JP7759972B2 - MELANOSOMAL TRANSPORT INHIBITOR AND EXTERNAL SKIN PREPARATION CONTAINING THE TRANSPORT INHIBITOR - Google Patents

Description

The present invention relates to a melanosome transport inhibitor and an external skin preparation containing the transport inhibitor.

Skin blemishes and freckles are caused by melanocytes localized in the basement membrane of the epidermis becoming abnormally activated due to stimuli such as ultraviolet light, hormonal abnormalities, genetic factors, etc., resulting in the synthesis of excess melanin pigment, which is then abnormally deposited in the skin (Non-Patent Document 1). Therefore, research and treatments have been conducted with a focus on inhibiting melanin synthesis in melanocytes.

Melanin is produced in intracellular organelles called melanosomes within melanocyte cells. The melanosomes, which turn black as the produced melanin accumulates, are transported by various transport-related proteins from the periphery of the melanocyte nucleus to the ends of the dendrites, where they are passed on to surrounding epidermal cells. Normally, the delivered melanosomes are excreted from the body as epidermal cells turn over. However, strong stimuli such as ultraviolet rays can induce excessive accumulation of melanosomes in epidermal cells, resulting in pigmentation known as age spots.

In recent years, it has been reported that intracellular transport of melanosomes is controlled by various proteins (Non-Patent Document 2).
Furthermore, as the identification of proteins involved in the intracellular transport of melanosomes progresses, it has been reported that compounds that quantitatively reduce these proteins inhibit the excessive accumulation of melanosomes in epidermal cells, thereby contributing to skin whitening effects. For example, inactivators of Rab27a, a low-molecular-weight G protein (Patent Document 1), Slp-2a, a binding molecule known as an effector of Rab27a (Patent Document 2), and synaptotagmin
-like protein homologue racking C2 domains-
Examples of such agents include a protein amount reducer for Slac2-a (also known as melanophilin or Mlph) (Patent Document 3).

Based on the above-mentioned previous research results, the present inventors have conducted extensive research into active compounds that have a skin-whitening effect.

Japanese Patent Application Laid-Open No. 2007-137821 JP 2010-229043 A JP 2011-132195 A

Prota, G. J., Invest. Dermatol. (1980), 75: 122-127. Kuroda, T. S et al., Nat. Cell Biol. (2004), 6: 1195-1203. Goda, Y et al., Biol.Pherm.Bull. (1999), 12:1319-1326.

An object of the present invention is to provide a melanosome transport inhibitor having a high whitening effect and an external skin preparation containing the same.

The present inventors have conducted extensive research to solve the above-mentioned problems and have found that rhamnadin, a flavonol, effectively inhibits melanosome transport. Based on this finding, the present inventors have furthered their research and have come to the conclusion that rhamnadin has a strong whitening effect.

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.
The rhamnadin is preferably a substance derived from a plant of the genus Rhamnus , such as natural Rhamnus petiolaris or Rhamnus saxatilis, or an organic synthetic product.

In addition, as a result of intensive research conducted by the inventors to solve the above-mentioned problems, they have come to the conclusion that a hydrolysate of an extract derived from a specific plant, etc., which is rich in rhamnadin (a second melanosome transport inhibitor) has a particularly high whitening effect.

That is, the second melanosome transport inhibitor of the present invention is a watercress (Nasturtium
Nasturtium (including Nasturtium officinale)
Genus Rhamnus, including Rhamnus saxailis
amnus, Viscum including Viscum coloratum, Nervilia fordii
) or clove (Syzygium arom)
It is preferable that the extract be a hydrolysate of an extract derived from a plant of the genus Syzygium, which includes Syzygium aticum.

The second melanosome transport inhibitor can be obtained by subjecting an extract extracted from a plant material by solvent extraction or the like to hydrolysis treatment.

