WO2013039056A1 - Composition contenant un hydrolysat de protéines de soja - Google Patents

Composition contenant un hydrolysat de protéines de soja Download PDF

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Publication number
WO2013039056A1
WO2013039056A1 PCT/JP2012/073170 JP2012073170W WO2013039056A1 WO 2013039056 A1 WO2013039056 A1 WO 2013039056A1 JP 2012073170 W JP2012073170 W JP 2012073170W WO 2013039056 A1 WO2013039056 A1 WO 2013039056A1
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Prior art keywords
collagen
hydrolyzate
soy protein
skin
adhesion
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PCT/JP2012/073170
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English (en)
Japanese (ja)
Inventor
正通 阿部
益徳 熊澤
康助 松本
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ロート製薬株式会社
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Priority to JP2013533668A priority Critical patent/JP6153469B2/ja
Priority to CN201280040545.4A priority patent/CN103747793A/zh
Publication of WO2013039056A1 publication Critical patent/WO2013039056A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • 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
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]

Definitions

  • the present invention relates to a composition for promoting adhesion between cells and collagen and / or promoting contraction of a collagen gel, and an invention related thereto.
  • fibroblasts adhere to collagen in the extracellular matrix, strengthening and stabilizing the lattice-like three-dimensional structure of collagen fiber bundles, and strengthening and elasticity of the skin Is one of the reasons for keeping It has been found that when the fibroblast adherence ability to collagen is reduced due to aging or the like, skin elasticity and elasticity are reduced. Furthermore, it is known that fibroblasts themselves become dull in their function (for example, production of various factors) unless they adhere to collagen fibers and grasp the scaffold. And the decline of dermal cell function for these reasons is not limited to the phenomenon of dermis alone, it also has an adverse effect on nutrient supply and migration of growth factors from the dermis to the epidermis. It is considered. For the above reasons, a component that promotes adhesion between fibroblasts and collagen has been searched.
  • the basement membrane existing on the boundary line between the epidermis and the dermis contains type IV collagen and type VII collagen as main components.
  • the adhesion between the basement membrane and epidermal keratinocytes gradually weakens and loosens due to ultraviolet rays, aging, etc., it not only causes sagging (eg, sagging pores) and wrinkles, but also horns.
  • the ability of the keratinocytes to grip the scaffold is reduced, and the ability of the keratinocytes to proliferate decreases, thereby causing poor turnover and causing spots (melanin accumulation) and dullness.
  • the basement membrane and the keratinocytes when loosening occurs between the basement membrane and the keratinocytes, the nutrient supply and the transfer of growth factors from the dermis to the epidermis, which are important roles of the basement membrane, are not successful, and the overall health of the skin is lost. There is concern that it may become defective and cause thinning of the epidermis. Therefore, by promoting the adhesion between collagen in the basement membrane and epidermal keratinocytes, the basement membrane and epidermal keratinocytes are firmly fixed, sagging and wrinkles, poor turnover, and accompanying stains and dullness, and more It is also important to prevent / improve thinning of the epidermis. For such a reason, a search for a component that promotes adhesion between keratinocytes and collagen is also required.
  • glycation of in vivo proteins has attracted attention as a kind of aging phenomenon. And it is also becoming clear that glycated collagen (also called AGE-modified collagen) produced by glycation of collagen protein in vivo becomes difficult to adhere to cells.
  • glycated collagen also called AGE-modified collagen
  • Patent Document 1 peptides having a specific sequence (Patent Document 1) and the like have been known as materials for promoting adhesion between skin cells and collagen.
  • the present invention has been made in view of the above prior art, and provides a useful novel material that is derived from natural products and is safe for living bodies, and that can highly exhibit adhesion promoting activity between cells and collagen.
  • the purpose is to do.
  • soy protein highly promotes adhesion between cells and collagen and has a collagen gel contraction promoting activity. It has been found that it can be used as a new material, and the present invention has been completed.
  • the present invention provides the following.
  • composition for promoting adhesion between cells and collagen containing a hydrolyzate of soy protein.
  • composition according to [1] or [2], wherein the hydrolyzate is a degradation product obtained by protease treatment of soybean protein.
  • the present invention provides a useful novel material that can effectively promote cell adhesion to collagen and has high collagen gel contraction promoting activity. Therefore, according to the present invention, it is possible to effectively prevent a decrease in adhesion ability between cells (particularly, fibroblasts and / or keratinocytes) and collagen caused by aging and the like, and the binding of fibroblasts to the extracellular matrix. Strengthening the skin, tightening the dermis, improving skin wrinkles and sagging, restoring skin elasticity and elasticity, and strengthening the adhesion between epidermal keratinocytes and basement membrane In addition to sagging and wrinkles, new materials are provided that are useful for preventing / improving reduced turnover, spots, dullness, and thinning of the epidermis.
