JP5968643B2 - Melanocyte differentiation induction inhibitor - Google Patents

Description

  The present invention relates to, for example, a differentiation induction inhibitor from stem cells to melanocytes and a whitening agent or an external preparation for skin containing the differentiation induction inhibitor.

In general, skin pigmentation, such as spots, freckles, and sunburn, is caused by excessive melanin formation in the skin due to hormonal abnormalities or ultraviolet light stimulation, which deposits in the skin. It is considered. As one method for preventing such pigmentation, a method for suppressing excessive production of melanin is known.
On the other hand, melanocytes have various differentiation stages (neural crest cells, pigment stem cells, melanoblasts, etc.), and various pigment abnormalities and spots are caused by abnormal differentiation. Therefore, identification of factors that control the differentiation itself of melanocytes is important for the resolution of fundamental dysplasia and spots. Conventionally, however, screening for melanocyte regulatory factors is performed mainly using melanoma cells (produced by melanocyte oncogenesis), terminally differentiated melanocytes and mashroom-derived tyrosinase. A system was used (Patent Document 1, Patent Document 2). Therefore, the search of the factor which controls the differentiation of a melanocyte has been hardly performed conventionally.

JP 2009-263282 A JP 2011-6462 A

As described above, the identification of factors that control the differentiation of melanocytes themselves is important for the resolution of fundamental dysplasia and spots.
Then, in view of the above-mentioned situation, the present invention aims to find a material having an activity of suppressing differentiation from stem cells to melanocytes, and to provide a fundamental whitening agent and an external preparation for skin that have never existed.

  As a result of intensive studies to solve the above problems, it has been found that the generation, differentiation, proliferation and melanin synthesis of melanocytes can be reproduced in vitro by using a differentiation induction system from stem cells to melanocytes. It was. Furthermore, by using this induction system as a screening system, the Ainu Wakame-derived glucose derivative has an excellent effect of suppressing melanocyte differentiation, and is found to be excellent as a whitening agent, thereby completing the present invention. It came to.

That is, the present invention contains a β-1,3-1,6-D-glucose derivative having all the following features (1) to (3), and a melanocyte differentiation induction inhibitor, It is.
(1) It originates from Ainu sea turtle (Alaria praelonga Kjellman).
(2) The ¹ H NMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent has two signals of 4.7 ppm and 4.5 ppm, and the area ratio is 7: 1 to 11: 1.
(3) D-mannitol is bound, and the content of D-mannitol is 0.1 to 10% by weight based on the total amount of the glucose derivative.

Furthermore, said substance is represented by the following general formula.
(Chemical formula 1)
Here, the β-1,3 bond / β-1,6 bond ratio is 7 to 11, and the mannitol content of the glucose derivative is 0.1 to 10% by weight. However, the arrangement of [] is in no particular order.

  According to the melanocyte differentiation induction inhibitor of the present invention, differentiation induction from stem cells to melanocytes can be remarkably suppressed, and it can be fundamentally treated / prevented in pigmentation disorders and spots.

Hereinafter, the present invention will be described in detail.
The Ainu-wakame-derived glucose derivative used in the present invention can be extracted, isolated and purified from Ainu-wakame (Alaria praelonga Kjellman). Ainu Wakame is a seaweed that grows naturally in eastern Hokkaido and can be collected in large quantities.

  Examples of the solvent used for the extraction include water (hot water), lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2- Butanol, etc.), liquid polyhydric alcohol (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.) and the like are preferable. These solvents may be used alone or in combination of two or more. Moreover, an acid or an alkali can be added to these solvents to adjust the pH. Most preferably, it is extracted with 0.01-1N, in particular 0.1N hydrochloric acid.

  Ainu Wakame is solvent-extracted using the above-mentioned solvent. The solvent extraction method is not particularly limited, but extraction by heating is preferable. For example, Ainu Wakame extract can be obtained by adding water to dried Ainu Wakame and performing hot water extraction at 95-100 ° C. Alternatively, an Ainu wakame extract can be obtained by adding an inorganic acid (for example, hydrochloric acid) to the dried Ainu wakame and performing extraction at about 4 ° C.

