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WO2022050819A1 - Composition anti-inflammatoire pour réduire les rides cutanées et hydrater et activer la régénération de la peau, contenant, en tant que principe actif, un extrait d'huile de krill traitée par une enzyme - Google Patents

Composition anti-inflammatoire pour réduire les rides cutanées et hydrater et activer la régénération de la peau, contenant, en tant que principe actif, un extrait d'huile de krill traitée par une enzyme Download PDF

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Publication number
WO2022050819A1
WO2022050819A1 PCT/KR2021/012139 KR2021012139W WO2022050819A1 WO 2022050819 A1 WO2022050819 A1 WO 2022050819A1 KR 2021012139 W KR2021012139 W KR 2021012139W WO 2022050819 A1 WO2022050819 A1 WO 2022050819A1
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Prior art keywords
skin
treated
enzyme
krill oil
composition
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English (en)
Korean (ko)
Inventor
박설웅
박상용
이근하
원경화
이충일
김재실
서슬아
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Sd Biotechnologies Co Ltd
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Sd Biotechnologies Co Ltd
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    • 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
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/612—Crustaceans, e.g. crabs, lobsters, shrimps, krill or crayfish; Barnacles
    • 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
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • 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
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A61K8/925—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of animal origin
    • 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
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95

Definitions

  • the present invention relates to a cosmetic composition comprising an enzyme-treated krill oil extract as an active ingredient, and more particularly, to a composition for anti-inflammatory, skin wrinkle improvement, moisturizing and skin regeneration promotion comprising a protease-treated krill oil extract as an active ingredient will be.
  • collagen exists in the dermal layer of the skin, and it is known as a major component that gives elasticity to the skin along with elastin, which accounts for about 70% to 80% of the total dry weight of the skin.
  • Collagen is reduced by internal factors such as decreased cellular activity due to natural aging, and biosynthesis is decreased or decomposition is accelerated by external factors such as increased stress in various harmful environments or increased reactive oxygen species caused by sunlight. there is.
  • ROS reactive oxygen species
  • Type I collagen is the most abundant protein in the extracellular matrix of connective tissue.
  • the extracellular matrix includes other types of proteins such as type III, V, and VII collagen, elastin, proteoglycans, and fibronectin.
  • a cosmetic composition for moisturizing generally used for the skin serves to maintain a certain amount of moisture in human hair or skin, thereby making it soft and lively, and preventing damage such as cracking and dryness. That is, the cosmetic composition for moisturizing the skin is used for the purpose of beautifying the skin or hair by supplying or retaining moisture to the skin or hair in a predetermined amount or more, and keeping them healthy.
  • the skin is an organ that has an essential function to maintain moisture, and it is known that it accounts for about 2/3 of the weight in moisture control. Therefore, in the cosmetic field, functional research related to supply and maintenance of moisture, that is, moisturizing, is being actively conducted.
  • the development of cosmetics that increase the moisturizing power of the skin by introducing physiologically active substances obtained from natural products into cosmetics to further strengthen the inherent defense function of the skin itself is an active trend.
  • the main object of the present invention is an enzyme that has excellent antioxidant activity and anti-inflammatory activity, and at the same time suppresses skin wrinkles, is effective in moisturizing the skin, and promotes the regeneration of damaged skin to comprehensively improve the skin condition.
  • the present invention provides a cosmetic composition for anti-inflammatory, skin wrinkle improvement, moisturizing and promoting skin regeneration comprising a protease-treated krill oil extract as an active ingredient.
  • the present inventors made intensive research efforts to discover natural ingredients that can be usefully used to improve skin condition. As a result, it was confirmed that the proteolytic enzyme-treated krill oil extract exhibits anti-inflammatory, skin wrinkle improvement, moisturizing and skin regeneration promoting activity and can be effectively applied to skin condition improvement.
