JP2021116279A - Melanosome transportation inhibitor and topical skin preparation containing the transportation inhibitor - Google Patents

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 of a following chemical formula (I) as an active ingredient.SELECTED DRAWING: None

Description

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

For skin spots and freckles, melanocytes localized in the basement membrane of the epidermis are abnormally activated due to stimuli such as ultraviolet rays, hormonal abnormalities, or genetic factors to synthesize excess melanin pigment, and the melanin pigment is the skin. It is caused by abnormal deposition in the skin (Non-Patent Document 1). Therefore, studies and treatments focusing on inhibiting melanin synthesis in melanocytes have been conventionally conducted.

Melanin is produced in intracellular organelles called melanosomes, which are intracellular melanocytes. The melanosomes that have turned black due to the accumulation of the produced melanin are transported from the periphery of the nucleus of melanocytes to the ends of dendrites by various transport-related proteins, and are transferred to the surrounding epidermal cells. Normally, the delivered melanosomes are excreted from the body with the turnover of epidermal cells. On the other hand, strong stimuli such as ultraviolet rays induce excessive accumulation of melanosomes in epidermal cells and cause pigmentation called spots.

In recent years, it has been reported that the intracellular transport of melanosomes is regulated by various proteins (Non-Patent Document 2).
In addition, as the identification of proteins involved in the intracellular transport of such melanosomes progresses, it has been reported that compounds that quantitatively reduce these proteins are useful for skin whitening effects because they suppress the excessive accumulation of melanosomes in epidermal cells. Has been done. For example, an inactivating agent for Rab27a, which is a low molecular weight G protein (Patent Document 1), Slp-2a (Patent Document 2), which is a binding molecule called an effector for Rab27a, and synaptotagmin-like protein homologue racking C2 domain-a (Slac2). -A, also known as: Melanofilin, Mlp) (Patent Document 3), a protein amount reducing agent and the like can be mentioned.

Based on the above-mentioned previous research results, the present inventors have diligently studied active compounds having a whitening effect on the skin.

Japanese Unexamined Patent Publication No. 2007-137821 Japanese Unexamined Patent Publication No. 2010-229043 Japanese Unexamined Patent Publication No. 2011-132195

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 melanosomes transport inhibitor having a high whitening effect and an external preparation for skin containing the same.

As a result of diligent studies to solve the above problems, the present inventors have found that rhamnazin, which is classified as a flavonol, effectively inhibits melanosome transport. Based on this finding, the present inventors proceeded with the study and came up with the idea that rhamnazin has a high whitening effect.

前記ラムナジンは、天然のラムナス・ペティオラリス（Ｒａｍｎｕｓ ｐｅｔｉｏｌａｒｉｓ）やラムナス・サクサティリス（Ｒａｍｎｕｓ ｓａｘａｔｉｌｉｓ）などラムナス（Ｒａｍｎｕｓ）属の植物由来物又は有機合成物であることが好適である。 The present invention provides a melanosome transport inhibitor (first melanosome transport inhibitor), which comprises ramnazin represented by the following chemical formula (I) as an active ingredient.

The ramnazin is preferably a plant-derived product or an organic compound of the genus Rhamnous, such as natural Rhamnus petiolaris and Ramnus saxatilis.

In addition, as a result of diligent studies to solve the above problems, the present inventors have made a hydrolyzate of an extract derived from a specific plant or the like containing abundant ramnazin (second melanosomes). It was conceived that the transport inhibitor) has a higher whitening effect.

That is, the second melanosomes transport inhibitor of the present invention includes Viscum, Viscum, Viscum, Viscum, Viscum, Viscum, Viscum, Viscum, Viscum, Viscum, Viscum, Viscum, Viscum, Viscum, Viscum, Viscum, Viscum. Is a hydrolyzate of an extract derived from a plant of the genus Viscum containing (), the genus Viscum containing Nerviria fordii, or the genus Viscum containing cloves (Syzygium aromaticum). Is preferable.

The second melanosome transport inhibitor can be obtained by hydrolyzing an extract extracted from a plant as a raw material by solvent extraction or the like.

The present invention also provides a skin external preparation containing the first or second melanosomes transport inhibitor. The concentration of rhamnazin contained in the external preparation for skin is preferably 0.0001 to 5% by mass.
It is preferable that the external preparation for skin further contains a whitening agent having no melanosome transport inhibitory effect.
The whitening agent is preferably an inhibitor of tyrosinase activity.
The tyrosinase activity inhibitor may be selected from the group consisting of ascorbic acid, ascorbic acid salt and its derivatives, hydroquinone, hydroquinone salts and glycosides, placenta extract, arbutin, ellagic acid, linoleic acid, or tranexamic acid. Suitable.

