JP5468757B2 - Composition having fibroblast proliferation promoting ability - Google Patents

Description

  The present invention relates to a novel composition for promoting fibroblast proliferation, and an invention related thereto.

The fibroblasts, which are the main cells of the dermis, are collagen that keeps the skin firm, elastin that keeps the skin elastic, and mucopolysaccharides that significantly affect the skin's moisture retention (e.g., hyaluronic acid, Chondroitin sulfate, etc.). However, the effects of certain external factors such as UV irradiation, significant drying of the air, excessive skin washing, etc., and aging reduce the ability of the fibroblasts to proliferate, resulting in a skin moisturizing function and elasticity. It is known that skin aging (for example, skin elasticity or elasticity decreases, skin wrinkles or tarmi, etc.) is accelerated.
Therefore, promoting the proliferation of fibroblasts is considered to contribute to the prevention and / or improvement against a decrease in skin elasticity or elasticity, and the prevention and / or improvement of skin wrinkles or tarmi. Various cosmetics and beauty foods and drinks containing plant extracts having a fibroblast proliferation promoting action have been proposed.

By the way, Patent Document 1 is characterized by hydrolyzing a bean (such as soybean) seed or a water extract of the processed product with a proteolytic enzyme, and using a fraction having a molecular weight of 10,000 or less as an active ingredient. A topical skin preparation is described. More specifically, an essence (formulation example 6) containing a soybean hydrolyzate, hyaluronic acid and the like by a protein hydrolase is described.
Patent Document 2 discloses hydrolyzed proteins and the like; at least one organic powder or surface-treated organic powder; and a dermatologically acceptable carrier, the carrier being in the form of an emulsion; A featured topical personal care composition is described. More specifically, topical creams (Table 5) containing sodium hyaluronate, hydrolyzed soy protein and the like are described.
However, Patent Documents 1 and 2 do not describe any product obtained by degrading soybean protein with a specific protease called thermolysin.

On the other hand, in Patent Document 3, a product obtained by degrading soybean protein with a specific protease called thermolysin has at least one production promoting ability selected from the group consisting of collagen and hyaluronic acid, and collagen and / or A therapeutic or preventive agent for diseases caused by a decrease in the amount of hyaluronic acid; a food for improving or preventing a condition caused by a decrease in the amount of collagen and / or hyaluronic acid; wrinkles or talmi of skin caused by UV exposure, aging, etc. Cosmetics for prevention and / or improvement of skin; cosmetics for prevention and / or improvement against reduction in skin elasticity or elasticity; and the like, and the product and hyaluronic acid There is no mention or suggestion of any combination use.
Japanese Patent Laid-Open No. 9-025225 Japanese translation of publication 2005-516048 International Publication No. 2007/049400 Pamphlet

  In view of the importance of the role played by fibroblasts in the living body as described above, development of a useful novel composition having a remarkable ability to promote fibroblast proliferation is desired. In view of such conventional problems, an object of the present invention is to provide a novel composition having remarkable fibroblast proliferation promoting ability.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have decomposed one or more selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives and salts thereof with a specific protease called thermolysin. In combination with the product obtained in this way, the inventors found that the proliferation of fibroblasts was remarkably promoted as compared with the case where each was used alone, and completed the present invention.

Accordingly, the present invention provides the following.
(1) (A) one or more selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, and salts thereof;
(B) A composition containing a thermolysin degradation product of soybean protein.
(2) The composition according to (1) above, which can be used to promote fibroblast proliferation.

  The present invention provides novel compositions that significantly promote fibroblast proliferation. The composition of the present invention is a composition for preventing or treating cosmetic problems, for example, a composition for preventing and / or improving skin wrinkles or talmi due to UV exposure, aging, etc., skin elasticity It is useful as a composition or the like for preventing and / or improving a decrease in sex or elasticity.

  Hereinafter, the present invention will be described in detail. It should be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

  The present invention is a composition comprising (A) one or more selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives and salts thereof, and (B) a thermolysin degradation product of soybean protein.

  In the present invention, at least one selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, and salts thereof is used (hereinafter, this may be collectively referred to as “hyaluronic acids” in the present specification). . The origin of the hyaluronic acid used in the present invention (chicken crown, microorganism, etc.) is not particularly limited, and those that can be usually used in the field of pharmaceuticals, quasi drugs, cosmetics or foods can be used without particular limitation.

