JP5955630B2 - Hyaluronic acid production promoter - Google Patents

Description

  The present invention relates to hyaluronic acid production promoters, hyaluronic acid production promoting foods and drinks, and hyaluronic acid production promoting cosmetics that are useful for preventing rough skin, wrinkles, reduced elasticity, and the like. More specifically, the present invention promotes hyaluronic acid production using as an active ingredient a milk basic protein fraction and / or a milk basic protein fraction degradation product obtained by degrading a milk basic protein fraction with a proteolytic enzyme. It relates to the agent.

  In recent years, research on the mechanism of the skin has been promoted, and the causes of skin dryness and rough skin are not only due to attenuation of metabolism due to aging, but also the effects of ultraviolet rays such as sunlight, drying, oxidation, etc. are involved in a complex manner (Non-Patent Documents 1 and 2). These factors have been shown to significantly reduce hyaluronic acid, which is the main matrix component of the dermis (Non-patent Document 3). Hyaluronic acid can retain moisture in the molecule, thereby keeping the skin moist. However, when hyaluronic acid is destroyed by these actions and the moisture retention mechanism of the skin is impaired, the skin becomes dry and rough, and wrinkles and sagging are increased.

  Many cosmetics containing hyaluronic acid, collagen, etc. have been proposed as an agent for improving the moisture retention function in the skin, but these only exert a moisturizing effect on the skin surface and reduce the skin function. It could not be improved essentially. In addition, vitamins and herbal medicines are used as skin cell activators, but the current situation is that they have not yet reached the point of treating a decrease in skin function. From the above, by promoting the biosynthesis of hyaluronic acid, which is one of the main components of the dermis layer, it is possible to prevent wrinkles and sagging of the skin, and there is no problem in terms of safety. Was desired.

  On the other hand, hyaluronic acid in synovial fluid covers the surface of articular cartilage and helps smooth operation of joint functions. The concentration of hyaluronic acid in normal human joint fluid is about 2.3 mg / mL. For example, in the case of rheumatoid arthritis, the concentration of hyaluronic acid in joint fluid decreases to about 1.2 mg / mL, and at the same time, The viscosity is also significantly reduced (Non-Patent Document 4). In addition, it is known that hyaluronic acid content decreases in pyogenic arthritis and gouty arthritis as in the case of rheumatoid arthritis (see Non-Patent Document 5).

  In the above diseases, the amount of hyaluronic acid in the joint fluid is increased in order to improve the lubrication function, cover / protect articular cartilage, suppress pain, and improve the properties of pathological joint fluid. For example, when rheumatoid arthritis patients are given joint injection therapy with sodium hyaluronate, the above improvement has been observed (Non-patent Document 6). Similarly, in traumatic arthritis, osteoarthritis and osteoarthritis, the improvement effect by the joint injection therapy of hyaluronic acid has been reported (Non-patent Document 7). From the above, promotion of hyaluronic acid production is effective for the prevention and treatment of skin diseases such as rough skin, joint diseases such as rheumatoid arthritis, traumatic arthropathy, osteoarthritis, and osteoarthritis. However, treatment of the above diseases is long-lasting and requires a doctor's prescription. Therefore, a cream or a food or drink containing a hyaluronic acid production promoter that can be easily treated in daily life has been desired.

Archives of Dermatology, 138, 11, 1462, 2002 American Journal of Clinical Dermatology, Vol. 4, No. 11, pp. 771, 2003 The American Journal of Pathology, 171 (5), 1451, 2007 Arthritis Rheumatism, 10, 357, 1967 The Lancet, 351, 197, 1998 Rheumatology International, Vol. 22, No. 4, 2002 Canadian Medical Association Journal, 172, 8, 1039, 2005

  This invention makes it a subject to provide the hyaluronic acid production promoter which is satisfactory in terms of safety. Moreover, this invention makes it a subject to provide the hyaluronic acid production promotion food-drinks and hyaluronic acid production promotion cosmetics which mix | blended such a substance.

  In order to solve these problems, the present inventors diligently searched for substances that promote hyaluronic acid production that are widely contained in food materials. As a result, the basic protein fraction derived from milk or Degradation product of the basic protein fraction obtained by degrading the basic protein fraction promotes the production of hyaluronic acid in the skin (including lips), joints, etc. in vivo (epidermal cells, dermal cells, etc.) As a result, the present invention has been completed.

