WO2015190808A2 - Whitening ability of small stem cell and use thereof - Google Patents

Abstract

The present invention relates to a whitening composition comprising, as an effective ingredient, a small stem cell, specifically, a small stem cell with a diameter of 8㎛or less, or a medium thereof, a preparation method therefor, and a use thereof, and relates to a use of the small cell with a diameter of 8㎛ or less for whitening by unusually remarkably reducing the amount of melanin.

Description

Whitening capacity of small stem cells and their uses

The present invention relates to a whitening composition containing a small stem cell, in particular, a small stem cell of 8 μm or less in diameter or a culture medium thereof as an active ingredient, a method for preparing the same, and a use thereof, wherein the size of 8 μm or less in diameter It relates to the use of the whitening function of exceptionally remarkably reduced melanin possessed by small stem cells.

Human skin color is determined by the concentration and distribution of melanin in the skin. In addition to genetic factors, it is also influenced by environmental or physiological conditions such as ultraviolet rays, fatigue and stress. Melanin is a type of amino acid tyrosine (tyrosinase) acts as an enzyme called tyrosinase (tyrosinase) is converted to dopa (DOPA), dopaquinone (dopaquinone) is produced through a non-enzymatic oxidation reaction.

If the melanin is generated more than necessary, such as blemishes, freckles, spots, such as hyperpigmentation (hyperpigmentation) is caused to bring a cosmetic or health consequences. In addition, as the leisure population increases, the number of people who enjoy being active outside increases the demand to prevent melanin pigmentation caused by ultraviolet rays. Therefore, by inhibiting the synthesis of melanin pigment in the skin, not only can brighten the skin tone to realize skin whitening, but also improve skin hyperpigmentation such as spots, freckles, etc. caused by ultraviolet rays, hormones and genetic causes. have.

Until now, the development of whitening agents has been mainly focused on finding substances that reduce the amount of melanin by inhibiting the activity of tyrosinase, a basic and most important enzyme in melanin biosynthesis. The developed whitening agents include hydro-quinone, thiol, kojic acid, arbutin, vitamin C, etc., but they do not have satisfactory whitening effects and have side effects such as allergy, skin absorption and rapid color change due to long-term use. Attempts to find and develop materials that induce or replace them are an integral part of materials research.

As part of such efforts, studies on cosmetic compositions using stem cells are being actively conducted. Korean Patent Publication No. 10-0848056 (2009.07.23) discloses a method for inhibiting melanin synthesis using adipose derived stem cell cultures, which are mesenchymal stem cells. Although it is described, Republic of Korea Patent Publication No. 10-2009-0116659 (2009.11.11) describes a whitening cosmetic composition comprising a cord blood-derived mesenchymal stem cell culture, but did not obtain a satisfactory whitening effect.

Accordingly, the present inventors have confirmed that when mesenchymal stem cells are cultured, cells of various sizes are usually gathered together, and the melanin amount according to the size of mesenchymal stem cells derived from various tissues is reduced by paying attention to the possibility of influence according to the cell size. Comparing the effect on the results, the present inventors have found that stem cells having a diameter of a specific size or less exhibit an exceptionally excellent whitening effect and have completed the present invention.

Prior art literature

Patent Literature

1. Republic of Korea Patent Publication No. 10-0848056 (2009.07.23)

2. Republic of Korea Patent Publication No. 10-2009-0116659 (2009.11.11)

Non Patent Literature

1.Kobayashi T, Urabe K, Winder A, Jimenez-Cervantes C, Imokawa G, Brewington T, Solano F, Garcia-Borron JC, and Hearing VJ., Tyrosinase related protein 1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis . EMBO J. (1994) 13, 5818-5825

2.Levy Carmit, Khaled Mehdi, David E Fisher., MITF: master regulator of melanocyte development and melanoma oncogene., Trends Mol Med. (2006) 12 (9), 406-414

Barbara Bellei, Enrica Flori, Enzo Izzo, Vittoria Maresca, Mauro Picardo., GSK3βinhibiton Promotes melanogenesis in B16 melanoma cells and normal human melanocytes. Cell. Signal. (2008) 20, 1750-1761

Barbara Bellei, Angela Pitisci, Caterina Catricala, Lionel Larue and Mauro Picardo., Wnt / β-catenin signaling is stimulated by α-melanocyte-stimulating hormone in melanoma and melanocyte cells: implication in cell differentiation., Pigment Cell Melanoma Res. (2010) 24; 309-325

Eun-Jung Lee, Yun Sang Lee, Soonho Hwang, Sanghee Kim, Jae Sung Hwang and Tae-Yoon Kim., N- (3,5-Dimethylphenyl) -3-Methoxybenzamide (A3B5) Targets TRP-2 and Inhibits Melanogenesis and Melanoma Growth., Journal of Investigative Dermatology (2011) 131, 1701-1709

Throughout this specification, numerous papers and patents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present invention uses the size of the stem cells to significantly reduce the amount of melanin, whitening function,

The main object of the present invention is to provide a cosmetic composition for whitening containing a small stem cell or a culture medium thereof of less than 8㎛ diameter as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of hyperpigmentation disorders containing small stem cells of 8 μm or less in diameter or a culture medium thereof as an active ingredient.

In order to solve the above problems, the present invention provides a specific whitening function of stem cells having a small diameter and various uses thereof.

In one embodiment, the present invention provides a cosmetic composition for whitening containing small stem cells or a culture medium thereof having a diameter of 8 μm or less as an active ingredient.

At this time, the small stem cells with a diameter of 8 ㎛ or less or the culture medium thereof has an effect of reducing the amount of melanin, which is a small stem cells with a diameter of 8 ㎛ or less melanocyte synthesis (melanocyte) melanocytes (melanocyte) cells This is because it inhibits melanin expression and promotes the degradation of synthesized melanin. In one embodiment of the present invention, the melanin-producing cells used Melan-A cells or B16F1 cells.

