KR102143542B1 - Composition for the production of melanin comprising plasma-activated media and use thereof - Google Patents

Abstract

The present invention relates to a composition for increasing melanin production comprising a plasma-activated culture medium (PAM) produced by irradiating plasma to a cell culture medium, a skin tanning cosmetic composition comprising the composition, a composition for treatment of melanin hypopigmentation comprising the composition, and a method for increasing melanin synthesis in cells by adding the PAM to cells. The method can increase melanin synthesis without killing or damaging melanocytes.

Description

Composition for the production of melanin comprising plasma-activated media and use thereof

The present invention relates to a composition for producing melanin comprising a plasma-activated culture medium and a method of culturing melanocytes using a plasma-activated culture medium, and a method of producing melanin using melanocytes.

Skin cells produce melanin in the melanosomes of melanocytes present in the base of the epidermis as a defense mechanism against stimulation of ultraviolet rays, environmental pollution, and other external factors. Melanin is an important factor in determining the color of animals' skin, eyes, and hair. Hypopigmentation is also known as a risk factor for skin cancer. Asians are sensitive to excessive production of melanin, so many studies related to whitening that inhibit melanin production have been conducted. However, in recent years, the demand for vitiligo, which appears due to inhibition of melanin production, is also increasing, and research on this is also underway.

Vitiligo is an acquired depigmentation disease in which white spots of various sizes and shapes appear on the skin due to the death or necrosis of melanocytes. It is a relatively common disease that occurs in about 1% of the population worldwide, and does not differ by race or region. The age of onset is most common between 10 and 30 years of age, 95% of cases develop before 40 years of age, and 30% of patients have a family history. Although the cause of vitiligo has not been accurately identified yet, various theories have been suggested, such as the theory of autoimmune, neuronal humor, and self-destruction of melanocytes. The autoimmune theory is that the expression of autoantibodies against melanocyte-based antigens causes destruction or dysfunction of melanocytes, or that melanocytes are destroyed by lymphokines secreted by cytotoxic or activated lymphocytes. Neural humoral hypothesis is that hydrogen peroxide related to stress is produced due to abnormalities in catecholamine biosynthesis and increased monoamine oxidase, and thus melanocytes are destroyed.Vitiligo may occur along the ganglion or after nerve damage or stress. The theory of melanocyte self-destruction is that the phenol complex, an intermediate metabolite or final metabolite, generated during the melanogenesis process accumulates in melanocytes and destroys the cell. In addition, various factors such as inherited cellular defects, genetic factors, apoptosis, and abnormal calcium metabolism have been suggested.

Melanin is synthesized from melanocytes and plays an important role in protecting the skin by absorbing UV rays or toxic substances and chemicals. Therefore, in people whose melanin synthesis does not normally occur, there is also an external problem in that all the skin becomes white rather than white, causing spots, and becoming sensitive to external stimuli is more problematic. Tyrosinase, tyrosinase related protein-1 (TRP-1), and TRP-2, which are important enzymes for melanin synthesis, act as catalysts in oxidative reactions (Pigment Cell Res. 14 (6): 43744). Tyrosinase acts to oxidize tyrosine to DOPA (L-3,4-dihydroxyphenylalanine), DOPA to DOPA quinone, and TRP-1 is dihydroxyindole carboxylic acid oxidase, which is 5,6-dihydroxyindole- It is involved in converting 2-carboxylic acid (DHICA) into indol-5,6-quinone-2-carboxylic acid. In addition, TRP-1 plays a role in stabilizing tyrosinase and regulating its activity.

TRP-2 is a DOPA chromium tautomerase, which converts DOPA chromium into DHICA to form eumelanon and pheomelanon, which make up melanocytes, and depending on their ratio, skin, hair loss, and pupils The color, etc. are determined.

Melanin synthesis is activated by UV irradiation and α-melanocyte stimulating hormone (MSH). α-MSH is a peptide hormone that is produced by ultraviolet rays and is known to be produced in various cells including the pituitary gland and skin. α-MSH is a tyrosinase that plays an important role in melanin synthesis by controlling the activity of the transcription factor MITF (microphthalmia-associated transcription factor) by acting on the melanocortin receptor (MCR) of melanogenic cells by paracrine. It regulates the activities of TRP-1 (DHICA oxidase) and TRP-2 (DOPAchrome tautomerase).

It has been reported that when melanocytes are stimulated by UV or α-MSH, tyrosinase is activated by p38 or protein kinase A (PKA), respectively. However, of these two pathways, the α-MSH → cAMP →> PKA process plays an important role in melanin synthesis. Increasing cAMP promotes CREB (cAMP-responsive element binding protein) phosphorylation, and this promotes the expression of the transcription factor MITF. Increase, it enhances the activity of tyrosinase, and there are reports that increase tyrosinase mRNA expression.

Meanwhile, Asians, including Korea, tend to prefer white skin, so studies on whitening ingredients that inhibit melanin production have been actively conducted. However, melanin produced by the skin's melanin-synthetic cells plays an important role in protecting the skin by absorbing toxic substances and chemicals from UV irradiation. When the normal synthesis of melanin does not appear, there is a need for treatment to normalize melanin synthesis because not only cosmetic problems caused by mottled skin but also problems such as skin becoming sensitive to external stimuli and becoming vulnerable to ultraviolet rays. Research is also ongoing. However, the development of technology for promoting melanin synthesis is still insufficient.

An object of the present invention is to provide a composition for increasing melanin production, comprising a plasma active culture medium (PAM).

Another object of the present invention is to provide a method of increasing melanin synthesis by using a plasma active culture medium.

Another object of the present invention is to provide a pharmaceutical composition for the treatment, alleviation or treatment of melanin hypopigmentation, comprising a plasma active culture medium.

Another object of the present invention is to provide a cosmetic composition for tanning skin, comprising a plasma active culture solution.

Another object of the present invention is to provide a method of screening for melanogenesis inhibitors.

Another object of the present invention is to provide a composition for inhibiting melanogenesis comprising an inhibitor selected by the screening method.

In order to achieve the object of the present invention, the present invention provides a composition for increasing melanogenesis of melanogenic cells, comprising a plasma-activated culture medium (PAM) prepared by irradiating plasma to a cell culture medium.

The present invention also comprises the steps of preparing a plasma-activated culture solution (PAM) by irradiating plasma to the culture solution; And it provides a method for increasing melanin synthesis comprising the step of culturing cells excluding cells in the human body with the plasma-activated culture medium.

The present invention also provides a pharmaceutical composition for improving or treating melanin hypopigmentation, comprising a plasma-activated culture solution (PAM) prepared by irradiating normal temperature and atmospheric pressure plasma for 4 minutes to 30 minutes while the electrode is in contact with the culture solution.

Hereinafter, the present invention will be described in more detail.

