JP2017035105A - Melanocyte differentiation induction inhibitor and method of using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a melanocyte differentiation induction inhibitor having the activity to inhibit differentiation induction from a stem cell to a melanocyte.SOLUTION: A melanocyte differentiation induction inhibitor comprises dihydroquercetin or dihydroquercetin derivatives or salts thereof as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to, for example, an agent for suppressing differentiation induction from stem cells to melanocytes and a composition for improving or treating dysplasia containing the agent for suppressing differentiation, or for improving stains.

  Melanocytes are cells that synthesize melanin pigments and determine the skin color and hair color of animals. Melanocytes are derived from neural crest cells derived from the neural tube that is the basis of vertebrate development. Neural crest cells migrate to the ventral side symmetrically from the neural tube into the dermis and mature while proliferating. Finally, neural crest cells settle into hair follicles, and further differentiate and mature to differentiate into melanocytes and mature.

  In the hair follicle, undifferentiated pigment stem cells are present in the vicinity of the bulge region together with mature melanocytes. The undifferentiated pigment stem cells are considered to be self-proliferating and supplying mature melanocytes to the epidermis and hair matrix through immature melanoblast as necessary. The supplied mature melanocytes supply pigments to the skin and hair by transferring melanosomes to the surrounding keratinocytes. Thus, there are various differentiation stages in melanocytes.

  Melanocyte differentiation and proliferation as well as abnormalities in the mechanism of melanin synthesis cause various pigment abnormalities and spots. For example, when an abnormality occurs in the migration of neural crest cells, neural crest cells that should move to the epidermis layer remain in the dermis. Remaining in the dermis causes symmetric dermal melanocytosis that is manifested late by various stimuli after birth. The lesion is brown and symmetrical. The disease is overwhelmingly common in women, and it often develops in the late 10s to 20s. Therefore, the involvement of female hormones in symmetric dermal melanocytosis is considered. However, the cause is still unclear.

  In addition, melasma is a stain that occurs predominantly in women, especially in women in their 30s. The clinical manifestations of melasma are symmetrical, brown brown spots that occur around the cheeks, around the mouth and forehead. The onset of melasma begins in the second or third month of pregnancy, gradually increases in severity, and gradually disappears with the onset of menstruation after delivery. However, melasma may persist for a long time and extend after menopause. Furthermore, it is known that melasma is induced by oral contraceptives containing female hormones.

  By the way, a typical external factor that acts on the proliferation and differentiation of melanocytes includes ultraviolet rays. Ultraviolet rays act on epidermal cells and promote the proliferation of various melanocytes and the secretion of differentiation promoting factors. It is also known that ultraviolet rays act directly on melanocytes and improve the expression of receptors for various melanocyte activators. Senile pigment spots are caused by many years of repeated increase in melanocytes and increased melanin synthesis caused by ultraviolet rays. In Japanese, almost 100% of people have senile pigment spots at the age of 60, and there is no gender difference in frequency. Sites that are relatively easily exposed to sunlight, such as the face, shoulders, back, hands, etc., are the most frequent sites of senile pigment spots, and produce brown to black-brown flat spots.

  As described above, when abnormalities occur in the generation, differentiation and proliferation of melanocytes and melanin synthesis, various dyschromia and spots are generated. Since these diseases appear directly as an external expression system, the patient's mental burden is heavy and is one of the factors that hinder quality of life (QOL). Therefore, the identification of melanocyte regulators is essential to fundamentally improve these diseases.

  On the other hand, as a whitening agent for controlling melanocytes, many whitening agents have been developed that focus on the activity inhibitory effect of tyrosinase, an enzyme involved in melanin synthesis. As substances having an inhibitory effect on tyrosinase activity, for example, kojic acid, hydroquinone, L-ascorbic acid and the like are well known. In addition, tyrosinase activity inhibitors and melanin production inhibitors containing natural ingredients such as an extract of a genus Pseudomonas plant (Patent Document 1) and an extract of an Ambudiaceae plant Embellia (Patent Document 2) have been developed. However, these tyrosinase inhibitors mainly act on mature melanocytes (express tyrosinase and have the ability to synthesize melanin) and cannot be said to fundamentally control melanocytes.

