WO2014203885A1

WO2014203885A1 - Collagen production promoter

Info

Publication number: WO2014203885A1
Application number: PCT/JP2014/066004
Authority: WO
Inventors: 高野　義彦; 晴彦加藤; 宏上野; 敏也小林
Original assignee: 雪印メグミルク株式会社
Priority date: 2013-06-17
Filing date: 2014-06-17
Publication date: 2014-12-24
Also published as: JP2015000862A; JP6259209B2

Abstract

In the present invention, a lipocalin family protein and/or a lipocalin family proteolytic product obtained by using a protease such as pepsin or pancreatin to decompose a lipocalin family protein is used as the active ingredient in a hyaluronic acid production promoter, a collagen production promoter, a food/beverage, and a collagen production promoting cosmetic. This lipocalin family protein and/or lipocalin family proteolytic product has the effect of increasing the amount of collagen.

Description

Collagen production promoter

The present invention relates to a collagen production promoter useful for preventing rough skin, wrinkles, reduced elasticity, reduced joint function, etc., a drink supplement for promoting collagen production, a food, and a cosmetic for promoting collagen production. More specifically, the present invention relates to a collagen production promoter comprising as an active ingredient a lipocalin family protein or a lipocalin family protein degradation product obtained by degrading the lipocalin family protein.

In recent years, much research has been conducted on the mechanism of the skin. Macroscopic causes of dryness and rough skin include a decrease in metabolism due to aging. In addition, it has been confirmed that actions such as sunlight (ultraviolet rays), drying, and oxidation are involved in complicated causes of dry skin and rough skin. It has been clarified that the action of these factors significantly reduces collagen fibers, which are the most major matrix components of the dermis. The tension retention mechanism such as the firmness and elasticity of the skin maintained by the collagen fibers is destroyed by the decrease of the collagen fibers, and the skin becomes wrinkled and sagging. Collagen also has a function of retaining moisture, and helps to keep the skin moist. When collagen is destroyed by external factors, the skin becomes dry and rough. From the above, it seems that wrinkles and sagging of the skin can be prevented by promoting the biosynthesis of collagen, which is one of the main components of the dermis layer. Therefore, a collagen production promoter has been desired.
As collagen production promoters, extracts from Euphorbiaceae Amla and Roman Chamomile are disclosed. (Patent Documents 1 and 2)

On the other hand, collagen in articular cartilage is useful for smooth operation of joint function. In the case of osteoarthritis (OA), the blood circulation around the joints deteriorates and the supply of oxygen decreases, thereby reducing the production of collagen and proteoglycans by chondrocytes, and dead chondrocytes stimulate the synovium and inflammation Cause pain in the joints. In addition, the release of cytokines during inflammation induces chondrocyte death and intensifies pain symptoms. Symptomatic treatment of OA includes administration of pain relievers and anti-inflammatory preparations, injection of high molecular hyaluronic acid (sodium hyaluronate) into the joint cavity, and the like. In recent years, approaches other than symptomatic treatment such as promotion of chondrocyte differentiation or suppression of chondrocyte hypertrophy, chondrocyte-mediated collagen and proteoglycan transcription, and synthesis promotion have begun to be taken.
From the above, promotion of collagen 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. Therefore, a supplement, cream or food or drink containing a collagen production promoter that can be easily taken in daily life has been desired.

Lipocalin 2 (LCN2) and β-lactoglobulin are proteins belonging to the lipocalin family having eight β-barrel structures.
Lipocalin 2 (LCN2), also called human neutrophil gelatinase-binding lipocalin (NGAL), has been identified as a protein that binds to a 92 kDa human neutrophil type IV collagenase. Lipocalin 2 secreted from the liver has an antibacterial action by selectively binding to diderophore-Fe3 + to inhibit bacterial iron utilization and growth. In addition, it has been reported that lipocalin 2 in plasma and blood can be used as a marker for inflammation and infection, and lipocalin 2 in urine can be used as a marker for malignant tumors such as breast cancer. Lipocalin 2 is known to be also contained in milk, but it has been reported that it is more contained in colostrum than in mature milk. However, there is no report about the function of lipocalin 2 in milk.
β-lactoglobulin is the major whey protein in milk. It has the ability to bind hydrophobic substances such as retinol, which is a useful physiologically active substance, and has excellent functional properties (emulsifying property, gelling property, foaming property).