The composition for increasing melanogenesis of melanin-producing cells provided in the present invention includes a plasma-activated culture medium (PAM) prepared by irradiating plasma to a cell culture medium.

Melanin of the present invention is a generic term for brown to black pigments present in tissues such as skin or eyes of animals. Melanin is known to block UV rays and maintain body temperature, and determines skin color and eye color. This melanin deficiency is the cause of albino or vitiligo. Unlike congenital albinism, vitiligo may appear congenital, and immunological, environmental or stress causes the disease.

Melanocytes are cells that synthesize melanosomes that store melanin, and account for about 5% of cells in the skin. It is mainly present in the basal layer at the bottom of the skin, and is characterized by having dendritic processes. Melanocytes synthesized from melanocytes move to keratinocytes through dendritic processes, and melanosomes are dissolved in the keratinocytes to secrete melanin to determine skin color.

Epidermal melanocytes divide at the same rate as the cell cycle of keratinocytes, and are known to be the same at 1200 to 1500 per 1 mm 2 regardless of race or skin color.

By using the plasma culture solution of the present invention, it is possible to compensate for the disadvantage that plasma activation effects cannot be obtained evenly on all cells when plasma is directly irradiated to cells in culture.

In the present invention, "plasma" refers to an ionized gas that satisfies Debye shielding. It is regarded as one of the three basic states of matter, gas, liquid, and solid, and is expressed as the fourth state. In the plasma of the present invention, a neutral gas is converted to a plasma by an external voltage, and electrons and cations may be generated by excitation and ionization of the neutral gas, and radicals excited by a molecular gas may exist.

The plasma of the present invention may be generated under normal pressure. For example, it may be a normal temperature and normal pressure plasma (NTAPP), but is not limited thereto. Room temperature atmospheric pressure plasma of the present invention may be used in the same meaning as the room temperature atmospheric pressure plasma. The plasma generating apparatus of the present invention can use a known plasma generating apparatus without limitation, as long as it can generate the plasma active culture solution according to the object of the present invention. In particular, the arc and static electricity are inhibited to generate plasma for biological purposes It features. For example, the plasma generating apparatus of the present invention comprises: an apparatus main body; A needle or rod-type power electrode in which a flat ground electrode provided on one side of the device body is disposed to face the flat ground electrode in the body; And a high voltage power supply for supplying power to the power electrode. In one embodiment of the present invention, plasma generation was performed at room temperature and atmospheric pressure using 3.0W microwave power and argon (Ar) gas, and the plasma generating apparatus and the medium were separated by 0.8cm. In the present invention, room temperature is a temperature range that does not correspond to high-temperature plasma, and can be applied without limitation to a temperature condition considered as room-temperature plasma in the art. It means to 45 ℃ or 20 to 40 ℃.

The carrier gas may be used without limitation as long as it is suitable for the purpose of producing a plasma and plasma active liquid that achieves the object of the present invention, but is preferably 1 selected from one or more combinations selected from the group consisting of helium, oxygen, neon, and argon. More than one species, more preferably argon may be used as a carrier gas, and plasma may be generated by supplying a high voltage current to such a gas.

Meanwhile, plasma activated water, which is a liquid activated using plasma, exhibits similar characteristics to gaseous plasma, and is used in agriculture and medical fields. Plasma activated water is produced by generating plasma on the surface of a liquid medium or in water.

The plasma-activated culture medium of the present invention may be used in the same sense as a plasma-conditioned medium (PCM). Such a plasma treatment medium has the same or improved or similar anticancer effect through the same mechanism as the irradiated plasma, and has an advantage of being easy to move and apply compared to plasma.

For the production of the plasma-activated culture medium of the present invention, the medium irradiated with plasma may use any known medium used for cell culture without limitation, and the medium may be used for culturing the corresponding cells according to the type of cells selected for the culture. It is preferable to select and use an appropriate medium. For example, in the case of a medium suitable for epithelial tissue of the skin, for example, melanocytes or keratinocytes, M254 medium may be used as a basic medium. The M254 medium may not contain calcium, and 20 amino acids, vitamins, inorganic salts, Adenine.HCl, D-Glucose (Dextrose), DL-alpha-lipoic Acid, Ethanolamine, HEPES, O-Phosphorylethanolamine , Putrescine 2HCl, Sodium Pyruvate, and Thymidine. The 20 amino acids are glycine, alanine, alanyl glutamine, arginine hydrochloride, asparagine, aspartic acid, cysteine, glutamic acid, histidine hydrochloride, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Includes. The vitamins include choline chloride, pantothenic acid, folic acid, myo-inositol, niacinamide, pyridoxal hydrochloride, vitamin B12, and d-Biotin. The inorganic salts are NaVO3, ((NH4)6(Mo7O24-4H2O), CuSo4-5H2O, FesO4-7H2O, MgCl2-6H2O, MnSO4-H2O, NiCl2-6H2O, KCl, NaCH3COO-3H2O, NaHCO3, NaCl, Na2SiO3-9H2O. , Ha2HPO4-7H2O, Na2SeO3, SnCl2-2H2O, and ZnSO4-7H2O.

In order to prepare the plasma-activated culture medium of the present invention, the medium to which plasma is irradiated may contain melanocyte growth supplement (HMGS).

The plasma may be generated using microwave power, and the microwave power may be appropriately selected and used by a person skilled in the art according to the purpose and method of the experiment, for example, 1.0W to 5.0W, 2.0W to 4.0 It may be W or 2.5W to 3.5W, preferably 3.0W.

The plasma may be irradiated in a state in which the culture medium to be irradiated with plasma and the plasma gas (plume) are in contact with each other or at a location separated by a predetermined distance. When the plasma electrode is separated from the medium and the plasma is irradiated, the separation distance is 0.1 to 15cm, 0.1 to 10cm, 0.1 to 5cm, 0.1 to 3cm, 0.1 to 1cm, 0.3 to 15cm, 0.3 to 10cm, 0.3 to 5cm, 0.3 to 3cm , 0.3 to 1cm, 0.5 to 15cm, 0.5 to 10cm, 0.5 to 5cm, or may be 0.5 to 3cm, preferably 0.5 to 1cm, more preferably 0.6 to 1cm. When the electrode is irradiated in contact with the culture solution, the electrode may be positioned on the surface of the culture solution or in the culture solution.

The time for irradiating the plasma to the culture medium is 4 minutes to 30 minutes, 4 minutes to 25 minutes, 4 minutes to 20 minutes, 5 minutes to 30 minutes, 5 minutes to 25 minutes, 5 minutes to 20 minutes, or 8 minutes to 20 minutes. It can be minutes.

