MELANOSOMAL TRANSPORT INHIBITOR AND EXTERNAL SKIN PREPARATION CONTAINING THE TRANSPORT INHIBITOR

Abstract

To provide a melanosome transportation inhibitor having high skin-whitening effect and a topical skin preparation containing the same.SOLUTION: A melanosome transportation inhibitor contains rhamnazin represented by the chemical formula (I) as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a melanosome transport inhibitor and an external skin preparation containing said transport inhibitor. [Background technology]

[0002] Skin blemishes and freckles are caused by irritation from ultraviolet rays, hormonal abnormalities, or genetic factors. This causes the melanocytes localized in the basement membrane of the epidermis to become abnormally activated and synthesize excessive melanin pigment. It occurs when the melanin pigment is abnormally deposited in the skin (Non-Patent Document 1). Therefore, research and treatments focusing on inhibiting melanin synthesis in melanocytes are being conducted. has traditionally been carried out.

[0003] Melanin is produced in organelles called melanosomes within melanocyte cells. The melanosomes, which turn black due to the accumulation of melanin produced, are involved in various transport-related events. It is transported by proteins from the perinuclear area of ​​melanocytes to the terminals of dendrites, and then transferred to the surrounding epidermal cells. Normally, the transferred melanosomes are transferred to the epidermal cells along with the turnover of the epidermal cells. On the other hand, strong stimuli such as ultraviolet rays cause excessive accumulation of melanosomes in epidermal cells. This induces pigmentation known as age spots.

[0004] Recently, it has been reported that intracellular transport of melanosomes is regulated by various proteins. (Non-Patent Document 2). Furthermore, as progress is being made in identifying proteins involved in the intracellular transport of such melanosomes, Compounds that reduce the amount of this protein suppress the excessive accumulation of melanosomes in epidermal cells. It has been reported that it is useful for whitening the skin. Inactivators of the protein Rab27a (Patent Document 1), effectors of Rab27a Slp-2a (Patent Document 2), a binding molecule called synaptotagmin -like protein homologue lacking C2 domains- a (Slac2-a, also known as Melanophilin, Mlph) (Patent Document 3) Examples of such agents include protein amount reducers.

[0005] Based on the results of the previous research, the present inventors have developed an activity that has a skin whitening effect. We have been conducting intensive research on functional compounds. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-137821 [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-229043 [Patent Document 3] Japanese Patent Application Laid-Open No. 2011-132195 [Non-patent literature]

[0007] [Non-Patent Document 1] Prota, GJ, Invest. Dermatol. (1980), 75: 122-127. [Non-patent document 2] Kuroda, T. S et al., Nat. Cell Biol. (2004), 6: 1195-1203. [Non-patent document 3] Goda, Y et al., Biol.Pherm.Bull. (1999), 12:1319-1326. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to a melanosome transport inhibitor having a high whitening effect and a topical skin treatment containing the same. The present invention aims to provide a drug. [Means for solving the problem]

[0009] As a result of intensive research into solving the above problems, the present inventors have found that flavonols The inventors have found that rhamnadins, which are classified as Based on this finding, they conducted further research and discovered that rhamnadin has a strong whitening effect. Ta.

[0010] The present invention provides a melanosome transport inhibitor (first melanosome transport inhibitor) characterized by containing rhamnadin represented by the following chemical formula (I) as an active ingredient. [ka] The rhamnagin is a naturally occurring compound found in Rhamnus petiolaris (R h amnus petiolaris and Rhamnus saxatilis (R h amnus saxatilis) and other rhamnus (R h It is preferable that the compound is derived from a plant of the genus amnus or an organic synthetic compound.

[0011] Furthermore, as a result of intensive research into solving the above problems, the present inventors have found that A hydrolyzate of extracts derived from specific plants etc. that are rich in munadin (the second melanoso We concluded that a soluble soluble hydroxybenzoate (a soluble hydroxybenzoate transport inhibitor) would have a greater whitening effect.

[0012] That is, the second melanosome transport inhibitor of the present invention is a watercress (Nasturtium sieboldii). Nasturitium (Nasturitium officinale) Genus Rhamnus, including Rhamnus saxailis amnus, including Viscum coloratum Viscum genus, Nervilia fordii ) or clove (Syzygium arom) Addition of extracts derived from plants of the genus Syzygium, including Syzygium aticum A hydrolysis product is preferred.

[0013] The second melanosome transport inhibitor is an enzyme extracted from the plant material by solvent extraction, etc. It can be obtained by hydrolyzing kiss.

