[1]Herein is a method for screening a composition and a skin-lightening effect material for skin whitening is provided.
BACKGROUND
[2]
Most animal cells are all biological, including different sizes and extracellular vesicles within the origin cell having a component which has the ability to secrete (extracellular vesicle), these extracellular follicle is blood, urine, saliva, and cell culture It is found in fluid (biological fluids) (Loyer X, Vion AC, Tedgui A, Boulanger CM Microvesicles as cell-cell messengers in cardiovascular diseases Circ Res 2014; 114:.. 345-53) (Ohno S, Ishikawa A, Kuroda M . Roles of exosomes and microvesicles in disease pathogenesis Adv Drug Deliv Rev 2013; 65:. 398-401).
[3]
Extracellular vesicles are smaller in diameter significantly from about 20 nm is a film structure vesicles body having a size of about 5 ㎛ diameter, there is heterogeneity in the size and configuration, the exo-bit (exosome), ekto bit (ectosome), micro-vesicles (microvesicle), it comprises a plurality of different species, such as microparticles (microparticle), apoptotic bodies (apoptotic body).
[4]
Such extracellular vesicles will reflect the state of the origin cells (donor cells) to secrete represents various biological activities depending on whether secretion in some cells, while moving the genetic material and protein between cells play an important role in interactions between cells to be carried out.
[5]
In addition, melanin (melanin) is a black pigment and a biopolymer material of phenols having a composite form of the protein, apple, potato, shell plume of browning or animals to occur when the cut surface of the bananas to be exposed to the air, skin, hair, It is observed in the eye. If melanin is over-produced are deposited on the skin spots, freckles, etc. is formed and promoting skin aging, skin cancer may also be induced.
[6]
Melanin-forming cells in humans (melanocyte) is a synthesis of melanin, as part of a defense mechanism for the ultraviolet radiation from the external cell, and serves for preventing the skin from being killed by the ultraviolet light. When the ultraviolet light from the outside will be exposed to the skin during the skin tissue cells melanin forming cells which synthesize melanin in response to ultraviolet light.
[7]
Melanin synthesis by UV light melanin is a melanin-stimulating hormone (melanin stimulating hormone, MSH) by ultraviolet secretion, MSH is to react the receptor of MC1R for increasing the cAMP in the melanin-forming cells induce reaction of the gene for melanin synthesis the synthesis and the synthesis of melanin is to serve to protect the skin from UV rays is secreted to the exterior of the melanin-forming cells. A protein involved in the biosynthesis of melanin are known include mitf, tyr, trp1, trp2.
[8]
However, it not has been reported about the association of cell death of the exo little secretion and melanin forming cells of secreted in the conventional melanin forming cells. The prior art relates to a method for screening a substance having a whitening active are disclosed in the Republic of Korea Patent No. 10-0840144 call.
Detailed Description of the Invention
SUMMARY
[9]
In one aspect, the present specification aims to provide a composition that is effective in lightening skin through the emission control of a bit in the exo melanin forming cells.
[10]
In another aspect, the present disclosure is to determine the secretion of the exo get secreted by the cells melanin formation is an object of the present invention to provide a method for screening a skin-lightening effect material.
Problem solving means
[11]
In one aspect, the techniques described herein provides a composition for skin whitening comprising the exo-bit inhibitor to inhibit the secretion of exo-bit (exosome) secreted from the melanin-forming cells (melanocytes), as an active ingredient.
[12]
In one exemplary embodiment, the exo-bit inhibitor may be one or more selected from the group consisting of GW4869 and pifithrin-μ.
[13]
In one exemplary embodiment, the active ingredient may be to inhibit melanogenesis.
[14]
In one exemplary embodiment, the active ingredient, spots, freckles, black point, nevi, melanoma, pigmentation caused by drugs, inflammation after pigmentation and at least one skin colorant selected from the group consisting of discoloration occurring in dermatitis the deposition disease may be to prevent, improve or treat.
[15]
In another aspect, the techniques described herein provides a way to promote skin lightening comprising applying a composition comprising an effective amount of exo-bit inhibitor to a subject.
[16]
In a further aspect, the melanin over-produce the prevention of disease caused by, comprising administering a composition comprising an effective amount of exo-bit inhibitors in improving or treating a subject in need, the prevention of disease caused by melanin over-produced, It provides an improvement or cure.
[17]
In one exemplary embodiment, the method may include applying the composition in the form of a pharmaceutical composition, a cosmetic composition or a food composition.
[18]
In one exemplary embodiment, the method may include applying the composition to the skin of the subject.
[19]
In another aspect, the techniques described herein provides an exo some inhibitors for promoting target skin whitening.
[20]
In another aspect, the techniques described herein provides an exo some inhibitors for the prevention, improvement or treatment of over-generation due to melanin.
[21]
In another aspect, the techniques disclosed herein includes the steps of processing after the test material irradiated with UV in the melanin-forming cells (melanocytes) is irradiated with ultraviolet rays, or melanin forming cells (melanocytes) after treatment with the test substance on; And it provides a method for screening a skin-lightening effect material comprising a step to determine the relative secretion of the exo-bit (exosome) secreted from the melanin-forming cells.
[22]
In one exemplary embodiment, the relative secretion of some the exo may be a melanin forming cells irradiated with UV without, but checked against the secretion of some exo secreted from a control group of melanin forming cells, the control group treated with the test substance .
[23]
In an exemplary embodiment, after confirming the relative secretion of the exo-bit, a test substance which reduces the relative secretion of some exo may include the step of screening a skin-lightening effect material.
[24]
In one exemplary embodiment, the UV light to UVB 20 mJ to about 30 / cm 2 may be to investigate.
[25]
In one exemplary embodiment, the time to process the test substances on the formation of melanin cell can be 10 to 24 hours.
[26]
In one exemplary embodiment, the secretion of some of the exo may be identified by expression of CD81 or HSP90.
[27]
In another aspect, the techniques described herein provides a composition for skin whitening comprising the substance identified as skin whitening effect material according to the above method as an active ingredient.
Effects of the Invention
[28]
In one aspect, the techniques disclosed herein is effective to control through the discharge of a bit in the exo melanin forming cells provide a composition that is effective in lightening skin.
[29]
In another aspect, the techniques disclosed herein is effective to verify the secretion of exo get secreted by the melanin forming cells provides a method for screening a skin-lightening effect material.
Brief Description of the Drawings
[30]
1 is a result of checking the average size of the exo some emitted from the melanin-forming cells in accordance with an experimental example of this disclosure.
[31]
2 is a result confirmed that happens secretion of an increased exo-bit during the ultraviolet ray irradiation according to the Experimental Example exo little decrease when inhibitor treatment in the present description.
[32]
Figure 3 is the result confirming the effect of exo-bit inhibitor on the viability of the ultraviolet ray irradiation melanin forming cells in accordance with an experimental example of this disclosure.
[33]
Figure 4 is the result confirming that the melanin forming cells without the UVB irradiation in accordance with an experimental example of the present specification there is no significant difference in cell viability in accordance with some exo.
Best Mode for Carrying Out the Invention
[34]
Hereinafter, the present invention will be described in detail.
[35]
In one aspect, the techniques described herein provides a composition for skin whitening comprising the exo-bit inhibitor to inhibit the secretion of exo-bit (exosome) secreted from the melanin-forming cells (melanocytes), as an active ingredient.
[36]
As that in the present specification means "exo-bit (exosome)" is secreted in the extracellular of the emitted nanoscale the extracellular space cell vesicles, i.e. extracellular vesicles (extracellular vesicles), exo inner bit are sieve film structure vesicles and is outside the segment, and passes the other cells and binds to tissue membrane components, proteins (growth hormones, cytokines, etc.), RNA such as cells are known to play a variety of roles, such as mediating the communication between the cells . On the other hand, the periphery of the melanin forming cells, there is a material between the cells and the cells that surround the melanin-forming cells. Specifically, a constant radius, for example, within 2 mm from the melanin-forming cells, and the material between the cells, such as keratinocytes or fibroblasts and / or collagen can be present, melanin forming cells secrete the exo-bit in the extracellular matrix.
[37]
In one aspect, the bit exo can keulil extracellular Bezier having a diameter of 20 to 400 nm.
[38]
In this specification, "bit exo inhibitor" as meaning a substance which suppresses the secretion of exo little is secreted from the melanin-forming cells, melanin-forming cells produced in the bit within the exo, to inhibit the secretion or release of some exo from the melanin-forming cells the concept of the broadest to mean a material.
[39]
In one exemplary embodiment, the exo-bit inhibitor may be one or more selected from the group consisting of GW4869 and pifithrin-μ.