  On the other hand, as described above, various differentiation stages (neural crest cells, pigment stem cells, melanoblasts, etc.) exist in melanocytes until reaching mature melanocytes. Spots are caused. Therefore, identification of factors that control the differentiation itself of melanocytes is important for the resolution of fundamental dysplasia and spots. However, in the past, screening systems that use melanoma cells (generated by canceration of melanocytes), melanocytes after final differentiation, mushroom-derived tyrosinase, and the like have been used to search for melanocyte regulatory factors. Therefore, the search of the factor which controls the differentiation of a melanocyte has not been performed conventionally.

  Patent Document 3 discloses an active oxygen scavenger characterized by containing taxifolin, which is a kind of dihydroquercetin. However, also in this document, the effect of the active oxygen scavenger on melanocyte differentiation itself has not been studied.

JP 2008-56587 JP 2005-289922 A JP-A-6-65074

  As described above, the identification of factors that control the differentiation of melanocytes themselves is important for the resolution of fundamental dysplasia and spots.

  Therefore, in view of the above-mentioned circumstances, the present invention finds a material having an activity of suppressing differentiation from stem cells to melanocytes, and provides an unprecedented radical pigmentation improvement or treatment composition and a stain improvement composition. The purpose is to do.

  As a result of intensive studies to solve the above problems, it was confirmed 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. Furthermore, by using this induction system as a screening system, it was found that dihydroquercetin has an excellent effect of suppressing melanocyte differentiation, and the present invention has been completed.

That is, the present invention includes the following.
(1) A melanocyte differentiation induction inhibitor containing dihydroquercetin or a dihydroquercetin derivative or a salt thereof as an active ingredient.
(2) A composition for improving or treating dysplasia, comprising the melanocyte differentiation induction inhibitor according to (1).
(3) A stain improving composition comprising the melanocyte differentiation induction inhibitor according to (1).

  According to the present invention, differentiation of melanocytes can be fundamentally controlled by remarkably suppressing differentiation induction from stem cells to melanocytes. Therefore, the present invention can greatly contribute to the field of treatment, prevention and improvement of dysplasia and stains involving melanocytes, and can be applied to the fields of medicine, pharmaceuticals, quasi drugs, beauty and health. is there.

Hereinafter, the present invention will be described in detail.
As mentioned above, the screening system uses differentiation induction system from undifferentiated stem cells to melanocytes instead of the conventional screening system that uses conventional melanoma cells, terminally differentiated melanocytes and mushroom-derived tyrosinase, etc. As a result of searching for a material that suppresses differentiation of melanocytes per se, it was found that dihydroquercetin has an activity to suppress differentiation induction from stem cells to melanocytes.

  Therefore, the melanocyte differentiation induction inhibitor according to the present invention contains dihydroquercetin as an active ingredient. Moreover, the dihydroquercetin derivative and the salt of dihydroquercetin or a dihydroquercetin derivative can be used as an active ingredient in the melanocyte differentiation induction inhibitor according to the present invention, similarly to dihydroquercetin.

  According to the melanocyte differentiation induction inhibitor according to the present invention, the differentiation of melanocytes can be controlled by suppressing the differentiation from stem cells to melanocytes. In addition, according to the melanocyte differentiation induction inhibitor according to the present invention, by controlling the differentiation of melanocytes, it is possible to fundamentally prevent, improve and treat diseases such as dysplasia associated with melanocytes and blemishes. Furthermore, the melanocyte differentiation induction inhibitor according to the present invention can also be used as a research reagent for suppressing the differentiation of stem cells into melanocytes.

Dihydroquercetin is a compound of formula I below:

Dihydroquercetin has four stereoisomers depending on the configuration at the 2nd and 3rd positions. One such compound of formula II below is called taxifolin:

  Examples of the dihydroquercetin derivative include glycosides in which a sugar is bonded to at least one hydroxyl group at the 3, 3 ′, 4 ′, 5 or 7 position of dihydroquercetin. For example, such a glycoside includes astilbine in which α-L-rhamnose is bonded to the hydroxyl group at the 3-position of taxifolin.