  • the present invention provides a composition for promoting adhesion between cells and collagen, which contains a hydrolyzate of soy protein.
  • the present invention also provides a composition for promoting contraction of a collagen gel, which contains a hydrolyzate of soy protein.
  • Soybean (Glycine max) is a leguminous annual plant with a height of about 60-70 cm. It is known that the seeds are often used as edible beans or processed into tofu, miso, soy sauce, etc.
  • the soybean protein used in the present invention may be any protein derived from the soybean plant described above, but may preferably be any protein derived from the seed of the soybean plant.
  • the protein derived from soybean plants, or the protein derived from soybean plant seeds is a protein contained in soybean plants or soybean plant seeds, and can be extracted, separated or purified by any method.
  • a protein that can be produced. Examples of soybean plants include whole plants or individual organs such as leaves, stems and roots.
  • proteins contained in soybean plant tissues and soybean cultured cells obtained by tissue culture can also be used. Examples of soy protein include, but are not limited to, glycinin and conglycinin (preferably ⁇ -conglycinin).
  • seeds of soybean plants themselves or crushed or pulverized products of the seeds may be used as soybean proteins, but preferably obtained by separating and purifying from all components in soybean plants.
  • a protein component more preferably, a protein component obtained by separating and purifying from all components in the seeds of soybean plants is used.
  • Soy protein obtained by separation and purification as described above is a soybean plant or soybean as long as the hydrolyzate has the ability to promote adhesion between cells and collagen and / or the ability to promote contraction to collagen gel. It may contain substantially all kinds of proteins contained in plant seeds, or may contain some kinds of proteins.
  • Separation / purification of soybean protein from soybean plants or soybean plant seeds can be performed by known protein separation / purification methods.
  • soy protein commercially available products can also be suitably used.
  • Nisshin Cosmo Foods Co., Ltd., ADM Far East Co., Ltd., Showa Sangyo Co., Ltd., Fuji Oil Co., Ltd., Koyo Shokai Co., Ltd., etc. Readily available from other manufacturers or suppliers.
  • Soy protein can be obtained, for example, as defatted soybean powder.
  • the seeds of soybean plants not only refer to the whole structure commonly referred to as soybean seeds but also include, for example, dehulled soybean seeds, defatted soybean seeds (powder), and snow flora (Okara) obtained from the whole soybean seeds. ) Etc.
  • any means known in the art can be used.
  • a method of hydrolyzing by treating with protease a method of hydrolyzing by treating with acid or alkali, etc.
  • a method of treating soybean protein with a protease can be preferably used.
  • the protease used in the method is not particularly limited, and an animal, plant, or microorganism-derived protease can be used. Examples include thermolysin, papain, caspase, bromelain, pepsin, cathepsin D, trypsin, chymotrypsin, pancreatin, subtilisin and the like. Moreover, you may use the enzyme formulation containing a commercially available protease.
  • Enzyme preparations include Sumiteam FL-G, Sumiteam AP, Sumiteam-LP, Sumiteam-FP, Sumiteam-LPL (Shin Nihon Chemical Industry), Protease A, Protease N, Protease S, Newase F3G, Newase A, Papain W-40, Promeline F, Protin SD-NY10, Protin SD-PC10F (above, Amano Enzyme), Protex 7L, Protex 14L, Promod 223LP, Papain (above, Genencor Kyowa), Alloase AP-10, Alloase NP-10 , Aroase NS, Aroase XA-10, Protease YP-SS, Pandidase NP-2, Pandase P (above Yakult Pharmaceutical), Papain F, TRYPSIN4.0T, COROLASE N, COROLASE L10, COROLASE 7089, VERON L10 (above, Higuchi Shokai), Orientase 20A, Orienta
  • protease is an acidic protease and / or a neutral protease.
  • Only one type of protease may be used in the present invention, or two or more types may be used in combination.
  • you may hydrolyze using combining 2 or more types of enzymes simultaneously, after hydrolyzing with one enzyme of 2 or more types of enzymes, and also hydrolyzing with another enzyme. Different enzymes may be used sequentially.
  • a commercially available product can be suitably used as the protease.
