JP5783484B2 - Skin collagen production promoter - Google Patents

Description

  The present invention relates to a skin collagen production promoter, a food and drink for promoting skin collagen production, and a cosmetic for promoting skin collagen production, which are useful for preventing rough skin, wrinkles, a decrease in elasticity, and the like. More specifically, the present invention relates to a skin collagen production promoter containing a milk protein fraction and / or a degradation product of the milk protein fraction as an active ingredient.

  In recent years, research on the mechanism of the skin has progressed, and macroscopically, the effects of sunlight (ultraviolet rays), drying, oxidation, etc. are complicated in addition to the deterioration of metabolism due to aging as a cause of skin dryness and rough skin. Has been confirmed to be involved. It has been clarified that collagen fibers, which are the main matrix components of the dermis, are significantly reduced by the action of these factors. When the tension retention mechanism such as skin elasticity and elasticity held by the collagen fibers is broken by the action of ultraviolet rays, the skin becomes wrinkled and sagging. Collagen can also retain moisture in its molecules, thereby helping to keep the skin moist, and when the collagen is destroyed by external factors, the skin becomes dry. And it becomes rough. From the above, it is possible to prevent skin wrinkles and sagging by promoting the biosynthesis of collagen, which is one of the main components of the dermis layer, and there is no problem in terms of safety. It was desired.

  Examples of substances having a skin collagen promoting action include a basic protein fraction present in milk or a basic peptide fraction obtained by degrading the basic protein fraction with a proteolytic enzyme (Patent Document 1), lactoferrin Alternatively, a lactoferrin degradation product obtained by degrading lactoferrin with a proteolytic enzyme (Patent Document 2), lactoperoxidase or a lactoperoxidase degradation product obtained by degrading lactoperoxidase with a proteolytic enzyme (Patent Document 3), angio An angiogenin degradation product obtained by degrading genin or angiogenin with a proteolytic enzyme (Patent Document 4) is already known.

JP 2003-144095 A JP 2004-331564 A JP 2004-331565 A JP-A-2004-331566

  The present invention provides a new skin collagen production promoter that promotes the biosynthesis of skin collagen that can be used as a food material that can prevent dryness, rough skin, wrinkles, and talmi, and that is safe in terms of safety. The issue is to provide. Moreover, this invention makes it a subject to provide the food / beverage products or cosmetics which mix | blended the skin collagen production promoter.

  In order to solve these problems, the inventors of the present invention have been diligently searching for substances that have a skin collagen production promoting action widely contained in food materials. A fraction and / or a degradation product of the milk protein fraction was found and the present invention was completed.

That is, the present invention includes the following aspects.
(1) A skin collagen production promoter comprising a milk protein fraction having the following characteristics (a) to (c) as an active ingredient.
(A) It is derived from milk.
(B) A fraction containing a protein having a molecular weight in the range of 12,000 to 16,000 daltons by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE).
(C) The basic amino acid is contained in the constituent amino acid composition in an amount of 18 to 20% by weight, and the basic amino acid / acidic amino acid ratio is in the range of 0.7 to 0.9.
(2) A skin collagen production promoter comprising a degradation product of a milk protein fraction having the characteristics (a) to (c) as an active ingredient.
(3) A food / drink product for promoting skin collagen production, comprising the milk protein fraction according to (1) or (2) or a degradation product of the milk protein fraction.
(4) A skin collagen production promoting cosmetic comprising the milk protein fraction according to (1) or (2) or a degradation product of the milk protein fraction.
(5) Production of skin collagen by orally ingesting a milk protein fraction having the following characteristics (a) to (c) by 10 μg or more per day, or by applying to 0.001 to 2% by weight Promotion method.
(A) It is derived from milk.
(B) A fraction containing a protein having a molecular weight in the range of 12,000 to 16,000 daltons by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE).
(C) The basic amino acid is contained in the constituent amino acid composition in an amount of 18 to 20% by weight, and the basic amino acid / acidic amino acid ratio is in the range of 0.7 to 0.9.
(6) Skin collagen obtained by orally ingesting the degradation product of the milk protein fraction having the characteristics (a) to (c) above by 10 μg or more per day or by applying to 0.001 to 2% by weight. Production promotion method.