  Hyaluronic acid derivatives include ester derivatives in which the hydrogen atom of the carboxyl group of hyaluronic acid is substituted with an alkyl group, alkenyl group, aryl group, etc .; ester derivatives in which the hydrogen atom of the hydroxyl group of hyaluronic acid is substituted with an acyl group; An ether derivative substituted with an alkyl group, an alkenyl group, an aryl group, etc .; the hydrogen atom of the amino group of hyaluronic acid was substituted with an amide derivative substituted with an acyl group, an alkyl group, an alkenyl group, an aryl group, or the like Amine derivatives; and the like. As the hyaluronic acid derivative, those having 1 to 6 substitutions per structural unit (disaccharide) are usually used, and those having 2 to 4 substitutions are preferred.

  As ester derivatives, the hydrogen atom of the carboxyl group of hyaluronic acid is substituted with an alkyl group, alkenyl group, aryl group, etc .; the hydrogen atom of the hydroxyl group of hyaluronic acid is substituted with an acyl group such as an alkanoyl group, arylcarbonyl group, etc. And the like.

As an alkyl group, a C1-C12 alkyl group is mentioned, Preferably it is a C1-C6 alkyl group. Specific examples include methyl group, ethyl group, propyl group, isopropyl group, butyl group, s-butyl group, t-butyl group, isobutyl group, pentyl group, hexyl group and the like.
Examples of the alkenyl group include alkenyl groups having 2 to 12 carbon atoms, preferably alkenyl groups having 2 to 6 carbon atoms. Specific examples include a vinyl group and an allyl group.
These alkyl group and alkenyl group may be substituted, and as the substituent, an alkoxy group having 1 to 6 carbon atoms (eg, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, s-butoxy group) Group, t-butoxy group, isobutoxy group, pentyloxy group, hexyloxy group, etc.), halogen atom (eg, fluorine atom, chlorine atom, bromine atom, iodine atom), amino group, hydroxy group and the like.

  Examples of the aryl group include aryl groups having 6 to 12 carbon atoms. Specific examples include a phenyl group and a naphthyl group (1-naphthyl group, 2-naphthyl group). In addition, the aryl group may be substituted, and examples of the substituent include alkyl groups having 1 to 6 carbon atoms (eg, methyl group, ethyl group, propyl group, isopropyl group, butyl group, s-butyl group, t -Butyl group, isobutyl group, pentyl group, hexyl group, etc.), C1-C6 alkoxy group (eg, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, s-butoxy group, t-butoxy group) Group, isobutoxy group, pentyloxy group, hexyloxy group, etc.), halogen atom (eg, fluorine atom, chlorine atom, bromine atom, iodine atom), amino group, hydroxy group and the like.

The alkanoyl group includes an alkanoyl group having 1 to 12 carbon atoms, and preferably an alkanoyl group having 1 to 7 carbon atoms. Specific examples include an acetyl group, a propionyl group, and a butyryl group. Further, the alkanoyl group may be substituted, and as the substituent, an alkoxy group having 1 to 6 carbon atoms (eg, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, s-butoxy group, t-butoxy group, isobutoxy group, pentyloxy group, hexyloxy group and the like), halogen atom (eg, fluorine atom, chlorine atom, bromine atom, iodine atom), amino group, hydroxy group and the like.
Examples of the arylcarbonyl group include an arylcarbonyl group having 7 to 15 carbon atoms, and an arylcarbonyl group having 7 to 12 carbon atoms is preferable. Specific examples include a benzoyl group and a naphthylcarbonyl group (1-naphthylcarbonyl group, 2-naphthylcarbonyl group). In addition, the arylcarbonyl group may be substituted, and examples of the substituent include alkyl groups having 1 to 6 carbon atoms (eg, methyl group, ethyl group, propyl group, isopropyl group, butyl group, s-butyl group, t-butyl group, isobutyl group, pentyl group, hexyl group, etc.), C1-C6 alkoxy group (eg, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, s-butoxy group, t- Butoxy group, isobutoxy group, pentyloxy group, hexyloxy group, etc.), halogen atom (eg, fluorine atom, chlorine atom, bromine atom, iodine atom), amino group, hydroxy group and the like.

Examples of ether derivatives include those in which the hydrogen atom of the hydroxyl group of hyaluronic acid is substituted with an alkyl group, alkenyl group, aryl group, or the like.
The “alkyl group”, “alkenyl group” and “aryl group” exemplified as the substituent of the ether derivative are the “alkyl group”, “alkenyl group” and “aryl group” exemplified as the substituent of the ester derivative, respectively. The same thing is mentioned.