[0014] In addition, the present invention provides a topical skin preparation containing the first or second melanosome transport inhibitor. The concentration of rhamnadin contained in the topical skin preparation is 0.0001 to 5% by mass. It is preferable that: The topical skin preparation further contains a whitening agent that does not have a melanosome transport inhibitory effect. is preferred. The whitening agent is preferably a tyrosinase activity inhibitor. The tyrosinase activity inhibitor includes ascorbic acid, ascorbic acid salts and derivatives thereof, Hydroquinone, hydroquinone salts and glycosides, placenta extract, arbutin, gills Preferably, the hydroxybenzoate is selected from the group consisting of lactic acid, linoleic acid, and tranexamic acid. [Effects of the Invention]

[0015] According to the present invention, a melanosome transport inhibitor having a high whitening effect and a skin agent containing the same are provided. A topical skin preparation is provided. [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a micrograph (magnification: 200x) showing a comparison of cells treated with watercress extract before hydrolysis and cells treated with extract after hydrolysis. [Figure 2] 1 is a graph showing the ratio of glycosides to aglycones before and after hydrolysis of the solution obtained in Example 1. [Figure 3] 1 is a graph showing the results of verifying the inhibitory effects of various flavonols on Mlph protein expression. [Figure 4] 1 is a graph showing the results of testing the cytotoxicity of rhamnagin. [Figure 5] These are micrographs (magnification: 200) showing the results of observing cells using a phase-contrast microscope to verify the inhibitory effect of rhamnadin on the intracellular transport of melanosomes. (a) shows cells without rhamnadin added, (b) shows cells with 5 μM rhamnadin added, and (c) shows cells with 10 μM rhamnadin added. [Figure 6] These are micrographs (magnification: 250) showing the results of observing cells with a confocal laser microscope to verify the inhibitory effect of rhamnadin on the intracellular transport of melanosomes. (a) shows cells without rhamnadin added, and (b) shows cells with 10 μM rhamnadin added. [Figure 7] 1 is a graph showing the results of calculating the percentage of cells in which melanosomes are aggregated near the nucleus, based on the results of observation using a confocal laser microscope. [Figure 8] 1 is a graph showing the results of verifying the inhibitory effect of rhamnagin on the extracellular secretion of melanin pigment. [Figure 9] 1 is a graph showing the results of verification of the inhibitory effect of rhamnadin on the expression of melanin synthesis-related proteins. [Figure 10] 1 is a graph showing the results of verification of the synergistic effect of combined use of rhamnagin and other drugs. DETAILED DESCRIPTION OF THE INVENTION

[0017] Hereinafter, the embodiments of the present invention will be described in more detail. The first melanosome transport inhibitor As mentioned above, the present inventors have found that rhamnadin, which is classified as a flavonol, binds to melanosomes. Based on this finding, the present inventors have furthered their investigations and found that the transport of It was verified that rhamnadin has a high whitening effect.

[0018] The first melanosome transport inhibitor according to the present invention contains rhamnadin represented by the following chemical formula (I) as an active ingredient. [ka] Rhamnadin is derived from natural products such as Rhamnus petiolaris (R h amnus petiolaris and Rhamnus saxatilis (R h amnus saxatilis) and other rhamnus (R h Rhamnus petiolaris and Rhamnus saxatilis are buckthorn plants native to Sri Lanka and Europe. Rhamnus saxatilis is particularly advantageous in that it contains a high amount of rhamnadin, allowing a larger amount to be obtained from a smaller amount of extract.

[0019] The first melanosome transport inhibitor according to the present invention is, for example, a melanosome transport inhibitor derived from a rhamnus (Rhamnus saxatilis) plant such as Rhamnus petiolaris or Rhamnus saxatilis. h The whole plant, roots, rhizomes, leaves, seeds, fruits, and flowers of the plant of the genus amnus can be prepared as a fine powder or crushed product. The first melanosome transport inhibitor of the present invention can also be prepared as an extract obtained by immersing such fine powder or crushed material in water or an organic solvent and filtering the residue, or as an extract obtained by removing the solvent from this extract (extracted extract). The first melanosome transport inhibitor according to the present invention can also be prepared in the form of a solution by further dissolving, dispersing or diluting the fine powder or the solvent-removed product in an appropriate solvent or the like.

[0020] When extracting with a solvent, the extraction solvent can be an alcohol such as methanol or ethanol, or It is preferable to use a low polarity solvent such as 1,3-butylene glycol. This is because the desired extract can be obtained if the solvent is used.

[0021] The rhamnadine contained in the first melanosome transport inhibitor of the present invention is a compound that can be easily dissolved in an organic solvent. In addition to or as an alternative to extraction, removal of solids by filtration or centrifugation may be performed, followed by silica gel column chromatography. The product can be purified by a purification process using chromatography such as HPLC.

[0022] Rhamnadin can also be obtained by utilizing organic synthesis reactions. Rhamnadins obtained by various synthesis (synthetic rhamnadins) can also be used in the present invention. It can be contained as an active ingredient of a cytomegalovirus transport inhibitor.

