KR101256588B1 - Composition including beta-carboline alkaloid for stimulating melanin production - Google Patents

Abstract

The present invention provides a composition for promoting melanin production comprising a material selected from the group consisting of beta-carboline alkaloids, derivatives thereof, salts thereof, hydrates thereof and solvates thereof as an active ingredient. More specifically, since the active ingredient of the present invention has a low side effect in vivo and has an excellent effect of safely promoting melanin production, the composition can be used for a pharmaceutical composition, health food composition or cosmetic composition for promoting melanin production.

Description

Composition including beta-carboline alkaloid for stimulating melanin production}

The present invention relates to a composition for promoting melanin production.

A person's hair or skin color is generally determined by the amount of melanin pigment present in the hair or skin.

It has been reported that melanogenesis is induced by the synthesis and accumulation of melanin, which is involved in human skin pigmentation (Sturm R A. Skin color and skin cancer-MC1R, the genetic link.Melanoma Res. 2002: 5: 405-16). Melanogenesis also contributes to the synthesis and transfer of melanin and to melanosome release (Boissy R E. Melanosome transfer to and trnslocation in the keratinocyte. Exp Derm 2003: 13: 5-2).

Melanin synthesis is affected by several factors, such as cyclic AMP (cAMP) elevating agents (forskolin, glycyrrhizin, isobutylmethylxanthine), such as α-melanocyte-stimulating hormone (α-MSH). In addition, melanin biosynthesis between melanocytes and melanoma cells is produced by tyrosinase, which is guided by tyrosine, and is involved in tyrosinase related protein-1 (TRP-1) and tyrosinase related protein-2 (Tyrosinase related protein-2). TRP-2) is known to be involved.

Tyrosinase, TRP-1, and TRP-2, which are involved in the synthesis of melanin, contain a binding site of the transcription factor Microphthalmia-associated transcription factor (MITF), which is why the tyrosinase, TRP-1, and TRP-2 Expression is regulated by the activation of MITF.

This MITF also modulates the important role involved in mealnogenesis through the transcription factor cAMP response element binding protein (CREB). There is a CREB binding site in the MITF, whereby CREB regulates MITF promoter activity and is involved in indirect activation of melanongenesis.

Mitogen-activated protein kinases (MAPKs) consist of extracellularly responsive kinases (ERK1 / 2), c-jun N-terminal or stress-regulated protein kinases (JNK / SAPK), and p38 MAPKs. These regulate important roles involved in mealnogenesis. p38 MAPK activity is reported to be involved in melanin biosynthesis by regulating the expression of molecules involved in melanogenesis. In contrast, ERK1 / 2 and JNK / SAPK are involved in inhibiting melanogensis. Therefore, inactivation of ERK signal transduction and activation of p38 MAPK signaling induces activation of CREB and MITF, which induce melanin biosynthesis by overexpression of tyrosinase, TRP-1, and TRP-2. .

Melanin is formed from L-tyrosine, an amino acid precursor in melnocytes, which plays an important role in protecting skin damage caused by UV light and other factors. Abnormal overproduction of melanin causes skin pigmentation abnormalities such as blemishes, freckles, pigmentation, etc. On the contrary, when less is produced, vitiligo, depigmentation, white nasal gangrene, rash, ecchymosis, idiopathic hypopigmentation, partial whitening Melanin hypopigmentation diseases such as hyperemia occur.

Among the melanin hypochromatosis diseases, vitiligo is a disease in which melanocytes are destroyed, and the other diseases are caused by a decrease in the amount of melanin produced in melanocytes.

Therapies for the treatment of hypopigmentation diseases can be largely divided into drug therapy and surgery. Pharmacotherapy is a method of taking or applying steroids, phototherapy with photosensitizers and UV irradiation is common, but the degree of pigment redeposition is low and there are risks from side effects of drugs. Epidermal transplantation is performed to remove the epidermis at the severity site and transplant the normal autologous epidermis into the area, but skin transplantation has a low success rate and frequently involves side effects.

Hypopigmentation can appear on the patient's face or on exposed skin, and worsening symptoms can significantly affect the patient's self-confidence or emotional state, which can lead to extreme physical discomfort. Although there may be a problem, there is no therapeutic agent with low side effects, stable and high therapeutic effects. Therefore, the present invention is to provide a composition for promoting melanin production and stable and high therapeutic effect, and a composition for improving and treating melanin hypopigmentation disease caused by lack of melanin production.

