JP4754178B2 - Promotion of melanin production by lignans - Google Patents

Description

  The present invention relates to a melanin production promoter, and more specifically, a melanin production promoter containing lignans as an active ingredient, a skin external preparation composition containing the melanin production promoter as an active ingredient, a hair cosmetic for improving white hair, and a back The present invention relates to a preventive and / or therapeutic agent for congenital depigmentation (white spot).

The most important factor that determines the color of skin and hair roots is a pigmented substance called melanin.
Melanin is produced in the skin by pigment-producing cells called melanocytes that are present in the basal epidermis and hair roots.

Acne vulgaris, which is acquired depigmentation (white spot), develops due to abnormal suppression of the production of melanin or disappearance of melanocytes. Acne vulgaris is a progressive depigmentation estimated to affect 1 to 2 million people in Japan. Conventionally, treatment of this disease has been extremely difficult. Due to the development of pathochemical and cell biological research on melanocytes and their abnormalities, this disease is not a single disease caused by a single causative factor. It has been found that the disease can be divided into types. At present, the treatment for this disease is performed by steroid therapy and photochemotherapy, and some results have been obtained, but the prognosis is often poor, and it is regarded as an intractable disease.
This disease is a selective loss of premelanosome generation in the pigment cells and subsequent loss of the pigment cells. Until now, it has been considered as a skin-only disease. Has also been found to cause lesions. However, there is currently no suitable treatment for such leukoplakia.

  Moreover, since the hair color is also due to melanin deposition, it is considered that white hair that occurs with aging is due to a decrease in melanin production in the hair root. The white hair is mostly dyed with a dye, and various hair cosmetics have been reported for improving white hair, but there is no fundamental white hair improving hair cosmetic.

On the other hand, lignan, in a narrow sense, refers to a compound in which two molecules of phenylpropanoid are bonded between β-carbons. In addition, there are three molecules of neolignan (neolignan) bonded between carbons other than β-carbon and oxygen. These are collectively called lignans, including sesquilignan to which phenylpropanoids are bound, and dilignan to which four molecules are bound.
Such lignans are widely distributed in higher plants, especially in timber, and have an antioxidant action (Japanese Patent Application Laid-Open No. 2004-002544), an anticancer action (Japanese Patent Publication No. 10-51185), and an antiviral action (specialty). (Kaihei 10-175861), an antibacterial action, a cholesterol metabolism promoting action, and other various physiological activities. Recently, it has been reported that it has an effect of regulating estrogen concentration in the living body, and has attracted particular attention.
JP-A-11-255539 discloses a carbon skeleton of lignans or norlignans, wherein at least one of the two benzene rings having a substituent is a 4-substituted resorcinol skeleton, followed by a carbon atom at the benzyl position. Discloses that lignan derivatives and / or norlignan derivatives having no substituents inhibit tyrosinase activity essential for melanin production in vivo in in vitro experiments.
However, it has not been known so far that when lignans are allowed to act on cells, melanin production is promoted through promoting expression of tyrosinase.
Japanese Patent Laid-Open No. 10-175861 Japanese National Patent Publication No. 10-511685 JP 11-255639 A Japanese Patent Laid-Open No. 2002-3381 JP 2004-2264 A JP 2004-2544 A

  The present invention provides a melanin production promoter, a skin external preparation composition containing the melanin production promoter as an active ingredient, a hair cosmetic for improving white hair, and an agent for preventing and / or treating acquired depigmentation (white spot). It was made for the purpose of providing.

  As a result of intensive studies, the present inventors have found that lignans represented by the following general formula (I) increase melanin production in human skin-derived melanocyte cells (HMVII) and human three-dimensional skin models. It is based on.

  The present inventors searched for substances that increase melanin production in human skin-derived melanocyte cells (HMVII) and human three-dimensional skin models. As a result, the lignans represented by the following general formula (I) are melanin production processes. Promotes the activity of the enzyme tyrosinase, which catalyzes the reaction from tyrosine to dopa and dopaquinone, which increases the rate of melanin production by melanocyte cells (HMVII), and improves white hair and acquired depigmentation (white spots) It was found useful for the prevention and / or treatment of the present invention, and the present invention was completed. That is, the present invention is as follows.

