比較例１］　カテキン類を含む水溶液組成物（液体試料Ｂ）の調製
　容量２００ｍｌのビーカー中に精製水として超純水約８０ｍＬ程度を量り取り、そのビーカーを同じくマグネチックスターラーにセットし、２００ｒｐｍの条件で回転し撹拌させた。
　この超純水中に、カテキン類として上記した粉末試料イ、即ちカテキン類の１種であるエピガロカテキンガレート（ＥＧＣｇ）粉末（太陽化学株式会社製商品名：サンフェノン（登録商標）ＥＧＣｇ）を、最終の組成物全重量に対する割合が１％となるように１ｇを計り、少しずつゆっくりと加えながら混合して溶解させた。そして、最終的に組成物全体が１００ｇとなるように超純水を追加して十分に混合させ、下記「表１」に示す比較例１の液体試料Ｂ（カテキン類含有組成物）を調製した。つまり、この比較例１は、カテキン類のみ溶解させたもので、サイクロデキストリンおよび抗酸化剤を含まない組成である。 [Comparative Example 1] Preparation of an aqueous solution composition containing catechins (liquid sample B) About 80 mL of ultrapure water as purified water was measured into a beaker with a capacity of 200 mL, and the beaker was similarly set in a magnetic stirrer and stirred at 200 rpm. It was rotated and stirred under the conditions of
In this ultrapure water, the powder sample A described above as catechins, that is, epigallocatechin gallate (EGCg) powder (manufactured by Taiyo Kagaku Co., Ltd., trade name: Sunfenon (registered trademark) EGCg), which is one type of catechins, 1 g was weighed out so that the ratio to the total weight of the final composition was 1%, and slowly added little by little while being mixed and dissolved. Then, ultrapure water was added and thoroughly mixed so that the total composition was 100 g, and liquid sample B (catechin-containing composition) of Comparative Example 1 shown in "Table 1" below was prepared. . In other words, Comparative Example 1 has a composition in which only catechins are dissolved and does not contain cyclodextrin and antioxidant.
　［比較例２］　カテキン類を含む水溶液組成物（液体試料Ｃ）の調製
　容量２００ｍｌのビーカー中に精製水として超純水約８０ｍｌを量り取り、このビーカーをマグネチックスターラーにセットし、２００ｒｐｍの条件で回転し撹拌させた。
　この超純水中に、β－サイクロデキストリン０．４ｇを、組成物全重量に対して０．１％となるように少しずつゆっくりと加えながら混合した。また、γ－サイクロデキストリン０．４ｇを、やはり組成物全重量に対して０．４％となるように、各々少しずつゆっくりと加えながら混合した。
　さらに、上記の粉末試料イ（ＥＧＣｇ）を、その重量が、組成物全重量に対して１％となるように１ｇを計り、少しずつゆっくりと混合して溶解した。その上で、最終的に組成物全体が１００ｇとなるように超純水を追加して十分に混合させ、下記「表１」に示す比較例２の液体試料Ｃ（カテキン類含有組成物）を調製した。つまり、この比較例２は抗酸化剤を含まない組成である。 [Comparative Example 2] Preparation of an aqueous solution composition containing catechins (liquid sample C) About 80 ml of ultrapure water as purified water was weighed into a beaker with a capacity of 200 ml, and this beaker was set in a magnetic stirrer and stirred at 200 rpm. to rotate and stir.
Into this ultrapure water, 0.4 g of β-cyclodextrin was slowly added little by little to 0.1% with respect to the total weight of the composition while mixing. Also, 0.4 g of γ-cyclodextrin was slowly added little by little while mixing so as to be 0.4% with respect to the total weight of the composition.
Further, 1 g of the above powder sample A (EGCg) was weighed so that its weight was 1% of the total weight of the composition, and slowly mixed little by little to dissolve. On top of that, ultrapure water was added so that the total composition was finally 100 g, and the liquid sample C (catechin-containing composition) of Comparative Example 2 shown in "Table 1" below was added. prepared. In other words, Comparative Example 2 has a composition that does not contain an antioxidant.
　［比較例３］　カテキン類を含む水溶液組成物（液体試料Ｄ）の調製
　容量２００ｍｌのビーカー中に超純水約８０ｍｌ量り取り、このビーカーをマグネチックスターラーにセットし、２００ｒｐｍの条件で回転し撹拌させた。
　この超純水中に、上記粉末試料イを１ｇ計り、組成物全重量に対して１％となるように少しずつゆっくりと混合して溶解した。
　さらにアスコルビン酸０．２ｇを、組成物全重量に対して０．２％となるように溶解させた。その上で、最終的に組成物全体が１００ｇとなるように超純水を追加して十分に混合させ、下記「表１」に示す比較例３の液体試料Ｄ（カテキン類含有組成物）を調製した。つまり、この比較例３は、サイクロデキストリンを含まない組成である。 [Comparative Example 3] Preparation of an aqueous solution composition containing catechins (liquid sample D) About 80 ml of ultrapure water was weighed into a beaker with a capacity of 200 ml, the beaker was set in a magnetic stirrer, and the beaker was rotated and stirred at 200 rpm. let me
Into this ultrapure water, 1 g of the above powder sample a was weighed and slowly mixed little by little to dissolve in 1% of the total weight of the composition.
Furthermore, 0.2 g of ascorbic acid was dissolved so as to be 0.2% with respect to the total weight of the composition. On top of that, ultrapure water was added and thoroughly mixed so that the total composition was finally 100 g, and liquid sample D (catechin-containing composition) of Comparative Example 3 shown in "Table 1" below was added. prepared. That is, this Comparative Example 3 is a composition that does not contain cyclodextrin.
　［実施例２］　カテキン類を含む水溶液組成物（液体試料Ｅ）の調製
　容量２００ｍｌのビーカー中に精製水として超純水約８０ｍｌを量り取り、そのビーカーをマグネチックスターラーにセットし、２００ｒｐｍの条件で回転し撹拌させた。
　この超純水中に、β-サイクロデキストリンを、最終の組成物全重量に対して１％となるように、１ｇを少しずつゆっくりと加えながら混合して溶解させた。
　さらに、上記の粉末試料イ（ＥＧＣｇ）を、その重量が、組成物全重量に対して１％となるように１ｇ計り、少しずつゆっくりと混合して溶解した。すなわち、カテキン類に対するサイクロデキストリン量を４０．４モル％とした。
　また、アスコルビン酸を、組成物全重量に対して０．２％となるように０．２ｇ溶解させて、最終的に組成物全体重量が１００ｇとなるように超純水を追加して十分に混合させ、下記「表１」に示す実施例２の液体試料Ｅ（カテキン類含有組成物）を調製した。 [Example 2] Preparation of an aqueous solution composition containing catechins (liquid sample E) About 80 ml of ultrapure water as purified water was weighed into a beaker with a capacity of 200 ml, and the beaker was set in a magnetic stirrer and stirred at 200 rpm. to rotate and stir.
In this ultrapure water, 1 g of β-cyclodextrin was slowly added little by little to 1% of the total weight of the final composition and dissolved by mixing.
Further, 1 g of the above powder sample A (EGCg) was weighed so that its weight was 1% of the total weight of the composition, and slowly mixed little by little to dissolve. That is, the amount of cyclodextrin to catechins was set to 40.4 mol %.
In addition, 0.2 g of ascorbic acid is dissolved so as to be 0.2% with respect to the total weight of the composition, and ultrapure water is added so that the total weight of the composition is finally 100 g. By mixing, liquid sample E (catechin-containing composition) of Example 2 shown in "Table 1" below was prepared.
　［比較例４］　カテキン類を含む水溶液組成物（液体試料Ｆ）の調製
　この比較例４は、上記実施例２に対応させたもので、やはり容量２００ｍｌのビーカー中に精製水として超純水約８０ｍｌを量り取り、そのビーカーをマグネチックスターラーにセットし、２００ｒｐｍの条件で回転し撹拌させた。
　この超純水中に、β-サイクロデキストリンを、最終の組成物全重量に対して１％となるように１ｇを、少しずつゆっくりと加えながら混合して溶解させた。
　　さらに、上記の粉末試料イ（ＥＧＣｇ）を、その重量が、組成物全重量に対して１％となるように１ｇ計り、少しずつゆっくりと混合して溶解させ、最終的に組成物全体重量が１００ｇとなるように超純水を追加して十分に混合させ、下記「表１」に示す比較例４の液体試料Ｆ（カテキン類含有組成物）を調製した。つまり、この比較例４は抗酸化剤を含まない組成である。 [Comparative Example 4] Preparation of an aqueous solution composition containing catechins (liquid sample F) This Comparative Example 4 corresponds to the above Example 2, in which about 200 ml of ultrapure water is added as purified water in a beaker having a capacity of 200 ml. 80 ml was weighed out, and the beaker was set in a magnetic stirrer and stirred by rotating at 200 rpm.
In this ultrapure water, 1 g of β-cyclodextrin was slowly added little by little so as to make 1% of the total weight of the final composition, and dissolved by mixing.
Furthermore, 1 g of the above powder sample A (EGCg) was weighed so that its weight was 1% with respect to the total weight of the composition, slowly mixed little by little to dissolve, and finally the total weight of the composition was Ultrapure water was added so as to make 100 g, and the mixture was sufficiently mixed to prepare a liquid sample F (catechin-containing composition) of Comparative Example 4 shown in "Table 1" below. In other words, this Comparative Example 4 has a composition that does not contain an antioxidant.
　［実施例３］　カテキン類を含む水溶液組成物（液体試料Ｇ）の調製
　容量２００ｍｌのビーカー中に精製水として超純水約８０ｍｌを量り取り、そのビーカーをマグネチックスターラーにセットし、２００ｒｐｍの条件で回転し撹拌させた。
　この超純水中に、γ-サイクロデキストリンを、最終の組成物全重量に対して１％となるように、１ｇを少しずつゆっくりと加えながら混合して溶解させた。
　さらに、上記の粉末試料イ（ＥＧＣｇ）を、その重量が、組成物全重量に対して１％となるように、１ｇを少しずつゆっくりと混合して溶解させた。すなわち、カテキン類に対するサイクロデキストリン量を３７．９モル％とした。
　また、アスコルビン酸を、組成物全重量に対して０．２％となるように０．２ｇ溶解させて、最終的に組成物全体重量が１００ｇとなるように超純水を追加して十分に混合させ、下記「表１」に示す実施例３の液体試料Ｇ（カテキン類含有組成物）を調製した。 [Example 3] Preparation of an aqueous solution composition containing catechins (liquid sample G) About 80 ml of ultrapure water as purified water was weighed into a beaker with a capacity of 200 ml, and the beaker was set in a magnetic stirrer and stirred at 200 rpm. to rotate and stir.
In this ultrapure water, 1 g of γ-cyclodextrin was slowly added little by little to 1% of the total weight of the final composition, and dissolved by mixing.
Further, 1 g of the above powder sample A (EGCg) was slowly mixed little by little and dissolved so that the weight thereof was 1% with respect to the total weight of the composition. That is, the amount of cyclodextrin to catechins was set to 37.9 mol %.
Further, 0.2 g of ascorbic acid is dissolved so as to be 0.2% with respect to the total weight of the composition, and ultrapure water is added so that the total weight of the composition is finally 100 g. By mixing, liquid sample G (catechin-containing composition) of Example 3 shown in "Table 1" below was prepared.
　［比較例５］　カテキン類を含む水溶液組成物（液体試料Ｈ）の調製
　この比較例５は上記実施例３に対応させたもので、容量２００ｍｌのビーカー中に精製水として超純水約８０ｍｌを量り取り、そのビーカーをマグネチックスターラーにセットし、２００rpmの条件で回転し撹拌させた。
　この超純水中に、γ-サイクロデキストリンを、最終の組成物全重量に対して１％となるように、１ｇを少しずつゆっくりと加えながら混合して溶解させた。
　さらに、上記の粉末試料イ（ＥＧＣｇ）を、その重量が、組成物全重量に対して１％となるように、１ｇを少しずつゆっくりと混合して溶解させ、最終的に組成物全体重量が１００ｇとなるように超純水を追加して十分に混合させ、下記「表１」に示す比較例５の液体試料Ｈ（カテキン類含有組成物）を調製した。つまり、この比較例５は抗酸化剤を含まない組成である。 [Comparative Example 5] Preparation of Aqueous Composition Containing Catechin (Liquid Sample H) This Comparative Example 5 corresponds to Example 3 above, in which about 80 ml of ultrapure water is added as purified water in a beaker having a capacity of 200 ml. The beaker was weighed, set in a magnetic stirrer, and stirred by rotating at 200 rpm.
In this ultrapure water, 1 g of γ-cyclodextrin was slowly added little by little to 1% with respect to the total weight of the final composition, and dissolved by mixing.