  • soy protein concentration is generally 0.1 to 30% (w / v), preferably about 1 to 10% (w / v) in a solvent such as water. Soy protein or a raw material containing soybean protein is suspended, and the suspension generally has a concentration of 0.001 to 5% (w / v), preferably 0.005 to 3% (w / v), and more preferably.
  • reaction temperatures of 30-80 ° C, preferably 40-70 ° C, more preferably 50-60 ° C may be used.
  • reaction time of 2 to 30 hours, preferably 3 to 24 hours, more preferably 10 to 20 hours may be used.
  • the pH of the reaction solution is preferably around the optimum pH of the protease to be used.
  • the reaction stopping means is not particularly limited, and known means can be used. Examples of such means include heat treatment.
  • the enzyme reaction is stopped by inactivating the protease contained in the reaction product by heat-treating the reaction product at a temperature of about 80 to 100 ° C. for 3 to 20 minutes, preferably 5 to 15 minutes. be able to. Specifically, heat treatment at 85 ° C. for 15 minutes, heat treatment at 100 ° C. for 5 minutes, and the like can be given.
  • the hydrolyzate obtained by the hydrolysis reaction as described above can be further processed by any method known to those skilled in the art, if necessary. For example, it is preferable to remove large solid particles in the hydrolyzate by a treatment such as filtration. Filtration conditions and the like are not particularly limited and may be appropriately selected by those skilled in the art according to common general technical knowledge. For example, when the filter paper is likely to be clogged, a filter aid or the like can be suitably used.
  • the hydrolyzate can be pulverized by concentrating under reduced pressure and then freeze-drying. Further, it may be pulverized by means such as spray drying.
  • the conditions and equipment used in vacuum concentration, freeze drying, spray drying and the like are not particularly limited and can be appropriately selected by those skilled in the art according to technical common sense.
  • the hydrolyzate thus powdered can be used as it is or after being dissolved in a solvent such as water.
  • Soy protein hydrolyzate includes a wide variety of peptides with different molecular weights and amino acid sequences.
  • the hydrolyzate used in the present invention may contain substantially all of a wide variety of peptides produced by hydrolyzing soy protein, or such a wide variety of peptides A portion obtained by further fractionation and purification by a known method using the presence / absence of adhesion promoting ability between cells and collagen as an index and / or the presence / absence of collagen gel contraction promoting ability as an index (for example, the adhesion promotion described above) And / or a peptide excluding a peptide that hardly influences the contraction promoting ability).
  • the average molecular weight of the hydrolyzate of soybean protein used in the present invention is preferably 300 to 50,000.
  • the average molecular weight is more preferably 500 to 30000, further preferably 700 to 20000, still more preferably 1000 to 10000, and particularly preferably from the viewpoint of enhancing the permeability to the skin and obtaining a higher effect. 2000 to 10,000.
  • the hydrolyzate of soy protein may be fractionated by molecular weight, the hydrolyzate contained in the above molecular weight range may be recovered, and the peptide fraction in the above molecular weight range may be used as the hydrolyzate of the soy protein of the present invention.
  • the average molecular weight of the hydrolyzate can be measured by any method known to those skilled in the art, and can be easily measured by, for example, the gel permeation chromatography (GPC) method described in the Examples below.
  • GPC gel permeation chromatography
  • the average molecular weight measured by the GPC method means “peak average molecular weight”, and “peak average molecular weight” is the molecular weight corresponding to the elution time of the peak top (the strongest peak) of the chromatogram. Means.
  • the molecular weight distribution of the hydrolyzate of soy protein used in the present invention is not particularly limited, but is preferably 70000 or less, more preferably from the viewpoint of enhancing the permeability to the skin and obtaining a higher effect. Is from 100 to 70000, more preferably from 100 to 50000, particularly preferably from 100 to 30000.
  • the molecular weight distribution of the hydrolyzate can be measured by any method known to those skilled in the art, and can be easily calculated from, for example, an elution curve obtained by the GPC method.
  • the soy protein hydrolyzate can promote adhesion between cells and collagen.
  • the cells are skin cells, more preferably fibroblasts and / or keratinocytes.
  • Various types of collagen such as types I to XIX have been known so far, and any type of collagen is also known.
  • the collagen may be saccharified and AGEd collagen.
  • the hydrolyzate of soy protein of the present invention is the adhesion between type I collagen (collagen type I) contained in the dermis of the skin and fibroblasts, and type IV collagen (collagen type contained in the skin basement membrane). IV) mainly promotes adhesion between keratinocytes.
