JP6259207B2 - Elastin production promoter - Google Patents

Description

The present invention relates to an elastin production promoter, a drink supplement for elastin production promotion, a food, and a cosmetic for promoting elastin production, which are useful for preventing rough skin, wrinkles, reduced elasticity, reduced joint function and the like. More specifically, the present invention relates to an elastin 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 collagen fibers, which are the main matrix components of the dermis, are significantly reduced by the action of these factors. 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. Elastin has a role to support the collagen fibers and is also called elastic fiber. Elastin is only about 2% of the dermis, but controlling elastin formation leads to improvements in skin elasticity and sagging. From the above, it seems that wrinkles and sagging of the skin can be prevented by promoting the biosynthesis of elastin, which is one of the main components of the dermis layer. Thus, an elastin production promoter has been desired.
As elastin production promoters, extracts from plants such as Kyomizuna, Kyojou Nana, Fushimi Togarashi, Manganji Togarashi and Kujo Negi, Ogaku, Ginseng, Altea, Yokuinin and Lily are disclosed. (Patent Documents 1 and 2)
On the other hand, elastin in articular cartilage and ligament 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, synthesis promotion, etc. have begun to be taken. There are not many studies.
From the above, promotion of elastin production is effective for the prevention and treatment of skin diseases such as rough skin, and joint diseases such as rheumatoid arthritis, traumatic arthropathy, osteoarthritis, and osteoarthritis. Therefore, a supplement, cream or food or drink containing an elastin 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-Fe ³⁺ 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).

JP2012-162487 JP2012-56933

  In the inventions disclosed in Patent Documents 1 and 2, because plant-derived raw materials are used, the cultivation of the raw material for the elastin production promoter 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, to provide an elastin production promoter that can be easily taken in daily life, and to provide an elastin production promotion supplement, a food and drink, and an elastin production promotion cosmetic containing such a substance. Let it be an issue.

In order to solve these problems, the present inventors diligently searched for a substance exhibiting elastin production promoting action. As a result, lipocalin family protein degradation obtained by degrading lipocalin family protein or its lipocalin family protein was investigated. It was found that the product increases the amount of elastin produced in the skin, and the present invention has been completed.
That is, the present invention includes the following aspects.
(1) An elastin production promoter containing a lipocalin family protein and / or a lipocalin family protein degradation product as an active ingredient.
(2) The elastin 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 elastin production promoter according to (1), wherein the lipocalin family and / or lipocalin family degradation product is β-lactoglobulin and / or β-lactoglobulin degradation product.
(4) The elastin 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 elastin production promoter according to (2), wherein the lipocalin 2 degradation product is obtained by degrading lipocalin 2 with a proteolytic enzyme.
(6) The elastin according to (5), wherein the proteolytic enzyme is at least one selected from trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. Production promoter.
(7) A skin care agent containing the elastin production promoter according to any one of (1) to (6) 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 supplement containing the elastin production promoter according to any one of (1) to (6).
(10) A food or drink containing the elastin production promoter according to any one of (1) to (6).
(11) A cosmetic comprising the elastin production promoter according to any one of (1) to (6).
(12) A method for improving skin quality by orally ingesting or applying the elastin production promoter according to any one of (1) to (6).
(13) The elastin production promoter according to any one of (1) to (6) is orally ingested in an amount of 10.0 μg or more per day, or a composition containing 0.05 to 0.5% by weight is applied. How to improve skin quality.

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

Shows the elastin production promoting effect of lipocalin 2.

  The elastin 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 elastin production promoter of the present invention exhibits an elastin production promoting effect by oral administration or application. When the elastin 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 according to 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 elastin production promoter of the present invention, depending on the purpose of use, it can be prepared into various dosage forms such as liquids, solids and semisolids by blending with commonly used known ingredients. Possible and preferred compositions include ointments, gels, creams, sprays, patches, lotions, powders and the like. For example, the elastin production promoter of the present invention includes hydrocarbons such as petrolatum, higher fatty acid lower alkyl esters such as stearyl alcohol and isopropyl myristate, animal fats and oils such as lanolin, polyhydric alcohols such as glycerin, glycerin fatty acid esters, monostearin Cosmetics and pharmaceuticals for promoting elastin production by mixing with surfactants such as acids, 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 elastin 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. As a result of animal experiments, it has been clarified that lipocalin 2 and / or lipocalin 2 degradation products show an elastin production promoting effect when ingested by 10 μg or more per 1 kg of rat body weight. Therefore, according to the extrapolation method, ingestion of 10 μg or more of lipocalin 2 and / or lipocalin 2 degradation product per adult per day can be expected to promote elastin 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 elastin production promoter of the present invention varies depending on the dosage form, but is preferably lipocalin 2 and / or lipocalin so as to be 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 was thoroughly washed with deionized water, passed through 10,000 L of skimmed milk, thoroughly washed 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 an elastin 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 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 and D can be used as elastin 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 elastin production promotion effect | action was investigated by the animal experiment using a rat. 7-week-old Wistar male rats were 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. Group (A-2 group) in which 100 μg of the obtained sample A was administered per kg of rat body weight, and Group B (groups B-1 to E-1) in which 10 μg of the samples B to E obtained in Example 2 were administered per kg of rat body weight Samples B to E 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 / kg of the rat body weight, and each was sampled once a day with a sonde. Orally administered and reared for 10 weeks. Regarding the amount of elastin in the skin, skin tissues (300 mg each) collected immediately after sacrifice of the shaved rat on the day before the test were subjected to measurement. An appropriate amount of 0.85% physiological saline was added to the skin tissue, and the tissue was homogenized with a homogenizer. Extracts from the skin tissue were collected, and after SDS-PAGE, Western blotting was performed using an anti-elastin antibody (abcam) to quantify the amount of elastin. The results are shown in Table 1.

