HK1187240A - Skin collagen production promoter - Google Patents

Description

Skin collagen production promoter

Technical Field

The present invention relates to a skin collagen production promoter, a skin collagen production promoting food or drink, and a skin collagen production promoting cosmetic useful for preventing skin deterioration such as skin roughness, wrinkles, and decreased elasticity. Specifically, the present invention relates to a skin collagen production promoter comprising, as an active ingredient, transforming growth factor beta (TGF-. beta.) and/or a TGF-. beta.degradation product obtained by degrading TGF-. beta.with a protease.

Background

Recent results in the study of the mechanism of skin deterioration have confirmed that, broadly speaking, dry feeling and skin roughness of the skin are caused not only by the slowing of aging metabolism but also complexly involving the action of factors such as sunlight (ultraviolet rays), dryness and oxidation. It was shown that due to the influence of these factors, collagen fibers, which are the most major matrix component of the dermis, are significantly reduced. If the mechanism of maintaining skin tension such as resilience and elasticity supported by collagen fibers is broken due to the action of ultraviolet rays or the like, wrinkles and sagging of the skin will increase. Since collagen can retain water in its molecules to help keep the skin moist, if collagen is damaged by external factors, the skin may be dry and rough. In view of the above, a skin collagen production promoter that prevents skin wrinkles and sagging without safety problems by promoting the biosynthesis of collagen, which is one of the main components of the dermal layer, is desired.

Transforming growth factor beta (TGF-beta) is a growth factor found in mammalian milk. The TGF-. beta.family includes five subtypes, which are proteins that form dimers of approximately 25kDa through disulfide bonds. The TGF- β family has functions of regulating promotion/inhibition of cell proliferation, biosynthesis, differentiation and apoptosis. It has been confirmed that the effect of inducing differentiation of animal cells is a potent effect of TGF-. beta.s (patent document 1). TGF-. beta.has also been reported as a growth factor for skin fibroblasts (non-patent document 1).

Reference list

Patent document

Patent document 1: japanese laid-open patent publication No. 2004-254674

Non-patent document

Non-patent document 1: J.Dermatol.Sci., Vol.24(Supple), p.70,2000

Disclosure of Invention

Problems to be solved by the invention

The problem to be solved by the present invention is to provide a skin collagen production promoter without safety problems. Another object of the present invention is to provide a food or beverage for promoting skin collagen production and a cosmetic for promoting skin collagen production, each containing the substance.

Means for solving the problems

As a result of intensive studies on substances contained in food materials and having an effect of promoting skin collagen production, the inventors of the present invention have found that TGF-beta or a TGF-beta decomposition product obtained by decomposing TGF-beta increases the collagen content in the skin, and thus have completed the present invention.

The present invention includes the following embodiments.

(1) A skin collagen production promoter contains TGF-beta and/or a TGF-beta decomposition product as an active ingredient.

(2) The skin collagen production promoter according to (1), wherein the TGF- β decomposition product is obtained by decomposing TGF- β with a protease.

(3) The agent for skin collagen production according to (2), wherein the protease is one or more selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin and V8 protease.

(4) The skin collagen production promoter according to any one of (1) to (3), wherein the TGF- β decomposition product has an average molecular weight of 500 or more and 8000 or less.

(5) A food or drink for promoting skin collagen production, which comprises the TGF- β and/or TGF- β decomposition product according to any one of (1) to (4).

(6) A cosmetic for promoting skin collagen production, which comprises the TGF- β and/or TGF- β decomposition product according to any one of (1) to (4).

(7) A method for improving the texture of a human body by orally administering or coating TGF-beta and/or a TGF-beta decomposition product to the human body.

(8) A method for improving the texture of a human body by orally administering a TGF-beta and/or a TGF-beta decomposition product at a dose of 10 [ mu ] g/day or more or coating the human body with 0.001 to 2 wt% of a TGF-beta and/or a TGF-beta decomposition product.

(9) A method for promoting skin collagen production, which comprises administering TGF-beta and/or a TGF-beta decomposition product to promote collagen production in skin.

(10) The method for promoting skin collagen production according to (9), wherein the TGF- β decomposition product is obtained by decomposing TGF- β with a protease.