Small stem cells of 8 μm or less in diameter are derived from bone marrow, cord blood, fat, blood, liver, skin, gastrointestinal tract, placenta, nerve, adrenal, epithelial, and uterine stems. Cells, and any one or more stem cells selected from the group consisting of embryonic stem cells, preferably derived from bone marrow, cord blood, or fat; More preferably, adult stem cells derived from cord blood, for example, mesenchymal stem cells derived from cord blood, are most preferred.

The culture medium may be any basal medium suitable for animal cell growth as a basal medium, and includes, but are not limited to, Minimal Essential Medium (MEM), Dulbecco modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), K-SFM (Keratinocyte Serum Free Medium) is mentioned. Most preferably, α-MEM (Minimal Essential Medium) medium is used.

In another aspect, the present invention can provide a pharmaceutical composition for the prevention or treatment of hyperpigmentation disease (hyperpigmentation disease) containing a small stem cell of 8 ㎛ or less in diameter or its culture medium as an active ingredient. The specific configuration thereof is as described above.

"Hyperpigmentation" means blackening or darkening relative to other areas due to excessive increase in melanin, including, for example, blemishes, freckles, senile plaques, or solarlentigines. do.

As such, the present invention is based on the finding that small stem cells of 8 μm or less in diameter or culture medium thereof have the most excellent ability to reduce the amount of melanin as compared with the case of conventional heterogeneous stem cells. It provides excellent melanin pigment synthesis inhibition ability and melanin pigment degradation promoting ability of the stem cells smaller than 8㎛ diameter and various uses thereof.

Figure 1 shows the melanin after treatment of various tissue-derived mesenchymal stem cell cultures to Melan-a cells treated with α-MSH increased melanin synthesis ability.

Figure 2 is a measure of melanin after treatment with a variety of tissue-derived mesenchymal stem cell culture to B16F1 cells treated with α-MSH increased melanin synthesis ability.

Figure 3 is a result showing that the culture of various tissue-derived mesenchymal stem cells are composed of cells of various sizes.

Figure 4 shows the change in cell shape according to the size of various tissue-derived mesenchymal stem cells.

Figure 5 is a graph showing the size distribution of stem cells contained in the culture medium after isolation culture according to the size of cord blood-derived mesenchymal stem cells.

6 is a result of observing the melanin amount reduction effect in the culture of mesenchymal stem cells of various tissues and cell size.

Figure 7 is a graph showing the amount of melanin changes according to the treatment of various tissue-derived mesenchymal stem cells sized to Melan-a cells.

8 is a graph showing the amount of changes in melanin according to the culture solution treatment of adipose tissue-derived mesenchymal stem cells in Melan-a cells.

Figure 9 is a graph showing the amount of melanin changes according to the treatment of bone marrow-derived mesenchymal stem cells sized to Melan-a cells.

Figure 10 is a graph showing the amount of melanin changes according to the treatment of the umbilical cord blood-derived mesenchymal stem cells sized to Melan-a cells.

11 is a result of the melanin synthesis inhibition and degradation promotion according to the treatment of umbilical cord blood-derived mesenchymal stem cells by size.

12 is a secreted protein related to the reduction of melanin, confirmed using Melan-a cells.

Figure 13 is a photograph confirming the melanin reduction results according to the cord blood-derived mesenchymal stem cell culture using artificial skin.

Definitions of terms used in the present invention are as follows.

"Stem cell" means a cell that can develop into any tissue. There are two basic features: first, self-renewal, which produces itself by repeating division, and the ability to differentiate into cells with specific functions according to the environment.

"(Medium) Mesenchymal Stem Cells" is a type of undifferentiated adult stem cells isolated from human or mammalian tissue and may be derived from various tissues. Among adult stem cells, hematopoietic stem cells are mainly non-adherent, but mesenchymal stem cells are mainly adherent cells. In particular, cord-derived mesenchymal stem cells, cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells, nerve-derived mesenchymal stem cells, skin-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells and It may be a placental-derived mesenchymal stem cell, preferably cord blood-derived mesenchymal stem cells. Techniques for separating stem cells from each tissue are already known in the art.

"Stem cell culture medium" is a substance containing a component contained in the culture medium obtained by culturing stem cells, the stem cells for producing the culture medium is not limited in its kind. For example, the stem cells from which the culture is made can be embryonic stem cells and can also be adult stem cells. Furthermore, adult stem cells can be derived from adult stem cells of all tissues. In a specific embodiment of the present invention, the culture medium was prepared using umbilical cord blood-derived adult stem cells.

The term "differentiation" refers to a phenomenon in which the structures or functions of each other are specialized during the division and proliferation of cells, that is, the shape or function of living cells, tissues, etc. changes in order to perform the tasks given to them. . Generally, a relatively simple system is divided into two or more qualitatively different sub systems.

"Medium" refers to a mixture for growth and proliferation of cells and the like in vitro including elements essential for the growth and proliferation of cells such as sugars, amino acids, various nutrients, serum, growth factors, minerals and the like. In particular, the medium of the present invention is a medium for growth and proliferation of stem cells.

"Basic medium" is a mixture containing essential sugars, amino acids, water, and the like necessary for a cell to survive, and is a mixture excluding serum, nutritional substances, and various growth factors. The basic medium of the present invention may be prepared by artificially synthesizing or using a commercially prepared medium. Commercially prepared media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Endothelial differentiation medium (EDM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and Iscove's Modified Dulbecco's Medium, but are not limited thereto. In one embodiment of the present invention, α-MEM (α-Minimal Essential Medium) was used.

The term "proliferation" or "growth" of a cell refers to a cell that divides and is homogeneous, usually increasing in the body of a multicellular organism. In time, it is common for traits to be changed (differentiated) and controlled at the same time.

A “control” cell, tissue, sample, or subject is a cell, tissue, sample, or subject of the same type as the test cell, tissue, sample, or subject.

"Whitening" is an element associated with preventing the darkening of the skin by inhibiting its ability to synthesize melanin pigment in melanocytes, the cells that determine the color of the skin, or by breaking down the synthesized melanin.

"Treatment" means an approach to obtain beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (partially or wholly), detectable or not detected. "Treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Such treatments include the treatments required for the disorders that have already occurred as well as the disorders to be prevented. "Palliating" a disease may reduce the extent of the disease state and / or undesirable clinical signs and / or slow or lengthen the time course of progression as compared to untreated treatment. It means losing.

