JP2019218268A - Melanin decomposition accelerator and skin-whitening cosmetics - Google Patents

Abstract

To provide a novel, useful melanin decomposition accelerator, and skin-whitening cosmetics useful for quickly relaxing and improving various pigmentary disorders including skin blotches and freckles.SOLUTION: Extract from leaves of Entada Phaseoloides, a Fabaceae evergreen plant, is used.SELECTED DRAWING: None

Description

The present invention relates to a melanin decomposition promoter useful for rapidly improving pigmentation caused by various factors such as ultraviolet rays and aging, and a whitening cosmetic containing the melanin decomposition promoter.

In areas that are routinely exposed to ultraviolet light, such as the face and arms, local pigmentation, so-called spots, form with age. This pigmentation is thought to be caused by excessive production of melanin due to activation of melanocytes present in the epidermis due to long-term exposure to ultraviolet light, resulting in an increase in the amount of melanin in the skin. (Non-Patent Document 1).

Therefore, as components that can be expected to improve stains, components that inhibit enzyme activities involved in melanin biosynthesis in melanocytes (Patent Document 1), components that inhibit signal transducers such as endothelin and prostaglandin that activate melanocytes, etc. Have been developed (Patent Documents 2 and 3).

It is clear that the use of cosmetics containing these components on a daily basis can prevent the formation of stains, but because a large amount of melanin is accumulated in the epidermis of the already formed stain site, In order to improve stains quickly, it is important to remove accumulated melanin in addition to the above-mentioned components having a melanin synthesis inhibitory effect.

As a method of removing melanin, a method of promoting epidermal replacement, that is, a method of promoting turnover has been proposed (Patent Document 4). However, since excessive melanin present in cells causes a decrease in cell division ability, intracellular It is difficult to exert a sufficient effect unless the amount of melanin present in the yeast is reduced.

In addition, a method of peeling the epidermis by a chemical method (chemical peeling) is also useful for improving pigment spots, and glycolic acid and salicylic acid are known. (See Non-Patent Document 2).

On the other hand, it is known that melanin accumulated in the epidermis of the stain portion is decomposed by the action of an enzyme in epidermal cells. For this reason, Patent Document 5 proposes a melanin decomposition accelerator using a boxweed extract, but almost no component having such an effect has been found.

As for the Modama leaf extract, an antioxidant effect (Patent Document 6) and a hair papilla cell activating effect (Patent Document 7) are known. However, the melanin decomposition promoting action of the modama leaf extract was not known at all.

Against this background, effective and highly safe melanin degradation promoters capable of decomposing melanin present in the epidermis of the spot area are strongly demanded in order to quickly improve pigmentation of spots and freckles. I was

JP-A-6-305978 JP-A-11-109766 JP 2006-143676 A JP 2008-247783 A JP-A-2015-51931 JP-A-11-5975 JP 2000128741 A

Moisturizing / Whitening / Anti-wrinkle / Antioxidant Evaluation / Experimental Method Manual February 1, 2012, Fragrance Journal Journal of the Japanese Dermatological Association 118 (3) 347-355, 2008

An object of the present invention is to provide a novel and useful melanin decomposition promoter and a whitening cosmetic which is useful for promptly alleviating and improving various pigmentation symptoms such as spots and freckles.

  As a result of intensive studies by the present inventors, the above problem was solved by using a modama leaf extract.

  According to the present invention, a new approach to whitening using a modama leaf extract can be provided. In addition, a novel melanin decomposition promoter can be provided. Further, by combining the modama leaf extract found in the present invention with a conventionally used component that suppresses the synthesis of melanin, it is possible to quickly improve already formed spots.

Modama (algae, scientific name: Entada Phaseoloides) used in the present invention is an evergreen plant of the legume family, widely distributed in Southeast Asia, and known as a plant that produces the largest beans in the world. It is a plant that is cultivated and used as a folk medicine (jam) on Java Island in Indonesia.

The melanin degradation promoter in epidermal cells of the present invention can be used in any of external and internal methods, but is preferably in the form of a skin external preparation. The dosage form can be arbitrarily selected according to the purpose, and can be cream, ointment, emulsion, lotion, solution, gel, pack, powder, stick, and the like. In addition, the melanin degradation promoter for epidermal cells of the present invention can be used as cosmetics, quasi-drugs, pharmaceuticals, and the like.