The present invention, in order to solve the problems of the prior art as described above, provides a composition comprising as an active ingredient a compound that is safe to the human body, has less side effects, and shows an excellent melanin production promoting effect, and also melanin as the compound as an active ingredient To provide a pharmaceutical composition, health food composition or cosmetic composition for promoting the production.

The present invention is to provide a composition for promoting melanin production comprising a substance selected from the group consisting of beta-carboline alkaloids, derivatives thereof, salts thereof, hydrates thereof and solvates thereof as an active ingredient.

In addition, beta-carboline alkaloid (beta-carboline alkaloid), derivatives thereof, salts thereof, hydrates thereof and solvates thereof, a pharmaceutical composition for promoting melanin production, health food composition or cosmetic composition comprising as an active ingredient To provide.

The inventors of the present invention have been using beta-carboline alkaloids such as harmaline and harmalol in search of compounds that can be used to treat hypopigmentation disorders, particularly melanin hypopigmentation disorders. The present invention was completed by discovering that melanin production may be promoted and hypopigmentation disease may be greatly improved.

Hereinafter, the present invention will be described in detail.

In the present invention, beta-carboline alkaloids are variously distributed, and may be present in the tissues of endogenous mammals as well as plants, microorganisms and cooked food alcohols and beverages (Allen JR, Holmested B R. The simple b-carboline alkaloids.Phytochemistry. 1980: 19: 15731582). Beta-carboline alkaloids play a wide range of pharmacological and biochemical roles. These kinds of β-carboline alkaloids, harmaline and harmalol, are effective as antioxidants and anti-cancer agents and anti-inflammatory drugs. In traditional pharmacy, plants containing these β-carboline alkaloids are used to treat cancer, malaria and asthma. There were no reports of hypopigmentation disease.

The present invention is to provide a composition for promoting melanin production comprising a substance selected from the group consisting of beta-carboline alkaloids, derivatives thereof, salts thereof, hydrates thereof and solvates thereof as an active ingredient.

The active ingredient of the present invention may include all compounds belonging to beta-carboline alkaloids, but may preferably be a compound selected from the group consisting of hamaline and hamalol. (Figure 1A). The compounds belonging to the beta-carboline alkaloids belong to the scope of the present invention, whether naturally separated or artificially synthesized, and the present invention is not limited to what the substance of the compound of the present invention is.

As an example of promoting melanin production of the present invention, the treatment of melanin cells with beta-carboline alkaloids harmaline and harmalol can increase melanogenesis and tyrosinase activity. In addition, the beta-carboline alkaloids harmaline and harmalol may also increase the expression of tyrosinase and proteins TRP-1 and TRP-2. In the case of tyrosinase, activity is regulated by MITF and CREB as transcription factors.Beta-carboline alkaloids harmaline and harmalol can also increase MITF expression and CREB activity, and also melanin through p38 MAPK during MAPK signaling. May increase production.

The present invention provides a composition for promoting melanin production comprising a beta-carboline alkaloid, a derivative thereof, a salt thereof, a hydrate thereof, and a solvate thereof as an active ingredient. It can be provided for the prevention and treatment of pigmentation disease.

The active ingredient may include all compounds belonging to beta-carboline alkaloids, but may preferably be a compound selected from the group consisting of hamaline and hamalol.

In this case, the hypopigmentation disease is a disease caused by the loss of melanin cells or a disease caused by inhibition of melanogenesis, whether caused by any cause, melanin production in all diseases caused by not reaching the normal level Although it is not particularly limited in its kind as a hypopigmentation disease caused by melanin, preferably vitiligo, albinism, leukemia, depigmentation nevus, white nasal mucus, rash, post-inflammatory bleaching, ecdyscopy , Partial leukemia, idiopathic red pigment hypopigmentation or spot leukemia.

In addition, the composition for promoting melanin production of the present invention may not only be used for the prevention and treatment of melanin hypopigmentation disease, but also for blackening or browning of hair, prevention of white hair, and blackening of skin for cosmetic purposes, such as skin, hair, etc. It can be used in vivo, and can be used in all applications that require melanin production promotion in vitro. Therefore, the composition for promoting melanin production of the present invention should be understood as including all of the composition for blackening or browning of hair, the composition for preventing gray hair and the composition for skin blackening, a composition for improving hypopigmentation and the like.

The present invention provides a pharmaceutical composition for promoting melanin production comprising a beta-carboline alkaloid, a derivative thereof, a salt thereof, a hydrate thereof, and a solvate thereof as an active ingredient.