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

It is a micrograph (magnification: 200) showing the cells treated with the Dutch glass extract, comparing the cells treated with the extract before the hydrolysis operation and the cells treated with the extract after the hydrolysis operation. .. It is a graph which shows the ratio of glycoside and aglycone before and after the hydrolysis treatment of the solution obtained in Example 1. FIG. It is a graph which shows the result of having verified the inhibitory effect on the expression of Mlp protein of various flavonols. It is a graph which shows the verification result about the cytotoxicity of ramnazin. It is a micrograph (magnification: 200) which shows the observation result of the cell by the phase contrast microscope regarding the verification result of the inhibitory effect of ramnadin on the intracellular transport of melanosomes. (A) shows that ramnazin is not added, (b) shows that 5 μM is added, and (c) shows that 10 μM is added. It is a micrograph (magnification: 250) which shows the observation result of the cell by the confocal laser scanning microscope regarding the verification result of the inhibitory effect of ramnadin on the intracellular transport of melanosomes. (A) shows that ramnazin is not added, and (b) shows that 10 μM is added. It is a graph which shows the result of having calculated the ratio of the cell which aggregates melanosomes in the vicinity of a nucleus based on the observation result by the confocal laser microscope. It is a graph which shows the verification result of the inhibitory effect on the extracellular excretion of melanin pigment of ramnazin. It is a graph which shows the verification result about the inhibitory effect on the expression of the melanin synthesis-related protein of ramnazin. It is a graph which shows the verification result about the synergistic effect by the combined use of ramnazin and other drugs.

Hereinafter, embodiments for carrying out the present invention will be described in more detail.
First Melanosome Transport Inhibitor As described above, the present inventors have found that rhamnazin, which is classified as a flavonol, effectively inhibits melanosome transport. Based on this finding, the present inventors proceeded with the study and verified that rhamnazin has a high whitening effect.

ラムナジンは、天然物由来のものとしては、ラムナス・ペティオラリス（Ｒａｍｎｕｓ ｐｅｔｉｏｌａｒｉｓ）やラムナス・サクサティリス（Ｒａｍｎｕｓ ｓａｘａｔｉｌｉｓ）などラムナス（Ｒａｍｎｕｓ）属（別名：クロウメモドキ属）の植物由来であることが好適である。ラムナス・ペティオラリスおよびラムナス・サクサティリスは、スリランカ及びヨーロッパ固有のクロウメモドキ属植物である。特にラムナス・サクサティリスはラムナジンを高含有し、より少量の抽出物から多くのラムナジンを得ることができる点で優れている。 The first melanosome transport inhibitor according to the present invention contains rhamnazin represented by the following chemical formula (I) as an active ingredient.

Rhamnazin is preferably derived from plants of the genus Rhamnus (also known as Buckthorn), such as Rhamnus petiolaris and Ramnus saxatilis, as natural products. .. Rhamnus petiolaris and Rhamnus saxatilis are buckthorn plants endemic to Sri Lanka and Europe. In particular, Rhamnazin saxatilis is excellent in that it contains a high content of Rhamnazin and a large amount of Rhamnazin can be obtained from a smaller amount of extract.

The first melanosome transport inhibitor according to the present invention finely powders whole plants, roots, rhizomes, leaves, seeds, fruits, and flowers of plants belonging to the genus Ramnus, such as Rhamnus petiolaris or Rhamnus saxatilis. Alternatively, it can be prepared as crushed.
The first melanosomes transport inhibitor according to the present invention is an extract obtained by dipping and extracting such fine powder or crushed material with water or an organic solvent and filtering out the residue, and the solvent removed from this extract ( It can also be prepared as an 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 solvent-removed product with an appropriate solvent or the like.

As the extraction solvent for extraction 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. This is because such an extraction solvent can obtain a desired extract.

The ramnazine contained in the first melanosomes transport inhibitor according to the present invention is purified by a chromatography method such as silica gel column or HPLC by removing solids by filtration or centrifugation in addition to or as an alternative to extraction with an organic solvent. It can be purified through the process.

Rhamnazin can also be obtained by utilizing an organic synthetic reaction. Rhamnazin (synthetic rhamnazin) obtained by such various synthesis can also be contained as an active ingredient of the melanosome transport inhibitor according to the present invention.