(11) The method for promoting skin collagen production according to (10), wherein the protease is one or more selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin and V8 protease.

(12) The method for promoting skin collagen production according to any one of (9) to (11), wherein the TGF- β decomposition product has an average molecular weight of 500 or more and 8000 or less.

(13) A method for preventing or ameliorating skin deterioration, which comprises administering TGF-beta and/or a TGF-beta decomposition product.

(14) The method for preventing or ameliorating skin deterioration according to (13), wherein the TGF- β decomposition product is obtained by decomposing TGF- β with a protease.

(15) The method for preventing or improving skin deterioration according to (14), wherein the protease is one or more selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin and V8 protease.

(16) The method for preventing or ameliorating skin deterioration according to any one of (13) to (15), wherein the TGF- β decomposition product has an average molecular weight of 500 or more and 8000 or less

(17) An agent for preventing or ameliorating skin deterioration, which comprises TGF-beta and/or a TGF-beta decomposition product as an active ingredient.

(18) The agent for preventing or ameliorating skin deterioration according to (17), wherein the TGF- β decomposition product is obtained by decomposing TGF- β with a protease.

(19) The skin deterioration preventing or improving agent according to (18), wherein the protease is one or more selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin and V8 protease.

(20) The agent for preventing or ameliorating skin deterioration according to any one of (17) to (19), wherein the TGF- β decomposition product has an average molecular weight of 500 or more and 8000 or less.

(21) A food or drink for preventing or improving skin deterioration, which comprises the TGF- β and/or the TGF- β decomposition product according to any one of (17) to (20).

(22) A cosmetic for preventing or improving skin deterioration, which comprises the TGF- β and/or the TGF- β decomposition product according to any one of (17) to (20).

(23) A method for improving the texture of a skin purely for cosmetic purposes, which improves the texture by orally administering or coating TGF-beta and/or TGF-beta decomposition products.

(24) A method for improving the texture of a skin purely for cosmetic purposes, which comprises orally administering a TGF-beta and/or TGF-beta decomposition product in an amount of 10 [ mu ] g/day or more, or applying a TGF-beta and/or TGF-beta decomposition product in an amount of 0.001 to 2 wt%.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention provides a skin collagen production promoter, a food and drink for promoting skin collagen production, and a cosmetic for promoting skin collagen production, each containing TGF-beta and/or a TGF-beta decomposition product as an active ingredient. The skin collagen production promoter, the skin collagen production promoting food and drink, and the skin collagen production promoting cosmetic have the skin collagen production promoting effect, and are useful for preventing and treating skin wrinkles, sagging, dry feeling, and roughness.

Detailed Description

The skin collagen production promoter of the present invention is characterized by containing TGF-beta and/or a TGF-beta decomposition product obtained by decomposing TGF-beta with a protease as an active ingredient. Any source of TGF-. beta.s may be used in the present invention. For example, the gene sequences of human-derived TGF- β and bovine-derived TGF- β have been clarified and can be obtained by gene recombination techniques, and TGF- β produced by genetic engineering techniques can be used in the present invention. TGF-. beta.is present in considerable amounts in bovine colostrum and can also be collected from milk. TGF-. beta.s may also be collected from the cell culture medium, and TGF-. beta.s derived from the cells may also be used. For example, TGF- β of milk origin can be produced according to known methods (see, e.g., j. protein chem., vol.10, pp. 565-575, 1991), and TGF- β can be produced from raw milk, milk powder, skim milk, reconstituted milk or other processed milk by: heat treatment, salt treatment, ethanol treatment, various chromatographic treatments such as ion exchange chromatography and gel filtration chromatography, and ultrafiltration treatment used in combination as needed.