Examples of the amide derivatives include those in which the hydrogen atom of the amino group of hyaluronic acid is substituted with an acyl group such as an alkanoyl group or an arylcarbonyl group.
Examples of the “alkanoyl group” and “arylcarbonyl group” exemplified as the substituent of the amide derivative include those similar to the “alkanoyl group” and “arylcarbonyl group” exemplified as the substituent of the ester derivative.

Examples of the amine derivative include those in which the hydrogen atom of the amino group of hyaluronic acid is substituted with an alkyl group, an alkenyl group, an aryl group, or the like.
Examples of the “alkyl group”, “alkenyl group” and “aryl group” exemplified as the substituent of the amine derivative are the “alkyl group”, “alkenyl group” and “aryl group” exemplified as the substituent of the ester derivative, respectively. The same thing is mentioned.

  As salts of hyaluronic acid and hyaluronic acid derivatives, pharmaceutically and physiologically acceptable salts are preferable. Examples include alkali metal salts such as sodium and potassium; alkaline earth metal salts such as magnesium and calcium; ammonium salts; alkanolamine salts such as monoethanolamine; and preferably alkali metal salts such as sodium salts It is.

  In the composition of the present invention, the average molecular weight of hyaluronic acid is not particularly limited, but is usually 1,000 to 4,000,000, preferably 1,000 to 1,200,000, more preferably 5,000 to 1,000,000, and particularly preferably 5,000 to 1,000,000. 400,000.

In the present invention, hyaluronic acids may be used alone or in any combination of two or more.
Moreover, as for hyaluronic acid, when there exists a commercial item, you may use a commercial item as it is.

Specific examples of hyaluronic acids used in the present invention include hyaluronic acid, sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, acetylated hyaluronic acid, sodium acetylated hyaluronate, and potassium acetylated hyaluronate. Acetylated magnesium hyaluronate, acetylated calcium hyaluronate, and the like.
The hyaluronic acid used in the present invention is preferably sodium hyaluronate from the viewpoint of solubility and stability.

  The amount of hyaluronic acid compounded in the composition of the present invention is not particularly limited as long as the effects of the present invention can be obtained, but is usually 0.0005% by weight or more, preferably 0.001% by weight or more based on the entire composition. More preferably 0.01% by weight or more, still more preferably 0.05% by weight or more, particularly preferably 0.1% by weight or more, and the upper limit of blending is preferably 5% by weight or less, more preferably 1% by weight or less. More preferably, it is 0.5% by weight or less, and particularly preferably 0.25% by weight or less.

  In the present invention, a thermolysin degradation product of soybean protein is further used (hereinafter, this product may be referred to as “soybean thermolysin degradation product”).

  Soybean (Glycin 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 can be any protein derived from the soybean plant described above, but preferably can be any protein derived from the seed of the soybean plant.

  Therefore, in the present invention, the soybean plant itself, the seed of the soybean plant itself, the plant or the crushed or pulverized product of the seed may be used as the soybean protein, but preferably the protein from all the components in the soybean plant. What separated and refine | purified the component, More preferably, what isolate | separated and refine | purified the protein component from all the components in the seed of a soybean plant is used. Soy protein obtained by separation and purification in this manner may contain substantially all kinds of proteins contained in soybean plants or seeds of soybean plants as long as the thermolysin degradation product can exert the effect of the present application. Moreover, some kinds of proteins may be included.

  As the soy protein, commercially available products can also be suitably used. For example, 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.

  In the present specification, 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.

  Thermolysin (EC3.4.24.27) used in the present invention is a heat-resistant protease produced by a heat-resistant bacterium called Bacillus thermoproteolyticus. Thermolysin is generally known to cleave peptide bonds on the amino group side of hydrophobic amino acid residues having large side chains (for example, isoleucine, leucine, valine, phenylalanine, methionine, alanine, etc.).

  A commercially available product can be suitably used for thermolysin, and for example, it can be easily obtained from a manufacturer such as Daiwa Kasei Co., Ltd. In the present invention, a protease known in the art as a protease having peptide cleavage characteristics (such as cleavage sequence specificity) equivalent to thermolysin can be used as thermolysin.

  The thermolysin used in the present invention may be one type of thermolysin or a protease having the same peptide cleavage characteristics (such as cleavage sequence specificity) as thermolysin, or may be used in combination of two or more types.