Regarding the melanosome transport inhibitor of the present invention, "containing ramnazine as an active ingredient" means that the melanosome transport inhibitor is composed of only the active ingredient ramnazine alone, the melanosome transport inhibitor alone, and the case where it contains trace impurities. In the case of fine powder or crushed whole plant, root, rhizome, leaf, seed, fruit, flower as described above, the fine powder or crushed product is immersed and extracted with water or an organic solvent, and the residue is filtered out. In the case of the obtained extract, in the case of the extract from which the solvent has been removed (extracted extract), the fine powder and the solvent-removed product are further dissolved, dispersed, and diluted with an appropriate solvent or the like to form a solution. It may be any of the cases prepared as the form of.

When the melanosome transport inhibitor of the present invention (including the second melanosome transport inhibitor described later) is prepared in the form of a solution, it is preferable to contain ramnazine in a proportion of 0.0001% by mass to 5% by mass. ..

Second Melanosome Transport Inhibitor As described above, the present inventors have conceived that a hydrolyzate of a naturally derived product such as a specific plant rich in rhamnazin has a high whitening effect.

The second melanosome transport inhibitor according to the present invention also contains rhamnazin represented by the chemical formula (I) as an active ingredient.
The second melanosomes transport inhibitor includes the genus Viscum, including the Dutch garashi (Nasturitum office), the genus Ramnus, including Ramnus saxilis, and the genus Viscum, including Viscum colorum. It is preferably a hydrolyzate of a naturally derived product derived from a plant of the genus Viscum, the genus Viscum including Nerviria fordii, or the genus Viscum including cloves (Syzygium aromaticum).

The second melanosome transport inhibitor can be obtained by hydrolyzing an extract extracted from a plant as a raw material by solvent extraction or the like.
For example, the above-ground part of a plant such as the Dutch sardine extract is lyophilized by immersing it in a solvent such as methanol to obtain an extract, then the extract is concentrated, and the obtained concentrate is column-purified to obtain methanol. An operation including a step of acid-hydrolyzing the fraction obtained by elution with a solvent such as the above is performed. This makes it possible to prepare a second melanosome transport inhibitor as a hydrolyzed solution of watercress or the like.
As described above, the first melanosome transport inhibitor includes the case where it is prepared in the form of an extract extracted from a plant as a raw material by solvent extraction or the like. The second melanosome transport inhibitor can be obtained by hydrolyzing such an extract, or more precisely, hydrolyzing the flavonoid glycosides contained in such an extract.

Furthermore, the present inventors investigated the mechanism of the high whitening effect of rhamnazin, and found that the addition of rhamnazin reduced the amount of Mlp protein associated with melanosomal transport. I came up with the idea of producing a strong whitening effect. The Mlp protein is a protein involved in melanosome transport in melanocytes. When the transport of melanosomes is inhibited, the transfer of melanin produced by melanocytes to keratinocytes is inhibited, so that skin pigmentation can be suppressed.

On the other hand, the present inventors have confirmed that conventional whitening agents, kojic acid, and arbutin do not have a melanosome transport inhibitory effect.
Therefore, the effect of suppressing skin pigmentation based on the melanosome transport inhibitor of the present invention is different from the whitening effect (effect of suppressing melanin synthesis) exerted by conventional whitening cosmetics containing kojic acid, arbutin, etc. It is different in the introduction.

By using the melanosome transport inhibitor of the present invention containing ramnazine in combination with another whitening agent that does not have a melanosome transport inhibitory effect, a synergistic effect that further enhances the whitening effect as compared with the use of the other whitening agent alone can be obtained. Can play.
The whitening agent that does not have the melanosome transport inhibitory action is preferably a tyrosinase activity inhibitor that inhibits melanin synthesis. Most of the commonly used whitening agents are tyrosinase activity inhibitors. Even if a whitening agent having the same function based on the same mechanism of action, for example, a tyrosinase activity inhibitor, is blended in a plurality or a large amount, the whitening effect is unlikely to be enhanced, and there is concern about the safety of the effect. However, as described above, the mechanism of action is different, for example, by combining a whitening agent that acts at different stages and different parts of the skin pigmentation process, that is, a tyrosinase activity inhibitor and a melanosomes transport inhibitor, a small amount of each By using them one by one, it is possible to obtain a high whitening effect and also provide excellent safety.

The tyrosinase activity inhibitor is not particularly limited, and includes ascorbic acid, ascorbic acid salt and its derivatives, hydroquinone, hydroquinone salts and glycosides, placenta extract, arbutin, ellagic acid, linoleic acid, tranexamic acid and the like. Can be mentioned.