The active ingredient may include all compounds belonging to beta-carboline alkaloids, but may preferably be a compound selected from the group consisting of hamaline and hamalol.

Although the above-mentioned substance itself may be used as the composition of the present invention, it may suitably be provided in the form of a pharmaceutical composition containing the substance and one or two or more pharmaceutically acceptable additives for the preparation. In the pharmaceutical composition, the ratio of the active ingredient of the present invention may be about 0.01% by weight to about 90% by weight.

The pharmaceutical of the present invention may be prepared in any formulation commonly prepared in the art (e.g., Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), for example granules, fines, powders, hard It may be administered as a pharmaceutical composition for oral administration, such as capsules, soft capsules, syrups, emulsions, suspensions or solutions, and may be intravenous, intramuscular, or subcutaneous injections, mucus, suppositories, transdermal absorbents, transdermally. It can also be administered as a pharmaceutical composition for parenteral administration, such as mucosal absorbents, nasal drops, ear drops, eye drops, inhalants, creams, ointments, and papules, and is prepared by dissolving the preparation prepared as a pharmaceutical composition in powder form at the time of use. Or as a mucus. In particular, the pharmaceutical composition of the present invention may be preferably in the form of a paste ointment, cream, milk, powder, powder, penetrating pad, solution, gel, spray, lotion or suspension, but the scope of the present invention is It is not limited by form.

For the preparation of pharmaceutical compositions, additives for the preparation of solids or liquids may be used. The preparation additive may be either organic or inorganic. In other words, when preparing oral solid preparations, excipients, binders, disintegrating agents, lubricants, coloring agents and the like are added to the main medicine, and then tablets, coating tablets, granules, powders, Preparations in the form of capsules and the like can be prepared. Examples of excipients to be used include lactose, sucrose, white sugar, glucose, corn starch, starch, talc, sorbet, crystalline cellulose, dextrin, kaolin, calcium carbonate, silicon dioxide and the like. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, Dextrin, pectin and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any coloring agent may be used as long as it is usually allowed to be added to pharmaceuticals. These tablets and granules can be appropriately coated according to sugar, gelatin coating, and other needs. If necessary, preservatives, antioxidants and the like may be added.

Inert diluents commonly used in the preparation of liquid preparations for oral administration, for example emulsions, syrups, suspensions, solutions, may be used, for example water or vegetable oils. In addition to the inert diluent, this preparation may be formulated with an adjuvant such as a wetting agent, suspending aid, sweetener, fragrance, colorant or preservative. After the liquid formulation is prepared, it may be filled into a capsule of absorbable material such as gelatin. As a solvent or suspending agent used for preparation of a parenteral administration agent, for example, an injection or a suppository, water, propylene glycol, polyethylene glycol, benzyl alcohol, ethyl oleate, lecithin are mentioned, for example. As a base used for manufacture of a suppository, a cacao butter, an emulsified cacao butter, a laurin butter, and a witezol are mentioned, for example. The preparation method of the formulation is not particularly limited, and any method commonly used in the art can be used.

In the case of injectable preparations, for example, diluents such as water, ethyl alcohol, macrogol, propylene glycol, citric acid, acetic acid, phosphoric acid, lactic acid, sodium lactate, sulfuric acid and sodium hydroxide; PH adjusters and buffers such as sodium citrate, sodium acetate and sodium phosphate; Stabilizers, such as sodium pyrosulfite, ethylenediamine tetraacetic acid, thioglycolic acid, and thio lactic acid, etc. can be used. In this case, in order to prepare an isotonic solution, a sufficient amount of salt, glucose, mannitol, or glycerin may be blended in the formulation, or a conventional dissolution aid, analgesic agent, or local anesthetic may be used.

In the case of ointments, for example, in the form of pastes, creams and gels, conventionally used bases, stabilizers, wetting agents, preservatives and the like can be incorporated as required, and the components can be mixed by a conventional method . As the base, for example, white petrolatum, polyethylene, paraffin, glycerin, cellulose derivatives, polyethylene glycol, silicon and bentonite can be used. As the preservative, methyl p-hydroxybenzoate, ethyl p-oxybenzoate, propyl p-hydroxybenzoate and the like can be used. In the case of the form of a sticking agent, the above ointment, cream, gel, paste or the like can be applied to a conventional support by a conventional method. As a support body, woven or nonwoven fabric which consists of cotton, staple fiber, and chemical fiber; Film or foam sheet such as soft vinyl chloride, polyethylene and polyurethane can be suitably used.