  • the hydrolyzate of soybean protein of the present invention can also promote adhesion between AGE collagen and cells, which are generally known to be difficult to adhere to cells. Fibroblasts adhere to collagen in the extracellular matrix within the dermis of living organisms, and collagen forms a fiber bundle structure, thereby strengthening the bond between fibroblasts and extracellular matrix and increasing the tensile strength of both. By tightening the dermis, the firmness and elasticity of the skin is maintained. Therefore, the hydrolyzate of soy protein is also beneficial for improving skin wrinkles and talmi caused by a decrease in the adhesion ability between the fibroblasts and collagen, or for restoring skin elasticity and elasticity. Can be used.
  • the turnover means that keratinocytes of the epidermis are produced in the basal layer of the skin, form a horny layer, and finally fall off in the skin. Skin cells are reborn as a new cell by turnover. The turnover time is shortened by promoting the turnover.
  • soy protein hydrolyzate is also beneficially used to improve tarmi and wrinkles, reduced turnover, spots, dullness, and thinning of the epidermis resulting from reduced adhesion between keratinocytes and collagen. Can be done.
  • cells for example, skin fibroblasts or skin keratinocytes
  • a culture medium containing the hydrolyzate in a culture plate coated with collagen. After washing the plate, it can be measured by counting the number of cells remaining in the collagen coat on the plate without being washed away, and in the case where no soy protein hydrolyzate is added, it remains in the collagen coat. When the number of cells obtained is significantly increased, it can be determined that adhesion between each cell and collagen has been promoted.
  • the hydrolyzate of soybean protein has a collagen gel contracting action and can promote the contraction of the collagen gel.
  • the hydrolyzate of soy protein of the present invention mainly promotes the contraction of a gel containing type I collagen (collagen type I) contained in a large amount in the dermis of the skin.
  • the collagen gel refers to a collagen gel containing cells (particularly fibroblasts).
  • the collagen gel contraction action of the hydrolyzate can be evaluated, and when the diameter of the collagen gel after the culture is significantly reduced as compared with that before the culture, it can be determined that the contraction of the collagen gel is promoted.
  • a collagen gel in which fibroblasts are embedded is used as a model of a living dermis.
  • the soy protein hydrolyzate of the present invention which can promote the contraction of collagen gels including fibroblasts, can promote the contraction of collagen gels formed by fibroblasts and collagen in living dermal connective tissue.
  • hydrolyzate capable of promoting adhesion between fibroblasts and collagen and / or a hydrolyzate capable of promoting contraction of collagen gel by such a method may be used as a fibroblast and an extracellular matrix. It can be understood that it has the effect of strengthening the bonding of the two, increasing the tensile strength of both, tightening the dermis, improving the skin wrinkles and talmi, or restoring the elasticity and elasticity of the skin.
  • the composition of the present invention contains the hydrolyzate of the above-mentioned soybean protein. Since the composition contains a hydrolyzate having the above-described characteristics, it serves as an adhesion promoter between cells and collagen to promote adhesion between cells and collagen, and to promote contraction of collagen gel. It can be beneficially used as a collagen gel shrinkage promoter.
  • the composition is, for example, a pharmaceutical composition, a cosmetic composition, a food composition, or a feed composition, and further for elucidation of a physiological state related to adhesion between cells and collagen, or for collagen gel contraction. It can be suitably used as a research reagent for state elucidation.
  • the pharmaceutical composition examples include a prophylactic and / or therapeutic agent for a disease caused by a decrease in adhesion ability between cells and collagen in mammals including humans.
  • the pharmaceutical composition of the present invention is, for example, a preventive agent for skin wrinkles or tarmi caused by a decrease in the ability to adhere between fibroblasts or keratinocytes and collagen caused by aging or the like and / or
  • a therapeutic agent as a prophylactic and / or therapeutic agent for skin elasticity or a decrease in elasticity due to the decrease in adhesion ability, or as a skin tightening agent, poor skin turnover due to the decrease in adhesion ability or It can be used as a prophylactic and / or therapeutic agent for accompanying stains or dullness, or as a prophylactic and / or therapeutic agent for thinning of the skin caused by the reduced adhesive ability.
  • the pharmaceutical composition of the present invention contains a hydrolyzate of soy protein that showed a high contraction effect in collagen gel contraction evaluation, which is also known as a model system for wound healing, wound healing, skin ulcer and keloid It can be beneficially used as a drug for promoting the fixation of a graft during the treatment of skin graft or the like.
  • the cosmetic composition can be used, for example, as a cosmetic for preventing and / or improving a state caused by a decrease in the adhesion ability between cells and collagen in mammals including humans.