In one embodiment of the present invention, a plasma-activated culture solution was prepared by applying normal temperature and atmospheric pressure plasma to the M254 medium. Plasma-activated cultures were prepared by irradiating plasma for 4 minutes, 8 minutes, 12 minutes, 16 minutes, or 20 minutes while the electrodes were 0.8cm apart from the surface of the 10ml M254 medium contained in a Petri dish. The conditions of the room temperature and atmospheric pressure plasma were performed at room temperature and atmospheric pressure using 3.0W microwave power and argon (Ar) gas, and the plasma generating apparatus and the medium were separated by 0.8 cm. 10 mL of cell-free medium was placed in a Petri dish, and plasma was irradiated for 4 minutes, 8 minutes, 12 minutes, 16 minutes, or 20 minutes, respectively, to prepare a final plasma active culture solution. Microwave accuracy was adjusted to ±2.0%, gas pressure was 0 to 3 Bar, and gas flow was adjusted to 0 to 5 SLM.

The emission spectrum from the injected argon gas atom was 690 to 990 nm and the peaks due to N2 and O were measured between 337.2 and 690 to 900 nm, respectively. The OH band was observed in the range of 307 to 309 nm.

The plasma-activated culture medium provided by the present invention has the advantage of increasing the amount of melanin production in melanocytes without causing death or cell damage of melanocytes.

The plasma-activated culture medium provided by the present invention has the ability to produce melanin of melanocytes by 1.0 to 4 times, 1.0 to 3 times, 1.1 to 4 times, 1.1 to 3 times, 1.1 to 2.5 times, 1.1 compared to the culture solution not irradiated with plasma. To 2 times, 1.3 to 4 times, 1.3 to 3 times, 1.3 to 2.5 times, 1.3 to 2 times, or 1.4 to 1.8 times.

In one embodiment of the present invention, when PAM was used, the expression levels of tyrosinase, TRP1 and TRP2 involved in melanin synthesis were all increased.

In an embodiment of the present invention, when a plasma-activated culture medium is used, the amount of melanin increases in proportion to the plasma irradiation time (FIG. 2). More specifically, it was confirmed that the amount of melanin increased in the group where the plasma irradiation time of the plasma-activated culture medium was 8 minutes, and the amount of melanin was rapidly increased in the 12-minute treatment group, and the same result was shown at 16 minutes and 20 minutes. . When compared with the control group, which is melanocytes cultured in a culture medium that was not treated with plasma, the 20-minute treatment group, which showed the result of the highest melanin amount, was 1.7 times as much as the melanin amount after 24 hours elapsed after treatment with the plasma-activated culture medium. , After 48 hours, the result was increased by 1.55 times.

Therefore, it can be seen that the plasma-activated culture medium of the present invention can increase the melanogenesis of melanocytes.

The culture composition according to the present invention is produced when melanocytes are cultured for 24 hours in a plasma-activated culture solution irradiated with plasma at room temperature and pressure for 20 minutes in a mammalian cell culture solution containing melanocyte growth factor (HMGS). The amount may be 1.1 to 4 times the amount of melanin generated when melanocytes are cultured in a mammalian cell culture medium that has not been subjected to plasma treatment. Specifically, the amount of melanin can be expressed in micrograms / (1 X 10 ⁶ cells).

The culture composition according to the present invention expresses tyrosinase mRNA when melanocytes are cultured for 24 hours in a plasma active culture medium irradiated with plasma at room temperature and pressure for 20 minutes in a mammalian cell culture medium containing human melanocyte growth supplement (HMGS). The amount is 1 to 4 times, 1.01 to 4 times, 1.01 to 3 times, 1.01 to 2.7 times, 1.02 to 4 times, 1.01 to 4 times, 1.01 to 3 times, based on the amount of tyrosinase mRNA expression when melanocytes are cultured in the mammalian cell culture medium not subjected to plasma treatment. It may be 4 times, 1.02 to 3 times, 1.02 to 2.7 times, 1.05 to 4 times, 1.05 to 3 times, 1.05 to 2.7 times.

The culture composition according to the present invention may be one to increase the expression of one or more genes selected from the group consisting of tyrosinase, TRP1, and TRP2 compared to a control group not treated with a plasma active culture medium.

Specifically, the expression level of TRP1 in melanocytes cultured in the plasma active culture medium is 1.01 to 4 times, 1.01 to 3.5 times, 1.01 to 3 times, 1.01 to 2.5 times, 1.01 compared to the expression level of the control group not containing the plasma active solution. To 2.3 times, 1.01 to 2.0 times, 1.05 to 4 times, 1.05 to 3.5 times, 1.05 to 3 times, 1.05 to 2.5 times, 1.05 to 2.3 times, or 1.05 to 2.0 times. The expression level of TRP2 in melanocytes cultured in the plasma active culture medium was 1.01 to 4 times, 1.01 to 3.5 times, 1.01 to 3 times, 1.01 to 2.5 times, 1.01 to 2.3 times compared to the expression level of the control without plasma active solution. , 1.01 to 2.0 times, 1.01 to 1.5 times, 1.05 to 4 times, 1.05 to 3.5 times, 1.05 to 3 times, 1.05 to 2.5 times, 1.05 to 2.3 times, 1.05 to 2.0 times, or 1.05 to 1.5 times .

The method for increasing melanin synthesis provided by the present invention,

Preparing a plasma-activated culture medium (PAM) by irradiating plasma to the cell culture medium; And

And culturing the plasma-activated culture medium as a cell culture medium.

The method for preparing the plasma active culture solution is as described above.

The plasma-activated culture medium is preferably used immediately after preparation, and specifically within 1 week, within 5 days, within 3 days, within 2 days, within 24 hours, within 12 hours, within 8 hours, within 4 hours, It may be used within 2 hours after manufacture, within 1 hour after manufacture, within 30 minutes after manufacture, within 15 minutes after manufacture, within 10 minutes after manufacture, or within 5 minutes after manufacture, but is not limited thereto. The time required for use after the preparation refers to the time required to replace the cell culture solution after plasma irradiation is completed on the plasma active culture solution.

The cells may be any one having melanin synthesis activity, excluding cells in the human body, for example, melanocytes, melanoma cells, or transformed cells having melanin synthesis ability. have. The melanocytes may be isolated from mammals including humans.

Cells other than cells in the human body may be cells being cultured in a culture medium. The culture medium in which the cells are cultivated may be a culture medium that has not been irradiated with plasma, and if necessary, a culture medium of the same or different types as the plasma active culture medium may be selected and used, and M254 medium may be preferably used.

In one embodiment of the present invention, 1% (v/v) of human melanocyte growth supplement (HMGS, Gibco Cascade biologics, USA), 100 U/ml penicillin and 50 ug/ml streptomycin were added to M254 in human skin melanocytes. After culturing in the medium, the culture medium was replaced with M254 medium irradiated with plasma for 4 minutes, 8 minutes, 12 minutes, 16 minutes or 20 minutes, respectively, and cultivation was performed thereafter.