The dosage of the pharmaceutical of the present invention is not particularly limited, but in the case of oral administration, it may be 1 µg / kg to 500 mg / kg, preferably 1 mg / kg to 100 mg / kg, as the weight of the substance as an active ingredient per adult. have. It is preferable to appropriately increase or decrease this dosage depending on the age, condition and symptoms of the patient. The daily dose may be administered once a day or divided into two to three times a day at appropriate intervals, or may be administered intermittently at intervals of several days. When used as an injection, the weight of the substance as an active ingredient per adult may be 1 μg / kg to 500 mg / kg, preferably 1 mg / kg to 100 mg / kg. However, the pharmaceutical composition of the present invention The dosage is determined in view of several related factors such as the route of administration, the age, sex, weight of the patient, the severity of the patient, etc. and therefore the dosage should not be understood as limiting the scope of the invention in any aspect.

In addition, the composition for promoting melanin production comprising a substance selected from the group consisting of beta-carboline alkaloid or derivatives thereof, salts thereof, hydrates thereof and solvates thereof as an active ingredient of the present invention is hypopigmented It can be used as a pharmaceutical composition for preventing and treating diseases. The hypopigmentation disease caused by the melanin is not particularly limited in its kind, preferably vitiligo, albinism, leukemia, depigmentation nevus, white nasal ganglia, stinging, post-inflammatory depigmentation, plaque scleroderma, partial leukemia , Idiopathic red hypopigmentation or mumps leukemia.

The present invention is to provide a health food composition for promoting melanin production comprising a substance selected from the group consisting of beta-carboline alkaloids, derivatives thereof, salts thereof, hydrates and solvates thereof as an active ingredient. .

The active ingredient may include all compounds belonging to beta-carboline alkaloids, but may preferably be a compound selected from the group consisting of hamaline and hamalol.

Examples of the food to which the compound can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to food or beverages for the purpose of promoting melanin production. At this time, the amount of the compound in the food or beverage may be added to 0.01 to 30% by weight of the total food weight, the health beverage composition may be added to 0.01 to 30 g based on 100 ml, but is not limited thereto.

   The health functional beverage composition of the present invention is not particularly limited to the other ingredients other than the above-mentioned compounds as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination, and the proportion of such additives is not so critical but is usually selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention is to provide a cosmetic composition for promoting melanin production comprising a material selected from the group consisting of beta-carboline alkaloids, derivatives thereof, salts thereof, hydrates thereof and solvates thereof as an active ingredient.

The active ingredient may include all compounds belonging to beta-carboline alkaloids, but may preferably be a compound selected from the group consisting of hamaline and hamalol.

There is no particular limitation on the application of the composition of the present invention, the composition of the present invention can be applied to all animals, including humans as well as animals, for example, breeding animals or pets.

The compositions of the present invention can be used by methods such as hair or scalp, direct application or spreading on the skin. The present invention can be used in cosmetics for the purpose of promoting melanin production or improving hypopigmentation diseases, preferably for the purpose of promoting melanin production of skin or preventing or treating bleaching of hair and white hair.

The form of the cosmetic of the present invention is not particularly limited and may be prepared in any form conventionally prepared in the art (eg, Remington's Pharmaceutical Science, the latest edition), such as emulsions, lotions, creams (oil-in-water) , Water-in-oil, multiphase), solutions, suspensions (anhydrous and aqueous), anhydrous products (oil and glycols), gels, masks, packs, powders, soaps and the like.

In addition, the cosmetics, hair bleach, hair whitening, hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nutrition cream, hair moisturizer cream, Hair Massage Cream, Hair Wax, Hair Aerosol, Hair Pack, Hair Nutrition Pack, Hair Soap, Hair Cleansing Foam, Hair Oil, Hair Dryer, Hair Preservative, Hair Dye, Hair Wave Agent, Hair Gel, Hair Glaze, Hair Dresser, Hair It can be used in hair products such as lacquers, hair moisturizers, hair mousses or hair sprays and can be used to prevent or treat hair discoloration and white hair.

 In the cosmetics of the present invention, conventional ingredients such as oils, surfactants, moisturizers, polyhydric alcohols, thickeners, water-soluble polymers, film-forming agents, water-insoluble polymers, powders, etc., may be used as long as they do not impair the effects of the present invention. , Pigments, dyes, lakes, lower alcohols, ultraviolet absorbers, metal ion chelating agents, organic amines, pH adjusting agents, active ingredients, sugars, preservatives, antioxidants, fragrances, water and the like can be added.