  Furthermore, examples of the salt of dihydroquercetin or a dihydroquercetin derivative include pharmaceutically acceptable salts. Examples of such salts include alkali metal salts such as sodium salts and potassium salts.

  Dihydroquercetin, dihydroquercetin derivatives and salts thereof can be produced, for example, by chemical synthesis or extraction from Siberian larch. Commercially available dihydroquercetin, dihydroquercetin derivatives and salts thereof can be used.

  Dihydroquercetin or a dihydroquercetin derivative or a salt thereof is used as an active ingredient of the melanocyte differentiation induction inhibitor according to the present invention. The melanocyte differentiation induction inhibitor according to the present invention can be used as a medicine, a quasi drug, a cosmetic, or a food or drink. In addition, the above-mentioned dihydroquercetin or dihydroquercetin derivative or a salt thereof can also be used for the production of a pharmaceutical, a quasi-drug, a cosmetic, or a food or drink. In particular, the melanocyte differentiation induction inhibitor according to the present invention can be used as a dysplasia improvement or treatment composition or a stain improvement composition, or for the production of the composition.

  Here, examples of dyschromia include symmetric dermal melanocytosis, liver spots, senile pigment spots, sparrow egg spot, Riehl melanosis, trichodermatosis, hereditary contralateral dyschromia, Addison disease , Photo-petal pigment spots, pigmented abnormal erythema, and the like.

  Furthermore, when the melanocyte differentiation induction inhibitor according to the present invention is used as a medicine, quasi-drug, cosmetic, or food or drink, dihydroquercetin, a dihydroquercetin derivative, or a salt thereof is added to the instruction manual or package. It can be displayed to the effect that it contains and has a melanocyte differentiation induction inhibitory activity.

  When the melanocyte differentiation induction inhibitor according to the present invention is used as a medicine or quasi-drug, the dosage form is not particularly limited, but for example, internal use (tablets, capsules, etc.), External preparations (ointments (creams), liquids, extracts, lotions, emulsions, etc.) and injections are mentioned. In addition to dihydroquercetin or dihydroquercetin derivatives or salts thereof, the drug or quasi-drug includes pharmaceutically acceptable agents such as auxiliaries, stabilizers, wetting agents, emulsifiers, absorption enhancers, and surfactants. These carriers can be combined in any combination.

  When the melanocyte differentiation induction inhibitor according to the present invention is used as a cosmetic, the dosage form is not particularly limited. For example, cream, lotion, foam, essence, foundation, pack, stick and powder Etc. In addition to dihydroquercetin or dihydroquercetin derivatives or salts thereof, the cosmetics include oils, surfactants, ultraviolet absorbers, alcohols, chelating agents, pH adjusters, antiseptics that are commonly used as cosmetic ingredients. Agents, thickeners, pigments, fragrances and the like can be combined in any combination.

  The amount of dihydroquercetin or dihydroquercetin derivative or a salt thereof in the melanocyte differentiation induction inhibitor according to the present invention is not particularly limited, but is preferably 0.00001 to 10% by weight of the total composition, 0.0001 to Most preferred is 1% by weight. If it is less than 0.00001% by weight of the total composition, the melanocyte differentiation induction suppressing effect may not be sufficiently exerted.

  By applying the melanocyte differentiation induction inhibitor according to the present invention described above to animals including humans, differentiation induction from stem cells to melanocytes can be suppressed, and pigmentation disorders or spots can be treated, prevented or improved. be able to. The activity of inhibiting differentiation induction from stem cells to melanocytes by the melanocyte differentiation induction inhibitor according to the present invention can be evaluated according to the following screening method.

  Specifically, the screening method comprises a step of bringing a stem cell into contact with a candidate substance in a melanocyte differentiation induction system, and the amount of melanin synthesis in the stem cell in contact with the candidate substance as compared with the stem cell in the absence of the candidate substance. And a step of determining that the candidate substance is a melanocyte differentiation induction regulator by regulating the expression of a melanocyte marker gene. In this screening method, after inducing differentiation of stem cells in the presence of various candidate substances in the melanocyte differentiation induction system, the amount of melanin synthesis and the number of cells were quantified, and the candidate substance promoted the amount of melanin synthesis per number of cells. In this case, a candidate substance is selected as a melanocyte differentiation induction promoter, or as a melanocyte differentiation induction inhibitor when the amount of melanin synthesis is suppressed. Alternatively, using the expression of the melanocyte marker gene as an index, select the candidate substance as a melanocyte differentiation induction promoter when the candidate substance promotes the expression of the gene, and as a melanocyte differentiation induction inhibitor when the gene expression is suppressed can do.