  • the cosmetic composition of the present invention can prevent and / or prevent wrinkles or talmi of the skin caused by, for example, a decrease in the adhesion ability between fibroblasts or keratinocytes caused by aging and the like and collagen.
  • the adhesive ability As a cosmetic for improvement, as a cosmetic for preventing and / or improving skin elasticity or a decrease in elasticity due to a decrease in the adhesive ability, or as a cosmetic for tightening the skin, the adhesive ability As a cosmetic for the prevention and / or improvement of skin turnover failure due to deterioration and the accompanying stains or dullness, or for the prevention and / or improvement of epidermis thinning due to the reduced adhesive ability It can be used as a fee. Furthermore, the cosmetic composition of the present invention can be beneficially used for promoting wound healing, improving conditions such as skin ulcers and keloids, or promoting graft establishment after skin transplantation.
  • the food composition for example, it can be used as a food for preventing and / or improving a state caused by a decrease in the adhesion ability between cells and collagen in mammals including humans.
  • the food composition of the present invention prevents and / or improves skin wrinkles or talmi caused by, for example, a decrease in the adhesion ability between fibroblasts or keratinocytes and collagen caused by aging or the like.
  • the food composition of the present invention can be beneficially used to promote wound healing, improvement of conditions such as skin ulcers and keloids, or promoting graft establishment after skin transplantation.
  • the food composition of the present invention includes health food, food for specified health use, nutritional functional food, health supplement food, and the like. These compositions include cosmetic health foods.
  • the food for specified health is a food that is ingested for the purpose of specific health in the diet, and indicates that the purpose of the health can be expected by the intake.
  • These food products can be provided as food products with a label indicating that they are used for the above-mentioned purposes in a specific embodiment. In other words, it is used to display the indication that it is used for beauty, the indication that it has a beautifying effect, the indication that it is used for improving the skin quality, and to restore the elasticity and elasticity of the skin.
  • An indication that it is used an indication that it is used for prevention or improvement of skin wrinkles and talmi, an indication that it is used for tightening the skin, poor skin turnover, and It is used for the prevention or improvement of accompanying stains or dullness, the indication that it is used for the prevention or improvement of epidermis thinning, and used for wound healing
  • feed for example, for the prevention and / or improvement of conditions caused by reduced adhesion between cells and collagen in livestock such as cattle, pigs, chickens, sheep and horses, and pet animals such as dogs and cats And feeds for improving conditions such as wound healing and skin ulcers and keloids.
  • the content of the soy protein hydrolyzate in the composition varies depending on the dosage form of the composition, etc., but generally, from the viewpoint of obtaining an effect of promoting adhesion between high cells and collagen, and / or From the viewpoint of obtaining a high collagen gel shrinkage promoting effect, it is preferably 0.00001 to 100% by weight, more preferably 0.0001 to 95% by weight, still more preferably 0.001 to 90% by weight, and particularly preferably 0.01 to 80% by weight.
  • composition of the present invention in addition to the soy protein hydrolyzate, carriers, bases, and / or additives that are usually used in fields such as the pharmaceutical field and food field are used to achieve the object of the present invention. It can mix
  • saccharides for example, saccharides, celluloses, poorly water-soluble gums, crosslinked vinyl polymers, lipids and the like can be used alone or in combination.
  • water, fats and oils, mineral oils, waxes, fatty acids, silicone oils, sterols, esters, metal soaps, alcohols and the like may be used alone or in combination of two or more.
  • additives include surfactants, solubilizing components, emulsifiers, oils, stabilizers, thickeners, preservatives, binders, lubricants, dispersants, pH adjusters, humectants, UV absorbers.
  • Chelating agents, percutaneous absorption enhancers, antioxidants, disintegrating agents, plasticizers, buffering agents, vitamins, amino acids, coloring agents, fragrances and the like may be used alone or in combination.
  • the composition of the present invention includes a whitening component, an anti-inflammatory component, an antibacterial component, a cell activation component, an astringent component, an antioxidant component, an acne improving component, Various components such as a biocomponent synthesis promoting component such as collagen, a blood circulation promoting component, a moisturizing component, and an anti-aging component may be used alone or in combination.
  • composition of the present invention may be in any dosage form such as an internal preparation (including food and feed) or an external preparation (including cosmetics).
  • the hydrolyzate can be appropriately blended by a method known to those skilled in the art depending on the properties of the target food.
  • the external preparation for example, it can be used in any form such as liquid, emulsion, cream, lotion, paste, mousse, gel, sheet (supported substrate), aerosol, and spray.
