JP6522286B2 - Hyaluronic acid production promoter - Google Patents

Description

  The present invention relates to a hyaluronic acid production promoter, a supplement for hyaluronic acid production promotion, a food and a drink, and a cosmetic for promoting hyaluronic acid production, which are useful for preventing roughening of the skin, wrinkles, elasticity reduction, joint function deterioration and the like.

In recent years, much research has been conducted on photoaging, for example, to prevent skin disorders such as stains, wrinkles, and sagging caused by continuous irradiation of ultraviolet rays on the skin for many years.
The skin is roughly composed of the epidermis present on the skin surface and the dermis present in the lower part of the epidermis. The epidermis mainly has a barrier function to protect from foreign substances and pathogens from the outside, ultraviolet rays, and a function to protect the leakage of substances from inside the living body. In addition, the dermis has a thickness of about 15 to 40 times that of the epidermis and plays a role in maintaining the mechanical strength of the skin. The dermis is a fiber component in which 70% of its dry weight is collagen, but there are substrates such as glycoproteins and proteoglycans between the fibers of the dermis and the skin fibroblasts. Proteoglycans are macromolecules with a molecular weight of 10 ⁵ to 10 ⁶ or more, in which glycoproteins and mucopolysaccharides such as glycosaminoglycans are bound, and glycosaminoglycans in the dermis are mainly composed of hyaluronic acid and dermatan sulfate. Acid is said to be the main matrix component in the dermis.
When the moisture content of the skin or the amount of hyaluronic acid in the skin decreases due to physiological factors such as aging, UV light such as sunlight, changes in external environment such as dryness, oxidation, etc., wrinkles are generated in the skin, resulting in a sagging condition cause. Since hyaluronic acid mainly has a function to retain skin moisture, it is thought that the reduction of skin moisture content can be prevented by increasing the hyaluronic acid content in the dermis, Many cosmetic compositions containing hyaluronic acid have been proposed as agents for improving the retention function. However, hyaluronic acid applied to these skin surfaces only exerts a moisturizing effect on the skin surface, and can not essentially improve the deterioration of the skin function.
Hyaluronic acid has also been reported to be poorly absorbed from the digestive tract when orally administered due to its high relative molecular weight, low lipid solubility, difficulty in passing through biological membrane barriers, and the presence of a large amount of polysaccharide degrading enzyme (Patent Document 1). Therefore, it is necessary to promote the biosynthesis of hyaluronic acid in the dermal layer in order to essentially improve the decrease in the function of the skin, which can prevent wrinkles and sagging of the skin, and is also a problem in terms of safety. A hyaluronic acid production promoter was desired.
So far, as hyaluronic acid production promoting agents, insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), plasma derived growth factor (PDGF) -BB, interleukin-1 (IL-1) Transforming growth factor (TGF) -β1 and the like are known, but none have been put to practical use as food and drink, cosmetics, medicines and the like.
On the other hand, hyaluronic acid in synovial fluid covers the surface of articular cartilage and helps smooth operation of articular function. Although the concentration of hyaluronic acid in normal human joint fluid is about 2.3 mg / mL, for example, in the case of rheumatoid arthritis, the concentration of hyaluronic acid in joint fluid drops to about 1.2 mg / mL, and at the same time The viscosity also decreases significantly. In addition, it is known that, in the cases of suppurative arthritis, gouty arthritis and the like as well as in the case of rheumatoid arthritis, a decrease in the content of hyaluronic acid occurs (Non-patent Document 1).
In the above-mentioned diseases, in order to improve the lubricating function, coat and protect articular cartilage, to suppress pain and to improve the properties of pathological joint fluid, it has been carried out to increase the amount of hyaluronic acid in the joint fluid. For example, when the joint injection therapy of sodium hyaluronate is performed on a patient with rheumatoid arthritis, the above-mentioned property improvement is observed (Non-patent Document 2). Similarly, in traumatic arthropathy, osteoarthritis and osteoarthritis, an improving effect by joint injection therapy of hyaluronic acid has been reported (Non-patent Document 3).
From the above, promotion of hyaluronic acid production is effective for prevention and treatment of joint diseases such as skin diseases such as rough skin, rheumatoid arthritis, traumatic arthropathy, osteoarthritis and osteoarthritis. However, treatment of the above-mentioned diseases is long-term and requires a doctor's prescription. Therefore, a supplement, a cream or a food / drink product containing a hyaluronic acid production promoter which can be easily treated in daily life has been desired.
The chemokine stromal cell derived factor 1 (SDF-1 or CXCL12, PBSF) is a ligand for the G protein coupled receptor CXCR4, but its SDF-1 / CXCR4 signaling system It is known to have physiological effects such as formation. Moreover, in recent years, SDF-1 has been reported to be expressed in the dermis of the back of mice and to promote cell proliferation of the epidermis. However, there is no report on the function of producing hyaluronic acid.