  The reaction conditions used when hydrolyzing soy protein with thermolysin are not particularly limited and may be appropriately selected by those skilled in the art according to common general technical knowledge. For example, when using commercially available thermolysin, it can be used according to the instruction manual. As a specific example, soy protein concentration is generally 0.1 to 30% (w / v), preferably 1 to 10% (w / v) in a solvent such as water. Soy protein or a raw material containing soy protein is suspended, and this suspension is generally about 0.001 to 3% (w / v), preferably about 0.01 to 0.125% (w / v). The aspect which adds thermolysin so that it may become and performs a hydrolysis reaction is mentioned. In general, reaction temperatures of 30-80 ° C, preferably 40-70 ° C, more preferably 50-60 ° C may be used. In general, a reaction time of 2 to 30 hours, preferably 3 to 24 hours, more preferably 10 to 20 hours, and even more preferably 12 to 18 hours may be used. The pH of the reaction solution is preferably in the vicinity of the optimum pH of the thermolysin used, and is preferably in the vicinity of 7.0 to 8.5, for example.

  The means for stopping the reaction is not particularly limited, and known means can be used. Examples of such means include heat treatment. For example, the thermolysin contained in the reaction product can be deactivated 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. For example, examples of such heat treatment include heat treatment at 85 ° C. for 15 minutes and heat treatment at 100 ° C. for 5 minutes.

  The thermolysin degradation product 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.

  Alternatively, the hydrolyzate can be pulverized by concentration under reduced pressure and then freeze-drying. The conditions and equipment used for concentration under reduced pressure and lyophilization are not particularly limited, and can be appropriately selected by those skilled in the art according to common technical knowledge. The hydrolyzate thus powdered can be used as it is or after being dissolved in a solvent such as water.

  The hydrolyzate used in the present invention may be in a state containing substantially all of a wide variety of peptides generated by decomposing soybean protein with thermolysin, or such a wide variety of peptides. Alternatively, it may be a part obtained by further fractionation and purification by a known method using the presence or absence of fibroblast growth promoting ability as an index. However, for convenience, the soy protein is used as it is in a state of containing substantially all of a wide variety of peptides obtained by decomposing the protein with thermolysin. The presence or absence of the ability to promote fibroblast proliferation can be confirmed, for example, as described in Examples below.

  In the present invention, the term “having the ability to promote fibroblast proliferation” means that when a test substance is allowed to act on fibroblasts, the proliferation of fibroblasts is less than when the test substance is not allowed to act. It means being promoted. In certain embodiments, fibroblasts in the term refer to dermal fibroblasts.

  The average molecular weight of the thermolysin decomposition product of soybean protein used in the present invention is preferably 300 to 10,000. The average molecular weight is more preferably 400 to 5000, still more preferably 500 to 3500, and even more preferably 550 to 3200, from the viewpoint of increasing the permeability to cells and obtaining a higher effect. Accordingly, the average molecular weight can be, for example, 1000 to 2000. The average molecular weight of the hydrolyzate can be measured by any method known to those skilled in the art. For example, it can be easily measured by the gel permeation chromatography (GPC) method described in the Examples below.

  The blending amount of the soybean thermolysin degradation product to be blended in the composition of the present invention is not particularly limited as long as the effect of the present invention can be obtained, but is usually 0.1% by weight or more, preferably 0.25% by weight with respect to the entire composition. % Or more, more preferably 0.5% by weight or more, still more preferably 0.75% by weight or more, particularly preferably 1.0% by weight or more, and the upper limit of blending is preferably 10% by weight or less, more preferably 7. It should be 5% by weight or less, more preferably 5% by weight or less, and particularly preferably 2.5% by weight or less.

  Further, the weight ratio of the hyaluronic acid and the soybean thermolysin degradation product in the composition of the present invention is not particularly limited as long as the effect of the present invention can be obtained, but the soybean thermolysin degradation product is usually 0 with respect to 1 part by weight of the hyaluronic acid. 0.02 to 20000 parts by weight, preferably 0.2 to 2000 parts by weight, more preferably 2 to 200 parts by weight, and even more preferably 20 to 50 parts by weight.

  In addition to the hyaluronic acids and soybean thermolysin degradation product as described above, the composition of the present invention has the purpose of enhancing or supplementing the action of hyaluronic acids or soybean thermolysin degradation product, or other useful effects on the composition of the present application. Whitening ingredients, anti-inflammatory ingredients, antibacterial ingredients, cell activation ingredients, astringent ingredients, antioxidant ingredients, acne improving ingredients, hyaluronic acid and other bio-component synthesis promoting ingredients, blood circulation promoting ingredients, moisturizing ingredients, anti-aging Various components, such as a component, can be blended in combination of one or more. Preferred are one or more of whitening components, anti-inflammatory components, antibacterial components, cell activation components, astringent components, antioxidant components, anti-aging components or moisturizing components. These components are not particularly limited as long as they can be used in the fields of pharmaceuticals, quasi-drugs, foods, and cosmetics, and arbitrary components can be appropriately selected and used.