The present invention also provides an external skin preparation containing the first or second melanosome transport inhibitor. The concentration of rhamnadine contained in the external skin preparation is preferably 0.0001 to 5% by mass.
The external preparation for skin preferably further contains a whitening agent that does not have a melanosome transport inhibitory effect.
The whitening agent is preferably a tyrosinase activity inhibitor.
The tyrosinase activity inhibitor includes ascorbic acid, ascorbic acid salts and derivatives thereof,
It is preferably selected from the group consisting of hydroquinone, salts and glycosides of hydroquinone, placenta extract, arbutin, ellagic acid, linoleic acid, and tranexamic acid.

According to the present invention, there are provided a melanosome transport inhibitor having a high whitening effect and an external skin preparation containing the same.

1 is a micrograph (magnification: 200x) showing a comparison of cells treated with watercress extract before hydrolysis and cells treated with extract after hydrolysis. 1 is a graph showing the ratio of glycosides to aglycones before and after hydrolysis of the solution obtained in Example 1. 1 is a graph showing the results of verifying the inhibitory effect of various flavonols on Mlph protein expression. 1 is a graph showing the results of testing the cytotoxicity of rhamnagin. 1 shows micrographs (magnification: 200) showing the results of observing cells using a phase-contrast microscope to verify the inhibitory effect of rhamnadin on intracellular melanosome transport, where (a) shows cells without rhamnadin added, (b) shows cells with 5 μM rhamnadin added, and (c) shows cells with 10 μM rhamnadin added. 1 shows micrographs (magnification: 250) showing the results of observing cells with a confocal laser microscope, in relation to the verification results of the inhibitory effect of rhamnadin on the intracellular transport of melanosomes, where (a) shows cells without rhamnadin added, and (b) shows cells with 10 μM rhamnadin added. 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. 1 is a graph showing the results of verifying the inhibitory effect of rhamnagin on the extracellular secretion of melanin pigment. 1 is a graph showing the results of verification of the inhibitory effect of rhamnadin on the expression of melanin synthesis-related proteins. 1 is a graph showing the results of verification of the synergistic effect of combined use of rhamnagin and other drugs.

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, effectively inhibits melanosome transport. Based on this finding, the present inventors have furthered their investigations and have found that
It was verified that rhamnadin has a high whitening effect.

The first melanosome transport inhibitor according to the present invention contains rhamnadin represented by the following chemical formula (I) as an active ingredient.
Rhamnadin derived from natural products is preferably derived from plants of the genus Rhamnus (also known as the genus Buckthorn), such as Rhamnus petiolaris and Rhamnus saxatilis. 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 and allows a larger amount of rhamnadin to be obtained from a smaller amount of extract.

The first melanosome transport inhibitor according to the present invention can be prepared, for example, as a fine powder or crushed product of the whole plant, roots, rhizomes, leaves, seeds, fruits, or flowers of a plant of the genus Rhamnus, such as Rhamnus petiolaris or Rhamnus saxatilis.
The first melanosome transport inhibitor of the present invention can also be prepared as an extract obtained by immersing and extracting 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.

When extracting with a solvent, it is preferable to use an alcohol such as methanol or ethanol, or a low-polarity solvent such as 1,3-butylene glycol, as the extraction solvent, since such an extraction solvent can give the desired extract.

The rhamnadin contained in the first melanosome transport inhibitor of the present invention can be purified by removing solid matter by filtration or centrifugation, in addition to or instead of extraction with an organic solvent, and then undergoing a purification process using chromatography methods such as silica gel column or HPLC.

Rhamnadin can also be obtained by utilizing organic synthesis reactions. Rhamnadin obtained by such various syntheses (synthetic rhamnadin) can also be contained as an active ingredient in the melanosome transport inhibitor of the present invention.

With regard to the melanosome transport inhibitors of the present invention, "containing rhamnasin as the active ingredient" means any of the following: a melanosome transport inhibitor consisting only of the active ingredient rhamnasin alone; a melanosome transport inhibitor containing trace amounts of impurities in addition to rhamnasin alone; a melanosome transport inhibitor that is finely powdered or crushed from the whole plant, roots, rhizomes, leaves, seeds, fruits, or flowers, as described above; an extract obtained by immersing the finely powdered or crushed material in water or an organic solvent and filtering the residue; an extract obtained by removing the solvent from this extract (extracted extract); and a melanosome transport inhibitor that is prepared in the form of a solution by further dissolving, dispersing, or diluting the finely powdered or solvent-removed material with an appropriate solvent or the like.