[0023] Regarding the melanosome transport inhibitor of the present invention, "rhamnagin as an active ingredient" This refers to the case of melanosome transport inhibitors that consist only of the active ingredient rhamnadin alone, and In addition to Munajin itself, trace amounts of impurities may be included, and as mentioned above, the whole plant may also be included. In the case of finely powdered or crushed roots, rhizomes, leaves, seeds, fruits, or flowers, the finely powdered or crushed material In the case of an extract obtained by immersion extraction with water or an organic solvent and filtering off the residue, In the case of the product obtained by removing the solvent from the powder (extract), the fine powder and the solvent-removed product are When prepared in the form of a solution by further dissolving, dispersing, or diluting with an appropriate solvent, etc. Either is acceptable.

[0024] The melanosome transport inhibitor of the present invention (including the second melanosome transport inhibitor described below) When prepared in the form of a solution, rhamnadin is contained in an amount of 0.0001% by mass to 5% by mass. It is preferable that:

[0025] A second melanosome transport inhibitor As mentioned above, the present inventors have discovered that rhamnadin is abundantly contained in natural products such as specific plants. It was discovered that the hydrolyzate of this compound has a high whitening effect.

[0026] The second melanosome transport inhibitor according to the present invention also contains rhamnadin represented by the above chemical formula (I) as an active ingredient. A second melanosome transport inhibitor is watercress (Nasturtium sieboldii). rt Nasturtium (Nastulium officinale) rt ium) genus, Rhamnus saxa Tee Squirrel (Rhamnus saxa t Rhamnus (R ilis) h It is preferable that the hydrolysate is a naturally occurring substance derived from a plant of the genus Viscum including Viscum coloratum, the genus Nervilia including Nervilia fordii, or the genus Syzygium including clove (Syzygium aromaticum).

[0027] The second melanosome transport inhibitor is an enzyme extracted from the plant material by solvent extraction, etc. It can be obtained by hydrolyzing kiss. For example, freeze-dried above-ground parts of plants such as watercress extract are dissolved in methanol or the like. The extract is then concentrated, and the resulting concentrate is purified by a column. The fraction obtained by elution with a solvent such as methanol is then subjected to acid hydrolysis. This allows the second melanin to be extracted as a hydrolyzed solution of watercress, etc. Somal transport inhibitors can be prepared. As mentioned above, the first melanosome transport inhibitor is obtained by solvent extraction from the plant as a raw material. Therefore, it also includes the case where it is prepared in the form of an extracted extract. The method involves hydrolyzing such extracts, or more precisely, the method involves the hydrolysis of the extracts. It can be obtained by hydrolyzing the flavonoid glycosides contained in the plant.

[0028] Furthermore, the present inventors investigated the mechanism of the high whitening effect of rhamnadin and found that The addition of rhamnadin reduced the amount of Mlph protein, which is involved in melanosome transport. This suggests that inhibiting melanosome transport in melanocytes can produce strong beauty products. The Mlph protein acts as a melanin receptor in melanocytes, which is thought to have a whitening effect. This protein is involved in melanosome transport. When this transport of melanosomes is inhibited, melanosomes This prevents the transfer of melanin produced by melanocytes to keratinocytes, resulting in the formation of It can suppress pigmentation.

[0029] On the other hand, conventional whitening agents, kojic acid and arbutin, do not have the effect of inhibiting melanosome transport. The present inventors have confirmed that: Therefore, the effect of suppressing skin pigmentation based on the melanosome transport inhibitor of the present invention is The whitening effect (melanin reduction) achieved by conventional whitening cosmetics containing kojic acid and arbutin, etc. Its mechanism of action differs from that of the steroid hormone (synthesis inhibitory effect).

[0030] The melanosome transport inhibitor of the present invention containing rhamnadin is used to treat a melanosome transport inhibitory effect. When used in combination with other whitening agents that do not contain EDTA, the whitening effect is greater than when the other whitening agents are used alone. This can produce a synergistic effect that further enhances the results. The whitening agent having no melanosome transport inhibitory effect is preferably a whitening agent having an inhibitory effect on melanin synthesis. It is a tyrosinase inhibitor that inhibits the activity of tyrosinase. These are tyrosinase activity inhibitors. For example, even if multiple or large amounts of tyrosinase inhibitors are mixed, the whitening effect is not enhanced. There are also concerns about the safety of its effectiveness. However, as mentioned above, For example, different whitening agents act on different areas of the skin, at different stages in the pigmentation process, That is, by combining a tyrosinase activity inhibitor with a melanosome transport inhibitor, Even using a small amount of each ingredient provides a high whitening effect and is also extremely safe. It is also possible to provide the following.