 Since the components commonly used in the preparation of the cosmetic composition are already known in the art, those skilled in the art can select and use appropriate components. For example, as the oil, olive oil, avocado oil, castor oil, palm oil, etc. As the surfactant, polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester, glycerin fatty acid ester, polyoxyethylene hardened castor oil and the like can be used, and as a moisturizing agent, glycerin, 1,3-butylene glycol, polyethylene glycol Carboxyvinyl polymer, carboxymethyl cellulose, etc. may be used as the thickener, and paraaminobenzoic acid, octylmethoxycinnamate, 2-ethoxyethyl-p-methoxycinnamate, etc. may be used as the sunscreen. As the amines, monoethanolamine, Triethanolamine etc. can be used, Tocopherols, dibutylhydroxytoluene, etc. can be used as antioxidant, Ethyl paraben, butyl paraben, sodium benzoate, etc. can be used as an antiseptic.

The blending amount of a substance selected from the group consisting of beta-carboline alkaloids, derivatives thereof, salts thereof, hydrates thereof and solvates thereof in the cosmetic composition is determined according to the type of the substance, etc. A person skilled in the art may appropriately select, but may be, for example, 0.001% to 30% by weight, but the weight ratio may vary depending on the use purpose, condition, and the like.

The present invention provides a composition for promoting melanin production with little side effects and stably used, which is useful for the prevention and treatment of hypopigmentation diseases, and helps to prevent and treat the discoloration and whitening of skin or hair caused by melanin. There is provided a composition.

The composition of the present invention will be particularly great because it can be utilized in various industrial fields, such as pharmaceutical compositions, health food compositions or cosmetic compositions.

Figure 1 shows the results of analyzing the degree of cytotoxicity and melanin biosynthesis by harmaline and harmalol in melanoma cells.
FIG. 2 shows the results of analyzing the effects of harmaline and harmalol on tyrosianse activity and the expression of proteins related to melanogenesis in melanoma cells.
3 is a result of analyzing the effects of harmaline and harmalol on the activity of MITF expression and phosphorylation of CREB in melanoma cells.
4 shows the results of analyzing the effects of harmaline and harmalol on p38 MAPK activation in melanoma cells.
Fig. 5 shows the results of analyzing the effects on melanin biosynthesis and tyrosinase activation after treatment with or alone with SB203580, a p38 MAPK-specific inhibitor in harmaline and harmalol in melanoma cells.

The present invention will be described in detail by the following Examples, Experimental Examples, and Formulation Examples, but the technical scope of the present invention is that these Examples, Experimental Examples, and Formulation Examples are merely illustrative of the present invention. It is not limited by the experiment example and formulation example.

< Example >

Example 1. Purchasing Reagents and Cell Culture

1) Harmaline and harmalol and α-MSH MTT [3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazolium bromide] and other reagents used in the examples of the present invention Sigma Chemical (St. Louis, Missouri) and SB203580 (p38 inhibitor) were purchased from A.Science, San Diego, California. phospho-p38, phospho-CREB and CREB were purchased from Cell Signaling Technology (Beverley, MA, USA) and p38, tyrosinase, TRP1, TRP2, and MITF were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

2) B16-F10 murine melanoma cell line purchased from American Type Culture Collection (ATCC Rockville, MD, USA), Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL, Carlsbad, CA, USA) and 10% heat-inactivated fetal bovine serum (FBS; GIBCO BRL) was incubated at 5% CO ₂ with 95% air and 37 ° C. To avoid altering cell-specific properties, 15 to 25 passages were used and cells were cultured once every two days.

Example 2. Cytotoxicity Assay

Cytotoxicity was assessed using the MTT colorimetric assay. B16-F10 murine melanoma cells were treated with various concentrations depending on the presence or absence of Harmaline and harmalol and incubated at 37 ° C and 5% CO ₂ incubator for 24-72 hours. After incubation for 3 hours with MTT (62.5 ㎍ / ml) reagent, the supernatant was removed, DMSO was added and analyzed at 570 nm using a microplate reader (Wallace, Boston, MA, USA).

Example 3. Intracellular Melanin Sum  analysis

To analyze the amount of melanin biosynthesis, 24-72 hours after 1-20 μM harmaline and harmalol treatment, the cells were washed twice with phosphate buffered saline and dissolved in 500 μl of 1 N NaOH in 10% DMSO. After vortexing to completely dissolve melanin after reaction at 80 ° C. for an hour, the amount of melain was analyzed at 475 nm using a microplate reader (Wallace, Boston, MA, USA). The degree of melanin biosynthesis was corrected using the protein content of the cell extract.