Furthermore, 1 g of the above powder sample A (EGCg) was slowly mixed little by little so that its weight was 1% with respect to the total weight of the composition, and finally the total weight of the composition was Ultrapure water was added so as to make 100 g, and the mixture was sufficiently mixed to prepare liquid sample H (catechin-containing composition) of Comparative Example 5 shown in "Table 1" below. In other words, this Comparative Example 5 has a composition that does not contain an antioxidant.
　［実施例４］　［保存安定性の確認試験１］
　上記実施例１で得られた液体試料Ａ（カテキン類含有組成物）、および比較例１－３で得られた液体試料Ｂ～Ｄについて、それぞれ３０ｍＬをサンプル瓶に入れ、４０℃の環境温度で１４日間保存した。その後、各試料について高速液体クロマトグラフィー（ＨＰＬＣ）によりＥＧＣｇ濃度を測定した。更に、このＥＧＣｇ濃度の経時変化を調べて経過日数に対するＥＧＣｇの残存率を求めた。
　また、上記各試料Ａ～Ｄの色調を評価する為に、黄色に見える波長である４２０ｎｍにおける吸光度を吸光度計（日本電色工業株式会社製）で測定した結果を、下記の「表１」に示す。 [Example 4] [Confirmation test 1 of storage stability]
30 mL of each of the liquid sample A (catechin-containing composition) obtained in Example 1 and the liquid samples B to D obtained in Comparative Examples 1-3 were placed in a sample bottle and placed at an ambient temperature of 40°C. Stored for 14 days. After that, EGCg concentration was measured for each sample by high performance liquid chromatography (HPLC). Furthermore, the change in the EGCg concentration over time was examined to determine the residual rate of EGCg with respect to the elapsed days.
In addition, in order to evaluate the color tone of each of the above samples A to D, the absorbance at 420 nm, which is a wavelength that looks yellow, was measured with an absorbance meter (manufactured by Nippon Denshoku Industries Co., Ltd.). The results are shown in "Table 1" below. show.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　上記の表１で明らかなように、本発明の実施例１に係るポリフェノール類製剤は、ＥＧＣｇの残存率が依然として１００％を維持しており、他の比較例１～３の数値に比して、成分安定性のすこぶる高いことが確認された。
　また、本発明の実施例１の吸光度は０．０８に過ぎず、他の比較例１～３の数値に比して、桁違いに褐変化しないことが確認された。 As is clear from Table 1 above, the polyphenols preparation according to Example 1 of the present invention still maintains a residual EGCg rate of 100%, which is higher than the values of other Comparative Examples 1 to 3. , it was confirmed that the component stability was extremely high.
In addition, the absorbance of Example 1 of the present invention was only 0.08, and it was confirmed that compared with the values of other Comparative Examples 1 to 3, browning did not occur by an order of magnitude.
　次に、本発明の上記実施例２、３についても同様に、アスコルビン酸のみを配合した比較例３と比較して、格段に高い成分安定性を示している。そして、サイクロデキストリンのみを配合した比較例４，５と比較しても、桁違いに高い色調安定性を示すことが確認できた。 Next, Examples 2 and 3 of the present invention also show remarkably high component stability compared to Comparative Example 3 in which only ascorbic acid is blended. In addition, it was confirmed that even when compared with Comparative Examples 4 and 5 in which only cyclodextrin was blended, an incomparably higher color tone stability was exhibited.
　［実施例５］　［スプレー用水溶液の保存安定性の確認試験］
＜ＥＧＣｇ含有水溶液組成物入りスプレー＞
　上記の水溶液組成物をノズルのついた容器に入れ、スプレーの形態により、ＥＧＣｇの効果と安定性を評価した。ノズルの一押しで、０．１ｍｌの水溶液が霧状になって噴霧できた。一押しで約１０リットル（Ｌ）の体積に霧状の水溶液が散布された。
　評価は次の通りとした。
・ＥＧＣｇ含有水溶液組成物を、製造直後の試料と４０℃で２週間保管後の試料とを比較した。評価項目は以下の通りとした。本発明試料に対する比較対象試料として、サイクロデキストリンを添加しない試料（対象試料１）、アスコルビン酸を添加しない試料（対象試料２）とした。
１）ＥＧＣｇの安定性
・沈殿の有無、白濁の有無を確認した。（以下のa.とb.）
　目視又は光学的な濁度測定により評価した。
　a.透明に溶解している
　b.白濁、又は沈殿物がある
２）水溶液組成物の色相
・分光光度計によりスペクトルを測定し、透明から着色が増加したかを測定した。
３）水溶液組成物の臭気（官能試験、以下のa.、b.、c.）
・水溶液を複数人によりその匂いを嗅いだ。
　a.無臭
　b.不快な匂いが若干ある
　c.不快な匂いが強い
４）綿製の布にスプレー噴射し、布の表面の感触を以下の基準で評価した（官能試験、以下のa.、b.）
　a.後のこり感がなく、サラッとしている
　b.べたつきが残り、不快感を感じる [Example 5] [Confirmation test of storage stability of aqueous solution for spray]
<Spray containing EGCg-containing aqueous solution composition>
The above aqueous solution composition was placed in a container with a nozzle, and the effect and stability of EGCg were evaluated according to the form of spray. With one push of the nozzle, 0.1 ml of the aqueous solution was atomized and could be sprayed. A single push dispensed a mist of the aqueous solution in a volume of approximately 10 liters (L).
The evaluation was as follows.
- The EGCg-containing aqueous solution composition was compared between a sample immediately after production and a sample stored at 40°C for 2 weeks. The evaluation items were as follows. As samples for comparison with the samples of the present invention, a sample to which cyclodextrin was not added (target sample 1) and a sample to which ascorbic acid was not added (target sample 2) were used.
1) The stability of EGCg, the presence or absence of precipitation, and the presence or absence of cloudiness were confirmed. (a. and b. below)
Evaluation was by visual or optical turbidity measurement.
a. transparent and dissolved b. white turbidity or precipitate 2) Hue of the aqueous solution composition The spectrum was measured with a spectrophotometer to determine whether the coloration increased from the transparency.
3) Odor of aqueous solution composition (sensory test, a., b., c. below)
・The aqueous solution was smelled by multiple people.
Odorless b. Slightly unpleasant odor c. Strong unpleasant odor b.)
a. There is no feeling of stiffness afterward, and it is smooth b. Stickiness remains and you feel uncomfortable
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　表２から分かるように、本発明試料は、安定性が良く、４０℃で２週間という過酷な条件下であっても色相の変化はなく、臭気もなく、さらに噴霧した際の対象物の表面も違和感なく、安定した品質を保持していることが分かる。
　これに対し、比較対象となる対象試料１および対象試料２では着色があり、臭気、表面感触の変化など、安定性に変化が認められた。
　このことから、スプレーという形態において本発明の水溶液組成物の安定性は優れていることが明らかとなった。 As can be seen from Table 2, the sample of the present invention has good stability, does not change color even under the severe conditions of 2 weeks at 40 ° C., has no odor, and when sprayed on the surface of the object. It can be seen that stable quality is maintained without any discomfort.
On the other hand, in Control Sample 1 and Control Sample 2, which are comparative objects, coloration was observed, and changes in stability such as changes in odor and surface feel were observed.
From this, it became clear that the aqueous composition of the present invention has excellent stability in the form of a spray.
　［実施例６］　［タブレットの保存安定性の確認試験］
＜ＥＧＣｇ含有水溶液組成物入りタブレット＞
　上記の水溶液組成物を、常法により固形錠剤（直径１ｃｍ、０．５ｃｍ（中央）、０．３ｃｍ（端部））を製造した。この錠剤について、ＥＧＣｇの安定性を評価した。
１）ＥＧＣｇの安定性（水への溶解性）
２）タブレットの色相
３）水溶液組成物の味覚（官能試験）
　評価は次の通りとした。
・ＥＧＣｇ含有水溶液組成物を、製造直後の試料と４０℃で２週間保管後の試料とを比較した。評価項目は以下の通りとした。本発明試料に対する比較対象試料として、サイクロデキストリンを添加しない試料（対象試料１）、アスコルビン酸を添加しない試料（対象試料２）とした。
１）ＥＧＣｇの安定性
・タブレットを水に溶解し、沈殿の有無、白濁の有無を確認した。（以下のa.とb.）
　目視又は光学的な濁度測定により評価した。
　a.透明に溶解している
　b.白濁、又は沈殿物がある
２）タブレットの色相、ひび割れ、崩壊の有無
・タブレット表面の色相を目視で評価した。
・その他ひび割れ、崩壊の有無を目視確認した。
３）タブレットの味覚（官能試験、以下のa.、b.、c.）
・水溶液を複数人によりその味覚を評価した。
　a.異常なく、香料成分によりさわやかな味を感じた。
　b.不快な味覚が若干ある
　c.不快な味覚が強い [Example 6] [Tablet storage stability confirmation test]
<Tablet containing EGCg-containing aqueous solution composition>
Solid tablets (diameter 1 cm, 0.5 cm (center), 0.3 cm (end)) were produced from the above aqueous composition by a conventional method. This tablet was evaluated for EGCg stability.
1) Stability of EGCg (solubility in water)
2) Color of tablet 3) Taste of aqueous solution composition (sensory test)
The evaluation was as follows.
- The EGCg-containing aqueous solution composition was compared between a sample immediately after production and a sample stored at 40°C for 2 weeks. The evaluation items were as follows. As samples for comparison with the samples of the present invention, a sample to which cyclodextrin was not added (target sample 1) and a sample to which ascorbic acid was not added (target sample 2) were used.
1) Stability of EGCg - The tablet was dissolved in water, and the presence or absence of precipitation and the presence or absence of cloudiness were confirmed. (a. and b. below)
Evaluation was by visual or optical turbidity measurement.
a. Dissolved transparently b. Cloudiness or presence of sediment 2) Presence or absence of hue, cracks, or collapse of the tablet The hue of the tablet surface was visually evaluated.
・Other cracks and collapses were visually checked.
3) Taste of tablet (sensory test, a., b., c. below)
・The taste of the aqueous solution was evaluated by multiple people.
a. There was no abnormality, and the fragrance component gave a refreshing taste.
b. Slightly unpleasant taste c. Strong unpleasant taste
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　表３から分かるように、本発明試料は、安定性が良く、４０℃で２週間という過酷な条件下であっても色相および味覚の変化はなく、安定した品質を保持していることが分かる。このことから、タブレット（錠剤）という形態において本発明の水溶液組成物を基に得られ得るタブレットの安定性は優れていることが明らかとなった。 As can be seen from Table 3, the samples of the present invention have good stability, and even under the severe conditions of 2 weeks at 40°C, there is no change in hue and taste, and it can be seen that they maintain stable quality. . From this, it became clear that the stability of the tablets obtained based on the aqueous composition of the present invention in the form of tablets (tablets) is excellent.
＜ＥＧＣｇの抗インフルエンザウイルス活性１＞
　はじめに
　緑茶にはカテキン類が多く含まれている。カテキン類の一種であるエピガロカテキンガレート（ＥＧＣｇ）はウイルスの増殖過程において、ウイルスが感染する細胞への吸着過程を阻害し、抗ウイルス効果を示すことが知られている。しかし、ＥＧＣｇは脂溶性が高いため、培養細胞を用いて直接抗ウイルス効果を評価することが困難であった。そこでＥＧＣｇの水溶性を高めた本発明の組成物、安定化した組成物としてＥＧＣｇを含む製剤を使用した。すなわち、ＥＧＣｇを含む製剤とＥＧＣｇ以外の成分を含む溶液（製剤）との抗インフルエンザウイルス活性を比較した。また、ＥＧＣｇを含む製剤の抗インフルエンザウイルス活性が、ウイルスの細胞への吸着あるいは侵入阻害によることを明らかにした。これにより、従来のＥＧＣｇと同様の抗ウイルス効果を示すことから、ＥＧＣｇの使用範囲を広げることが可能となる。
　そこで、ｉｎ　ｖｉｔｒｏにおいてＭＤＣＫ（Ｍａｄｉｎ－Ｄａｒｂｙ　Ｃａｎｉｎｅ　Ｋｉｄｎｅｙ）細胞を用いたプラーク減少法を行うことでＥＧＣｇの抗インフルエンザウイルス効果を検討した。 <EGCg anti-influenza virus activity 1>
Introduction Green tea contains a lot of catechins. Epigallocatechin gallate (EGCg), which is a kind of catechin, is known to inhibit the process of adsorption to virus-infected cells during the process of viral proliferation and exhibit an antiviral effect. However, since EGCg is highly lipid-soluble, it has been difficult to directly evaluate its antiviral effect using cultured cells. Therefore, the composition of the present invention in which the water solubility of EGCg is enhanced, and the formulation containing EGCg as a stabilized composition were used. That is, the anti-influenza virus activity was compared between a formulation containing EGCg and a solution (formulation) containing components other than EGCg. In addition, it was clarified that the anti-influenza virus activity of preparations containing EGCg is due to the adsorption of viruses to cells or inhibition of their entry. As a result, since it exhibits the same antiviral effect as conventional EGCg, it is possible to expand the range of use of EGCg.