[0031] The tyrosinase activity inhibitor is not particularly limited, and examples thereof include ascorbic acid, Corbic acid salts and their derivatives, hydroquinone, hydroquinone salts and glycosides, pla Examples include centaury extract, arbutin, ellagic acid, linoleic acid, and tranexamic acid. .

[0032] topical skin preparations The external skin preparation according to the present invention contains the first or second melanosome transport inhibitor. The external skin preparation of the present invention preferably contains rhamnadine in an amount of 0.00 based on the total amount of the external skin preparation. If it contains at least 0.0001% by mass, it is considered melanosomes. This corresponds to the concentration at which the transport inhibitory effect is minimal, and if it is 5% by mass or less, This is because it provides an inhibitory effect on nosomal transport and is also safe.

[0033] The melanosome transport inhibitor of the present invention is applied to the skin in cosmetics, pharmaceuticals, quasi-drugs, etc. Therefore, it can be used as a topical skin preparation. Ointment-based, solubilized, emulsified (W / O, O / W), powder-based, ointment-based, oil-based, gel-based It can be used in a wide range of forms, such as a water-oil two-layer system, a water-oil-powder three-layer system, etc. The topical skin preparation of the present invention contains, in addition to the melanosome transport inhibitor, In the range, other ingredients commonly used in skin care products, such as water, alcohol, oil, surfactants, Antiseptics, thickeners, powders, chelating agents, preservatives, pH adjusters, various medicinal agents, animal and plant-derived and microbial-derived Natural extracts, colorings, fragrances, etc. can be blended as needed. By using medicinal ingredients such as anti-inflammatory agents, cell activators, anti-inflammatory agents, and UV protection agents in combination, The effect can be further enhanced or other effects can be added.

[0034] The external skin preparation of the present invention may be in the form of, for example, a lotion, oil, emulsion, cream, softener, or the like. Also, emulsions, creams, lotions, serums, sheet masks, cleansers, It is also possible to provide the composition as a cosmetic in various forms, such as a makeup cosmetic.

[0035] The topical skin preparation according to the present invention further contains a whitening agent that does not have a melanosome transport inhibitory effect. Such a whitening agent may include the above-mentioned tyrosinase activity inhibitors. The melanosome transport inhibitor of the present invention containing rhamnadin and the melanosome Skin topical preparations containing other whitening agents that do not have a steroid transport inhibitory effect are This can produce a synergistic effect that further enhances the whitening effect compared to topical skin preparations containing

[0036] As described above, the topical skin preparation of the present invention contains rhamnadin as an active ingredient in the melanosome transport inhibitor. It acts as a component and is particularly effective as a whitening cosmetic for the purpose of beautifying the skin. [Example]

[0037] The present invention will be specifically explained below using examples and formulation examples, but the present invention is not limited to these. It is not something that is done.

[0038] Example 1 The freeze-dried aerial parts of watercress were immersed in methanol and left overnight at room temperature. The extract was then concentrated using an evaporator. The concentrate was purified on an HP-20 column and eluted with methanol to obtain a high content of flavonoids. After concentrating the fraction with an evaporator, 6N hydrochloric acid was added. Acid hydrolysis was carried out by heating at 100°C for 90 minutes. After the reaction, After neutralization, extraction was performed with ethyl acetate. The obtained extract was concentrated using an evaporator, The solution obtained by redissolving in methanol was prepared as a hydrolyzed solution of watercress extract. did. On the other hand, after concentrating the above fraction with an evaporator, no acid hydrolysis was carried out and no other operations were carried out. A similar procedure was carried out to prepare a watercress extract. Mouse B16 melanoma cells were cultured in 10% fetal bovine serum (FBS) After pre-cultivation, the cells were pre-cultured in a D-MEM medium containing PEG-400 for 24 hours. Watercress extract (before hydrolysis) or watercress extract hydrolysis solution (after hydrolysis) After decomposition, the mixture was added to a predetermined final concentration and cultured for another 72 hours. After completion of the incubation, the cell morphology was observed using a phase contrast microscope. The results are shown in Figure 1. The watercress extract (before hydrolysis) had a 5.0% concentration of This result suggests that watercress extract has the potential to inhibit the transport of melanosomes. It was revealed that watercress extract has an inhibitory effect on the transport of cereals. It was found that the same effect could be obtained at a concentration of 0.5% in the solution after hydrolysis. The results showed that the effect was amplified by hydrolyzing the watercress extract. It was. Watercress contains rhamnagin, rhamnetin, isorhamnetin, and kaempferol. It is known to contain glycosides of flavonols such as quinone and quercetin (non-specific In addition, a stronger inhibitory effect can be obtained by hydrolysis treatment, so Flavonol glycosides contained in Dagarashi extract are converted to aglycones by hydrolysis. This reminded me that this may be due to the In fact, Figure 2 shows the distribution of flavonol glycosides and aglycones. Therefore, it can be seen that the amount of aglycones of flavonols increases.

