JP5955632B2 - 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 relates to a hyaluronic acid production promoter containing angiogenin and / or angiogenin degradation product obtained by degrading angiogenin with a proteolytic enzyme as an active ingredient.

  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, but in the case of rheumatoid arthritis, the concentration of hyaluronic acid in joint fluid decreases to about 1.2 mg / mL, and the viscosity of joint fluid is also reduced. Remarkably 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 the case of traumatic arthritis, osteoarthritis and osteoarthritis, the above-described improvement effect by joint injection therapy with 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, angiogenin or its angiogenin was converted into pepsin or Angiogenin degradation products obtained by degrading with proteolytic enzymes such as pancreatin promote the production of hyaluronic acid in the skin (including lips), joints, etc. in vivo (epidermal cells, dermal cells, etc.) The headline and the present invention were completed.

That is, the present invention includes the following aspects.
(1) Hyaluronic acid production promoter containing angiogenin and / or angiogenin degradation product as an active ingredient.
(2) The hyaluronic acid production promoter according to (1), wherein the angiogenin degradation product has a molecular weight of 500 or more and 8000 or less.
(3) The hyaluronic acid production promoter according to (1), wherein the angiogenin degradation product is obtained by degrading angiogenin with a proteolytic enzyme.
(4) The hyaluron according to (3), wherein the proteolytic enzyme is at least one selected from trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. Acid production promoter.
(5) A skin care agent containing angiogenin and / or an angiogenin degradation product as an active ingredient.
(6) The skin care agent according to (5), wherein the skin care is prevention and / or improvement of rough skin.
(7) A food and drink for promoting hyaluronic acid production containing the angiogenin and / or angiogenin degradation product according to any one of (1) to (4).
(8) A hyaluronic acid production promoting cosmetic comprising the angiogenin and / or angiogenin degradation product according to any one of (1) to (4).
(9) A method for improving skin quality by orally ingesting or applying angiogenin and / or angiogenin degradation products.
(10) A method for improving skin quality by orally ingesting 9.4 μg or more of angiogenin and / or angiogenin degradation products per day, or by applying a composition formulated to be 0.001 to 2% by weight .

  The present invention provides hyaluronic acid production promoters, hyaluronic acid production promoting foods and beverages and hyaluronic acid production promoting cosmetics containing angiogenin and / or angiogenin degradation products as active ingredients. 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 angiogenin and / or an angiogenin degradation product is an active ingredient. Angiogenin is an angiogenic factor, and its angiogenic activity has been suggested for use in diseases such as trauma, ulcers, organ transplantation, and circulatory dysfunction. JP-A-4-210618) and an example of use as a whitening agent are known (JP-A-6-312922). However, it is not known that angiogenin and its degradation products have a skin hyaluronic acid production promoting action and are useful as hyaluronic acid production promoters.

  The angiogenin of the present invention is prepared from mammalian milk. Examples of the source include milk such as cows, buffalos, humans, pigs, sheep, goats and horses. Angiogenin is a known substance and is commercially available. To produce it, a known method, for example, cation exchange chromatography as described in JP-A-2-296000 is used. A method for purifying angiogenin by combining gel filtration chromatography and gel filtration can be advantageously used industrially. In the present invention, angiogenin produced by genetic engineering techniques can also be used.

  An angiogenin degradation product is a peptide obtained by limiting and degrading the above angiogenin with a proteolytic enzyme such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease so that the molecular weight is 8,000 or less. It is possible to use a mixture. 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 orally administering the hyaluronic acid production promoter of the present invention, the active ingredient angiogenin and / or angiogenin degradation products can be used as they are, but according to conventional methods, powders, granules, tablets In addition, it can also be used in the form of 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 angiogenins and / or angiogenin degradation products can be blended in nutrients, foods and drinks, etc. as they are or after preparation. In addition, if angiogenin and / or angiogenin degradation products are added 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, Hyaluronic acid production promoting action can be expected. In addition, since angiogenin and / or angiogenin degradation products are relatively stable to heat, it is possible to heat sterilize raw materials containing angiogenin and / or angiogenin degradation products under conditions that are normally performed. It is.

  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 found that angiogenin and / or an angiogenin degradation product must be ingested in an amount of 9.4 μg or more per 1 kg body weight in order to show hyaluronic acid production promoting action. Therefore, according to the extrapolation method, since an effect can be expected by ingesting 9.4 μg or more of angiogenin and / or angiogenin degradation products per adult per day, it is formulated in foods and drinks to ensure this necessary amount. Or it may be administered as a medicine. 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 is preferably from 0.001 to 2% by weight based on the total amount of the composition to be applied, and angiogenin and / or What is necessary is just to mix | blend an angiogenin degradation product. 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 eluted with 0.02 M carbonate buffer (pH 7.0) containing 2.0 M sodium chloride. Then, the elution fraction containing angiogenin was adsorbed on an S-Sepharose FF column (Amersham Biosciences), sufficiently washed with deionized water, equilibrated with 10 mM phosphate buffer (pH 7.0), The adsorbed fraction was eluted with a linear gradient of 0 to 2.0 M sodium chloride, and the fraction containing angiogenin was collected. Then, the fraction was subjected to gel filtration chromatography using HiLoad 16/60 Superdex 75 pg (manufactured by Amersham Biosciences) to obtain 1.8 g of a fraction rich in angiogenin. In addition, the angiogenin content in the fraction rich in angiogenin obtained in this way is 10%, and can be used as it is as a hyaluronic acid production promoter.