Example 4 Activity Analysis of Intracellular Tyrosinase

After 24-72 hours of 1-20 μM harmaline and harmalol treatment, wash the cells twice with phosphate buffered saline and add 1% (w / v) phosphate buffered saline containing protonase inhibitor with Triton X-100 After harvesting, the supernatant was taken after centrifugation. Tyrosinase substrate L-DOPA (2 mg / ml) reaction was carried out using this supernatant as an enzyme source, and tyrosinase activity was analyzed at 405 nm using a microplate reader (Wallace, Boston, MA, USA). The degree of tyrosinase activity was corrected using the protein content of the cell extract. In case of in-situ L-DOPA reactivity, cells were first cultured in a slide glass chamber, treated with harmaline and harmalol, and then fixed with cells in 3.5% paraformaldehyde in PBS for 10 minutes, followed by permeabilization of cells in 100% methanol for 10 minutes. After washing fixed and permeabilization cells, L-DOPA (2 mg / ml) was added, followed by reaction at 37 ° C for 4 hours, followed by digital video camera system (InFinity CAPTURE application version 4.6.0 Lumenera, Ottawa, Ontario, Canada) Pictures were taken using an attached Axiovert 40CFL inverted microscope (Carl Zeiss, Jena, Germany).

Example 5. Western Brotting

After lysing the cells with lysate solution, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed using lysate. The protein was transferred to PVDF membrane. After the desired primary antibody is added to the membrane, the reaction is carried out for 1 hour to 16 hours, and after the secondary antibody is added, the reaction is performed for 30 minutes to 1 hour. Then, the expression level of the protein was measured using a chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) using a mageQuant 350 analyzer (Amersham Biosciences) and analyzed using ImageQuant TL software (Amersham Biosciences).

Example  6. Immunofluorescence Analysis and Multi-photon Air Conditioning Laser Microscope  (Immunofluorescence confocal microscopy )

After fixing the cells in RT with 3.5% paraformaldehyde for 10 minutes, the cells were reacted with 100% methanol for 10 minutes at RT. Then, MITF and phospho-CREB antibodies are added, followed by reaction for 16 hours, followed by washing in cold PBS three times, followed by addition of secondary antibody bound to FITC for 2 hours at RT. After staining for 10 minutes in a 1 μg / ml DAPI solution to stain the nucleus, it was observed using a confocal microscopy (Zeiss LSM 510 Meta).

Example  7. Statistical Analysis.

All results are expressed as mean ± SE. Each experiment was based on at least three iterations. Statistical significance indicated Student's t-test difference values for non-pairwise observations between control and experimental samples. P values of 0.05 or less were considered statistically significant.

< Experimental Example >

Experimental Example  1. Cytotoxicity and melanin  Biosynthesis

Experimental Example 1 -One

Melanoma cells were treated with 1 μM, 5 μM, and 10 μM harmaline and 5 μM, 10 μM and 20 μM harmalol for 72 hours, and then the melanin biosynthesis was analyzed. a-MSH (1μM) is provided as a positive control. Data presented represent ± SE of three independent observations in 3 replicates. (*, p <0.05 compared to control cells).

At this time, Figure 1D shows the difference in appearance after treatment of harmaline and harmalol to melanoma cells, and in Figure 1B, Harmaline and harmalol increase melanin biosynthesis in a concentration-dependent manner using B16F10 cells, a mouse melanoma cell line. It was confirmed. 5 μM harmaline and 20 μM harmalol increased melanin production by 2.3 and 2.1 times as compared with the control group.

Experimental Example 1 -2

The melanoma cells were treated with 1 μM, 5 μM and 10 μM harmaline and 5 μM, 10 μM and 20 μM harmalol for 24 hours, 48 hours and 72 hours, respectively, followed by MTT assay. Data presented represent ± SE of three independent observations made in 3 replicates (*, p <0.05 compared to control cells), with-in front of the table in FIG. 1C indicating the control without addition.

As shown in FIG. 1C, the necrosis of melanoma cells by harmaline and harmalol was not achieved, but a slight decrease in the proliferative capacity of the melanoma cells by the drug was observed.