  • krill Eupausia superba
  • 'krill oil' refers to an oil component separated from krill
  • 'krill oil extract' refers to an extract obtained by removing water-soluble components from krill oil.
  • extract is an extract obtained by extraction of krill oil, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a prepared or purified product of the extract, or a mixture thereof, etc. , including extracts of all formulations that can be formed using the extract itself and the extract.
  • the extract of the present invention may be prepared and used in the form of a liquid or dry powder, preferably after extraction.
  • the term "enzyme-treated krill oil” refers to an oil separated from krill pulverized product treated with protease.
  • the protease is used in the same sense as protease, and refers to an enzyme that hydrolyzes proteins and peptide bonds, alkalase, esperase, protease NL, protease ( Protease A, Protease P, Protease S, Evelase 6.0 and the like can be used as enzymes.
  • a krill oil extract was obtained by separating the oil component from the enzymatically-treated krill pulverized product using Alkalase.
  • the method of extracting the extract is not particularly limited, and may be extracted according to a method commonly used in the art.
  • Non-limiting examples of the extraction method include a solvent fractionation method, a centrifugation method, and the like, and these may be performed alone or in combination of two or more methods.
  • the term “improving skin condition” refers to antioxidant activity, improvement of inflammatory skin disease symptoms, anti-wrinkle effect by reducing or preventing skin wrinkles, replenishing moisture to the skin, and improving skin blood circulation. It is used in the meaning of helping and promoting the regeneration of skin tissue or skin cells.
  • the cosmetic composition of the present invention is characterized in that it has antioxidant activity.
  • the antioxidant activity of the enzyme-treated krill oil extract according to the present invention increased in a concentration-dependent manner, and it was confirmed that it exhibited an excellent antioxidant activity at the same level as compared to the control biotin (Fig. see 1).
  • the cosmetic composition of the present invention is characterized in that it exhibits anti-inflammatory activity by inhibiting the production of nitric oxide.
  • the enzyme-treated krill oil extract exhibited an excellent effect in inhibiting nitric oxide production (see FIGS. 2 and 3).
  • the cosmetic composition of the present invention suppresses MMP-1 gene expression and promotes type 1 procollagen protein expression to exhibit skin wrinkle improvement activity.
  • Collagen of dermal fibroblasts is directly related to skin regeneration, skin elasticity, skin wrinkle formation, and tissue repair or regeneration during skin damage. That is, when the synthesis of collagen or procollagen in skin fibroblasts is promoted or the synthesis of matrix metallo-proteinase (hereinafter referred to as 'MMP') that decomposes collagen is inhibited, skin elasticity improvement, skin regeneration, It is possible to obtain effects such as skin wrinkle improvement, wound healing, repair and regeneration of damaged skin tissue, and prevention of skin aging.
  • 'MMP' matrix metallo-proteinase
  • the cosmetic composition of the present invention is characterized in that it exhibits skin moisturizing activity by promoting the synthesis of hyaluronic acid.
  • the amount of hyaluronic acid (Hyaluronan) production is increased in a concentration-dependent manner by the treatment of the enzyme-treated krill oil extract, and in all concentration sections, the hyaluronic acid production promoting effect is further increased compared to the biotin-treated positive control group. It has been confirmed that (see FIG. 7).
  • the cosmetic composition of the present invention is characterized in that it promotes the expression of vascular endothelial growth factor (VEGF) factor to exhibit skin blood circulation improvement activity.
  • VEGF vascular endothelial growth factor
  • the experimental group treated with the enzyme-treated krill oil extract significantly increased the expression of the VEGF factor in all concentration sections compared to the control group (Con) only scratched, so that it is effective in improving skin blood circulation. It has been confirmed (see FIG. 8).
  • the cosmetic composition of the present invention does not harm the human body by using an edible krill-derived oil material, and does not impair the quality of the cosmetic material due to its addition, so it is suitable for being included in the cosmetic composition.