  Since the skin collagen production promoter of the present invention promotes collagen biosynthesis in the skin by oral administration or application, it is useful for preventing dryness of the skin, rough skin, wrinkles, reduced elasticity, and the like.

  The skin collagen production promoter of the present invention is (a) derived from milk, (b) by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis, and having a molecular weight in the range of 12,000 to 16,000 daltons. A fraction containing protein; (c) a composition containing amino acids containing 18 to 20% by weight of basic amino acids, and a basic amino acid / acidic amino acid ratio in the range of 0.7 to 0.9; The active ingredient is a milk protein fraction that satisfies all three conditions, or a degradation product of the milk protein fraction. Such a milk protein fraction is obtained by, for example, bringing milk materials such as skim milk and whey into contact with a cation exchange resin, washing with a 0.3 M salt solution, and then adding 0. It can be obtained by eluting with a 6M salt eluate. As salts, potassium salt, ammonia salt, phosphate, acetate, carbonate, etc. can be used in addition to sodium chloride, the ionic strength of the washing solution is 0.3 to 0.4, and the ionic strength of the elution solution Can be appropriately adjusted to 0.55 to 0.65 to obtain the milk protein fraction of the present invention. Further, this elution fraction can be collected, desalted and concentrated by reverse osmosis (RO) membrane, electrodialysis (ED) method or the like, and dried if necessary. Examples of the reverse osmosis (RO) membrane include Desal-3 (manufactured by Desalination), HR-95 (manufactured by Dow Danmark), NTR-729HF (manufactured by Nitto Denko), and the like. Examples of the electrodialysis (ED) device include electrodialysis devices manufactured by Yuasa Ionics and Nippon Ruisui.

  Moreover, as a method for obtaining a protein fraction derived from milk, after bringing milk or a milk-derived raw material into contact with a cation exchanger, the basic protein fraction adsorbed on the cation exchanger exceeds pH 5, A method of elution with an eluent with an ionic strength exceeding 0.5 (Japanese Patent Laid-Open No. 5-202098), a method of using an alginate gel (Japanese Patent Laid-Open No. 61-246198), and milk using inorganic porous particles A method for obtaining from milk (Japanese Patent Laid-Open No. 1-86839), a method for obtaining from milk using a sulfated ester compound (Japanese Patent Laid-Open No. 63-255300), etc. are known. Can use the milk protein fraction obtained by such a method. In addition, the milk protein fraction collected in this way can be used after being pulverized by freeze-drying or the like.

  The milk protein fraction having a skin collagen production promoting action used in the present invention contains 18-20% by weight of basic amino acids in its constituent amino acid composition, and the basic amino acid / acidic amino acid ratio is 0.7-0. The range is 9. Outside this range, the effects of the present invention cannot be exhibited. The product of the present invention is a mixture of various proteins having a molecular weight of 12,000 to 16,000 daltons and an isoelectric point of 10 or more.

  The degradation product of the milk protein fraction has the same amino acid composition as that of the milk protein fraction. For example, a protease such as pepsin, trypsin or chymotrypsin acts on the milk protein fraction obtained by the above method. Furthermore, if necessary, a degradation product having an average molecular weight of 4,000 or less can be obtained by acting a proteolytic enzyme such as pancreatin. The degradation product of the milk protein fraction can also be used after being pulverized by freeze drying or the like.

  As milk or a milk-derived raw material that serves as a source of the milk protein fraction that is the active ingredient of the skin collagen production promoter of the present invention, milk such as cow's milk, human milk, goat milk, and sheep milk can be used. These milks can be used as they are, or reduced milk, skim milk, whey and the like of these milks can be used.