  The Ainu Wakame-derived glucose derivative used in the present invention can be purified from the above extract by using column chromatography such as gel filtration or anion exchange, preparative high-performance liquid chromatography, or the like. The Ainu Wakame-derived glucose derivative used in the present invention may be a purified product or a crude product, or may contain a solvent.

The structural analysis of the glucose derivative derived from Ainu Wakame used in the present invention can be performed according to the method of Abu et al. (Basics of Glycochemistry, 1984, Kodansha, Kimiko Abu, Nobuko Seno). Specifically, methods such as constituent sugar analysis using GC-MS, infrared absorption spectrum analysis, molecular weight analysis using HPLC, methylation analysis, and ¹ H-NMR analysis can be used.

(1) Component (constituent sugar) analysis The glucose derivative of the present invention obtained by chromatography or the like is hydrolyzed, and after reduction, the sugar constituting the glucose derivative is identified by measurement with GC-MS. be able to.
The constituent sugars of the glucose derivative are glucose and mannitol, and the content of glucose is preferably 80 to 95% by weight, and most preferably 85 to 90% by weight based on the total amount of glucose derivative. The mannitol content is preferably 0.1 to 10% by weight, and most preferably 1 to 4% by weight.

(2) Infrared absorption spectrum analysis By performing infrared absorption spectrum analysis (KBr method), the anomeric configuration of α-bond or β-bond can be determined. Absorption near 844 cm ⁻¹ represents the presence of α bonds, and absorption near 891 cm ⁻¹ represents the presence of β bonds.

(3) Molecular weight analysis The molecular weight can be determined by gel filtration chromatography. Pullulan can be used as a standard substance (molecular weight marker).

(4) Methylation analysis By thoroughly methylating the glucose derivative of the present invention, the partially methylated sugar obtained by hydrolysis is analyzed, and by knowing where the hydroxyl group that is not methylated exists. The sugar 1,3 bond and 1,6 bond can be confirmed.

(5) ¹ H-NMR analysis
By measuring ¹ H-NMR, the ratio of β-1,3 bonds to β-1,6 bonds can be determined. When the glucose derivative of the present invention is used and dissolved in a 1N sodium hydroxide aqueous solution and measured, signals are observed at about 4.7 ppm and about 4.5 ppm, respectively. Since NMR measurement values change due to subtle changes in conditions, and it is a well-known fact that there is an error, “about 4.7 ppm” and “about 4.5 ppm” are within the normally expected range. It means a numerical value including the fluctuation range of the measured value (for example, ± 0.2 ppm). The ratio of β-1,3 bond / β-1,6 bond of the Ainu Wakame-derived glucose derivative used in the present invention is preferably 7 to 11, and most preferably 8 to 10.

By the structural analysis of (1) to (5) above, the Ainu Wakame-derived glucose derivative used in the present invention can be represented by the following general formula.
(Chemical formula 1)
The ratio of β-1,3 bonds / β-1,6 bonds is 7 to 11, and the mannitol content of the glucose derivative is 0.1 to 10% by weight. In the chemical formula ₁ , glucose having a binding mode in [] (β-D-Glc bound at the 6-position of ₃ β-D-Glc ₁ , ₃ β-D ₆ -Glc ₁ , ₃ β-D ₆ those β-D-glc2 mol 6-position of -Glc ₁ is _bonded, such as ₆ β-D-Glc ₁₎ or mannitol Ainu wakame derived gluco - more in a range that satisfies the molecular weight of the scan derivative stoichiometrically It is present in the individual molecule.
Moreover, it means that the above-mentioned sequences such as glucose and mannitol in [] are not specified. This can be described as out of order in the sequence of [].

  The Ainu-wakame-derived glucose derivative thus obtained is used as an active ingredient of the melanocyte differentiation induction inhibitor according to the present invention. As the melanocyte differentiation-inducing inhibitor of the present invention, Ainu Wakame-derived glucose derivatives may be used as they are, and within the range not impairing the effects of Ainu Wakame-derived glucose derivatives, external preparations, injections, and internal preparations. Fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, perfumes, moisturizers, powders, UV absorption Agent, thickener, pigment, antioxidant, whitening agent, chelating agent, excipient, extender, binder, wetting agent, disintegrant, lubricant, dispersant, preservative, solubilizer, solvent, etc. These components can be blended.

