JP2009040688A - Melanogenesis inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new melanogenesis inhibitor, a tyrosinase inhibitor and a skin care preparation composition for external use containing the melanogenesis inhibitor, and a cosmetic. <P>SOLUTION: The melanogenesis inhibitor comprises N-trans-feruloyltyramine as an active ingredient. The tyrosinase inhibitor comprises N-trans-feruloyltyramine as an active ingredient. The skin care preparation composition for external use comprises the same. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a melanin production inhibitor, a tyrosinase inhibitor, a skin external preparation composition containing the same, and a cosmetic.

Conventionally, whitening cosmetic compositions containing vitamin C and derivatives thereof, melanin production inhibitors such as kojic acid, arbutin, and ellagic acid are known. However, there are few things that have a sufficient melanin production inhibitory effect.
(Patent Documents 1 to 4).

JP2007-22960 JP2007-106675A JP2007-131602 JP2007-137844

An object of the present invention is to provide a novel melanin production inhibitor.
Another object of the present invention is to provide a novel tyrosinase inhibitor.
Still another object of the present invention is to provide a skin external preparation composition containing the melanin production inhibitor or the tyrosinase inhibitor.
Still another object of the present invention is to provide a cosmetic, particularly a whitening cosmetic, containing the melanin inhibitor or tyrosinase inhibitor.

The present invention provides the following melanin production inhibitor, tyrosinase inhibitor, external skin composition containing the same, and cosmetics.
1. Melanin production inhibitor containing N-trans-feruloyl tyramine as an active ingredient.
2. A tyrosinase inhibitor containing N-trans-feruloyl tyramine as an active ingredient.
3. A skin external preparation composition comprising the melanin production inhibitor described in 1 above.
4). Cosmetics containing the melanin production inhibitor described in 1 above.
5). 3. A skin external preparation composition containing the tyrosinase inhibitor according to 2 above.
6). Cosmetics containing the tyrosinase inhibitor according to 2 above.

  N-trans-feruloyl tyramine, which is an active ingredient of the present invention, has an inhibitory effect on the expression of “tyrosinase”, which is the initial stage of melanin synthesis. For this reason, it shows much stronger melanin production inhibitory action than kojic acid which is a conventionally known tyrosinase inhibitor. That is, N-trans-feruloyl tyramine remarkably inhibits the expression of tyrosinase in B16 cells, which are melanin-producing cultured cell lines, and can significantly reduce the amount of melanin in B16 cells.

The present inventor has found that N-trans-feruloyl tyramine represented by the following formula (I) suppresses melanin production on melanoma cells, and has a new mechanism of action compared to conventionally known tyrosinase inhibitors. It has been found that it has a working melanin production inhibitory effect, and the present invention has been completed.

The present inventor has found that N-trans-feruloyltyramine has a much higher inhibitory action on melanin production than kojic acid, which is conventionally known as a melanin production inhibitor.
This N-trans-feruloyl tyramine is a compound having the structure shown in the formula (I).
Melanin production goes through the process of tyrosine->DOPA-> DOPA quinone-> leuco DOPA chrome-> DOPA chrome-> melanin. It has been found that it suppresses the production of “tyrosinase” itself.

Therefore, the N-trans-feruloyl tyramine of the formula (I) according to the present invention is useful as a melanin production inhibitor and a tyrosinase inhibitor.
The melanin production inhibitor or tyrosinase inhibitor of the present invention is N-trans-feruloyl tyramine alone or in combination with a skin external preparation base or other ingredients (for example, therapeutic agents, cosmetics). Can be used as

The blending amount of N-trans-feruloyl tyramine when used as a skin external preparation composition is preferably 0.05 ppm (mass) to 10 mass%, more preferably 0.2 ppm (mass) to the total mass of the composition. It is 0.5 mass%, Most preferably, it is 0.5-50 ppm (mass).
As a skin external preparation base suitable for constituting the skin external preparation according to the present invention, for example, a solvent, water, oil (for example, glycerin, petrolatum, olive oil, peppermint oil, stearyl alcohol, conventionally used for skin external preparations, A cyclodextrin, etc. can be used The skin external preparation can be in the form of a solution, dispersion, emulsion, ointment (agent), patch (agent), or the like.