Patent document 1: JP-A 2009-500503

Journal of Cell Science, 118, 1981, 2005

  An object of the present invention is to provide a hyaluronic acid production promoter that does not have a problem in terms of safety. Moreover, this invention makes it a subject to provide the supplement for hyaluronic acid production promotion which compounded such a substance, food-drinks, and cosmetics for hyaluronic acid production promotion.

In order to solve these problems, the present inventors have intensively searched for substances having a hyaluronan production promoting action widely contained in food materials, and as a result, SDF-1 produces a quantity of hyaluronan produced. It has been found to increase, and the present invention has been completed.
That is, the present invention includes the following aspects.
(1) A hyaluronic acid production promoter containing SDF-1 as an active ingredient.
(2) A skin care agent containing SDF-1 as an active ingredient.
(3) The skin care agent according to (2), wherein the skin care is the prevention and / or improvement of rough skin.
(4) The supplement for hyaluronic acid production promotion which mix | blended SDF-1 in any one of (1)-(3).
(5) Food-drinks for hyaluronic acid production promotion which mix | blended SDF-1 in any one of (1)-(3).
(6) A cosmetic for promoting hyaluronic acid production, which contains SDF-1 according to any one of (1) to (3).
(7) A method of improving skin quality by orally taking or applying SDF-1.
(8) A method for improving skin quality by orally ingesting 10 μg or more of SDF-1 per day, or applying a product adjusted to 0.05 to 0.5% by weight.

  According to the present invention, a hyaluronic acid production accelerator, a food / beverage product for hyaluronic acid production promotion, and a hyaluronic acid production promotion cosmetic comprising SDF-1 as an active ingredient are provided. The hyaluronic acid production promoter of the present invention, the supplement for hyaluronic acid production promotion, the food and drink, and the cosmetic for promoting hyaluronic acid production have an action to promote the hyaluronic acid production of the skin, and the wrinkles and slackness of the skin, the feeling of dryness and It is useful for prevention and treatment of rough skin.

It compares about the influence which SDF-1 exerts on the mRNA expression of a hyaluronic acid synthetase gene (Has2).

  The feature of the hyaluronic acid production promoter of the present invention is that SDF-1 is an active ingredient. The SDF-1 of the present invention may be of any origin. For example, SDF-1 derived from human and bovine has already been clarified its gene sequence, and recombinant production is possible, but in the present invention, SDF-1 produced by genetic engineering techniques is also used It is also possible to use those derived from cells recovered from cell culture broth. In addition, SDF-1 is contained in bovine colostrum and may be recovered from milk, and raw milk, milk powder, skimmed milk, reduced milk, etc., heat treatment, salting treatment, ethanol treatment, ion exchange It is also possible to obtain by various chromatography processes such as chromatography and gel filtration chromatography, ultrafiltration process and the like.