Therefore, the anti-influenza virus effect of EGCg was examined by conducting an in vitro plaque reduction method using MDCK (Madin-Darby Canine Kidney) cells.
　方法
　Ｉｎ　ｖｉｔｒｏでのプラーク減少法：ＭＤＣＫ細胞を培養後、Ａ型株のＢａｎｇｋｏｋ（Ｈ１Ｎ１）、ＰＲ８（Ｈ１Ｎ１）、とＡｉｃｈｉ（Ｈ３Ｎ２）、Ｂ型株のＳｉｎｇａｐｏｒｅの４種のインフルエンザウイルス株を用いて、プラーク数を５０％まで減少させる濃度（以下「ＥＣ５０」とする。）を調べた。
　ＥＧＣｇのウイルスの細胞への吸着前の阻害については、ＥＧＣｇ溶液（ＥＧＣｇ／水溶液溶液）、または、ＥＧＣｇを白酒で溶解した溶液（ＥＧＣｇ／白酒溶液）でウイルスを希釈し、ＭＤＣＫ細胞にウイルスを吸着・感染させた。
　同様に、溶解液（ＥＧＣｇを溶解した水溶液で添加剤等が含まれているもの）、または白酒でウイルスを希釈し、ＭＤＣＫ細胞にウイルスを吸着・感染させた。
　ウイルス吸着後の阻害については、ウイルスのＭＤＣＫ細胞への吸着・感染後に感染済MDCK細胞に対してＥＧＣｇ処理を上記希釈濃度で行った。また、ＥＧＣｇ溶液をウイルス感染時と同じ条件で希釈し、単層培養したＭＤＣＫ細胞に処理を行った（ＥＧＣｇ前処理)後に、ウイルスの吸着・感染を行った。 Method In vitro plaque reduction method: After culturing MDCK cells, using four influenza virus strains: type A strain Bangkok (H1N1), PR8 (H1N1), and Aichi (H3N2), type B strain Singapore , the concentration that reduces the number of plaques by 50% (hereinafter referred to as " EC50 ") was investigated.
For the inhibition of EGCg on the virus before adsorption to cells, the virus was diluted with an EGCg solution (EGCg/aqueous solution) or a solution of EGCg dissolved in white liquor (EGCg/white liquor solution), and the virus was adsorbed on MDCK cells.・Infected
Similarly, the virus was diluted with a lysate (an aqueous solution in which EGCg was dissolved and containing additives, etc.) or white sake, and the virus was allowed to adsorb and infect MDCK cells.
Regarding the inhibition after virus adsorption, EGCg treatment was performed on the infected MDCK cells at the above-mentioned dilution concentration after virus adsorption/infection to MDCK cells. In addition, the EGCg solution was diluted under the same conditions as in virus infection, and monolayer-cultured MDCK cells were treated (EGCg pretreatment), followed by virus adsorption and infection.
　［実施例７］
〔実験方法〕
　以下の表４に、シャーレ１枚当たり（３ｍｌ）のプラークアッセイ用培地の組成を示す。
Figure JPOXMLDOC01-appb-T000010
 [Example 7]
〔experimental method〕
Table 4 below shows the composition of plaque assay medium per petri dish (3 ml).
Figure JPOXMLDOC01-appb-T000010
　用いた細胞の調整方法は以下の通りである。
　解凍したＭＤＣＫ細胞を７５ｃｍ２フラスコにて培養した。７５ｃｍ２フラスコの隅まで細胞が増殖していることが確認できたものを用い、継代を２～３回行った。その後、７５ｃｍ２フラスコからシャーレに継代し、均等に細胞が単層に増殖しているものを実験に用いた。 The method for adjusting the cells used is as follows.
Thawed MDCK cells were cultured in 75 cm 2 flasks. Cells were confirmed to have proliferated to the corners of the 75 cm 2 flask, and were subcultured 2 to 3 times. After that, the cells were subcultured from the 75 cm 2 flask to a petri dish, and the cells in which the cells were evenly proliferating into a monolayer were used in the experiment.
　本実施例において用いたプラーク減少法（抗インフルエンザウイルスアッセイ）は以下の通りである。
　以下には、例示条件に沿って、その方法と結果を示す。 The plaque reduction method (anti-influenza virus assay) used in this example is as follows.
The method and results will be shown below along with the exemplified conditions.
　［実施例８］
　ＥＧＣｇ溶液でインフルエンザウイルス原液を希釈した場合
　６０ｍｍシャーレに単層培養したＭＤＣＫ細胞を、５ｍｌのＰＢＳ（リン酸緩衝生理食塩水）でリンス（洗浄）した。
　インフルエンザウイルス(原液)は、５０００ＰＦＵ／ｍｌとなるようにＰＢＳで希釈した種々の濃度のＥＧＣｇ水溶液（原液：１００００ｐｐｍ）と混合し（対照液はＰＢＳ、ｃｏｎｔｒｏｌと表示)、さらに、それぞれ種々の濃度のＥＧＣｇを含むＰＢＳ（０，０．１，０．３，１，３，１０，３０ｐｐｍ）で１０倍希釈した５００ＰＦＵ／ｍｌのウイルス液２００μｌ（１００ＰＦＵ／２００μｌ／ｐｌａｔｅ）を、単層培養してＰＢＳでリンスしたＭＤＣＫ細胞に１時間、室温で吸着した。このとき、ウイルスが均一に細胞に感染するように１５分ごとにシャーレを傾ける作業を繰り返した。
　感染したＭＤＣＫ細胞を５ｍｌのＰＢＳでリンス後、プラークアッセイ用培地（上記、表４）と２％ａｇａｒｏｓｅを３：２の割合で混合し、混合した溶液を５ｍＬずつシャーレに加えた。コントロール、各濃度は３枚ずつシャーレを作製した。寒天培地が固まった後シャーレを３７度，５％ＣＯ２で２日間インキュベートした。
　コントロールのシャーレにプラークができていることを確認した後、ホルマリンで固定しプラーク数をカウントした。プラークを数えた結果をもとにプラーク数を５０％まで減少させる濃度（ＥＣ５０）を算出した。
　以上の結果を表５に示す。 [Example 8]
When Influenza Virus Stock Solution Was Diluted with EGCg Solution MDCK cells monolayer-cultured in a 60-mm petri dish were rinsed (washed) with 5 ml of PBS (phosphate-buffered saline).
Influenza virus (stock solution) was mixed with various concentrations of EGCg aqueous solution (stock solution: 10000 ppm) diluted with PBS to 5000 PFU / ml (control solution is PBS, indicated as control), and further, each of various concentrations 200 μl (100 PFU/200 μl/plate) of a 500 PFU/ml virus solution diluted 10-fold with PBS containing EGCg (0, 0.1, 0.3, 1, 3, 10, 30 ppm) was cultured in a monolayer and cultured in PBS. was adsorbed for 1 hour at room temperature to MDCK cells rinsed with At this time, the operation of tilting the petri dish every 15 minutes was repeated so that the cells were uniformly infected with the virus.
After rinsing the infected MDCK cells with 5 ml of PBS, the plaque assay medium (Table 4 above) and 2% agarose were mixed at a ratio of 3:2, and 5 ml of the mixed solution was added to each Petri dish. Three plates were prepared for the control and each concentration. After the agar medium solidified, the dishes were incubated at 37°C, 5% CO2 for 2 days.
After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated.
Table 5 shows the above results.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
　表５では、Ａ型株のＢａｎｇｋｏｋ（Ｈ１Ｎ１、表中では「バンコク」と表示）、ＰＲ８（Ｈ１Ｎ１、表中では「ＰＲ８」と表示）、とＡｉｃｈｉ（Ｈ３Ｎ２、表中では「愛知」と表示）、Ｂ型株のＳｉｎｇａｐｏｒｅ（表中では「シンガポール」と表示）の４種のインフルエンザウイルス株を用いている。以下の表４～表８においてもこれらの表示は同じである。
　上記の表３では、上から下へ、バンコク株、ＰＲ８株、愛知株、シンガポール株による結果を示した。また左から右へ、各株のControl、各ＥＧＣｇ量に対するプラーク数、プラーク数の割合、各々の近似線の傾き、Ｙ切片、ＥＣ５０の平均値および標準偏差、t検定によるＰ値を示した。
　表５によれば、プラークはＥＧＣｇの３ｐｐｍ以上添加で、いずれのインフルエンザウイルス株であってもウイルス量が５０％以上減少し、つまりウイルスの増殖が抑制された。
　上記データの統計的解析として、ｔ検定による解析によればいずれもｐ＜０．０５であり有意、つまりカテキン類とウイルス株とが接触することによりウイルス増殖の抑制効果が統計的にも有意であることが示された。
　従って、インフルエンザウイルス株をＥＧＣｇ含有液で希釈、すなわち細胞に吸着前にＥＧＣｇとウイルスを接触させることで、ウイルスの細胞への吸着あるいは侵入が阻害されて、インフルエンザウイルス株の増殖を抑制できることが分かる。 In Table 5, type A strains Bangkok (H1N1, labeled "Bangkok" in the table), PR8 (H1N1, labeled "PR8" in the table), and Aichi (H3N2, labeled "Aichi" in the table) , type B strain Singapore (indicated as “Singapore” in the table). These indications are the same in Tables 4 to 8 below.
In Table 3 above, from top to bottom, the results for the Bangkok strain, the PR8 strain, the Aichi strain, and the Singapore strain were shown. In addition, from left to right, the control of each strain, the number of plaques for each EGCg amount, the ratio of the number of plaques, the slope of each approximation line, the Y intercept, the average value and standard deviation of EC50 , and the P value by t test are shown. .
According to Table 5, when 3 ppm or more of EGCg was added to the plaques, the virus amount was reduced by 50% or more for any influenza virus strain, that is, virus growth was suppressed.
As a statistical analysis of the above data, according to the analysis by t-test, both p < 0.05 and significant, that is, the effect of suppressing virus growth by contacting catechins and virus strains is statistically significant. It was shown that there is
Therefore, by diluting the influenza virus strain with a solution containing EGCg, that is, by contacting the virus with EGCg before adsorption to the cells, adsorption or entry of the virus into the cells is inhibited, and the proliferation of the influenza virus strain can be suppressed. .
　［実施例９］
　溶解液（ＥＧＣｇを溶解した水溶液で、ＥＧＣｇを含まない添加剤等だけが含まれている）でインフルエンザウイルスを希釈した場合
　６０ｍｍシャーレに単層培養したＭＤＣＫ細胞を、５ｍｌのＰＢＳでリンスした。上記「ＥＧＣｇ溶液でインフルエンザウイルス原液を希釈した場合」と同様に、溶解液を希釈して最終的に５００ＰＦＵ/ｍｌ（１００ＰＦＵ／２００μｌ／ｐｌａｔｅ）のウイルス液を調整した。
　溶解液で種々に希釈したウイルス液２００μｌを、単層培養してＰＢＳでリンスしたＭＤＣＫ細胞に１時間室温で吸着した。このとき、ウイルスが均一に細胞に感染するように１５分ごとにシャーレを傾ける作業を繰り返した。
　感染したＭＤＣＫ細胞を５ｍｌのＰＢＳでリンス後、プラークアッセイ用培地（上記、表２）と２％ａｇａｒｏｓｅを３：２の割合で混合し、混合した溶液を５ｍＬずつシャーレに加えた。コントロール、各濃度は３枚ずつシャーレを作製した。寒天培地が固まった後シャーレを３７℃，５％ＣＯ２で２日間インキュベートした。コントロールのシャーレにプラークができていることを確認した後、ホルマリンで固定しプラーク数をカウントした。プラークを数えた結果をもとにプラーク数を５０%まで減少させる濃度（ＥＣ５０）を算出した。
　以上の結果を表６に示す。 [Example 9]
In the case of diluting influenza virus with a lysate (an aqueous solution in which EGCg is dissolved, containing only additives, etc. that do not contain EGCg): MDCK cells monolayer-cultured in a 60 mm petri dish were rinsed with 5 ml of PBS. The lysate was diluted in the same manner as in the case of diluting the influenza virus stock solution with the EGCg solution to finally prepare a virus solution of 500 PFU/ml (100 PFU/200 μl/plate).
200 μl of the virus solution diluted variously with the lysate was adsorbed to MDCK cells which had been monolayer-cultured and rinsed with PBS for 1 hour at room temperature. At this time, the operation of tilting the petri dish every 15 minutes was repeated so that the virus would uniformly infect the cells.