  • Cosmetics include, for example, basic cosmetics such as lotions, emulsions, creams, oils, packs, makeup cosmetics such as foundations, blushers and lipsticks, as well as cleansers such as facial cleansers, cleansings, body cleansers, and bath preparations. Etc. may be used in any form.
  • the feed is not particularly limited because it can be used in any form.
  • the present invention further provides a method for promoting adhesion between cells and collagen and / or a method for promoting contraction of collagen gel, wherein the hydrolyzate of the soy protein is used.
  • the soy protein hydrolyzate is used in an amount that is at least an effective amount that provides an effect of promoting adhesion between cells and collagen and / or an amount that is at least an effective amount that provides an effect of promoting contraction on collagen gel. Use it.
  • the amount of the soy protein hydrolyzate used in the method of the present invention is usually about 0.001 to 10,000 mg / day, more preferably about 1 to 1000 mg per day for an adult, about 50 kg body weight in the case of internal use. / Day, more preferably about 1 to 100 mg / day.
  • the amount used is usually preferably about 1 ng to 2 g / day, more preferably about 0.01 ⁇ g to 2 g / day, more preferably about 0.1 ⁇ g to 2 g / day per 50 kg body weight of an adult. .
  • the amount of soy protein hydrolyzate applied to the skin is preferably about 0.01 ng to 500 ⁇ g / cm 2 , more preferably about 0.1 ng to 50 ⁇ g / cm 2 , and more preferably about 1 ng. ⁇ 10 ⁇ g / cm 2 .
  • the present invention further provides the use of the soy protein hydrolyzate for the manufacture of a composition for promoting adhesion between cells and collagen.
  • the present invention also provides use of the soy protein hydrolyzate for the production of a composition for promoting the shrinkage of a collagen gel.
  • the amount of the hydrolyzate of the soybean protein may be used so as to be the content in the composition.
  • the present invention relates to a method of administering a composition containing a hydrolyzate of soy protein to mammals including humans, and promoting adhesion between cells and collagen in the mammals including humans, And a composition containing a hydrolyzate of soy protein to a mammal including a human, and a method of promoting contraction of a collagen gel including cells in the mammal including the human.
  • the present invention also includes a soy protein hydrolyzate used to promote adhesion between cells and collagen, and a soy protein hydrolyzate used to promote the shrinkage of collagen gel containing cells. To do.
  • the present invention relates to the use of a soy protein hydrolyzate for the manufacture of a medicament for promoting adhesion between cells and collagen, and the manufacture of a medicament for promoting contraction of a collagen gel containing cells.
  • a soy protein hydrolyzate for the manufacture of a medicament for promoting adhesion between cells and collagen, and the manufacture of a medicament for promoting contraction of a collagen gel containing cells.
  • Example 1 Preparation of hydrolyzate of soy protein (1) Soy protein is suspended so that the concentration of defatted soybean powder (trade name Profam 974, manufactured by ADM Far East Co., Ltd.) is 5% (w / v), and 0.05% (w / v) is added to this suspension.
  • Pepsibio1 10,000 (manufactured by Nippon Biocon Co., Ltd.), a kind of pepsin, was added so that the decomposition reaction was carried out at 50 ° C. for 18 hours. The pH of the reaction solution was 3.5, which is near the optimum pH of the protease.
  • the reaction product was heat treated at 85 ° C. for 15 minutes to deactivate the enzyme contained in the reaction product, and then neutralized with 10N sodium hydroxide.
  • the Buchner funnel was pre-coated with diatomaceous earth (Fuso Kagaku, Radiolite # 200) and subjected to suction filtration.
  • the filtrate was concentrated under reduced pressure and freeze-dried to obtain a powdered soybean protein hydrolyzate in a yield of 42.8%.
  • Example 2 Preparation of soy protein hydrolyzate (2) Soy protein is suspended so that the concentration of defatted soybean powder (trade name Profam 974, manufactured by ADM Far East Co., Ltd.) is 5% (w / v), and 0.05% (w / v) is added to this suspension. Trypsin was added so as to obtain a decomposition reaction at 50 ° C. for 18 hours. The pH of the reaction solution was set at pH 7.5, which is around the optimum pH of the protease.
  • defatted soybean powder trade name Profam 974, manufactured by ADM Far East Co., Ltd.
  • the reaction product was heat treated at 85 ° C. for 15 minutes to inactivate the enzyme contained in the reaction product.
  • the Buchner funnel was pre-coated with diatomaceous earth (Fuso Chemical, Radiolite # 200) and subjected to suction filtration. Subsequently, the filtrate was concentrated under reduced pressure and freeze-dried to obtain a powdered soybean protein hydrolyzate in a yield of 33.8%.
