JP7205880B2 - MELANOCYTE DIFFERENTIATION INDUCTIVE AND USAGE THEREOF - Google Patents

Description

The present invention relates to a differentiation-inducing agent from stem cells to melanocytes, a composition for improving or treating pigmentary disorders containing the differentiation-inducing agent, and a melanocyte differentiation-inducing method using the differentiation-inducing agent.

Melanocytes are cells that synthesize melanin pigments and determine skin and coat colors of animals. Melanocytes are derived from neural crest cells that derive from the neural tube, which is fundamental to vertebrate development. Neural crest cells migrate symmetrically ventrally from the neural tube into the dermis and mature while proliferating. Finally, after the neural crest cells settle in the hair follicle, they further differentiate and mature into melanocytes and mature.

In addition, in the hair follicle, undifferentiated melanocyte stem cells exist near the bulge region together with mature melanocytes. The undifferentiated melanocyte stem cells are believed to self-proliferate, undergo immature melanoblasts as necessary, and supply mature melanocytes to the epidermis and hair matrix. The supplied mature melanocytes supply pigments to the skin and hair by transferring melanosomes to surrounding keratinocytes. Thus, melanocytes have various stages of differentiation.

Differentiation and proliferation of melanocytes and abnormal melanin synthesis mechanisms cause various diseases. For example, mutations in genes that play an important role in early differentiation of melanocytes cause Waardenburg syndrome, a triad characterized by vitiligo skin, heterochromia iris, and deafness. In addition, when tyrosinase, a protein involved in melanin synthesis, is mutated and the activity of the enzyme is lost, melanin is not produced at all, and the individual develops oculocutaneous albinism type I: OCA1) (Non-Patent Document 1).

Furthermore, vitiligo vulgaris is an acquired and progressive depigmentation that is estimated to affect 1-2.5 million people in Japan. Vitiligo vulgaris is clinically classified into a localized type that develops only in one part of the body, a segmental type that develops unilaterally in a certain innervated area, and a generalized type that is scattered over a relatively wide area. . The cause of vitiligo vulgaris is thought to be autoimmunity against melanocytes or melanin, neuropathy, etc., but the details are unknown.

As a treatment for these pigmentary disorders, various ultraviolet therapies (PUVA therapy that irradiates long wavelength ultraviolet rays, narrow band UVB therapy that selectively irradiates only the effective wavelength (311 nm) for treatment among medium wavelength ultraviolet rays, etc.) and steroid drugs. and other topical treatments, with some success. However, these treatment methods for pigmentary disorders cannot be said to be complete treatments, and pigmentary disorders are still regarded as intractable diseases. Therefore, it is essential to identify factors that can fundamentally solve pigmentary disorders and to develop therapeutic methods.

Stem cells, on the other hand, are cells that have both pluripotency capable of differentiating into various cells and self-proliferation capability capable of maintaining pluripotency (undifferentiated state) even after cell division. Stem cells are present in each tissue in vivo. Stem cells play a role in maintaining tissue homeostasis by supplying new cells when tissue cells are lost due to injury, disease, aging, or the like. In recent years, in the fields of cell transplantation therapy and tissue engineering (regenerative medicine and regenerative cosmetics), active research has been conducted to apply the properties of these stem cells to the regeneration of organs and tissues (Non-Patent Document 2).

In addition, through analysis using a differentiation-inducing system from undifferentiated stem cells to specific cells, agents for promoting the induction of differentiation into various cells have been developed. For example, in Patent Document 1, pioglitazone was discovered as a differentiation induction promoting agent using a differentiation induction system from mesenchymal stem cells to cardiomyocytes. Furthermore, in Patent Document 2, a calcium antagonist was discovered as a differentiation-inducing agent using a differentiation-inducing system from stem cells to osteoblasts. In addition, agents for promoting the induction of differentiation of stem cells into nerve cells, adipocytes and the like have been developed.

Similarly, regarding melanocytes, in the differentiation induction system from stem cells to melanocytes, if a material that promotes the differentiation induction is found, it will be a therapeutic agent for diseases involving melanocytes such as pigmentary disorders, which are more fundamental than ever before. and the development of therapeutic methods are expected.