  • it since it has functions related to antioxidant, anti-inflammatory, wrinkle improvement, moisturizing, blood circulation improvement, and skin regeneration activity, it is preferably included in a functional cosmetic composition.
  • the cosmetic composition of the present invention includes skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, mask pack, It may be prepared in any one formulation selected from soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser, but is not limited thereto.
  • the cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, humectant, lower alcohol, and thickener. , a chelating agent, a colorant, a preservative, a fragrance, and the like may be appropriately mixed, but the present invention is not limited thereto.
  • the cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation of the cosmetic composition.
  • the formulation of the present invention is an ointment, paste, cream or gel
  • a carrier component animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used, but is not limited thereto. These may be used alone or in combination of two or more.
  • lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohard propellants such as, but not limited to, locarbon, propane/butane or dimethyl ether. These may be used alone or in combination of two or more.
  • a solvent, solubilizer or emulsifier may be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butylglycol oil, etc. can be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, fatty acid ester of polyethylene glycol or sorbitan may be used, but is not limited thereto. These may be used alone or in combination of two or more.
  • the formulation of the present invention is a suspension
  • a liquid diluent such as water, ethanol or propylene glycol
  • a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters
  • Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used, but is not limited thereto. These may be used alone or in combination of two or more.
  • alkali metal salts of fatty acids When the formulation of the present invention is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionate, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugar, etc. are used as carrier components may be, but is not limited thereto. These may be used alone or in combination of two or more.
  • the enzyme-treated krill oil extract is specifically, by dry weight, 0.0001% to 50% by weight of the total weight of the cosmetic composition, more specifically 0.0005% to 10% by weight can be included as Within the above range, there is an advantage of exhibiting excellent efficacy for antioxidant, anti-inflammatory, skin wrinkle and blood circulation improvement, moisturizing, and skin regeneration activity, and there is an advantage in that the formulation of the composition is stabilized.
  • the present invention provides a food composition for anti-inflammatory, skin wrinkle improvement, moisturizing and skin regeneration promotion comprising an enzyme-treated krill oil extract as an active ingredient.
  • the enzyme-treated krill oil extract of the present invention can be usefully used as a food composition for improving skin condition by showing excellent effects in antioxidant, anti-inflammatory, skin wrinkle and blood circulation improvement, moisturizing, and skin regeneration.
  • the enzyme-treated krill oil extract of the present invention and the effects on antioxidant, anti-inflammatory, moisturizing, wrinkle improvement, blood circulation improvement, and skin regeneration are the same as described above.
  • the food composition may be used in the form of a health functional food, but is not limited thereto.
  • the food composition of the present invention may be included in the form of an enzyme-treated krill oil extract and a processed product thereof.
  • the composition may include a food pharmaceutically acceptable food additive in addition to the active ingredient.
  • food supplementary additive means a component that can be added to food as an auxiliary, added to the manufacture of health functional food of each formulation, and can be appropriately selected by those skilled in the art.
  • food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of the food additive of the present invention.
  • the food composition of the present invention may include a health functional food.
  • health functional food refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful for the human body.
  • functionality refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological actions with respect to the structure and function of the human body.
  • the health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art.
  • the formulation of the health functional food is also recognized as a health functional food, it may be manufactured without limitation.
  • the food composition of the present invention can be prepared in various types of dosage forms, and unlike general drugs, edible krill oil is used as a raw material and has the advantage that there are no side effects that may occur during long-term intake of food, excellent portability, and antioxidant , anti-inflammatory, anti-wrinkle, moisturizing, blood circulation improvement, and can be taken as an adjuvant for enhancing the effect on skin regeneration activity.
  • the health functional food of the present invention can take, and it may include all foods in a conventional sense, and may be used in combination with terms known in the art, such as functional food.
  • the health functional food of the present invention can be prepared by mixing known additives with other suitable auxiliary ingredients that may be included in food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the enzyme-treated krill oil extract according to the present invention as a main component to juice, tea, jelly and juice. Also included are foods used as feed for animals.