  • Example 3 Preparation of soy protein hydrolyzate (3) Soy protein is suspended so that the concentration of defatted soybean powder (trade name Profam 974, manufactured by ADM Far East Co., Ltd.) is 5% (w / v), and 5% (w / v) is added to this suspension. Trypsin was added and reacted at pH 7.5 near the optimum pH of the protease at 50 ° C. for 18 hours, and then 0.05% (w / v) of Sumiteam FL-G (manufactured by Shin Nippon Chemical Co., Ltd.) was added. The degradation reaction was carried out at 60 ° C. for 18 hours at 7.0 near the optimum pH of the protease.
  • Soy protein is suspended so that the concentration of defatted soybean powder (trade name Profam 974, manufactured by ADM Far East Co., Ltd.) is 5% (w / v) is added to this suspension. Trypsin was added and reacted at pH 7.5 near the optimum pH of the
  • the reaction product was heat treated at 85 ° C. for 15 minutes to inactivate the enzyme contained in the reaction product.
  • the Buchner funnel was pre-coated with diatomaceous earth (Fuso Chemical, Radiolite # 200) and subjected to suction filtration.
  • the filtrate was concentrated under reduced pressure and freeze-dried to obtain a powdered soybean protein hydrolyzate in a yield of 43.9%.
  • Example 4 Measurement of Molecular Weight of Soy Protein Hydrolyzate The average molecular weight of the soy protein hydrolyzate obtained in Examples 1 to 3 was measured by gel permeation chromatography (GPC).
  • the soy protein hydrolyzate of each Example was dissolved in 25 mM Tris-HCl buffer solution (containing 150 mM NaCl, pH 7.5) to prepare a 2 mg / mL test solution.
  • An HPLC column Superdex peptide HR (10 mm ID ⁇ 30 cm, manufactured by GE Healthcare Japan) was equilibrated with the same buffer, and 50 ⁇ L of the test solution was injected into this column.
  • the flow rate of the column was 0.5 mL / min, the column temperature was room temperature, the peptide was detected at 214 nm, and the molecular weight distribution and peak average molecular weight were estimated from the elution time.
  • Cytochrome C (Sigma, molecular weight 12327), Aprotinin (Roche, molecular weight 6518), Hexaglycine (Sigma, molecular weight 360), Triglycine (Sigma, molecular weight 189), Glycine (manufactured by Wako Pure Chemical Industries, Ltd., molecular weight 75) was used.
  • the average molecular weights (peak average molecular weights) of the soy protein hydrolysates of Examples 1 to 3 are 2233 (Example 1), 9274 (Example 2), and 3407 (Example 3), respectively. confirmed. Chromatograms showing the molecular weight distribution of each hydrolyzate are shown in FIGS.
  • Example 5 Evaluation of adhesion of fibroblasts to collagen coat Using the various soybean protein hydrolysates prepared in Examples 1 to 3 above, the effect of promoting adhesion of fibroblasts to the collagen coat was evaluated.
  • human normal skin-derived fibroblasts (Kurabo: KF-4009) were first seeded at a concentration of 2 ⁇ 10 4 cells / well on a cell culture plate (96 well) coated with collagen type I. Next, 200 ⁇ l of Dulbecco's Modified Eagle Medium (DMEM; containing AB 1%) diluted so that each soy protein hydrolyzate has a concentration of 0.5 mg / ml was added to each well, 37 ° C., 5% carbon dioxide gas and 95% Incubation was performed for 4 hours in an air environment. On the other hand, what added 200 microliters of D-MEM culture medium which has not added soybean protein hydrolyzate was prepared, and it was set as control.
  • DMEM Dulbecco's Modified Eagle Medium
  • Example 6 Evaluation of adhesion of keratinocytes to collagen coat Using the soybean protein hydrolyzate prepared in Example 2 above, the effect of promoting adhesion of epidermal keratinocytes to the collagen coat was evaluated.
  • human normal skin-derived keratinocytes (Kurabo: kk-4009) were seeded at a concentration of 7.5 ⁇ 10 4 cells / well on a cell culture plate (96 well) coated with collagen type IV.
  • 200 ⁇ l of epidermal keratinocyte basal medium (Kurabo: kk-2350S) diluted so that the soy protein hydrolyzate has a concentration of 5 ⁇ g / ml was added to each well at 37 ° C., 5% carbon dioxide and 95%. Incubation was performed for 4 hours in an air environment.