Cordyceps sinensis (Berkeley) Saccardo (scientific name: Cordyceps sinensis (Berkeley) Saccardo) is a kind of mushroom belonging to the phylum Ascomycota, the order Hypocreales, and the Clavicipitaceae family. It contains sterols and antibacterial ingredients, and has been used in Chinese medicine since ancient times as a miracle drug for longevity, nourishment, and recovery from fatigue. Its effectiveness as an adjuvant drug for cancer treatment is being evaluated by alleviating the side effects of therapy and improving the QOL of cancer patients. Cordyceps sinensis also has angiogenesis-promoting action (Patent Document 3), hepatocyte growth factor production-inducing action (Patent Document 4), antidepressant action (Patent Document 5), and metabolic syndrome prevention/improving action by promoting adiponectin production (Patent Document 6). ) have been reported to exist. However, nothing has been known about the effect of cordyceps to induce differentiation from stem cells to melanocytes.

JP 2009-153514 A WO 06/123699 Japanese Patent Application Laid-Open No. 2008-143868 Japanese Patent Application Laid-Open No. 2008-201749 Japanese Patent Application Laid-Open No. 2007-210898 Japanese Patent Application Laid-Open No. 2007-31302

Bennett DC, Lamoreux ML, Pigment Cell Research, 2003, Vol. 16, No. 4, pp. 333-344 Shinichi Nishikawa et al., Experimental Medicine Special Edition, 2008, Vol.26, No.5, pp.74-80

SUMMARY OF THE INVENTION In view of the above circumstances, the present invention aims to find a material having an activity of promoting the induction of differentiation from stem cells to melanocytes, and to provide a composition for improving or treating an unprecedented fundamental pigmentary disorder. and

In order to solve the above problems, the present inventors have searched for a material that promotes differentiation induction from stem cells to melanocytes by using a differentiation induction system from undifferentiated stem cells to melanocytes as a screening system. , that an extract of cordyceps sinensis has an activity of promoting the differentiation induction from stem cells to melanocytes, leading to the completion of the present invention.

That is, the present invention includes the following.
(1) A differentiation-inducing agent from stem cells to melanocytes, containing an extract of Cordyceps sinensis as an active ingredient.
(2) A composition for ameliorating or treating pigmentary disorders, which contains the differentiation-inducing agent from stem cells to melanocytes according to (1).
(3) A method for promoting the induction of differentiation from stem cells to melanocytes, comprising the step of culturing stem cells in the presence of the agent for promoting the induction of differentiation from stem cells to melanocytes according to (1).

ADVANTAGE OF THE INVENTION According to this invention, melanocyte can be produced from a stem cell very efficiently by promoting the differentiation induction from a stem cell to a melanocyte remarkably. Therefore, the present invention can greatly contribute to the field of treatment, prevention and improvement of pigmentary disorders associated with abnormal differentiation of melanocytes and the field of tissue regeneration using melanocytes. And application to the health field is possible.

The present invention will be described in detail below.
The agent for promoting induction of differentiation from stem cells to melanocytes of the present invention (hereinafter referred to as "agent for promoting differentiation of melanocyte") contains an extract of Cordyceps sinensis as an active ingredient. According to the melanocyte differentiation induction promoting agent according to the present invention, differentiation of melanocytes can be controlled by promoting differentiation from stem cells to melanocytes.

In addition, according to the melanocyte differentiation induction promoting agent according to the present invention, by controlling the differentiation of melanocytes, it is possible to fundamentally prevent, improve and treat diseases such as pigmentary disorders associated with abnormal differentiation of melanocytes. Furthermore, the melanocyte differentiation induction promoting agent according to the present invention can also be used as a research reagent for promoting the differentiation of stem cells into melanocytes.