  The melanocyte differentiation induction inhibitor of the present invention can be used in any of cosmetics, quasi-drugs, and pharmaceuticals. Examples of the dosage form include lotions, creams, emulsions, gels, aerosols, Essences, packs, cleaning agents, bath preparations, foundations, powders, lipsticks, ointments, poultices, etc., or powders, granules, tablets, sugar-coated tablets, capsules when used orally Agents, syrups, pills, suspensions, solutions, emulsions and the like. In addition, injection solutions, suppositories and the like can be mentioned.

  The compounding amount of the Ainu Wakame-derived glucose derivative used in the present invention is 0.00001% by weight or more, preferably 0.0001 to 10% by weight in terms of solids, based on the total amount of the differentiation induction inhibitor of the present invention. good. If it is less than 0.00001% by weight, a sufficient effect is hardly desired. When the blending amount exceeds 10% by weight, the effect is hardly recognized and it is uneconomical. In addition, the addition method may be added in advance or during the production, and may be appropriately selected in consideration of workability.

  Next, in order to explain the present invention in detail, production examples, formulation examples and experimental examples of Ainu seaweed-derived glucose derivatives used in the present invention will be given as examples, but the present invention is not limited thereto. In the examples, the part of the amount is part by weight, and% is% by weight.

Production Example 1 Purification of Ainu Wakame-derived Glucose Derivatives 800 ml of 0.1N hydrochloric acid was added to 20 g of dried Ainu wakame and extracted at 4 ° C. for 7 days. After extraction, the extract was filtered, and the obtained filtrate was concentrated and lyophilized to obtain 3.1 g of Ainu Wakame HCl extract. This extract was subjected to gel filtration column chromatography (column: Sephadex G-50, 2 × 100 cm) to collect fractions having a molecular weight of about 500 to about 10,000. Non-adsorbed fractions were collected by anion exchange column chromatography (column: Ecteola Cellulose, 3 × 50 cm), and further purified by preparative high performance liquid chromatography (column: Develosil 100 Diol, 8 × 30 cm). 10 mg of (derived glucose derivative) was obtained.

Experimental example 1
Analysis of the purified product (1) Analysis of component (component sugar) 2N-trifluoroacetic acid was added to the purified product, and hydrolysis was performed at 100 ° C. for 4 hours. The hydrolyzate was reduced by reacting with sodium borohydride and further reacted with acetic anhydride to obtain alditol acetate. This sample was subjected to GC-MS analysis. As a result of analysis, it was found that the main component was glucose and contained a small amount of mannitol (Table 1).

(2) Infrared absorption spectrum analysis As a result of measuring the infrared absorption spectrum (KBr), a characteristic absorption was observed in the β-anomer appearing in the vicinity of 891 cm ⁻¹ . Further, absorption near 844 cm ⁻¹ characteristic of α-anomer was not observed. From this result and the result of said structural component, it turned out that this purified product is (beta) -glucose (FIG. 1).

(3) Molecular weight analysis When measured by high performance liquid chromatography (column: Develosil 100 Diol, 8 x 30 cm), a molecular weight distribution of 1,700 to 9,500 was shown. The peak top was 4,500. Pullulan was used as a standard substance (molecular weight marker).

(4) Methylation analysis The purified product was completely methylated with methyl iodide. 2N-trifluoroacetic acid was added to the methylated product, and hydrolysis was performed at 100 ° C. for 4 hours. The hydrolyzate was reduced by reacting with sodium borohydride and further reacted with acetic anhydride to obtain alditol acetate. This sample was subjected to GC-MS analysis. As a result of analysis, β-1,3-bonded glucose is the main chain, and among these, glucose has a structure in which the 1-position of glucose is bonded to the 6-position of some glucose as a side chain. It was suggested that glucose having a bond at the 6-position has a structure in which a small amount is bound in the molecule (Table 2).

(5) ¹ H-NMR analysis The purified product was dissolved in ¹ N sodium hydroxide aqueous solution so as to be 2%, and ¹ H-NMR analysis was performed. As a result, it was found from the two signal results of about 4.7 ppm and about 4.5 ppm that the ratio of β-1,3 bond / β-1,6 bond was 9.42 (FIG. 2).