After rinsing the infected MDCK cells with 5 ml of PBS, the plaque assay medium (above, Table 2) and 2% agarose were mixed at a ratio of 3:2, and 5 ml of the mixed solution was added to each Petri dish. For the control and each concentration, 3 petri dishes were prepared. After the agar medium had solidified, the dishes were incubated at 37° C., 5% CO 2 for 2 days. After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated.
Table 6 shows the above results.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
　上記の表６では、上から下へ、バンコク株、ＰＲ８株、愛知株、シンガポール株による結果を示した。また左から右へ、各株のControl、Ｂ１０またはＢ３０（各ＥＧＣｇ量）に対するプラーク数、プラーク数の割合を示した。
　表４によれば、プラークはＥＧＣｇ溶液の３０ｐｐｍに相当するＥＧＣｇを含まない溶解液の添加で、いずれのインフルエンザウイルス株であってもウイルス量が５０％以上に減少することはなかった。つまりＥＧＣｇを含まない溶解液だけでは、ウイルスの増殖が抑制されなかった。
　従って、表３と比較すると、ＥＧＣｇを含まない溶解液だけでは抗インフルエンザウイルス活性は認められないが、ＥＧＣｇを含む溶解液では顕著な抗インフルエンザウイルス活性が認められたことになる。したがって、ＥＧＣｇ単独には、抗インフルエンザウイルス活性があることが明らかとなった。 In Table 6 above, results from Bangkok strain, PR8 strain, Aichi strain, and Singapore strain are shown from top to bottom. From left to right, the number of plaques and the ratio of the number of plaques to Control, B10 or B30 (each EGCg amount) of each strain are shown.
According to Table 4, the addition of EGCg-free lysate corresponding to 30 ppm of the EGCg solution to plaques did not reduce the viral load by more than 50% for any of the influenza virus strains. In other words, the lysate without EGCg alone did not inhibit virus growth.
Therefore, when compared with Table 3, anti-influenza virus activity was not observed in the lysate containing no EGCg alone, but significant anti-influenza virus activity was observed in the lysate containing EGCg. Therefore, it was revealed that EGCg alone has anti-influenza virus activity.
　［実施例１０］
　ＥＧＣｇで前処理を行ったＭＤＣＫ細胞にインフルエンザウイルスを吸着・感染した場合
　６０ｍｍシャーレに単層培養したＭＤＣＫ細胞を５ｍｌのＰＢＳでリンスした後、ＰＢＳで希釈した種々の濃度のＥＧＣｇ水溶液（０，０．１，０．３，１，３，１０，３０ｐｐｍ）を各シャーレに２００μｌずつ加え、１５分おきにシャーレを傾ける作業を繰り返しながら、１時間、４℃で前処理を行った。
　ＥＧＣｇ水溶液による前処理後、５ｍｌのＰＢＳで２回リンスしたＭＤＣＫ細胞に、ＰＢＳで希釈したインフルエンザウイルスを１００ＰＦＵ／２００μｌ／ｐｌａｔｅで1時間、４℃で吸着・感染した。このとき、ウイルスが均一に細胞に感染するように１５分ごとにシャーレを傾ける作業を繰り返した。
　吸着後、５ｍｌのＰＢＳで１回リンスして、プラークアッセイ用培地（上記表２）と２%ａｇａｒｏｓｅを３：２の割合で混合した溶液を５ｍＬずつシャーレに加えた。コントロール、各濃度は3枚ずつシャーレを作製した。
　寒天培地が固まった後シャーレを37℃,5％CO2で2日間インキュベートした。コントロールのシャーレにプラークができていることを確認した後、ホルマリンで固定しプラーク数をカウントした。プラークを数えた結果をもとにプラーク数を５０%まで減少させる濃度（ＥＣ５０）を算出した。
　以上の結果を表７に示す。 [Example 10]
When MDCK cells pretreated with EGCg were adsorbed and infected with influenza virus After rinsing MDCK cells monolayer-cultured in a 60 mm petri dish with 5 ml of PBS, EGCg aqueous solutions of various concentrations diluted with PBS (0, 0 1, 0.3, 1, 3, 10, 30 ppm) was added to each petri dish, and pretreatment was performed at 4°C for 1 hour while repeating the operation of tilting the petri dish every 15 minutes.
After pretreatment with an EGCg aqueous solution, MDCK cells were rinsed twice with 5 ml of PBS, and PBS-diluted influenza virus was adsorbed and infected at 100 PFU/200 μl/plate for 1 hour at 4°C. At this time, the operation of tilting the petri dish every 15 minutes was repeated so that the cells were uniformly infected with the virus.
After adsorption, the plate was rinsed once with 5 ml of PBS, and 5 ml of a solution obtained by mixing plaque assay medium (Table 2 above) and 2% agarose at a ratio of 3:2 was added to the petri dish. Three plates were prepared for the control and each concentration.
After the agar medium solidified, the dishes were incubated at 37°C, 5% CO 2 for 2 days. After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated.
Table 7 shows the above results.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
　上記の表７では、上から下へ、バンコク株、ＰＲ８株、愛知株、シンガポール株による結果を示した。また左から右へ、各株のControl、各ＥＧＣｇ量に対するプラーク数、プラーク数の割合を示した。
　表によれば、プラークはＥＧＣｇの３０ｐｐｍ添加で、いずれのインフルエンザウイルス株であってもウイルス量が５０％以上に減少することはなかった。つまりＥＧＣｇを含む水溶液の細胞への前処理では、ウイルス量は抑制されなかった。
　従って、インフルエンザウイルス株を細胞に吸着・感染する前に細胞にＥＧＣｇ処理が行われてもウイルス増殖が抑制されなかった。このため、ＥＧＣｇは細胞に直接作用してウイルスの吸着・侵入を抑制することはないと考えられた In Table 7 above, from top to bottom, the results for the Bangkok strain, the PR8 strain, the Aichi strain, and the Singapore strain were shown. In addition, from left to right, the control of each strain, the number of plaques with respect to each amount of EGCg, and the ratio of the number of plaques are shown.
According to the table, plaques with 30 ppm of EGCg did not reduce viral load by more than 50% for any of the influenza virus strains. Thus, pretreatment of cells with an aqueous solution containing EGCg did not suppress viral load.
Therefore, even if the cells were treated with EGCg before the cells were adsorbed and infected with the influenza virus strain, virus proliferation was not suppressed. Therefore, it was thought that EGCg does not directly act on cells to suppress virus adsorption/invasion.
　［実施例１１］
　インフルエンザウイルス吸着・感染後にＥＧＣｇ処理をした場合
　６０ｍｍシャーレに単層培養したＭＤＣＫ細胞を５ｍｌのＰＢＳでリンスした。
　インフルエンザウイルス(原液)をＰＢＳで希釈して１００ＰＦＵ／２００μｌ／ｐｌａｔｅでＭＤＣＫ細胞に１時間、室温で吸着・感染した。このとき、ウイルスが均一に細胞に感染するように１５分ごとにシャーレを傾ける作業を繰り返した。
　５ｍｌのＰＢＳで１回リンスした後、種々の濃度のＥＧＣｇを含むプラークアッセイ用培地（上記表２）と２%ａｇａｒｏｓｅを３：２の割合で混合し、混合した溶液を５ｍＬずつシャーレに加えた。各培地には、ＥＧＣｇの最終濃度が０，０．３，１，３，１０，３０ｐｐｍになるよう加え、コントロール、各濃度は３枚ずつシャーレを作製した。
　寒天培地が固まった後シャーレを３７℃，５%ＣＯ２で２日間インキュベートした。コントロールのシャーレにプラークができていることを確認した後、ホルマリンで固定しプラーク数をカウントした。プラークを数えた結果をもとにプラーク数を５０%まで減少させる濃度（ＥＣ５０）を算出した。
　以上の結果を表８に示す。 [Example 11]
EGCg Treatment After Influenza Virus Adsorption/Infection MDCK cells monolayer-cultured in a 60 mm petri dish were rinsed with 5 ml of PBS.
Influenza virus (stock solution) was diluted with PBS and adsorbed and infected to MDCK cells at 100 PFU/200 μl/plate for 1 hour at room temperature. At this time, the operation of tilting the petri dish every 15 minutes was repeated so that the virus would uniformly infect the cells.
After rinsing once with 5 ml of PBS, plaque assay medium containing various concentrations of EGCg (Table 2 above) and 2% agarose were mixed at a ratio of 3:2, and 5 mL of the mixed solution was added to each Petri dish. . EGCg was added to each medium at final concentrations of 0, 0.3, 1, 3, 10, and 30 ppm, and 3 petri dishes were prepared for each concentration as a control.
After the agar medium had solidified, the dishes were incubated at 37°C, 5% CO2 for 2 days. After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated.
Table 8 shows the above results.
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
　上記の表８では、上から下へ、バンコク株、ＰＲ８株、愛知株、シンガポール株による結果を示した。また左から右へ、各株のControl、各ＥＧＣｇ量に対するプラーク数、プラーク数の割合、各々の近似線の傾き、Ｙ切片、平均値および標準偏差、t検定によるＰ値を示した。
　表８によれば、いずれのインフルエンザウイルス株におけるEC５０値は表５の吸着前のＥＧＣｇ処理した場合のＥＣ５０値と比較して８．３～１３．７倍高かった。（後記する表１１参照のこと。）
　従って、ＥＧＣｇの抗インフルエンザウイルス活性は、主にウイルスの細胞への吸着・侵入阻害によると考えられた。また、感染増殖して細胞外に放出された子孫ウイルスの細胞への再吸着・侵入が、ウイルス感染後の高濃度のＥＧＣｇ処理により抑制されたと考えられた。 In Table 8 above, from top to bottom, the results for the Bangkok strain, the PR8 strain, the Aichi strain, and the Singapore strain were shown. Also shown from left to right are the control of each strain, the number of plaques to each EGCg amount, the ratio of the number of plaques, the slope of each approximation line, the Y-intercept, the mean and standard deviation, and the P-value by t-test.
According to Table 8, the EC50 values for all influenza virus strains were 8.3 to 13.7 times higher than the EC50 values for EGCg treatment before adsorption in Table 5. (See Table 11 below.)
Therefore, it was considered that the anti-influenza virus activity of EGCg is mainly due to the inhibition of virus adsorption and entry into cells. In addition, it was considered that re-adsorption/invasion of progeny viruses released outside the cells after infection and multiplication into cells was suppressed by high-concentration EGCg treatment after virus infection.
　［実施例１２］
　ＥＧＣｇ溶液（白酒で溶解したもの）でインフルエンザウイルスを希釈した場合
　６０ｍｍシャーレに単層培養したＭＤＣＫ細胞を５ｍｌのＰＢＳでリンスした。
　インフルエンザウイルス（原液）は５０００ＰＦＵ／ｍｌとなるようにＰＢＳで希釈した種々の濃度のＥＧＣｇ／白酒溶液（原液：１００００ｐｐｍ）と混合し（対照液はＰＢＳ）、さらに、それぞれ種々の濃度のＥＧＣｇを含むＰＢＳ（０，０．１，０．３，１，３，１０，３０ｐｐｍ）で１０倍希釈した５００ＰＦＵ／ｍｌ（１００ＰＦＵ／２００μｌ／ｐｌａｔｅ）のウイルス液２００μｌを単層培養してＰＢＳでリンスしたＭＤＣＫ細胞に１時間、室温で吸着した。このとき、ウイルスが均一に細胞に感染するように15分ごとにシャーレを傾ける作業を繰り返した。
　なお、用いた白酒はＷＵＬＩＡＮＧＹＥ　五粮液（商品名）、ＷＵＬＩＡＮＧＹＥ　ＹＩＢＩＮ　ＣＯ，ＬＴＤ製であり、後記する実施例１０も同じである。
　感染したＭＤＣＫ細胞を５ｍｌのＰＢＳでリンス後、プラークアッセイ用培地（上記表２）と２%ａｇａｒｏｓｅを３：２の割合で混合し、混合した溶液を５ｍＬずつシャーレに加えた。コントロール、各濃度は３枚ずつシャーレを作製した。
　寒天培地が固まった後シャーレを３７℃，５%ＣＯ２で２日間インキュベートした。コントロールのシャーレにプラークができていることを確認した後、ホルマリンで固定しプラーク数をカウントした。プラークを数えた結果をもとにプラーク数を５０%まで減少させる濃度（ＥＣ５０）を算出した。
　以上の結果を表９に示す。 [Example 12]
When Influenza Virus Was Diluted with EGCg Solution (Dissolved in Shirojiu) MDCK cells monolayer-cultured in a 60-mm petri dish were rinsed with 5 ml of PBS.