[0039] Example 2 (Evaluation 1: Examination of the inhibitory effect of various flavonols on the expression of Mlph protein proof) Mlph protein is a protein that plays a central role in intracellular transport of melanosomes. The solution obtained after hydrolysis in Example 1 contains rhamnadin and rhamnadin. Contains the aglycones of netin, isorhamnetin, kaempferol, and quercetin as its main components. Therefore, it was suggested that these flavonols are involved in the production of Mlph in pigment cells. To examine the effect on the expression of Mlph, Western blotting was performed using anti-Mlph antibody. Various flavonols were obtained by dissolving commercially available purified products in dimethyl sulfoxide. and used it.

[0040] Mouse B16 melanoma cells were pre-cultured in D-MEM medium containing 10% FBS for 24 hours. After culturing, the final concentrations of the various flavonols were 10 μM rhamnadin and 10 μM rhamnocitrin. 25μM, rhamnetin 5μM, isorhamnetin 50μM, kaempferol 20μM, Kel The final concentration of each flavonol was set at 50 μM. Before determining the concentration, a cytotoxicity test was conducted for each flavonol to determine whether the cell viability was 80% or higher. After 3 days of culture, the cells were washed three times with PBS. , cell lysis buffer (25mM Tris-HCl (pH7.5), 150mM Na Cl, 1% NP-40, 1% sodiumdeoxycholate and proteases The samples diluted in SDS-PAGE sample buffer were boiled for 5 minutes. The samples were separated by 10% SDS-PAGE and analyzed by semi-dry blotter (B Polyvinylidene difluoride membrane ( After transferring to a PVDF membrane, the membrane was blocked with 5% skim milk in phosphate buffer at room temperature for 1 hour. The PVDF membrane was washed three times with TBS-T and then incubated with anti-Mlph antibody. The PVDF membrane was incubated overnight at 4°C. After washing with TBS-T three times, Immunoprecipitation with a secondary antibody conjugated with seradish peroxidase for an additional hour The plate was incubated for 3 hours and then washed three times with TBS-T before detection. West Femto Maximum Sensitivity Substrate( Detection was performed using a Thermo Scientific (manufactured by Thermo Scientific) and LAS-3000. Quantitative assessment of protein was performed by comparing the intensity of the detected band with that of the unspiked sample. The effects of various flavonoids on the expression of Mlph protein were evaluated. , Glyceraldehyde-3-phosphate dehydrogenas e(Gapdh) was similarly detected as a standard protein.

[0041] The results are shown in Figure 3. Flavonols other than rhamnadin had no effect on the expression of Mlph protein. Rhamnagin significantly reduced the expression of Mlph protein, whereas rhamnagin had little effect on Mlph. These results suggest that rhamnagin suppresses the expression of Mlph protein in pigment cells. It has been shown to be effective and is clear that the active ingredient is the hydrolyzed watercress extract. It became clear.

[0042] Example 3 (Evaluation 2: Verification of rhamnagin cytotoxicity) To evaluate the toxicity of rhamnagin to pigment cells, we used a Cell Count™ (Dojindo) Cytotoxicity tests were performed using the Binding Kit-8.

[0043] Mouse B16 melanoma cells were pre-cultured in D-MEM medium containing 10% FBS for 24 hours. After culturing, rhamnagin was added to the cells to a final concentration of 5 and 10 μM, respectively. After culturing for 3 days, the cells were counted using the Cell Counting Kit-8 manufactured by Dojindo Laboratories. The cell viability was calculated according to the manual provided by the manufacturer.

[0044] The results are shown in Figure 4. When rhamnadin was added at concentrations of 5 and 10 μM, there was almost no decrease. From these results, it was found that rhamnagin was not a cytotoxic agent at concentrations of 10 μM or less. It was found that the cell viability rate exceeded the standard of 80% or more, and within the same concentration range, It was shown to be non-cytotoxic.

[0045] Example 4 (Evaluation 3: Verification of the inhibitory effect of rhamnadin on intracellular transport of melanosomes) The results of Evaluation 1 showed that rhamnagin suppressed the expression of Mlph protein in pigment cells. This protein binds melanosomes produced in pigment cells to the Since rhamnadin is a protein that plays a central role in transporting melanosomes, It was thought that rhamnagin may have the effect of inhibiting intracellular transport. In order to verify whether or not it has an inhibitory effect on the intracellular transport of melanosomes in To this end, we observed cell morphology and melanosomes using a phase contrast microscope and a confocal laser microscope. The percentage of cells in which the nuclei were aggregated was calculated.