[40]
"GW4869" is a molecular formula C 30 H 30 Cl 2 N 6 O 2 to a sphingomyelinase (Smase) inhibitors having the general formula is represented by 1 (CAS 6823-69-4). Sphingomyelinase is sphingomyelin to produce ceramide and phosphocholine to hydrolyze sphingomyelin - a specific phospholipid Piaget (phospholipase) C.
[41]
Formula 1

[42]
"Phi trim (pifithrin) -μ" is a molecular formula C 8 H 7 NO 2 to a p53 inhibitor has the S is represented by the formula 2 (CAS 64984-31-2). cancer gene p53 is controlled so as to control cell proliferation unnecessarily. However, the critical to the survival of the cells when the abnormal transient with p53 activation, pifithrin during radiotherapy normal cells was identified as a compound to increase cancer cell death while being protected.
[43]
[Formula 2]

[44]
In one exemplary embodiment, the active ingredient is effective for preventing, treating or ameliorating skin damage or skin disorders that are associated, directly or indirectly, and melanin forming cells increased. That is, the active ingredient has the effect that it effectively suppresses the melanin synthesis, for preventing, treating or improving the diseases caused by excessive generation of melanin.
[45]
Due to the melanin over-produce disease is liver spots, freckles, age spots, blemishes, skin melanocyte lesions (Epidermal melanocytic lesion), milk coffee spots (Cafe's au lait macules), Becker Nevus (Becker's Nevus), questioned the nevus (Nevus Spilus ), black (Lentigines), dermal melanocyte lesions (dermal melanocytic lesions), monggoban (Mongolian spot), Ota nevus (nevus of Ota), acquired bilateral Ota nevus positive half (acquired bilateral nevus of Ota-like macules), Ito nevus (nevus of Ito), the blue nevus (blue nevus), melanin formation cellular nevus (Melanocytic nevus), boundary nevus (Junctional nevus), compound nevus (compound nevus), the dermis within the nevus (Intradermal nevus), unryun nevus ( Halo nevus), congenital melanocyte St. nevus (congenital nevocytic nevus), Spitz nevus (Spitz nevus), dysplastic nevi (dysplastic nevus), melanoma (melanoma), malignant surplus melanoma (Lentigo maligna melanoma), superficial scalability melanoma (Superficial spreading melanoma), leading black St. melanoma (Acral lentiginous melanoma), nodular melanoma (Nodular melanoma), pigmented basal cell carcinoma (pigment basal cell carcinoma), pigmented skin fibrosis (dermatofibromas), pigmented skin cysts (dermoid cyst),
[46]
In one exemplary embodiment, the pigmentation of the composition occurs in pigmentation, after inflammation pigmentation and dermatitis due to spots, freckles, black point, nevi, melanoma, drugs that occurs locally on the skin by increasing melanin synthesis at least one skin pigmentation disorder is selected from the group consisting of may be to prevent, improve or treat.
[47]
In another aspect, the techniques disclosed herein includes the steps of processing, and then irradiated with UV in the melanin-forming cells irradiated with UV light after the treatment with the test substance in (melanocytes), or melanin forming cells (melanocytes) the test substance; And it provides a method for screening a skin-lightening effect material comprising a step to determine the relative secretion of the exo-bit (exosome) secreted from the melanin-forming cells.
[48]
In one exemplary embodiment, the relative secretion of some the exo may be a melanin forming cells irradiated with UV without, but checked against the secretion of some exo secreted from a control group of melanin forming cells, the control group treated with the test substance .
[49]
In an exemplary embodiment, after confirming the relative secretion of the exo-bit, a test substance which reduces the relative secretion of some exo may include the step of screening a skin-lightening effect material.
[50]
In one exemplary embodiment, the UV light to UVB 20 mJ to about 30 / cm 2 may be to investigate.
[51]
In one exemplary embodiment, the time to process the test substances on the formation of melanin cell can be 10 to 24 hours.
[52]
In one exemplary embodiment, the secretion of some of the exo, for example, the same cell quantitation some discrete exo using the same separation from the equipment and Q-Nano or using NanoSight device, nanoparticle tracking analysis (NTA) can the particle to to the target to three or exo some proteins on the surface, a lipid or exo little inside of the proteins, peptides, RNA, miRNA (microRNA), lncRNA (long noncoding RNA), to check over the amount of such metabolites, to quantitatively depending Western blotting (western blot), dot blot (dot blot), ELISA, Northern blot (northern blot), PCR, RT-qPCR, GC-MS, LC-MS, NMR, radiation immunoassay (radioimmunoassay), immunoprecipitation reaction analysis methods such as (immunoprecipitation assay), radiation immunodiffusion (radioimmunodiffusion), FACS, and protein chip (protein chip) are those skilled in the art can be selected appropriately. For example, embodiments of the herein example but not exo although some surfaces of the CD81 or exo visually assess the relative amount of the exo-bit via bit internal Western blot using an HSP90 as a quantitative marker of limitation.
[53]
Exo little released from the melanin-forming cells to the extracellular matrix suppresses the apoptosis of a melanin forming cells ultraviolet irradiation. Thus, it is possible to increase the apoptosis of melanin forming cells by inhibiting the secretion of exo get secreted by the melanin-forming cells, to inhibit Consequently melanogenesis due to apoptosis of these melanin-forming cells, an effect of the whitening efficacy demonstrated It is (Pigment Cell Melanoma Res 2009 Jun; 22 (3):. 307-318).
[54]
In addition, substances that inhibit or increase the release of some exo melanin formation in the cell can be formed by melanin promoting apoptosis of cells (secretion inhibitory substance) or decrease (increase secreted substances) to control the melanin production. Accordingly, there is an effect that it is possible to screen the ultraviolet treatment before or after the skin lightening efficacy candidates by processing the material to a melanin-forming cells to control by confirming the exo secretion little is secreted from the melanin-forming cells skin pigmentation.
[55]
In another aspect, the techniques described herein provides a composition for skin whitening comprising the substance identified as skin whitening effect material according to the above method as an active ingredient.
[56]
According to an exemplary embodiment, the composition may be a pharmaceutical composition.
[57]
The pharmaceutical composition may further contain preservatives, stabilizers, wetting or emulsifying accelerators, pharmaceutical adjuvants and other therapeutically useful substances such as salt and / or buffer for the osmotic pressure in addition to exo-bit inhibitors, conventional methods wide variety of oral administration may hwahal claim or parenteral dosage form in accordance with the second aspect.
[58]
The orally administered agent is, for example, and the form of tablets, pills, hard and soft capsules, solutions, suspensions, emulsions, syrups, powders, powders, fine granules, granules, pellets and the like, these formulations in addition to active ingredient, a surfactant the diluent may contain: (silica, talc, stearic acid and its magnesium or calcium salt, and polyethylene glycol for example) (for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants . Tablets may also contain magnesium aluminum silicate, starch paste, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose and polyvinylpyrrolidine and may contain the same binder, a starch, agar, alginic acid or its sodium salt, as appropriate a disintegrating agents, absorbents, colorants, flavors pharmaceutical additives, such as agents, and sweetening agents, such as may contain. The tablets may be prepared by conventional mixing, granulating or coating methods.
[59]
Further, in the parenteral administration form can be a transdermal dosage forms, such as injections, drops, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories (坐 劑), dosage forms, such as the patch Although one can, without being limited thereto.
[60]
The pharmaceutical composition may be parenteral, administration to the rectal, topical, transdermal, subcutaneous and the like.
[61]
Dose determination of the active ingredient is within the level of ordinary skill, daily doses of the drug varies depending on various factors such as the severity, onset of destination, age, health condition, complications, to be administered, adults On the one side, based on the composition to 1㎍ / kg to 200mg / kg, may be administered to 50㎍ / kg to about 50mg / kg to 1 day divided to three times a day in another aspect, the dosage is any method also not intended to limit the scope of the invention.
[62]
The pharmaceutical composition may be a skin preparation for external use may be, the topical is any would also be included in the various formulations as a generic drug that is applied from the outer skin to be included herein.
[63]
According to an exemplary embodiment, the composition may be a cosmetic composition.
[64]
The cosmetic composition may contain additional components included in addition to some inhibitors exo functional additives and typical cosmetic composition. In the functional additive can include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamin, peptide polymer, polysaccharide polymer, Scotland pinggo lipid and seaweed extract. Maintaining As the component ingredients contained in addition, a moisturizing agent, emollient agent, surfactant, organic and inorganic pigments, organic powder, ultraviolet absorbent, antiseptic, fungicide, antioxidant, plant extract, pH adjuster, alcohol, pigments, perfumes, blood circulation in there may be mentioned promoters, the feeling of cold, limits (制 汗) agents, such as purified water.