  In addition to the above components, the composition of the present invention may be appropriately mixed with components usually used in the field of pharmaceuticals, quasi drugs, cosmetics or foods, depending on the use or dosage form of the composition. The components that can be blended are not particularly limited, but include, for example, amino acids, alcohols, polyhydric alcohols, saccharides, high molecular compounds such as gums and polysaccharides, surfactants, solubilizing components, fats and oils, transdermal Absorption-promoting ingredients, antiseptic / antibacterial / bactericidal agents, pH adjusters, chelating agents, antioxidants, enzyme components, binders, disintegrating agents, lubricants, fluidizing agents, cooling agents, minerals, cell activation Agents, nourishing tonics, excipients, thickeners, stabilizers, preservatives, tonicity agents, dispersants, adsorbents, disintegration aids, wetting agents or wetting regulators, dampening agents, coloring agents, wearing Perfume or fragrance, fragrance, reducing agent, solubilizer, solubilizer, foaming agent, thickener or thickener, solvent, base, emulsifier, plasticizer, buffer, brightener, sweetener , Sour seasonings, dietary fiber, fruit juice powder, organic acids, seasonings, umami ingredients, swelling agents, bleaching agents, coloring agents, etc. It is possible.

The composition of the present invention can take a dosage form usually used for pharmaceuticals, quasi drugs, cosmetics or foods, and is usually a solid, semi-solid or liquid. Specifically, tablets (including oral disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, coated tablets, chewable tablets, etc.), pills, granules, fine granules, powders, Hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), gels, liposomes, extracts, tinctures, lemonades, ointments, jellys, etc. Can take form. In addition, pastes, mousses, gels, liquids, emulsions, creams, sheets (substrate support), aerosols can be added by blending other solvents and commonly used bases as necessary. It can be prepared in various desired forms such as spray.
These dosage forms can be produced by conventional methods in the art.

The composition of the present invention can be a composition for internal use or a composition for external use.
In addition to pharmaceuticals for internal use (including quasi-drugs), the composition for internal use includes confectionery, beverages, health foods, nutritional supplements (including balanced nutritional foods, supplements, etc.), functional nutritional foods, and special health use Includes food such as food. In food, specifically, milk, milk drinks, lactic acid bacteria drinks, soft drinks with fruit juice, carbonated drinks, vegetable juice drinks, tea drinks, ionic drinks, sports drinks, functional drinks, vitamin supplement drinks, balanced nutrition drinks, Beverages such as jelly beverages, alcoholic beverages, soups; puddings such as custard pudding, milk pudding and pudding with fruit juice; gel foods such as jellys, dressings and creams; gums such as chewing gum and bubble gum (plates) Gum, sugar-coated grain gum, etc.), chocolates (solid chocolate, coated chocolate, chocolate with flavors such as strawberry chocolate, blueberry chocolate, melon chocolate), soft candy (caramel, nougat, gummy candy, marshmallow, etc.) ), Caramels such as toffee, biscuits (Hard biscuits, soft biscuits, soft cookies, crackers, semi-biscuit biscuits, etc.), cakes and other confectionery; ice cream, ice milk, lacto ice, sorbet, frozen confectionery such as ice confectionery; breads; noodles; liquid (water, milk , Juice, etc.) can be easily converted into a drink, a reconstituted food such as a powdered beverage, a paste or a tablet; a supplement having a form such as a powder, a capsule or a tablet;

  Moreover, as an external composition, cosmetics are contained in addition to external medicine (including quasi drugs). For cosmetics, specifically, makeup cosmetics such as foundation, lipstick, mascara, eye shadow, eyeliner, eyebrows and beauty nails; Basic cosmetics such as oils and packs; cleansing agents such as facial cleansers, cleansing and body cleaning agents, bathing agents, and the like.

  Since the composition of the present invention has the ability to promote fibroblast proliferation, for example, a composition for preventing or treating cosmetic problems caused by, for example, reduction or inactivation of fibroblasts (for example, exposure to ultraviolet rays) Composition for prevention and / or improvement of skin wrinkles or tarmi caused by decrease or inactivation of fibroblasts due to aging or aging, etc., or skin caused by reduction or inactivation of fibroblasts The composition can be suitably used as a composition for preventing and / or improving the decrease in elasticity or elasticity.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1
Preparation of Thermolysin Degradation Product of Soy Protein 50 g of defatted soybean powder (trade name Profam 974, manufactured by ADM Far East Co., Ltd.) was dispersed in 2 L of distilled water and adjusted to pH 8.5 with 0.1 N NaOH. 500 mg of thermolysin (product name “Samoase PC10F”, manufactured by Daiwa Kasei Co., Ltd.) was added thereto, and decomposition was performed at 60 ° C. for 15 hours. After the reaction, thermolysin was inactivated by boiling at 100 ° C. for 10 minutes. After allowing to cool, 25 g of a filter aid (Radiolite 500, Showa Chemical Industry Co., Ltd.) was added and stirred, followed by filtration.