The melanosome transport inhibitor of the present invention (including the second melanosome transport inhibitor described below)
When prepared in the form of a solution, it is preferable that rhamnadin is contained in a proportion of 0.0001% to 5% by weight.

Second Melanosome Transport Inhibitor As mentioned above, the present inventors have discovered that hydrolysates of natural products such as specific plants that are rich in rhamnadin have a high whitening effect.

The second melanosome transport inhibitor according to the present invention also contains rhamnadin represented by the above chemical formula (I) as an active ingredient.
The second melanosome transport inhibitor is preferably a hydrolysate of a naturally occurring product derived from a plant of the genus Nasturtium, including watercress ( Nasturtium officinale ), the genus Rhamnus, including Rhamnus saxatilis , the genus Viscum , including Viscum coloratum, the genus Nervilia, including Nervilia fordii, or the genus Syzygium, including clove (Syzygium aromaticum).

The second melanosome transport inhibitor can be obtained by subjecting an extract extracted from a plant material by solvent extraction or the like to hydrolysis treatment.
For example, a procedure including steps of immersing freeze-dried aerial parts of a plant such as watercress extract in a solvent such as methanol to obtain an extract, concentrating the extract, purifying the resulting concentrate through a column, and eluting the fraction with a solvent such as methanol to obtain a fraction is subjected to acid hydrolysis, thereby preparing a second melanosome transport inhibitor as a hydrolyzed solution of watercress or the like.
As described above, the first melanosome transport inhibitor may be prepared in the form of an extract extracted from a plant material by solvent extraction, etc. The second melanosome transport inhibitor can be obtained by hydrolyzing such an extract, more precisely, by hydrolyzing the flavonoid glycoside contained in such an extract.

Furthermore, the present inventors investigated the mechanism of the strong whitening effect of rhamnadin and found that the addition of rhamnadin reduced the amount of Mlph protein, which is involved in melanosome transport. This led them to believe that a strong whitening effect can be achieved by inhibiting melanosome transport in melanocytes. Mlph protein is a protein involved in melanosome transport in melanocytes. Inhibition of melanosome transport inhibits the transfer of melanin produced by melanocytes to keratinocytes, thereby suppressing skin pigmentation.

On the other hand, the present inventors have confirmed that conventional whitening agents, kojic acid and arbutin do not have the effect of inhibiting melanosome transport.
Therefore, the effect of inhibiting skin pigmentation based on the melanosome transport inhibitor of the present invention differs in terms of mechanism of action from the whitening effect (melanin synthesis inhibitory effect) achieved by conventional whitening cosmetics containing kojic acid, arbutin, etc.

By using the melanosome transport inhibitor of the present invention, including rhamnadin, in combination with other whitening agents that do not have melanosome transport inhibitory activity, a synergistic effect can be achieved that further enhances the whitening effect compared to using the other whitening agents alone.
The whitening agent without melanosome transport inhibitory activity is preferably a tyrosinase inhibitor that inhibits melanin synthesis. Most of the commonly used whitening agents are tyrosinase inhibitors. Even if multiple or large amounts of whitening agents that work in the same way based on the same mechanism of action, such as tyrosinase inhibitors, are compounded, the whitening effect is unlikely to be enhanced, and there are concerns about the safety of the effect. However, as mentioned above, whitening agents that have different mechanisms of action, such as those that act on different stages or different parts of the skin pigmentation process,
That is, by combining a tyrosinase activity inhibitor and a melanosome transport inhibitor, it is possible to achieve a high whitening effect even when using small amounts of each, and also to provide excellent safety.

Examples of tyrosinase activity inhibitors include, but are not limited to, ascorbic acid, ascorbic acid salts and derivatives thereof, hydroquinone, hydroquinone salts and glycosides, placenta extract, arbutin, ellagic acid, linoleic acid, and tranexamic acid.