JP2011-229111 JP 2010-180179 A

In the inventions disclosed in Patent Documents 1 and 2, because plant-derived raw materials are used, the cultivation of raw materials for collagen production promoters is limited to a specific region, and stable supply such as large fluctuations in supply due to the season There is a problem that it cannot be done. Therefore, the present invention provides a collagen production promoter that can be easily ingested in daily life, and a collagen production promotion supplement, food and drink, and a cosmetic for promoting collagen acid production, containing such substances. It is an issue to provide.

In order to solve these problems, the present inventors diligently searched for a substance exhibiting a skin collagen production promoting action, and found that the lipocalin family protein or a lipocalin family protein obtained by degrading the lipocalin family protein. The degradation product has been found to increase the amount of collagen production in the skin, and the present invention has been completed.
That is, the present invention includes the following aspects.
(1) A collagen production promoter comprising a lipocalin family and / or a lipocalin family degradation product as an active ingredient.
(2) The collagen production promoter according to (1), wherein the lipocalin family and / or lipocalin family degradation product is lipocalin 2 and / or lipocalin 2 degradation product.
(3) The collagen production promoter according to (1), wherein the lipocalin family and / or lipocalin family degradation product is β-lactoglobulin and / or a β-lactoglobulin degradation product.
(4) The collagen production promoter according to (2), wherein the lipocalin 2 degradation product has a molecular weight of 300 or more and 8000 or less.
(5) The collagen production promoter according to (2), wherein the lipocalin 2 degradation product is obtained by degrading lipocalin 2 with a proteolytic enzyme.
(6) The collagen production promoter according to (5), wherein the proteolytic enzyme is at least one selected from trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. (7) A supplement containing the collagen production promoter according to any one of claims 1 to 6.
(8) A food or drink comprising the collagen production promoter according to any one of claims 1 to 6.
(9) A cosmetic comprising the collagen production promoting agent according to any one of claims 1 to 6.

According to the present invention, a collagen production promoter having improved safety can be provided by using a lipocalin family protein and / or a lipocalin family protein degradation product as an active ingredient.

Shows the collagen production promoting effect of lipocalin 2.

The collagen production promoter of the present invention is characterized in that lipocalin 2 and / or lipocalin 2 degradation product obtained by degrading lipocalin 2 with a proteolytic enzyme is an active ingredient. The lipocalin 2 of the present invention can be used from any origin. For example, the lipocalin 2 derived from human and bovine has already been clarified in gene sequence and can be produced by gene recombination, but in the present invention, lipocalin 2 produced by genetic engineering techniques can also be used. In addition, cells derived from the cell culture medium can also be used. Lipocalin 2 is contained in bovine colostrum and may be recovered from milk. From raw milk, powdered milk, skim milk, reduced milk, etc., heat treatment, salting treatment, ethanol treatment, ion exchange chromatography can be used. It can also be obtained by various chromatographic treatments such as chromatography and gel filtration chromatography, ultrafiltration treatment and the like.

The lipocalin 2 degradation product is a peptide mixture obtained by limiting degradation of lipocalin 2 with a proteolytic enzyme such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease to a molecular weight of 8,000 or less. It is possible to use. However, the lower limit of the molecular weight is preferably 300 or more.

The collagen production promoter of the present invention exhibits an effect of promoting collagen production by oral administration or application. When the collagen production promoter of the present invention is orally administered, the active ingredient lipocalin 2 or lipocalin 2 degradation product can be used as it is, but in accordance with conventional methods, powders, granules, tablets, capsules In addition, it can be formulated into a 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 is possible to In this type of preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a colorant, a fragrance, and the like may be used as appropriate in addition to the excipient. 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, crystalline cellulose and the like. In addition, as surfactant, soybean lecithin, sucrose fatty acid ester, etc., as lubricant, talc, wax, sucrose fatty acid ester, hydrogenated vegetable oil, etc., as fluidity promoter, silicic anhydride, dry aluminum hydroxide, Examples include magnesium silicate. Furthermore, these lipocalin 2 and lipocalin 2 degradation products can be blended in nutrients, foods and drinks, etc. as they are or after preparation. Since lipocalin 2 or a lipocalin 2 degradation product is relatively stable to heat, it is possible to heat sterilize the raw material containing the lipocalin 2 or the lipocalin 2 degradation product under conditions that are usually performed.

When applying the collagen production promoter of the present invention, it can be prepared into various dosage forms such as liquids, solids and semi-solids by blending with commonly used known ingredients according to the purpose of use. Possible and preferred compositions include ointments, gels, creams, sprays, patches, lotions, powders and the like. For example, the collagen production promoter of the present invention is a hydrocarbon such as petrolatum, a higher fatty acid lower alkyl ester such as stearyl alcohol, isopropyl myristate, an animal fat such as lanolin, a polyhydric alcohol such as glycerin, a glycerin fatty acid ester, a monostearin Cosmetics and pharmaceuticals for promoting collagen production by mixing with acids, surfactants such as polyethylene glycol, inorganic salts, waxes, resins, water and, if necessary, preservatives such as methyl paraoxybenzoate and butyl paraoxybenzoate Can be manufactured.