External preparation for skin The external preparation for skin according to the present invention contains the first or second melanosome transport inhibitor. The external preparation for skin according to the present invention preferably contains 0.0001 to 5% by mass of ramnazin with respect to the total amount of the external preparation for skin. This is because if it is contained at least 0.0001% by mass, it corresponds to a concentration at which the melanosome transport inhibitory effect can be obtained to a minimum, and if it is 5% by mass or less, the melanosome transport inhibitory effect can be obtained and safety can be obtained. Because it is possible.

The melanosome transport inhibitor of the present invention can be used as a skin external preparation applied to the exodermis such as cosmetics, pharmaceuticals, and quasi-drugs. Therefore, the dosage forms are water-soluble (lotion-based), solubilized-based, emulsified-based (W / O-type, O / W-type), powder-based, ointment-based, oil-liquid-based, gel-based, and water-oil two-layer. It can be used in a wide range of forms such as lotion, water-oil-powder three-layer system. In addition to the melanosomes transport inhibitor, the external preparation for skin of the present invention includes other components usually used for external preparation for skin, such as water, alcohol, oil, and surfactant, as long as the effects of the present invention are not impaired. Agents, thickeners, powders, chelating agents, preservatives, pH adjusters, various medicinal agents, animals and plants, extracts derived from microorganisms, pigments, fragrances and the like can be appropriately blended as needed. Further, by using a medicinal ingredient such as an antioxidant, a cell activator, an anti-inflammatory agent, and an ultraviolet inhibitor in combination, the effect of the present invention can be further enhanced or other effects can be further added.

Examples of the dosage form of the external preparation for skin of the present invention include lotions, oils, emulsions, creams, ointments and the like. It can also be provided as various forms of cosmetics such as milky lotions, creams, lotions, beauty essences, sheet masks, cleaning agents, and makeup cosmetics.

The external preparation for skin according to the present invention can further contain a whitening agent having no melanosome transport inhibitory effect. As such a whitening agent, a tyrosinase activity inhibitor as described above can be used. A skin external preparation containing a melanosome transport inhibitor containing ramnazine and another whitening agent having no melanosome transport inhibitory action has a whitening effect more than a skin external preparation containing only the other whitening agent. A synergistic effect that further enhances can be achieved.

As described above, in the external preparation for skin of the present invention, ramnazin acts as an active ingredient of the melanosomes transport inhibitor, and is particularly effective as a whitening cosmetic for the purpose of skin beautification and the like.

Hereinafter, the present invention will be specifically described with reference to Examples, Formulation Examples, etc., but the present invention is not limited thereto.

Example 1
The above-ground part of watercress (watercress) was lyophilized and immersed in methanol and shaken overnight at room temperature to obtain an extract. Then, the extract was concentrated with an evaporator. The obtained concentrate was purified on an HP-20 column, and a fraction having a high flavonoid content was obtained by elution with methanol. After concentrating the fraction with an evaporator, acid hydrolysis was carried out by adding hydrochloric acid of 6N and heating at 100 ° C. for 90 minutes. After the reaction, the mixture was neutralized with sodium hydroxide and extracted with ethyl acetate. The obtained extract was concentrated with an evaporator and then redissolved in methanol to prepare a solution obtained as a hydrolyzed solution of watercress extract.
On the other hand, the fraction was concentrated with an evaporator, and then acid hydrolysis was not carried out, and other operations were carried out in the same manner to prepare a watercress extract.
Mouse B16 melanoma cells were precultured in D-MEM medium containing 10% fetal bovine serum (FBS) for 24 hours. After pre-culture, the Dutch glass extract obtained in Example 1 (before hydrolysis) or the hydrolyzed solution of Dutch glass extract (after hydrolysis) is added so that the final concentration becomes a predetermined concentration, and further 72 Incubated for hours. After completion of the culture, the cell morphology was observed with a phase-contrast microscope.
The results are shown in FIG. The watercress extract (before hydrolysis treatment) showed inhibition of melanosome transport at a concentration of 5.0%. From this result, it was clarified that the watercress extract has an inhibitory effect on the transport of melanosomes. It was also clarified that the same effect can be obtained at a concentration of 0.5% in the hydrolyzed solution of the watercress extract. From this result, it was shown that the same effect was amplified by hydrolyzing the watercress extract.
Watercress is known to contain flavonol glycosides such as ramnazin, ramnetin, isorhamnetin, kaempferol, and quercetin (Non-Patent Document 3). In addition, since the hydrolysis treatment provides a stronger inhibitory effect, we recalled the possibility that the flavonol glycoside contained in the watercress extract was converted to aglycone by hydrolysis.
In fact, FIG. 2 shows the distribution of flavonol glycosides and aglycones. It is understood that hydrolysis increases the amount of flavonols aglycones.