External Skin Preparation The external skin preparation according to the present invention contains the first or second melanosome transport inhibitor.
The external skin preparation according to the present invention preferably contains rhamnadine in an amount of 0.00 based on the total amount of the external skin preparation.
The content is 0.01 to 5% by mass. A content of at least 0.0001% by mass corresponds to a concentration at which the melanosome transport inhibitory effect is minimally obtained, and a content of 5% by mass or less provides the melanosome transport inhibitory effect and also ensures safety.

The melanosome transport inhibitor of the present invention can be used as an external preparation for skin that is applied to the outer skin, such as a cosmetic, pharmaceutical, or quasi-drug. Therefore, it can be used in a wide range of dosage forms, including water-soluble systems (lotion systems), solubilized systems, emulsion systems (W/O type, O/W type), powder systems, ointments, oil systems, gel systems, water-oil two-layer systems, and water-oil-powder three-layer systems.
In addition to the melanosome transport inhibitor, the topical skin preparation of the present invention can contain other ingredients commonly used in topical skin preparations, such as water, alcohol, oils, surfactants, thickeners, powders, chelating agents, preservatives, pH adjusters, various medicinal agents, extracts derived from animals, plants, or microorganisms, pigments, fragrances, etc., as needed, within the scope of not impairing the effects of the present invention. Furthermore, the effects of the present invention can be further enhanced or other effects can be added by using medicinal ingredients such as antioxidants, cell activators, anti-inflammatory agents, and ultraviolet protection agents in combination.

Examples of the dosage form of the external skin preparation of the present invention include lotions, oils, emulsions, creams, ointments, etc. In addition, emulsions, creams, lotions, serums, sheet masks, cleansers,
It is also possible to provide the composition as various types of cosmetics, such as makeup cosmetics.

The topical skin preparation of the present invention can further contain a whitening agent that does not have melanosome transport inhibitory activity. As such a whitening agent, the above-mentioned tyrosinase activity inhibitor can be used. A topical skin preparation containing the melanosome transport inhibitor of the present invention containing rhamnadin and another whitening agent that does not have melanosome transport inhibitory activity can exhibit a synergistic effect that further enhances the whitening effect compared to a topical skin preparation containing only the other whitening agent.

As described above, the topical skin preparation of the present invention contains rhamnadine as an active ingredient that inhibits melanosome transport, and is particularly effective as a whitening cosmetic for the purpose of improving skin condition.

The present invention will be specifically explained below using examples, formulation examples, etc., but the present invention is not limited to these.

Example 1
The freeze-dried aboveground parts of watercress were immersed in methanol and shaken overnight at room temperature to obtain an extract. The extract was then concentrated using an evaporator. The resulting concentrate was purified using an HP-20 column, and a fraction with a high flavonoid content was obtained by elution with methanol. After concentrating this fraction using an evaporator, 6N hydrochloric acid was added and heated at 100°C for 90 minutes to carry out acid hydrolysis. After the reaction, the mixture was neutralized with sodium hydroxide and extracted with ethyl acetate. The resulting extract was concentrated using an evaporator, and then
The solution obtained by redissolving it in methanol was prepared as a hydrolyzed solution of watercress extract.
On the other hand, the above fraction was concentrated using an evaporator, and the remaining operations were carried out in the same manner as above, but without acid hydrolysis, to prepare a watercress extract.
Mouse B16 melanoma cells were cultured in 10% fetal bovine serum (FBS).
The cells were pre-cultured for 24 hours in D-MEM medium containing HCl. After pre-culture, the watercress extract (before hydrolysis) or the watercress extract hydrolyzed solution (after hydrolysis) obtained in Example 1 was added to a predetermined final concentration, and the cells were further cultured for 72 hours. After the culture was completed, the cell morphology was observed using a phase-contrast microscope.
The results are shown in Figure 1. Watercress extract (before hydrolysis) inhibited melanosome transport at a concentration of 5.0%. These results demonstrate that watercress extract has an inhibitory effect on melanosome transport. Furthermore, it was revealed that the same effect could be obtained at a concentration of 0.5% in the solution after hydrolysis of watercress extract. These results demonstrate that this effect is amplified by hydrolyzing the watercress extract.
Watercress is known to contain glycosides of flavonols, such as rhamnadin, rhamnetin, isorhamnetin, kaempferol, and quercetin (Non-Patent Document 3). Furthermore, since a stronger inhibitory effect was obtained by hydrolysis, we suspected that this was due to the conversion of the flavonol glycosides contained in watercress extract to aglycones by hydrolysis.
In fact, Figure 2 shows the distribution of flavonol glycosides and aglycones, and it can be seen that the amount of aglycones of flavonols increases with hydrolysis.