    The hyaluronic acid production promoter of the present invention exerts a hyaluronic acid production promoting effect by oral administration or application. When orally administering the hyaluronic acid production promoter of the present invention, SDF-1 which is an active ingredient can be used as it is, but according to a conventional method, powders, granules, tablets, capsules, drinks It can also be formulated and used. In the present invention, oral preparations such as powders, granules, tablets, capsules and the like are formulated by a conventional method using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like. It is possible to In this type of preparation, in addition to the above-mentioned excipients, binders, disintegrants, surfactants, lubricants, flow promoters, colorants, perfumes and the like may be used as appropriate. Binders include, for example, starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, carboxymethylcellulose sodium, methylcellulose, crystalline cellulose, ethylcellulose, polyvinyl pyrrolidone, and disintegrants include, for example, starch, hydroxypropyl starch And carboxymethylcellulose, sodium carboxymethylcellulose, crosslinked sodium carboxymethylcellulose, crystalline cellulose and the like. Further, as a surfactant, soybean lecithin, sucrose fatty acid ester, etc., as a lubricant, talc, wax, sucrose fatty acid ester, hydrogenated vegetable oil, etc., as a fluidity promoter, anhydrous silicic acid, dry aluminum hydroxide, Magnesium silicate etc. are mentioned.

Furthermore, it is also possible to mix | blend these SDF-1 with a supplement, a nutrient agent, food-drinks, etc., after preparing as it is or formulation. In addition, since SDF-1 is relatively stable to heat, it is also possible to heat-sterilize the raw material containing SDF-1 on the conditions normally performed.

  When applying the hyaluronic acid production promoter of the present invention, it may be prepared into various dosage forms such as liquid agent, solid agent, semi-solid agent, etc. by blending with known components generally used depending on the purpose of use. Possible compositions and ointments, gels, creams, sprays, patches, lotions, powders and the like as preferred compositions. For example, the hyaluronic acid production promoter of the present invention may be a hydrocarbon such as vaseline, a higher fatty acid lower alkyl ester such as stearyl alcohol and isopropyl myristate, an animal oil and fat such as lanolin, a polyhydric alcohol such as glycerin, glycerin fatty acid ester, mono Cosmetics for promoting hyaluronic acid production by mixing with surfactants such as stearic acid and polyethylene glycol, inorganic salts, waxes, resins, water and preservatives such as methyl parahydroxybenzoate and butyl parahydroxybenzoate if necessary. And can produce medicines.

  The effective dose of the hyaluronic acid production promoter of the present invention by oral administration is appropriately determined depending on 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 was revealed that SDF-1 exhibits a hyaluronic acid production promoting action by taking 10 μg or more per kg of rat body weight. Therefore, according to the extrapolation method, the hyaluronic acid production promoting effect can usually be expected by taking in 10 μg or more of SDF-1 per adult per day, so it should be added to the food or drink so that the necessary amount can be secured or It may be administered as a medicine. In addition, it is also possible to divide administration into several times a day, as needed.

  Although the effective amount by application of the hyaluronic acid production promoter of the present invention varies depending on the dosage form, it is preferable to blend SDF-1 so as to be 0.001 to 2% by weight based on the total amount of the composition to be applied. Just do it. However, what is diluted at the time of use like a bathing agent can further increase a compounding quantity.

[Test Example 1]
A sample of Recombinant Human CXCL12 (SDF-1α) (carrier-free) purchased from Biolegend was used as sample A, and its hyaluronic acid production promoting action was examined by animal experiments using rats. Seven-week-old Wistar male rats in the saline administration group (control group), a group to which 10 μg of sample A is administered per kg of rat weight (group A-1), a group to which 100 μg of sample A is administered per kg of rat weight Group A-2), each was orally administered once daily with a sonde and bred for 10 weeks. Regarding the amount of hyaluronic acid in the skin, skin tissues (300 mg each) collected immediately after slaughtering of the shaved rat on the day before the test were subjected to measurement. The heat-denatured skin tissue was proteolytically degraded by actinase. The resulting degradation product was further degraded into hyaluronic acid with hyaluronidase. Hyaluronic acid was measured by HPLC method.
The results are shown in Table 1.