An "effective amount" is an appropriate amount that affects a beneficial or desirable clinical or biochemical result. An effective amount can be administered once or more. For the purposes of the present invention, an effective amount of inhibitor compound is an amount suitable to temporarily alleviate, ameliorate, stabilize, reverse, slow or slow the progression of a disease state. If the recipient animal can tolerate the administration of the composition, or if the administration of the composition to that animal is suitable, the composition indicates "pharmaceutically or physiologically acceptable". If the amount administered is physiologically important, it can be said that the agent has been administered in a "therapeutically effective amount." The agent is physiologically meaningful if the presence of the agent has resulted in a physiologically detectable change in the recipient patient.

"About" means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for reference quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths. , Amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, varying by about 3, 2 or 1%.

Throughout this specification, the terms “comprises” and “comprising”, unless otherwise indicated in the context, include a given step or element, or group of steps or elements, but any other step or element, or step or element It should be understood that this group is not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to the use of specific whitening functions of stem cells having a small diameter.

Stem cells are cells that have the ability to self-replicate and differentiate into two or more cells. Embryonic stem cells or adult stem cells can be used depending on the source, and in the present invention, various origins are preferred. Adult stem cells derived from tissues, for example, tissue derived stem cells such as fat, uterus, bone marrow, muscle, placenta, umbilical cord blood or skin (epithelial) can be used. In particular, the mesenchymal stem cells (MSCs) are preferred. The mesenchymal stem cells are generally hematopoietic supporting cells (stroma), and have the ability to differentiate into various mesodermal cells including bone, cartilage, fat, and muscle cells, and can easily proliferate while maintaining undifferentiated state. There is a characteristic. In one embodiment of the present invention, adipose, bone marrow and umbilical cord blood-derived mesenchymal stem cells (MSCs) were used.

Most preferably, cord blood-derived mesenchymal stem cells are used.

Umbilical cord blood refers to umbilical cord blood that comes out of the umbilical cord at birth, and contains hematopoietic stem cells and vascular endothelial progenitor cells that make white blood cells, red blood cells, and platelets, and mesenchymal stem cells that make cartilage, bones, muscles, and nerves. Very high value. The characteristics of umbilical cord blood are that the number of hematopoietic stem cells is higher than that of bone marrow or peripheral blood, and their maturity is more immature and much better than the hematopoietic stem cells found in bone marrow. In addition, since it can be obtained through a simple procedure in the discarded umbilical cord (Umbilical cord), and includes a number of hematopoietic stem cells and stem cells compared to the amount, a preferred embodiment of the present invention is mesenchymal stem cells isolated from human umbilical cord blood-derived blood To use.

On the other hand, stem cells according to the invention can be proliferated and cultured according to methods known in the art.

Suitable media are any use that can be developed in the laboratory for the cultivation of animal cells and especially mammalian cells, or can be prepared in the laboratory with the appropriate components necessary for animal cell growth, such as anabolic carbon, nitrogen and / or micronutrients. Possible media can be used.

The medium may be any basic medium suitable for animal cell growth, and as a non-limiting example, a basic medium generally used for culture includes Minimal Essential Medium (MEM), Dulbecco modified Eagle Medium (DMEM), and Roswell Park Memorial Institute. Medium) and K-SFM (Keratinocyte Serum Free Medium), and any medium used in the art can be used without limitation. Preferably, α-MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM / F12 medium, PCM medium, M199 / F12 (mixture) (GIBCO), and MSC expansion medium (Chemicon). To this basal medium, anabolic sources of carbon, nitrogen and micronutrients can be added, including but not limited to serum sources, growth factors, amino acids, antibiotics, vitamins, reducing agents, and / or sugar sources. However, it will be apparent that one of ordinary skill in the art can appropriately culture by a known method by selecting or combining a medium most suitable for stem cells derived from various tissues. In an embodiment of the present invention, α-MEM medium, K-SFM medium and the like were used.

In addition, it is apparent that the culture can be carried out while adjusting conditions such as a suitable culture environment, time, temperature, and the like based on common knowledge in this field.

In one embodiment of the present invention, the mesenchymal stem cells are cultured in α-MEM medium until cell confluence is propagated at about 80-90%, preferably about 90%, for example, PBS. And the like, followed by further incubation in K-SFM medium for about 20-25 hours, preferably 24 hours. The term "confluence (%)" is a term commonly used in the art that expresses 'cell concentration per area (saturation)', and relatively indicates the number of cells per unit area (cell concentration) in cell culture. It is a unit frequently used by those skilled in the art in experiments. The present invention also includes a method for producing a stem cell and a culture medium of such a small size of less than 8㎛.

On the other hand, it may further comprise the step of treating the stem cells cultured in the medium according to the invention with trypsin. Treatment of trypsin with cultured stem cells yields stem cells in the form of a single cell. In this case, trypsin inhibits aggregation between cells so that the cells are treated to have a single cell form. It can be used as long as it can be used.

The present invention relates to the use of the exceptionally excellent whitening function of stem cells having a diameter of 10 μm or less, most preferably 8 μm or less, of the stem cells described above.

In particular, the whitening function of the stem cells having a diameter of less than 8 μm in diameter not only reduces the amount of melanin expression in melanocytes, which are melanocytes, but also promotes the breakdown of the synthesized melanin, thereby reducing the total amount of melanin. It is due to the efficacy of reducing.

Melanin, which determines the color of a person's skin, is caused by an imbalance of hormone balance in the human body, stress, or ultraviolet (UV) exposure.

This production of melanin is made mainly from skin cells called melanocytes, which are melanocytes, which are located in the stratum basale layer, which is the lowest of the epidermis, and is called epidermal cells called keratinocytes. Move. Melanosite is a cell located in the stratum basale of the skin's epidermis and the uvea of the middle layer of the eye, and is a pigment found in skin, eyes and hair through a process called melanogenesis. It is a cell that produces melanin. In general there are between 1000 and 2000 melanocytes per mm 2 of skin and account for 5-10% of the bottom face of the epidermis. Here, melanin plays an important role such as forming a hat-like structure around the nucleus to protect genes from ultraviolet rays and to remove free radicals to protect intracellular proteins. These melanin-degrading enzymes are not present in vivo but are removed by falling off the skin as keratinocytes are separated from the epidermis.