Example 2
(Evaluation 1: Verification of the inhibitory effect of various flavonols on the expression of Mlph protein)
Mlph protein is known to play a central role in intracellular transport of melanosomes. It was assumed that the hydrolysis solution obtained in Example 1 contained the aglycones of rhamnadin, rhamnetin, isorhamnetin, kaempferol, and quercetin as its main components. Therefore, it was suggested that these flavonols are involved in the Mlph transport in pigment cells.
To examine the effect of Mlph on the expression of flavonols, Western blotting was performed using anti-Mlph antibody. Commercially available purified flavonols were dissolved in dimethyl sulfoxide and used.

Mouse B16 melanoma cells were pre-cultured in D-MEM medium containing 10% FBS for 24 hours, and then various flavonols were added to the cells at final concentrations of 10 μM rhamnadin, 25 μM rhamnocitrin, 5 μM rhamnetin, 50 μM isorhamnetin, 20 μM kaempferol, and 50 μM quercetin, respectively, and then cultured. The final concentrations of the various flavonols were determined by conducting a cytotoxicity test for each flavonol in advance, and the cell viability was determined to be 80% or higher.
After 3 days of culture, the cells were washed three times with PBS and lysed in a cell lysis buffer (25 mM Tris-HCl (pH 7.5), 150 mM Na
The samples were dissolved in 10% SDS-PAGE sample buffer (1% NP-40, 1% sodium deoxycholate, and protease inhibitors). The diluted samples were boiled for 5 minutes. The samples were separated by 10% SDS-PAGE and then analyzed by semi-dry blotter (B
Polyvinylidene difluoride membrane (
After transferring the antibody to a PVDF membrane, blocking was performed in 5% skim milk 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 at 4°C overnight.
The sections were then incubated with a secondary antibody labeled with seradish peroxidase for an additional hour. Before detection, the sections were washed three times with TBS-T.
West Femto Maximum Sensitivity Substrate (
Detection was carried out using a chromatograph (manufactured by Thermo Scientific) and LAS-3000.
The quantitative evaluation of the Mlph protein was carried out by comparing the intensity of the detected band with that of the untreated sample, and the effect of various flavonoids on the expression of the Mlph protein was evaluated.
e(Gapdh) was similarly detected as a standard protein.

The results are shown in Figure 3. Flavonols other than rhamnagin had almost no effect on Mlph protein expression, whereas rhamnagin significantly reduced Mlph protein expression. These results demonstrate that rhamnagin has the effect of suppressing Mlph protein expression in pigment cells, and are therefore an active ingredient in the watercress extract hydrolysate.

Example 3
(Evaluation 2: Verification of rhamnagin cytotoxicity)
To evaluate the toxicity of rhamnagin to pigment cells, a cytotoxicity test was carried out using Cell Counting Kit-8 manufactured by Dojindo Laboratories.

Mouse B16 melanoma cells were pre-cultured in 10% FBS-containing D-MEM medium for 24 hours, then rhamnadin was added to final concentrations of 5 and 10 μM, followed by further culture for 3 days. After the culture was completed, the cell viability was calculated using the Cell Counting Kit-8 (Dojindo Laboratories) according to the accompanying manual.