  • the present invention provides an anti-inflammatory pharmaceutical composition
  • an enzyme-treated krill oil extract as an active ingredient.
  • the anti-inflammatory activity is performed through inhibition of nitric oxide (NO) production. It has been confirmed that it can be used for the prevention or treatment of inflammatory diseases by effectively blocking the inflammatory-related intracellular signaling system by inhibiting the production of nitric oxide (NO).
  • NO nitric oxide
  • the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; it is not going to be
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally. According to one embodiment of the present invention, it is administered by a parenteral method, and according to another embodiment of the present invention, it is administered by a transdermal administration method.
  • a suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity of the patient.
  • a preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.0001-100 mg/kg for adults.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains. or it may be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.
  • the present invention provides a composition for anti-inflammatory, anti-wrinkle improvement, skin moisturizing, skin blood circulation and skin regeneration promotion composition comprising an enzyme-treated krill oil extract as an active ingredient.
  • the enzyme-treated krill oil extract of the present invention has excellent antioxidant activity and anti-inflammatory activity, and at the same time suppresses skin wrinkles, is effective in moisturizing the skin, and promotes the regeneration of damaged skin to improve the skin condition comprehensively Because it is effective, it can be usefully used in cosmetics, food, and pharmaceutical fields.
  • composition of the present invention is a material isolated from edible krill, and is not only very safe for the human body, but also has excellent stability.
  • 1 is a graph showing the change in antioxidant activity according to the enzyme-treated kile oil extract treatment.
  • Figure 2 is a graph showing the measurement of the cell viability of RAW 264.7 according to the enzyme-treated krill oil extract treatment.
  • 3 is a graph showing the change in NO production after the enzyme-treated krill oil extract was treated in RAW 264.7 cells.
  • Figure 4 is a graph showing the measurement of the cell viability of human dermal fibroblasts (NHDF, normal human dermal fibroblasts) according to the enzyme-treated krill oil extract treatment.
  • FIG. 5 is a graph showing changes in the expression level of MMP-1 protein by ELISA method after the enzyme-treated krill oil extract was treated on human skin fibroblasts.
  • FIG. 6 is a graph showing the change in the expression level of type 1 procollagen protein by ELISA after the enzyme-treated krill oil extract was treated on human skin fibroblasts.
  • Figure 7 is a graph showing the change in the amount of hyaluronic acid (Hyaluronan) synthesis by the ELISA method after treating the enzyme-treated krill oil extract to human keratinocytes.
  • FIG. 8 is a graph showing the change in the expression level of VEGF factor by ELISA after the enzyme-treated krill oil extract was treated on human keratinocytes.
  • FIG. 9 is a photograph of the results of cell staining in the plate using a cell stain solution after the enzyme-treated krill oil extract was treated on human keratinocytes.
  • a proteolytic enzyme reaction was performed by adding 1,500 g of purified water to the mature krill and adding 100 g of a proteolytic enzyme, Alkalase. The enzymatic reaction was performed at about 55° C. for about 2 hours to hydrolyze the krill protein.
  • enzyme inactivation was performed to stop the enzymatic reaction after the enzymatic reaction.
  • the temperature of the enzyme inactivation was 90° C., and 100 g of citric acid was added to the hydrolyzate of the krill and left still for 30 minutes to adjust the pH to 4.0 to 5.0.
  • the hydrolyzate was filtered through a decanter to obtain a filtrate. Filtration was performed at 10,000 rpm at 70° C. to 99° C. for 2 hours using the decanter.
  • the filtrate was centrifuged at a speed of 10,000 rpm at 70° C. to 99° C. for 2 hours to separate the filtrate into light oil, heavy oil, and protein.
  • the light oil is a supernatant of the oil obtained by centrifugation
  • the heavy oil is a supernatant of the oil obtained by centrifugation.