Influenza virus (stock solution) was mixed with various concentrations of EGCg/white liquor solution (stock solution: 10000 ppm) diluted with PBS to 5000 PFU/ml (control solution was PBS), and each contained EGCg at various concentrations. MDCK obtained by monolayer culture of 200 μl of virus solution of 500 PFU/ml (100 PFU/200 μl/plate) diluted 10-fold with PBS (0, 0.1, 0.3, 1, 3, 10, 30 ppm) and rinsed with PBS The cells were adsorbed for 1 hour at room temperature. At this time, the operation of tilting the petri dish every 15 minutes was repeated so that the virus would uniformly infect the cells.
The white liquor used was WULIANGYE Goryui (trade name) manufactured by WULIANGYE YIBIN CO, LTD.
After rinsing the infected MDCK cells with 5 ml of PBS, the plaque assay medium (Table 2 above) and 2% agarose were mixed at a ratio of 3:2, and each 5 ml of the mixed solution was added to the petri dish. For the control and each concentration, 3 petri dishes were prepared.
After the agar medium had solidified, the dishes were incubated at 37°C, 5% CO2 for 2 days. After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated.
Table 9 shows the above results.
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
　上記の表９では、上から下へ、バンコク株、ＰＲ８株、愛知株、シンガポール株による結果を示した。また左から右へ、各株のControl、各ＥＧＣｇ溶液（白酒で溶解したもの）に対するプラーク数、プラーク数の割合、各々の近似線の傾き、Ｙ切片、平均値および標準偏差、t検定によるＰ値を示した。
　表９によれば、プラークはＥＧＣｇの１ｐｐｍ以上添加で、いずれのインフルエンザウイルス株であってもウイルス量が５０％以上減少し、つまりウイルスの増殖が抑制された。
　上記データの統計的解析として、ｔ検定による解析によれば一部を除きｐ＜０．０５であり有意、つまりカテキン類とウイルス株とが接触することによるウイルス増殖の抑制効果は統計的にも有意であることが示された。
　従って、インフルエンザウイルス株をＥＧＣｇ含有液で希釈、すなわち細胞に吸着前に白酒中のＥＧＣｇとウイルスを接触させることで、ウイルスの細胞への吸着あるいは侵入が阻害されて、インフルエンザウイルス株の増殖を抑制できることが分かる。さらに、表５のEC５０値に比べ表９のＥＣ５０値は低いためＥＧＣｇは水溶液より白酒中の方が抗インフルエンザウイルス活性は高まるように思われた In Table 9 above, from top to bottom, the results for the Bangkok strain, the PR8 strain, the Aichi strain, and the Singapore strain were shown. From left to right, the control of each strain, the number of plaques in each EGCg solution (dissolved in white sake), the percentage of plaques, the slope of each approximation line, the Y-intercept, the mean and standard deviation, the P by the t-test showed the value.
According to Table 9, when 1 ppm or more of EGCg was added to plaques, the amount of virus was reduced by 50% or more for any influenza virus strain, that is, virus proliferation was suppressed.
As a statistical analysis of the above data, according to the t-test analysis, p < 0.05 is significant except for some, that is, the effect of suppressing virus growth due to contact between catechins and virus strains is statistically shown to be significant.
Therefore, by diluting the influenza virus strain with a solution containing EGCg, that is, by contacting the virus with EGCg in white sake before adsorption to the cells, the adsorption or entry of the virus into the cells is inhibited, and the proliferation of the influenza virus strain is suppressed. I know you can. Furthermore, since the EC50 values in Table 9 are lower than the EC50 values in Table 5, it seems that EGCg has a higher anti-influenza virus activity in white liquor than in aqueous solution.
　［実施例１３］
　ＥＧＣｇを含まない白酒でインフルエンザウイルスを希釈した場合
　６０ｍｍシャーレに単層培養したＭＤＣＫ細胞を、５ｍｌのＰＢＳでリンスした。上記「ＥＧＣｇ溶液（白酒で溶解したもの）でインフルエンザウイルスを希釈した場合」と同様に、白酒をＰＢＳで希釈して最終的に５００ＰＦＵ/ｍｌ（１００ＰＦＵ／２００μｌ／ｐｌａｔｅ）のウイルス液を調整した。
　ＰＢＳで種々に希釈したウイルス液２００μｌを、単層培養してＰＢＳでリンスしたＭＤＣＫ細胞に１時間室温で吸着した。このとき、ウイルスが均一に細胞に感染するように１５分ごとにシャーレを傾ける作業を繰り返した。
　感染したＭＤＣＫ細胞を５ｍｌのＰＢＳでリンス後、プラークアッセイ用培地（上記、表４）と２％ａｇａｒｏｓｅを３：２の割合で混合し、混合した溶液を５ｍＬずつシャーレに加えた。コントロール、各濃度は３枚ずつシャーレを作製した。寒天培地が固まった後シャーレを３７℃，５％ＣＯ２で２日間インキュベートした。コントロールのシャーレにプラークができていることを確認した後、ホルマリンで固定しプラーク数をカウントした。プラークを数えた結果をもとにプラーク数を５０%まで減少させる濃度（ＥＣ５０）を算出した。
　以上の結果を表１０に示す。 [Example 13]
When Influenza Virus Was Diluted with Shirojiu Not Containing EGCg MDCK cells monolayer-cultured in a 60-mm petri dish were rinsed with 5 ml of PBS. Shirozake was diluted with PBS to finally prepare a virus solution of 500 PFU/ml (100 PFU/200 µl/plate) in the same manner as in the case of diluting influenza virus with EGCg solution (dissolved in white sake).
200 μl of virus solution diluted variously with PBS was adsorbed to MDCK cells which had been monolayer-cultured and rinsed with PBS for 1 hour at room temperature. At this time, the operation of tilting the petri dish every 15 minutes was repeated so that the virus would uniformly infect the cells.
After rinsing the infected MDCK cells with 5 ml of PBS, the plaque assay medium (Table 4 above) and 2% agarose were mixed at a ratio of 3:2, and 5 ml of the mixed solution was added to each petri dish. For the control and each concentration, 3 petri dishes were prepared. After the agar medium had solidified, the dishes were incubated at 37° C., 5% CO 2 for 2 days. After confirming the formation of plaques in the control dish, the dish was fixed with formalin and the number of plaques was counted. Based on the results of plaque counting, the concentration (EC 50 ) at which the number of plaques was reduced by 50% was calculated.
Table 10 shows the above results.
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
　上記の表１０では、上から下へ、バンコク株、ＰＲ８株、愛知株、シンガポール株による結果を示した。いずれのウイルス株も白酒だけで希釈したものを用いた。いずれのウイルス株もControl、１０ｐｐｍ、３０ｐｐｍに当するように白酒で希釈したときのプラーク数とその割合を示した
　表１０によれば、プラークは３０ppmに相当するように白酒で希釈したときもほとんど減少しなかった。したっがって、白酒の抗インフルエンザウイルス活性に対する影響はないと考えられた。 In Table 10 above, results from Bangkok strain, PR8 strain, Aichi strain, and Singapore strain are shown from top to bottom. All virus strains were diluted with white liquor only. All virus strains were diluted with white sake to correspond to Control, 10 ppm, and 30 ppm. did not decrease. Therefore, it was considered that baijiu had no effect on the anti-influenza virus activity.
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
　上記の表１１は、表５～表１０の結果をまとめたものである。 Table 11 above summarizes the results of Tables 5 to 10.
　結果・考察
　ＥＧＣｇの吸着前阻害について、Ａ型株のＢａｎｇｋｏｋ（Ｈ１Ｎ１）、ＰＲ８（Ｈ１Ｎ１）、とＡｉｃｈｉ（Ｈ３Ｎ２）、Ｂ型株のＳｉｎｇａｐｏｒｅの４種のインフルエンザウイルス株に対するＥＣ５０は、以下の結果となった。
　ＥＧＣｇ／水溶液溶液で０．９３±０．３５～２．７６±０．２３ｐｐｍ、ＥＧＣｇ／白酒溶液希釈で０．４０±０．０５～０．８０±０．０７ｐｐｍであった。溶解液、白酒でウイルス希釈を行った場合、ＥＣ５０＞３０ｐｐｍであったことから、上記の４種のウイルスに対する抗ウイルス効果は認められなかった。 Results and considerations Regarding pre-adsorption inhibition of EGCg, the EC 50 against 4 influenza virus strains of type A strains Bangkok (H1N1), PR8 (H1N1), and Aichi (H3N2), type B strain Singapore were as follows. became.
It was 0.93±0.35 to 2.76±0.23 ppm for the EGCg/water solution and 0.40±0.05 to 0.80±0.07 ppm for the EGCg/shirozake diluted solution. When the virus was diluted with the dissolution solution and white sake, the EC 50 was >30 ppm, so no antiviral effect was observed against the above four viruses.
　ＥＧＣｇのウイルス吸着後阻害についてのＥＣ５０は、Ｂａｎｇｋｏｋでは１９．３２±１．７９ｐｐｍ、ＰＲ８は３０ｐｐｍ以上、Ａｉｃｈｉは２２．８９±１．４４ｐｐｍ、Ｓｉｎｇａｐｏｒｅは１１．０８±１．５６ｐｐｍとなった。これは、４種のウイルスの吸着前阻害に対して、それぞれ、約１４倍、１０倍以上、約８倍、約１２倍高い結果となった。また、ＥＧＣｇ前処理を行った場合の４種のインフルエンザウイルスに対するＥＣ５０は、３０ｐｐｍ以上となった。 The EC 50 for post-virus adsorption inhibition of EGCg was 19.32±1.79 ppm in Bangkok, ≧30 ppm for PR8, 22.89±1.44 ppm for Aichi, and 11.08±1.56 ppm for Singapore. This resulted in about 14-fold, more than 10-fold, about 8-fold, and about 12-fold higher pre-adsorption inhibition of the four viruses, respectively. In addition, the EC50 for the four types of influenza viruses when EGCg pretreatment was performed was 30 ppm or more.
　以上の結果から、ＥＧＣｇの抗ウイルス効果は、吸着以前の過程においてインフルエンザウイルス粒子に対して阻害効果が認められた。したがって、ＥＧＣｇにはインフルエンザウイルス粒子の細胞への吸着または侵入の過程において阻害作用を示すことで抗ウイルス活性があるのではないかと考えられ、これ他の結果は以前の結果をカテキン水溶液においても確認できた。
　さらに、インフルエンザウイルスはコロナウイルスと同様にＲＮＡウイルスであり、コロナウイルス（Ｃｏｖｉｄ－１９）及びその変異株についてもウイルスが宿主となるヒト、動物などの細胞に入り込むメカニズムを防御する可能性があると考えられる。
　このことは、抗体によるウイルス感染防止効果のような特異的な作用ではないものの、逆にウイルス一般などにおいて感染防御できる作用を期待できるものである。 From the above results, the antiviral effect of EGCg was found to have an inhibitory effect on influenza virus particles in the process prior to adsorption. Therefore, EGCg may have antiviral activity by exhibiting an inhibitory effect on the process of adsorption or entry of influenza virus particles into cells. did it.
In addition, influenza virus is an RNA virus like coronavirus, and coronavirus (Covid-19) and its mutant strains may also be protected by the mechanism by which the virus enters host cells such as humans and animals. Conceivable.
Although this is not a specific action such as the effect of preventing viral infections by antibodies, it is conversely expected to have the effect of preventing infection with viruses in general.
＜ＥＧＣｇの抗ウイルス活性２＞
　前記のＥＧＣｇの抗インフルエンザウイルス活性１に加え、ＳＡＲＳ－ＣｏＶ－２（ＣＯＶＩＤ－１９）に近縁で、ヒトに風邪症状を引き起こすｈｕｍａｎ　ｃｏｒｏｎａｖｉｒｕｓ（ＨＣｏＶ）－２２９Ｅに対するＥＧＣｇの抗ウイルス効果を５０％　Ｔｉｓｓｕｅ　Ｃｕｌｔｕｒｅ　Ｉｎｆｅｃｔｉｏｕｓ　Ｄｏｓｅ（ＴＣＩＤ５０）を指標に測定した。さらに、ＨＣｏＶ－２２９Ｅの宿主細胞であるＭＲＣ－５細胞、ＳＡＲＳ－ＣｏＶ－２の宿主細胞であるＶｅｒｏ－Ｅ６／ＴＭＰＲＳＳ２細胞、インフルエンザウイルスの宿主細胞であるＭＤＣＫ細胞に対する細胞毒性も評価して、抗ウイルス効果を評価する試験において、細胞毒性による影響のない、あるいは評価可能な条件で抗ウイルス効果を測定することができる条件を検討した。 <EGCg antiviral activity 2>
In addition to the anti-influenza virus activity 1 of EGCg described above, the antiviral effect of EGCg against human coronavirus (HCoV)-229E, which is closely related to SARS-CoV-2 (COVID-19) and causes cold symptoms in humans, is reduced by 50%. Culture Infectious Dose (TCID 50 ) was used as an indicator. Furthermore, cytotoxicity against MRC-5 cells, which are host cells for HCoV-229E, Vero-E6/TMPRSS2 cells, which are host cells for SARS-CoV-2, and MDCK cells, which are host cells for influenza virus, were also evaluated. In tests to evaluate virus effects, we investigated conditions under which antiviral effects can be measured without cytotoxic effects or under conditions that can be evaluated.