The extraction site of the Modama extract used in the present invention is a leaf, and the extraction solvent is not particularly limited. For example, water, methanol, ethanol, lower alcohols such as propyl alcohol, or polyhydric alcohols such as propylene glycol, 1,3-butylene glycol and the like, and in particular, water, ethanol and 1,3-butylene glycol It is preferable to select one kind or two or more kinds.

The extraction method of the modama leaf extract used in the present invention is not particularly limited. For example, using a mixed solution of 1 to 100 parts by mass of water and 1,3 butylene glycol or ethanol with respect to 1 part by mass of a dried plant, at a temperature of 5 to 70 ° C., preferably 10 to 60 ° C., and 1 to 7 parts It is preferable to extract for 3 days, especially 3 to 4 days. After the extraction, it is filtered and can be used as it is. However, if necessary, further purification treatment such as deodorization and decolorization can be performed within a range that does not affect the effect. Furthermore, it can be used in a powder state by freeze-drying or the like.

Examples of the present invention will be specifically described below, but the present invention is not limited to these examples. Unless otherwise specified, the amounts are shown in mass%.

[Test 1] Melanin degradation promoting action confirmation test The present inventors evaluated the melanin degradation promoting action in human epidermis-derived cells.
(Preparation of sample)
Modama leaves and perilla leaves, Melinjo, known as Indonesian native plants as comparative controls, and perilla leaves, which are known to have a high antioxidant effect and a melanin production-suppressing effect, were dried to obtain a dry substance.
The mixture is mixed at a mass ratio of the dry substance 1: extraction solvent 10 and the extraction solvent is extracted at room temperature for 1 week when the extraction solvent is 50% aqueous ethanol, and at 60 ° C. for 5 hours when the extraction solvent is water. An extraction was performed. After extraction, a solid was obtained by freeze-drying.

(Preparation of melanin solution)
Human-derived melanoma cells HM3KO were cultured in a 37 ° C. incubator under 5% CO ₂ using a D-MEM medium containing 5% FBS (Gibco manufactured by Invitrogen).
The cells that have become nearly confluent and have increased melanin production are collected in an Eppendorf tube using trypsin to a concentration of 7.0 × 10 ⁶ cells / tube, and centrifuged (1000 g, 4 ° C., 3 minutes) to pellet the cells. It was created. Thereafter, the pellet was washed with PBS (-).

 To the cell pellet, 1 mL of cold lysis buffer (1% octylphenoxy poly (ethylenoxy) ethanol (IGEPAL CA-630), 0.1 M Tris-HCl solution (pH 7.5) containing 0.01% SDS was added. This dispersion was centrifuged (1000 g, 4 ° C., 3 minutes) to precipitate unwanted substances, and the melanosome-containing supernatant was collected while stirring at room temperature for 20 minutes with stirring every 10 minutes. The supernatant was centrifuged again (1000 g, 4 ° C., 3 minutes) and the supernatant was collected, and the supernatant was centrifuged (20000 g, 4 ° C., 3 minutes), and the obtained precipitate was separated into a melanosome-rich fraction. The supernatant was removed by suction, and the melanosome pellet was washed twice with PBS (-) (20,000 g, ° C., 3 min) which in PBS (-.) Was added and the suspension was melanin solution was dispersed melanosomes by pipetting 50 times.