Example 2
(Evaluation 1: Verification of the inhibitory effect of various flavonols on the expression of Mlp protein)
The Mlp protein is known as a protein that plays a central role in the intracellular transport of melanosomes. It was recalled that the hydrolyzed solution obtained in Example 1 contained aglycones of rhamnazin, rhamnetin, isorhamnetin, kaempferol, and quercetin as main components. Therefore, in order to investigate the effect of these flavonols on the expression of Mlp in pigment cells, a Western blotting method was performed using an anti-Mlf antibody. For various flavonols, a commercially available refined product was dissolved in dimethyl sulfoxide and used.

After pre-culturing mouse B16 melanoma cells in D-MEM medium containing 10% FBS for 24 hours, the final concentrations of various flavonols are rhamnazin 10 μM, ramnocitrin 25 μM, rhamnetin 5 μM, isorhamnetin 50 μM, kaempferol 20 μM, and quercetin 50 μM, respectively. Was added and cultured. In setting the final concentrations of various flavonols, a cytotoxicity test of each flavonol was conducted in advance, and the maximum concentration at which the cell viability was 80% or more was adopted. After culturing for 3 days, cells were washed 3 times with PBS and with buffer for cell lysis (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and protease inhibitor). It was dissolved. Samples diluted with SDS-PAGE sample buffer were boiled for 5 minutes. Samples were separated by 10% SDS-PAGE, transferred to a Polyvinylidene fluoride membrane (PVDF membrane) using a semi-drive rotor (manufactured by Bio-Rad), and then at room temperature for 1 hour in a phosphate buffer solution of 5% skim milk. Blocking was performed at. PVDF membranes were washed 3 times with TBS-T and then incubated with anti-Mlp antibody overnight at 4 ° C. The PVDF membrane was washed 3 times with TBS-T and then incubated with a secondary antibody labeled with Horseradish peroxidase for an additional hour. It was washed 3 times with TBS-T before detection. Detection was performed using SuperSignal West Femto Maximum Sensitivity Substrate (manufactured by Thermo Scientific) and LAS-3000. The quantitative evaluation of the Mlf protein was performed by comparing the concentration of the detected band with the band of the unadded sample, and the effect of various flavonoids on the expression of the Mlf protein was evaluated. In addition, Glyceraldehyde-3-phosphate dehydogenase (Gapdh) was similarly detected as a standard protein.

The results are shown in FIG. Flavonols other than rhamnazin had little effect on the expression of the Mlp protein, whereas rhamnazin significantly reduced the expression of the Mlp protein. From this result, it was shown that Rhamnazin has an effect of suppressing the expression of Mlp protein in pigment cells, and it was clarified that it is an active ingredient of the hydrolyzate of watercress extract.

Example 3
(Evaluation 2: Verification of the cytotoxicity of rhamnazin)
To evaluate the toxicity of rhamnazin to pigment cells, cytotoxicity tests were performed using Cell Counting Kit-8 manufactured by Dojin Chemical Co., Ltd.

Mouse B16 melanoma cells were pre-cultured in D-MEM medium containing 10% FBS for 24 hours, then rhamnazin was added so that the final concentrations were 5 and 10 μM, respectively, and the cells were cultured for another 3 days. After completion of the culture, the cell viability was calculated according to the accompanying manual using Cell Counting Kit-8 manufactured by Dojin Chemical Co., Ltd.

The results are shown in FIG. Almost no decrease was observed at the addition concentration of rhamnazin at 5 and 10 μM. From this result, it was clarified that rhamnazin cleared the cytotoxicity standard of 80% or more at a concentration of 10 μM or less, and it was shown that there was no cytotoxicity within the same concentration range.

Example 4
(Evaluation 3: Verification of the inhibitory effect of rhamnazin on intracellular transport of melanosomes)
From the result of evaluation 1, it was clarified that rhamnazin suppresses the expression of Mlp protein in pigment cells. Since this protein is a protein that plays a central role in the intracellular transport of melanosomes produced in pigment cells, it is possible that ramnazine has the effect of inhibiting the intracellular transport of melanosomes. .. Therefore, in order to verify whether ramnazine has an inhibitory effect on intracellular transport of melanosomes in pigment cells, cell morphology was observed with a phase-contrast microscope and a confocal laser microscope, and melanosomes aggregated near the nucleus. The proportion of cells in the cell was calculated.