[0046] Mouse B16 melanoma cells were cultured on collagen-coated coverslips. The cells were seeded onto a dish and pre-cultured in D-MEM for 24 hours. After pre-culture, the final concentration Rhamnadin was added to the culture medium to achieve a predetermined concentration, and the culture was continued for another 72 hours. The cell morphology was observed under a phase-contrast microscope. The cells were fixed by placing them in a phosphate buffer solution of methylaldehyde and leaving them at room temperature for 15 minutes. The cover glass was washed three times with PBS and then soaked in 0.1% Triton X-100. The cells were placed in PBS containing 3% CO₂O₂ and left at room temperature for 10 minutes to permeabilize them. After washing, place in PBS containing 3% bovine serum albumin and leave at room temperature for 1 hour. To stain actin, which is a cytoskeleton, the cells were blocked with PBS. After washing twice, the cells were incubated in PBS containing 0.01% Alexafluor 594-labeled phalloidin. The cells were then placed in PBS and left for 20 minutes. Next, the cells were washed three times with PBS to stain the nuclei. 2 μg / ml of 4',6-diamidino-2-phenylindole (DAP After washing three times with PBS, the cells were placed in fluoromount. After drying, the specimen was mounted using a confocal microscope (Diagnostic Biosystems). Cells were imaged using a laser scanning microscope (TCS SP2 AOBS, Leica Microsystems). The percentage of cells in which melanosomes were aggregated near the nucleus was 1.0%. I-stained nuclei were counted as an indicator of total cell number, and phalloidin-stained actin was counted as an indicator of total cell number. Counting the number of cells in which melanosomes are concentrated near the nucleus, taking the whole cell into account The total number of cells counted per sample was approximately 100.

[0047] The results of observation using a phase contrast microscope are shown in Figures 5(a) to 5(c). In untreated cells, melanosomes are uniformly distributed to the extremities. In cells treated with rhamnadin, melanosomes were distributed near the nucleus. Aggregation was observed (arrow).

[0048] The results of observation using a confocal laser microscope are shown in Figure 6(a) and (b). Based on the results of observation using a laser microscope, cells in which melanosomes are aggregated near the nucleus are predominant. The calculated percentage is shown in Figure 7. As observed under a phase contrast microscope, the melanosomes were more concentrated near the nucleus than in untreated cells. A significant aggregation of melanosomes was observed in the cells treated with rhamnagin. The percentage of cells clumped near the nucleus was about 1% in untreated cells, whereas in the lamina propionate treatment, In cells treated with rhamnagin, the rate was approximately 80%. It has been shown to have an inhibitory effect on the intracellular transport of cytosolic steroids.

[0049] Example 5 (Evaluation 4: Verification of the inhibitory effect of rhamnagin on the extracellular secretion of melanin pigment) The results of Evaluation 3 show that rhamnadin has the effect of inhibiting intracellular transport of melanosomes. This effect was found to be due to the suppression of melanin pigment secretion from pigment cells. Therefore, we investigated whether rhamnagin inhibits the extracellular secretion of melanin pigment. To verify whether or not the inhibitory effect of the extracellular medium on pigment cell culture was observed, Ranin levels were quantified.

[0050] Mouse B16 melanoma cells were pre-cultured in D-MEM medium containing 10% FBS for 24 hours. After culturing, the medium was changed to D-MEM medium containing 10% FBS and 50 μM forskolin. The medium was replaced with a new one, and rhamnagin was added to a final concentration of 10 μM, followed by culturing for 3 days. The medium was collected and counted using an Arvo-MX1420 multilabel counter (PerkinElmer). ) was measured at 405 nm, which is the absorption wavelength specific to black melanin, and the melanin in the culture medium was measured. The amount of extracellular melanin was measured in a cell culture medium without rhamnadin. The calculated value was set at 100%. The cell culture medium cultured in the same manner served as a negative control.

[0051] The results are shown in Figure 8. Rhamnadin reduced the amount of extracellular melanin by approximately 85%. These results indicate that rhamnagin has an inhibitory effect on the extracellular secretion of melanin pigment. was shown.

[0052] Example 6 (Evaluation 5: Inhibitory effect of rhamnagin on the expression of melanin synthesis-related proteins verification) From the results of evaluations 1, 3, and 4, it was found that rhamnadin inhibited the production of melanosomes in pigment cells. It was revealed that the drug has an inhibitory effect on the intracellular transport of steroids. Regarding the effect of rhamnagin in inhibiting the extracellular secretion of melanin pigment, One of the reasons for this is the inhibitory effect on intracellular transport of melanosomes, as well as the This may be due to the suppression of melanin synthesis in the lamina propria. To verify whether gin has an inhibitory effect on melanin synthesis in pigment cells To achieve this, microphthalmia-as, a type of melanin synthesis-related protein, Specific for associated transcription factor (Mitf) Western blotting was performed using specific antibodies.