　ＥＧＣｇの抗ウイルス効果の評価には次の方法により行ない、結果を得た。 The following method was used to evaluate the antiviral effect of EGCg, and the results were obtained.
　試験［１］　ＥＧＣｇの添加によるＨＣｏＶ－２２９Ｅの直接不活化試験
　ＥＧＣｇのＨＣｏＶ－２２９Ｅへの添加による不活化を検証するために、９０μＬの１０，０００　ＴＣＩＤ５０／ｍｌのＨＣｏＶ－２２９Ｅに、最終濃度が０、０．１、１、１０ｐｐｍになるように１０μＬのＥＧＣｇを混合し、３０秒後にリン酸緩衝液（ＰＢＳ）で２倍希釈した。その後、ＰＢＳで２倍段階希釈を行い、９６ウエル（ｗｅｌｌ）のプレート（ｐｌａｔｅ）中のＭＲＣ－５細胞に1時間吸着させた。ＨＣｏＶ－２２９Ｅの細胞への吸着後、ウイルス液を取り除き、培地を加え、５日間培養した。ウイルスの力価は細胞変性を指標にＴＣＩＤ５０法で算出した。また、ＥＧＣｇが０ｐｐｍの力価をもとに感染性を算出した。各試験は４ウエルで２度行った。
　その結果、１ｐｐｍおよび１０ｐｐｍのＥＧＣｇと混合することにより、ＨＣｏＶ－２２９Ｅの感染性がそれぞれ平均で４５％および５５％減少することが確認された。また、２回の試験結果から、この実験系におけるＥＧＣｇのＨＣｏＶ－２２９Ｅに対する５０％効果濃度（ＥＣ５０）は０．５４ｐｐｍであることが明らかとなった。 Test [1] Direct inactivation study of HCoV-229E by addition of EGCg To verify the inactivation by addition of EGCg to HCoV-229E, 90 μL of 10,000 TCID50/ml HCoV-229E was added to a final concentration of 10 μL of EGCg was mixed so as to have concentrations of 0, 0.1, 1 and 10 ppm, and after 30 seconds, it was diluted twice with phosphate buffered saline (PBS). After that, 2-fold serial dilution was performed with PBS and allowed to adsorb to MRC-5 cells in a 96-well plate for 1 hour. After adsorption of HCoV-229E to the cells, the virus solution was removed, medium was added, and the cells were cultured for 5 days. The virus titer was calculated by the TCID 50 method using cytopathicity as an index. In addition, the infectivity was calculated based on the EGCg titer of 0 ppm. Each test was performed in duplicate with 4 wells.
The results confirmed that mixing with 1 ppm and 10 ppm EGCg reduced the infectivity of HCoV-229E by an average of 45% and 55%, respectively. Also, the results of two tests revealed that the 50% effective concentration (EC 50 ) of EGCg against HCoV-229E in this experimental system was 0.54 ppm.
　上記の試験プロトコールは以下の通りである。
１）水溶性カテキンを０、１、１０、１００ｐｐｍになるようにＰＢＳで希釈。
２）１０μＬの０、１、１０、１００ｐｐｍの水溶性カテキンを１．５ｍＬチューブに分注。
３）９０μＬの１．０×１０３ＴＣＩＤ５０／ｍｌのＨＣｏＶ－２２９Ｅを加える。
４）ピペッティングし、混合。
５）混合後、速やかにＰＢＳで２倍希釈
６）その後、ＰＢＳで２倍段階希釈
７）前日に１．０×１０４Ｃｅｌｌｓ／ｗｅｌｌで播種したＭＲＣ－５細胞にウイルス液を加える。
８）室温で１時間吸着
９）ウイルス液を除き、ＰＢＳで洗浄し、培地を加え、５日間培養
１０）ＴＣＩＤ５０を測定 The above test protocol is as follows.
1) Water-soluble catechins were diluted with PBS to 0, 1, 10 and 100 ppm.
2) Dispense 10 μL of 0, 1, 10, 100 ppm water-soluble catechins into 1.5 mL tubes.
3) Add 90 μL of 1.0×10 3 TCID 50 /ml HCoV-229E.
4) Pipette and mix.
5) Immediately after mixing, dilute 2-fold with PBS 6) Then serially dilute 2-fold with PBS 7) Add virus solution to MRC-5 cells seeded at 1.0×10 4 cells/well the day before.
8) Adsorption for 1 hour at room temperature 9) Remove virus solution, wash with PBS, add medium, culture for 5 days 10) Measure TCID 50
　最終濃度が０ｐｐｍ、０．１ｐｐｍ、１ｐｐｍ、１０ｐｐｍ、になるようにカテキンを１０００ＴＣＩＤ５０／ｍｌのＨＣｏＶ－２２９Ｅと混合し、速やかに２倍にＰＢＳを希釈した。その後、ＰＢＳで２倍段階希釈を行った。細胞にウイルスを室温で１時間吸着し、ＴＣＩＤ５０を測定した。各試験を２回４ウエルで行った。
　２回の平均で、１ｐｐｍおよび１０ｐｐｍにおいて、それぞれ平均で４５％および５５％の力価の減少が確認された。
　また、２回の試験をまとめると、ＥＣ５０＝０．５４ｐｐｍであることが分かる。
この結果から、ＥＧＣｇのＨＣｏＶ－２２９Ｅの直接不活化効果が明らかとなったが。その減少はそれほど高いとは言えない。 Catechin was mixed with 1000 TCID 50 /ml HCoV-229E at final concentrations of 0 ppm, 0.1 ppm, 1 ppm, 10 ppm, and rapidly diluted 2-fold in PBS. After that, 2-fold serial dilution was performed with PBS. Virus was adsorbed to the cells for 1 hour at room temperature and TCID50 was measured. Each test was performed in duplicate with 4 wells.
An average of 45% and 55% reduction in potency was observed at 1 ppm and 10 ppm, respectively, on average in duplicate.
In addition, it can be seen that the EC 50 =0.54 ppm when the two tests are combined.
This result revealed a direct inactivating effect of EGCg on HCoV-229E. The decline is not very high.
　試験［２］　ＥＧＣｇによるＨＣｏＶ－２２９Ｅの吸着阻害試験
　試験［１］において、ＥＧＣｇとＨＣｏＶ－２２９Ｅを混合することにより、ＨＣｏＶ－２２９Ｅの力価が５５％減少することから、直接不活化効果が明らかとなったが、その減少はそれほど高いとは言えない。ＥＧＣｇによるウイルスの感染阻害には、ウイルスの不活化だけでなく、吸着阻害が関与していると考えられる。そこで、試験１の直接不活化効果に加えて、細胞への吸着阻害を加えた効果を検証するために、９０μＬの２，５００ＴＣＩＤ５０／ｍｌのＨＣｏＶ－２２９Ｅに、最終濃度が０、０．１、０．２５、０．５、１、２．５、５、１０ｐｐｍになるように１０μＬのＥＧＣｇを混合し、速やかに同濃度のＥＧＣｇを含むＰＢＳで２倍希釈した。その後、ＥＧＣｇによるＨＣｏＶ－２２９ＥのＭＲＣ－５細胞への吸着阻害効果を確認するために、同濃度のＥＧＣｇを含むＰＢＳで２倍段階希釈を行い、９６ウエルプレート中のＭＲＣ－５細胞に１時間吸着させた。ＨＣｏＶ－２２９Ｅの細胞への吸着後、ウイルス液を取り除き、ＰＢＳで洗浄し、培地を加え、５日間培養した。ウイルスの力価は細胞変性を指標にＴＣＩＤ５０法で算出した。また、ＥＧＣｇが０ｐｐｍの力価をもとに感染性を算出した。各試験は４ウエルで３度行った。
　その結果、ＥＧＣｇ濃度依存的なＨＣｏＶ－２２９Ｅの感染性の減少が確認された。特に２．５ｐｐｍにおいて４７％、５ｐｐｍおよび１０ｐｐｍにおいて８２％と顕著な感染性の減少が確認された。また、３度の試験の結果から、この実験系におけるＥＧＣｇのＨＣｏＶ－２２９Ｅに対するＥＣ５０は１．６６ｐｐｍであることが確認された。 Test [2] Adsorption inhibition test of HCoV-229E by EGCg In test [1], mixing EGCg and HCoV-229E reduced the titer of HCoV-229E by 55%, indicating a direct inactivation effect. However, the decrease cannot be said to be very high. Inhibition of virus infection by EGCg is considered to involve not only virus inactivation but also adsorption inhibition. Therefore, in addition to the direct inactivation effect of Test 1, in order to verify the effect of inhibiting adsorption to cells, 90 μL of 2,500 TCID 50 /ml HCoV-229E was added to final concentrations of 0 and 0.1. , 0.25, 0.5, 1, 2.5, 5 and 10 ppm of EGCg was mixed, and quickly diluted twice with PBS containing the same concentration of EGCg. After that, in order to confirm the inhibitory effect of EGCg on adsorption of HCoV-229E to MRC-5 cells, a 2-fold serial dilution was performed with PBS containing the same concentration of EGCg, and MRC-5 cells in a 96-well plate were added for 1 hour. adsorbed. After adsorption of HCoV-229E to the cells, the viral fluid was removed, the cells were washed with PBS, medium was added, and the cells were cultured for 5 days. The virus titer was calculated by the TCID 50 method using cytopathicity as an index. In addition, the infectivity was calculated based on the EGCg titer of 0 ppm. Each test was performed in triplicate with 4 wells.
As a result, an EGCg concentration-dependent reduction in HCoV-229E infectivity was confirmed. In particular, a significant reduction in infectivity was confirmed, 47% at 2.5 ppm and 82% at 5 ppm and 10 ppm. The results of three tests also confirmed that the EC50 of EGCg against HCoV-229E in this experimental system was 1.66 ppm.
　上記の試験プロトコールは以下の通りである。
１）水溶性カテキンを０、１、２．５、５、１０、２５、５０、１００ｐｐｍになるようにＰＢＳで希釈。
２）４０μＬの０、１、２．５、５、１０、２５、５０、１００ｐｐｍの水溶性カテキンを１．５ｍｌチューブに分注。
３）３６０μＬの２．５×１０２ＴＣＩＤ５０／ｍｌのＨＣｏＶ－２２９Ｅを加える。
４）ピペッティングし、混合。
５）混合後、速やかに同濃度のカテキン入りＰＢＳで２倍希釈
６）その後、同濃度のカテキン入りＰＢＳで２倍段階希釈
７）前日に１．０×１０４Ｃｅｌｌｓ／ｗｅｌｌで播種したＭＲＣ－５細胞にウイルス液を加える。
８）室温で１時間吸着
９）ウイルス液を除き、ＰＢＳで洗浄し、培地を加え、５日間培養
１０）ＴＣＩＤ５０を測定 The above test protocol is as follows.
1) Water-soluble catechins were diluted with PBS to 0, 1, 2.5, 5, 10, 25, 50 and 100 ppm.
2) Dispense 40 μL of 0, 1, 2.5, 5, 10, 25, 50, 100 ppm water-soluble catechin into a 1.5 ml tube.
3) Add 360 μL of 2.5×10 2 TCID 50 /ml HCoV-229E.
4) Pipette and mix.
5) After mixing, immediately dilute 2-fold with PBS containing catechin at the same concentration 6) Then serially dilute 2-fold with PBS containing catechin at the same concentration 7) MRC- 5 Add the virus solution to the cells.
8) Adsorption for 1 hour at room temperature 9) Remove virus solution, wash with PBS, add medium, culture for 5 days 10) Measure TCID 50
　最終濃度が０ｐｐｍ、０．１ｐｐｍ、０．２５ｐｐｍ、０．５ｐｐｍ、１ｐｐｍ、２．５ｐｐｍ、５ｐｐｍ、１０ｐｐｍになるようにカテキンを２５０ＴＣＩＤ５０／ｍｌのＨＣｏＶ－２２９Ｅと混合し、速やかに２倍にカテキン入りのＰＢＳに希釈した。その後、カテキン入りのＰＢＳで２倍段階希釈を行った。細胞にウイルスを室温で１時間吸着し、ＴＣＩＤ５０を測定した。各試験を3回４ウエルで行った。
　３回の試験の平均で、２．５ｐｐｍにおいて４７％、５ｐｐｍと１０ｐｐｍにおいて、８２％と顕著な力価の減少が確認された。
　また、３回の試験をまとめると、ＥＣ５０＝１．６６ｐｐｍであることが分かる。
　この結果から、直接不活化効果だけでなく、細胞への吸着阻害効果が示唆された。 Catechin was mixed with 250 TCID 50 /ml HCoV-229E at final concentrations of 0 ppm, 0.1 ppm, 0.25 ppm, 0.5 ppm, 1 ppm, 2.5 ppm, 5 ppm, 10 ppm, and rapidly doubled catechin diluted in PBS. After that, 2-fold serial dilution was performed with PBS containing catechin. Virus was adsorbed to the cells for 1 hour at room temperature and TCID50 was measured. Each test was performed in triplicate in 4 wells.