  • the light oil and the heavy oil both contain EPA, DHA, phospholipids, and astaxanthin.
  • the astaxanthin-containing krill oil was concentrated to a dry moisture content of less than 2% using a TFE (Thin Film Evaporator).
  • the krill oil containing the concentrated astaxanthin was purified with fermented alcohol and then filtered under pressure to prepare an enzyme-treated krill oil extract.
  • a krill oil extract was prepared in the same manner as in Example 1, except for the process of performing the proteolytic enzyme reaction using the proteolytic enzyme alkalase and the enzyme inactivation process.
  • the antioxidant activity of the krill oil extract not treated with enzyme was found to be weak, but the enzyme-treated krill oil extract according to the present invention showed an increase in antioxidant activity in a concentration-dependent manner, and compared with the control biotin, It was also observed to exhibit excellent antioxidant activity at the same level (see FIG. 1).
  • RAW 264.7 cells a macrophage cell line
  • KTCC Korea Cell Line Bank
  • FBS penicillin
  • FBS Fetal bovine serum
  • the RAW 264.7 cells were inoculated in a 96-well plate at a concentration of 1 ⁇ 10 6 cells/mL (in DMEM) and incubated for 24 hours, each of the enzyme-treated krill oil extract prepared in Example 1 was 1, 10, and 50 ⁇ g/
  • the sample diluted to a concentration of mL and a fresh medium containing LPS (1 ⁇ g/mL) known as endotoxin were simultaneously treated and cultured for 24 hours. Thereafter, the concentration of the MTT reagent was treated to be 0.1 mg/mL, and 3 hours later, formazan generated by MTT treatment was dissolved in DMSO in the cells to measure the absorbance at 595 nm.
  • dexamethasone 1 ⁇ g/mL treatment group and Biotin 1, 10, and 50 ⁇ g/mL treatment group were used as positive controls. In addition, all experiments were repeated three times.
  • nitric oxide a representative cytotoxic substance involved in inducing inflammation
  • RAW 264.7 cells a macrophage cell line
  • DMEM Dulbecco's modified Eagle's medium
  • FBS Fetal bovine serum
  • the cells were inoculated into a medium supplemented with mL penicillin and 100 ⁇ g/mL streptomycin, and cultured at 37° C., 5% CO 2 conditions.
  • the RAW 264.7 cells were inoculated into a 96-well plate at a concentration of 1 ⁇ 10 6 cells/mL (in DMEM) and incubated for 24 hours, each of the enzyme-treated krill oil extract prepared in Example 1 was 1, 10, and a sample diluted to a concentration of 50 ⁇ g/mL and a fresh medium containing LPS (1 ⁇ g/mL) known as an endotoxin were simultaneously treated and cultured for 24 hours.
  • nitric oxide production was reduced by 51.8% in the positive control group treated with dexamethasone compared to the group treated with LPS (1 ⁇ L/mL) alone.
  • the krill oil extract treatment group of the comparative example without enzyme treatment increased the production of nitric oxide compared to the LPS alone treatment group at the concentrations of 1 and 10 ⁇ g/mL, respectively, but at the concentration of 50 ⁇ g/mL, the amount of nitric oxide was reduced by 19.4%. .
  • nitric oxide production was observed to be reduced by 12.2%, 37%, and 40.7% at concentrations of 1, 10, and 50 ⁇ g/mL, even compared to the biotin-treated positive control group Overall, it was found that the nitric oxide production inhibitory effect was excellent.
  • the enzyme-treated krill oil extract of the present invention exhibits an excellent effect in inhibiting nitric oxide production (see FIG. 3 ).