Cordyceps sinensis (Berkeley) Saccardo (scientific name: Cordyceps sinensis (Berkeley) Saccardo) is an ascomycete (mushroom) belonging to the phylum Ascomycota, the order Hypocreales, and the Clavicipitaceae family. It produces endophytic sclerotia and forms sessile, club-shaped or globular fruiting bodies. Cordyceps sinensis (also known as Cordyceps sinensis) that parasitizes bat moth larvae (Hepialus armoricanus Oberthur) is sometimes called Cordyceps sinensis, but in Japan, it is generally called Clavicipitaceae. Cordyceps belongs to the genus Cordyceps (also referred to as the genus Cordyceps), and ascomycetes that parasitize insects or their larvae and form fruiting bodies (mushrooms) are positioned as Cordyceps sinensis. Therefore, cordyceps used in the present invention include Cordyceps sinensis parasitic on bat moth larvae (Hepialus armoricanus Oberthur), Cordyceps militaris Link, Cordyceps sobolifera B parasitic on cicada larvae, Examples include Cordyceps nutans Pat, which parasitizes stink bugs, and Cordyceps sinensis is preferred. It may also be a complex of the Cordyceps sinensis and a host insect or its larvae.

In the preparation of the cordyceps extract, the portion of the cordyceps to be used includes the fruiting body and the mycelia, but the fruiting body is preferred. Cordyceps sinensis may be used as it is, or may be dried, pulverized, cut into small pieces, or the like.

The extraction method for obtaining the cordyceps extract is not particularly limited, and may be, for example, heat extraction or normal temperature extraction. Examples of solvents used for extraction include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-heptanol, 2-heptanol, etc.), and liquid polyhydric alcohols. (propylene glycol, glycerin, 1,3-butylene glycol, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.) , ethers (ethyl ether, propyl ether, tetrahydrofuran, etc.) and the like. Among these solvents, polar solvents such as water, lower alcohols and liquid polyhydric alcohols are preferred, and water, ethanol, propylene glycol and 1,3-butylene glycol are more preferred. These solvents may be used alone or in combination of two or more. Moreover, pH can be adjusted by adding acid or alkali to these solvents. Furthermore, when ethanol or 1,3-butylene glycol is used as a solvent, ethanol or 1,3- Butylene glycol can be used.

Cordyceps sinensis is subjected to solvent extraction using the solvent described above. The ratio of the solvent to Cordyceps is, for example, 1-50% (w/w), preferably 5-20% (w/w). For example, an extract of Cordyceps sinensis can be obtained by adding water to Cordyceps sinensis (fruiting body etc.) and performing hot water extraction at 95 to 100°C. Alternatively, a lower alcohol (e.g., ethanol, etc.) or liquid polyhydric alcohol (e.g., propylene glycol, 1,3-butylene glycol, etc.) is added to Cordyceps sinensis (fruiting body, etc.) and extracted at room temperature (e.g., 5-35°C). By performing, it is possible to obtain an extract of Cordyceps sinensis.

After solvent extraction, the resulting solvent phase itself can be an extract of Cordyceps sinensis. Alternatively, if necessary, the obtained solvent phase is subjected to treatments such as concentration, dilution, filtration, drying, etc., decolorization treatment with activated carbon, etc., deodorization treatment, etc., and the resulting product is used as an extract of Cordyceps sinensis. be able to. In particular, it is preferable to subject the extracted solution to a treatment such as concentration to dryness, spray drying, or freeze drying, and use the resulting dried product as an extract of Cordyceps sinensis.

The Cordyceps sinensis extract thus obtained is used as an active ingredient of the melanocyte differentiation-inducing and promoting agent according to the present invention. The melanocyte differentiation induction promoting agent according to the present invention can be used as a drug, quasi-drug, cosmetic, or food and drink. Moreover, the extract of Cordyceps sinensis described above can also be used for the production of pharmaceuticals, quasi-drugs, cosmetics, or food and drink. Specifically, the agent for promoting differentiation induction according to the present invention is used as a composition for improving or treating dyschromia or deafness, particularly dyschromia, which is a disease associated with abnormal differentiation of melanocytes, or for manufacturing the composition. can be used for Furthermore, the differentiation-inducing promoter according to the present invention can be used as, for example, cell culture additives, research reagents, medical reagents, cell transplantation agents, and the like.

Here, as pigmentary disorders, for example, Waardenburg syndrome, oculocutaneous albinism type I (OCA1), vitiligo vulgaris, focal albinism, centrifugal acquired vitiligo, Vogt - Koyanagi-Harada disease, senile vitiligo, depigmented nevus, pseudosyphilitic vitiligo, etc.