[65]
The cosmetic composition may be appropriately selected, as the formulation is not particularly limited, it is an object. For example, skin lotion, skin softening, skin toner, astringent lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, pack, soap, Cleansing foams, cleansing lotion, cleansing cream, but can be made of any one or more dosage forms selected from the group consisting of a body lotion and body cleansers, but is not limited thereto.
[66]
When the formulation of a paste, cream or gel, the animal fiber, plant fiber, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc or zinc oxide and the like be used as the support component can.
[67]
When the formulations of the present invention is powder or spray, lactose, talc, silica, as the support component of aluminum hydroxide, calcium silicate, or and the polyamide powders may be used, particularly if a spray-in, hydrocarbons in addition chlorofluorocarbons, propane / it may comprise propellants such as butane or dimethyl ether.
[68]
If the formulation is a solution or emulsion of the present invention, the solvent, and the solvent agent or emulsifying agent as a carrier component is used, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , a 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester.
[69]
If the dosage form of the invention the suspension has a support component liquid diluent, such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan suspension, such as sorbitan esters claim, microcrystalline such as cellulose, aluminum meta-hydroxide, bentonite, agar or tragacanth may be used.
[70]
The formulation of the present invention a surface-if the surfactant-containing cleansing, the aliphatic alcohol sulfate as carrier components, aliphatic alcohol ether sulfate, sulfosuccinate monoester, isethionate, imidazolinium iodonium derivatives, methyl taurates, sarcoidosis when sulfonates, fatty acid amide ether sulfates, and the like alkyl amido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, Lee Quinoline derivative or ethoxylated glycerol fatty acid esters may be used.
[71]
According to an exemplary embodiment, the composition may be a food composition.
[72]
The food composition is a liquid or may be in solid dosage forms, e.g., various types of foods, drinks, gum, tea, vitamin complex, health supplement foods, etc., and powder, granule, tablet, capsule or beverage used in the form can. Food composition of the respective formulations can be any person skilled in the art by blending the enemy selected without difficulty by a conventional the formulation or use components that are used in the art in addition to the active ingredient, may take place is a synergistic effect when applying at the same time as the other raw material.
[73]
The liquid component, which may contain in addition to the active ingredient disclosed herein has no particular limitations, and the like various flavors or natural carbohydrates as additional ingredients, such as conventional beverage. Examples of the natural carbohydrate is a monosaccharide, glucose, disaccharide, maltose, shoe sugar alcohols such as polysaccharides, dextrin, ordinary sugar and xylitol, sorbitol, erythritol, such as cyclodextrin erythritol, such as a cross, such as fructose and the like. The flavor agent can be used to advantage to natural flavors (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (e. G., Such as saccharin and aspartame) . the content of the natural carbohydrate may be described herein generally range from about 1 to about 20g, about 5 to 12g in one aspect a composition per 100ml.
[74]
The food composition comprises various nutrients in one aspect, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents, coloring agents and mogul agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof such as natural flavorants and it may include an organic acid, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonated agents, and the like used in soft drinks. On the other side it may include pulp for the production of natural fruit and vegetable juice drinks. The components may be used independently or in combination. The content of the additive, but may vary, it is generally selected from the range of composition 100 parts by weight of 0.001 to about 20 parts by weight per disclosed herein.
Mode for the Invention
[75]
The present invention will be described in further detail with reference to the following examples. These embodiments will provide for only illustrative of the present invention, self-evident to a person of ordinary skill in the art, the scope of the present invention is not construed as being limited to these examples.
[76]
Experimental Example.
[77]
(1) Cell culture and materials
[78]
Human melanin forming cells (normal human melanocytes, Cascade Biologics, Portland, OR, USA) was stored in M-254 medium (Cascade) containing the human melanin forming cells growth supplement (Cascade). GW4869 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Phi trim (pifithrin) -μ (Sigma, St. Louis, MO, USA) was used and dissolved in DMSO.
[79]
(2) exo-bit (exosome) separation
[80]
For some separation exo washing confluent melanin forming cells with PBS and incubated for 48 hours in M-254. Centrifuged at 100,000 × g for 10 minutes to collect the conditioned media 500 × a (conditioned medium) g, 20 bungan 3,000 × g, and 2 hours. The obtained exo-bit-containing pellets were stored at -80 ℃ resuspended in PBS. In addition, the conditioned medium was filtered to give a 0.45 ㎛ pore filter (Millipore, Billerica, MA, USA) and, 100 kDa cut-off and concentrated using a spin column (Millipore). Exo some are using ExoQuick-TC kit (SBI, San Jose, CA, USA) were isolated according to the manufacturer's instructions. Separate exo some 20 ㎕ the Western blot and silver staining was analyzed.
[81]
(3) DLS(Dynamic light scattering) 분석
[82]
Exo bit diameter was measured using a Zetasizer Nano S instrument (Malvern Instruments Ltd., Worcestershire, UK) in the scattering intensity 10 × 30 s provided with a 633 nm laser.
[83]
(4) Gel protein separation
[84]
Exo some proteins were separated by SDS-PAGE. How the SDS-PAGE gel cut into 10 slices and group known (Bahk YY, Kim SA, Kim JS, et al (2004) Antigens secreted from Mycobacterium tuberculosis:. Identification by proteomics approach and test for diagnostic marker Proteomics 4:. 3299 -307.) it was isolated within the gel using trypsin (Promega, Madison, WI, USA) according to the. In short, the gel pieces 1: 1 acetonitrile (ACN) / 25 mM ammonium bicarbonate (ABC) to 4-5 times washed, dehydrated, and dried at 100% ACN. In 45 minutes 56 ℃ from the reduction and 30 minutes light blocking at room temperature in 100 mM DTT with 100 mM ABC then Corporate alkyl in 55 mM iodine acetamide with 100 mM ABC, and drying the pieces at 100% ACN 37 ℃ again it was hydrated in 325 mM ABC containing 20 ng of trypsin for 20 hours. Peptide to move the liquid to a fresh tube and the residual by using a 50% (v / v) aqueous acetonitrile containing 0.1% at 30 ℃ 40 bungan (v / v) formic acid was extracted from the gel. Evaporating the combined supernatant and 0.1% for the mass spectrometric analysis (v / v) formic acid containing 5% (v / v) was dissolved in aqueous acetonitrile solution.
[85]
5. Protein Identification by LC-MS / MS
[86]
Separate peptides group known methods (Zuo X, Echan L, Hembach P, et al. (2001) Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble . and large proteins Electrophoresis 22:. was added to slight variations in 1603-15) Finnigan LCQ ion-analyzed using a reversed-phase capillary HPLC directly connected to the trap mass spectrometry (LC-MS / MS). Peptide was coupled for 10 min trapping column and eluted at 0.2 ㎕ / min flow rate of 50 minutes with 0.1% (v / v) 5 ~ 80% (v / v) aqueous acetonitrile containing formic acid gradient. Tandem MS was handled using a was performed at m / z = 450 ~ 2000 Da, each spectra TurboSEQUEST software (Thermo Quest, San Jose, CA, USA). The resulting peak lists were used to query the NCBI database using the MASCOT MSDB or program (http://www.matrixscience.com).
[87]
(6) Western blot and silver staining
[88]
Cell or separate the exo protease inhibitor bit (Sigma) containing a lysis buffer (1% NP-40, 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.01 M MgCl 2 was dissolved using a). The protein concentration was analyzed by BCA assay, the sample is separated by SDS-PAGE, which was moved to the PVDF membrane and analyzed with antibodies to CD81 (SBI). On the other hand, experiments were applied to a SDS-PAGE gel according to the manufacturer's instructions for the dye is staining kit (Thermo Fisher Scientific, Rockford, IL, USA).
[89]
7 Cytotoxicity test
[90]
1 × 10 melanin-forming cells in 6-well plates 4 were dispensed at a density of cell / well. Followed by day, processing a bit 10 ㎕ the exo-isolated from the same cell. Fixing the cells with 4% formaldehyde in PBS and 15 minutes, and stained with 0.1% of 20 minutes, washed with PBS, and crystal violet. After washing with PBS, the cells were dried and dissolved in 10% acetic acid. By using a spectrophotometer to measure the absorbance at 590 nm.
[91]
(8) Quantitative real-time PCR
[92]
Using Trizol reagent (Invitrogen) to remove the total RNA and using the ReverTra Ace (Toyobo, Osaka, Japan) was reverse transcribed into cDNA. Gene expression analysis of TaqMan Universal Master Mix and TaqMan Gene Expression Assays: was performed using Hs00365052_m1 (Applied Biosystems, Foster City, CA, USA).