The method of increasing melanin synthesis according to the present invention is that the amount of melanin production is 1.2 to 4 times, 1.2 to 3.5 times, 1.2 to 3 times, 1.2 to 2.5 times, 1.2 to 2 times, 1.2 to 1.9 times, 1.2 to 1.8 times compared to the control group. Times, 1.3 to 4 times, 1.3 to 3.5 times, 1.3 to 3 times, 1.3 to 2.5 times, 1.3 to 2 times, 1.3 to 1.9 times, 1.3 to 1.8 times, 1.4 to 4 times, 1.4 to 3.5 times, 1.4 to 3 times Times, 1.4 to 2.5 times, 1.4 to 2 times, 1.4 to 1.9 times or 1.4 to 1.8 times.

The method of increasing melanin synthesis according to the present invention may be that the plasma-activated culture medium increases the expression of one or more genes selected from the group consisting of tyrosinase, TRP1 and TRP2, thereby increasing melanin synthesis.

In one embodiment of the present invention, the amount of melanin increased in proportion to the plasma irradiation time (FIG. 2), and more specifically, the amount of melanin increased rapidly in the 12-minute treatment group. In the case of the 20-minute treatment group, where the amount of melanin was highest, the amount of melanin increased by 1.7 times after 24 hours and 1.55 times after 48 hours, compared to the control without treatment.

In one embodiment of the present invention, as a result of analyzing the amount of mRNA of tyrosinase, TRP1, and TRP2 involved in melanin synthesis using a plasma active culture medium as a cell culture medium, in the case of tyrosinase, each The amount of mRNA of the gene was high (FIGS. 3 to 5 ). More specifically, in the case of tyrosinase, the amount of mRNA increased in proportion to the plasma irradiation time from 8 minutes to 20 minutes, and the amount of mRNA increased 2.5 times compared to the control at 20 minutes (Fig. 3). ), TRP1 and TRP2 did not show an increase in expression in proportion to the plasma irradiation time, but the expression levels of TRP1 and TRP2 increased compared to the control in all cells cultured in the plasma-activated culture medium (FIGS. 4 and 5).

In one embodiment of the present invention, as a result of using the plasma active culture medium as a cell culture medium, no damage or death of melanocytes was observed (Example 3). More specifically, as a result of examining melanocytes under a microscope, no damage to the cells appeared in appearance, such as dendritic processes peculiar to melanocytes were confirmed (FIG. 6 ). Therefore, the method of increasing melanin synthesis according to the present invention can increase melanin without damaging normal cells.

The method of increasing melanin synthesis according to the present invention increases melanin production in melanocytes, increases the expression of tyrosinase, an enzyme that regulates melanin synthesis, and expression of TRP1 and TRP2, which are factors involved in melanin formation. Increase (Example 2). The results of Example 2 indicate that the plasma-activated culture medium of the present invention has an effect of alleviating melanin hypopigmentation by increasing melanin production. Therefore, the plasma-activated culture medium of the present invention can be used for prevention, improvement and treatment of melanin hypopigmentation.

In the present invention, melanin hypopigmentation refers to vitiligo, albinism, depigmented nevus, white nasal nevus, corpus callosum, post-inflammatory depigmentation, ecchymosis, partial leukocytosis, idiopathic red pigment hypopigmentation or petechiae leukoplakia.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating melanin hypopigmentation comprising the plasma active culture medium of the present invention as an active ingredient.

When the composition of the present invention is prepared as a pharmaceutical composition, a pharmaceutically effective amount of the plasma active culture solution of the present invention and a pharmaceutically acceptable carrier may be included. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the composition comprising the plasma active culture medium.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.

The pharmaceutical composition of the present invention is preferably administered parenterally, and may be administered, for example, by topical skin administration.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, Usually the skilled practitioner can readily determine and prescribe the dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-1000 mg/kg.

The pharmaceutical composition of the present invention is prepared in a unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs. Or it can be prepared by placing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule or gel (eg, hydrogel), and may additionally include a dispersant or a stabilizer. .

In the pharmaceutical composition according to the present invention, the plasma active culture medium may be contained in an amount of 0.001 to 99.9% by weight, preferably 0.01 to 50% by weight.

The present invention also provides a cosmetic composition for increasing melanin production comprising the plasma-activated culture medium. The cosmetic composition according to the present invention can be used for the prevention or improvement of skin tanning or melanin hypopigmentation.

The plasma-activated culture medium has a surface of the culture medium and a plasma electrode of 0.1 to 15cm, 0.1 to 10cm, 0.1 to 5cm, 0.1 to 3cm, 0.1 to 1cm, 0.3 to 15cm, 0.3 to 10cm, 0.3 to 5cm, 0.3 to 3cm, 0.3 To 1cm, 0.5 to 15cm, 0.5 to 10cm, 0.5 to 5cm, 0.5 to 3cm, 0.5 to 1cm, or 0.6 to 1cm apart from 4 minutes to 30 minutes or 4 minutes to 20 minutes at room temperature and atmospheric pressure plasma It may be prepared, and the culture medium may be M254 medium. The M254 medium may be one to which human melanocyte growth supplement (HMGS) is added.

In the present invention, "tanning" refers to any cosmetic action that makes the skin dark for cosmetic purposes, and skin tanning using the sun and/or artificial ultraviolet rays, and temporary, semi-permanent, or permanent skin tanning using a tanning cream or lotion Includes all coloring.

The melanin hypopigmentation refers to vitiligo, albinism, depigmented nevus, white nasal nevus, corpus callosum, post-inflammatory depigmentation, ecchymosis, partial leukocytosis, idiopathic redness hypopigmentation, or petechiae.

In one embodiment of the present invention, since no cell damage or killing effect on normal melanocytes was observed (Example 3), the cosmetic composition provided by the present invention is different from skin tanning using ultraviolet rays or the sun. You can expect an effective tanning effect with less risk.

The cosmetic composition of the present invention is a solution, external ointment, cream, foam, nutritional lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleaning agent, bathing agent, sunscreen cream, sun oil, suspension, emulsion It can be prepared in a formulation selected from the group consisting of liquid, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch, and spray, but is limited thereto. no.

In addition, the cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers formulated in general skin cosmetics, and for example, oils, water, surfactants, moisturizers, lower alcohols, etc. Thickeners, chelating agents, pigments, preservatives, fragrances, etc. may be appropriately blended, but are not limited thereto.

The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation.

When the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures of these can be used.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component, and in particular, in the case of a spray, additional chloro Propellants such as fluorohydrocarbon, propane/butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent, or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3 -Butylglycol oil, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspensions such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, micro Crystalline cellulose, aluminum metahydroxide, bentonite, agar or trakant, etc. can be used.

When the formulation of the present invention is a soap, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolyzate, isethionate, a lanolin derivative, an aliphatic alcohol, vegetable oil, glycerol, sugar, etc. may be used as a carrier component. I can.

The cosmetic composition of the present invention may contain 0.001 to 30% by weight of the plasma active culture solution.