Furthermore, when the melanocyte differentiation induction promoting agent according to the present invention is used as a drug, quasi-drug, cosmetic, or food or drink, the instruction manual or package contains an extract of cordyceps sinensis to induce melanocyte differentiation. It can be labeled as being characterized by having promoting activity.

When the agent for inducing melanocyte differentiation according to the present invention is used as a drug or quasi-drug, the dosage form is not particularly limited. Examples include external preparations (ointments (creams), solutions, extracts, lotions, emulsions, etc.) and injections. The drug or quasi-drug may optionally be combined with a pharmaceutically acceptable carrier such as an adjuvant, a stabilizer, a wetting agent, an emulsifier, an absorption enhancer and a surfactant, in addition to the cordyceps extract. can be blended together.

When the melanocyte differentiation induction promoter according to the present invention is used as a cosmetic, the dosage form is not particularly limited, but examples include creams, lotions, foams, essences, foundations, packs, sticks and powders. etc. In addition to the Cordyceps sinensis extract, the cosmetic contains oils, surfactants, ultraviolet absorbers, alcohols, chelating agents, pH adjusters, preservatives, thickeners, and other commonly used cosmetic ingredients. Colorants, perfumes and the like can be arbitrarily combined and blended.

The amount of cordyceps extract in the melanocyte differentiation induction promoting agent according to the present invention is, for example, in the case of a skin external preparation, 0.0001% by weight or more in terms of solid matter with respect to the total amount of the melanocyte differentiation induction promoting agent. It is preferably 0.001 to 10% by weight. If it is less than 0.0001% by weight, it is difficult to expect a sufficient effect. On the other hand, if the content exceeds 10% by weight, it is difficult to increase the effect and it is uneconomical. As for the method of addition, it may be added in advance or may be added during the production, and may be appropriately selected in consideration of workability.

By applying the melanocyte differentiation induction promoting agent according to the present invention described above to animals including humans, the induction of differentiation from stem cells to melanocytes can be promoted. In addition, by allowing the melanocyte differentiation induction promoting agent according to the present invention to act directly on in vivo stem cells, it is possible to treat diseases for which promotion of differentiation into melanocytes is effective. In particular, the agent for promoting melanocyte differentiation induction according to the present invention can treat, prevent, or improve pigmentation disorders. Furthermore, by allowing the melanocyte differentiation induction promoting agent according to the present invention to act directly on in vivo stem cells, there is no need for in vitro differentiation of stem cells into melanocytes and transplantation. Thereby, the time required for differentiation into melanocytes can be shortened, and the number of cells required for transplantation can be reduced.

The melanocyte differentiation induction promotion activity from stem cells of the melanocyte differentiation induction promotion agent according to the present invention can be evaluated according to the evaluation method described below with respect to the melanocyte differentiation induction promotion method according to the present invention.

On the other hand, the method for promoting differentiation induction from stem cells to melanocytes according to the present invention (hereinafter referred to as "this method") comprises culturing stem cells in the presence of the melanocyte differentiation-inducing agent according to the present invention. It is a method for promoting the induction of differentiation from cells to melanocytes. According to this method, melanocytes can be produced by promoting differentiation induction from stem cells to melanocytes. In other words, the method can also be used as a method for producing melanocytes.

The stem cells used in this 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, Cells in an undifferentiated state present in muscle and other tissues (collectively referred to as somatic stem cells) (Young H.E. et al., The Anatomical Record Part A, 2004, 276A: 75-102; Alison M.R. et al. ., Journal of Pathology 2009, 217: 144-160); includes stem cells and the like artificially produced by gene introduction 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. For example, iPS cells (induced pluripotent stem cells) (Yang R. et al., Generation of Melanocytes from Induced Pluripotent Stem Cells, Journal of Investigative Dermatology, advance online publication, 11 August 2011), dermal tissue-derived stem cells (Li L. et al., Journal of Cell Science, 2010, 123(Pt 6): 853-60) and differentiation induction from adipose tissue-derived stem cells to melanocytes are known, and these stem cells are used in this method. It can be a stem cell. In addition, stem cells derived from all mammals can be used as long as they have similar characteristics regarding the direction of differentiation and the process of differentiation of stem cells. 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 this method, the stem cell culture medium, the medium for inducing the differentiation of stem cells into melanocytes, and the additives used simultaneously with these mediums are not limited, but include, for example, the following.