[93]
(9) Statistical analysis
[94]
The two-tailed Student's t-test was used to analyze the differences between the two groups. Data are mean ± SD for three experiments expressed as (***; p <0.05; p <0.005, **).
[95]
1. The results confirm the secretion of exo get from melanin forming cells
[96]
Melanin forming cells was found to have a mean diameter of the size of some secreted some exo and was released, and separate the exo analysis (dynamic light scattering analysis) as a result, the exo-bit is 206.90 ± 22.18 nm emitted from the melanin-forming cells (see Fig. 1).
[97]
2. Experimental results secreted from melanocytes according to the ultraviolet light irradiation to form the cell bit exo changes in the secretion
[98]
A melanin forming cells UVB (20 mJ / cm 2 irradiated with a), and was found to increase the secretion of some exo when a result, ultraviolet light exposure for 24 hours.
[99]
In addition, when treated with UVB exposure before, GW4869 (Smase (sphingomyelinase) inhibitors) with skin of the trim, or 10 μM (pifithrin) -μ (p53 inhibitor), 10 μM of some of the exo generation / secretion inhibitor for 12 hours, some of the exo It has been found that reducing the amount of CD81 expression markers (see Fig. 2).
[100]
In this way, melanin forming cells was confirmed that an increase in the amount of exo-bit that is released on exposure to ultraviolet rays, and even when exposed to UV light reduces the amount of exo some emitted when applied to the generation of a bit exo / secretion inhibitor.
[101]
3. Experimental results exo little effect of inhibitors on the viability of the ultraviolet irradiation melanin forming cells
[102]
UVB (20 mJ / cm with a little each exo and melanin forming cells from UVB unprocessed a melanin-forming cells, UVB-treated melanin forming cells, UVB + GW4869 treatment melanin forming cells 2 is irradiated with) were incubated for 3 days result, it was found that some of the melanin-forming cells derived from the received ultraviolet exposure exo as shown in Figure 3 is to increase the viability of melanin forming cells receiving the UV damage. The survival promoting effects of a melanin-forming cells was reduced line by a melanin-forming cells derived from exo-bit processing with the GW4869, pifithrin-bit μ secretion inhibitor exo UV. On the other hand, UVB untreated melanin in the same way as the forming cells, UVB each exo-bit obtained from the treated melanin forming cells, but incubated with melanin forming cells when cultured under conditions that do not examine the UVB melanin forming cells, melanocytes there was no significant difference in viability of cells formed (see Fig. 4).
[103]
Thus, the efficacy of inhibiting apoptosis (apoptosis) of sikimyeo melanin receiving the UV exposure to form the cell bit-derived exo increases the viability of a melanin forming cells receiving the UV damage, some exo exiting the UV irradiation during the melanin forming cells melanin forming cells for it was found that it has.
[104]
Accordingly, substances which inhibit or increase the release of some exo from melanin forming cells and promote the apoptosis of melanin forming cells (discharge suppressing material) or decreased (increased emissions), it was confirmed that to control the melanin production.
[105]
One an example formulation of a composition according to an aspect of the invention from below, and also applicable to various other formulations, which are only intended to describe in detail, not intended to limit the invention.
[106]
[ Formulation Example 1] The soft capsules
[107]
By GW4869 or pifithrin-μ 50mg, L- carnitine 80 ~ 140mg, 180mg soybean oil, palm oil 2mg, vegetable hydrogenated oil mixture of 8mg, and 4mg hwangnap lecithin 6mg, and by filling 400mg per capsule by a conventional method to prepare soft capsules .
[108]
[ Formulation Example 2] Tablets
[109]
A mixture of GW4869 or pifithrin-μ 50mg, galacto-oligosaccharide 200mg, Lactose 60mg and 140mg maltose, was added to the ester (sugar ester) per then granulated using a fluid bed dryer 6mg was prepared in the other appointed give the tablet press.
[110]
[ Formulation Example 3 Granules
[111]
GW4869 or pifithrin-μ 50mg, then a solution of the anhydrous crystalline glucose, 250mg and 550mg of starch, and formed into granules using a fluid bed granulated by filling a capsule to prepare a granule.
[112]
[ Formulation Example 4] Drink
[113]
After mixing GW4869 or pifithrin-μ 50mg, glucose 10g, 0.6g citric acid, and a liquid oligosaccharide 25g was added to 300ml of purified water were charged to each bottle by 200ml. After filling the bottle and sterilized at 130 ℃ 4 ~ 5 seconds to produce a beverage drinks.
[114]
[ Formulation Example 5] Lotion
[115]
To prepare a lotion in a conventional manner according to the composition shown in Table 1.
[116]
TABLE 1
ingredient Content (% by weight)
GW4869 or pifithrin-μ 2.00
L- ascorbic acid 2-phosphate magnesium salt 1.00
Soluble collagen (1% aqueous solution) 1.00
Sodium citrate 0.10
Citric acid 0.05
Licorice Extract 0.20
1,3-butylene glycol 3.00
Purified water Remaining
Sum 100.00

[117]
[ Formulation Example 6] Cream
[118]
To prepare a cream in a conventional manner according to the composition shown in Table 2.
[119]
TABLE 2
ingredient Content (% by weight)
GW4869 or pifithrin-μ 2.00
Polyethylene glycol monostearate 2.00
A self-emulsifiable glycerin monostearate 5.00
Cetyl alcohol 4.00
Squalene 6.00
Tree 2-ethylhexanoate glyceryl 6.00
Sphingoglycolipids 1.00
1,3-butylene glycol 7.00
Purified water Remaining
Sum 100.00

[120]
[ Formulation Example 7] Pack
[121]
To prepare a pack in a conventional manner according to the proportion described in Table 3.
[122]
TABLE 3
ingredient Content (% by weight)
GW4869 or pifithrin-μ 2.00
Polyvinyl alcohol 13.00
L- ascorbic acid 2-phosphate magnesium salt 1.00
Lauroyl hydroxyproline 1.00
Soluble collagen (1% aqueous solution) 2.00
1,3-butylene glycol 3.00
ethanol 5.00
Purified water Remaining
Sum 100.00

[123]
Above, that according to a person of the present invention a particular ordinary skill in the art, bar, hayeotneun detail the portion of the content, this specific technique, rather than being just only a preferred embodiment, whereby the limits of the invention it will be apparent. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

[Claim 1]Composition for skin whitening comprising the exo-bit inhibitor to inhibit the secretion of exo-bit (exosome) secreted from the melanin-forming cells (melanocytes), as an active ingredient.
[Claim 2]
The method of claim 1 wherein said exo-bit inhibitor is 1 or more, a composition for skin whitening is selected from the group consisting of GW4869 and pifithrin-μ.
[Claim 3]
The method of claim 1 wherein the active ingredient is a composition for skin whitening to suppress melanin production.
[Claim 4]
The method of claim 1 wherein the active ingredient is liver spots, freckles, black point, nevi, melanoma, pigmentation, inflammation after pigmentation and at least one skin pigmentation is selected from the group consisting of discoloration occurring in dermatitis due to drug prevention, improvement or treatment of disease, the composition for skin whitening.
[Claim 5]
And then it was treated with the test substance in the melanin-forming cells (melanocytes) is irradiated with ultraviolet rays, or melanin irradiated with ultraviolet rays to form cells (melanocytes) treating the test substance; of exo-bit (exosome) secreted from the melanin-forming cells method for screening a skin-lightening effect material comprising a step to determine the relative secretion.
[Claim 6]
The method of claim 5 wherein the relative secretion of some the exo is but checked against the secretion of some exo secreted from a control group of melanin forming cells, the control group, the skin whitening melanin forming cells irradiated with UV, without treatment with the test substance method for screening the efficacy material.
[Claim 7]
6. The method of claim 5, after checking the relative secretion of the exo-bit, in which the test substance reducing the relative secretion of some exo comprising screening a skin-lightening effect material, screening the skin lightening efficacy material.
[Claim 8]
The method of claim 5, wherein the ultraviolet radiation is a UVB 20 mJ to about 30 / cm 2 Method of screening for irradiating a skin whitening effect material.
[Claim 9]
The method of claim 5, wherein time to process the test substances on the formation of melanin cells are screened for 10 to 24 hours in skin whitening effect material.
[Claim 10]
The method of claim 5, wherein the secretion of the exo-bit is a method for screening, skin whitening efficacy substance to determine the expression level of CD81 or HSP90.
[Claim 11]
Of claim 5 to claim 10 wherein the composition for skin whitening comprising the substance identified as skin whitening efficacy material according to any one of the active ingredients. [1]
Herein is a method for screening a composition and a skin-lightening effect material for skin whitening is provided.