  The skin collagen production promoter of the present invention exhibits an effect of promoting skin collagen production by oral administration or application. When orally administering the skin collagen production promoter of the present invention, the active ingredient milk protein fraction or the degradation product of the milk protein fraction can be used as it is. It can also be formulated into a medicine, tablet, capsule, drink or the like. In the present invention, oral preparations such as powders, granules, tablets and capsules are formulated by conventional methods using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts. It becomes. In this type of preparation, in addition to the above-mentioned excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, colorants, fragrances and the like can be appropriately used. More specifically, examples of the binder include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, sodium carboxymethylcellulose, methylcellulose, crystalline cellulose, ethylcellulose, and polyvinylpyrrolidone. Examples of the disintegrant include starch, hydroxypropyl starch, carboxymethylcellulose, sodium carboxymethylcellulose, crosslinked sodium carboxymethylcellulose, and crystalline cellulose. As surfactant, soybean lecithin, sucrose fatty acid ester, etc., as lubricant, talc, wax, sucrose fatty acid ester, hydrogenated vegetable oil, etc., as fluidity promoter, anhydrous silicic acid, dry aluminum hydroxide, Examples include magnesium silicate.

  Furthermore, it is also possible to mix these milk protein fractions or degradation products of the milk protein fractions as they are or into preparations, and then add them to nutrients, foods and drinks, and the like. Moreover, if a milk protein fraction or a degradation product of a milk protein fraction is blended together with components that are conventionally considered to have an effective action for collagen production such as vitamin C, further skin collagen production promoting action can be expected. . In addition, since the milk protein fraction or the degradation product of the milk protein fraction is relatively stable to heat, it can be sterilized by heating under the conditions usually used.

  When applying the skin collagen production promoter of the present invention, it is prepared into various dosage forms such as a liquid agent, a solid agent, a semi-solid agent, etc., by blending with commonly used known components according to the purpose of use. Preferred compositions include ointments, gels, creams, sprays, patches, lotions, powders and the like. For example, the skin collagen production promoter of the present invention is a hydrocarbon such as petrolatum, higher fatty acid lower alkyl esters such as stearyl alcohol, isopropyl myristate, animal fats such as lanolin, polyhydric alcohols such as glycerin, glycerin fatty acid esters, mono Cosmetics for promoting skin collagen production by mixing with surfactants such as stearic acid and polyethylene glycol, inorganic salts, waxes, resins, water and, if necessary, preservatives such as methyl paraoxybenzoate and butyl paraoxybenzoate And can produce medicines.

  The effective amount of the skin collagen production promoter of the present invention by oral administration is appropriately defined by the formulation form, administration method, purpose of use, and age, weight, and medical condition of the patient to which it is applied, but is not constant. According to the results of animal experiments, it was found that it was necessary to ingest 10 μg or more of the milk protein fraction or the degradation product of the milk protein fraction per kg body weight of the rat in order to show the skin collagen production promoting action. Therefore, according to the extrapolation method, the effect can be expected by ingesting a milk protein fraction or a degradation product of milk protein fraction of 10 μg or more per adult per day. What is necessary is just to mix | blend or administer as a pharmaceutical. The administration can be divided into several times a day as necessary.

  The effective amount by application of the skin collagen production promoter of the present invention varies depending on the dosage form, but is preferably from 0.001 to 2% by weight based on the total amount of the composition to be applied. What is necessary is just to mix | blend the decomposition product of a milk protein fraction. However, what is diluted at the time of use like a bath agent can increase a compounding quantity further.

  Hereinafter, the present invention will be described in detail with reference to reference examples, examples and test examples. However, these are merely examples of the present invention, and the present invention is not limited thereto. .

[Reference Example 1]
By the following method (see Japanese Patent Application Laid-Open No. 2003-144095), a milk protein fraction that is recognized to promote skin collagen production was prepared. A 10 cm diameter column packed with 0.5 liter of a cation exchange resin sulfonated chitopearl (Fujibo) was thoroughly washed with deionized water. After 50 liters of non-sterilized skim milk was passed through the column at a flow rate of 100 ml / min, the column was thoroughly washed with deionized water, and 0.05 M phosphate buffer (pH 7.0) containing 0.95 M sodium chloride. The protein adsorbed on the resin was eluted by passing 2.5 liters. The eluate was desalted and concentrated by reverse osmosis (RO) membrane treatment, and then freeze-dried to obtain a powdered milk protein fraction. This operation was repeated twice to obtain 104 g of protein fraction. The isoelectric point of this protein fraction was 7.0 to 8.5, and the basic amino acid contained in this protein fraction was 17.8%.