  In addition to the above-mentioned skin external preparation base, additives and bases that have been conventionally blended in various skin external preparations in the skin external preparation composition according to the present invention do not impair the purpose of the present invention. Can be used in ranges and amounts. Specifically, moisturizers (for example, xylitol, dipropylene glycol, propylene glycol, mannitol, produ (DL-pyrrolidone carboxylic acid + L-proline + sodium lactate + sorbitol + collagen aqueous solution), isomerized sugar / pentabaiten), ultraviolet light Absorbents (eg oxybenzone, guaiazulene, phenyl salicylate, synoxate, paraaminobenzoate, 2- (2-hydroxy-5-methylphenyl) -benzotriazole), vitamins (eg vitamin A, vitamin B, vitamin C, Vitamin E, vitamin D), antioxidants (eg, CoQ 10 (CoQ10), carotenoids, dibutylhydroxytoluene, butylhydroxyanisole, rosemary, sage, oregano, sesaminol, catechol , Superoxide dismutase (SOD), catalase, glutathione), fats and oils (eg corn oil, olive oil), surfactants (eg lauryltrimethylammonium chloride, polyoxine ethylenelanolin alcohol, cetyltrimethylammonium bromide, sodium cetyl sulfate, linear Sodium alkylbenzenesulfonate, polyoxyethylene lauryl ether sulfates, polyoxyethylene lanolin, polyoxyethylene lanolyl alcohol, lauric sulfate), powder (eg cyclodextrin), colorant (eg dihydroxyacetone (DHA)) Etc.

Hereinafter, the present invention will be described more specifically with reference to examples. However, it goes without saying that the technical scope of the present invention is not limited to these examples.
Production Example 1
Extraction and purification of N-trans-feruloyl tyramine
Methanol from branches of Enicosanthum membranifolium Sinclair by the method described in Efdi M., Itoh T., Akao Y., Nozawa Y., Koketsu M., Ishihara H., Bioorg. Med. Chem., 15, 36673671 (2007) The extract (369 g) was poured into water and fractionated sequentially with n-hexane, ethyl acetate, and n-butanol to obtain N-trans-feruloyltyramine.

Production Example 2
Enicosanthum cupulare (King) Airy-Shaw air-dried bark (5 Kg) was finely ground and immersed in methanol at room temperature. The mixture was filtered and concentrated in vacuo to give a methanol extract (185 g). This was fractionated sequentially with n-hexane, ethyl acetate, and n-butanol. The ethyl acetate extract (20 g) was fractionated on a silica gel 60 (Kanto Chemical Co., Ltd., 40-50 μm) column (φ 60 mm x 500 mm, 300 g) and eluted sequentially with chloroform-acetone and methanol. Fractions A to J were obtained. Fraction D (1.18 g) was separated by HPLC (Wakosil-II 5C18 HG Prep, φ 20 mm x 250 mm, Wako Pure Chemical Industries, Ltd., Tokyo), and N-trans-feruloyl tyramine (40.9 mg) was separated. Obtained.

The physical properties of the obtained N-trans-feruloyl tyramine are shown below.
Mp: 92-93 ° C.
UV (EtOH) ν _max 207, 321 nm.
_{IR (KBr) v max: 3410} , 1651 cm -.
¹ H NMR (500 MHz, CD ₃ OD): δ 2.75 (2H, t, J = 7.2 Hz, H-β), 3.46 (2H, t, J = 7.2 Hz, H-α), 3.86 (3H, s, OCH ₃ ), 6.40 (1H, d, J = 15.5 Hz, H-2), 6.72 (2H, d, J = 8.6 Hz, H-3 ', H-5'), 6.78 (1H, d, J = 8.6 Hz, H-8), 7.00 (1H, dd, J = 2.3, 8.6 Hz, H-9), 7.04 (2H, d, J = 8.6Hz, H-2 ', H-6'), 7.09 (1H, d, J = 2.3 Hz, H-5), 7.43 (1H, d, J = 15.5 Hz, H-3).
¹³ C NMR (125 MHz, CD ₃ OD): δ35.8 (C-β), 42.5 (C-α), 56.3 (OCH ₃ ), 111.5 (C-5), 116.3 (C-3 ', C- 5 '), 116.5 (C-8), 118.6 (C-2), 123.3 (C-9), 128.0 (C-4), 130.7 (C-2', C-6 '), 131.3 (C-1 '), 142.1 (C-3), 149.4 (C-6), 150.2 (C-7), 156.9 (C-4'), 169.2 (C-1).
EIMS m / z: [313] ⁺ .

Test example 1
This test example is a test example of the inhibitory activity of N-trans-feruloyl tyramine compared with kojic acid, which is conventionally known as a tyrosinase inhibitor.
The culture conditions, melanin content measurement method, tyrosinase activity measurement method, protein assay, and Western blot analysis method are as follows.

Cell culture Mouse B16 melanoma cells were purchased from Riken Cell Bank (Tsukuba, Japan). This was cultured at 37 ° C. in a CO ₂ (5%) incubator in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v / v) fetal bovine serum, penicillin 100 units / ml, and streptomycin 100 μg / ml.

Measurement of melanin content
B16 cells were washed with PBS, dissolved in 2 N NaOH and kept at 60 ° C. for 1 hour. Using a standard synthetic melanin, the absorbance at 470 nm was measured to determine the melanin content. Cell survival was determined by trypan blue exclusion test.

Measurement of tyrosinase activity
B16 cells in RIPA buffer containing protease inhibitors (10 mM Tris-HCl, pH 7.5, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA) at 4 ° C. for 30 Incubate for minutes to dissolve. This was centrifuged at 15,000 × g for 30 minutes, and the supernatant was used as a tyrosinase source. The reaction mixture contained 50 mM phosphate buffer, pH 6.8, 0.05% L-dopa and supernatant. After 20 minutes incubation at 37 ° C., dopachrome formation was monitored by measuring absorbance at 470 nm.