As the TGF- β decomposition product, a peptide mixture having an average molecular weight of 8000 or less obtained by the TGF- β limited proteolysis with a protease such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin and V8 protease is usable. The lower limit of the average molecular weight is preferably 500 or more. For example, the TGF- β decomposition product has an average molecular weight of 500 or more and 8000 or less, 1500 or more and 8000 or less, 2500 or more and 8000 or less, 3500 or more and 8000 or less, 4500 or more and 8000 or less, 5500 or more and 8000 or less, 6500 or more and 8000 or less, 7500 or less, 1500 or more and 7500 or less, 2500 or more and 7500 or less, 3500 or more and 7500 or less, 4500 or more and 7500 or less, 5500 or more and 7500 or less, 6500 or more and 7500 or less, 500 or more and 6500 or less, 1500 or more and 6500 or less, 2500 or more and 6500 or less, 3500 or more and 6500 or less, 4500 or more and 6500 or less, 5500 or more and 6500 or less, 500 or more and 5500 or less, 2500 or more and 5500 or less, 3500 or more and 5500 or less, 4500 or more and 4500 or less, 2500 or more and 4500 or less, 500 to 3500, 1500 to 3500, 2500 to 3500, 500 to 2500, 1500 to 2500, 500 to 1500, and 500 to 1500.

The skin collagen production promoter of the present invention exerts the effect of promoting skin collagen production by oral administration or application. When the skin collagen production-promoting agent of the present invention is orally administered, the active ingredient, i.e., TGF- β or TGF- β decomposition product, can be used as it is, or formulated in a conventional manner and used as an oral agent such as powder, granule, tablet, capsule and drink. For example, in the present invention, excipients such as starch, lactose, sucrose, mannitol, carboxymethyl cellulose, corn starch, and inorganic salts are used to formulate oral agents such as powders, granules, tablets, and capsules in a conventional manner. Such a dosage form can be obtained by using excipients and, as required, pharmaceutical additives such as binders, disintegrants, surfactants, lubricants, flowability improvers, colorants, perfumes, and the like. More specifically, the binder includes, for example, starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, sodium carboxymethyl cellulose, methyl cellulose, crystalline cellulose, ethyl cellulose, and polyvinyl pyrrolidone. Disintegrants include, for example, starch, hydroxypropyl starch, carboxymethyl cellulose, sodium carboxymethyl cellulose, croscarmellose sodium and crystalline cellulose. The surfactant comprises soybean lecithin, sucrose fatty acid ester, etc.; lubricants include talc, wax, sucrose fatty acid esters, hydrogenated vegetable oils, and the like; the fluidity improver includes anhydrous silicic acid, dried aluminum hydroxide, magnesium silicate, etc.

TGF-beta or TGF-beta decomposition products may be combined with nutrients, foods, drinks, etc. either directly or after being formulated. Further promotion of skin collagen production may be expected if TGF-beta or a TGF-beta decomposition product is included together with a component generally recognized as being effective for collagen production, such as vitamin C. Since TGF-beta or a TGF-beta decomposition product is relatively stable to heat, a raw material containing TGF-beta or a TGF-beta decomposition product can be sterilized by heating under ordinary use conditions.

When the skin collagen production-promoting agent of the present invention is used, the skin collagen production-promoting agent may be combined with commonly used known ingredients and prepared into various dosage forms such as a liquid, solid or semi-solid according to the purpose of use, and examples of preferred compositions include ointments (ointment), gels (gel), creams (cream), sprays (spray), patches (patch), lotions (lotion), powders (powder), and the like. For example, the skin collagen production promoter in the present invention may be mixed with a hydrocarbon such as vaseline, stearyl alcohol, a higher fatty acid lower alkyl ester such as isopropyl myristate, an animal fat such as lanolin, a polyhydric alcohol such as glycerin, a glycerin fatty acid ester, monostearate, a surfactant such as polyethylene glycol, an inorganic salt, a wax, a resin, water, and a preservative such as methyl paraben and butyl paraben added as needed, to produce cosmetic and pharmaceutical preparations for promoting skin collagen production.

Although the orally effective dose of the skin collagen production-promoting agent of the present invention is not constant but is appropriately determined depending on the dosage form, administration mode, purpose of use and age, body weight and condition of a patient to which the agent is applied, it has been found as a result of animal experiments using rats that the skin collagen production-promoting effect is expected to be produced by ingestion of 10. mu.g or more of TGF- β and/or TGF- β decomposition product per 1kg of body weight of rats. Therefore, according to extrapolation, the effect is expected by taking in TGF- β and/or TGF- β decomposition product at 10 μ g/day or more per adult, and therefore, TGF- β and/or TGF- β decomposition product may be administered in combination with food and drink or as a drug to ensure the necessary amount. Administration may be in separate forms several times per day as desired.