The results are shown in Figure 4. Almost no decrease was observed when rhamnagin was added at concentrations of 5 and 10 µM. These results demonstrate that rhamnagin at concentrations of 10 µM or less meets the cytotoxicity standard of a cell viability of 80% or more, indicating that it is not cytotoxic within this concentration range.

Example 4
(Evaluation 3: Verification of the inhibitory effect of rhamnadin on intracellular transport of melanosomes)
The results of Evaluation 1 revealed that rhamnagin suppresses the expression of the Mlph protein in pigment cells. Since this protein plays a central role in the intracellular transport of melanosomes produced in pigment cells, it was thought that rhamnagin may have the effect of inhibiting the intracellular transport of melanosomes. Therefore, to verify whether rhamnagin has an inhibitory effect on the intracellular transport of melanosomes in pigment cells, we observed cell morphology using a phase-contrast microscope and a confocal laser microscope and calculated the proportion of cells in which melanosomes aggregated near the nucleus.

Mouse B16 melanoma cells were seeded onto a culture dish containing a collagen-coated cover glass and pre-cultured in D-MEM for 24 hours. After pre-culture, rhamnadin was added to a predetermined final concentration, and the cells were further cultured for 72 hours.
Cell morphology was observed under a phase-contrast microscope. The collected cover glass was placed in 4% paraformaldehyde phosphate buffer and left at room temperature for 15 minutes to fix the cells. The cover glass was then washed three times with PBS, and then placed in PBS containing 0.1% Triton X-100 and left at room temperature for 10 minutes to permeabilize the glass.
After washing the cells twice, they were placed in PBS containing 3% bovine serum albumin and left at room temperature for 1 hour for blocking.
After washing twice, the cells were incubated in PB containing 0.01% Alexafluor 594-labeled phalloidin.
The cells were then placed in PBS and left for 20 minutes. Next, to stain the nuclei, the cells were washed three times with PBS.
2 μg/ml of 4′,6-diamidino-2-phenylindole (DAP
The cells were placed in PBS containing DAP and left for 3 minutes. After washing three times with PBS, they were mounted using Fluoromount (Diagnostic Biosystems). After drying, the cell morphology was observed using a confocal laser microscope (TCS SP2 AOBS, Leica Microsystems). The percentage of cells in which melanosomes aggregated near the nucleus was determined by the DAP concentration.
The number of cells with melanosomes concentrated near the nucleus was calculated by counting I-stained nuclei as an index of the total cell number, and by regarding phalloidin-stained actin as the whole cell image, the number of cells with melanosomes concentrated near the nucleus was calculated. The total number of cells counted per sample was approximately 100 cells.

The results of observation using a phase-contrast microscope are shown in Figures 5(a) to (c). In the images, melanosomes are observed as black spots. In untreated cells, melanosomes were uniformly distributed to the extremities, whereas in cells treated with rhamnadin, melanosomes were observed to aggregate near the nucleus (arrows).

The results of observation using a confocal laser microscope are shown in Figures 6(a) and 6(b). Based on the results of observation using the confocal laser microscope, the percentage of cells in which melanosomes aggregate near the nucleus was calculated and the results are shown in Figure 7.
As observed under a phase-contrast microscope, cells treated with rhamnazine showed significant aggregation of melanosomes near the nucleus compared to untreated cells. Furthermore, the percentage of cells in which melanosomes were aggregated near the nucleus was approximately 1% in untreated cells, whereas it was approximately 80% in rhamnazine-treated cells. These results demonstrate that rhamnazine has an inhibitory effect on intracellular melanosome transport.

Example 5
(Evaluation 4: Verification of the inhibitory effect of rhamnagin on the extracellular secretion of melanin pigment)
The results of Evaluation 3 revealed that rhamnadin has the effect of inhibiting the intracellular transport of melanosomes. This effect is thought to be linked to the suppression of the extracellular secretion of melanin pigment. Therefore, to verify whether rhamnadin has an inhibitory effect on the extracellular secretion of melanin pigment, the amount of extracellular melanin was quantified during the culture of pigment cells.