The medium for culturing stem cells should be a basic medium containing ingredients necessary for cell survival (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, etc.), with growth factors such as Leukemia Inhibitory Factor (LIF) added. media. Examples of the basal 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. In addition, if necessary, the medium contains 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, the medium preferably contains serum such as FBS (fetal bovine serum) at a content of 1 to 20%. However, since the components of serum differ depending on the lot, and the effect varies, it is preferable to use serum after lot check.

In the culture of stem cells or the induction of differentiation of stem cells into melanocytes, medium exchange is preferably performed once every 2 to 3 days, more preferably every day.

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

In this method, stem cells are first prepared. In stem cell culture (preculture), mouse embryonic fibroblasts (MEFs) treated with mitomycin C (MMC) are used as feeder cells in order to maintain an undifferentiated state. The medium preferably contains LIF, L-glutamine and the like. For example, feeder cells are cultured on the bottom surface of a medium-containing container to a confluent state, stem cells are seeded thereon, and the stem cells are cultured for a predetermined time and culture temperature. After culturing, the stem cells separated from the feeder cells are collected and used for induction of melanocyte differentiation.

Next, in this method, the stem cells are cultured in the presence of the melanocyte differentiation induction promoting agent according to the present invention to promote induction of differentiation from stem cells to melanocytes. In particular, culturing is performed in a melanocyte differentiation induction system. As a melanocyte differentiation induction system, for example, seeding of stem cells on feeder cells (feeder cell monolayer), FBS, DEX (Dexamethasone: dexamethasone), bFGF (basic Fibroblast Growth Factor: basic fibroblast growth factor), CT (Cholera Toxin), EDN3 (Endothelin-3: endothelin-3) and other mediums supplemented with factors promoting melanocyte differentiation (Yamane T., Hayashi S., Mizoguchi M., Yamazaki H. , Kunisada T., Developmental Dynamics, 1999, Vol. 216, Issue 4-5, pp. 450-458). Examples of feeder cells include mouse and human stromal cells such as ST2 cells, OP9 cells, PA6 cells, StromaNKtert, and fibroblasts. The number of stem cells seeded onto confluent feeder cells is, for example, 100 to 1000, preferably 300 to 600 per well of a 24-well plate. Also, FBS is added to the medium at a concentration of, for example, 1-20 (v/v)%, preferably 5-15 (v/v)%. DEX (dexamethasone) is added to the medium at a concentration of, for example, 10-500 nM, preferably 50-200 nM. bFGF (basic fibroblast growth factor) is added to the medium at a concentration of, for example, 1-100 pM, preferably 10-40 pM. CT (cholera toxin) is added to the medium at a concentration of, for example, 1-100 pM, preferably 5-20 pM. EDN3 (endothelin 3) is added to the medium at a concentration of, for example, 10-1000 ng/mL, preferably 50-200 ng/mL.

On the other hand, the concentration of the melanocyte differentiation induction promoting agent according to the present invention to the medium used in the melanocyte differentiation induction system is appropriately determined according to the amount of Cordyceps sinensis extract in the melanocyte differentiation induction promoting agent according to the present invention described above. For example, concentrations of 1 to 500 μg/mL, preferably 10 to 100 μg/mL can be mentioned. During the melanocyte differentiation induction period, the melanocyte differentiation induction promoting agent according to the present invention is periodically added to the medium, for example, once every 1 to 5 days (preferably once a day).

Culture conditions in the melanocyte differentiation induction system include, for example, 35 to 38° C. (preferably 36 to 37° C.) for 20 to 30 days (preferably 22 to 26 days).