Protein assay The protein concentration was determined using a BSA-containing DC protein assay reagent (Bio-Rad) as a standard.
Western blot analysis Cells were lysed by incubation in RIPA buffer containing protease inhibitors for 30 minutes at 4 ° C. and centrifuged at 15,000 × g for 30 minutes. The supernatant (lysed protein) was subjected to SDS-PAGE on 7.5% polyacrylamide gel. The protein was electrophoretically transferred to a PVDF membrane. Blocked in Tris buffered saline containing 5% skim milk powder. The blot was incubated with the first antibody and then incubated with horseradish peroxidase-conjugated second antibody. The protein was visualized with an ECL detector.

Mouse B16 melanoma cells were cultured for 3 days in the presence of N-trans-feruloyltyramine (hereinafter also referred to as “FA”). Melanin content in cells treated with FA was reduced compared to control cells.
FIG. 1 shows micrographs of B16 melanoma cells treated with 10 μM N-trans-feruloyltyramine and untreated. It can be seen that melanoma cells treated with N-trans-feruloyltyramine clearly suppressed melanin production compared to untreated cells.

Test example 2
Melanin production when treated for 3 days in the presence of 0-20 μM N-trans-feruloyl tyramine, and the number of viable cells in the presence of melanin, and N-trans-feruloyl tyramine (10 μM) or kojic acid (10 μM) FIG. 2 shows the result of a comparative experiment when culturing for 3 days in the presence of
A: Amount of melanin produced upon treatment with various concentrations of N-trans-feruloyl tyramine B: Number of viable cells in the presence of various concentrations of N-trans-feruloyl tyramine (% of control)
C: Comparative experiment when cultured for 3 days in the presence of N-trans-feruloyl tyramine (10 μM) and kojic acid (10 μM) (FA: N-trans-feruloyl tyramine, KA: kojic acid)

As is clear from FIG. 2A, the amount of melanin produced was suppressed in a concentration-dependent manner when treated with N-trans-feruloyltyramine. At that time, it acted almost without reducing the number of melanoma cells, and safety was confirmed (B in FIG. 2).
Further, from FIG. 2C, it can be seen that N-trans-feruloyl tyramine exhibits a much stronger inhibitory action than kojic acid.

Test example 3
The inhibitory activity of FA on mouse tyrosinase was examined by measuring the formation of dopachrome using L-dopa (3,4-dihydroxyphenylalanine) as a substrate. The results are shown in FIG. It can be seen that FA inhibits mouse tyrosinase activity.

Test example 4
Mouse B16 melanoma cells were cultured for 3 days in a medium containing 0-10 μM FA, and the expression level of tyrosinase protein was examined by Western blot analysis using an antibody specific for tyrosinase. The results are shown in FIG. It can be seen that the FA treatment inhibits the expression of tyrosinase protein.

Examples of the external preparation for skin of the present invention are shown below.
Example 1 Cream (Prescription)
Stearic acid 4.0% by mass
Stearyl alcohol 4.0
Isopropyl myristate 15.0
Glycerol monostearate 3.0
Propylene glycol 10.0
FA 0.01
Preservative Appropriate amount of perfume Appropriate amount

Example 2 Emulsion (prescription)
Stearic acid 2.0% by mass
Cetyl alcohol 2.5
Vaseline 5.0
Liquid paraffin 10.0
Polyethylene glycol 1200 3.0
Triethanolamine 1.0
Carboxyvinyl polymer 0.05
FA 0.01
Sodium bisulfite 0.01
Ethylparaben 0.3
Perfume Appropriate amount of ion-exchanged water

It is a figure which shows the microscope picture of the thing which processed 10 micromol N-trans-feruloyl tyramine to B16 melanoma cell, and a thing which is not processed. The amount of melanin produced when treated for 3 days in the presence of N-trans-feruloyl tyramine (A), the number of viable cells in the presence of melanin (B), and N-trans-feruloyl tyramine (10 μM) or kojic acid ( It is drawing which shows the comparative experiment result (C) when it culture | cultivates for 3 days in presence of 10 micromol). It is drawing which shows the inhibitory activity of FA with respect to mouse tyrosinase. It is a drawing showing that the expression of tyrosinase protein is inhibited by FA treatment. I understand.

Claims (6)

  Melanin production inhibitor containing N-trans-feruloyl tyramine as an active ingredient.   A tyrosinase inhibitor containing N-trans-feruloyl tyramine as an active ingredient.   A skin external preparation composition comprising the melanin production inhibitor according to claim 1.   Cosmetics containing the melanin production inhibitor according to claim 1.   A skin external preparation composition containing the tyrosinase inhibitor according to claim 2.   A cosmetic comprising the tyrosinase inhibitor according to claim 2.