Although the coating amount of the skin collagen production-promoting agent of the present invention varies depending on the dosage form, TGF-beta and/or TGF-beta decomposition product may be preferably controlled to 0.001 to 2% by weight based on the total dosage of the composition to be used. However, if the composition is diluted when used as a bath additive, the content may be further increased.

Examples

The present invention will be described in detail hereinafter with reference to examples and test examples; however, these examples merely illustrate embodiments of the present invention, and the present invention is not limited to these examples.

Example 1

The column filled with 3,000g S-sepharose was washed thoroughly with deionized water, after passing 10,000 liters of skim milk through the column, the column was washed thoroughly with deionized water, and then eluted with a linear gradient of 0.1 to 1.0M sodium chloride. The fractions eluted containing TGF-. beta.were re-fractionated by phenyl-S sepharose hydrophobic column chromatography. This fraction was further successively treated in an HPLC system with reverse phase chromatography using C4 and C8 and gel filtration chromatography to give 412mg of TGF-. beta.s (fraction A), and the TGF-. beta.s obtained in this manner can be used directly as a skin collagen production promoter.

Example 2

After suspending 25mg of fraction A obtained in example 1 in 100ml of water, pancreatin was added to a final concentration of 1% to conduct enzyme treatment at 37 ℃ for 5 minutes to 6 hours. After heat treatment at 90 ℃ for 5 min to inactivate the enzyme, 24mg of TGF-. beta.lysate (fractions B, C and D) was obtained by lyophilization. The average molecular weights of TGF- β decompositions B, C and D obtained in this way are about 8000, about 500 and about 300, respectively. Fractions B and C can be used directly as skin collagen production promoters.

[ test example 1]

The collagen production promoting effects of fraction a obtained in example 1 and fractions B to D obtained in example 2 were examined by an animal experiment using rats. Male rats of vista (Wistar) of 7 weeks old were divided into nine test groups (n =6) consisting of a group administered with physiological saline (control group), a group administered with 10 μ g of fraction a obtained in example 1 per kg of body weight of rat (group a-1), a group administered with 100 μ g of fraction a obtained in example 1 per kg of body weight of rat (group a-2), a group administered with 10 μ g of fractions B to D obtained in example 2 per kg of body weight of rat (group B-1 to D-1), and a group administered with 100 μ g of fractions B to D obtained in example 2 per kg of body weight of rat (group B-2 to D-2), and the rats were orally administered with a probe once a day for ten days each. Regarding the amount of collagen in the skin, the amount of hydroxyproline in the soluble fraction can be determined after treating the dermis of rats according to the method of Nimni et al (see arch, biochem, biophysis, p 292, 1967). Since hydroxyproline is a specific amino acid contained only in collagen and accounts for about 10% of all amino acids constituting collagen, the amount of collagen can be estimated (see Ryuji Asano et al, BioIndustry, p 12, 2001). The results are shown in Table 1.

[ Table 1]

Each value represents mean ± standard deviation (n = 6).

Indicates significant difference (p <0.05) compared to control group.

The results show that the amount of hydroxyproline in the soluble fraction was significantly higher after ten weeks in all the test groups compared to the control group. Therefore, TGF-beta and TGF-beta decomposition products having an average molecular weight of 500 to 8000 have a skin collagen production promoting effect and can be used as skin collagen production promoters. Skin collagen production promoting effects are observed when TGF-beta and TGF-beta decomposition products are administered to rats in an amount of at least 10. mu.g per kg body weight.

[ test example 2]

The collagen production promoting effect of fraction a obtained in example 1 and fraction B obtained in example 2 was determined by using a human fibroblast cell line (CCD45SK (ATCCRL1506) taken from the skin of a white human female). Normal human fibroblast cell lines were cultured at 4X 10 using Modified Eagle Medium (MEM) containing 10 vol% fetal bovine serum (hereinafter abbreviated as FBS) ("10-101" manufactured by Dainippon Pharmaceutical Co., Ltd.)⁴Cells/well/0.4 ml were seeded into 24-well plates and saturated with 5% CO at 37 ℃ under steam₂The cells were cultured for 24 hours and replaced with MEM medium containing 0.6 vol% FBS. The fraction a obtained in example 1 and the fraction B obtained in example 2 were added to each well (at a final concentration of 0.1 vol%) (n =6) and cultured for 24 hours, and then β -aminopropionitrile and tritiated L-proline (final concentrations of 50 μ g/ml and 1 μ Ci/ml, respectively) were added, and the cells were cultured for another 24 hours to obtain a culture solution. The collagen fraction was fractionated from the culture solution obtained in this manner according to the method of Webster et al (see Analytical Biochemistry, page 220, 1979), and the radioactivity absorbed in the collagen fraction was measured. The same experiment without addition of TGF-beta and TGF-beta decomposition products was performed as a control. The results are shown in Table 2.