  A column packed with 3,000 g of a cation exchange resin sulfonated chitopearl (Fuji Boseki) was thoroughly washed with deionized water, and then 100 L (pH 6.7) of unsterilized skim milk was passed through this column. did. The column was then washed thoroughly with deionized water and then eluted with a linear concentration gradient of 0.1-2.0 M sodium chloride. And the elution fraction containing angiogenin is fractionated by S-Sepharose cation exchange chromatography (manufactured by Amersham Biosciences), and the obtained angiogenin-containing fraction is heated at 90 ° C. for 10 minutes, and centrifuged. The precipitate was removed by separation. Further, this angiogenin-containing fraction was sequentially treated with Mono S cation exchange chromatography, Superose 12 gel filtration chromatography, hydroxyapatite chromatography and C4 reverse phase chromatography to obtain 55 mg of angiogenin (sample A). The angiogenin thus obtained has a purity of 99% and can be used as it is as a hyaluronic acid production promoter.

  Angiogenin (5.0 mg) obtained in Example 2 was suspended in 10 ml of water, pancreatin was added to a final concentration of 0.01%, 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) and deactivating an enzyme, it lyophilized | freeze-dried and 4.0 mg (samples B, C, and D) of angiogenin degradation products were obtained. The average molecular weight of the angiogenin degradation product thus obtained was about 3,000 for B, about 500 for C, and about 300 for D. Fractions B, C, and D can be used as hyaluronic acid production promoters as they are.

[Test Example 1]
About the sample A obtained in Example 2, and the samples B thru | or D obtained in Example 3, the hyaluronic acid production promotion effect was investigated by the animal experiment using a rat. A 7-week-old Wistar male rat was obtained in Example 1 with a physiological saline-administered group (control group), a group in which sample A obtained in Example 1 was administered at 5 μg per kg of body weight of the rat (Group A-1). Group (A-2 group) administered 50 μg of the obtained sample A per kg body weight of the rat, Group (B-1 to D-1 group) administered 5 μg of the samples B to D obtained in Example 2 per kg of the rat body weight Samples B to D obtained in Example 2 were divided into 9 test groups (n = 6) of groups (B-2 to D-2 groups) administered with 50 μg / kg of rat body weight, and each was sampled once a day with a sonde. Orally administered and reared for 10 weeks. 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 Table 1.

  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. From this, it was revealed that angiogenin and / or an angiogenin degradation product has a hyaluronic acid production promoting action, and it was shown to be useful as a hyaluronic acid production promoter. It was also revealed that this hyaluronic acid production promoting action was observed when angiogenin and / or an angiogenin degradation product was administered at a minimum of 5.0 μg / kg of rat body weight.

[Test Example 2]
Experiment using sample A obtained in Example 2 and samples B, C, and D obtained in Example 3 using a normal human fibroblast cell line [CCD45SK (ATCCRL 1506) collected from white female skin] The hyaluronic acid production promoting effect was examined by the above. 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, Sample A obtained in Example 2 and Samples B, C, and D obtained in Example 3 were added to each well so as to be 0.1% by volume (n = 6), and 72 hours Culture was obtained by culturing. 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 angiogenin or an angiogenin degradation product. The results are shown in Table 2.

  According to this, the group to which angiogenin and / or angiogenin degradation product was added increased hyaluronic acid production more than twice as much as the group to which angiogenin and / or angiogenin degradation product was not added (control). Showed the ability. This reveals that angiogenin and / or angiogenin degradation products act on dermal fibroblasts and promote hyaluronic acid production, indicating that they are useful as hyaluronic acid production promoters. It was.

  Beverages for promoting hyaluronic acid production with the formulation shown in Table 3 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 4 was produced by a conventional method, molded, and then baked to produce hyaluronic acid production promoting biscuits.

  Hyaluronic acid production promoters having the composition shown in Table 5 were produced by a conventional method.

  A lotion having the composition shown in Table 6 was produced by a conventional method.

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

[Test Example 3]
Using the lotion obtained in Example 7 and the cream obtained in Example 8, an actual use test was conducted. As a comparative product, the same composition as in Examples 7 and 8 was used except that angiogenin and / or angiogenin degradation products 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 8.

  From the results of Table 8, it was demonstrated that the lotion of the product of the present invention is significantly improved in dry feeling, rough skin and the like, and excellent in hyaluronic acid production promoting effect compared with the comparative lotion. 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 3 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 9.

  From the results shown in Table 9, it was demonstrated that the test food of the present invention was 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 (5)

  A hyaluronic acid production promoter containing angiogenin and / or an angiogenin degradation product as an active ingredient.    The hyaluronic acid production promoter according to claim 1, wherein the angiogenin degradation product has a molecular weight of 500 or more and 8000 or less.    The hyaluronic acid production promoter according to claim 1, wherein the angiogenin degradation product is obtained by degrading angiogenin with a proteolytic enzyme.   The hyaluronic acid production promotion according to claim 3, wherein the proteolytic enzyme is at least one selected from trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. Agent. A method for improving skin quality by promoting hyaluronic acid production by oral administration of angiogenin and / or angiogenin degradation products in an amount of 5.0 to 50 μg per day.