However, sometimes, due to genetic or acquired causes, melanin-producing cells are abnormally present in the dermis and melanin overexpression is caused, such as ota's nevus. If melanin is produced more than necessary, it causes cosmetic hyperpigmentation such as blemishes, freckles, and spots, which may result in cosmetically bad results. As such, normalizing or regulating melanin synthesis and expression is very important and can be beneficially utilized.

Small stem cells of less than 8㎛ diameter of the present invention or their cultures are characterized by exhibiting a remarkable whitening effect as compared with the case where the heterogeneous stem cells of the prior art were mixed. This is because stem cells or their culture medium have excellent melanin synthesis inhibitory ability and melanin pigmentation promoting ability.

Small stem cells less than 8㎛ in diameter of the present invention inhibits the expression and activity of tyrosinase, a key enzyme of melanin synthesis, that is, has an excellent ability to inhibit melanin synthesis by participating in melanogenesis inhibitory mechanism. . It is already known that stem cells have the ability to inhibit tyrosinase activity, but it is not known that stem cells are particularly excellent in their capacity.

In addition, small stem cells of less than 8㎛ diameter of the present invention is characterized in that involved in promoting the degradation of the already synthesized melanin.

Regarding the reduction of melanin, conventionally known stem cell secretion proteins include TGF-β2, TNF-α, IFN-γ, ERK-1, ERK-2, Tyrosinase, IL-1, 2, 3, 4, 5, 6, 10, 17, EGF-R, G-CSF, GM-CSF and TRP-1, etc., but the present inventors have found that CD32 / Fcg Receptor II, Progesterone receptor (Progesterone Receptor), Ferritin (Ferritin), Prohibitin (Prohibitin), Laminin B1 (LamininB1), Thrombospondin (Thrombospondin), C-ERB-4 / HER-4, etc. It was confirmed that more secretion . Proteins secreted from these small stem cells are believed to affect better melanin synthesis inhibition and melanin degradation.

In one embodiment of the present invention, melanin-producing melanin cells (Melan-a) and B16F1 cells are treated with α-MSH (melanozyten stimulierende hormone) to induce melanin expression and mesenchymal stem cells of various tissue sizes By distinction, the melanin amount reduction function was observed. As one of the comparative controls, the effect was compared with RSV (antimelanogenesis activity of resveratrol, antimelanogenic agent) which is known as the most representative substance to reduce melanin.

As is known in the art, culturing mesenchymal stem cells is mixed with stem cells having various size distributions. When only small cells of 8 μm or less are extracted and cultured, tyrosinase activity is suppressed and autophagy is performed. It was confirmed that the melanin amount was most remarkably reduced by promotion, whereas mesenchymal stem cells larger than 20 μm did not affect the melanin amount reduction effect.

In other words, the small stem cells or their culture medium of less than 8㎛ diameter of the present invention compared to the case of the conventional stem cell culture (heterogeneous) mixed with conventional stem cells of various sizes or RSV known to have an excellent melanin reduction effect In addition, the degree to which the amount of melanin is reduced is markedly different.

As for the melanin reduction effect, the "size" of stem cells is the most important factor, and stem cells having a small size of less than 8 μm in diameter are excellent in melanin reduction regardless of the type of stem cell-derived tissue such as adipose tissue, bone marrow or cord blood. Although retaining efficacy, it is particularly preferred to use cord blood derived stem cells. That is, the most excellent melanin synthesis inhibitory ability and the ability to promote degradation are those containing small cord blood-derived mesenchymal stem cells having a diameter of 8 μm or less or their culture solution.

Therefore, the present invention relates to a cosmetic composition for whitening and a method for producing the same comprising a small stem cell or a culture medium thereof having a diameter of 8 ㎛ or less as an active ingredient in one aspect.

'Cosmetic composition' is a composition comprising a small stem cell culture medium of 8 ㎛ or less in diameter, the formulation may be in any form.

Cosmetic compositions of the invention may be prepared in any formulation conventionally prepared in the art, including, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, It may be formulated as an oil, powder foundation, emulsion foundation, wax foundation, spray, and the like, but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be. When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular, in the case of spray, additionally chloro fluorohydrocarbon, propane Propellant such as butane or dimethyl ether. When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan. When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, and microcrystals are used as carrier components. Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used. When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The components included in the cosmetic composition of the present invention may include components conventionally used in cosmetic compositions in addition to the active ingredient, and include conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers. It may include.

Preparation of the cosmetic composition may be prepared by any method commonly used.

In addition, the present invention provides a cosmetic method comprising applying to the human skin a cosmetic composition containing small stem cells or a culture medium thereof of 8 μm or less in diameter of the present invention.

The cosmetic method of the present invention refers to all cosmetic methods for applying the cosmetic composition of the present invention to human skin. That is, all the methods known in the art for applying the cosmetic composition to the skin belong to the cosmetic method of the present invention. The cosmetic composition of the present invention may be used alone or in duplicate, or may be used in combination with other cosmetic compositions other than the present invention. In addition, the cosmetic composition with excellent skin protection effect according to the present invention can be used according to a conventional method of use, the number of times of use can be varied according to the user's skin condition or taste.

When the cosmetic composition of the present invention is a soap, surfactant-containing cleansing or surfactant-free cleansing formulation, it may be wiped off, peeled off or washed with water after application to the skin. As a specific example, the soap is liquid soap, powdered soap, solid soap and oil soap, the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towels and cleansing pack, the surfactant-free cleansing formulation is a cleansing cream , Cleansing lotion, cleansing water and cleansing gel, but are not limited thereto.

In another aspect, the present invention may provide a pharmaceutical composition for preventing or treating hyperpigmentation disease, which contains small stem cells having a diameter of 8 μm or less or a culture medium thereof as an active ingredient.