The promotion of differentiation induction from stem cells to melanocytes is, for example, compared to stem cells cultured in the absence of the melanocyte differentiation induction promoter according to the present invention, in the stem cells cultured in the presence of the promoter, melanin synthesis amount or melanocyte marker It can be evaluated by determining whether the expression of the gene is increased. Melanin synthesis and expression of melanocyte marker genes are indicators of differentiation of stem cells into melanocytes. Here, an increase in melanocyte marker gene expression means an increase in melanocyte marker gene expression at the mRNA level or protein level in stem cells. Examples of melanocyte marker genes 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 culturing, the amount of melanin synthesis and the number of cells are quantified, and the amount of melanin synthesis per cell number is calculated. When measuring cell counts, it is desirable to use a kit that can collect cells even after measurement, such as the commercially available Cell Counting Kit-8 (manufactured by Dojindo Laboratories). Methods for quantifying melanin include, but are not limited to, a method of lysing the cells whose number was measured after induction of differentiation with 2N NaOH at 60°C for 2 hours and measuring the absorbance at 475 nm of the lysate. not something. Next, the obtained melanin synthesis amount per cell number is compared with the melanin synthesis amount per cell number in stem cells cultured in the absence of the melanocyte differentiation induction promoting agent according to the present invention.

On the other hand, in a method for evaluating melanocyte marker gene expression, mRNA or protein is extracted from cultured cells. Next, the melanocyte marker gene expression level in the resulting mRNA or protein is compared with the gene expression level in stem cells cultured in the absence of the melanocyte differentiation induction and promoting agent according to the present invention. At the mRNA level, for example, confirmation methods include RT-PCR, quantitative PCR, and Northern blotting using primers and probes specific to melanocyte marker genes. At the protein level, for example, immunological methods such as ELISA, flow cytometry, and Western blotting using antibodies specific to proteins encoded by melanocyte marker genes can be used.

Compared to stem cells cultured in the absence of the melanocyte differentiation induction promoting agent according to the present invention, in stem cells cultured in the presence of the melanocyte differentiation induction promoting agent according to the present invention, the amount of melanin synthesis or the expression of melanocyte marker genes is significant (e.g. , 1.05 to 10-fold), it can be judged that the induction of differentiation from stem cells to melanocytes was promoted and melanocytes could be produced.

The melanocytes obtained by the present method described above can generally be cultured outside the body and then transplanted by direct injection or the like to a wound site or a site where tissue regeneration is desired. That is, the obtained melanocytes are used as cell medicines and pharmaceutical compositions, and can be used, for example, for the treatment of pigmentary disorders such as vitiligo.

EXAMPLES The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.
[Example 1] Production Example of Solvent Extract from Cordyceps An example of production of a solvent extract using Cordyceps is shown below.

(Production Example 1) Preparation of Hot Water Extract of Cordyceps 2 L of water was added to 100 g of the dried fruiting body of Cordyceps, and the mixture was extracted at 90-100° C. for 2 hours. After filtering the resulting extract, the filtrate was concentrated under reduced pressure and freeze-dried to obtain 2.9 g of a hot water extract of Cordyceps sinensis.

(Production Example 2) Preparation of 50% Ethanol Extract of Cordyceps 2 L of 50% (V/V) ethanol aqueous solution was added to 100 g of the dried fruiting body of Cordyceps, and extracted at room temperature for 1 week. After filtering the resulting extract, the filtrate was concentrated under reduced pressure and freeze-dried to obtain 2.4 g of a 50% ethanol extract of Cordyceps sinensis.

(Production Example 3) Preparation of Ethanol Extract of Cordyceps 2 L of ethanol was added to 100 g of dried fruit bodies of Cordyceps, and extracted at room temperature for 1 week. After filtering the resulting extract, the filtrate was concentrated under reduced pressure and freeze-dried to obtain 2.3 g of an ethanol extract of Cordyceps sinensis.

(Production Example 4) Preparation of 1,3-butylene glycol extract of cordyceps 100 mL of 1,3-butylene glycol was added to 5 g of the dried fruiting body of cordyceps, and extracted at room temperature for 1 week. By filtering the resulting extract, 96.0 g of a 1,3-butylene glycol extract of Cordyceps sinensis was obtained.