Mouse B16 melanoma cells were seeded in a culture dish containing a collagen-coated cover glass in advance, and pre-cultured with D-MEM for 24 hours. After pre-culture, rhamnazin was added so that the final concentration became a predetermined concentration, and the cells were further cultured for 72 hours. After completion of the culture, the cell morphology was observed with a phase-contrast microscope. The recovered cover glass was placed in a phosphate buffer solution of 4% paraformaldehyde and left at room temperature for 15 minutes to fix cells. The cover glass was washed with PBS three times, then placed in PBS containing a concentration of 0.1% Triton X-100, and allowed to stand at room temperature for 10 minutes for permeation treatment. After washing 3 times with PBS, the cells were placed in PBS containing 3% bovine serum albumin and left at room temperature for 1 hour for blocking. After washing 3 times with PBS to stain actin, which is a cytoskeleton, the cells were placed in PBS containing 0.01% Alexafluor 594-labeled phalloidin and left for 20 minutes. The nuclei were then washed 3 times with PBS and then placed in PBS containing 2 μg / ml 4', 6-diamidino-2-phenylindole (DAPI) and left for 3 minutes. After washing with PBS three times, encapsulation was performed using a fluoromount (manufactured by Diagnostics). After drying, the cell morphology was observed with a confocal laser scanning microscope (TCS SP2 AOBS, manufactured by Leica Microsystems). The proportion of cells in which melanosomes are aggregated near the nucleus is counted in the DAPI-stained nucleus as an index of the total number of cells, and the phalloidin-stained actin is regarded as the whole cell image, and the melanosomes are biased toward the cell nucleus. It was calculated by counting the number of cells. The total number of cells counted in one sample was about 100 cells.

The observation results by the phase contrast microscope are shown in FIGS. 5 (a) to 5 (c). In the image, melanosomes are observed as black spots. Melanosomes were uniformly distributed to the terminal in untreated cells, whereas aggregation of melanosomes near the nucleus was observed in cells to which rhamnazin was added (arrow part).

The observation results by the confocal laser scanning microscope are shown in FIGS. 6 (a) and 6 (b). In addition, FIG. 7 shows the result of calculating the ratio of cells in which melanosomes are aggregated in the vicinity of the nucleus based on the observation results by this confocal laser scanning microscope.
Rhamnazin-supplemented cells showed marked aggregation of melanosomes near the nucleus, as compared to untreated cells, as observed with a phase-contrast microscope. The proportion of cells in which melanosomes were aggregated in the vicinity of the cell nucleus was about 1% in untreated cells, whereas it was about 80% in ramnazin-treated cells. From the above results, it was shown that rhamnazin has an inhibitory effect on the intracellular transport of melanosomes.

Example 5
(Evaluation 4: Verification of the inhibitory effect of rhamnazin on the extracellular excretion of melanin pigment)
From the results of Evaluation 3, it was clarified that rhamnazin has an effect of inhibiting the intracellular transport of melanosomes. It is considered that this effect may lead to suppression of the excretion of melanin pigment to the outside of pigment cells. Therefore, in order to verify whether or not ramnadin has an inhibitory effect on the extracellular excretion of melanin pigment, the amount of extracellular melanin during culture of pigment cells was quantified.

Mouse B16 melanoma cells were precultured in D-MEM medium containing 10% FBS for 24 hours. After culturing, the medium was replaced with a D-MEM medium containing 10% FBS and 50 μM forskolin, rhamnazin was added to a final concentration of 10 μM, and the cells were cultured for 3 days. After culturing, the medium was collected and measured with an Arvo-MX1420 multi-label counter (manufactured by PerkinElmer) at an absorption wavelength specific to black melanin at 405 nm to evaluate the amount of melanin in the medium. The amount of extracellular melanin was calculated assuming that the measured value of the cell culture medium to which ramnazin was not added was 100%. In addition, a cell medium similarly cultured in a D-MEM medium containing no forskolin was used as a negative control.

The results are shown in FIG. Rhamnazin reduced the amount of extracellular melanin by about 85%. From this result, it was shown that rhamnazin has an inhibitory effect on the extracellular excretion of melanin pigment.