Experimental Example 2 . tyrosianse  Active and Melanogenesis  Expression of related proteins

Experiment 2 -One tyrosianse  activation

Melanoma cells were treated with 5 μM of harmanlin for 0 hours, 24 hours, 48 hours, 72 hours, and 10 μM of harmalol for 24 hours, 48 hours, 72 hours, and then analyzed for their tyrosinase activity. a-MSH (1μM) is provided as a positive control. Data presented represent ± SE of three independent observations made in 3 replicates (*, p <0.05 compared to control cells).

As shown in FIG. 2A, the degree of activity of tyrosinase in melanoma cells by harmaline and harmalol was increased in a time-dependent manner.

Experimental Example  2-2 Melanogenesis  Expression of related proteins

1) In situ tyrosinase activity was measured after 5 μM of harmanlin and 10 μM of harmalol in melanoma cells (FIG. 2B).

2) Melanoma cells were analyzed for their melanin levels after 5 hours of harmanlin and 10 μM of harmalol at 0, 24, 48 and 72 hours (FIG. 2C).

3) The expression levels of tyrosinase, TRP-1 and TRP-2 were analyzed after 5 μM of harmanlin and 10 μM of harmalol in melanoma cells at 0, 12, 24, 48 and 72 hours. The same protein was loaded to indicate the amount of α-tubulin protein expression (FIG. 2D). In addition, the expression level of the protein for the control group versus the treatment group is shown in the lower part of FIG.

As shown in FIG. 2B, the results of tyrosinase activity in the treatment of Harmaline and harmalol using In situ tyrosinase activity can be confirmed to increase tyrosinase activity in the same manner. FIG. 2C As shown in Fig. 2, the time-dependent increase of melanin synthesis was confirmed by Harmaline and harmalol. 2D, Western blotting was performed to analyze the expression level of the protein in order to confirm that Tyrosinase, TRP-1 and TRP-2 are involved in tyrosinase activity. As a result, Tyrosinase, TRP-1 by Harmaline and harmalol was performed. It was confirmed that the expression of TRP-2 increased in time dependent manner.

Experimental Example 3 . MITF  Expression and CREB Phosphorylation of

After 72 hours of 5 μM of harmanlin and 10 μM of harmalol, the degree of MITF and phospho-CREB expression was analyzed by Western blotting and confocal microscopy to determine the level of protein expression (the bar shows 10 μm) (FIG. 3A). ). After treatment with 5 μM of harmanlin and 10 μM of harmalol at 0 hours, 4 hours, 8 hours, 12 hours, and 24 hours, it was analyzed by Western blotting. The same protein was loaded, indicating the amount of a-tubulin and CREB protein expression. The numbers shown below in Figure 3B are relative indications of the expression level of the protein relative to the control group versus the treatment group.

As shown in Figure 3, it was confirmed that Harmaline and harmalol increase the expression of MITF in a time-dependent manner, and also phosphorylation of CREB also increases in a time-dependent manner. From these results, it was confirmed that Harmaline and harmalol induced melnanogenesis through increased expression of MITF and phosphorylation of CREB, and to confirm these results, Immunofluorescence was performed. In addition to the expression, the phosphorylation of CREB was increased through the difference of green and dark, and in the case of blue, staining the nucleus, staining the control group that distinguishes the difference between the nucleus and the cell membrane.

Experimental Example 4 . p38 MAPK  Activation

The expression levels of phospho-p38 MAPK were analyzed between 0 and 12 hours after 5 μM of harmanlin and 10 μM of harmalol. Total p38 MAPK was analyzed as a control. The numbers shown below FIG. 4 indicate the expression level of the protein relative to the control group versus the treatment group.

As shown in Figure 4, after treating the harmaline and harmalol in B16F10 melanoma cells, it can be seen that the phosphorylation of p38 MAPK is the most at about 4 hours, through which, harmaline and harmalol is active through p38 MAPK phosphorylation Increasingly, it was found to be involved in melanogenesis.

Experimental Example 5 . p38 MAPK  Specific inhibitors SB203580 With or without addition  Melanin biosynthesis and activation of tyrosinase according to

The harmaline and harmalol increase the activity through p38 MAPK phosphorylation, and together with or alone with the p38 MAPK specific inhibitor SB203580 to confirm that it is involved in melanogenesis.

1) Melanin biosynthesis was analyzed after 72 hours of 5 μM of harmanlin and 10 μM of harmalol with or without SB203580 (20 μM) (FIG. 5A).

2) Tyrosinase activity was analyzed after 5 hours of harmanlin and 10 μM of harmalol treated with SB203580 (20 μM) together or alone for 72 hours (FIG. 5B).