"Hyperpigmentation" means blacking or darkening relative to other areas by excessive increase of melanin in certain areas of the skin or nails. Preferably, the disease caused by hyperpigmentation includes, but is not limited to, blemishes, freckles, senile plaques, or solar blackig.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, may be administered by intravenous infusion, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like. It is preferable that the route of administration of the pharmaceutical composition of the present invention is determined according to the type of the disease to be applied. For example, the pharmaceutical composition of the present invention is most preferably administered in a manner applied topically to the skin because it is used for the treatment of skin diseases caused by overdeposition of melanin pigment.

As another embodiment, the present invention provides a cosmetic composition and a method for producing a pharmaceutical composition comprising the step of separating the small stem cells of less than 8㎛ diameter. The preparation of the composition may be prepared by any method commonly used, and the process of isolating, culturing and separating small stem cells having a diameter of 8 μm or less is not limited to the method of the present invention and is commonly performed in the art. It can be implemented by the method.

As described above, small stem cells having a diameter of 8 μm or less according to the present invention, or their culture solutions may be usefully used as raw materials for the development of functional cosmetics and pharmaceutical (outside) products that can prevent and treat pigmentation of the skin.

In the present specification, a functional cosmetic composition for whitening and a pharmaceutical composition for treating hyperpigmentation diseases have been described, but the present invention has a whitening effect including small stem cells having a diameter of 8 μm or less or their culture solution as an active ingredient. It will be apparent to those skilled in the art that the present invention may be used in various forms as compositions and methods of using the same.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Materials and methods

In the present invention, human cord blood-derived mesenchymal stem cells provided by MediPost Co., Ltd. (Korea) were used. The cells may be obtained from the umbilical cord blood collection step and the step of separating and culturing the mesenchymal stem cells from the umbilical cord blood. Details of each step are as follows.

In the umbilical cord blood collection phase, the umbilical cord vein is collected from the umbilical vein that is left out of the uterus after birth, and in the case of cesarean section, the umbilical cord is delivered out of the uterus. Collect from vein.

In the present invention, when the cord blood is collected from the umbilical vein extracted from the uterus after delivery, the umbilical vein is collected from the umbilical vein connecting the placenta and the fetus after the newborn is born by aseptic manipulation. After securing the umbilical vein, the cord blood is collected in a cord blood collection bag (bag) containing anticoagulant using a sampling needle.

As a method for isolating and culturing mesenchymal stem cells from the cord blood collected as described above, any of the conventional methods, including the method of Korean Patent No. 10-0494265, can be used (Pittinger MF, Mackay AM, et al., Science, 284: 143-7, 1999; Lazarus HM, Haynesworth SE, et al., Bone Marrow Transplant, 16: 557-64,1995), for example:

The collected cord blood is centrifuged to separate mononuclear cells, and then washed several times to remove foreign substances. After washing, the mononuclear cells are planted in a culture vessel at an appropriate density and cultured. The cells proliferate in a monolayer, of which homogeneous and spindle-shaped long cells are observed. The cells that proliferate in colony form are mesenchymal stem cells. Subsequently, when cells grow to a confluent level, subcultures are performed and grown until the required number of cells is reached.

1.Melanocyte and mesenchymal stem cell culture

The B16F1 mouse melanoma cell line was purchased from American type of culture collection (ATCC), Manassas, VA, USA, and Melan-a melanocyte cells were obtained from Dr. It was sold by Dorothy C.Bennett (St. George's Hospital Medical School, Lodon, UK).

B16F1 cells were incubated at 37 ° C. in 5% CO ₂ incubation, DMEM (dulbecco's modified eagle's medium) containing 10% FBS (fetal bovine serum: Invitrogen, Calsbd, CA) and P / S (penicillin / streptomycin) as medium. : Hyclone, Thermoscientific, Logan, UT) were used. Melan-a cells were RPMI 1640 medium (Hyclone) containing 10% FBS, 1% P / S, and 200 mM PMA (phorbol-12-myristate-13-acetate: Sigma, St. Louis, Mo.) Incubated at.

In addition, cord blood-derived mesenchymal stem cells [hUCB-MSC (umbilical cord blood mesenchymal stem cell)] and bone marrow-derived mesenchymal stem cells [BM (bone maroow) -MSC], which are cultured and stored in Medipost (Korea), Adipose-derived mesenchymal stem cells [Adipo (Adipose derived) -MSC] were cultured in α-MEM (GIBCO) containing 10% FBS and 1% P / S.

2. Preparation of Sample Group Media

Sample group conditioned media was prepared from hUCB-MSC, BM-MSC, Adipo-MSC.

In a 37 ° C., 5% CO ₂ incubator, thaw and culture cells in storage (stored in an LN2 tank), at which time cell confluence is approximately 90% in α-MEM (GIBCO) medium containing 2% FBS. Incubate until propagated.

Then, washed three times with PBS (phosphate buffered saline) and incubated in keratinocyte medium (K-SFM, Keratinocyte Serum Free Medium) without phenol red for 24 hours, and the culture was collected. This was repeated for three days. In addition, the collected culture solution was filtered (Top Filer System, Nunc) and then refrigerated and frozen and used.

3. Preparation of Stem Cell Cultures by Cell Size

The culture of mesenchymal stem cells by cell size was performed as follows.

In a 37 ° C., 5% CO ₂ incubator, thaw and culture the cells in a stored state (stored in an LN2 tank),

After culturing at 37 ° C. and 5% CO ₂ incubator, thawing and culturing the cells in the stored state (stored in LN2 tank), stem cells of small size were collected by using 8 μm and 20 μm membrane filters. . At this time, cells having a diameter of 8 to 20 µm were obtained by using a 20 µm membrane filter, followed by two successive 8 µm membrane filters.

Cultures were incubated in α-MEM (GIBCO) medium containing 10% FBS until cell confluence grew to around 70% and washed three times with PBS. Then, after incubating for 24 hours in keratinocyte (Keratinocyte) medium (K-SFM) to which phenol red was not added, the culture solution was collected and repeated for 3 days. The collected culture solution was filtered (Top Filer System, Nunc) and then refrigerated and frozen.