The effective amount of the collagen production promoter of the present invention by oral administration is appropriately defined according to the formulation form, administration method, purpose of use, and age, weight, and medical condition of the patient to which it is applied, but rats are used. As a result of animal experiments, it has been clarified that lipocalin 2 and / or lipocalin 2 degradation products show a collagen production promoting effect when ingested at least 10 μg per kg of rat body weight. Therefore, according to the extrapolation method, ingestion of lipocalin 2 and / or lipocalin 2 degradation product of 10 μg or more per adult per day can be expected to promote collagen production. 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 collagen production promoter of the present invention varies depending on the dosage form, but is preferably lipocalin 2 and / or lipocalin so that it is preferably 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 2 decomposition products. 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 more detail with reference to examples and test examples. “%” In Examples and Test Examples means “% by weight” unless otherwise specified.
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 packed with 3,000 g of S Sepharose cake is thoroughly washed with deionized water, passed through 10,000 L of skimmed milk, washed thoroughly with deionized water, and then a straight line of 0.1 to 1.0 M sodium chloride. Elute with a gradient. Thereafter, the eluted fraction containing lipocalin 2 was fractionated again by phenyl S Sepharose hydrophobic column chromatography. Furthermore, this fraction was sequentially treated with C4 and C8 reverse phase chromatography and gel filtration chromatography using an HPLC system to obtain 2400 mg of lipocalin (fraction A). In addition, the lipocalin 2 obtained in this way can be used as a collagen production promoter as it is.

Fraction A 25 mg 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 deactivated, it lyophilized | freeze-dried and 24 mg (fractions B, C, D) of lipocalin 2 degradation products were obtained. The average molecular weight of the lipocalin 2 degradation product thus obtained was about 8,000 for B, about 500 for C, and about 300 for D. Further, 25 mg of the fraction A obtained in Example 1 was suspended in 100 ml of water, pepsin was added so that the final concentration was 0.01%, the pH was adjusted to 2 to 3, and then 12 ° C at 37 ° C. After carrying out enzyme treatment for a time and adjusting the pH to 8, trypsin was added so that the final concentration was 0.01%, and the enzyme treatment was carried out at 37 ° C. for 12 hours. And after heat-processing at 90 degreeC for 5 minute (s) and deactivating an enzyme, it lyophilized | freeze-dried and 24 mg (fraction E) of lipocalin 2 degradation products were obtained. Fractions B, C, D and E can be used as collagen production promoters as they are.

[Test Example 1]
About the sample A obtained in Example 1, and the samples B thru | or E obtained in Example 2, the collagen production promotion effect | action was investigated by the animal experiment using a rat. A 7-week-old Wistar male rat was obtained in the physiological saline-administered group (control group), the sample A obtained in Example 1 was administered in 10 μg per kg body weight of the rat (Group A-1), and in Example 1. The group in which 100 μg of the obtained sample A was administered per kg of rat body weight (Group A-2), and the samples B to D obtained in Example 2 were administered in the amount of 10 μg per kg of rat body weight (Groups B-1 to E-1) The samples B to D obtained in Example 2 were divided into 11 test groups (n = 6) in a group (B-2 to E-2 group) administered with 100 μg per kg of body weight of rats, and each sample was subjected to a sonde once a day. Orally administered and reared for 10 weeks. The amount of collagen in the skin was determined according to the following procedure. 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 the collagen, so the amount of collagen was estimated (see Takashi Asano et al., Bio Industry, p. 12, 2001). The results are shown in Table 1.

As a result, the collagen content 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 lipocalin 2 and / or lipocalin 2 degradation products have a collagen production promoting action, and were shown to be useful as a collagen production promoter. Further, it has been clarified that this collagen production promoting action is observed when lipocalin 2 and / or lipocalin 2 degradation product is administered at least 10.0 μg per kg body weight of the rat.