BACKGROUND
[2]
Most animal cells are all biological, including different sizes and extracellular vesicles within the origin cell having a component which has the ability to secrete (extracellular vesicle), these extracellular follicle is blood, urine, saliva, and cell culture It is found in fluid (biological fluids) (Loyer X, Vion AC, Tedgui A, Boulanger CM Microvesicles as cell-cell messengers in cardiovascular diseases Circ Res 2014; 114:.. 345-53) (Ohno S, Ishikawa A, Kuroda M . Roles of exosomes and microvesicles in disease pathogenesis Adv Drug Deliv Rev 2013; 65:. 398-401).
[3]
Extracellular vesicles are smaller in diameter significantly from about 20 nm is a film structure vesicles body having a size of about 5 ㎛ diameter, there is heterogeneity in the size and configuration, the exo-bit (exosome), ekto bit (ectosome), micro-vesicles (microvesicle), it comprises a plurality of different species, such as microparticles (microparticle), apoptotic bodies (apoptotic body).
[4]
Such extracellular vesicles will reflect the state of the origin cells (donor cells) to secrete represents various biological activities depending on whether secretion in some cells, while moving the genetic material and protein between cells play an important role in interactions between cells to be carried out.
[5]
In addition, melanin (melanin) is a black pigment and a biopolymer material of phenols having a composite form of the protein, apple, potato, shell plume of browning or animals to occur when the cut surface of the bananas to be exposed to the air, skin, hair, It is observed in the eye. If melanin is over-produced are deposited on the skin spots, freckles, etc. is formed and promoting skin aging, skin cancer may also be induced.
[6]
Melanin-forming cells in humans (melanocyte) is a synthesis of melanin, as part of a defense mechanism for the ultraviolet radiation from the external cell, and serves for preventing the skin from being killed by the ultraviolet light. When the ultraviolet light from the outside will be exposed to the skin during the skin tissue cells melanin forming cells which synthesize melanin in response to ultraviolet light.
[7]
Melanin synthesis by UV light melanin is a melanin-stimulating hormone (melanin stimulating hormone, MSH) by ultraviolet secretion, MSH is to react the receptor of MC1R for increasing the cAMP in the melanin-forming cells induce reaction of the gene for melanin synthesis the synthesis and the synthesis of melanin is to serve to protect the skin from UV rays is secreted to the exterior of the melanin-forming cells. A protein involved in the biosynthesis of melanin are known include mitf, tyr, trp1, trp2.
[8]
However, it not has been reported about the association of cell death of the exo little secretion and melanin forming cells of secreted in the conventional melanin forming cells. The prior art relates to a method for screening a substance having a whitening active are disclosed in the Republic of Korea Patent No. 10-0840144 call.
Detailed Description of the Invention
SUMMARY
[9]
In one aspect, the present specification aims to provide a composition that is effective in lightening skin through the emission control of a bit in the exo melanin forming cells.
[10]
In another aspect, the present disclosure is to determine the secretion of the exo get secreted by the cells melanin formation is an object of the present invention to provide a method for screening a skin-lightening effect material.
Problem solving means
[11]
In one aspect, the techniques described herein provides a composition for skin whitening comprising the exo-bit inhibitor to inhibit the secretion of exo-bit (exosome) secreted from the melanin-forming cells (melanocytes), as an active ingredient.
[12]
In one exemplary embodiment, the exo-bit inhibitor may be one or more selected from the group consisting of GW4869 and pifithrin-μ.
[13]
In one exemplary embodiment, the active ingredient may be to inhibit melanogenesis.
[14]
In one exemplary embodiment, the active ingredient, spots, freckles, black point, nevi, melanoma, pigmentation caused by drugs, inflammation after pigmentation and at least one skin colorant selected from the group consisting of discoloration occurring in dermatitis the deposition disease may be to prevent, improve or treat.
[15]
In another aspect, the techniques described herein provides a way to promote skin lightening comprising applying a composition comprising an effective amount of exo-bit inhibitor to a subject.
[16]
In a further aspect, the melanin over-produce the prevention of disease caused by, comprising administering a composition comprising an effective amount of exo-bit inhibitors in improving or treating a subject in need, the prevention of disease caused by melanin over-produced, It provides an improvement or cure.
[17]
In one exemplary embodiment, the method may include applying the composition in the form of a pharmaceutical composition, a cosmetic composition or a food composition.
[18]
In one exemplary embodiment, the method may include applying the composition to the skin of the subject.
[19]
In another aspect, the techniques described herein provides an exo some inhibitors for promoting target skin whitening.
[20]
In another aspect, the techniques described herein provides an exo some inhibitors for the prevention, improvement or treatment of over-generation due to melanin.
[21]
In another aspect, the techniques disclosed herein includes the steps of processing after the test material irradiated with UV in the melanin-forming cells (melanocytes) is irradiated with ultraviolet rays, or melanin forming cells (melanocytes) after treatment with the test substance on; And it provides a method for screening a skin-lightening effect material comprising a step to determine the relative secretion of the exo-bit (exosome) secreted from the melanin-forming cells.
[22]
In one exemplary embodiment, the relative secretion of some the exo may be a melanin forming cells irradiated with UV without, but checked against the secretion of some exo secreted from a control group of melanin forming cells, the control group treated with the test substance .
[23]
In an exemplary embodiment, after confirming the relative secretion of the exo-bit, a test substance which reduces the relative secretion of some exo may include the step of screening a skin-lightening effect material.
[24]
In one exemplary embodiment, the UV light to UVB 20 mJ to about 30 / cm 2 may be to investigate.
[25]
In one exemplary embodiment, the time to process the test substances on the formation of melanin cell can be 10 to 24 hours.
[26]
In one exemplary embodiment, the secretion of some of the exo may be identified by expression of CD81 or HSP90.
[27]
In another aspect, the techniques described herein provides a composition for skin whitening comprising the substance identified as skin whitening effect material according to the above method as an active ingredient.
Effects of the Invention
[28]
In one aspect, the techniques disclosed herein is effective to control through the discharge of a bit in the exo melanin forming cells provide a composition that is effective in lightening skin.
[29]
In another aspect, the techniques disclosed herein is effective to verify the secretion of exo get secreted by the melanin forming cells provides a method for screening a skin-lightening effect material.
Brief Description of the Drawings
[30]
1 is a result of checking the average size of the exo some emitted from the melanin-forming cells in accordance with an experimental example of this disclosure.
[31]
2 is a result confirmed that happens secretion of an increased exo-bit during the ultraviolet ray irradiation according to the Experimental Example exo little decrease when inhibitor treatment in the present description.
[32]
Figure 3 is the result confirming the effect of exo-bit inhibitor on the viability of the ultraviolet ray irradiation melanin forming cells in accordance with an experimental example of this disclosure.
[33]
Figure 4 is the result confirming that the melanin forming cells without the UVB irradiation in accordance with an experimental example of the present specification there is no significant difference in cell viability in accordance with some exo.
Best Mode for Carrying Out the Invention
[34]
Hereinafter, the present invention will be described in detail.
[35]
In one aspect, the techniques described herein provides a composition for skin whitening comprising the exo-bit inhibitor to inhibit the secretion of exo-bit (exosome) secreted from the melanin-forming cells (melanocytes), as an active ingredient.
[36]
As that in the present specification means "exo-bit (exosome)" is secreted in the extracellular of the emitted nanoscale the extracellular space cell vesicles, i.e. extracellular vesicles (extracellular vesicles), exo inner bit are sieve film structure vesicles and is outside the segment, and passes the other cells and binds to tissue membrane components, proteins (growth hormones, cytokines, etc.), RNA such as cells are known to play a variety of roles, such as mediating the communication between the cells . On the other hand, the periphery of the melanin forming cells, there is a material between the cells and the cells that surround the melanin-forming cells. Specifically, a constant radius, for example, within 2 mm from the melanin-forming cells, and the material between the cells, such as keratinocytes or fibroblasts and / or collagen can be present, melanin forming cells secrete the exo-bit in the extracellular matrix.
[37]
In one aspect, the bit exo can keulil extracellular Bezier having a diameter of 20 to 400 nm.
[38]
In this specification, "bit exo inhibitor" as meaning a substance which suppresses the secretion of exo little is secreted from the melanin-forming cells, melanin-forming cells produced in the bit within the exo, to inhibit the secretion or release of some exo from the melanin-forming cells the concept of the broadest to mean a material.
[39]
In one exemplary embodiment, the exo-bit inhibitor may be one or more selected from the group consisting of GW4869 and pifithrin-μ.