  Stem cells used in the screening method may be any stem cells that differentiate into melanocytes, such as embryonic stem cells (ES cells); bone marrow, blood, skin, fat, brain, liver, pancreas, kidney, Examples include undifferentiated cells existing in muscle and other tissues (collectively referred to as somatic stem cells); stem cells artificially prepared by gene transfer and the like. These stem cells may be primary cultured cells, subcultured cells, or frozen cells. Preferably, ES cells or stem cells derived from bone marrow, blood, skin or adipose tissue can be used. In addition, stem cells derived from all mammals can be used as long as they have equivalent characteristics regarding the direction of differentiation of the stem cells and the process of differentiation. For example, stem cells derived from mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats and pigs can be used.

  In the screening method, examples of candidate substances include nucleic acids, peptides, proteins, synthetic compounds, microbial culture supernatants, natural components derived from plants and marine organisms, plant extracts, animal tissue extracts, and the like.

  In the screening method, the stem cell culture medium, the differentiation induction medium from stem cells to melanocytes, and the additive used simultaneously with these media are not limited, and examples thereof include the following.

  As a medium for culturing stem cells, basic ingredients including components necessary for cell survival (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, etc.) added with Leukemia Inhibitory Factor (LIF) as growth factors A culture medium is mentioned. Examples of the basic medium include Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), Hank's balanced salt solution, MCDB153 medium and the like. If necessary, the medium may be Epidermal Growth Factor (EGF), Tumor Necrosis Factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27- It may contain supplements, N2-supplements, ITS-supplements, antibiotics, and the like.

  In addition to the above, it is preferable that serum such as FBS (fetal bovine serum) is contained in the medium at a content of 1 to 20%. However, since serum has different components depending on the lot and there are variations in its effects, it is preferably used after lot check.

  The medium exchange in stem cell culture or differentiation induction from stem cells to melanocytes is preferably performed once every 2 to 3 days, more preferably daily. In addition, differentiation induction from stem cells to melanocytes in the melanocyte differentiation induction system is preferably performed for 24 days or more.

  Examples of the container used for stem cell culture or differentiation induction from stem cells to melanocytes include disposable petri dishes, microtiter plates, and the like.

  In stem cell culture (preculture), Mouse Embryonic Fibroblast (MEF) or the like treated with Mitomycin C (MMC) is used as a feeder cell in order to maintain an undifferentiated state. The medium preferably contains LIF, L-glutamine and the like. For example, feeder cells are cultured to a confluent state on the bottom surface of the medium-containing container, stem cells are seeded thereon, and the stem cells are cultured at a predetermined time and a culture temperature. After culturing, the stem cells separated from the feeder cells are collected and used for induction of melanocyte differentiation.

  On the other hand, melanocyte differentiation induction systems include, for example, seeding of stem cells on ST2 cells, which are mouse stromal cells, and FBS, Dexamethasone (DEX), basic Fibroblast Growth Factor (bFGF), Cholera Toxin (CT), Endothelin-3 ( EDN3) and other media containing factors that promote differentiation of melanocytes can be mentioned (Yamane T., Hayashi S., Mizoguchi M., Yamazaki H., Kunisada T., Developmental Dynamics, 1999, Vol. 216, Issue 4-5, pp. 450-458).

  In the screening method, stem cells are first brought into contact with a candidate substance in a melanocyte differentiation induction system. Here, the contact means a state in which the candidate substance affects the differentiation from stem cells to melanocytes. For example, the candidate substance may be simply added to the melanocyte differentiation induction medium. Alternatively, the candidate substance may be added to the melanocyte differentiation induction medium together with a certain kind of carrier substance (protein, lipid, etc.). In addition, candidate substances may be directly introduced into stem cells by microinjection or the like.