The present invention also provides a method of screening for melanogenesis inhibitors.

The melanin production inhibitor screened by the above screening method can be used for skin whitening.

The method of screening for a substance that inhibits melanin production of the present invention comprises the steps of preparing a plasma-activated culture solution (PAM) by irradiating plasma to a cell culture solution, culturing the plasma-activated culture solution as a cell culture solution, and the plasma activity Treating the candidate material to the cell culture in which the culture medium is used; And analyzing the content of at least one selected from the group consisting of melanin, tyrosinase mRNA, tyrosinase protein, TRP1 mRNA, TRP1 protein, TRP2 mRNA, and TRP2 protein in the cell.

In one embodiment of the present invention, melanin production was increased by using a plasma-activated culture medium (PAM) as a cell culture medium (Example 1), and therefore, a candidate substance was treated on the cells before or after the treatment of the plasma-activated culture medium to treat intracellular melanin. , Tyrosinase mRNA, tyrosinase protein, TRP1 mRNA, TRP1 protein, TRP2 mRNA, and TRP2 protein.By checking the content of one or more selected from the group consisting of, it is possible to screen for substances involved in skin pigmentation or whitening. There is an effect.

After the step of analyzing the content of at least one selected from the group consisting of melanin, tyrosinase mRNA, tyrosinase protein, TRP1 mRNA, TRP1 protein, TRP2 mRNA, and TRP2 protein in the cell, the analysis result, the candidate substance When the content of at least one selected from the group consisting of intracellular melanin, tyrosinase mRNA, tyrosinase protein, TRP1 mRNA, TRP1 protein, TRP2 mRNA and TRP2 protein decreases compared to the absence of It may further include the step of determining the compound.

For the measurement of the amount of melanin in the cell, those skilled in the art can freely select and use a method known in the art, for example, absorbance measurement, or western blot, immunohistochemistry, confocal microscopy, and It may be one or more methods selected from the group consisting of polymerase chain reaction (PCR), but is not limited thereto. The absorbance measurement may be performed by measuring the absorbance of a solution obtained by dissolving a cell lysate in concentrated sulfuric acid or sodium hydroxide.

In one embodiment of the present invention, the measurement of the amount of melanin in cells was performed by dissolving melanocytes in 1N NaOH at 60° C. for 1 hour to measure absorbance for a wavelength of 470 nm (Example 1).

One or more methods for measuring intracellular content selected from the group consisting of tyrosinase mRNA, TRP1 mRNA, and TRP2 mRNA can be freely selected and used by those skilled in the art. For example, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time PCR, RNase protection assay (RPA), microarray , And Northern blotting (northern blotting) may be performed by measuring the transcription level level of one or more mRNA selected from the group consisting of, but is not limited thereto.

The method for measuring the intracellular content of one or more selected from the group consisting of melanin, tyrosinase protein, TRP1 protein, and TRP2 protein includes western blotting, radioimmunoassay (RIA), and radioimmunodiffusion. , Enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and protein chip ( protein chip) may be performed by measuring the expression or activity level of one or more proteins selected from the group consisting of, but is not limited thereto.

The candidate substance or a compound that inhibits melanin production may be one that inhibits the expression of one or more selected from the group consisting of tyrosinase, TRP1, and TRP2.

The present invention also provides a composition for skin whitening comprising, as an active ingredient, a material selected to have melanin production inhibition or skin whitening effect according to the screening method.

The composition may be a pharmaceutical composition. The pharmaceutical composition may be used for prevention, treatment, or alleviation of diseases caused by overproduction of melanin.

Diseases caused by the overproduction of melanin include melasma, freckles, age spots, blemishes, epidermal melanocytic lesions, milk coffee spots (Cafe's au lait macules), Becker's Nevus, and Nevus Spilus ), Lentigines, Dermal melanocytic lesions, Mongolian spot, Nevus of Ota, Acquired bilateral nevus of Ota-like macules, Nevus of Ito, Blue nevus, Melanocytic nevus, Junctional nevus, Compound nevus, Intradermal nevus, Ilium nevus Halo nevus), Congenital nevocytic nevus, Spitz nevus, Dysplastic nevus, Melanoma, Lentigo maligna melanoma, Superficial dilated melanoma (Superficial spreading melanoma), Acral lentiginous melanoma, Nodular melanoma, pigment basal cell carcinoma, dermatofibromas, pigmented skin cyst (dermoid) cyst), pigmented keloid, and pigmented keratoacanthomas.

In addition to the melanin production inhibitor, the pharmaceutical composition may further contain pharmaceutical adjuvants such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts and/or buffers for controlling osmotic pressure, and other therapeutically useful substances. Depending on the method, it can be formulated into various oral or parenteral dosage forms.

The oral administration agents include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, powders, powders, fine granules, granules, pellets, etc., and these formulations include surfactants other than active ingredients. , Diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (e.g. silica, talc, stearic acid and magnesium or calcium salts thereof and polyethylene glycol) . Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally starch, agar, alginic acid or sodium salts thereof. It may contain pharmaceutical additives such as disintegrants, absorbents, colorants, flavors, and sweeteners such as. The tablets can be prepared by conventional mixing, granulating or coating methods.

In addition, the parenteral dosage form may be a transdermal dosage form, for example, injections, drops, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, patches, etc. May be, but is not limited thereto.

The pharmaceutical composition may be administered parenterally, rectal, topically, transdermally, subcutaneously, etc. The determination of the dosage of the active ingredient is within the level of a person skilled in the art, and the daily dose of the drug is the progress of the subject to be administered. Although it depends on various factors such as degree, onset time, age, health condition, complications, etc., the composition is 1㎍ / kg to 200mg / kg in one aspect, and in the other aspect, 50㎍ / kg to 50mg on the basis of an adult /kg may be divided and administered 1 to 3 times a day, and the dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition may be an external preparation for skin, and the external preparation for skin is a generic term that may include anything applied outside the skin, and pharmaceuticals of various formulations may be included therein.

According to one embodiment of the present invention, the composition may be a cosmetic composition for whitening skin.

In addition to the exosome inhibitor, the cosmetic composition may further include functional additives and components included in general cosmetic compositions. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingo lipids, and seaweed extract. Other components included include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation Accelerators, cooling agents, limiting agents, purified water, and the like.

The formulation of the cosmetic composition is not particularly limited, and may be appropriately selected depending on the intended purpose. For example, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing Foam, cleansing lotion, cleansing cream, body lotion, and may be prepared in one or more formulations selected from the group consisting of a body cleanser, but is not limited thereto.

By adding the plasma-activated culture medium provided by the present invention to cultured melanocytes, it is possible to increase the amount of melanin production in melanocytes without damaging cells or leading to cell death. By using a composition containing the plasma-activated culture medium, it can be used for the treatment, alleviation or treatment of skin diseases such as melanin hypopigmentation, or cosmetics for tanning skin with reduced risk of skin cancer.