[40]
"GW4869" is a molecular formula C 30 H 30 Cl 2 N 6 O 2 to a sphingomyelinase (Smase) inhibitors having the general formula is represented by 1 (CAS 6823-69-4). Sphingomyelinase is sphingomyelin to produce ceramide and phosphocholine to hydrolyze sphingomyelin - a specific phospholipid Piaget (phospholipase) C.
[41]
Formula 1

[42]
"Phi trim (pifithrin) -μ" is a molecular formula C 8 H 7 NO 2 to a p53 inhibitor has the S is represented by the formula 2 (CAS 64984-31-2). cancer gene p53 is controlled so as to control cell proliferation unnecessarily. However, the critical to the survival of the cells when the abnormal transient with p53 activation, pifithrin during radiotherapy normal cells was identified as a compound to increase cancer cell death while being protected.
[43]
[Formula 2]

[44]
In one exemplary embodiment, the active ingredient is effective for preventing, treating or ameliorating skin damage or skin disorders that are associated, directly or indirectly, and melanin forming cells increased. That is, the active ingredient has the effect that it effectively suppresses the melanin synthesis, for preventing, treating or improving the diseases caused by excessive generation of melanin.
[45]
Due to the melanin over-produce disease is liver spots, freckles, age spots, blemishes, skin melanocyte lesions (Epidermal melanocytic lesion), milk coffee spots (Cafe's au lait macules), Becker Nevus (Becker's Nevus), questioned the nevus (Nevus Spilus ), black (Lentigines), dermal melanocyte lesions (dermal melanocytic lesions), monggoban (Mongolian spot), Ota nevus (nevus of Ota), acquired bilateral Ota nevus positive half (acquired bilateral nevus of Ota-like macules), Ito nevus (nevus of Ito), the blue nevus (blue nevus), melanin formation cellular nevus (Melanocytic nevus), boundary nevus (Junctional nevus), compound nevus (compound nevus), the dermis within the nevus (Intradermal nevus), unryun nevus ( Halo nevus), congenital melanocyte St. nevus (congenital nevocytic nevus), Spitz nevus (Spitz nevus), dysplastic nevi (dysplastic nevus), melanoma (melanoma), malignant surplus melanoma (Lentigo maligna melanoma), superficial scalability melanoma (Superficial spreading melanoma), leading black St. melanoma (Acral lentiginous melanoma), nodular melanoma (Nodular melanoma), pigmented basal cell carcinoma (pigment basal cell carcinoma), pigmented skin fibrosis (dermatofibromas), pigmented skin cysts (dermoid cyst),
[46]
In one exemplary embodiment, the pigmentation of the composition occurs in pigmentation, after inflammation pigmentation and dermatitis due to spots, freckles, black point, nevi, melanoma, drugs that occurs locally on the skin by increasing melanin synthesis at least one skin pigmentation disorder is selected from the group consisting of may be to prevent, improve or treat.
[47]
In another aspect, the techniques disclosed herein includes the steps of processing, and then irradiated with UV in the melanin-forming cells irradiated with UV light after the treatment with the test substance in (melanocytes), or melanin forming cells (melanocytes) the test substance; And it provides a method for screening a skin-lightening effect material comprising a step to determine the relative secretion of the exo-bit (exosome) secreted from the melanin-forming cells.
[48]
In one exemplary embodiment, the relative secretion of some the exo may be a melanin forming cells irradiated with UV without, but checked against the secretion of some exo secreted from a control group of melanin forming cells, the control group treated with the test substance .
[49]
In an exemplary embodiment, after confirming the relative secretion of the exo-bit, a test substance which reduces the relative secretion of some exo may include the step of screening a skin-lightening effect material.
[50]
In one exemplary embodiment, the UV light to UVB 20 mJ to about 30 / cm 2 may be to investigate.
[51]
In one exemplary embodiment, the time to process the test substances on the formation of melanin cell can be 10 to 24 hours.
[52]
In one exemplary embodiment, the secretion of some of the exo, for example, the same cell quantitation some discrete exo using the same separation from the equipment and Q-Nano or using NanoSight device, nanoparticle tracking analysis (NTA) can the particle to to the target to three or exo some proteins on the surface, a lipid or exo little inside of the proteins, peptides, RNA, miRNA (microRNA), lncRNA (long noncoding RNA), to check over the amount of such metabolites, to quantitatively depending Western blotting (western blot), dot blot (dot blot), ELISA, Northern blot (northern blot), PCR, RT-qPCR, GC-MS, LC-MS, NMR, radiation immunoassay (radioimmunoassay), immunoprecipitation reaction analysis methods such as (immunoprecipitation assay), radiation immunodiffusion (radioimmunodiffusion), FACS, and protein chip (protein chip) are those skilled in the art can be selected appropriately. For example, embodiments of the herein example but not exo although some surfaces of the CD81 or exo visually assess the relative amount of the exo-bit via bit internal Western blot using an HSP90 as a quantitative marker of limitation.
[53]
Exo little released from the melanin-forming cells to the extracellular matrix suppresses the apoptosis of a melanin forming cells ultraviolet irradiation. Thus, it is possible to increase the apoptosis of melanin forming cells by inhibiting the secretion of exo get secreted by the melanin-forming cells, to inhibit Consequently melanogenesis due to apoptosis of these melanin-forming cells, an effect of the whitening efficacy demonstrated It is (Pigment Cell Melanoma Res 2009 Jun; 22 (3):. 307-318).
[54]
In addition, substances that inhibit or increase the release of some exo melanin formation in the cell can be formed by melanin promoting apoptosis of cells (secretion inhibitory substance) or decrease (increase secreted substances) to control the melanin production. Accordingly, there is an effect that it is possible to screen the ultraviolet treatment before or after the skin lightening efficacy candidates by processing the material to a melanin-forming cells to control by confirming the exo secretion little is secreted from the melanin-forming cells skin pigmentation.
[55]
In another aspect, the techniques described herein provides a composition for skin whitening comprising the substance identified as skin whitening effect material according to the above method as an active ingredient.
[56]
According to an exemplary embodiment, the composition may be a pharmaceutical composition.
[57]
The pharmaceutical composition may further contain preservatives, stabilizers, wetting or emulsifying accelerators, pharmaceutical adjuvants and other therapeutically useful substances such as salt and / or buffer for the osmotic pressure in addition to exo-bit inhibitors, conventional methods wide variety of oral administration may hwahal claim or parenteral dosage form in accordance with the second aspect.
[58]
The orally administered agent is, for example, and the form of tablets, pills, hard and soft capsules, solutions, suspensions, emulsions, syrups, powders, powders, fine granules, granules, pellets and the like, these formulations in addition to active ingredient, a surfactant the diluent may contain: (silica, talc, stearic acid and its magnesium or calcium salt, and polyethylene glycol for example) (for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants . Tablets may also contain magnesium aluminum silicate, starch paste, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose and polyvinylpyrrolidine and may contain the same binder, a starch, agar, alginic acid or its sodium salt, as appropriate a disintegrating agents, absorbents, colorants, flavors pharmaceutical additives, such as agents, and sweetening agents, such as may contain. The tablets may be prepared by conventional mixing, granulating or coating methods.
[59]
Further, in the parenteral administration form can be a transdermal dosage forms, such as injections, drops, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories (坐 劑), dosage forms, such as the patch Although one can, without being limited thereto.
[60]
The pharmaceutical composition may be parenteral, administration to the rectal, topical, transdermal, subcutaneous and the like.
[61]
Dose determination of the active ingredient is within the level of ordinary skill, daily doses of the drug varies depending on various factors such as the severity, onset of destination, age, health condition, complications, to be administered, adults On the one side, based on the composition to 1㎍ / kg to 200mg / kg, may be administered to 50㎍ / kg to about 50mg / kg to 1 day divided to three times a day in another aspect, the dosage is any method also not intended to limit the scope of the invention.
[62]
The pharmaceutical composition may be a skin preparation for external use may be, the topical is any would also be included in the various formulations as a generic drug that is applied from the outer skin to be included herein.
[63]
According to an exemplary embodiment, the composition may be a cosmetic composition.
[64]
The cosmetic composition may contain additional components included in addition to some inhibitors exo functional additives and typical cosmetic composition. In the functional additive can include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamin, peptide polymer, polysaccharide polymer, Scotland pinggo lipid and seaweed extract. Maintaining As the component ingredients contained in addition, a moisturizing agent, emollient agent, surfactant, organic and inorganic pigments, organic powder, ultraviolet absorbent, antiseptic, fungicide, antioxidant, plant extract, pH adjuster, alcohol, pigments, perfumes, blood circulation in there may be mentioned promoters, the feeling of cold, limits (制 汗) agents, such as purified water.