As a result, the amount of elastin in the soluble fraction after 10 weeks was significantly higher in all test groups than in the control group. From this, it became clear that lipocalin 2 and / or lipocalin 2 degradation product has an elastin production promoting action, and was shown to be useful as an elastin production promoter. Further, it has been clarified that this elastin 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]
About the sample A obtained in Example 1, and the samples B, C, and D obtained in Example 2, the elastin production promoting action was examined by an experiment using normal human neonatal foreskin skin fibroblasts (NHDF (NB)). It was. NHDF (NB) cells were seeded in a 12-well plate to 0.6 × 10 ⁵ cells / well and cultured in MEDIUM 106 medium (GIBCO) at 37 ° C. in a 5% CO ² environment for 2 days. Thereafter, the medium was replaced with serum-free MEDIUM 106 medium and further cultured for 24 hours. Then, the sample A obtained in Example 1 and the samples B, C, and D obtained in Example 2 were adjusted with serum-free MEDIUM 106 medium so that each well had a volume of 0.1% by volume. Added (n = 3) and cultured for 24 hours. The cultured cells were washed with PBS, detached by trypsin treatment, and then collected. The amount of elastin produced in the cells was measured with a colorimetric kit (biocolor, Funakoshi). 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, the group to which lipocalin 2 and / or lipocalin 2 degradation product was added was significantly more effective in promoting the production of elastin than the group to which lipocalin 2 and / or lipocalin 2 degradation product was not added (control). It was a lot. From this, it was revealed that lipocalin 2 and / or lipocalin 2 degradation product has an action of acting on dermal fibroblasts and promoting elastin production, and was shown to be useful as an elastin production promoter.

[Test Example 3]
Regarding the effect of NGAL on elastin mRNA expression by cell experiments using commercially available human lipocalin 2 recombinant protein (NGAL) (PROSPEC) and normal human neonatal foreskin dermal fibroblasts (NHDF (NB)), real-time PCR The method was examined.
Specifically, NHDF (NB) cells are seeded in a 24-well plate so as to be 0.5 × 10 ⁵ cells / well, and are placed in MEDIUM 106 medium (GIBCO) at 37 ° C. in a 5% CO ² environment. For 3 days. After 3 days of culture period, NGAL dissolved in MEDIUM106 medium to 10 nM was added to and retained in cells for 2 hours, 4 hours, 6 hours, 9 hours, 12 hours and 24 hours, respectively. Total RNA was recovered by the procedure described above and cDNA was synthesized.
After 0.5 ml of ISOGEN (manufactured by Nippon Gene) as an RNA extractant was added to the cultured cell solution and allowed to stand for 5 minutes, the cell solution solubilized by pipetting was collected in a 1.5 ml tube. After 0.1 ml of chloroform was added to the cell solution and stirred sufficiently, the upper layer (aqueous layer) separated into two layers was collected in a new 1.5 ml tube. 0.25 ml of 2-propyl alcohol was added to the recovered solution, allowed to stand for 10 minutes, and then centrifuged at 15,000 rpm, 4 ° C. for 15 minutes to obtain a total RNA precipitate. The obtained precipitate was washed with 70% ethanol and then dissolved in DEPC water to obtain an RNA solution. CDNA was synthesized from 1 μg of RNA using Takara PrimeScript ^™ RT reagent Kit. Real-time PCR using SYBR Green (Takara SYBR Prime Ex TaqII) was performed using the obtained cDNA as a template. The reaction conditions were 95 ° C., 30 seconds of initial denaturation, 95 ° C., 5 seconds of denaturation, 57 ° C., 15 seconds of annealing, 72 ° C., 20 seconds of extension, for a total of 40 cycles. Primers for ELN listed in Table 3 were used. The results are shown in FIG.

    As shown in FIG. 1, ELN mRNA expression by NGAL was significantly enhanced in 2 to 24 hours.

Elastin production-promoting beverages having the formulations shown in Table 4 were 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 5 was prepared by a conventional method, molded, and then baked to produce a biscuit for elastin production promotion.

The elastin production promoter having the composition shown in Table 6 was produced by a conventional method.

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

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

[Test Example 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 shown in Table 9, it was demonstrated that the skin lotion of the present invention is significantly improved in dry feeling, rough skin, etc., and excellent in promoting elastin production, compared to the skin lotion of the comparative product. . In addition, the cream of the present invention also has an improvement in dry feeling and a marked improvement in rough skin, as compared with the comparative cream, and has been found to have an effect of suppressing natural deterioration such as rough skin.
[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 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 10.

From the results of Table 10, it can be seen that both the VAS and WMAC indices are significantly improved from 6 months after the ingestion compared to the values at the start of ingestion. Therefore, it was demonstrated that the test food of the product of the present invention is significantly improved in joint pain and function and superior in elastin production promoting effect as compared to the comparative test food.

The present invention relates to an elastin production promoter, an elastin production promoting supplement, a food and drink, and a cosmetic for promoting elastin production, which are useful for preventing rough skin, wrinkles, reduced elasticity, reduced joint function, and the like.

Claims (6)

An elastin production promoter comprising lipocalin 2 and / or lipocalin 2 degradation product as an active ingredient.   The elastin production promoter according to claim 1, wherein the lipocalin 2 degradation product has a molecular weight of 300 or more and 8000 or less.   The elastin production promoter according to claim 2, wherein the lipocalin 2 degradation product is obtained by degrading lipocalin 2 with a proteolytic enzyme.   The elastin production promoter 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. .   The supplement for elastin production promotion which mix | blended the elastin production promoter in any one of Claims 1-4.   Elastin production promotion food and drink containing the elastin production promoter according to any one of claims 1 to 4.

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