(Cell culture)
The human epidermal cell line (HaCaT) is treated with trypsin, the cells are dispersed in a D-MEM (Invitrogen) medium containing 10% fetal bovine serum, and the cells are dispersed in a 48-well plate so as to be 1 × 10 ⁴ cells / well. And cultured at 37 ° C. for 1 day. After culturing for one day, the melanin solution prepared by the method described above was added.
In order to incorporate melanin into the human epidermal cell line, the cells were further cultured for one day. afterwards,
By removing the medium and washing with PBS (-), melanin that was not taken up by the cells was removed. Thereto, a D-MEM medium containing 2% fetal calf serum and a sample were added.
After the addition, the cells were cultured for 4 days. Thereafter, the medium was removed, the cells were washed with PBS (-), and treated with mild form (Wako) for 10 minutes to fix the cells. After fixation, the fixative was completely removed by washing with purified water, and Fontana ammonia silver solution (Mutoh Chemical) was added (fontana-Masson stain, melanin stain). After overnight (18 hours) treatment at normal temperature, the ammonia silver fontana solution was removed, and the mixture was washed with purified water. A solution of Triton X-100 (Wako) diluted 1000-fold with PBS (-) was added, and the mixture was treated for 15 minutes and then washed with PBS (-). DAPI (DOJINDO) was diluted 1000-fold with PBS (−), added in 400 μL aliquots, and allowed to react at 37 ° C. for 45 minutes to stain cell nuclei. After washing, an image of the cells was obtained with a microscope (Keyence, magnification: 10).

(Quantification of residual amount of melanin)
The obtained image was binarized by ImageJ (open source) to select a melanin existing part. The amount of melanin in the image was calculated by obtaining the total area of the melanin existing part. Similarly, the number of cells in the image was calculated using ImageJ. From these results, the amount of melanin per cell was determined.

Assuming that the amount of melanin per cell when purified water is added is 100, the amount of melanin when a modama leaf extract and a plant extract to be compared are added is shown in the following table. The amount of melanin per cell was calculated from the formula 100 × (melanin amount of sample / number of cells) / (melanin amount of purified water / number of cells).

[Test 2] DPPH test [Preparation of DPPH solution]
The DPPH solution was prepared by mixing the following components at a ratio of (A) :( B) :( C) = 1: 4: 3 by volume.
(A) 5.52 g of ES (2- (N-morpholino) ethanesulfonic acid) was dissolved in 100 mL of water, and adjusted to pH 6.1 with 1N-NaOH.
(B) 15.7 mg of DPPH (1,1-diphenyl-2-picrylhydrazyl) was dissolved in 100 ml of ethanol.
(C) Purified water [Preparation of Trolox solution]
25 mg of Trolox was dissolved in 10 ml of DMSO (dimethyl sulfoxide) to prepare a 10 mM solution.
[DPPH test]
For the measurement, 10 μL of a 12.5-fold diluted sample was added to 1 well of a 96-well plate, and then 190 μl of a DPPH solution was quickly added and mixed. After 30 minutes, the absorbance of each well was measured at 540 nm. The Trolox equivalent of the test solution was prepared by taking 10 μl of 0.156 mM, 0.313 mM, 0.625 mM, 1.25 mM, 2.5 mM, and 5.0 mM Trolox solution, adding 190 μl of DPPH solution, mixing, and 540 nm after 30 minutes. Was measured to prepare a calibration curve of the Trolox concentration and the absorbance value at 540 nm. The Trolox equivalent was calculated from the absorbance value of the test solution at 540 nm using a calibration curve. Trolox used as a standard is a substance having a structure similar to tocopherol. When Trolox is 1, α-tocopherol, γ-tocopherol, and δ-tocopherol are said to show 0.50, 0.74, and 1.36, respectively, so if the Trolox equivalent is 0.5 or more, It can be said that the antioxidant effect is sufficient.

As shown in Table 1, the amount of melanin per cell in the Modama leaf extract was 63.1%, which was about 37% smaller than that of purified water compared to purified water, and the degradation of melanin in cells was significantly promoted. It was confirmed that. On the other hand, a perilla leaf extract is well known for its high antioxidant activity and melanin production inhibitory effect, but no melanin degradation promoting effect was observed. Therefore, it was considered that the antioxidant effect and the melanin production inhibitory effect were not linked to the melanin degradation promoting effect.

INDUSTRIAL APPLICABILITY The modama leaf extract of the present invention has an effect of promoting intracellular melanin degradation in epidermal cells, and can be expected to promptly improve pigmentation caused by ultraviolet rays and aging.

Claims (3)

  A melanin decomposition accelerator containing a modama leaf extract as an active ingredient.   A whitening cosmetic comprising the melanin decomposition promoter according to claim 1 as an active ingredient. A cosmetic method for promoting the decomposition of melanin present in epidermal cells, wherein a composition containing a modama leaf extract is applied to the skin.