[Test Example 2]
Experiments using sample A obtained in Example 1 and samples B, C, and D obtained in Example 2 using a normal human fibroblast cell line [CCD45SK (ATCCRL 1506) collected from white female skin] Thus, the collagen production promoting action was examined. 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 MEM medium containing 0.6% by volume FBS. Then, Sample A obtained in Example 1 and Samples B, C, and D obtained in Example 2 were added to each well so as to be 0.1% by volume (n = 6), and 24 hours After culturing, β-aminopropionitrile was added at 50 μg / ml and tritium-L-proline was added at 1 μCi / ml, and further cultured for 24 hours to obtain a culture solution. According to the method of Webster et al. (Analytical Biochemistry, p. 220, 1979), the radioactivity incorporated into the collagen fraction of the culture solution thus obtained was measured. As a control, the same test was performed without adding lipocalin 2 or lipocalin 2 degradation product. The results are shown in Table 2.

As a result of Table 2, in the group to which lipocalin 2 and / or lipocalin 2 degradation product was added, collagen production was promoted twice or more compared to the group to which lipocalin 2 and / or lipocalin 2 degradation product was not added (control). Showed the ability. From this, it was revealed that lipocalin 2 and / or lipocalin 2 degradation products act on dermal fibroblasts and promote collagen production, indicating that they are useful as collagen production promoters.

[Test Example 3]
The collagen production-promoting effect of lipocalin 2 was examined by cell experiments using commercially available human lipocalin 2 recombinant protein (NGAL) (PROSPEC) and normal human neonatal foreskin skin fibroblasts (NHDF (NB)). NHDF (NB) cells were seeded in a 12-well plate at 8 × 10 ³ cells / well and cultured in MEDIUM 106 medium (GIBCO) at 37 ° C. in a 5% CO ² environment for 3 days. What was melt | dissolved in MEDIUM106 culture medium so that NGAL might be 10 nM was added to the cultured cell, and it culture | cultivated for 72 hours. The amount of human procollagen type I C-terminal peptide (PIP) in the culture broth thus obtained was measured as a collagen amount using a measurement kit Procollagen Type I C-peptide (PIP) EIA kit (Takara Bio). The result is shown in FIG.

As a result, it was confirmed that lipocalin 2 promotes collagen production in the sample to which NGAL was added as compared with the control because the amount of collagen was significantly increased.

A beverage for promoting collagen production having the composition shown in Table 3 was produced by a conventional method. The flavor of the produced beverage was good and there were no problems such as precipitation.

A dough having the composition shown in Table 4 was prepared and molded by a conventional method, and then baked to produce a biscuit for promoting collagen production.

A collagen production promoter having the composition shown in Table 5 was 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 4]
Using the lotion obtained in Example 6 and the cream obtained in Example 7, an actual use test was conducted. As a comparative product, the same formulation as in Examples 6 and 7 was used except that lipocalin 2 and / or lipocalin 2 degradation product was 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 skin lotion of the present invention is significantly improved in dry feeling, rough skin, etc., and excellent in promoting collagen production, compared with 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.
[Test Example 5]
For 20 patients with mild pain due to osteoarthritis, 100 g of the beverage shown in Example 3 was drunk once a day, and a one-year clinical trial was conducted. Assess joint pain and function with the visual analog scale (VAS) for pain and 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 in Table 9, it can be seen that both the VAS and WMAC indices are significantly improved after 6 months of ingestion compared to the values at the start of ingestion. Therefore, it was demonstrated that the test food of the present invention is significantly improved in joint pain and function and superior in collagen production promoting effect as compared to the comparative test food.

The present invention relates to a collagen production promoter, a collagen production promotion supplement, a food and drink, and a collagen production promotion cosmetic useful for preventing rough skin, wrinkles, a decrease in elasticity, a decrease in joint function, and the like.

Claims

A collagen production promoter comprising a lipocalin family and / or a lipocalin family degradation product as an active ingredient.
The collagen production promoter according to claim 1, wherein the lipocalin family and / or lipocalin family degradation product is lipocalin 2 and / or lipocalin 2 degradation product.
The collagen production promoter according to claim 1, wherein the lipocalin family and / or lipocalin family degradation product is β-lactoglobulin and / or β-lactoglobulin degradation product.
The collagen production promoter according to claim 2, wherein the lipocalin 2 degradation product has a molecular weight of 300 or more and 8000 or less.
The collagen production promoter according to claim 2, wherein the lipocalin 2 degradation product is obtained by degrading lipocalin 2 with a proteolytic enzyme.
The collagen production promoter according to claim 5, wherein the proteolytic enzyme is at least one selected from trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease.
A supplement containing the collagen production promoter according to any one of claims 1 to 6.
Food or drink containing the collagen production promoter according to any one of claims 1 to 6.
A cosmetic comprising the collagen production promoting agent according to any one of claims 1 to 6.