  • the skin fibroblasts were inoculated at a concentration of 1.25 in a 40 mm cell culture dish in DMEM (Dulbeccos modified Eagles medium) medium, which is a dedicated medium containing 10% FBS (fetal bovine serum, Cambrex), for 24 hours, 37°C , 5% CO 2 Incubated under humid conditions. Thereafter, the medium was removed, 300 ⁇ l of PBS was added, and the enzyme-treated krill oil extract diluted with serum-free DMEM medium was treated with each concentration (1, 10, and 50 ⁇ g/ ⁇ l) and cultured for 72 hours. After 72 hours, 1 mg/mL of MTT was treated and 2 hours later, formazan generated by MTT treatment in cells was dissolved in DMSO and absorbance was measured at 570 nm.
  • DMEM Dulbeccos modified Eagles medium
  • FBS fetal bovine serum, Cambrex
  • DMEM culture solution 2ml of DMEM culture solution was put in a 40mm cell culture dish, and human fibroblasts were inoculated at a concentration of about 1.2x10 5 , and then cultured at 37° C., 5% CO 2 environment for 24 hours. Thereafter, the culture medium was replaced with a medium containing the enzyme-treated krill oil extract (1, 10, and 50 ⁇ g/ml, respectively) prepared in Example 1 and cultured for 3 days. After that, the culture medium was harvested and centrifuged for 5 minutes at 4° C., 7,500 rpm, and MMP-1 (Human Total MMP-1 kit, R&D Systems, Inc., Minneapolis, MN, USA) using an ELISA method. ) of the protein expression level was confirmed. For the comparative experiment, TGF- ⁇ 1 10ng/mL treatment group and Biotin 1, 10, and 50 ⁇ g/mL treatment group were used as positive controls. In addition, all experiments were repeated three times.
  • the enzyme-treated krill oil extract treatment group significantly reduced the amount of MMP-1 protein production was measured compared to the krill oil treatment group of the comparative example that was not treated with the enzyme.
  • the enzyme-treated krill oil extract treatment group of the present invention reduced MMP-1 protein production by 20.2%, 31.8% and 45.4%, respectively, at 1, 10, and 50 ⁇ g/ml doses compared to the normal control group (Nor).
  • the enzyme-treated krill oil of the present invention reduced MMP-1 protein production by 33.6%, 35.9% and 40.8% at 1, 10, and 50 ⁇ g/ml dose, respectively. It was confirmed that the enzyme treatment was more effective in inhibiting the production of MMP-1 protein (see FIG. 5).
  • the effect of the enzyme-treated krill oil extract treatment group of the present invention was superior in all concentration ranges, and 10,536.29 pg in the enzyme-treated krill oil extract 50 ⁇ g/ml dose treatment group.
  • MMP-1 protein at /ml was measured, and it was observed that the TGF- ⁇ 1 10ng/mL treatment group showed an equivalent level of effect compared to the measured MMP-1 protein value of 9,219.86 pg/ml.
  • DMEM culture solution 2ml of DMEM culture solution was put in a 40mm cell culture dish, and human fibroblasts were inoculated at a concentration of about 1.2x10 5 , and then cultured at 37° C., 5% CO 2 environment for 24 hours. Thereafter, the culture medium was replaced with a medium containing the enzyme-treated krill oil extract (1, 10, and 50 ⁇ g/ml, respectively) prepared in Example 1. After that, the culture medium was harvested, centrifuged for 5 minutes at 4°C and 7,500 rpm, and protein expression level of type 1 procollagen (Procollagen Type IC Peptide EIA Kit, Takara, Shiga, Japan) using ELISA method. Change was confirmed.
  • type 1 procollagen Procollagen Type IC Peptide EIA Kit, Takara, Shiga, Japan
  • human fibroblasts treated with the krill oil extract of Comparative Example without biotin or enzyme treatment did not show a significant change in the amount of type 1 procollagen protein produced, and rather type 1 procollagen compared to the untreated normal control group. A decrease in protein production was observed.
  • the enzyme-treated krill oil extract of the present invention can be effectively used to prevent and improve skin wrinkles by effectively promoting the production of type 1 procollagen protein that inhibits wrinkle formation.