  Next, in the screening method, it is determined whether or not the amount of melanin synthesis or the expression of the melanocyte marker gene is regulated in the stem cells in contact with the candidate substance, as compared with the stem cells cultured in the absence of the candidate substance. Melanin synthesis and melanocyte marker gene expression are indicators of differentiation from stem cells to melanocytes. Here, the regulation of the amount of melanin synthesis means a decrease or increase in the amount of melanin synthesis in the stem cells. The regulation of the expression of the melanocyte marker gene means a decrease or increase in the expression of the melanocyte marker gene at the mRNA level or protein level in the stem cell. Examples of the melanocyte marker gene include Mitf-M (Melanocyte-specific Microphthalmia-associated transcription factor) (Tachibana M. Pigment Cell Res. (2000) 13 (4): 230-40. ) And Tyrp1 (Tyrosinase-related protein 1) (Sarangarajan R, Boissy RE. Pigment Cell Res. (2001) 14 (6): 437-44.).

  In the method for evaluating the amount of melanin synthesis, after the culture, the amount of melanin synthesis and the number of cells are quantified, and the amount of melanin synthesis per number of cells is calculated. In measuring the number of cells, it is desirable to use a kit that can collect cells even after measurement, such as a commercially available Cell Counting Kit-8 (manufactured by Dojindo Laboratories). Examples of the quantification method for melanin include, but are not limited to, a method in which the number of cells after differentiation induction is lysed with 2N NaOH at 60 ° C. for 2 hours, and the lysate is measured at an absorbance of 475 nm. It is not something. Next, the amount of melanin synthesis per number of cells obtained is compared with the amount of melanin synthesis per number of cells in stem cells cultured in the absence of the candidate substance.

  On the other hand, in the method for evaluating melanocyte marker gene expression, mRNA or protein is extracted from the cultured cells. Next, the expression level of the melanocyte marker gene in the obtained mRNA or protein is compared with the expression level of the gene in stem cells cultured in the absence of the candidate substance. At the mRNA level, for example, RT-PCR using a primer or probe specific for the melanocyte marker gene, quantitative PCR, or Northern blotting may be used. At the protein level, for example, immunological methods such as ELISA, flow cytometry, and Western blotting using an antibody specific for the protein encoded by the melanocyte marker gene can be mentioned.

  Compared with stem cells cultured in the absence of the candidate substance, the amount of melanin synthesis or the expression of the melanocyte marker gene is significantly increased (for example, 1.5 to 100 times, preferably 2 to 5 times) in the stem cells contacted with the candidate substance Or when it significantly decreases (for example, 1/2 to 1/1000, preferably 1/4 to 1/10), the candidate substance can be determined to be a melanocyte differentiation induction regulator. .

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
[Example 1] Evaluation of inhibitory effect on differentiation induction of dihydroquercetin from stem cells to melanocytes Below, using dihydroquercetin, the effect of inhibiting differentiation induction from stem cells to melanocytes was evaluated, and the induction of differentiation induction of dihydroquercetin into melanocytes was inhibited. It confirmed about the effect as an agent.

  The dihydroquercetin (taxifolin) used in the following examples was a general one available from Wako Pure Chemical Industries, but one obtained from Santa cruz biotechnology or Sigma Aldrich may be used. .

1. Experimental Example 1 Evaluation of the inhibitory effect of dihydroquercetin on induction of differentiation into melanocytes (Melanin quantitative test)
In this experimental example, melanocyte differentiation induction system from mouse ES cells previously developed by Yamane et al. (Yamane T., Hayashi S., Mizoguchi M., Yamazaki H., Kunisada T., Developmental Dynamics, 1999, Vol. 216, Issue 4-5, pp. 450-458), the effect of dihydroquercetin was examined. By using this induction system, it is possible to evaluate materials at all stages from the generation of melanocytes to maturity. Details will be described below.

MEF treated with MMC in a 35 mm dish was cultured in a confluent state, and 1 × 10 ⁵ to 2 × 10 ⁵ mouse ES cells were seeded thereon, and precultured at 37 ° C. in a 5% CO ₂ incubator. The medium used was the addition of chemicon additive factors for ES cells (L-glutamine solution, 2-mercaptoethanol solution, nucleoside solution, non-essential amino acid solution and ESGRO) to DMEM at the recommended concentration, and then 15% FBS. It was what was added.