That is, the present invention includes the following aspects.
(1) A hyaluronic acid production promoter containing a milk basic protein fraction and / or a milk basic protein fraction degradation product as an active ingredient.
(2) The hyaluronic acid production promoter according to (1), wherein the milk basic protein fraction is a fraction containing 15% by weight or more of basic amino acids in its amino acid composition.
(3) The basic protein fraction derived from milk brings milk or a milk-derived raw material into contact with a cation exchange resin to adsorb basic protein, and the fraction adsorbed on this resin has a salt concentration of 0.1M to 1M. The hyaluronic acid production promoter according to (1), which is a fraction obtained by elution with an eluate.
(4) The degradation product of the milk basic protein fraction is obtained by decomposing the milk basic protein fraction according to any one of (1) to (3) with a proteolytic enzyme. The hyaluronic acid production promoter according to (1).
(5) The hyaluron according to (4), wherein the proteolytic enzyme is at least one selected from trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. Acid production promoter.
(6) The hyaluronic acid production promoter according to any one of (1) to (4), wherein the milk basic protein fraction degradation product has a molecular weight of 500 or more and 8000 or less.
(7) A skin care agent comprising a milk basic protein fraction and / or a milk basic protein fraction degradation product as an active ingredient.
(8) The skin care agent according to (7), wherein the skin care is prevention and / or improvement of rough skin.
(9) A hyaluronic acid production promoting food or drink comprising the milk basic protein fraction and / or the milk basic protein fraction degradation product according to any one of (1) to (6).
(10) A hyaluronic acid production promoting cosmetic comprising the milk basic protein fraction and / or the milk basic protein fraction degradation product according to any one of (1) to (6).
(11) A method for improving skin quality by orally ingesting or applying a milk basic protein fraction and / or a milk basic protein fraction degradation product.
(12) Skin quality by orally ingesting 10 μg or more of milk basic protein fraction and / or milk basic protein fraction degradation product or applying a composition containing 0.001 to 2% by weight per day How to improve.

  According to the present invention, a hyaluronic acid production promoter, a hyaluronic acid production promoting food and drink, and a hyaluronic acid production promoting cosmetic comprising a milk-derived basic protein fraction and / or a milk-derived basic protein fraction degradation product as active ingredients Fees are provided. The hyaluronic acid production promoter, the hyaluronic acid production promoting food and drink and the hyaluronic acid production promoting cosmetic of the present invention have an action of promoting hyaluronic acid production.

  The hyaluronic acid production promoter of the present invention is characterized in that a basic protein fraction derived from milk and / or a degradation product of basic protein fraction derived from milk is used as an active ingredient.

The milk basic protein fraction of the present invention can be used from any origin. For example, this basic protein fraction derived from milk has the following properties.
1) According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it consists of several proteins having a molecular weight in the range of 3,000-80,000.
2) 95% by weight or more is protein and contains a small amount of other fat and ash.
3) Protein mainly consists of lactoferrin and lactoperoxidase.
4) The amino acid composition of the protein contains 15% by weight or more of basic amino acids such as lysine, histidine and arginine.

  Such a basic protein fraction is obtained by, for example, bringing a milk material such as skim milk or whey into contact with a cation exchange resin to adsorb the basic protein, and the basic protein fraction adsorbed on the resin is reduced to 0.00. Elution with an eluate having a salt concentration of 1M to 1M, collecting this elution fraction, desalting and concentrating with a reverse osmosis (RO) membrane, electrodialysis (ED) method, etc., and drying if necessary be able to. Examples of the source include milk such as cows, buffalos, humans, pigs, sheep, goats and horses.

  As a method of obtaining a milk-derived basic protein fraction, milk or a milk-derived raw material is brought into contact with a cation exchanger to adsorb a basic protein, and then the basic protein adsorbed on the cation exchanger is used. A method for obtaining a protein fraction by elution with an eluent exceeding pH 5 and an ionic strength exceeding 0.5 (Japanese Patent Laid-Open No. 5-202098), a method using alginate gel (Japanese Patent Laid-Open No. 61-246198) ), A method of obtaining from whey using inorganic porous particles (Japanese Patent Laid-Open No. 1-86839), a method of obtaining from milk using a sulfated ester compound (Japanese Patent Laid-Open No. 63-255300), and the like. In the present invention, the basic protein fraction obtained by such a method can be used.