A significant potency reduction of 47% at 2.5ppm and 82% at 5ppm and 10ppm was confirmed on average of three tests.
Moreover, it turns out that it is EC50 =1.66ppm when the test of 3 times is put together.
This result suggested not only a direct inactivation effect but also an inhibitory effect on cell adsorption.
　試験［３］　１時間のＥＧＣｇ処理によるＨＣｏＶ－２２９Ｅの直接不活化および吸着阻害試験
　試験［１］と試験［２］の結果から、ＨＣｏＶ－２２９ＥはＥＧＣｇによって、直接不活化されるだけでなく、細胞への吸着も阻害されると考えられる。そこで、ＥＧＣｇによるＨＣｏＶ－２２９Ｅの不活化の機序をより詳細に明らかにすることを目的に、長時間のＥＧＣｇ処理によるＨＣｏＶ－２２９Ｅに対する直接不活化能を測定するために、９０μＬの２，５００ＴＣＩＤ５０／ｍｌのＨＣｏＶ－２２９Ｅに、最終濃度が０、０．１、０．５、１、２．５、５、１０ｐｐｍになるように１０μＬのＥＧＣｇを混合し、１時間室温で置いた後に、同濃度のＥＧＣｇを含むＰＢＳで２倍希釈した。その後、同濃度のＥＧＣｇを含むＰＢＳで２倍段階希釈を行い、９６ウエルプレート中のＭＲＣ－５細胞に１時間吸着させた。ＨＣｏＶ－２２９Ｅの細胞への吸着後、ウイルス液を取り除き、ＰＢＳで洗浄し、培地を加え、５日間培養した。ウイルスの力価は細胞変性を指標にＴＣＩＤ５０法で算出した。また、ＥＧＣｇが０ｐｐｍの力価をもとに感染性を算出した。各試験は４ウエルで３度行った。
　その結果、ＥＧＣｇ濃度依存的なＨＣｏＶ－２２９Ｅの感染性の減少が確認された。特に２．５ｐｐｍにおいては６２％、５．０ｐｐｍにおいては７６％、１０ｐｐｍにおいては８８％と顕著な感染性の減少が確認された。また、３度の試験の結果から、この実験系におけるＥＧＣｇのＨＣｏＶ－２２９Ｅに対するＥＣ５０は１．１１ｐｐｍであることが確認された。 Test [3] Direct Inactivation of HCoV-229E by EGCg Treatment for 1 Hour and Adsorption Inhibition Test From the results of Tests [1] and [2], HCoV-229E is not only directly inactivated by EGCg, but also Adsorption to cells is also thought to be inhibited. Therefore, for the purpose of clarifying the mechanism of HCoV-229E inactivation by EGCg in more detail, 90 μL of 2,500 TCID 10 μL of EGCg was mixed with 50 /ml of HCoV-229E to a final concentration of 0, 0.1, 0.5, 1, 2.5, 5, 10 ppm, and left at room temperature for 1 hour. It was diluted twice with PBS containing the same concentration of EGCg. After that, 2-fold serial dilution was performed with PBS containing the same concentration of EGCg and allowed to adsorb to MRC-5 cells in a 96-well plate for 1 hour. After adsorption of HCoV-229E to the cells, the virus fluid was removed, washed with PBS, added with medium, and cultured for 5 days. The virus titer was calculated by the TCID 50 method using cytopathicity as an index. In addition, the infectivity was calculated based on the EGCg titer of 0 ppm. Each test was performed in triplicate with 4 wells.
As a result, an EGCg concentration-dependent reduction in HCoV-229E infectivity was confirmed. In particular, a significant reduction in infectivity was confirmed, with 62% at 2.5 ppm, 76% at 5.0 ppm, and 88% at 10 ppm. The results of three tests also confirmed that the EC50 of EGCg against HCoV-229E in this experimental system was 1.11 ppm.
　上記の試験プロトコールは以下の通りである。
１）水溶性カテキンを０、１、２．５、５、１０、２５、５０、１００ｐｐｍになるようにＰＢＳで希釈。
２）４０μＬの０、１、２．５、５、１０、２５、５０、１００ｐｐｍの水溶性カテキンを１．５ｍｌチューブに分注。
３）３６０μＬの２．５×１０２ＴＣＩＤ５０／ｍｌのＨＣｏＶ－２２９Ｅを加える。
４）ピペッティングし、混合。
５）混合後、1時間室温で静置
６）その後、同濃度のカテキン入りＰＢＳで２倍段階希釈
７）前日に１．０×１０４Ｃｅｌｌｓ／ｗｅｌｌで播種したＭＲＣ－５細胞にウイルス液を加える。
８）室温で１時間吸着
９）ウイルス液を除き、ＰＢＳで洗浄し、培地を加え、５日間培養
１０）ＴＣＩＤ５０を測定 The above test protocol is as follows.
1) Water-soluble catechins were diluted with PBS to 0, 1, 2.5, 5, 10, 25, 50 and 100 ppm.
2) Dispense 40 μL of 0, 1, 2.5, 5, 10, 25, 50, 100 ppm water-soluble catechin into a 1.5 ml tube.
3) Add 360 μL of 2.5×10 2 TCID 50 /ml HCoV-229E.
4) Pipette and mix.
5) After mixing, let stand at room temperature for 1 hour 6) Then serially dilute 2-fold with PBS containing catechin at the same concentration 7) Add the virus solution to MRC-5 cells seeded at 1.0×10 4 cells/well the day before. Add.
8) Adsorption for 1 hour at room temperature 9) Remove virus solution, wash with PBS, add medium, culture for 5 days 10) Measure TCID 50
　最終濃度が０ｐｐｍ、０．１ｐｐｍ、０．５ｐｐｍ、１ｐｐｍ、２．５ｐｐｍ、５ｐｐｍ、１０ｐｐｍになるようにカテキンを２５０ＴＣＩＤ５０／ｍｌのＨＣｏＶ－２２９Ｅと混合し、１時間室温で静置した。その後、カテキン入りのＰＢＳで２倍段階希釈を行った。細胞にウイルスを室温で１時間吸着し、ＴＣＩＤ５０を測定した。各試験を３回４ウエルで行った。
　３回の試験のすべての試験において、濃度依存的な力価の減少が確認された。特に２．５ｐｐｍでは６２％、５．０ｐｐｍでは７６％、１０ｐｐｍでは８８％の力価の減少が確認された。
　また、３回の試験をまとめると、ＥＣ５０＝１．１１ｐｐｍであることが分かった。 Catechin was mixed with 250 TCID 50 /ml HCoV-229E to final concentrations of 0 ppm, 0.1 ppm, 0.5 ppm, 1 ppm, 2.5 ppm, 5 ppm and 10 ppm, and allowed to stand at room temperature for 1 hour. After that, 2-fold serial dilution was performed with catechin-containing PBS. Cells were adsorbed with virus for 1 hour at room temperature and TCID50 was measured. Each test was performed in triplicate in 4 wells.
A concentration-dependent decrease in potency was confirmed in all three tests. In particular, a decrease in potency of 62% at 2.5 ppm, 76% at 5.0 ppm, and 88% at 10 ppm was confirmed.
It was also found that the EC50 = 1.11 ppm when the three tests were combined.
　試験［４］　ＥＧＣｇの細胞毒性の測定
　ＥＧＣｇの細胞毒性を測定するために、２４ウエルプレート中のＶｅｒｏ－Ｅ６／ＴＭＰＲＳＳ２細胞、ＭＲＣ－５細胞、ＭＤＣＫ細胞に、最終濃度が０、０．１、１、１０、１００、３００ｐｐｍになるようにＥＧＣｇを滴下し、２日間培養した。細胞毒性は、ＷＳＴ―８試薬を加え、３７℃Ｃで１．５時間静置し、吸光度をもとに生細胞数を測定することで評価した。また、５０％細胞毒性濃度（ＣＣ５０）は、ＥＧＣｇが０ｐｐｍの細胞の生細胞数をもとに算出した。
　その結果、試験したすべての細胞において、１０ｐｐｍから１００ｐｐｍにかけて、生細胞数の減少が確認された。このことから、高濃度のＥＧＣｇにおいて、細胞毒性が存在することが明らかとなった。また、Ｖｅｒｏ－Ｅ６／ＴＭＰＲＳＳ２細胞、ＭＲＣ－５細胞、ＭＤＣＫ細胞のＣＣ５０はそれぞれ、６７．５３ｐｐｍ、６５．３４ｐｐｍ、８５．６１ｐｐｍであることが明らかとなった。 Test [4] Determination of EGCg Cytotoxicity To determine the cytotoxicity of EGCg, Vero-E6/TMPRSS2, MRC-5, MDCK cells in 24-well plates were added to final concentrations of 0, 0.1, EGCg was added dropwise to 1, 10, 100 and 300 ppm, and cultured for 2 days. Cytotoxicity was evaluated by adding WST-8 reagent, allowing to stand at 37°C for 1.5 hours, and measuring the number of viable cells based on absorbance. The 50% cytotoxic concentration (CC 50 ) was calculated based on the viable cell count of cells with an EGCg of 0 ppm.
As a result, a decrease in the number of viable cells was confirmed from 10 ppm to 100 ppm in all cells tested. This revealed that cytotoxicity exists at high concentrations of EGCg. In addition, CC 50 of Vero-E6/TMPRSS2 cells, MRC-5 cells and MDCK cells were found to be 67.53 ppm, 65.34 ppm and 85.61 ppm, respectively.
　上記の試験プロトコールは以下の通りである。
水溶化カテキン（ＥＧＣｇ）の毒性試験
１）Ｖｅｒｏ－Ｅ６－ＴＭＰＲＳＳ２細胞あるいはＭＲＣ－５細胞を１．０×１０５ｃｅｌｌｓ／ｍｌになるようウエルプレートに播種（培地は５００μＬ）
２）１７時間培養
３）カテキンとその溶媒を０～１０００ｐｐｍになるように、５０μＬ加える。
４）４８時間培養
５）ＷＳＴ―８試薬を３０μＬ加え、１．５時間３７℃で加温
６）９６ウエルプレートに上清を１００μＬずつ移し、ＯＤ４５０を測定 The above test protocol is as follows.
Toxicity Test of Solubilized Catechin (EGCg) 1) Vero-E6-TMPRSS2 cells or MRC-5 cells were seeded in a well plate at 1.0×10 5 cells/ml (500 μL medium).
2) Incubate for 17 hours 3) Add 50 μL of catechin and its solvent so that the concentration is 0 to 1000 ppm.
4) Culture for 48 hours 5) Add 30 μL of WST-8 reagent and heat at 37° C. for 1.5 hours 6) Transfer 100 μL of the supernatant to a 96-well plate and measure OD450
　全ての細胞において、１００ｐｐｍ、３００ｐｐｍのＥＧＣｇでＷＳＴ試薬の反応が弱くなることから、ＥＧＣｇに細胞毒性があることが分かった。 　In all cells, 100 ppm and 300 ppm of EGCg weakened the reaction of the WST reagent, indicating that EGCg is cytotoxic.
　以上の試験［１］～［４］の結果を下記の表１２にまとめた。
Figure JPOXMLDOC01-appb-T000018
 The results of the above tests [1] to [4] are summarized in Table 12 below.