  • 200 ⁇ l of epidermis keratinocyte basic medium not added with soy protein hydrolyzate was prepared and used as a control.
  • soy protein hydrolyzate also promotes the adhesion of keratinocytes to a collagen coat coated with type IV collagen. From this result, by using soybean protein hydrolyzate, adhesion between type IV collagen stretched in the basement membrane and keratinocytes is promoted, and keratinization caused by ultraviolet rays, aging, etc. Since the loosening of the cell-basement membrane can be suppressed, in addition to tarmi and wrinkles, poor turnover and the accompanying stains and dullness (skin color improvement), as well as thinning of the epidermis are effectively prevented or It can be seen that it can be improved.
  • Example 7 Evaluation of adhesion of fibroblasts to AGE-modified collagen coat Using the soybean protein hydrolyzate prepared in Example 2 above, the effect of promoting adhesion of fibroblasts to AGE-modified collagen was evaluated. .
  • glyoxal was added to a cell culture plate (96 well) coated with collagen with type I collagen at a concentration of 2 mM / PBS, and cultured at 37 ° C. for 9 days to make collagen AGE.
  • human normal skin-derived fibroblasts (Kurabo: KF-4009) were seeded at a concentration of 2 ⁇ 10 4 cells / well on a cell culture plate coated with the aforementioned AGE type I collagen.
  • DMEM Dulbecco's Modified Eagle Medium
  • Example 8 Evaluation of Collagen Gel Shrinkage Promotion Using the various soybean protein hydrolysates prepared in Examples 2 to 3 above, the collagen gel shrinkage promoting effect was evaluated.
  • Collagen gel contraction acceleration evaluation was performed using a collagen gel culture kit (Nitta Gelatin). Specifically, first, 30 ml of collagen type IA solution was placed in 50 ml vials on ice. Separately, a 10-fold concentrated solution (MEM) containing no NaHCO 3 added so that the final concentration of fibroblasts was 5 ⁇ 10 4 cells / ml was prepared and slowly pipetted. Next, the MEM solution to which the fibroblasts were added was added to the collagen type IA solution and slowly pipetted, and then the reconstitution buffer was added to quickly pipet. The collagen type IA solution added with the fibroblasts was placed in each well of the 24-well plate so as not to be bubbled, and allowed to stand at 37 ° C.
  • MEM 10-fold concentrated solution
  • Collagen gel shrinkage (%) (diameter of collagen gel after 1 week of culture / initial value of collagen gel diameter) ⁇ 100
  • composition containing the hydrolyzate of soy protein of the present invention can be used as a new material for improving skin wrinkles and talmi and restoring skin firmness and elasticity.

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Abstract

La présente invention concerne une substance inédite ayant la capacité de favoriser l'adhérence entre une cellule et le collagène. La présente invention concerne, donc, la préparation d'une composition visant à favoriser l'adhérence entre une cellule et du collagène au moyen d'un hydrolysat de protéines de soja.
PCT/JP2012/073170 2011-09-12 2012-09-11 Composition contenant un hydrolysat de protéines de soja WO2013039056A1 (fr)

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CN103719534A (zh) * 2013-12-23 2014-04-16 江南大学 一种对大豆分离蛋白进行酶水解的方法
JP2018525360A (ja) * 2015-07-22 2018-09-06 テンプル ユニヴァーシティ − オブ ザ コモンウェルス システム オブ ハイアー エデュケーション 創傷治癒、組織工学、及び再生医療のための組成物及び方法における使用のための大豆由来の生物活性ペプチド
CN112451433A (zh) * 2019-09-06 2021-03-09 广东丸美生物技术股份有限公司 一种毛豆提取物及其制备方法和应用
JPWO2023281657A1 (fr) * 2021-07-07 2023-01-12

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CN111875671A (zh) * 2020-07-23 2020-11-03 西北民族大学 一种用于无血清培养基的大豆蛋白水解添加物及制备方法

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103719534A (zh) * 2013-12-23 2014-04-16 江南大学 一种对大豆分离蛋白进行酶水解的方法
JP2018525360A (ja) * 2015-07-22 2018-09-06 テンプル ユニヴァーシティ − オブ ザ コモンウェルス システム オブ ハイアー エデュケーション 創傷治癒、組織工学、及び再生医療のための組成物及び方法における使用のための大豆由来の生物活性ペプチド
CN112451433A (zh) * 2019-09-06 2021-03-09 广东丸美生物技术股份有限公司 一种毛豆提取物及其制备方法和应用
JPWO2023281657A1 (fr) * 2021-07-07 2023-01-12

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