In addition, by using the screening method for melanin production inhibitors provided by the present invention, an inhibitor that effectively inhibits melanin production in cells with increased melanin production can be selected, and the prevention of diseases caused by overproduction of melanin using the inhibitor as an active ingredient, It is possible to provide a pharmaceutical composition for alleviation or treatment or a pharmaceutical composition for whitening or a cosmetic composition.

1 is a result of visual observation of the color of a solution obtained by dissolving cell lysate of each melanocyte in NaOH.
2 is a graph showing the result of measuring the amount of melanin production after culturing for 24 hours or 48 hours after adding a plasma active culture medium, a-MSH, or arbutin to melanocytes.
3 is a graph showing the results of measuring the amount of tyrosinase mRNA after adding each of the indicated additives to the melanocyte culture solution and then culturing for 24 hours.
4 is a graph showing the results of measuring the amount of TRP1 mRNA after adding each of the indicated additives to the melanocyte culture solution and culturing for 24 hours.
5 is a graph showing the results of measuring the amount of TRP2 mRNA after adding each of the indicated additives to the melanocyte culture medium and culturing for 24 hours.
6 is a photograph of melanocytes cultured for 24 hours or 48 hours by adding a plasma-activated culture medium irradiated with plasma for 20 minutes under a microscope.
7 is a graph showing the results of measuring the number of viable cells after culturing for 24 hours or 48 hours by adding each plasma active culture solution.
8A is a schematic diagram of a normal temperature and normal pressure plasma generator.
8B is a graph showing an optical emission spectrum in the range of 200 to 100 nm during discharge of the plasma jet at room temperature and pressure according to Example 1-1.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention and do not limit the scope of the present invention.

Example 1. Confirmation of the effect of increasing the amount of melanin in cells of the plasma active culture medium

1-1: Preparation of plasma activated media (PAM)

In order to compensate for the disadvantages such as the inability to obtain the plasma activation effect evenly on all cells when the plasma is directly irradiated to the cells in culture, a plasma-activated culture solution was prepared and used in the experiment by directly irradiating plasma to the liquid medium for cell culture. .

M254 medium (GIBCO Cascade Biologics, Portland, OR, USA) was used as the melanocyte culture medium, and a plasma active medium was prepared by applying normal temperature and atmospheric pressure plasma to the M254 medium.

Specifically, the room temperature and atmospheric pressure plasma conditions according to the present invention were performed at room temperature and atmospheric pressure using 3.0W microwave power and argon (Ar) gas, and the plasma generating device and the medium were separated by 0.8 cm. The plasma irradiation temperature did not exceed 40°C. 10 mL of cell-free medium was placed in a Petri dish, and plasma was irradiated for 4 minutes, 8 minutes, 12 minutes, 16 minutes, or 20 minutes, respectively, to prepare a final plasma active culture solution. Microwave accuracy was adjusted to ±2.0%, gas pressure was 0 to 3 Bar, and gas flow was adjusted to 0 to 5 SLM. Fig. 8A shows a schematic diagram of the normal temperature and normal pressure plasma generator, and Fig. 8C shows the distance between the plasma generator electrode and the medium.

8B shows the optical emission spectrum during discharge of the plasma jet at room temperature and pressure of the present invention in the range of 200 to 1000 nm. The emission spectrum from the injected argon gas atom was 690 to 990 nm and the peaks due to N2 and O were measured between 337.2 and 690 to 900 nm, respectively. The OH band was observed in the range of 307 to 309 nm.

After culturing the melanocytes first, the experiment was carried out by replacing the culture medium with a plasma-activated medium, and plasma-activated medium (PAM) was used for culturing melanocytes immediately after plasma irradiation.

1-2: culture of melanocytes

The melanocytes of normal human skin used in the present invention were purchased from ATTC (Manassas, VA, USA) and used. The melanocytes used in the present invention were M254 Medium (GIBCO Cascade Biologics, USA) in a medium to which 1% of human melanocyte growth supplement (HMGS, Gibco Cascade biologics, USA), 100 U/ml penicillin and 50 ug/ml streptomycin were added. Cultured.

Dispense the cultured cells into a 60mm cell culture dish at 1 Х 10 ⁶ cells/dish and adapatation for 24 hours, followed by 4 minutes, 8 minutes, 12 minutes, 16 minutes, or 20 minutes of plasma irradiation in 10 mL of plasma activated culture medium. Or 48 hours incubation. As a positive control, α-MSH (α-melanocyte stimulating hormone), which is known to have an effect of increasing melanin production instead of plasma active culture, was treated in 10 mL of medium to a final concentration of 1 μM, and as a negative control Arbutin, which is known to inhibit melanin production, was added to 1 mM.

After incubation, cells were harvested after treatment with trypsin-EDTA solution (JBI, Seoul, Korea) and washing with PBS containing 5% (v/v) FBS. All experiments were repeated 3 times.

1-3. Color change of cell lysate

The amount of melanin contained in melanocytes was measured using absorbance. Specifically, first, each melanocyte was dissolved in 1N NaOH at 60° C. for 1 hour to prepare a sample for measuring absorbance. The color of the absorbance measurement sample obtained by dissolving the obtained cell lysate in NaOH was visually observed.

Figure 1 shows the results of dissolving each melanin cell lysate in NaOH. When the plasma-activated culture medium irradiated with plasma for 20 minutes was used, the color of the solution was darker than that of the control group. Control refers to the case where the plasma-activated culture medium is not treated, Arbutin refers to the cell lysate treated with arbutin, and Plasma refers to the case of using the plasma culture medium irradiated with normal temperature and atmospheric pressure plasma for 20 minutes. In the case of arbutin treatment, the color of the NaOH solution of the cell lysate was the lightest, indicating that melanin production was suppressed, as is known, and when the plasma-activated culture medium was treated, the color was dark enough to be visible with the naked eye compared to the control. This indicates that the amount of melanin in the melanocytes treated with the plasma-activated culture medium was increased (FIG. 1).

1-4. Measurement of melanin amount using absorbance

The amount of melanin contained in melanocytes was measured using absorbance. Specifically, first, each melanocyte was dissolved in 1N NaOH at 60° C. for 1 hour to prepare a sample for measuring absorbance. The absorbance of each sample was measured using a microplate reader (Spark 10M, Tecan, Austria) at 37° C. for a wavelength of 470 nm, and all experiments were repeated three times.

The results of measuring the absorbance are shown in FIG. 2 and Table 1 below. The upper graph of FIG. 2 shows as an example that melanocytes were cultured in a plasma-activated culture medium obtained by irradiating plasma for 4, 8, 12, 16 or 20 minutes, and after adding a-MSH or arbutin to M254 medium It is a graph showing the result of measuring the amount of melanin produced after incubation for 24 hours. The lower graph of FIG. 2 is a graph showing the results of measuring the amount of melanin produced after culturing for 48 hours. Control is M254 medium without treatment of plasma-activated culture medium, and as negative control, arbutin is added to M254 medium to be 1 mM, and positive control is α-MSH in M254 medium. Is added.