[0053] Mouse B16 melanoma cells were pre-cultured in D-MEM medium containing 10% FBS for 24 hours. After culturing, the medium was changed to D-MEM medium containing 10% FBS and 50 μM forskolin. The medium was replaced with a new one, and rhamnagin was added to a final concentration of 10 μM, followed by culturing for 3 days. After the culture, verification was performed in the same manner as in Evaluation 1 using an anti-Mitf antibody.

[0054] The results are shown in Figure 9. Rhamnadin inhibited the expression of Mitf induced by forskolin. The expression of Mlph was similarly evaluated, and the expression of Mlph was not suppressed at all. The presence or absence of forskolin did not affect the expression of Mlph. These results suggest that rhamnadin inhibits the melanin synthesis-related proteins. It was shown that it specifically inhibits melanosome transport-related proteins without suppressing the expression of proteins. was done.

[0055] Example 7 (Evaluation 6: Verification of synergistic effects when used in combination with other drugs) Our results so far suggest that rhamnagin reduces the expression of Mlph, a melanin synthesis-related protein. While it specifically suppresses the expression of melanin synthesis-related proteins, it does not suppress the expression of melanin synthesis-related proteins. Thus, the secretion of extracellular melanin suppresses the expression of melanin synthesis-related proteins. Therefore, the expression of melanin synthesis-related proteins may be suppressed by The combined use of rhamnagin with a known whitening agent characterized by inhibiting the excretion of extracellular melanin Therefore, we have already investigated the effect of the enzyme, which is one of the melanin synthesis enzymes. When used in combination with arbutin, which is known to have the effect of inhibiting the enzyme activity of tyrosinase, We examined whether there was an inhibitory effect on extracellular melanin secretion.

[0056] Mouse B16 melanoma cells were pre-cultured in D-MEM medium containing 10% FBS for 24 hours. After culturing, the medium was changed to D-MEM medium containing 10% FBS and 50 μM forskolin. The final concentrations were 5 or 10 μM rhamnadin, 50 μM arbutin, and 5 μM rhamnadin. The cells were cultured for 3 days after adding 50 μM of nasin and 50 μM of arbutin. The amount of extracellular melanin was evaluated in the same manner as in Evaluation 4. The measured value of the cell culture medium to which neither arbutin nor erythritol was added was set as 100%.

[0057] The results are shown in Figure 10. Addition of arbutin alone significantly increased the amount of extracellular melanin compared to when no arbutin was added. The decrease when rhamnadin was added alone at a concentration of 5 or 10 μM was 84%. The results were approximately 41% and 20%, respectively. The decrease when either was added was 21%, which was less than when either was added alone. These results suggest that the combined use of rhamnagin and a known skin whitening agent synergistically promotes the excretion of extracellular melanin. This suggests that the compound may suppress pigmentation of the skin more efficiently than ever before. It has been shown that this can be provided.

[0058] Formulation Example 1 :Lotion The following components (3), (4), and (8) to (11) were mixed and dissolved (mixture A). The following components (1), (2), (5) to (7), and (12) were mixed and dissolved (Mixture B). The obtained mixtures A and B were mixed uniformly to obtain a lotion. (composition) (%) (1) Glycerin 7.0 (2) 1,3-butylene glycol 6.5 (3) Polyoxyethylene sorbitan Monolaurate (20E.O.) 1.2 (4) Ethanol 5.0 (5) Lactic acid 0.05 (6) Sodium lactate 0.1 (7) Collagen 1.0 (8) Hydrolyzed watercress extract 0.05 (9) Ascorbic acid 2-glucoside 0.5 (10) Preservatives 0.1 (11)Fragrance 0.1 (12) Remaining purified water

[0059] Formulation Example 2 :Emulsion The following component (10) was heated and maintained at 70°C (Solution A). ) to (9) and (11) were heated and mixed and kept at 70°C (mixture B). Solution A was added to B and mixed to form a uniform emulsion (mixture C). The resulting mixture C was cooled. Then, the following ingredients (12) to (15) were added and mixed uniformly to obtain an emulsion. (composition) (%) (1) Polyoxyethylene sorbitan Monostearate (10 E.O.) 1.0 (2) Polyoxyethylene sorbitol Tetraoleate (60E.O.) 0.5 (3) Glyceryl monostearate 1.0 (4) Stearic acid 0.5 (5) Behenyl alcohol 0.5 (6) Squalane 8.0 (7) Ethanol 5.0 (8) Hydrolyzed watercress extract 0.001 (9) Arbutin 1.0 (10) Remaining purified water (11) Preservatives 0.1 (12) Carboxyvinyl polymer 0.2 (13) Sodium hydroxide 0.1 (14) Hyaluronic acid 0.1 (15)Fragrance 0.1