  The filtrate obtained as described above was passed through a column packed with a strongly acidic ion exchange resin (trade name “Dowex 50W × 2, H + form, 50-100 mesh”, manufactured by Dow Chemical Company), and then 5 times the column. Wash with a volume of deionized water to remove non-peptide components. A 2M ammonia solution was passed through to elute the column adsorbing component, and the peptide fraction was collected. Ammonia was removed using an evaporator and further concentrated to dryness. Water was added thereto to dissolve the dried solid, and then centrifuged (10,000 rpm, 30 minutes) to remove insoluble matters. As a result of freeze-drying the supernatant, about 26 g of soybean protein thermolysin hydrolyzate was finally obtained as a thermolysin hydrolyzate of soybean protein.

  The average molecular weight of the decomposition product thus obtained was measured by the GPC method. 100 mg of a thermolysin degradation product of soybean protein after lyophilization was dissolved in 2.0 ml of 0.1 M sodium phosphate buffer (pH 7.0) to prepare a test solution. A column (φ2.6 × 100 cm) packed with Sephadex G25 (Medium type, manufactured by Amersham Biosciences) was equilibrated with the same 0.1 M sodium phosphate buffer (pH 7.0). The column was loaded with 2.0 ml of the test solution and eluted at a flow rate of 1.0 ml / min. Insulin (bovine pancreas origin, Sigma, molecular weight 5733), Insulin A chain (bovine pancreas origin, Sigma, molecular weight 2532), and Bradykinin (Sigma, molecular weight 1050) are used as peptide preparations with known molecular weights. It was. Peptides were detected at 214 nm, and the molecular weight distribution and average molecular weight were estimated from the elution time. As a result, the average molecular weight of the thermolysin degradation product of soybean protein obtained by the above method was estimated to be about 1500.

Example 2
Fibroblast proliferation promotion test Human normal skin-derived fibroblasts (CRL-2089; ATCC, Lot # 204741) were cultured in 96-well culture plates. More specifically, the cells were seeded at a density of 5.0 × 10 ³ cells / cm ² and cultured at 37 ° C. in an environment of 5% carbon dioxide gas and 95% air for 48 hours. As the culture solution, Dulbecco's Modified Eagle Medium (D-MEM) was used in an amount of 200 μl per well. Thereafter, the culture solution was removed, and the test drug shown in Table 1 below was replaced with a medium in which each concentration was dissolved and cultured. On the other hand, a medium supplemented with 200 μl of medium to which nothing was added was used as a control. After culturing for 48 hours, the number of viable cells in each well was measured by a general cell number measuring method (Cell Counting Kit-8; manufactured by Dojindo Laboratories). Based on the measurement results, the number of viable cells (%) in each test drug addition group was calculated with the number of viable cells in the control as 100%.

From this result, in the cells cultured in the culture solution containing both hyaluronic acid and soybean thermolysin degradation product, the proliferation of fibroblasts is significantly increased compared to the case of culturing in the culture solution of the component alone, and the soybean protein is called thermolysin. It was demonstrated that degradation products obtained by treating with specific proteases can dramatically enhance the action of ECM components.
Moreover, it was simultaneously recognized that the fibroblast proliferation promoting action obtained by combining hyaluronic acid and soybean thermolysin degradation product was synergistic.

  Formulation examples are given below, but the present invention is not limited to these examples.

Formulation Example 1: Emulsion [Ingredient] [Ratio]
Hyaluronic acid 0.1
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.5
Squalane 2.0
Liquid paraffin 5.0
Cetanol 0.5
Glyceryl monostearate 2.0
POE (25) cetyl ether 2.0
Triethanolamine 0.8
Glycerin 4.0
1,3-butylene glycol 6.0
Preservative (Methylparaben, Propylparaben) Appropriate amount Perfume Appropriate amount
Purified water
100.0% by weight