4. Reagents for Melanin Expression

In order to activate melanin expression, α-MSH (melanozyten stimulierende hormone) (St. Louis, MO, USA) at a concentration of 1 μM in B16F1 mouse melanoma cell line and melan-a melanocytes Treated.

In addition, 50 μM of RSV (antimelanogenesis activity of resveratrol, antimelanogenic agent, Sigma, St. Louis, MO, USA), which is known as the most representative substance to reduce the amount of melanin, was prepared for the comparison control to compare the degree of melanin synthesis reduction. The concentration was treated.

5. Melanin Assay

Both B16F1 melanoma cells and Melan-a cells were thawed in storage to initiate culture. After 24 hours, α-MSH was treated for 24 hours, RSV was used as a reference experiment for the melanin synthesis reduction effect, and each culture was treated for 24 hours for comparison.

Thereafter, the cells were trypsinized to separate and collected from the cell culture plate, and melanin in the cells was separated at 100 ° C. for 30 minutes using a buffer solution in which 1 N NaOH was dissolved in 10% DMSO.

After the melanin separation, the melanin amount was measured at 415 nm using an ELISA plate reader (Victor X3, Perkin Elmer, Waltham, Mass., USA).

6. Determination of melanin synthesis inhibition and degradation promoting ability by stem cell origin and cell size

After separating the mesenchymal stem cells such as bone marrow, adipose, cord blood-derived mesenchymal stem cells into 8, 8-20, and 20 μm by size, how the culture medium cultured from these cells reduces the amount of melanin synthesized by the mechanism. To find out.

6-1. Melanin synthesis inhibition experiment

Umbilical cord blood-derived mesenchymal stem cell culture was treated with α-MSH treated melanocytes by cell size, and then proteins were extracted from melanocytes.

All protein extracts for each condition were protein sample buffer [62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.01% bromophenol blue] (BioRad , Hercules, CA). Each sample was then electrophoresed on SDS-PAGE, transferred to PVDF membrane (BioRad), and anti-tyrosinase antibody (VJ Hearing (provided by NIH, Bethesda, MD)) at 4 ° C. Reacted overnight.

For protein detection, the PVDF membrane was reacted with an HRP-conjugated secondary antibody (Pierce, Rockford, IL), followed by AE-9300 Ez-Capture MG Hours Image Saver HR image capture tool (ATTO, Tokyo, Japan). Was confirmed the activity of tyrosinase.

6-2. Melanin decomposition promotion experiment

In order to confirm whether cord blood-derived mesenchymal stem cell cultures promote melanin synthesis synthesized by α-MSH treatment, autophagy of melanocytes according to the culture solution treatment by cell size was confirmed.

For this experiment, B16F1 cells were transfected with lipofectamine 2000 with the pEGFP-LC3 vector and treated with G418 (1 mg / ml) for 7 days. After single cell dropping into 96 wells, cell lines were prepared by growing viable and fluorescence clones, and the cells were identified using a fluorescence microscope (IX71, Olympus, Japan).

In autophagy, the conversion of GFP-LC3 I to GFP-LC3 II occurs. When this conversion to GFP-LC3 II occurs, GFP-LC3 is targeted to autophagosomes. This can be confirmed through punctate. In order to confirm the autophagy phenomenon, the cells showing the GFP-LC3 punctate among 100 cells identified under the microscope were counted and measured. At this time, 50 μM of RSV (Sigma, St. Louis, MO), which is an autophagy inducer and an anti-melanogenic agent, was used as a positive control.

7. Growth Factors assay

1% protease inhibitor cocktail (Sigma, St. Louis, Mo), 1% phosphotase inhibitor cocktail (Sigma, St. Louis, Mo), and lysis beads in each mesenchymal stem cell Proteins were extracted using beads (Fullmoon biosystems, Sunnyvale, CA), and pure proteins were purified using antibody array analysis kits (Fullmoon biosystems, Sunnyvale, CA). Protein analysis was performed using antibody microarray slides (Fullmoon biosystems, Sunnyvale, CA) to measure expression and analysis using GenePix 4000B scanner (Axon Instrument, USA), Genowiz 4.0TM (Ocimum Biosolutions, India) and UniProt DB. It was.

8. 3D artificial skin experiment method

The present inventors confirmed the melanin amount reduction ability of the stem cell culture at the cellular level, and further confirmed the melanin amount reduction using artificial skin.

3D artificial skin was cultured using MEL-300-B-MelanoDerm tissues (MatTek Co. Kr) and EPI-100-LLMM-NMM-NMM-133 at 37 ° C. and 5% CO 2 incubator conditions. After treatment with DW as a negative control, 2% kojic acid as a positive control, and then subjected to a comparative experiment by treating the culture medium (conditioned media, CM) after separation by cell size. The degree of melanin expression of melanocytes was observed through a microscope.

Example 1 Effect of Melanin Reduction on Melan-a Melanosite

Melan-a cells are melanin-synthesizing cells during culture, which are black when melanin synthesis is activated. Melan-a cells have a large number of cell storage times, that is, cell passage, the treatment of α-MSH increases melanin synthesis in Melan-a cells.

The inventors treated the α-MSH 1μM for 24 hours while culturing the Melan-a cells to double the melanin synthesis ability, and then treated melanin synthesis inhibitors and RSV 50μM known as melanin amount-reducing substance, melano A system was established to reduce the amount of melanin in the site by half. RSV is known to be the most effective of the melanin synthesis inhibitors and melanin amount reducing substance known to date.

On the other hand, the present inventors treated the culture medium containing hUCB-MSC, BM-MSC, Adipo-MSC culture solution for 24 hours to the Melan-a cells and measured the change in melanin amount.

The results are shown in FIG. This is the result obtained by repeating the experiment three times.

As can be seen from Figure 1, when treated with α-MSH induction of melanin synthesis in the control (Control) state when the amount of melanin expression based on 200%, BM-MSC, Adipo-MSC-derived culture was treated In some cases, the amount of melanin synthesis was measured at about 180%, resulting in little melanin content.