Mouse B16 melanoma cells were pre-cultured for 24 hours in D-MEM medium containing 10% FBS. After incubation, the medium was replaced with D-MEM medium containing 10% FBS and 50 μM forskolin. Rhamnadin was added to a final concentration of 10 μM and the cells were cultured for 3 days. After incubation, the medium was collected and measured at 405 nm, an absorption wavelength specific to black melanin, using an Arvo-MX1420 multilabel counter (PerkinElmer) to assess the amount of melanin in the medium. The amount of extracellular melanin was calculated based on the value measured for the cell culture medium without rhamnadin. A cell culture medium similarly cultured in D-MEM medium without forskolin served as a negative control.

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.

Example 6
(Evaluation 5: Verification of the inhibitory effect of rhamnagin on the expression of melanin synthesis-related proteins)
The results of evaluations 1, 3, and 4 revealed that rhamnagin has an inhibitory effect on the intracellular transport of melanosomes produced in pigment cells. On the other hand, the effect of rhamnagin on the suppression of extracellular secretion of melanin pigment, as shown in evaluation 4, was thought to be due in part to the suppression of melanin synthesis in pigment cells, in addition to the inhibitory effect on the intracellular transport of melanosomes. Therefore, in order to verify whether rhamnagin has an inhibitory effect on melanin synthesis in pigment cells, microphthalmia-as, a type of melanin synthesis-related protein, was examined.
Western blotting was performed using a specific antibody against associated transcription factor (Mitf).

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, and rhamnadin was added to a final concentration of 10 μM. The cells were then cultured for 3 days. After culturing for 3 days, verification was performed using an anti-Mitf antibody in the same manner as in Evaluation 1.

The results are shown in Figure 9. Rhamnagin did not suppress the expression of Mitf induced by forskolin at all. The expression of Mlph was similarly evaluated, and the results showed that the expression of Mlph was not affected by the presence or absence of forskolin. On the other hand, the inhibitory effect of rhamnagin on the expression of Mlph was significant. These results indicate that rhamnagin does not suppress the expression of melanin synthesis-related proteins, but specifically suppresses melanosome transport-related proteins.

Example 7
(Evaluation 6: Verification of synergistic effects when used in combination with other drugs)
From the results so far, rhamnagin specifically inhibits the expression of Mlph, a melanin synthesis-related protein, but does not inhibit the expression of melanin synthesis-related proteins. As mentioned above, extracellular melanin excretion may also be inhibited by the mechanism of inhibiting the expression of melanin synthesis-related proteins. Therefore, the combined use of rhamnagin with a known skin-whitening agent characterized by inhibiting the expression of melanin synthesis-related proteins is expected to have a synergistic effect on the excretion of extracellular melanin. Therefore, we investigated whether or not the combined use of rhamnagin with arbutin, which is already known to inhibit the enzymatic activity of tyrosinase, a melanin synthesis enzyme, has an inhibitory effect on the excretion of extracellular melanin.

Mouse B16 melanoma cells were pre-cultured for 24 hours in D-MEM medium containing 10% FBS. After culturing, the medium was replaced with D-MEM medium containing 10% FBS and 50 μM forskolin, and rhamnagin at final concentrations of 5 or 10 μM, 50 μM arbutin, or 5 μM rhamnagin and 50 μM arbutin were added, followed by culturing for 3 days. The amount of melanin in the medium was then evaluated in the same manner as in Evaluation 4. The amount of extracellular melanin was calculated by setting the measured value for the cell culture medium without the addition of either rhamnagin or arbutin as 100%.

The results are shown in Figure 10. Addition of arbutin alone reduced the amount of extracellular melanin to approximately 84% compared to when no arbutin was added. Addition of rhamnadin alone at a concentration of 5 or 10 µM reduced the amount of extracellular melanin by approximately 84%.
The reduction was approximately 41% and 20%, respectively. Furthermore, when both arbutin and rhamnadin (5 μM) were added, the reduction was 21%, which was less than when either was added alone. These results suggest that the combined use of rhamnadin and a known skin-lightening agent synergistically inhibits extracellular melanin secretion, demonstrating that a more efficient means of inhibiting skin pigmentation than previously possible can be provided.