数値は、平均値±標準偏差（ｎ＝６）を示す。
※は対照群と比較して有意差があることを示す（ｐ＜０．０５）。

Numerical values indicate mean value ± standard deviation (n = 6).
※ indicates that there is a significant difference compared to the control group (p <0.05).

As a result, the amount of hyaluronic acid in the soluble fraction after 10 weeks showed significantly higher values in all test groups compared to the control group. From this, it became clear that SDF-1 has hyaluronic acid production promoting action, and it was shown that it is useful as a hyaluronic acid production promoting agent. In addition, it was revealed that this hyaluronic acid production promoting action was observed when SDF-1 was administered at least 10.0 μg / kg of rat body weight.

[Test Example 2]
Sample A was examined for its hyaluronic acid production promoting effect by an experiment using a normal human fibroblast cell line (CCD45SK (ATCCRL 1506) collected from the skin of a white female). Using a modified Eagle's medium (MEM, 10-101, manufactured by Dainippon Pharmaceutical Co., Ltd.) containing 10% by volume of fetal bovine serum (hereinafter abbreviated as FBS), 4 × 10 ⁴ normal human fibroblast cell lines / well / 0 The cells were seeded in a 24-well plate to 4 ml, cultured for 24 hours at 37 ° C. under 5% carbon dioxide gas, saturated steam, and then replaced with MEM medium containing 0.6% by volume of FBS. Then, the sample A was added to each well to a volume of 0.1% by volume (n = 6), and cultured for 72 hours to obtain a culture solution. The amount of hyaluronic acid (Biotech Trading Partners, Inc.) in the culture solution thus obtained was measured. As a control, the same test was performed without adding SDF-1. The results are shown in Table 2.

数値は、平均値±標準偏差（ｎ＝６）を示す。
※は対照と比較して有意差があることを示す（ｐ＜０．０５）。

Numerical values indicate mean value ± standard deviation (n = 6).
※ indicates that there is a significant difference compared to the control (p <0.05).

As a result of Table 2, the group which added SDF-1 showed 2 times or more hyaluronan production promotion ability compared with the group (control) which does not add SDF-1. From this, it became clear that SDF-1 acts on skin fibroblasts and has an action of promoting hyaluronic acid production, and it was shown that it is useful as a hyaluronic acid production promoter.

[Test Example 3]
The effect of SDF-1 on the mRNA expression of the hyaluronic acid synthetase gene (Has2) was confirmed using cell experiments using normal human neonatal foreskin skin fibroblasts (NHDF (NB)) and a real-time PCR method. Specifically, NHDF (NB) cells are seeded at 0.5 × 10 ⁵ cells / well in a 24-well plate, and are used at 5 ° C. in a 5% CO ² environment at 37 ° C. in MEDIUM 106 medium (GIBCO). Culture for 7 days. For the final 24 hours of the 7-day culture period, after dissolving the sample A in MEDIUM 106 medium so as to be 100 nM, 500 nM and 1 μM, respectively, total RNA was recovered and cDNA was synthesized. 0.5 ml of ISOGEN (manufactured by Nippon Gene Co., Ltd.), which is an RNA extraction agent, was added to the cultured cells and allowed to stand for 5 minutes, and then the solubilized cell solution was collected by pipetting in a 1.5 ml tube. After 0.1 ml of chloroform was added to the cell solution and thoroughly stirred, the upper layer (aqueous layer) separated into two layers was collected into a new 1.5 ml tube. To the collected solution, 0.25 ml of 2-propyl alcohol was added, and after standing for 10 minutes, it was centrifuged at 15,000 rpm and 4 ° C. for 15 minutes to obtain a precipitate of total RNA. The obtained precipitate was washed with 70% ethanol and then dissolved in DEPC water to prepare an RNA solution. CDNA was synthesized from 1μg amount of RNA using a Takara PrimeScript ^TM RT reagent Kit. Real-time PCR using SYBR Green (Takara SYBR Prime Ex Taq II) was performed using the obtained cDNA as a template. The reaction conditions were initial denaturation at 95 ° C. for 30 seconds, denaturation at 95 ° C. for 5 seconds, annealing at 57 ° C. for 15 seconds, and elongation at 72 ° C. for 20 seconds, for a total of 40 cycles of reaction. The primers used were the Has2 gene expression confirmation primers described in Table 3. The results are shown in FIG.