3) The protein expression levels of tyrosinase and TRP-1 were analyzed after 72 hours treatment with 5 μM of harmanlin and 10 μM of harmalol with or without SB203580 (20 μM). The same protein was loaded to indicate the amount of a-tubulin protein expression. The numbers shown below are relative to the expression level of the protein in the control group versus the treatment group.

Figure 5 A, B, C through the treatment of harmaline and harmalol alone compared with the result of treatment with p 38 MAPK specific inhibitor SB203580, melanin biosynthesis and tyrosinase activity and treatment in the group treated with SB203580 The protein expression of tyrosinase and TRP-1 was markedly reduced, and once again, it was confirmed that harmaline and harmalol are involved in melanognesis through p38 MAPK.

< Formulation example >

Formulation Example 1 . Pharmaceutical composition

Formulation example  1-1 ointment

 Beta-carboline alkaloids 0.020 g

 81.700 g of isopropyl myristate

 Mineral oil fluid 9.1 g

9.180 g of silica

Formulation example  1-2 cream

 0.100 g of beta-carboline alkaloids

 Lanolin emulsifying alcohol, wax 39.900 g

 0.075 g of methyl para-hydroxybenzoate

 0.075 g of propyl para-hydroxybenzoate

Sterile demineralized water balance 100 g

Formulation example  1-3 lotions

 0.100 g of beta-carboline alkaloids

 69.900 g of polyethylene glycol (PEG 400)

30.000 g of 95% ethanol

Formulation example  2. Health food composition

Formulation example  2-1 Health food

Beta-carboline alkaloids ..... 1000 mg

Vitamin Mix ............

Vitamin A Acetate ......... 70 μg

Vitamin E ................................. 1.0 mg

Vitamin B1 .................. 0.13 mg

Vitamin B2 ........................ 0.15 mg

Vitamin B6 ............... 0.5 mg

Vitamin B12 ........................ 0.2 μg

Vitamin C ................................. 10 mg

Biotin ......................................... 10 μg

Nicotinic Acid Amide ... 1.7 mg

Folic Acid ... 50 μg

Calcium Pantothenate ......................................... 0.5 mg

Mineral mixture ........................

Ferrous Sulfate ............... 1.75 mg

Zinc Oxide ............... 0.82 mg

Magnesium Carbonate ........................... 25.3 mg

Potassium monophosphate ......................................... 15 mg

Dicalcium Phosphate Dibasic ............... 55 mg

Potassium Citrate ... 90 mg

Calcium Carbonate ... 100 mg

Magnesium Chloride ........................... 24.8 mg

Formulation example  2-2 Manufacturing of Healthy Drinks

Beta-carboline alkaloids 100 mg

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Water quantification

Formulation Example 3 . Cosmetics  Composition

Formulation example  3-1 Softening Cosmetics (Content: Weight%)

 Beta-Carboline Alkaloids 0.01

 Glycerin 3.0

 Butylene Glycol 2.0

 Propylene Glycol 2.0

 Carboxy Vinyl Polymer 0.1

 Ethanol 10.0

 Triethanolamine 0.1

 Preservatives, trace pigments, trace fragrances, trace amount of purified water

Total 100.0

Formulation example  3-2 Preparation of Hair Tonic (Content: Weight%)

 Beta-Carboline Alkaloids 10.0

 Resorcinol 0.1

 Menthol 0.05

 Panthenol 0.2

 Salicylic Acid 0.1

 Tocopheryl Acetate 0.1

 Pyridoxine hydrochloride 0.1

 Castor Oil 5.0

 Pigment amount

 Spices

 Ethanol

 Purified water balance

Total 100.0%

Formulation example  3-3 Hair soap  Manufacture (content: wt%)

Beta-Carboline Alkaloids 0.1

Titanium Dioxide 0.2

Polyethylene Glycol 0.8

 Glycerin 0.5

Ethylenediaminetetraacetic Acid 0.05

Sodium 1.0

Pigment amount

Soap flavor

Cosmetic soap base (moisture 13, parts by weight)

Total 100.0

Claims (12)

A skin external preparation cosmetic composition for skin blackening, comprising at least one beta-carboline alkaloid selected from the group consisting of hamaline and hamalol as an active ingredient. delete delete delete delete delete delete delete The method of claim 1,
The composition is a cosmetic composition, characterized in that it is formulated in the form of paste ointment, cream, milk, powder, pape, penetrating pad, solution, gel, spray, lotion or suspension. delete delete delete

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