  Milk-derived basic protein fraction degradation product and lactoferrin degradation product are obtained by converting milk-derived basic protein fraction and lactoferrin into trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, V8 protease, etc. It is possible to use a peptide mixture that has been limitedly decomposed to an enzyme with a molecular weight of 8,000 or less. However, the lower limit of the molecular weight is preferably 500 or more.

  The hyaluronic acid production promoter of the present invention exhibits hyaluronic acid production promoting effect by oral administration or application. When the hyaluronic acid production promoter of the present invention is orally administered, the basic protein fraction derived from milk or the degradation product of basic protein fraction derived from milk as an active ingredient can be used as it is. According to the method, it can be formulated into powders, granules, tablets, capsules, drinks and 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, 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, these basic protein fractions derived from milk and the basic protein fractions derived from milk can be used as they are or after preparation, and then blended with nutrients, foods and drinks, and the like. In addition, the basic protein fraction derived from milk and the basic protein fraction derived from milk together with components that have been considered to have an effective action for hyaluronic acid production, such as N-acetylglucosamine and N-methyl-L-serine. If a decomposed product is blended, further hyaluronic acid production promoting action can be expected. Since the milk-derived basic protein fraction or the milk-derived basic protein fraction degradation product is relatively stable to heat, the milk-derived basic protein fraction or the milk-derived basic protein fraction It is also possible to heat sterilize the raw material containing the decomposed product under conditions that are normally performed.

  When applying the hyaluronic acid production promoter of the present invention, various dosage forms such as liquids, solids, semisolids, etc. are prepared by blending with commonly known components according to the purpose of use. Preferred compositions include ointments, gels, creams, sprays, patches, lotions, powders and the like. For example, the hyaluronic acid 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 hyaluronic acid 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 hyaluronic acid 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. According to the results of animal experiments, it was necessary to ingest at least 10 μg of milk-derived basic protein fraction and / or milk-derived basic protein fraction per kg body weight of the rat in order to show hyaluronic acid production promoting action. It turns out that there is. Therefore, according to the extrapolation method, an effect can be expected by usually ingesting 10 μg or more of a milk-derived basic protein fraction and / or a milk-derived basic protein fraction degradation product per day per adult. What is necessary is just to mix | blend with food-drinks so that a required amount can be ensured, or just to administer as a pharmaceutical. The administration can be divided into several times a day as necessary.

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

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

  A column (5 cm in diameter x 30 cm in height) packed with 400 g of a cation exchange resin sulfonated chitopearl (Fujibo Co., Ltd.) was thoroughly washed with deionized water, and then 40 liters of unsterilized skim milk (pH 6) was added to this column. 7) was passed at a flow rate of 25 ml / min. After passing through the column, the column was thoroughly washed with deionized water, and the basic protein fraction adsorbed on the resin was eluted with a 0.02 M carbonate buffer solution (pH 7.0) containing 0.98 M sodium chloride. This eluate was desalted with a reverse osmosis (RO) membrane, concentrated, and then freeze-dried to obtain 21 g of a powdered basic protein fraction (Example product A). When the obtained milk-derived basic protein fraction was measured by sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight was distributed in the range of 3,000-80,000, and the component composition Was as shown in Table 1. Table 2 shows the results of analyzing the amino acid composition with an amino acid analyzer (L-8500 type, manufactured by Hitachi, Ltd.) after hydrolysis with 6N hydrochloric acid at 110 ° C. for 24 hours. Furthermore, when the protein composition was analyzed by the ELISA method, as shown in Table 3, 40% or more of lactoferrin and lactoperoxidase were contained. The milk-derived basic protein fraction thus obtained can be used as it is as a hyaluronic acid production promoter.

  A cheese whey sterilized by heating at 121 ° C. for 30 seconds after thoroughly washing a column (diameter 100 cm × height 10 cm) packed with 30 kg of a cation exchange resin SP Toyopearl (Tosoh Corporation) with deionized water 3 t (pH 6.2) was passed at a flow rate of 10 liters / min. After the pain solution, this column was thoroughly washed with deionized water, and the basic protein fraction adsorbed on the resin was eluted with 0.1 M citrate buffer (pH 5.7) containing 0.9 M sodium chloride. The eluate was desalted by electrodialysis (ED), concentrated, and lyophilized to obtain 183 g of a powdered milk-derived basic protein fraction (Example product B).