  • Hyaluronic acid (HA, Hyaluronan, Hyaluronic acid) is mainly synthesized by epidermal keratinocytes and dermal fibroblasts, and is a substance that plays an important role in creating an environment in which cells can function normally by combining with water.
  • a decrease in hyaluronic acid in the skin is one of the direct causes of a decrease in skin elasticity and a decrease in moisture content. Therefore, in this experimental example, the amount of hyaluronic acid produced in human keratinocytes was confirmed.
  • human keratinocytes were placed in 1 mL of a culture medium in a 24-well cell culture dish and inoculated at a concentration of about 1 ⁇ 10 5 cells/mL, and then cultured at 37° C., 5% CO 2 environment for 24 hours. Thereafter, the medium containing the enzyme-treated krill oil extract prepared in Example 1 and the krill oil extract of Comparative Example without enzyme treatment (1, 10, and 50 ⁇ g/ml, respectively) was replaced and cultured for 24 hours.
  • Hyaluronan kit (Hyaluronan kit, R&D Systems, Inc., Minneapolis, MN, USA) was performed using an ELISA method. was confirmed.
  • a normal control group (Nor, 100% DMEM) and a control group (Con, solvent + sample-free medium) were used, and a biotin-treated group was used as a positive control group. In addition, all experiments were repeated three times.
  • the enzyme-treated krill oil extract of the present invention effectively increases the production of hyaluronic acid in skin cells and can be very effectively used for skin moisturizing purposes.
  • VEGF vascular endothelial growth factor
  • Example 1 First, put 1 mL of DMEM culture solution in a 24-well cell culture dish, and inoculate human keratinocytes (HaCaT) cells at a concentration of about 1.2x10 5 cells/mL, 37°C, 5% CO 2 environment for 24 hours. incubated during Then, the enzyme-treated krill oil extract prepared in Example 1 was replaced with a medium containing a sample diluted to a concentration of 1, 10, and 50 ⁇ g/ml, respectively, and cultured for 24 hours.
  • HaCaT human keratinocytes
  • VEGF antibody Human VEGF kit, R&D Systems, Inc., Minneapolis, MN, USA
  • VEGF antibody Human VEGF kit, R&D Systems, Inc., Minneapolis, MN, USA
  • the expression of the VEGF factor was increased in a concentration-dependent manner up to a concentration of 10 ⁇ g/ml, and 1, 10, and 50 ⁇ g compared to the control group (Con) with only scratches. At the /ml concentration, it was observed that the expression of the VEGF factor increased by 106.4%, 155.5%, and 152.4%, respectively.
  • the enzyme-treated krill oil extract of the present invention can be effectively used to improve skin blood circulation by promoting the expression of VEGF factors that promote blood vessel formation and increase microvascular permeability.

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Abstract

La présente invention concerne une composition anti-inflammatoire pour réduire les rides cutanées et hydrater et activer la régénération de la peau, contenant, en tant que principe actif, un extrait d'huile de krill traitée par une protéase. L'extrait d'huile de krill traitée par une enzyme selon la présente invention présente une excellente activité antioxydante et une excellente activité anti-inflammatoire, et possède en même temps un excellent effet d'amélioration complète d'états cutanés par la prévention de rides cutanées, est efficace pour hydrater la peau et active la régénération de peau endommagée, et peut ainsi être efficacement utilisé dans les domaines cosmétique, alimentaire et pharmaceutique. De plus, la composition selon la présente invention est une substance isolée à partir de krill comestible, est sans danger pour le corps humain et présente une excellente stabilité.
PCT/KR2021/012139 2020-09-07 2021-09-07 Composition anti-inflammatoire pour réduire les rides cutanées et hydrater et activer la régénération de la peau, contenant, en tant que principe actif, un extrait d'huile de krill traitée par une enzyme Ceased WO2022050819A1 (fr)

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