  Next, ST2 cells were cultured until they became confluent on a 24-well plate, and 250-500 cultured ES cells separated from MEF were seeded there. The culture was cultured in a differentiation-inducing medium in which 10% FBS, 100 nM DEX, 20 pM bFGF, 10 pM CT and 100 ng / mL EDN3 were added to α-MEM, and ES cells were induced to differentiate into melanocytes. Microscopic observation revealed that ES cell colonies formed on ST2 cells began to spread on the 6th day after induction, and melanocytes appeared around the colonies around 18th day. By the 24th day after induction, it was observed that the melanocytes that emerged further synthesize melanin. From the above, it was confirmed that the generation of melanocytes, differentiation and melanin synthesis can all be reproduced by using this induction system.

In this induction system, dihydroquercetin was continuously added at the respective concentrations (12.5, 25, 50 μg / mL) to the differentiation induction medium described above, and differentiation induction was performed for 24 days. Then, the relative cell number was quantified using Cell Counting Kit-8. On the other hand, after quantifying the number of cells, the cells were washed 3 times with PBS (−) and then dissolved in 2N NaOH at 60 ° C. for 2 hours. The absorbance of the lysate was measured at 475 nm. As a result, the amount of melanin synthesis per cell number decreased in a concentration-dependent manner.
The test results are shown in Table 1 below.

  As shown in Table 1, dihydroquercetin showed a remarkable melanocyte differentiation induction inhibitory effect. From the above, it was revealed that dihydroquercetin has an excellent inhibitory effect on melanocyte differentiation induction from stem cells, and the effect of dihydroquercetin as a melanocyte differentiation induction inhibitor was confirmed.

2. Experimental Example 2 Evaluation of inhibitory effect of dihydroquercetin on induction of differentiation into melanocytes (gene expression analysis)
In the same manner as in Test Example 1, differentiation induction from mouse ES cells into melanocytes was performed, and the efficiency of differentiation into melanocytes was evaluated using the expression of melanocyte specific marker genes (Mitf-M and Tyrp1) as an index.

Specifically, dihydroquercetin was continuously added to the above-described differentiation induction medium at a concentration of 50 μg / mL, and differentiation induction was performed for 24 days. On the 24th day of differentiation induction, the cells were collected, washed twice with PBS (−), and RNA was extracted from the cells with Trizol Reagent (Invitrogen). Next, using 2-STEP real-time PCR kit (Applied Biosystems), the extracted RNA was reverse transcribed into cDNA, and then the obtained cDNA was used as a template by ABI7300 (Applied Biosystems). Real-time PCR (95 ° C .: 15 seconds, 60 ° C .: 30 seconds, 40 cycles) was performed. Other operations were carried out in accordance with established methods.
Primer set for Mitf-M:
5'-TGCCTTGTTTATGGTGCCTTCT-3 '(SEQ ID NO: 1), and
5'-TCCCTCTACTTTCTGTAATTCCAATTC-3 '(SEQ ID NO: 2)
Primer set for Tyrp1:
5'-CAACGCTATGCTGAGGACTATGA-3 '(SEQ ID NO: 3), and
5'-GCGGCTATCAGACCATGGA-3 '(SEQ ID NO: 4)
Primer set for Gapdh:
5'-TGCACCACCAACTGCTTAGC-3 '(SEQ ID NO: 5), and
5'-TCTTCTGGGTGGCAGTGATG-3 '(SEQ ID NO: 6)

Regarding the differentiation induction efficiency of each cell into melanocytes, the expression level of the melanocyte marker genes (Mitf-M and Tyrp1) in the cells induced to differentiate without adding dihydroquercetin as a ratio to the expression level of Gapdh mRNA as an internal standard The value of the relative expression level of the calculated melanocyte-specific marker gene was set to 100, and the value of the relative expression level of the melanocyte-specific marker gene in each cell induced to differentiate by adding dihydroquercetin was calculated and evaluated.
The results are shown in Table 2 below.

  As shown in Table 2, dihydroquercetin showed a remarkable inhibitory effect on induction of melanocyte differentiation. On the other hand, arbutin, which is a conventional whitening agent, did not show an inhibitory effect on melanocyte differentiation induction.