(1) The following general formula (I)
(I)
Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are the same or different and are a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 4 carbon atoms, or a hydroxyalkyl group having 1 to 4 carbon atoms. Or an alkenyl group having 1 to 4 carbon atoms).
(2) R1, R2, R5 and R6 are the same or different and are a hydrogen atom, a hydroxyl group or an alkoxy group having 1 to 4 carbon atoms, and R3 and R4 are the same or different and are an alkyl group having 1 to 4 carbon atoms, The melanin production promoter of Claim 1 which is a C1-C4 hydroxyalkyl group or a C1-C4 alkoxy group.
(3) R1, R2, R5, and R6 are hydroxyl groups, and R3 and R4 are the same or different, and are an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, or an alkyl group having 1 to 4 carbon atoms. The melanin production promoter according to (1), which is an alkoxy group.
(4) The melanin production promoter according to (1), wherein R1, R2, R5, and R6 are hydroxyl groups, and R3 and R4 are the same or different and are alkyl groups having 1 to 4 carbon atoms.
(5) The melanin production promoter according to (1), wherein the compound is nordihydroguaiaretic acid represented by the following general formula (II).
(II)
(6) A skin external preparation composition containing the melanin production promoter according to any one of (1) to (5) as an active ingredient.
(7) The external composition for skin according to (6), which is a hair cosmetic for improving white hair.
(8) The external preparation for skin according to (6), which is a preventive and / or therapeutic agent for acquired depigmentation (white spots).

  The lignans of the present invention promote the activity of tyrosinase, increase the amount of melanin produced by melanocyte cells (HMVII), and can be used for skin external preparation compositions, hair cosmetics for improving white hair, and acquired depigmentation (white spots). It can be used as a prophylactic and / or therapeutic agent. This new physiology of lignans, which has never been known before, can be applied to the improvement of aging phenomena and pathological conditions with depigmentation such as vulgaris, white hair, etc. It becomes possible.

Melanin production promoter of the present invention The melanin production promoter of the present invention contains lignans, which are compounds represented by the above general formula (I), as active ingredients. In formula (I), R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are the same or different and are a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 4 carbon atoms, or a hydroxy group having 1 to 4 carbon atoms. An alkyl group or an alkoxy group having 1 to 4 carbon atoms is shown.
From the viewpoint of reactivity, R ₁ , R ₂ , R ₅ , and R ₆ are the same or different and are a hydrogen atom, a hydroxyl group, or an alkoxy group having 1 to 4 carbon atoms, and R ₃ and R ₄ are the same or different, An alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 1 to 4 carbon atoms, or an alkoxy group having 1 to 4 carbon atoms is preferable.
Further, from the viewpoint of reactivity, R ₁ , R ₂ , R ₅ and R ₆ are hydroxyl groups, and R ₃ and R ₄ are the same or different, and are an alkyl group having 1 to 4 carbon atoms and 1 to 4 carbon atoms. It is more preferable that it is a C1-C4 alkoxy group.
Further, R ₁ , R ₂ , R ₅ and R ₆ are hydroxyl groups, and R ₃ and R ₄ are the same or different and are alkyl groups having 1 to 4 carbon atoms. preferable.

Examples of the lignans represented by the general formula (I) include nordihydroguaiaretic acid, meso-dihydroguaiaretic acid, that is, (2R, 3S) -1,4-bis- (4-hydroxy-3-methoxyphenyl) -2,3-dimethylbutane) and the like. Among these, nordihydroguaiaretic acid represented by the following general formula (II) is preferable.