Formulation Example 1 : Lotion The following ingredients (3), (4), and (8) to (11) were mixed and dissolved (Mixture A). Then, the following ingredients (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 (20 E.O.) 1.2
(4) Ethanol 5.0
(5) Lactic acid 0.05
(6) Sodium lactate 0.1
(7) Collagen 1.0
(8) Watercress extract hydrolysate 0.05
(9) Ascorbic acid 2-glucoside 0.5
(10) Preservative 0.1
(11) Fragrance 0.1
(12) Remaining purified water

Formulation Example 2 : Emulsion The following component (10) was heated and maintained at 70°C (solution A).
Components (12) to (15) were added to the obtained mixture C, and mixed uniformly to obtain a emulsion.
(Composition) (%)
(1) Polyoxyethylene sorbitan monostearate (10 E.O.) 1.0
(2) Polyoxyethylene sorbitan tetraoleate (60 E.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) Watercress extract hydrolysate 0.001
(9) Arbutin 1.0
(10) Purified water remaining amount (11) Preservative 0.1
(12) Carboxyvinyl polymer 0.2
(13) Sodium hydroxide 0.1
(14) Hyaluronic acid 0.1
(15) Fragrance 0.1

Formulation Example 3 : Cream The following component (11) was heated and maintained at 70°C (solution A).
) to (8) were heated and mixed, and the mixture was maintained at 70°C (Mixture B). Solution A was added to the resulting Mixture B, and mixed to form a uniform emulsification (Mixture C). After cooling the resulting Mixture C, the following ingredients (9) to (10) were added and mixed to form a uniform 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
(20 E.O.)
(8) Preservatives 0.1
(9) Flavoring 0.15
(10) Hydrolyzed watercress extract 1.0
(11) Remaining purified water

Formulation Example 4 : Liquid Foundation The following components (1) to (7) were mixed and dissolved (Solution A).
The following ingredients (8) to (12) were then added and mixed uniformly, and the mixture was maintained at 70°C (Mixture B). Next, the following ingredients (8) to (12) were added and mixed uniformly, and the mixture was maintained 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 Glyceryl Monostearate 1.0
(6) 2-ethylhexyl paramethoxycinnamate 8.0
(7) Preservatives 0.1
(8) Glycerin 5.0
(9) Triethanolamine 1.0
(10) Carboxymethyl cellulose 0.2
(11) Bentonite 0.5
(12) Purified water remaining amount (13) Titanium oxide 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

Formulation Example 5 : Sunscreen emulsion The following ingredients (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 uniformly emulsified (Mixture C). The following components (16) and (17) were added to the obtained Mixture C to obtain a sunscreen emulsion.
(Composition) (%)
(1) Polyoxyalkylene-modified organopolysiloxane 1.0
(2) Dimethylpolysiloxane 5.0
(3) Octamethylcyclotetrasiloxane 20.0
(4) Isotridecyl isononanoate 5.0
(5) 2-Ethylhexyl paramethoxycinnamate 10.0
(6) Preservatives 0.1
(7) Fragrance 0.1
(8) Silicone-treated titanium dioxide fine particle 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) Purified water remaining (15) Salt 0.2
(16) Watercress extract hydrolysate 0.02
(17) L-Ascorbyl phosphate
Magnesium 3.0

It was confirmed that the cosmetics of Formulation Examples 1 to 5 had a high whitening effect when applied to the skin.

Claims (4)

The present invention relates to a method for treating a rhamnus disease, comprising: extracting a plant of the genus Rhamnus; or hydrolyzing an extract of a plant selected from the group consisting of the genus Nasturtium, the genus Rhamnus, the genus Viscum, and the genus 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:
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. 2. The expression-inhibiting composition of 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 . A method for inhibiting the expression of Mlph protein in pigment cells, comprising the step of contacting pigment cells with the composition described in any one of claims 1 to 3.