Figure JPOXMLDOC01-appb-T000018
　表１２に示す通り、試験［１］～［３］の結果から、ＥＧＣｇは直接不活化効果と吸着阻害効果の二つの作用により、ＨＣｏＶ―２２９Ｅの感染性を減少させることが明らかとなった。その結果、１０ｐｐｍのＥＧＣｇによって、５５％～８５％のＨＣｏＶ－２２９Ｅの力価の減少が認められ、一定の抗ウイルス効果を示すことが明らかとなった。また、そのＥＣ５０は０．５４ｐｐｍ～１．１１ｐｐｍであり、非常に低濃度で抗ウイルス効果を示すことが示唆された。
　一方で、試験［４］の細胞毒性の結果から、ＥＧＣｇはＶｅｒｏ－Ｅ６／ＴＭＰＲＳＳ２細胞、ＭＲＣ－５細胞、ＭＤＣＫ細胞に対して、細胞毒性を有することが明らかとなった。しかし、そのＣＣ５０は６５．５３ｐｐｍ～８５．６１ｐｐｍとＥＣ５０と比較して、高濃度であることから、細胞、例えばヒト、動物等の細胞への影響はほとんどなく、人体等に悪影響はないと考えられる。
　さらに、ＳＡＲＳ－ＣｏＶ－２に近縁なＨＣｏＶ－２２９Ｅに対して、抗ウイルス効果を示したことから、ＥＧＣｇはＳＡＲＳ－ＣｏＶ－２に対しても抗ウイルス効果を示すことが期待される。
　特にカテキン類の抗ウイルス効果がインフルエンザ及びＨＣｏＶ－２２９Ｅ（ウイルス）にあることが示されていることから、近縁のＳＡＲＳ－ＣｏＶ－２（ＣＯＶＩＤ－１９）及びその各種の変異株にも抗ウイルス効果が認められることが期待できる。 As shown in Table 12, the results of Tests [1] to [3] revealed that EGCg reduces the infectivity of HCoV-229E through two actions: a direct inactivation effect and an adsorption inhibition effect. As a result, 10 ppm EGCg decreased the HCoV-229E titer by 55% to 85%, demonstrating a certain antiviral effect. Also, its EC 50 was 0.54 ppm to 1.11 ppm, suggesting that it exhibits antiviral effects at very low concentrations.
On the other hand, the cytotoxicity results of test [4] revealed that EGCg has cytotoxicity to Vero-E6/TMPRSS2 cells, MRC-5 cells and MDCK cells. However, since its CC 50 is 65.53 ppm to 85.61 ppm, which is a high concentration compared to EC 50 , it has almost no effect on cells such as human and animal cells, and has no adverse effect on the human body. it is conceivable that.
Furthermore, since it showed an antiviral effect against HCoV-229E, which is closely related to SARS-CoV-2, EGCg is expected to show an antiviral effect against SARS-CoV-2 as well.
In particular, catechins have been shown to have an antiviral effect on influenza and HCoV-229E (virus), so it is also antiviral for the closely related SARS-CoV-2 (COVID-19) and its various mutant strains. It is expected that the effect will be recognized.
［実施例１４］
＜マウスによるｉｎ　ｖｉｖｏ評価（抗インフルエンザウイルス活性）＞
　各群４匹のマウス両鼻の穴から３μｌのカテキン溶液(０，１０００，１００００ｐｐｍ)を垂らした後、５分以内にインフルエンザウイルス液２μｌ（２ｘ１０７ＰＦＵｍｌ）を両鼻の穴に垂らした。感染３日後に肺を摘出して、重さを測り、２ｍｌのＰＢＳを加えてホモジナイズした。ホモジナイズした懸濁液を遠心して上清中のウイルス量をプラーク法で測定した。
　その結果、以下の表１３に示す通り、カテキンを加えることで、統計的に有意にウイルス抑制効果が認められた。なお表１３－１はｉｎ　ｖｉｖｏ評価の基礎データを、表１３－２はその結果をＴテスト（Ｔ検定）により評価した結果である。 [Example 14]
<In vivo evaluation using mice (anti-influenza virus activity)>
After dropping 3 μl of catechin solution (0, 1000, 10000 ppm) from both nostrils of four mice in each group, 2 μl of influenza virus solution (2×10 7 PFU ml) was dropped into both nostrils within 5 minutes. Lungs were removed 3 days after infection, weighed, and homogenized with 2 ml of PBS. The homogenized suspension was centrifuged and the amount of virus in the supernatant was determined by the plaque method.
As a result, as shown in Table 13 below, addition of catechin was found to have a statistically significant virus suppressing effect. Table 13-1 shows basic data for in vivo evaluation, and Table 13-2 shows the results of evaluation by T-test (T-test).
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000021
Figure JPOXMLDOC01-appb-T000021
＜ＥＧＣｇ含有水溶液組成物入りタブレット（錠剤）の製造＞
［実施例１５］
　上記の水溶液組成物を、以下の条件で1個当たり１２００ｍｇの固形錠剤を製造した。 <Production of tablet (tablet) containing EGCg-containing aqueous solution composition>
[Example 15]
Solid tablets of 1200 mg each were produced from the above aqueous solution composition under the following conditions.
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-T000022
　上記配合１００ｇにつきカテキンを含む水溶液組成物６ｍｌ／１００ｇ添加した。水溶液組成物は以下で示す造粒用のバインダー液として、水溶液組成物を投入した。
・造粒用原料と、混合用原料に分けて秤量した。
・造粒工程では以下の材料に、カテキンを含む水溶液組成物を投入、撹拌した。
乳糖
でん粉
ブドウ果皮色素
甘味料
・その後、５５℃で３時間乾燥した。
・乾燥後、各錠剤の径が１０００μｍ以下になるよう篩（ふるい）を通して整粒した。
・整流した整粒物と、残りの原料を投入し混合した。
・混合物を打錠機により打錠した。 6 ml/100 g of an aqueous composition containing catechin was added per 100 g of the above formulation. The aqueous composition was charged as a binder liquid for granulation shown below.
- The raw material for granulation and the raw material for mixing were separately weighed.
- In the granulation step, an aqueous solution composition containing catechin was added to the following materials and stirred.
Lactose starch Grape peel pigment Sweetener ・Then, dried at 55°C for 3 hours.
- After drying, the tablets were sized through a sieve so that the diameter of each tablet was 1000 μm or less.
・The rectified granules and the remaining raw materials were added and mixed.
• The mixture was compressed using a tablet press.
＜ＥＧＣｇ含有水溶液組成物入りタブレット（錠剤）によるインフルエンザウイルス抑制試験＞
［実施例１６］
　タブレットの効果を確認するため、ＭＤＣＫ細胞を用いたｉｎ ｖｉｔｒｏ試験を行なった。
　Ｉｎ　ｖｉｔｒｏ実験では、１００００ＰＦＵのウイルス液とＰＢＳに溶かしたＥＧＣｇ溶液を同量混ぜて、ＥＧＣｇ各濃度で５０００ＰＦＵウイルス量が処理された。ウイルスとＥＧＣｇ混合液をＭＤＣＫ細胞に感染してプラーク法を行い、形成されたプラーク数からウイルス増殖を５０％抑制する濃度としてＥＣ５０値を算出した。Ｉｎ　ｖｉｔｒｏ実験ではＥＧＣｇが含まれていないタブレットを対照にして行なった。対照では全くウイルス抑制作用はなかった。 <Influenza virus inhibition test with tablets (tablets) containing EGCg-containing aqueous solution composition>
[Example 16]
To confirm the effect of the tablet, an in vitro test using MDCK cells was performed.
In the in vitro experiment, equal amounts of 10000 PFU virus solution and EGCg solution dissolved in PBS were mixed, and 5000 PFU virus amount was treated at each concentration of EGCg. MDCK cells were infected with a mixture of virus and EGCg, and the plaque method was performed. From the number of plaques formed, the EC50 value was calculated as the concentration that inhibits virus growth by 50%. In vitro experiments were performed with tablets containing no EGCg as a control. Controls had no virus-suppressing effect.
Figure JPOXMLDOC01-appb-T000023
Figure JPOXMLDOC01-appb-T000023
　本発明によるカテキン類を含む組成物、製剤は、特に優れた保存安定性を有し、しかも経時的な褐変化等の色調変化を伴わず、また実質的に低級アルコールや界面活性剤を含まないので、生体への安全性が極めて高く、しかも保存安定性に優れている。したがって、食品、化粧料、医薬品等に配合すると、カテキン類の有益な健康効果や抗酸化効果を期待でき、産業上に極めて有用な技術である。 Compositions and formulations containing catechins according to the present invention have particularly excellent storage stability, are not accompanied by changes in color tone such as browning over time, and are substantially free of lower alcohols and surfactants. Therefore, it has extremely high safety to the living body and excellent storage stability. Therefore, when blended in foods, cosmetics, pharmaceuticals, etc., the beneficial health effects and antioxidant effects of catechins can be expected, and this is an extremely useful technology in industry.

Claims (12)

  1. 　カテキン類、サイクロデキストリン及び抗酸化剤を含む水溶液組成物であって、
    　前記カテキン類は溶解度以下の濃度に溶解され、
    　水溶液組成物中、前記サイクロデキストリンは、前記カテキン類に対して１／１０以上のモル量であり、
    　４０℃、１４日間で、水溶液組成物中のカテキン類が９６重量％以上残存している、
    　保存安定性に優れた水溶液組成物。     An aqueous solution composition containing catechins, cyclodextrin and an antioxidant,
    The catechins are dissolved at a concentration below the solubility,
    In the aqueous solution composition, the cyclodextrin is in a molar amount of 1/10 or more with respect to the catechins,
    96% by weight or more of catechins in the aqueous solution composition remains at 40° C. for 14 days,
    An aqueous solution composition with excellent storage stability.
  2. 　カテキン類、サイクロデキストリン及び抗酸化剤を含む水溶液組成物であって、
    　前記カテキン類は前記水溶液組成物中に１０００ｐｐｍ以上の濃度で溶解しており、
    　前記水溶液組成物中、前記サイクロデキストリンは、前記カテキン類に対して１／１０以上のモル量であり、
    　前記水溶液組成物を４０℃、１４日間保存したときに、水溶液組成物中のカテキン類が５０重量％以上残存している、
    　保存安定性に優れた水溶液組成物。     An aqueous solution composition containing catechins, cyclodextrin and an antioxidant,
    The catechins are dissolved in the aqueous solution composition at a concentration of 1000 ppm or more,
    In the aqueous solution composition, the cyclodextrin is in a molar amount of 1/10 or more with respect to the catechins,
    When the aqueous composition is stored at 40° C. for 14 days, 50% by weight or more of catechins remain in the aqueous composition.
    An aqueous solution composition with excellent storage stability.
  3. 　水に包接化剤としてのサイクロデキストリンを溶解させた後、カテキン類を溶解し、さらに抗酸化剤を加えて溶解させてなる、請求項１又は請求項２に記載の組成物。 The composition according to claim 1 or 2, which is obtained by dissolving cyclodextrin as an encapsulating agent in water, dissolving catechins, and then adding and dissolving an antioxidant.
  4. 　包接化剤としてのサイクロデキストリンとカテキン類とを混合した後、水に加えて溶解させ、さらに抗酸化剤を加えて溶解させてなる、請求項１又は請求項２に記載の組成物。 The composition according to claim 1 or claim 2, which is obtained by mixing cyclodextrin as an encapsulating agent and catechins, adding water to dissolve the mixture, and adding and dissolving an antioxidant.
  5. 　カテキン類が、エピガロカテキンガレートである、請求項１～４のいずれか一項に記載の組成物。 The composition according to any one of claims 1 to 4, wherein the catechin is epigallocatechin gallate.
  6. 　サイクロデキストリンが、α-サイクロデキストリン、β-サイクロデキストリン及びγ-サイクロデキストリンからなる群より選ばれる１種または２種以上の混合物である、請求項１～５のいずれか一項に記載の組成物。 The composition according to any one of claims 1 to 5, wherein the cyclodextrin is one or a mixture of two or more selected from the group consisting of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin. .
  7. 　抗酸化剤が、アスコルビン酸もしくはその誘導体、またはフィトン酸もしくはその誘導体である、請求項１～６のいずれか一項に記載の組成物。 The composition according to any one of claims 1 to 6, wherein the antioxidant is ascorbic acid or a derivative thereof, or phytonic acid or a derivative thereof.
  8. 　請求項１～７のいずれか一項に記載の組成物を、内容物を噴射可能な容器に充填してなる、カテキン類を含むスプレー。 A spray containing catechins, obtained by filling the composition according to any one of claims 1 to 7 into a container capable of spraying the contents.
  9. 　請求項１～７のいずれか一項に記載の組成物が、固形物に含侵されており、錠剤の形態とされてなる、カテキン類を含む錠剤。 A tablet containing catechins, in which the composition according to any one of claims 1 to 7 is impregnated in a solid material and formed into a tablet form.
  10. 　カテキン類もしくはこれの薬学的に許容可能な塩、サイクロデキストリン及び抗酸化剤を含む水溶液組成物であって、カテキン類を有効成分として含む抗ウイルス剤。 An antiviral agent that is an aqueous solution composition containing catechins or a pharmaceutically acceptable salt thereof, cyclodextrin, and an antioxidant, which contains catechins as an active ingredient.
  11. 　ウイルスがコロナウイルスである、請求項１０に記載の抗ウイルス剤。 The antiviral agent according to claim 10, wherein the virus is a coronavirus.
  12. 　ウイルスが新型コロナウイルス（ＣＯＶＩＤ－１９）である、請求項１０に記載の抗ウイルス剤。 The antiviral agent according to claim 10, wherein the virus is the novel coronavirus (COVID-19).
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