(II)

  These lignans represented by the general formula (I), for example, nordihydroguaiaretic acid, can be purchased from Sigma Chemical Co. These can also be obtained by the method disclosed in JP-A-2-180846. That is, a compound-rich fraction obtained by repeatedly subjecting guaiac fat (Guaiacum officinale L. resin) to silica gel column chromatography is subjected to column chromatography using ODS silica gel, or n-hexane, ether, It can be obtained by recrystallization using acetone, ethanol, methanol, water alone or a mixed solvent thereof. If necessary, the compound may be appropriately methylated with a common methylating agent such as methyl iodide, dimethyl sulfate or diazomethane, or a mixed solvent of acetic anhydride and pyridine or a general acetylation such as acetyl chloride. It can be obtained by acetylation with an agent or demethylation with a demethylating agent such as boron tribromide, boron trichloride or aluminum trichloride. Further, it can be synthesized by the method disclosed in JP-T-10-511699.

As the lignans used in the present invention, one kind may be used alone, or two or more kinds may be used in combination. In addition, as shown in Examples below, it has an excellent tyrosinase activity promoting action and melanin production promoting action, and a melanin production promoter containing this as an active ingredient, and a skin external preparation composition containing the melanin production promoter as an active ingredient Products, hair cosmetics for improving white hair, and preventive and / or therapeutic agents for acquired depigmentation (white spots).
The content of lignans when used as a skin external preparation composition or a hair cosmetic for improving white hair is preferably 0.1 to 50% by mass, more preferably 5 to 20% by mass, based on the total mass of the composition.

The skin external preparation composition of the present invention may be the same as the normal skin external preparation composition as long as the effects of the present invention are not impaired, except that a melanin production promoter is used as an active ingredient. That is, additives and base materials that have been generally blended in various external preparations for skin can be used in ranges and amounts that do not impair the object of the present invention. Specifically, xylitol, dipropylene glycol, propylene glycol, mannitol, produ (DL-pyrrolidone carboxylic acid, L-proline, sodium lactate, sorbitol, collagen aqueous solution), humectant such as isomerized sugar / pentabaiten, oxybenzone UV absorbers such as guaiazulene, phenyl salicylate, sinoxate, paraaminobenzoate, 2- (2-hydroxy-5-methylphenyl) -benzotriazole, vitamin A, vitamin B, vitamin C, vitamin E, vitamin D Vitamins such as CoQ10, carotenoids, dibutylhydroxytoluene, butylhydroxyanisole, rosemary, sage, oregano, sesaminol,
Antioxidants such as catechol, superoxide dismutase (SOD), catalase, glutathione, fats and oils such as corn oil and olive oil, lauryltrimethylammonium chloride, polyoxynethylenelanolin alcohol acetate, cetyltrimethylammonium bromide, sodium cetylsulfate, linear type Surfactants such as sodium alkylbenzene sulfonate, polyoxyethylene lauryl ether sulfates, polyoxyethylene lanolene, polyoxyethylene lanolene alcohol, lauryl sulfate, powders such as cyclodextrin, dihydroxyacetone (DHA), etc. And coloring agents.

  The hair cosmetic composition for improving gray hair according to the present invention comprises a melanin production promoter as an active ingredient, medicinal ingredients blended in normal hair cosmetics, etc., for example, plant extract extracts such as assembly extract and carrot extract, vitamin E and its derivatives Further, vitamins such as biotin, nicotinic acid esters and the like can be contained.

  The hair cosmetic composition for improving gray hair of the present invention can be used as, for example, a cream, lotion, emulsion, ointment, gel, hair tonic, hair liquid, liniment, hair rinse, hair shampoo, hair treatment, hair conditioner, aerosol, mousse, etc. preferable.

The amount of the hair cosmetic composition for improving white hair and the external preparation for skin of the present invention varies depending on the content of the active ingredient. For example, in the case of cream or ointment, ² mg to 1000 mg is used per 1 cm ^{2 of} the skin layer surface. Preferably, 100 mg to 400 mg is used. In the case of a liquid preparation, it is preferable to use ² mg to 1000 mg, more preferably 100 mg to 400 mg, per 1 cm ^{2 of} the skin layer surface.