As a result of the analyzes of (1) to (5) above, it was estimated that the purified product had the structure shown below.
(Chemical formula 2)
Here, the ratio of β-1,3 bond / β-1,6 bond is 9.42, and the content of mannitol in the glucose derivative is about 3% by weight. However, the arrangement of [] is in no particular order.

Formulation Example 1 Lotion Prescription Formulation Amount (parts)
1. Ainu Wakame-derived glucose derivative (Production Example 1) 0.1
2. 1,3-butylene glycol 8.0
3. Glycerin 2.0
4). Xanthan gum 0.02
5. Citric acid 0.01
6). Sodium citrate 0.1
7). Ethanol 5.0
8). Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1
10. Perfume proper amount11. [Manufacturing method] Components 1 to 6 and 11 and components 7 to 10 are uniformly dissolved in purified water, and both are mixed and filtered to obtain a product.

Comparative Example 1 Conventional lotion In Formulation Example 1, a conventional lotion was prepared by replacing the Ainu Wakame-derived glucose derivative with purified water.

Formulation Example 2 Cream Formulation Blending amount (parts)
1. Ainu Wakame-derived glucose derivative (Production Example 1) 0.05
2. Squalane 5.5
3. Olive oil 3.0
4). Stearic acid 2.0
5. Beeswax 2.0
6). Octyldodecyl myristate 3.5
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Behenyl alcohol 1.5
9. Glycerol monostearate2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.2
12 Ethyl paraoxybenzoate 0.05
13.1,3-Butylene glycol 8.5
14 [Manufacturing method] Components 2 to 9 are heated and dissolved and mixed with purified water to a total amount of 100, and kept at 70 ° C to obtain an oil phase. Ingredients 1 and 11 to 14 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring. The component 10 is added at 45 ° C., and further cooled to 30 ° C. to obtain a product.

Comparative Example 2 Conventional Cream In Formulation Example 2, the Ainu Wakame-derived glucose derivative was replaced with purified water to obtain a conventional cream.

Formulation Example 3 Tablet Formulation Blending amount (parts)
1. Ainu Wakame-derived glucose derivative (Production Example 1) 5.0
2. Dry corn starch 25.0
3. Carboxymethylcellulose calcium 20.0
4). Microcrystalline cellulose 40.0
5. Polyvinylpyrrolidone 7.0
6). Talc 3.0
[Production method] Components 1 to 4 are mixed, and then an aqueous solution of component 5 is added as a binder to form granules. Ingredient 6 is added to the molded granules and compressed. One tablet is 0.52 g.

Formulation Example 4 Beverage Formulation Blending amount (parts)
1. Ainu Wakame-derived glucose derivative (Production Example 1) 0.05
2. Stevia 0.05
3. Malic acid 5.0
4). Fragrance 0.1
5. Bring the total amount to 100 with purified water.
[Production Method] Components 2 and 3 are dissolved in a small amount of water. Components 1, 4 and 5 are then added and mixed.

  Next, experimental examples will be given to explain the effects of the present invention in detail.

Experimental Example 2 Evaluation of Inhibitory Effect on Melanin Synthesis by Inhibition of Differentiation of Stem Cell-Derived Glucose Derivatives from Stem Cells to Melanocytes In this experimental example, mouse embryonic stem cells (ES cell: embryonic) reported by Yamane et al. stem cell to differentiation from stem cells to melanocytes (Yamane T., Hayashi S., Mizuguchi M., Yamazaki H., Kunisada T., Developmental 21, V, 99, Developmental Dynamics, 19) 450-458), the effect of inhibiting the induction of differentiation of Ainu seaweed-derived glucose derivatives into melanocytes and the effect of inhibiting melanin synthesis associated therewith were investigated. In this experiment, arbutin, which has been confirmed to have an inhibitory effect on melanin synthesis, was also evaluated using a similar experimental system. Details will be described below.