  A 10 cm diameter column packed with 0.5 liter of a cation exchange resin sulfonated chitopearl (Fujibo) was thoroughly washed with deionized water. After 50 liters of non-sterilized skim milk was passed through this column at a flow rate of 100 ml / min, the column was thoroughly washed with 0.05 M phosphate buffer (pH 7.0) containing 0.3 M sodium chloride, and then 0.55 M chloride. The protein adsorbed on the resin was eluted by passing 2.5 liters of 0.05M phosphate buffer (pH 7.0) containing sodium. The eluate was desalted and concentrated by reverse osmosis (RO) membrane treatment, and then freeze-dried to obtain a powdered milk protein fraction. This operation was repeated 5 times to obtain 37.4 g of a milk protein fraction. This fraction contains a protein having a molecular weight of 12,000 to 16,000 daltons and has an isoelectric point of 10 or more. Among the constituent amino acids contained in this protein fraction, the basic amino acid is 18 to 20%. there were. The basic amino acid / acidic amino acid ratio was 0.7 to 0.9. The obtained milk protein fraction can be used as it is as the skin collagen production promoter of the present invention.

  A 10 cm diameter column packed with 0.5 liter of a cation exchange resin sulfonated chitopearl (Fujibo) was thoroughly washed with deionized water. After 50 liters of non-sterilized skim milk was passed through the column at a flow rate of 100 ml / min, the column was sufficiently washed with 0.05 M phosphate buffer (pH 7.0) containing 0.4 M sodium chloride to obtain 0.65 M. The protein adsorbed on the resin was eluted by passing 2.5 liters of 0.05 M phosphate buffer (pH 7.0) containing sodium chloride. The eluate was desalted and concentrated by reverse osmosis (RO) membrane treatment, and then freeze-dried to obtain a powdered milk protein fraction. This operation was performed 3 times to obtain 22.8 g of milk protein fraction. This fraction contains a protein having a molecular weight of 12,000 to 16,000 daltons and has an isoelectric point of 10 or more. Among the constituent amino acids contained in this milk protein fraction, basic amino acids are 18 to 20%. Met. The basic amino acid / acidic amino acid ratio was 0.7 to 0.9. The obtained milk protein fraction can be used as it is as the skin collagen production promoter of the present invention.

  After 37.4 g of the milk protein fraction obtained in Example 1 was dissolved in 10 liters of distilled water, pepsin (manufactured by Kanto Chemical) was added to a concentration of 2%, and hydrolysis was performed while stirring at 37 ° C. for 1 hour. did. Subsequently, after neutralizing to pH 6.8 with a sodium hydroxide solution, 1% pancreatin (manufactured by Sigma) was added and reacted at 37 ° C. for 2 hours. After the reaction, the enzyme was inactivated by heat treatment at 80 ° C. for 10 minutes to obtain 36.2 g of a degradation product of the milk protein fraction. The degradation product of the obtained milk protein fraction can be used as it is as the skin collagen production promoter of the present invention.

  22.8 g of the milk protein fraction obtained in Example 2 was dissolved in 8 liters of distilled water, and then trypsin (manufactured by Kanto Chemical) was added to a concentration of 2%, followed by hydrolysis with stirring at 37 ° C. for 1 hour. did. Subsequently, after neutralizing to pH 6.6 with a sodium hydroxide solution, 1% pancreatin (manufactured by Sigma) was added and reacted at 37 ° C. for 2 hours. After the reaction, the enzyme was inactivated by heat treatment at 80 ° C. for 10 minutes to obtain 21.1 g of a degradation product of the milk protein fraction. The degradation product of the obtained milk protein fraction can be used as it is as the skin collagen production promoter of the present invention.