24 hours 48 hours Melanin production (ug/1x10 ⁶ ) Standard Deviation Melanin production (ug/1x10 ⁶ ) Standard Deviation control 26.41 0.18 45.60 0.07 α-MSH (1μM) 31.59 0.47 48.07 0.49 Arbutin (1mM) 24.79 0.97 41.12 0.42 PAM 4min 26.32 0.11 50.44 0.32 PAM 8min 32.99 0.24 56.90 0.28 PAM 12min 39.77 0.21 66.39 0.13 PAM 16min 43.08 0.55 68.70 0.17 PAM 20min 44.45 0.11 70.15 0.12

In the case of using the plasma-activated culture medium, the amount of melanin increased in proportion to the plasma irradiation time (FIG. 2). More specifically, it was confirmed that the amount of melanin was increased in the group treated with the plasma-activated culture medium for 8 minutes, and the amount of melanin was rapidly increased in the group treated for 12 minutes, and the same results were shown at 16 minutes and 20 minutes. Based on the treatment group with a plasma irradiation time of 20 minutes, the amount of melanin reached the highest level. When the culture time of melanocytes in the plasma-activated culture medium elapsed for 24 hours, the amount of melanin was treated, and the plasma was treated in M254 medium. The amount of melanin was increased by 1.7 times compared to the control which was not used and after 48 hours by 1.55 times.

Therefore, it can be seen that the plasma-activated culture medium of the present invention can increase the melanogenesis of melanocytes.

Example 2. Confirmation of the amount of mRNA of genes related to melanin synthesis

2-1. Cell culture

Melanocytes were dispensed into a 100mm cell culture dish to be 1.5 Х 10 ⁶ cells/dish, and cultured for 72 hours. A control cultured using only a general culture medium, a positive control with 10 mL of 1uM a-MSH, a negative control with 10 mL of 1mM arbutin , plasma irradiation time of 4 minutes, 8 minutes, 12 minutes, 16 minutes, or 20 minutes The experimental group cultured in the plasma activated culture solution was further cultured for 24 hours after treatment with each additive.

2-2. RNA extraction and RT-PCR

It is known that melanogenesis in melanocytes is regulated by tyrosinase, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2). have. Therefore, the expression levels of the three genes involved in improving the melanin production ability of melanocytes by the plasma-activated culture medium were examined using real-time PCR.

Total RNA was extracted from each of the melanocytes using TRIzol reagent. Specifically, 1 mL of Trizol reagent was added to each test group and control cells to a 100 mm dish to homogenize the cells, then harvested, 0.2 mL of chloroform was added, and strongly mixed for 15 seconds. Subsequently, after reacting at room temperature for 2-3 minutes, centrifugation was performed at 4° C. for 15 minutes at 12,000 g, and 500 μl of 100% isopropanol was added to the resulting supernatant, mixed, homogenized, and centrifuged at 4° C. and 12,000 g for 10 minutes. After washing by adding 1 mL of 75% Ethanol to the obtained precipitate, water was removed at room temperature for 10 minutes, and then centrifuged at 4°C for 5 minutes at 75,000 g to collect RNA, and then DEPC distilled water was added and stored.

CDNA synthesis was performed using 1 μg of total RNA extracted by the above-described method as a template. Specifically, cDNA was synthesized by primer annealing at 25°C for 10 minutes, reverse transcriptase (RT) reaction at 42°C for 60 minutes, and transcriptase denaturation at 95°C for 5 minutes. Then using a Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), 1 μl cDNA, 1 μl primer solutions (solutions of both the reverse and forward primers), 10 μl Power SYBR Green PCR Master Mix, and 7 Add 20 μl of each reaction extract to which μl PCR-grade water was added, and initial denaturation at 95˚C for 10 minutes, denaturation at 95˚C for 15 seconds and annealing at 60˚C and 1 minute for real-time gene expression. Investigated.

The primer solution is a solution containing a primer pair of SEQ ID NOs: 1 and 2, a primer pair of SEQ ID NOs: 3 and 4, or a primer pair of SEQ ID NOs: 5 and 6 shown in Table 2 below.

Sequence number name Base sequence (5'→ 3') One tyrosinase_F 5'-TTGGCAGATTGTCTGTAGCC-3' 2 tyrosinase_R 5'-AGGCATTGTGCATGCTGCTT-3'' 3 TRP1_F 5'-AGAGATGATCGGGAGGTCTG-3'' 4 TRP1_R 5′-CTGTGCCATGTGAGAAAAGC-3′' 5 TRP2_F 5′-GCCGGCTATAATTGTGGAGA-3′' 6 TRP2_R 5'-AGGGCAAAGCAAAAGACTCA-3''

It is known that melanogenesis in melanocytes is regulated by tyrosinase, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2). have. Therefore, the expression levels of three genes involved in improving the melanin production ability of melanocytes by the plasma-activated culture medium were measured, and the results are shown in FIGS. 3 to 5 and Table 3. Table 3 shows the relative expression levels of each sample when the mRNA expression level of the control group is set to 1.

Tyrosinase TRP1 TRP2 Control One One One a-MSH 1uM 1.39 1.49 1.21 Arbutin 1mM 0.90 0.94 0.78 PAM 4min 1.08 1.14 1.06 PAM 8min 1.07 1.10 1.11 PAM 12min 1.31 1.15 1.07 PAM 16min 2.04 1.93 1.10 PAM 20min 2.57 1.58 1.35

Tyrosinase showed a result of increasing in proportion to the exposure time from the time when the exposure time to the plasma-activated culture medium was 8 minutes.In particular, the mRNA expression level increased sharply in the plasma 16-minute treatment group compared to the positive control treated with a-MSH, The maximum was reached in 20 minutes (about 2.5 times compared to the control without treatment of the plasma active culture medium in M254) (Fig. 3).

In the case of TRP1 and TRP2, the expression was not increased in proportion to the plasma irradiation time, but it was confirmed that the expression level was increased in all cells cultured in the plasma-activated culture medium compared to the control (FIGS. 4 and 5).

Therefore, it can be predicted that tyrosinase, TRP1, and TRP2, which are involved in melanin synthesis, are all increased by culturing in a plasma-activated culture medium, thereby contributing to increasing the amount of melanin in melanocytes.