Formulation Example 2: Cream [Ingredient] [Ratio]
Acetylated sodium hyaluronate 0.3
Soybean protein thermolysin degradation product (average molecular weight 1500) 1.0
Vaseline 1.0
Squalane 5.0
Liquid paraffin 10.0
Stearic acid 1.5
Stearyl alcohol 2.0
Glyceryl monostearate 2.0
POE (20) cetyl ether 3.0
Triethanolamine 1.0
Glycerin 6.0
1,3-butylene glycol 8.0
Preservative (Ethylparaben, Propylparaben) Appropriate amount Perfume Appropriate amount
Purified water
100.0% by weight

Formulation Example 3: Lotion [Ingredient] [Ratio]
Acetylated hyaluronic acid 0.05
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.5
POE (20) sorbitan monoisostearate 0.3
Succinic acid 0.2
Sodium succinate 0.5
Edetate trisodium 0.05
1,3-butylene glycol 6.0
Preservative (Methylparaben, Propylparaben) Appropriate amount Perfume Appropriate amount
Purified water
100.0% by weight

Formulation Example 4: Soft capsule [Ingredient] [Ratio]
Sodium hyaluronate 1.0
Thermolysin degradation product of soybean protein (average molecular weight 1500) 7.5
Melon extract 5.0
Ceramide-containing material 1.0
Haematococcus alga pigment extract (containing astaxanthin) 1.0
Gelatin 30.0
Glycerin 15.0
Safflower oil
100.0% by weight

Formulation Example 5: Beverages [Ingredients] [Ratio]
Sodium hyaluronate 0.01
Soybean protein thermolysin degradation product (average molecular weight 1500) 2.0
Royal Jelly Extract 1.0
Lemon juice 1.0
Vitamin B2 0.005
Vitamin B6 0.05
Vitamin C 0.5
Erythritol 5.0
Reduced maltose starch syrup 5.0
Water soluble dietary fiber 5.0
Stabilizer (pectin) 0.2
Sweetener (acesulfame potassium, sucralose) 0.008
Calcium lactate 0.05
Magnesium chloride 0.015
Potassium chloride 0.015
Perfume 0.025
Preservatives (sodium benzoate, butylparaben) appropriate amount acidulant appropriate amount
(PH adjusted to 3.5)
Purified water remaining
100.0% by weight

Formulation Example 6: Beverages [Ingredients] [Ratio]
Soybean protein thermolysin degradation product (average molecular weight 1500) 2.0
Sodium hyaluronate 0.8
Methylsulfonylmethane 2.0
Fish collagen 0.1
Glucosamine 1.0
Honey 6.0
Maltitol 4.0
Calcium gluconate 0.3
Sweetener (acesulfame potassium, sucralose) 0.02
Preservatives (sodium benzoate, butylparaben) 0.05
Antioxidant (Berberry extract) 0.04
Vitamin B2 0.005
Perfume 0.025
Sour seasoning
(Adjust pH to 3.6)
Purified water remaining
100.0% by weight

Formulation Example 7: Lotion [Ingredient] [Ratio]
Soybean protein thermolysin degradation product (average molecular weight 1500) 1.0
Acetylated sodium hyaluronate 0.25
Whey / BG mixture 1.0
Dipropylene glycol 10.0
Polyoxyethylene hydrogenated castor oil 50 0.25
Triethanolamine appropriate amount perfume appropriate amount
Purified water remaining
100.0% by weight

Formulation Example 8: Lotion [Ingredient] [Ratio]
Soybean protein thermolysin degradation product (average molecular weight 1500) 2.0
Sodium hyaluronate 0.5
Concentrated glycerin 5.0
Polyoxypropylene methyl glucoside 3.0
Water-soluble collagen 0.5
Polyethylene glycol monostearate 0.2
Preservative appropriate amount Fragrance proper amount
Purified water remaining
100.0% by weight

Formulation Example 9: Emulsion [Ingredient] [Ratio]
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.2
Acetylated sodium hyaluronate 1.0
Dipropylene glycol 3.0
Ascorbine tetra-2-hexyldecanoate 0.05
Glyceryl tri-2-ethylhexanoate 8.0
Shea fat 1.5
Sodium edetate 0.5
Polysorbate 60 5.0
Preservative (Methylparaben, Propylparaben) Appropriate amount Perfume Appropriate amount
Purified water remaining
100.0% by weight

Formulation Example 10: Cream [Ingredient] [Ratio]
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.7
Sodium hyaluronate 0.5
Concentrated glycerin 10.0
1,3-butylene glycol 5.0
Terminaria extract 0.01
Squalane 4.0
Shea fat 2.0
Hydrolyzed elastin 0.05
Polyoxyethylene hydrogenated castor oil 60 0.2
(Hydroxyethyl acrylate /
Acryloyldimethyltaurine Na) copolymer 1.5
Preservative (Ethylparaben, Propylparaben) Appropriate amount Perfume Appropriate amount
Purified water remaining
100.0% by weight