[Test Example 1]
Collagen production of the milk protein fraction obtained in Reference Example 1, the milk protein fraction obtained in Example 1, and the degradation product of the milk protein fraction obtained in Example 3 by animal experiments using rats The promoting effect was examined. Seven-week-old Wistar male rats were obtained in the physiological saline administration group (Group A), the group (Group B) in which the milk protein fraction obtained in Reference Example 1 was administered at 10 μg / kg of the rat body weight, and Reference Example 1. The group (group C) in which 100 μg of the obtained milk protein fraction was administered per kg of rat body weight, the group (group D) in which the milk protein fraction obtained in Example 1 was administered at 10 μg per kg of rat body weight were obtained in Example 1. Group (group E) in which 100 μg of the obtained milk protein fraction was administered per kg body weight of the rat (group E), group (F group) in which the degradation product of the milk protein fraction obtained in Example 3 was administered at 10 μg per kg of body weight of the rat, Example The degradation product of the milk protein fraction obtained in 3 was divided into 7 test groups (n = 6) in a group (group G) administered with 100 μg / kg of rat body weight, and each was administered once daily with a sonde for 10 weeks. It was. Regarding the amount of collagen in the skin, the rat dermis was treated according to the method of Nimni et al. (See Arch. Biochem. Biophys., Page 292, 1967), and then the amount of hydroxyproline contained in the soluble fraction was measured. Hydroxyproline is a special amino acid contained only in collagen and accounts for about 10% of all amino acids constituting collagen, so that the amount of collagen can be estimated (see Takashi Asano et al., Bio Industry, p. 12, 2001). The results are shown in Table 1.

  According to this, the amount of hydroxyproline in the soluble fraction after 10 weeks was significantly higher in the B group, the C group, the D group, the E group, the F group and the G group than in the A group. In addition, the milk protein fraction obtained in Example 1 and the degradation product of the milk protein fraction obtained in Example 3 were about twice as effective as the milk protein fraction obtained in Reference Example 1. I found out. This revealed that the milk protein fraction of the present invention or a degradation product of the milk protein fraction has a skin collagen production promoting action, and was shown to be useful as a skin collagen production promoter. It was also revealed that this skin collagen production promoting action was observed when a milk protein fraction or a degradation product of the milk protein fraction was administered at least 10 μg per kg body weight of the rat.

  The drink which mix | blended the skin collagen production promoter with the mixing | blending shown in Table 2 was manufactured by the conventional method. The flavor of the manufactured beverage was good, and the flavor did not deteriorate even after storage at room temperature for 1 year, and there was no problem such as precipitation.

  A dough having the composition shown in Table 3 was prepared and molded by a conventional method, and then baked to produce a biscuit containing a skin collagen production promoter.

  With the formulation shown in Table 4, a skin collagen production promoter was produced by a conventional method.

  Each component was mixed with the composition shown in Table 5, emulsified at an emulsification temperature of 85 ° C., and processed cheese containing a skin collagen production promoter was prepared.

[Test Example 2]
For the milk protein fraction obtained in Example 2 (Example Product 2) and the degradation product of the milk protein fraction obtained in Example 4 (Example Product 4), normal human fibroblast cell line [white female Skin collagen production promoting action was examined by an experiment using CCD45SK (ATCCRL 1506)] collected from the skin. Using a modified Eagle's medium (MEM, 10-101, manufactured by Dainippon Pharmaceutical Co., Ltd.) containing 10% by volume fetal bovine serum (hereinafter abbreviated as FBS), 4 × 10 ⁴ normal human fibroblast cell lines / well / 0 After seeding in a 24-well plate so as to be 4 ml and culturing at 37 ° C. under 5% carbon dioxide gas and saturated steam for 24 hours, the medium was replaced with a 0.6 volume% FBS-containing MEM medium. Then, the milk protein fraction obtained in Example 2 (Example Product 2) and the degradation product of the milk protein fraction obtained in Example 4 (Example Product 4) were added to each well in an amount of 0.1% by volume. (N = 6) and culturing for 24 hours, then adding β-aminopropionitrile to 50 μg / ml and tritium-L-proline to 1 μCi / ml, and further for 24 hours Culture was obtained by culturing. The collagen fraction was fractionated from the culture solution thus obtained according to the method of Webster et al. (See Analytical Biochemistry, page 220, 1979), and the radioactivity incorporated into the collagen fraction was measured. As a control, the same test was performed without adding the milk protein fraction and the degradation product of the milk protein fraction. The results are shown in Table 6.