However, hUCB-MSC-derived culture medium was found to reduce the amount of melanin by about 2 times than RSV and about 4 times than the control based on the amount of melanin synthesis in the control group treated with α-MSH.

That is, in the conventional cell culture in which cells of various sizes are mixed without classification according to the cell size, the umbilical cord blood-derived mesenchymal stem cells among the stem cells derived from various tissues have the best ability to reduce the amount of melanin. there was.

Example 2 Melanin Reduction Effect in B16F1 Cells

The inventors further performed experiments in the same manner as in Example 1 in B16F1 cells, and the results are shown in FIG. 2.

As a result, when melanin synthesis was induced after B16F1 cells were treated with α-MSH, the melanin content was 200%, and when RSV was treated, 120%, BM-MSC-culture and Adipo-MSC Cultures were measured at 180% and 200% melanin levels, respectively. In other words, the culture medium made from bone marrow and adipose (Adipocyte) -derived mesenchymal stem cells did not reduce melanin levels.

However, as in Example 1, the melanin content was observed to be 110% when the UCB-MSC-culture solution was treated, and this observation also revealed that the melanin content was higher than that of RSV, which is known to be a good melanin synthesis inhibitor. It was confirmed that it can be reduced.

Example 3: Confirmation of cell size distribution and growth morphology

The present inventors attempted to confirm the distribution state of the grown mesenchymal stem cells in various tissue-derived, i.e., fat, bone marrow, and cord blood-derived mesenchymal stem cell cultures.

As a result, as can be seen through Figure 3, it was confirmed that the culture fluid of various tissue-derived mesenchymal stem cells are essentially mesenchymal stem cells of various sizes gathered. In particular, umbilical cord blood-derived mesenchymal stem cells were found to be more diverse in size after cell growth by cell culture than when they were immediately thawed in a stock state. This phenomenon has been shown to be similar in cord blood-derived mesenchymal stem cells as well as bone marrow and fat-derived mesenchymal stem cells.

On the other hand, the present inventors further observed the cell shape change according to the size of various tissue-derived mesenchymal stem cells are shown in Figure 4.

As a result, it was confirmed that stem cells of small size did not show a large change in cell morphology compared to hematogenous cells regardless of tissue origin.

Example 4 Observation of Cell Distribution According to Separation and Culture by Cell Size

The size distribution of mesenchymal stem cells cultured for 6-7 days after cell separation was reconfirmed in order to observe whether the stem cells separated by diameter changed into a heterogenous population of various sizes after culture. The results are shown in FIG.

The size distribution of stem cells contained in the culture medium after heterogeneous cell culture is shown in FIG. 5. A, and the cell size distribution after isolation culture of cells having a diameter of 8 μm or less is shown in FIG. Cell size distribution after separation culture of ˜20 μm cells is shown in FIG. 5. C, and cell size distribution after separation culture of cells having a diameter of 20 μm or more is shown in FIG.

After the mesenchymal stem cells were separated and cultured by diameter, the cell size was analyzed. As a result, the cells were not cultured into heterogenous populations, but only cells of similar sizes exist.

Example 5 Efficacy of Reducing Melanin According to Stem Cell Size

After separating the cells by size as described above, prepare a culture medium (conditioned media), and reduce the melanin amount by treating the culture medium with melan-a cells that stimulated melanin synthesis with α-MSH The effect on the was observed.

The results are shown in FIGS. 6 and 7 by various tissue origins.

First, we observed the effect of melanin reduction effect on the melanin reduction effect of the cell size of fat, bone marrow and umbilical cord blood-derived mesenchymal stem cells by 8 µm or less, 8-20 µm and 20 µm or more. When the melanin reduction effect was increased to confirm that the color fades (Fig. 6).

More specifically, it is 8 μm or less than the case where heterogenous cells are cultured without separation of stem cells, that is, a culture solution made from stem cells including various cell sizes is treated with melan-a cells. When the culture solution of cells having a small diameter size of the melanin amount was reduced by about 12% (Fig. 7).

However, when the cell culture medium having a large diameter of 20 μm or more was treated to melan-a cells, there was no significant difference from that of the control group. This means that mesenchymal stem cells of 20 µm or more in size do not significantly affect melanin amount.

In addition, the mesenchymal stem cell culture medium having a diameter of 8 ~ 20㎛ also did not show a significant melanin reduction effect compared to the control of the mixed state (heterogenous population) (see p value of Figs. 8 to 10).

Example 6 Effect of Reduction of Melanin Amount According to Mesenchymal Stem Cells Derived from Various Tissues

The present inventors separated the fat, bone marrow and umbilical cord blood-derived mesenchymal stem cells by size in order to determine whether the melanin reduction function is largely influenced by the size of the mesenchymal stem cells as well as the type of tissue derived from the tissue. .

The results are shown in FIGS. 7 to 10, and the results of fat, bone marrow, and cord blood-derived mesenchymal stem cells, respectively, are shown in separate graphs.

Umbilical cord blood-derived mesenchymal stem cell culture showed an excellent melanin reduction even when compared with other adult stem cells (eg, fat, bone marrow) derived culture (FIG. 10). In particular, it showed an effect of reducing melanin by about twice as much as when treated with RSV 50μM, which is known to have an excellent effect on reducing the amount of melanin.

Results of the same experiment on bone marrow and adipose-derived mesenchymal stem cell cultures (FIGS. 8 and 9), although not as much as the melanin reduction effect of cord blood-derived mesenchymal stem cell cultures (FIG. 10), control stem cells ( Heterogeneous) showed a 10% reduction in melanin levels when treated with a cell culture medium of 8 μm or less, indicating that the "size" of stem cells was greater than that of stem cells. It was found to be an important determinant.

Example 7: Confirmation of melanin synthesis inhibition and degradation promoting ability

Using the cord blood-derived mesenchymal stem cell culture of the present invention, the melanin synthesis inhibition ability was confirmed by Western blotting analysis of tyrosinase, and the autophagy phenomenon analysis was performed, and the results are shown in FIG. 11. Shown.