Example 6
(Evaluation 5: Verification of the inhibitory effect of rhamnazin on the expression of melanin synthesis-related proteins)
From the results of evaluations 1, 3 and 4, it was clarified that rhamnazin has an inhibitory effect on the intracellular transport of melanosomes produced in pigment cells. On the other hand, regarding the effect of suppressing the extracellular excretion of melanin pigment of ramnadin shown in Evaluation 4, melanin synthesis in pigment cells is one of the factors that brought about the effect, in addition to the factor due to the inhibitory effect on intracellular transport of melanosomes. It was considered possible that this was due to the suppression of. Therefore, in order to verify whether or not rhamnazin has an inhibitory effect on melanin synthesis in pigment cells, Western blotting was performed using a specific antibody against microphthalmia-associated transcription factor (Mitf), which is a kind of melanin synthesis-related protein. Made the law.

Mouse B16 melanoma cells were precultured in D-MEM medium containing 10% FBS for 24 hours. After culturing, the medium was replaced with a D-MEM medium containing 10% FBS and 50 μM forskolin, rhamnazin was added to a final concentration of 10 μM, and the cells were 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 FIG. Rhamnazin did not suppress forskolin-induced expression of Mitf at all. As a result of the same evaluation of the expression of Mlp, the expression of Mlp was not affected by the presence or absence of forskolin. On the other hand, the inhibitory effect of rhamnazin on the expression of Mlp was remarkable. From these results, it was shown that rhamnazin does not suppress the expression of melanin synthesis-related protein, but specifically suppresses the transport-related protein of melanosomes.

Example 7
(Evaluation 6: Verification of synergistic effect by combined use with other drugs)
From the results so far, rhamnazin specifically suppresses the expression of Mlp, which is a melanin synthesis-related protein, but does not suppress the expression of the melanin synthesis-related protein. As mentioned above, extracellular melanin excretion may also be suppressed by a mechanism that suppresses the expression of melanin synthesis-related proteins. Therefore, the combined use of a known whitening agent characterized by suppressing the expression of melanin synthesis-related proteins and rhamnazin can be expected to have a synergistic effect on the excretion of extracellular melanin. Therefore, we examined the presence or absence of an inhibitory effect on extracellular melanin excretion when used in combination with arbutin, which is already known to have an effect of inhibiting the enzymatic activity of tyrosinase, which is one of the melanin synthases.

Mouse B16 melanoma cells were precultured in D-MEM medium containing 10% FBS for 24 hours. After culturing, the medium was replaced with a D-MEM medium containing 10% FBS and 50 μM forskolin, and ramnazin having a final concentration of 5 or 10 μM, 50 μM arbutin, 5 μM ramnazin, and 50 μM arbutin were added and cultured for 3 days. .. Subsequent evaluation of the amount of melanin in the medium proceeded in the same manner as in Evaluation 4. The amount of extracellular melanin was calculated assuming that the measured value of the cell culture medium to which neither rhamnazin nor arbutin was added was 100%.

The results are shown in FIG. The addition of arbutin alone reduced the amount of extracellular melanin to approximately 84% as compared to the non-addition. The decrease when ramnazin was added alone at a concentration of 5 or 10 μM was about 41 and 20%, respectively. In addition, the decrease when both arbutin and rhamnazin (5 μM) were added was 21%, which was a result of the decrease as compared with the case where each was added alone. From this result, it was suggested that the combined use of rhamnazin and a known whitening agent synergistically suppresses extracellular melanin excretion, and it was shown that a means for suppressing skin pigmentation can be provided more efficiently than before. ..

Pharmaceutical example 1 : Toner The following components (3), (4) and (8) to (11) were mixed and dissolved (mixture A). Then, the following components (1), (2), (5) to (7) and (12) were mixed and dissolved (mixture B). The obtained mixture A and the mixture B were uniformly mixed to obtain a lotion.
(Composition) (%)
(1) Glycerin 7.0
(2) 1,3-butylene glycol 6.5
(3) Polyoxyethylene sorbitan monolauric acid ester (20EO) 1.2
(4) Ethanol 5.0
(5) Lactic acid 0.05
(6) Sodium lactate 0.1
(7) Collagen 1.0
(8) Hydrolyzed product of watercress extract 0.05
(9) Ascorbic acid 2-glucoside 0.5
(10) Preservative 0.1
(11) Fragrance 0.1
(12) Remaining amount of purified water