[Example 2] Formulation example of dihydroquercetin Dihydroquercetin was formulated as a formulation example as follows.

1. Formulation Example 1 Lotion

[Production method]
Components 1 to 6 and 12 and components 7 to 11 were uniformly dissolved, and then both were mixed and filtered to obtain a product.

2. Comparative Formulation Example 1 Conventional Lotion In Formulation Example 1, a product obtained by replacing dihydroquercetin with purified water was used as a conventional lotion.

3. Formulation Example 2 Cream

[Production method]
Ingredients 2-9 were dissolved by heating and mixed, and kept at 70 ° C. to obtain an oil phase. In addition, Component 1 and Components 11 to 14 were heated and dissolved and mixed, and kept at 75 ° C. to obtain an aqueous phase. Next, the aqueous phase was added to the oil phase to emulsify, and the mixture was cooled while stirring.

4). Comparative Formulation Example 2 Conventional Cream In Formulation Example 2, a product obtained by replacing dihydroquercetin with purified water was used as a conventional cream.

5). Formulation Example 3 Tablet

[Production method]
Components 1-5 were mixed, then 10% water was added as a binder, subjected to extrusion granulation and then dried. Ingredient 6 was added to the molded granules, mixed and tableted. One tablet was 0.52 g.

6). Comparative Formulation Example 3 Conventional Tablet In Formulation Example 3, a product obtained by replacing dihydroquercetin with purified water was used as a conventional tablet.

7). Formulation Example 4 Beverage

[Production method]
Ingredients 1 to 4 were dissolved in a part of the ingredient 5 water with stirring. The remaining water of component 5 was then added and mixed.

8). Comparative Formulation Example 4 Conventional Beverage In Formulation Example 4, a product obtained by replacing dihydroquercetin with water was used as a conventional beverage.

[Example 3] Evaluation of stain and dullness improving effect of preparation containing dihydroquercetin Preparation Example 1 lotion and Formulation 2 cream prepared in Example 2, Comparative Formulation Example 1 lotion and Comparative Formulation Example 2 Using the cream, a 30-month female (18 to 50 years old) suffering from spots and dullness was used for a 2-month use test.
After use, the effect of improving spots and dullness was determined by a questionnaire.
The results are shown in Table 7.

  As shown in Table 7, the lotion of Formulation Example 1 and the cream of Formulation Example 2 were superior in the effect of preventing and improving spots and dullness compared to the lotion of Comparative Formulation Example 1 and the cream of Comparative Formulation Example 2. During the test period, there were no skin problems and no safety problems. Moreover, there was no problem also about deterioration of the prescription component of the lotion of prescription example 1 and the cream of prescription example 2.

  Similarly, the tablets of Formulation Example 3 and the beverage of Formulation Example 4 were tested for use by oral ingestion, and showed a safe and excellent anti-smudge and dullness-improving action. During the test period, there were no subjects who were unwell, and there was no problem in safety. Moreover, there was no problem about deterioration of the prescription component of the tablet of prescription example 3 and the drink of prescription example 4.

  According to the melanocyte differentiation induction inhibitor according to the present invention, it is possible to treat, prevent, and improve dyschromia and stains. For example, by using dihydroquercetin found in the present invention, it leads to the solution of fundamental pigment abnormalities and spots. By introducing the dihydroquercetin found in the present invention by oral administration, direct injection into the skin, application, sticking, etc., differentiation of undifferentiated cells present in the tissue into melanocytes can be suppressed.

Claims (2)

Contacting stem cells with a candidate substance in a melanocyte differentiation induction system;
Compared with the stem cells in the absence of the candidate substance, the amount of melanin synthesis and / or the expression of the melanocyte marker gene is regulated in the stem cells in contact with the candidate substance, whereby the candidate substance is regulated to induce melanocyte differentiation. Determining that it is an agent;
A screening method for a melanocyte differentiation induction regulator.   The method of claim 1, wherein the melanocyte differentiation induction system comprises seeding of stem cells on ST2 cells and the use of a medium containing fetal bovine serum, dexamethasone, basic fibroblast growth factor, cholera toxin and endothelin-3. .