[65]
The cosmetic composition may be appropriately selected, as the formulation is not particularly limited, it is an object. For example, skin lotion, skin softening, skin toner, astringent lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, pack, soap, Cleansing foams, cleansing lotion, cleansing cream, but can be made of any one or more dosage forms selected from the group consisting of a body lotion and body cleansers, but is not limited thereto.
[66]
When the formulation of a paste, cream or gel, the animal fiber, plant fiber, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc or zinc oxide and the like be used as the support component can.
[67]
When the formulations of the present invention is powder or spray, lactose, talc, silica, as the support component of aluminum hydroxide, calcium silicate, or and the polyamide powders may be used, particularly if a spray-in, hydrocarbons in addition chlorofluorocarbons, propane / it may comprise propellants such as butane or dimethyl ether.
[68]
If the formulation is a solution or emulsion of the present invention, the solvent, and the solvent agent or emulsifying agent as a carrier component is used, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , a 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester.
[69]
If the dosage form of the invention the suspension has a support component liquid diluent, such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan suspension, such as sorbitan esters claim, microcrystalline such as cellulose, aluminum meta-hydroxide, bentonite, agar or tragacanth may be used.
[70]
The formulation of the present invention a surface-if the surfactant-containing cleansing, the aliphatic alcohol sulfate as carrier components, aliphatic alcohol ether sulfate, sulfosuccinate monoester, isethionate, imidazolinium iodonium derivatives, methyl taurates, sarcoidosis when sulfonates, fatty acid amide ether sulfates, and the like alkyl amido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, Lee Quinoline derivative or ethoxylated glycerol fatty acid esters may be used.
[71]
According to an exemplary embodiment, the composition may be a food composition.
[72]
The food composition is a liquid or may be in solid dosage forms, e.g., various types of foods, drinks, gum, tea, vitamin complex, health supplement foods, etc., and powder, granule, tablet, capsule or beverage used in the form can. Food composition of the respective formulations can be any person skilled in the art by blending the enemy selected without difficulty by a conventional the formulation or use components that are used in the art in addition to the active ingredient, may take place is a synergistic effect when applying at the same time as the other raw material.
[73]
The liquid component, which may contain in addition to the active ingredient disclosed herein has no particular limitations, and the like various flavors or natural carbohydrates as additional ingredients, such as conventional beverage. Examples of the natural carbohydrate is a monosaccharide, glucose, disaccharide, maltose, shoe sugar alcohols such as polysaccharides, dextrin, ordinary sugar and xylitol, sorbitol, erythritol, such as cyclodextrin erythritol, such as a cross, such as fructose and the like. The flavor agent can be used to advantage to natural flavors (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (e. G., Such as saccharin and aspartame) . the content of the natural carbohydrate may be described herein generally range from about 1 to about 20g, about 5 to 12g in one aspect a composition per 100ml.
[74]
The food composition comprises various nutrients in one aspect, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents, coloring agents and mogul agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof such as natural flavorants and it may include an organic acid, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonated agents, and the like used in soft drinks. On the other side it may include pulp for the production of natural fruit and vegetable juice drinks. The components may be used independently or in combination. The content of the additive, but may vary, it is generally selected from the range of composition 100 parts by weight of 0.001 to about 20 parts by weight per disclosed herein.
Mode for the Invention
[75]
The present invention will be described in further detail with reference to the following examples. These embodiments will provide for only illustrative of the present invention, self-evident to a person of ordinary skill in the art, the scope of the present invention is not construed as being limited to these examples.
[76]
Experimental Example.
[77]
(1) Cell culture and materials
[78]
Human melanin forming cells (normal human melanocytes, Cascade Biologics, Portland, OR, USA) was stored in M-254 medium (Cascade) containing the human melanin forming cells growth supplement (Cascade). GW4869 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Phi trim (pifithrin) -μ (Sigma, St. Louis, MO, USA) was used and dissolved in DMSO.
[79]
(2) exo-bit (exosome) separation
[80]
For some separation exo washing confluent melanin forming cells with PBS and incubated for 48 hours in M-254. Centrifuged at 100,000 × g for 10 minutes to collect the conditioned media 500 × a (conditioned medium) g, 20 bungan 3,000 × g, and 2 hours. The obtained exo-bit-containing pellets were stored at -80 ℃ resuspended in PBS. In addition, the conditioned medium was filtered to give a 0.45 ㎛ pore filter (Millipore, Billerica, MA, USA) and, 100 kDa cut-off and concentrated using a spin column (Millipore). Exo some are using ExoQuick-TC kit (SBI, San Jose, CA, USA) were isolated according to the manufacturer's instructions. Separate exo some 20 ㎕ the Western blot and silver staining was analyzed.
[81]
(3) DLS(Dynamic light scattering) 분석
[82]
Exo bit diameter was measured using a Zetasizer Nano S instrument (Malvern Instruments Ltd., Worcestershire, UK) in the scattering intensity 10 × 30 s provided with a 633 nm laser.
[83]
(4) Gel protein separation
[84]
Exo some proteins were separated by SDS-PAGE. How the SDS-PAGE gel cut into 10 slices and group known (Bahk YY, Kim SA, Kim JS, et al (2004) Antigens secreted from Mycobacterium tuberculosis:. Identification by proteomics approach and test for diagnostic marker Proteomics 4:. 3299 -307.) it was isolated within the gel using trypsin (Promega, Madison, WI, USA) according to the. In short, the gel pieces 1: 1 acetonitrile (ACN) / 25 mM ammonium bicarbonate (ABC) to 4-5 times washed, dehydrated, and dried at 100% ACN. In 45 minutes 56 ℃ from the reduction and 30 minutes light blocking at room temperature in 100 mM DTT with 100 mM ABC then Corporate alkyl in 55 mM iodine acetamide with 100 mM ABC, and drying the pieces at 100% ACN 37 ℃ again it was hydrated in 325 mM ABC containing 20 ng of trypsin for 20 hours. Peptide to move the liquid to a fresh tube and the residual by using a 50% (v / v) aqueous acetonitrile containing 0.1% at 30 ℃ 40 bungan (v / v) formic acid was extracted from the gel. Evaporating the combined supernatant and 0.1% for the mass spectrometric analysis (v / v) formic acid containing 5% (v / v) was dissolved in aqueous acetonitrile solution.
[85]
5. Protein Identification by LC-MS / MS
[86]
Separate peptides group known methods (Zuo X, Echan L, Hembach P, et al. (2001) Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble . and large proteins Electrophoresis 22:. was added to slight variations in 1603-15) Finnigan LCQ ion-analyzed using a reversed-phase capillary HPLC directly connected to the trap mass spectrometry (LC-MS / MS). Peptide was coupled for 10 min trapping column and eluted at 0.2 ㎕ / min flow rate of 50 minutes with 0.1% (v / v) 5 ~ 80% (v / v) aqueous acetonitrile containing formic acid gradient. Tandem MS was handled using a was performed at m / z = 450 ~ 2000 Da, each spectra TurboSEQUEST software (Thermo Quest, San Jose, CA, USA). The resulting peak lists were used to query the NCBI database using the MASCOT MSDB or program (http://www.matrixscience.com).
[87]
(6) Western blot and silver staining
[88]
Cell or separate the exo protease inhibitor bit (Sigma) containing a lysis buffer (1% NP-40, 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.01 M MgCl 2 was dissolved using a). The protein concentration was analyzed by BCA assay, the sample is separated by SDS-PAGE, which was moved to the PVDF membrane and analyzed with antibodies to CD81 (SBI). On the other hand, experiments were applied to a SDS-PAGE gel according to the manufacturer's instructions for the dye is staining kit (Thermo Fisher Scientific, Rockford, IL, USA).
[89]
7 Cytotoxicity test
[90]
1 × 10 melanin-forming cells in 6-well plates 4 were dispensed at a density of cell / well. Followed by day, processing a bit 10 ㎕ the exo-isolated from the same cell. Fixing the cells with 4% formaldehyde in PBS and 15 minutes, and stained with 0.1% of 20 minutes, washed with PBS, and crystal violet. After washing with PBS, the cells were dried and dissolved in 10% acetic acid. By using a spectrophotometer to measure the absorbance at 590 nm.
[91]
(8) Quantitative real-time PCR
[92]
Using Trizol reagent (Invitrogen) to remove the total RNA and using the ReverTra Ace (Toyobo, Osaka, Japan) was reverse transcribed into cDNA. Gene expression analysis of TaqMan Universal Master Mix and TaqMan Gene Expression Assays: was performed using Hs00365052_m1 (Applied Biosystems, Foster City, CA, USA).