    According to FIG. 1, when SDF-1 was added to NHDF (NB) cells, the mRNA expression level of the Has2 gene was significantly enhanced depending on the concentration of SDF-1.

The hyaluronic acid production promoting beverage formulated as shown in Table 4 was produced by a conventional method. The flavor of the produced beverage was good and there was no problem such as precipitation.

Doughs having the formulations shown in Table 5 were produced by the usual method, and then molded and roasted to produce biscuits for promoting hyaluronic acid production.

The hyaluronic acid production promoter of the composition shown in Table 6 was manufactured by a conventional method.

The lotion of the composition shown in Table 7 was manufactured by a conventional method.

The cream of the composition shown in Table 8 was manufactured by a conventional method.

[Test Example 5]
An actual use test was conducted using the lotion obtained in Example 4 and the cream obtained in Example 5. As a comparative product, the thing of the same composition as Example 4 and 5 was used except having removed SDF-1. Ten adult females with sagging face and small wrinkles and with a sense of dryness are randomly placed in 2 groups (groups A and B), and 20 females with rough skin in their hands. 10 people each randomly divided into 2 groups (C, D groups), 2 g of the lotion of the present invention on the face of group A, 2 g of the lotion of the comparative product on the face of group B, C group 2 g of the cream of the present invention was applied to the fingers of the present invention, and 2 g of the comparative cream was 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 9.

＋＋；１０日間塗布後に顕著な改善効果あり。
＋；１０日間塗布後に改善効果あり。
±；１０日間塗布後に改善効果なし（１０日前と変わらない）。

++; There is a remarkable improvement effect after 10 days application.
+; Improves after 10 days application.
±; no improvement after application for 10 days (same as 10 days before).

From the results of Table 9, it is demonstrated that the cosmetic lotion of the present invention has remarkable improvement in dryness, wrinkles and the like as compared with the cosmetic lotion of the comparative product, and is excellent in the hyaluronic acid production promoting effect. The In addition, the cream of the present invention also has an improvement in the feeling of dryness, a remarkable improvement in skin roughening, and a natural exacerbation suppressing effect such as skin roughening, as compared with the cream of the comparative product.

[Test Example 6]
The beverage of Example 3 was taken 100 g once a day for 20 patients having mild pain caused by osteoarthritis, and a one-year clinical trial was conducted. Joint pain and function are evaluated using the Visual Analog Scale for Pain (VAS) and the Western Ontario and McMaster Universities (WOMAC) Index of Pain, Function, and Stiffness in Arthritic Joints for osteoarthritis. The The results are shown in Table 10.

数値は、平均値±標準偏差（ｎ＝２０）を示す。
※ は初期値と比較して有意差があることを示す（ｐ＜０．０５）。

Numerical values indicate mean value ± standard deviation (n = 20).
※ indicates that there is a significant difference compared to the initial value (p <0.05).

The present invention relates to a hyaluronic acid production promoter, a supplement for hyaluronic acid production promotion, a food and a drink, and a cosmetic for promoting hyaluronic acid production, which are useful for preventing roughening of the skin, wrinkles, elasticity reduction, joint function deterioration and the like.

Claims (4)

The supplement for hyaluronic acid production promotion which uses milk-derived SDF-1 as an active ingredient.   The hyaluronic acid production supplement according to claim 1, wherein the supplement is for preventing a decrease in joint function. Food-drinks for hyaluronic acid production promotion which use milk-derived SDF-1 as an active ingredient. The said food-drinks are for food-drinks for hyaluronic acid production promotion of Claim 3 which are for joint function fall prevention.

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