Example 3. Confirmation of cell death or damage

3-1. Cell culture

Dispense melanocytes into a 60mm cell culture dish at 5 Х 10 ⁵ cells/dish and incubate for 24 hours, then plasma-activated culture solution (plasma irradiation time of 4 minutes, 8 minutes, 12 minutes, 16 minutes or 20 minutes, respectively) or 1uM a -MSH, 1mM arbutin was added, hydrogen peroxide (H ₂ O ₂ ), which is known to form ROS, was treated to a final concentration of 100 μM or 200 μM, followed by incubation for 3 hours. Then, each cell was washed with PBS, and M254 containing 20 uM 2, 7-dichlorofluorescin diacetate (2, 7-dichlorofluorescin diacetate; DCFH-DA) (Sigma-Aldrich, St. Louis, MO, USA). The medium was treated with HMGS at 37°C for 30 minutes and used for flow cytometry.

3-2. Microscopic observation

The results of microscopic observation of cells cultured for 24 hours or 48 hours in a plasma-activated medium irradiated with plasma for 20 minutes are shown in FIG. 6, respectively. As can be seen in FIG. 6, as the culture time increases, the number of melanocytes increased, and dendritic processes peculiar to melanocytes were identified, indicating that almost no damaged cells were observed. Therefore, it can be seen that the plasma-activated culture medium of the present invention does not cause apoptosis or damage of melanocytes, unlike when the active oxygen is increased by irradiating plasma to cancer cells.

3-3. Cell count

Apoptosis or cell damage was confirmed by measuring the number of cells. Specifically, after attaching 1×10 ⁶ cells to a 100 mm culture dish for 24 hours, the culture solution was removed, and melanocytes were cultured using PAM treated for 4 minutes, 8 minutes, 12 minutes, 16 minutes, and 20 minutes, respectively. Subsequently, after 24 and 48 hours, the cells were removed by treatment with trypsin-EDTA solution (JBI, Seoul, Korea), and the enzyme was inhibited with 5% FBS. Then, it was washed twice with phosphate buffered saline (PBS, pH 7.4) and the number of cells was measured using an automatic cytometer ADAM-MC cell counter (NanoEnTeK, Seoul, Korea).

To determine whether melanocytes are killed or damaged, the plasma exposure time of 0 minutes, 4 minutes, 8 minutes, 12 minutes, 16 minutes, or 20 minutes of plasma activated water is used. The number was measured. The results are shown in FIG. 7. As a result of measuring the number of cells, as the plasma exposure time increased, the number of harvested cells gradually decreased, but there was no decrease in the number of cells compared to the amount of inoculated cells, indicating that there was no cell death or cell damage. That is, the plasma-activated culture medium of the present invention has the effect of somewhat inhibiting cell proliferation, but does not cause cell death or cell damage.

<110> UNIVERSITY-INDUSTRY COOPERATION GROUP OF KYUNG HEE UNIVERSITY <120> Composition for the production of melanin comprising plasma-activated media and use thereof <130> DPP20184004KR <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> tyrosinase_F <400> 1 ttggcagatt gtctgtagcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> tyrosinase_R <400> 2 aggcattgtg catgctgctt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRP1_F <400> 3 agagatgatc gggaggtctg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRP1_R <400> 4 ctgtgccatg tgagaaaagc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRP2_F <400> 5 gccggctata attgtggaga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRP2_R <400> 6 agggcaaagc aaaagactca 20

Claims (15)

A culture composition for melanin production of melanocytes, comprising a plasma activated media (PAM) prepared by irradiating plasma to a mammalian cell culture medium containing melanocyte growth factor (HMGS). The composition of claim 1, wherein the plasma is a normal temperature and normal pressure plasma, and the normal pressure is atmospheric pressure. The composition of claim 1, wherein the plasma is irradiated for 4 to 30 minutes. The composition of claim 1, wherein the melanin-producing cells are melanocytes, melanoma cells, or transformed cells that produce melanin. The method of claim 1, wherein the culture medium is D-glucose, DL-alpha-lipoic acid, ethanolamine, Hepes, O-phosphorylethanol Amine (O-Phosphorylethanolamine), putrescine 2HCl, sodium pyruvate, and adenine HCl containing The composition. The composition of claim 1, wherein the plasma active culture medium increases the expression of one or more genes selected from the group consisting of tyrosinase, TRP1 and TRP2. The method of claim 1, wherein the culture composition is a case where melanocytes are cultured for 24 hours in a plasma-activated culture solution in which a mammalian cell culture solution containing human melanocyte growth supplement (HMGS) is irradiated with plasma at room temperature for 20 minutes. A culture composition in which the amount of melanin produced is 1.1 to 4 times the amount of melanin produced when the melanocytes are cultured in a mammalian cell culture medium not subjected to the plasma treatment. The method of claim 1, wherein the culture composition is a case where melanocytes are cultured for 24 hours in a plasma-activated culture solution in which a mammalian cell culture solution containing human melanocyte growth supplement (HMGS) is irradiated with plasma at room temperature for 20 minutes. A culture composition wherein the expression amount of tyrosinase mRNA is 1.1 to 4 times the amount of expression of tyrosinase mRNA when melanocytes are cultured in the mammalian cell culture medium not subjected to plasma treatment, wherein the normal pressure is atmospheric pressure. Preparing a plasma activated media (PAM) prepared by irradiating a plasma to a mammalian cell culture medium containing melanocyte growth factor (HMGS); And
A method for increasing melanin production of melanin producing cells, comprising the step of culturing melanin producing cells using the plasma-activated culture medium. The method of claim 9, wherein the plasma is a normal temperature and normal pressure plasma, and the normal pressure is atmospheric pressure. The method of claim 9, wherein the plasma is irradiated for 4 to 30 minutes. The method of claim 9, wherein the melanin producing cells are melanocytes, melanoma cells, or transformed cells that produce melanin. The method of claim 9, wherein the plasma-activated culture medium has increased expression of one or more genes selected from the group consisting of tyrosinase, TRP1 and TRP2. Preparing a plasma activated media (PAM) prepared by irradiating a plasma to a mammalian cell culture medium containing melanocyte growth factor (HMGS);
Culturing melanocytes using the plasma-activated culture medium as a cell culture medium;
Treating the cell culture cultured in the plasma-activated culture medium with a candidate substance for an inhibitor of melanogenesis; And
A method for screening a melanogenesis inhibitor comprising analyzing the content of one or more selected from the group consisting of melanin, tyrosinase mRNA, tyrosinase protein, TRP1 mRNA, TRP1 protein, TRP2 mRNA, and TRP2 protein in the melanocyte. . According to claim 14, After the step of analyzing the content of at least one selected from the group consisting of melanin, tyrosinase mRNA, tyrosinase protein, TRP1 mRNA, TRP1 protein, TRP2 mRNA, and TRP2 protein in the cell,
When the content of one or more selected from the group consisting of intracellular melanin, tyrosinase mRNA, tyrosinase protein, TRP1 mRNA, TRP1 protein, TRP2 mRNA, and TRP2 protein decreases compared to the case where no candidate substance is present, the above The method further comprising determining the candidate substance as an inhibitor of melanogenesis.

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