MEF cells (mouse embryonic fibroblast) treated with mitomycin C were cultured on a 6 cm plastic dish, and 10 mouse cells (10 × 10 ^{4 to} 20 × 10 ⁴ cells) were seeded thereon, and 5% CO ₂ incubate at 37 ° C. It was precultured in a cultivator. The medium used was the addition of ES cell additive factors (L-glutamine solution, 2-mercaptoethanol solution, nucleoside solution, non-essential amino acid solution and ESGRO, all manufactured by Chemicon) to DMEM, What added 15% of FBS was used.
Next, ST2 cells were cultured until they became confluent on a 24-well plate, and 250-500 cultured ES cells separated from MEF cells were seeded there. The culture is cultured in a differentiation-inducing medium (α-MEM supplemented with 10% fetal bovine serum, 100 nM dexamethasone, 20 pM basic fibroblast growth factor, 10 pM cholera toxin and 100 ng / mL endothelin 3), and ES. Cells were induced to differentiate into melanocytes. In 24 days after induction of differentiation, melanocytes appeared and melanin synthesis was observed.
In this induction system, Ainu seaweed-derived glucose derivatives were continuously added to the differentiation induction medium at the respective concentrations (12.5, 25, 50 μg / mL) to induce differentiation for 24 days. The same experiment was conducted for arbutin. Thereafter, the relative cell number was quantified using Cell Counting Kit-8, and the cells were washed 3 times with PBS (−), and then lysed with 2N NaOH at 60 ° C. for 2 hours. Absorbance at 475 nm was measured for the lysate, and the amount of melanin synthesis was quantified. As a result, by adding the Ainu Wakame-derived glucose derivative, differentiation into melanocytes was suppressed, and the amount of melanin synthesis per number of cells was significantly reduced in a concentration-dependent manner.

These test results are shown in Table 3. A remarkable effect of inhibiting the induction of melanocyte differentiation was observed in the Ainu Wakame-derived glucose derivative (Production Example 1). From the above, it was clarified that the Ainu Wakame-derived glucose derivative has extremely excellent inhibitory effects on melanocyte differentiation induction and melanin synthesis from stem cells, and the Ainu Wakame-derived glucose derivative induces melanocyte differentiation induction inhibitor and melanin synthesis. The effect as an inhibitor (whitening agent) was recognized. Arbutin, which has been confirmed to have a conventional whitening effect, did not show an inhibitory effect on melanocyte differentiation induction in this test system.

Experimental Example 3 Use Test 20 females (22 to 51 years old) using the lotion of Formulation Example 1, the cream of Formulation Example 2, the conventional lotion of Comparative Example 1 and the conventional cream of Comparative Example 2. ) Was used for 3 months. After use, the effect of improving skin spots and dullness was determined by questionnaire.

The test results are shown in Table 4. As a result, the external preparation for skin containing the Ainu Wakame-derived glucose derivative of the present invention showed excellent stain and dullness improving effects. During the test period, there was no skin problem and there was no problem with safety. There was also no problem with the deterioration of the prescription ingredients.

According to the melanocyte differentiation induction inhibitor of the present invention, it is possible to treat, prevent, and improve dyschromia and stains. For example, the use of the Ainu wakame-derived glucose derivative found in the present invention leads to the resolution of pigmentation disorders and stains from the root. Inhibiting differentiation of undifferentiated cells present in tissues into melanocytes by introducing the Ainu Wakame-derived glucose derivative found in the present invention by oral administration, direct injection into the skin, coating, sticking, etc. Can do.

It is an infrared absorption spectrum chart of a glucose derivative derived from Ainu Wakame. It is a ¹ H-NMR chart of a glucose derivative derived from Ainu Wakame.

Claims (3)

A β-1,3-1,6-D-glucose derivative having all the following features (1) to (3).
(1) It originates from Ainu sea turtle (Alaria praelonga Kjellman).
(2) The ¹ H NMR spectrum of a solution using 1N sodium hydroxide heavy aqueous solution as a solvent has two signals of 4.7 ppm and 4.5 ppm, and the area ratio is 7: 1 to 11: 1.
(3) D-mannitol is bound, and the content of D-mannitol is 0.1 to 10% by weight based on the total amount of the glucose derivative. Β-1,3-1,6-D-glucose derivative represented by the following general formula.
(Chemical formula 1)
Here, the β-1,3 bond / β-1,6 bond ratio is 7 to 11, and the mannitol content of the glucose derivative is 0.1 to 10% by weight. However, the arrangement of [] is in no particular order. A composition for treating dysplasia associated with abnormal differentiation of melanocytes, comprising the β-1,3-1,6-D-glucose derivative according to claim 1 or 2.