TABLE 2

Each value represents mean ± standard deviation (n = 6).

Indicates significant difference (p <0.05) compared to control group.

The results showed that all the groups to which TGF-beta and TGF-beta decomposition products were added exhibited collagen production promoting ability twice or more as high as that of the group to which TGF-beta and TGF-beta decomposition products were not added (control). Therefore, TGF-beta and TGF-beta decomposition products have been shown to have an effect of promoting collagen production by skin fibroblasts and to be useful as skin collagen production promoters.

Example 3

A skin collagen production promoting drink having the composition shown in Table 3 was prepared by a conventional method. The produced beverage has good flavor, does not deteriorate even after being stored at room temperature for 1 year, and has no problems such as precipitation.

TABLE 3

Example 4

Dough (dough) having the composition shown in table 4 was prepared, shaped and baked according to a conventional method to produce a biscuit for promoting skin collagen production.

TABLE 4

Example 5

Skin collagen production promoters having the compositions of table 5 were prepared according to conventional methods.

TABLE 5

Example 6

Lotions containing the components of table 6 were prepared according to conventional methods.

TABLE 6

Example 7

A cream having the composition shown in table 7 was prepared according to a conventional method.

TABLE 7

[ test example 3]

The makeup water obtained in example 6 and the cream obtained in example 7 were used to conduct practical use tests. A comparative sample having the same composition as in example 6 or 7 was used except that TGF- β and a TGF- β degradation product were not added. 20 adult women with sagging facial skin, fine wrinkles and dry skin were randomly divided into 2 groups per group of 10 persons (groups E and F) and 20 women with rough hand skin were randomly divided into 2 groups per group of 10 persons (groups G and H), 2G of the lotion of the present invention was applied to the face of group E, 2G of the comparative lotion was applied to the face of group F, 2G of the cream of the present invention was applied to the fingers of group G, and 2G of the comparative cream was applied to the fingers of group H, twice daily in a similar manner for 10 days in the case of normal usage. The results are shown in Table 8.

TABLE 8

++: a significant improvement was observed 10 days after coating.

+: the improvement effect was observed 10 days after coating.

+ -: no improvement was observed 10 days after coating (no change compared to ten days ago).

As seen from table 8, a significant improvement effect was exhibited in the dry feeling and the skin roughness particularly in the groups E and G using the toning lotion of example 6 and the cream of example 7, as compared with the groups F and H using the toning lotion and the cream of the comparative product.

Claims (8)

1. A skin collagen production promoter contains TGF-beta and/or a TGF-beta decomposition product as an active ingredient.

2. The skin collagen production promoter according to claim 1, wherein the TGF- β decomposition product is obtained by decomposing TGF- β with a protease.

3. The skin collagen production promoter according to claim 2, wherein the protease is one or more selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin and V8 protease.

4. The skin collagen production promoter according to any one of claims 1 to 3, wherein the TGF- β decomposition product has an average molecular weight of 500 or more and 8000 or less.

5. A food or drink for promoting skin collagen production, which contains the TGF- β and/or TGF- β decomposition product according to any one of claims 1 to 4.

6. A cosmetic for promoting skin collagen production, which comprises the TGF- β and/or the TGF- β decomposition product according to any one of claims 1 to 4.

7. A method for improving the texture of a human body by orally administering or coating TGF-beta and/or a TGF-beta decomposition product to the human body.

8. A method for improving the texture of a human body by orally administering a TGF-beta and/or a TGF-beta decomposition product at a dose of 10 [ mu ] g/day or more or coating the human body with 0.001 to 2 wt% of a TGF-beta and/or a TGF-beta decomposition product.