[Example 2] Evaluation of differentiation-inducing and promoting effect of Cordyceps sinensis extract from stem cells to melanocytes Differentiation from stem cells to melanocytes is performed using the cordyceps extracts of Production Examples 1 to 4 produced in Example 1 below. The induction promotion effect was evaluated, and the effect of the extract as a melanocyte differentiation induction promotion agent was confirmed.

In this example, a melanocyte differentiation induction system from mouse ES cells (Yamane T., Hayashi S., Mizoguchi M., Yamazaki H. ., Kunisada T., Developmental Dynamics, 1999, Vol. 216, Issue 4-5, pp. 450-458) was used to examine the effect of Cordyceps sinensis extract.

MMC (mitomycin C)-treated MEF cells (mouse embryonic fibroblasts) were cultured in a 35 mm Petri dish in a confluent state, and 1×10 ⁵ to 2×10 ⁵ mouse ES cells were seeded thereon. Precultured in a _CO2 incubator. The culture medium was prepared by adding ES cell additives (L-glutamine solution, 2-mercaptoethanol solution, nucleoside solution, non-essential amino acid solution, and ESGRO) manufactured by Chemicon to DMEM at recommended concentrations, and then adding 15% FBS. used something.

Then, the ST2 cells were cultured on a 24-well plate until confluent, and 250 to 500 of the cultured ES cells separated from the MEFs were seeded there. The cultures were added to α-MEM with 10% fetal bovine serum (FBS), 100 nM dexamethasone (DEX), 20 pM basic fibroblast growth factor (bFGF), 10 pM cholera toxin (CT) and 100 ng/mL endothelin 3 (EDN3). ) to induce differentiation of the ES cells into melanocytes. Induction of differentiation was carried out for 24 days by continuously adding the Cordyceps sinensis extracts of Production Examples 1 to 4 at various concentrations (25, 50, 100 μg/mL) to the differentiation induction medium. Microscopic observation revealed that ES cell colonies formed on the ST2 cells began to spread 6 days after induction, and melanocytes appeared around the colonies around 18 days after induction. By 24 days after induction, the emergent melanocytes were observed to further synthesize melanin.

On day 24 of induction of differentiation, the relative cell number was quantified using Cell Counting Kit-8 (Dojindo Laboratories). On the other hand, after quantifying the number of cells, the cells were washed three times with PBS(-) and then lysed with 2N NaOH at 60°C for 2 hours, and the absorbance at 475 nm of the lysate was measured. The results of these tests are shown in Table 1 below.

As shown in Table 1, all extracts of Cordyceps sinensis (Production Examples 1 to 4) increased the amount of melanin synthesis per cell number in a concentration-dependent manner, and a remarkable melanocyte differentiation-inducing and promoting effect was observed. From the above, it was clarified that the cordyceps extract has an extremely excellent melanocyte differentiation-inducing and promoting effect from stem cells, and the effect of the cordyceps extract as a melanocyte differentiation-inducing promoting agent was confirmed.

According to the melanocyte differentiation induction promoting agent according to the present invention, it is possible to treat, prevent and improve melanocyte-related diseases such as pigmentation disorders. For example, use of the Cordyceps sinensis extract found in the present invention leads to the resolution of pigmentation disorders (white spots, etc.) from the root. In addition, the Cordyceps sinensis extract found in the present invention can be introduced by oral administration, direct injection, application, application, or the like, and can act directly on stem cells present in tissues, thereby promoting induction of differentiation from stem cells to melanocytes. can be made Furthermore, with this promotion, direct regeneration of skin tissue can also be expected.

Claims (3)

A melanin synthesis accelerator containing a cordyceps hot water or water extract, an ethanol aqueous solution extract, an ethanol extract, or a 1,3-butylene glycol extract as an active ingredient , wherein the final concentration of the active ingredient is 0 A melanin synthesis accelerator characterized by being used in the range of 0.0025% by weight to 0.01% by weight . A composition for promoting melanin synthesis, comprising the melanin synthesis promoting agent according to claim 1. A method for promoting melanin synthesis in melanocytes , comprising the step of culturing stem cells in the presence of the melanin synthesis promoting agent according to claim 1 and inducing differentiation into melanocytes.