  According to this, the milk protein fraction and the group to which the degradation product of the milk protein fraction was added were twice as much as the group to which the milk protein fraction and the degradation product of the milk protein fraction were not added (control). The above collagen production promoting ability was demonstrated. This reveals that the milk protein fraction and the degradation product of the milk protein fraction of the present invention have an action of acting on dermal fibroblasts and promoting collagen production, and are useful as a skin collagen production promoter. It was shown that there is.

  A lotion having the composition shown in Table 7 was produced by a conventional method.

  A cream having the composition shown in Table 8 was produced by a conventional method.

[Test Example 3]
Using the lotion obtained in Example 8 and the cream obtained in Example 9, an actual use test was conducted. As a comparative product, the same formulation as in Examples 8 and 9 was used except that the milk protein fraction and the degradation product of the milk protein fraction were excluded. Twenty adult girls with dry skin with sagging and fine wrinkles on the face, each with 10 people randomly divided into 2 groups (Groups A and B), and 20 girls with rough skin on the hands, 10 people randomly divided into 2 groups (Group C, D), 2g of the product of the present invention was applied to the face of Group A, 2g of the comparison product was applied to the face of Group B, and fingers of Group C 2 g of the product of the present invention and 2 g of the comparative product were applied to the fingers of group D twice a day for 10 days in the same manner as in normal use. Thereafter, the degree of improvement was investigated for dryness, rough skin, wrinkles and sagging. The results are shown in Table 9.

  From Table 9, it was clarified that the skin lotion of the product of the present invention is significantly improved in dry feeling, rough skin, etc., and excellent in promoting skin collagen production, compared to the skin lotion of the comparative product. . In addition, the cream of the present invention also has an improvement in dry feeling and a marked improvement in rough skin, as compared with the comparative cream, and has been found to have an effect of suppressing natural deterioration such as rough skin.

Claims (5)

A skin collagen production promoter comprising a milk protein fraction having the following characteristics (a) to (c) as an active ingredient (except for the form of food and drink).
(A) It is derived from milk.
(B) A fraction containing a protein having a molecular weight in the range of 12,000 to 16,000 daltons by electrophoresis with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE).
(C) The basic amino acid composition contains 18 to 20% by weight, and the basic amino acid / acidic amino acid ratio is in the range of 0.7 to 0.9. A skin collagen production promoter comprising a degradation product of a milk protein fraction having the following characteristics (a) to (c) as an active ingredient (except for the form of food and drink).
(A) It is derived from milk.
(B) A fraction containing a protein having a molecular weight in the range of 12,000 to 16,000 daltons by electrophoresis with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE).
(C) The basic amino acid composition contains 18 to 20% by weight, and the basic amino acid / acidic amino acid ratio is in the range of 0.7 to 0.9. A cosmetic for promoting skin collagen production, comprising the milk protein fraction according to claim 1 or a degradation product of the milk protein fraction. Non-therapeutic skin collagen obtained by orally ingesting a milk protein fraction having the following characteristics (a) to (c) in an amount of 10 μg or more per day or by application to 0.001 to 2% by weight: Production promotion method (except for the form of food and drink for oral consumption) .
(A) It is derived from milk.
(B) A fraction containing a protein having a molecular weight in the range of 12,000 to 16,000 daltons by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE).
(C) The basic amino acid is contained in the constituent amino acid composition in an amount of 18 to 20% by weight, and the basic amino acid / acidic amino acid ratio is in the range of 0.7 to 0.9. Non-therapeutic by orally ingesting 10 μg or more of the degradation product of the milk protein fraction having the following characteristics (a) to (c) or applying to 0.001 to 2% by weight per day A method for promoting the production of skin collagen (except for the form of food and drink for oral consumption) .
(A) It is derived from milk.
(B) A fraction containing a protein having a molecular weight in the range of 12,000 to 16,000 daltons by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE).
(C) The basic amino acid is contained in the constituent amino acid composition in an amount of 18 to 20% by weight, and the basic amino acid / acidic amino acid ratio is in the range of 0.7 to 0.9.