WO2023026867A1 - Composition de solution aqueuse contenant de la catéchine présentant une excellente stabilité au stockage, et son utilisation - Google Patents

Composition de solution aqueuse contenant de la catéchine présentant une excellente stabilité au stockage, et son utilisation Download PDF

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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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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

Le problème décrit par la présente invention est de fournir une préparation de polyphénol dans laquelle des catéchines sont présentes dans de l'eau de façon stable sur une longue période de temps et qui ne subit pas de changement de ton de couleur tel que le brunissement au cours du temps, et par conséquent, est utile pour de nombreuses applications telles que des boissons et des aliments, des produits cosmétiques, des médicaments et similaires, et est utile en tant que matière première pour sa production ; et un procédé de production de la préparation de polyphénol. La solution selon l'invention porte sur une préparation de catéchine qui comprend de l'eau purifiée, au moins un composant de catéchine, au moins une cyclodextrine et au moins un agent anti-oxydant, et qui présente une excellente stabilité au stockage.
PCT/JP2022/030616 2021-08-24 2022-08-10 Composition de solution aqueuse contenant de la catéchine présentant une excellente stabilité au stockage, et son utilisation WO2023026867A1 (fr)

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WO2021256473A1 (fr) * 2020-06-15 2021-12-23 京都府公立大学法人 Agent anti-coronavirus
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JP2008148588A (ja) * 2006-12-14 2008-07-03 Taiyo Kagaku Co Ltd ポリフェノール組成物
JP2010168318A (ja) * 2009-01-23 2010-08-05 Pure Green Kk 保存安定性に優れたポリフェノール類製剤
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