Formulation Example 11: Pack cream [Ingredient] [Ratio]
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.5
Sodium hyaluronate 0.2
Whey / BG mixture 1.0
α-olefin oligomer 5.0
Dipropylene glycol 10.0
Tetra-2-ethylhexanoic acid pentaerythrit 5.0
Tormentilla extract 0.01
Tocopherol acetate 0.05
POE (20) cetyl ether 4.0
Preservative (Ethylparaben, Propylparaben) Appropriate amount Perfume Appropriate amount
Purified amount
100.0% by weight

Formulation Example 12: Soft capsule [Ingredient] [Ratio]
Thermolysin degradation product of soybean protein (average molecular weight 1500) 10.0
Acetylated sodium hyaluronate 2.0
Collagen tripeptide 1.5
Lycopene 2.0
Vitamin C 1.0
Gelatin 35.0
Glycerin 10.0
Evening Primrose Oil 30.0
Vegetable oil
100.0% by weight

Formulation Example 13: Tablet [Ingredient] [Ratio]
Thermolysin degradation product of soybean protein (average molecular weight 1500) 5.0
Sodium hyaluronate 5.0
Ceramide 1.5
Vitamin B2 0.5
Vitamin C 4.0
Sucrose fatty acid ester 3.0
Crystalline cellulose
100.0% by weight

Formulation Example 14: Coated Tablet [Ingredient] [Ratio]
Thermolysin degradation product of soybean protein (average molecular weight 1500) 0.1
Sodium hyaluronate 1.0
Grape seed extract 5.0
Melon extract 2.0
Magnesium stearate 3.0
Corn starch 30.0
Shellac 3.0
Lactose remaining
100.0% by weight

Formulation Example 15: Chewable tablet [ingredients] [ratio]
Soybean protein thermolysin degradation product (average molecular weight 1500) 1.0
Hyaluronic acid 0.1
Collagen 5.0
Elastin 3.0
Sweetener 0.1
Perfume Trace calcium stearate 1.0
Reduced maltose water candy
100.0% by weight

Formulation Example 16: Granules [Ingredients] [Ratio]
Thermolysin degradation product of soybean protein (average molecular weight 1500) 3.0
Acetylated sodium hyaluronate 0.5
Rose extract 5.0
Chestnut tannin 5.0
Sweetener 0.1
Citric acid 0.3
Fragrance Trace amount Lactose 35.0
Crystal Cellulos
100.0% by weight

Formulation Example 17: Beverages [Ingredients] [Ratio]
Thermolysin degradation product of soybean protein (average molecular weight 1500) 2.5
Sodium hyaluronate 0.1
Collagen 2.0
Royal Jelly 0.3
Lotus germ extract 0.01
Sweetener (sucralose) 0.05
Citric acid 0.5
Trisodium citrate 0.02
Perfume
Purified water remaining
100.0% by weight

Formulation Example 18: Carbonated beverage [Ingredient] [Ratio]
Soybean protein thermolysin degradation product (average molecular weight 1500) 1.0
Sodium hyaluronate 0.1
Swallow's nest extract 0.1
Pectin 0.3
Vitamin B1 0.01
Vitamin C 1.0
Fructose glucose liquid sugar 12.0
Citric acid 0.5
Perfume
Purified water remaining
100.0% by weight

Claims (5)

(A) one or more selected from the group consisting of hyaluronic acid, ester derivatives of hyaluronic acid, ether derivatives of hyaluronic acid, amide derivatives of hyaluronic acid, amine derivatives of hyaluronic acid , and salts thereof;
(B) Thermolysin degradation product having an average molecular weight of soybean protein of 300 to 10000 (wherein the thermolysin is at least one large side chain selected from the group consisting of isoleucine, leucine, valine, phenylalanine, methionine and alanine) It is a protease that cleaves peptide bonds on the amino group side of hydrophobic amino acid residues)
A composition containing   2. The composition of claim 1, which can be used to promote fibroblast proliferation. The composition according to claim 1 or 2, wherein the component (A) contains the component (B) in a proportion of 0.02 to 20000 parts by weight with respect to 1 part by weight. (A) The composition in any one of Claims 1-3 whose compounding quantity of a component is 0.0005 to 5 weight% with respect to the whole composition. (B) The composition in any one of Claims 1-4 whose compounding quantity of a component is 0.1 to 10 weight% with respect to the whole composition.