As shown in FIG. 11A, when the cord blood-derived mesenchymal stem cell culture medium was treated, especially when the cell culture medium of 8 μm or less was treated, the tyrosinase activity of melanin cells was inhibited as much as that of the RSV treatment group as a positive control group. Was observed.

In addition, as shown in FIG. 11B, when treated with the culture medium of cord blood-derived mesenchymal stem cells, autophagy activity in melanocytes was significantly increased. Furthermore, it was confirmed that autophagy activity in melanocytes was increased by 40% compared to the negative control group, when treated with cell culture medium of 8 μm or less, similar to the RSV treated group.

Through these results, the treatment of the culture solution of cord blood-derived mesenchymal stem cells of 8 μm or less of the present invention promotes melanin by inhibiting melanin synthesis and autophagy activity by inhibiting tyrosinase activity in melanocytes. It was found to reduce.

Example 8 Secretory Proteins Involved in Melanin Reduction

Secreting proteins of small-sized stem cells involved in the reduction of the melanin amount while culturing the cultured Melan-a cells as in Example 1 were examined (FIG. 12). At this time, 650 proteins secreted in the culture were analyzed using the Fullmoon assay method.

The results are shown in the table below.

TABLE 1

In particular, in addition to the protein known in the prior art, CD32 / Fcg Receptor II, Progesterone Receptor, Ferritin, Prohibitin, Laminin B1 ), The thrombospondin (Thrombospondin), C-ERB-4 / HER-4 protein was confirmed that more secretion.

Therefore, it is believed that these proteins secreted from the stem cells of small size affect the ability to inhibit melanin synthesis and promote melanin degradation.

Example 9 Reduction of Melanin in Artificial Skin

The present inventors further treated the cord blood-derived mesenchymal stem cell culture according to the present invention to artificial skin and observed the results.

As a result, as shown in Fig. 13, in the group treated with the cell culture medium of 8 µm or less of the present invention, it was observed that the cells showing melanin pigment decreased significantly after 5 days of the culture solution treatment.

Through these results, regardless of the type of stem cell-derived tissue, stem cells having a small diameter of less than 8 μm secrete various protein-related proteins to inhibit melanin synthesis and promote melanin degradation, thereby reducing melanin amount. It shows the most excellent function, which was found to exert exceptionally remarkable whitening effect as compared to the case of conventional culture (heterogenous) without simply separating the stem cells by size. In addition, when using a cell culture medium having a large diameter of 20 ㎛ or more does not have a significant effect on the melanin reduction effect, it was found that the whitening function of stem cells is greatly affected by the size of stem cells .

Small stem cells having a diameter of 8 μm or less according to the present invention, or a culture medium thereof, exhibit a very excellent whitening effect by significantly reducing the amount of melanin by inhibiting melanin expression in melanin synthesis cells and promoting degradation of the synthesized melanin. It can be used as raw materials, such as functional cosmetics and quasi-drugs, so will be very useful in the field of whitening applications.

Claims (15)

1. A whitening cosmetic composition containing small stem cells or a culture medium thereof having a diameter of 8 μm or less as an active ingredient.
2. The method of claim 1,

   Small stem cells of 8 μm or less in diameter are derived from bone marrow, cord blood, fat, blood, liver, skin, gastrointestinal tract, placenta, nerve, adrenal, epithelial, and uterine stems. Whitening cosmetic composition comprising a cell, and any one or more stem cells selected from the group consisting of embryonic stem cells.
3. The method of claim 2,

   The small stem cells of less than 8㎛ diameter whitening cosmetic composition, characterized in that derived from bone marrow, cord blood, or fat.
4. The method of claim 3,

   The small stem cells of less than 8㎛ diameter whitening cosmetic composition, characterized in that derived from umbilical cord blood.
5. The method of claim 4, wherein

   The small stem cells of less than 8㎛ diameter whitening cosmetic composition, characterized in that the cord blood-derived mesenchymal stem cells.
6. The method of claim 1,

   Whitening cosmetic composition, characterized in that the culture medium using K-SFM (Keratinocyte Serum Free Medium) medium as a basal medium.
7. The method of claim 1, wherein the small stem cells of 8㎛ or less in diameter or its culture medium inhibits melanin synthesis in melanocytes (melanocyte) while reducing the amount of melanin expression, whitening characterized in that the degradation of the synthesized melanin Cosmetic composition for.
8. The method of claim 7, wherein

   The melanin-producing cells are whitening cosmetic composition, characterized in that the Melan-A (Melan-a) cells or B16F1 cells.
9. A pharmaceutical composition for preventing or treating hyperpigmentation disease containing small stem cells or a culture medium thereof having a diameter of 8 μm or less, or a culture medium thereof as an active ingredient.
10. The method of claim 9,

    The small stem cells of less than 8㎛ diameter is a bone marrow-derived, umbilical cord blood, or fat-derived, characterized in that the prevention or treatment of hyperpigmentation disease (hyperpigmentation disease).
11. The method of claim 10,

    The pharmaceutical composition for the prevention or treatment of hyperpigmentation disease, characterized in that the small stem cells of less than 8㎛ diameter are derived from umbilical cord blood.
12. The method of claim 11,

    The small stem cell of less than 8㎛ diameter is a medicinal composition for the prevention or treatment of hyperpigmentation disease (hyperpigmentation disease), characterized in that the cord blood-derived mesenchymal stem cells.
13. The method of claim 9,

    The culture medium is a pharmaceutical composition for the prevention or treatment of hyperpigmentation disease, characterized in that using the K-SFM (Keratinocyte Serum Free Medium) medium as a basal medium.
14. Small stem cells with a diameter of 8 μm or less were incubated and washed in α-MEM (Minimal Essential Medium) medium until confluence was increased to about 80-90%, and then washed in K-SFM (Keratinocyte Serum Free Medium) medium. Method for producing a small stem cell culture medium of less than 8㎛ diameter of claim 1, characterized in that it comprises a step of further incubation for ˜25 hours.
15. 15. The method of claim 14, wherein the washing is performed using PBS (phosphate buffered saline).

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