[0060] Formulation Example 3 :cream The following component (11) was heated and maintained at 70°C (Solution A). ) to (8) were heated and mixed and kept at 70°C (mixture B). Solution A was added to the obtained mixture B. The resulting mixture C was cooled and then mixed with the following ingredients. Ingredients (9) and (10) were added and mixed uniformly to obtain a cream. (composition) (%) (1) Liquid paraffin 23.0 (2) Vaseline 7.0 (3) Behenyl alcohol 1.0 (4) Stearic acid 2.0 (5) Beeswax 2.0 (6) Sorbitan monostearate 1.5 (7) Polyoxyethylene sorbitan monostearate 2.5 (20E.O.) (8) Preservatives 0.1 (9)Fragrance 0.15 (10) Hydrolyzed watercress extract 1.0 (11) Remaining purified water

[0061] Formulation Example 4 : Liquid foundation The following components (1) to (7) were mixed and dissolved (Solution A). ) to (18) were added, mixed uniformly, and maintained at 70°C (Mixture B). Components (8) to (12) were dissolved uniformly and kept at 70°C (mixture C). Mixture B was added to the mixture and emulsified uniformly (mixture D). After cooling the obtained mixture D, The following ingredients (19) and (20) were added to obtain a liquid foundation. (composition) (%) (1) Liquid Lanolin 2.0 (2) Liquid paraffin 5.0 (3) Stearic acid 2.0 (4) Cetyl alcohol 1.0 (5) Self-emulsifying type Glyceryl Monostearate 1.0 (6) para-methoxycinnamic acid -2-Ethylhexyl 8.0 (7) Preservatives 0.1 (8) Glycerin 5.0 (9) Triethanolamine 1.0 (10) Carboxymethylcellulose 0.2 (11) Bentonite 0.5 (12) Remaining purified water (13) Titanium dioxide 6.0 (14) Fine particle titanium dioxide 2.0 (15) Fine particle zinc oxide 5.0 (16) Mica 2.0 (17) Talc 4.0 (18) Color pigment 4.0 (19) Hydrolyzed watercress extract 0.001 (20)Fragrance 0.1

[0062] Formulation Example 5 : Sunscreen lotion The following components (1) to (11) were mixed and dispersed (dispersion A). ) to (15) were mixed and dispersed (Dispersion B). Dispersion B was added to the obtained Dispersion A, and the mixture was homogenized. The resulting mixture was emulsified (mixture C). The following ingredients (16) and (17) were added to the resulting mixture C. Thus, a sunscreen lotion was obtained. (composition) (%) (1) Polyoxyalkylene-modified Organopolysiloxane 1.0 (2) Dimethylpolysiloxane 5.0 (3) Octamethylcyclotetrasiloxane 20.0 (4) Isotridecyl isononanoate 5.0 (5) para-methoxycinnamic acid -2-Ethylhexyl 10.0 (6) Preservatives 0.1 (7)Fragrance 0.1 (8) Silicone-treated titanium dioxide microparticles 8.0 (9) Silicone-treated zinc oxide microparticles 7.0 (10) Polystyrene powder 3.0 (11) Trimethylsiloxysilicate 0.5 (12) Dipropylene glycol 3.0 (13) Ethanol 10.0 (14) Remaining purified water (15) Salt 0.2 (16) Hydrolyzed watercress extract 0.02 (17) L-Ascorbyl Phosphate Magnesium 3.0

[0063] The cosmetics of Formulation Examples 1 to 5 were confirmed to have a high whitening effect when applied to the skin. .

Claims

1. A method for treating a rhombic ulcer comprising the steps of: (a) extracting a plant of the genus Rhamnus; or (b) hydrolyzing an extract of a plant selected from the group consisting of the genera Nasturtium, Rhamnus, Viscum, and Nervilia; A composition for inhibiting the expression of Mlph protein in pigment cells, wherein the extract or hydrolysate contains rhamnadin represented by the following chemical formula (I) as an active ingredient: 【Chemical 1】

2. The expression-inhibiting composition according to claim 1, wherein the extract of a plant of the genus Rhamnus is an extract of a plant selected from the group consisting of Rhamnus petiolaris and Rhamnus saxatilis.

3. The expression-inhibiting composition according to claim 1, wherein the hydrolysate is a hydrolysate of an extract of a plant selected from the group consisting of Nasturtium officinale, Rhamnus saxatilis, Viscum coloratum, and Nervilia fordii.

4. A method for inhibiting expression of Mlph protein in pigment cells, comprising the step of contacting the composition according to any one of claims 1 to 3 with pigment cells.

Images