[93]
(9) Statistical analysis
[94]
The two-tailed Student's t-test was used to analyze the differences between the two groups. Data are mean ± SD for three experiments expressed as (***; p <0.05; p <0.005, **).
[95]
1. The results confirm the secretion of exo get from melanin forming cells
[96]
Melanin forming cells was found to have a mean diameter of the size of some secreted some exo and was released, and separate the exo analysis (dynamic light scattering analysis) as a result, the exo-bit is 206.90 ± 22.18 nm emitted from the melanin-forming cells (see Fig. 1).
[97]
2. Experimental results secreted from melanocytes according to the ultraviolet light irradiation to form the cell bit exo changes in the secretion
[98]
A melanin forming cells UVB (20 mJ / cm 2 irradiated with a), and was found to increase the secretion of some exo when a result, ultraviolet light exposure for 24 hours.
[99]
In addition, when treated with UVB exposure before, GW4869 (Smase (sphingomyelinase) inhibitors) with skin of the trim, or 10 μM (pifithrin) -μ (p53 inhibitor), 10 μM of some of the exo generation / secretion inhibitor for 12 hours, some of the exo It has been found that reducing the amount of CD81 expression markers (see Fig. 2).
[100]
In this way, melanin forming cells was confirmed that an increase in the amount of exo-bit that is released on exposure to ultraviolet rays, and even when exposed to UV light reduces the amount of exo some emitted when applied to the generation of a bit exo / secretion inhibitor.
[101]
3. Experimental results exo little effect of inhibitors on the viability of the ultraviolet irradiation melanin forming cells
[102]
UVB (20 mJ / cm with a little each exo and melanin forming cells from UVB unprocessed a melanin-forming cells, UVB-treated melanin forming cells, UVB + GW4869 treatment melanin forming cells 2 is irradiated with) were incubated for 3 days result, it was found that some of the melanin-forming cells derived from the received ultraviolet exposure exo as shown in Figure 3 is to increase the viability of melanin forming cells receiving the UV damage. The survival promoting effects of a melanin-forming cells was reduced line by a melanin-forming cells derived from exo-bit processing with the GW4869, pifithrin-bit μ secretion inhibitor exo UV. On the other hand, UVB untreated melanin in the same way as the forming cells, UVB each exo-bit obtained from the treated melanin forming cells, but incubated with melanin forming cells when cultured under conditions that do not examine the UVB melanin forming cells, melanocytes there was no significant difference in viability of cells formed (see Fig. 4).
[103]
Thus, the efficacy of inhibiting apoptosis (apoptosis) of sikimyeo melanin receiving the UV exposure to form the cell bit-derived exo increases the viability of a melanin forming cells receiving the UV damage, some exo exiting the UV irradiation during the melanin forming cells melanin forming cells for it was found that it has.
[104]
Accordingly, substances which inhibit or increase the release of some exo from melanin forming cells and promote the apoptosis of melanin forming cells (discharge suppressing material) or decreased (increased emissions), it was confirmed that to control the melanin production.
[105]
One an example formulation of a composition according to an aspect of the invention from below, and also applicable to various other formulations, which are only intended to describe in detail, not intended to limit the invention.
[106]
[ Formulation Example 1] The soft capsules
[107]
By GW4869 or pifithrin-μ 50mg, L- carnitine 80 ~ 140mg, 180mg soybean oil, palm oil 2mg, vegetable hydrogenated oil mixture of 8mg, and 4mg hwangnap lecithin 6mg, and by filling 400mg per capsule by a conventional method to prepare soft capsules .
[108]
[ Formulation Example 2] Tablets
[109]
A mixture of GW4869 or pifithrin-μ 50mg, galacto-oligosaccharide 200mg, Lactose 60mg and 140mg maltose, was added to the ester (sugar ester) per then granulated using a fluid bed dryer 6mg was prepared in the other appointed give the tablet press.
[110]
[ Formulation Example 3 Granules
[111]
GW4869 or pifithrin-μ 50mg, then a solution of the anhydrous crystalline glucose, 250mg and 550mg of starch, and formed into granules using a fluid bed granulated by filling a capsule to prepare a granule.
[112]
[ Formulation Example 4] Drink
[113]
After mixing GW4869 or pifithrin-μ 50mg, glucose 10g, 0.6g citric acid, and a liquid oligosaccharide 25g was added to 300ml of purified water were charged to each bottle by 200ml. After filling the bottle and sterilized at 130 ℃ 4 ~ 5 seconds to produce a beverage drinks.
[114]
[ Formulation Example 5] Lotion
[115]
To prepare a lotion in a conventional manner according to the composition shown in Table 1.
[116]
TABLE 1
ingredient Content (% by weight)
GW4869 or pifithrin-μ 2.00
L- ascorbic acid 2-phosphate magnesium salt 1.00
Soluble collagen (1% aqueous solution) 1.00
Sodium citrate 0.10
Citric acid 0.05
Licorice Extract 0.20
1,3-butylene glycol 3.00
Purified water Remaining
Sum 100.00

[117]
[ Formulation Example 6] Cream
[118]
To prepare a cream in a conventional manner according to the composition shown in Table 2.
[119]
TABLE 2
ingredient Content (% by weight)
GW4869 or pifithrin-μ 2.00
Polyethylene glycol monostearate 2.00
A self-emulsifiable glycerin monostearate 5.00
Cetyl alcohol 4.00
Squalene 6.00
Tree 2-ethylhexanoate glyceryl 6.00
Sphingoglycolipids 1.00
1,3-butylene glycol 7.00
Purified water Remaining
Sum 100.00

[120]
[ Formulation Example 7] Pack
[121]
To prepare a pack in a conventional manner according to the proportion described in Table 3.
[122]
TABLE 3
ingredient Content (% by weight)
GW4869 or pifithrin-μ 2.00
Polyvinyl alcohol 13.00
L- ascorbic acid 2-phosphate magnesium salt 1.00
Lauroyl hydroxyproline 1.00
Soluble collagen (1% aqueous solution) 2.00
1,3-butylene glycol 3.00
ethanol 5.00
Purified water Remaining
Sum 100.00

[123]
Above, that according to a person of the present invention a particular ordinary skill in the art, bar, hayeotneun detail the portion of the content, this specific technique, rather than being just only a preferred embodiment, whereby the limits of the invention it will be apparent. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Claims
[Claim 1]
Composition for skin whitening comprising the exo-bit inhibitor to inhibit the secretion of exo-bit (exosome) secreted from the melanin-forming cells (melanocytes), as an active ingredient.
[Claim 2]
The method of claim 1 wherein said exo-bit inhibitor is 1 or more, a composition for skin whitening is selected from the group consisting of GW4869 and pifithrin-μ.
[Claim 3]
The method of claim 1 wherein the active ingredient is a composition for skin whitening to suppress melanin production.
[Claim 4]
The method of claim 1 wherein the active ingredient is liver spots, freckles, black point, nevi, melanoma, pigmentation, inflammation after pigmentation and at least one skin pigmentation is selected from the group consisting of discoloration occurring in dermatitis due to drug prevention, improvement or treatment of disease, the composition for skin whitening.
[Claim 5]
And then it was treated with the test substance in the melanin-forming cells (melanocytes) is irradiated with ultraviolet rays, or melanin irradiated with ultraviolet rays to form cells (melanocytes) treating the test substance; of exo-bit (exosome) secreted from the melanin-forming cells method for screening a skin-lightening effect material comprising a step to determine the relative secretion.
[Claim 6]
The method of claim 5 wherein the relative secretion of some the exo is but checked against the secretion of some exo secreted from a control group of melanin forming cells, the control group, the skin whitening melanin forming cells irradiated with UV, without treatment with the test substance method for screening the efficacy material.
[Claim 7]
6. The method of claim 5, after checking the relative secretion of the exo-bit, in which the test substance reducing the relative secretion of some exo comprising screening a skin-lightening effect material, screening the skin lightening efficacy material.
[Claim 8]
The method of claim 5, wherein the ultraviolet radiation is a UVB 20 mJ to about 30 / cm 2 Method of screening for irradiating a skin whitening effect material.
[Claim 9]
The method of claim 5, wherein time to process the test substances on the formation of melanin cells are screened for 10 to 24 hours in skin whitening effect material.
[Claim 10]
The method of claim 5, wherein the secretion of the exo-bit is a method for screening, skin whitening efficacy substance to determine the expression level of CD81 or HSP90.
[Claim 11]
Of claim 5 to claim 10 wherein the composition for skin whitening comprising the substance identified as skin whitening efficacy material according to any one of the active ingredients.