  25 mg of Example product A obtained in Example 1 was suspended in 100 ml of water, pancreatin was added to a final concentration of 1%, and enzyme treatment was performed at 37 ° C. for 5 minutes to 6 hours. And after heat-processing at 90 degreeC for 5 minute (s), the enzyme was inactivated, it lyophilized | freeze-dried and 24 mg (Example goods C, D, and E) of basic protein fraction degradation products derived from milk were obtained. In addition, the average molecular weight of the milk basic protein fraction degradation product thus obtained was about 8,000 for C, about 500 for D, and about 300 for E. Example products C, D, and E can be used as hyaluronic acid production promoters as they are.

[Reference Example 1]
(Purification of lactoferrin)
A column (5 cm in diameter x 30 cm in height) packed with 400 g of a cation exchange resin sulfonated chitopearl (Fujibo Co., Ltd.) was thoroughly washed with deionized water, and then 40 liters of unsterilized skim milk (pH 6) was added to this column. 7) was passed at a flow rate of 25 ml / min. After passing through the column, the column was thoroughly washed with deionized water and eluted with 0.02 M carbonate buffer (pH 7.0) containing 2.0 M sodium chloride. The eluted fraction containing lactoferrin is adsorbed on an S-Sepharose FF column (Amersham Biosciences), washed thoroughly with deionized water and equilibrated with 10 mM phosphate buffer (pH 7.0). The fraction adsorbed with a linear gradient of ˜2.0 M sodium chloride was eluted, and the fraction containing lactoferrin was collected. Then, the fraction was treated by gel filtration chromatography using HiLoad 16/60 Superdex 75 pg (manufactured by Amersham Biosciences) to obtain 8.0 g of lactoferrin (Comparative Example Product 1). The lactoferrin thus obtained had a purity of 96%.

[Test Example 1]
Example product A obtained in Example 1, Example product B obtained in Example 2, and Example products C to E obtained in Example 3 produced hyaluronic acid by animal experiments using rats. The promoting effect was examined. Moreover, the same test was implemented using the lactoferrin of the comparative example product 1 as a comparison object. 7-week-old Wistar male rats were treated with a physiological saline administration group (control group), Example product A obtained in Example 1 and Example product B obtained in Example 2 at 10 μg per kg body weight of the rat. Group (A-2, Group B-1) to be administered, Example product A obtained in Example 1 and Example product B obtained in Example 2 were administered at a dose of 100 μg / kg rat body weight (A-2) , B-2 group), a group (C-1 to E-1 group) in which 10 μg of the example products C to E obtained in Example 3 are administered per kg of rat body weight, and the example product obtained in Example 3. Group (C-2 to E-2 group) administered with 100 μg of C to E per kg body weight of rat, Group (F-1 group) administered with 10 μg of lactoferrin of Comparative Example Product 1 per kg of rat body weight, Group of Comparative Example Product 1 Lactoferrin is administered at 100 μg / kg rat body weight (F-2 group) of the 13 test group divided into (n = 6), were respectively housed orally administered to 10 weeks in one sonde day. Regarding the amount of hyaluronic acid in the skin, skin tissues (each 300 mg) collected immediately after sacrifice of the shaved rat on the day before the test were subjected to measurement. The skin tissue denatured by heating was subjected to protein degradation with actinase, and hyaluronic acid degraded with hyaluronidase was measured by HPLC. The results are shown in Tables 4 and 5.

  As a result, the amount of hyaluronic acid in the soluble fraction after 10 weeks was significantly higher in all test groups than in the control group. This reveals that the milk-derived basic protein fraction and the milk-derived basic protein fraction degradation product have a hyaluronic acid production-promoting action and are useful as hyaluronic acid production-promoting agents. It was done. Moreover, it became clear that it has the hyaluronic acid production promotion effect predominantly also compared with the F-1 group and F-2 group which administered lactoferrin. It has been clarified that this hyaluronic acid production promoting action is observed when a milk-derived basic protein fraction or a milk-derived basic protein fraction degradation product is administered at least 10 μg per kg body weight of the rat.