Pharmaceutical example 2 : Emulsion The following component (10) was heated and held at 70 ° C. (solution A). Next, the following components (1) to (9) and (11) were heated and mixed and held at 70 ° C. (mixture B). Solution A was added to the obtained mixture B, mixed, and uniformly emulsified (mixture C). After cooling the obtained mixture C, the following components (12) to (15) were added and mixed uniformly to obtain a milky lotion.
(Composition) (%)
(1) Polyoxyethylene sorbitan monostearate (10EO) 1.0
(2) Polyoxyethylene sorbitol tetraoleate (60EO) 0.5
(3) Glyceryl monostearate 1.0
(4) Stearic acid 0.5
(5) Behenyl alcohol 0.5
(6) Squalene 8.0
(7) Ethanol 5.0
(8) Hydrolyzate of watercress extract 0.001
(9) Arbutin 1.0
(10) Remaining amount of purified water (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 held at 70 ° C. (solution A). Next, the following components (1) to (8) were heated and mixed and held at 70 ° C. (mixture B). Solution A was added to the obtained mixture B, mixed, and uniformly emulsified (mixture C). After cooling the obtained mixture C, the following components (9) to (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
(20EO)
(8) Preservative 0.1
(9) Fragrance 0.15
(10) Hydrolyzate of watercress extract 1.0
(11) Remaining amount of purified water

Pharmaceutical example 4 : Liquid foundation The following components (1) to (7) were mixed and dissolved (solution A). The following components (13) to (18) were added to this solution A, mixed uniformly, and held at 70 ° C. (mixture B). Then, the following components (8) to (12) were uniformly dissolved and held at 70 ° C. (mixture C). Mixture B was added to the obtained mixture C and uniformly emulsified (mixture D). After cooling the obtained mixture D, the following components (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) Cetanol 1.0
(5) Self-emulsifying glycerin monostearate 1.0
(6) Paramethoxycinnamate -2-ethylhexyl 8.0
(7) Preservative 0.1
(8) Glycerin 5.0
(9) Triethanolamine 1.0
(10) Carboxymethyl cellulose 0.2
(11) Bentonite 0.5
(12) Remaining amount of purified water (13) Titanium oxide 6.0
(14) Fine particle titanium oxide 2.0
(15) Fine particle zinc oxide 5.0
(16) Mica 2.0
(17) Talc 4.0
(18) Color pigment 4.0
(19) Hydrolyzate of watercress extract 0.001
(20) Fragrance 0.1

Pharmaceutical example 5 : Sunscreen lotion The following components (1) to (11) were mixed and dispersed (dispersion liquid A). Next, the following components (12) to (15) were mixed and dispersed (dispersion liquid B). The 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) Paramethoxycinnamate -2-ethylhexyl 10.0
(6) Preservative 0.1
(7) Fragrance 0.1
(8) Silicone-treated fine particle titanium oxide 8.0
(9) Silicone-treated fine particle zinc oxide 7.0
(10) Polystyrene powder 3.0
(11) Trimethylsiloxysilicic acid 0.5
(12) Dipropylene glycol 3.0
(13) Ethanol 10.0
(14) Remaining amount of purified water (15) Salt 0.2
(16) Hydrolyzate of watercress extract 0.02
(17) Phosphate-L-ascorbic acid
Magnesium 3.0

When the cosmetics of Pharmaceutical Examples 1 to 5 were applied to the skin, a high whitening effect was confirmed.

Claims (7)

A melanosome transport inhibitor characterized by containing rhamnazin represented by the following chemical formula (I) as an active ingredient.

The melanosome transport inhibitor according to claim 1, which is derived from one plant selected from the group consisting of plants of the genus Rhamnous, such as Rhamnus petiolaris and Rhamnus saxatilis. Viscum (Viscum), including Viscum (Viscum), Viscum (Viscum), Viscum (Viscum), Viscum (Viscum), Viscum (Viscum), Viscum (Viscum), Viscum (Viscum), Viscum (Viscum), Viscum (Viscum) The first aspect of claim 1, which is a hydrolyzate of a naturally derived product derived from a plant or the like selected from the group consisting of plants of the genus Viscum including Syzygium and plants of the genus Viscum including clove (Syzygium aromaticum). Melanosome transport inhibitor. An external preparation for skin, which comprises the melanosomes transport inhibitor according to any one of claims 1 to 3. The external preparation for skin according to claim 4, further containing a whitening agent having no melanosome transport inhibitory action. The external preparation for skin according to claim 5, wherein the whitening agent is a tyrosinase activity inhibitor. The tyrosinase activity inhibitor is selected from the group consisting of ascorbic acid, ascorbic acid salt and its derivatives, hydroquinone, hydroquinone salts and glycosides, placenta extract, arbutin, ellagic acid, linoleic acid, or tranexamic acid. The skin external preparation according to claim 6.

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