[Test Example 2]
About Example product A obtained in Example 1, Example product B obtained in Example 2, and Example products C, D, and E obtained in Example 3, normal human fibroblast cell line [white The hyaluronic acid production promoting action was examined by an experiment using CCD45SK (ATCCRL 1506)] collected from female skin. As a comparison object, the same test was performed using the lactoferrin of Comparative Example Product 1. 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, Example Product A obtained in Example 1, Example Product B obtained in Example 2, Example Product C, D, E obtained in Example 3, and lactoferrin of Comparative Example Product 1 It added to each well so that it might become 0.1 volume% (n = 6), and it culture | cultivated for 72 hours, and obtained the culture solution. The amount of hyaluronic acid (manufactured by Biotech Trading Partners) was measured from the culture solution thus obtained. As a control, the same test was performed without adding the milk-derived basic protein fraction and the milk-derived basic protein fraction degradation product. The results are shown in Table 5.

  According to this, milk-derived basic protein fraction and milk-derived basic protein fraction degradation products are added to milk-derived basic protein fraction and milk-derived basic protein fraction degradation products. The hyaluronic acid production promoting ability was more than twice as high as that of the non-treated group (control), which was higher than the group administered with lactoferrin. This reveals that the milk-derived basic protein fraction and the milk-derived basic protein fraction degradation product have an action of acting on dermal fibroblasts and promoting hyaluronic acid production. It has been shown to be useful as an accelerator.

  Beverages for promoting hyaluronic acid production with the formulation shown in Table 6 were produced by conventional methods. 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 7 was prepared by a conventional method, molded, and then baked to produce a hyaluronic acid production promoting biscuits.

  Hyaluronic acid production promoters having the composition shown in Table 8 were produced by a conventional method.

  A lotion having the composition shown in Table 9 was produced by a conventional method.

  Creams having the composition shown in Table 10 were produced by a conventional method.

[Test Example 3]
Using the lotion obtained in Example 9 and the cream obtained in Example 10, an actual use test was conducted. As comparative products, those having the same composition as in Examples 7 and 8 and those to which lactoferrin was added were used except that the milk-derived basic protein fraction and the milk-derived basic protein fraction degradation product were excluded. 20 adult women with dry skin where facial sagging and fine wrinkles are recognized, 10 people each randomly into 2 groups (Groups A and B), and 20 women with rough skin on the hands, respectively 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. The results are shown in Table 11.

  From the results shown in Table 11, it was demonstrated that the skin lotion of the present invention is significantly improved in dry feeling, rough skin, etc., and superior in hyaluronic acid production promoting effect as compared with the skin lotion of the comparative product. It was. 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.

[Test Example 4]
For 20 patients with mild pain due to osteoarthritis, the test diet shown in Example 6 was drunk once a day and a one-year clinical trial was conducted. Assess joint pain and function with visual analog scale (VAS) for pain and with the Western Ontario and McMaster Universities (WOMAC) index for pain, function and stiffness in arthritic joints It was. The results are shown in Table 12.

  From the results of Table 12, it was demonstrated that the test food of the present invention is significantly improved in joint pain and function and superior in hyaluronic acid production promoting effect as compared to the test food of the comparative product.

Claims (7)

Promotion of hyaluronic acid production with a milk basic protein fraction and / or a milk basic protein fraction degradation product mainly composed of lactoferrin and lactoperoxidase as an active ingredient and a dose of 10 μg to 0.1 mg per day Agent.   The hyaluronic acid production promoter according to claim 1, wherein the milk basic protein fraction is a fraction containing 15% by weight or more of basic amino acids in its amino acid composition.   The basic protein fraction derived from milk makes milk or a milk-derived raw material contact a cation exchange resin to adsorb basic protein, and the fraction adsorbed on this resin is an eluate having a salt concentration of 0.1M to 1M. The hyaluronic acid production promoter according to claim 1, which is a fraction obtained by elution.   The milk basic protein fraction degradation product is obtained by degrading the milk basic protein fraction according to any one of claims 1 to 3 with a proteolytic enzyme. The hyaluronic acid production promoter as described.   The hyaluronic acid production promotion according to claim 4, wherein the proteolytic enzyme is at least one selected from trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. Agent.   The hyaluronic acid production promoter according to any one of claims 1 to 4, wherein the milk basic protein fraction degradation product has a molecular weight of 500 or more and 8000 or less. Skin quality improvement method through the promotion of hyaluronic acid production by 10μg or 0.1mg following oral administration per day of the milk basic protein fraction and / or milk basic protein fraction degradation product.