  The preventive and / or therapeutic agent for acquired depigmentation (white spots) of the present invention can be administered orally or parenterally with a melanin production promoter as an active ingredient. What is necessary is just to select a dosage form suitable for the target disease. Examples of the preparation to be administered orally include dosage forms such as tablets, capsules, powders, granules, fine granules, and liquids for internal use. In addition, preparations for parenteral administration include injections, extracts, spirits, suppositories, suspensions, tinctures, ointments, poultices, liniments, lotions, aerosols, plasters, etc. Mention may be made of a skin external preparation in dosage form. In the present invention, a skin external preparation that is easy to administer is preferred.

  Formulation can be performed by known formulation techniques, and appropriate formulation additives can be added to the formulation. Formulation additives include excipients, suspending agents, emulsifiers, moisturizing (wetting) agents, preservatives, surfactants, thickeners, pigments, fragrances, propellants, etc. What is necessary is just to add suitably in the range which does not impair the effect of this invention.

  The prophylactic and / or therapeutic agent for acquired depigmentation (white spot) of the present invention may further contain a known tyrosinase activity promoter, melanin production promoter, and the like. Examples of these components include florecithin glycoside (Japanese Patent Laid-Open No. 8-259448), shellfish essence (Japanese Patent Laid-Open No. 7-285874), diisopropyl 1,3-dithiol-2-ylidene malonate (Japanese Patent Laid-Open No. 7-256829), basidiomycete culture solution or bacterial cell extract (JP-A-7-316026), ω-alkoxycarbonylalkyltrialkylammonium and / or salt thereof (JP-A-7-316048) Etc. are disclosed. One or more of these components may be added to the lignans according to the present invention.

The blending amount of the lignans in the preparation may be appropriately examined in consideration of the target disease, sex, age, symptoms, etc., but the lignans are preferably blended in an amount of 0.1 to 50% by weight in the preparation.
It is more preferable to mix 5 to 20% by mass.

  EXAMPLES Next, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to these Examples.

    Hereinafter, the present invention will be described specifically by way of examples.

Effect of Lignan on Melanin Content of HMVII Cells The following shows the effect of nordihydroguaiaretic acid, which is a lignan, on the melanin content of HMVII cells. The nordihydroguaiaretic acid subjected to the test is a commercially available product (1,4-Bis (3,4-dihydroxyphenyl) -2,3-dimethylbutane) obtained from Sigma Chemical Co.

First, human skin-derived melanocyte cells (HMVII, Riken Genebank (RCB0777)) and melanocyte cells (HMVII) added with nordihydroguaiaretic acid (final concentration 20 μM), to which DMSO was added as a control, were compared.
As shown in FIG. 1, dark brown color was observed in melanocyte cells (HMVII) to which nordihydroguaiaretic acid (final concentration 20 μM) was added compared to human skin-derived melanocyte cells (HMVII) to which control DMSO was added. It was clarified that the pigmented substance to be deposited was markedly deposited. Since this cell line (HMVII) is a cell having melanin producing ability, it was predicted that the pigment increased and deposited by addition of nordihydroguaiaretic acid was melanin.

Therefore, in fact, control (no addition), control (DMSO addition) and nordihydroguaiaretic acid (final concentrations 1 μM, 5 μM, 10 μM, 20 μM) were added to the cells (HMVII), and then the cells (HMVII) were added. ) And the amount of melanin contained in the cells (HMVII) after 1, 3, 5, and 7 days have been measured by adding nordihydroguaiaretic acid at a final concentration of 20 μM.
For the measurement, about 1 × 10 ⁶ human skin-derived melanocyte cells (HMVII) treated above were washed with phosphate buffer (pH 7.4), ultrasonically disrupted with 500 μl of 1M NaOH, and left overnight. Cells were lysed. The absorbance (475 nm) of this cell lysate was measured using SPECTRAmax 250 microplate reader (Molecular Device Co. USA), and calculated from the absorbance of a known amount of synthetic melanin to determine the amount of melanin. The results are shown in FIGS. 1, 2a and 2b.
As shown in FIG. 2a, when the amount of nordihydroguaiaretic acid added was changed (final concentrations 1 μM, 5 μM, 10 μM, 20 μM), the amount of melanin production increased depending on the final concentration of nordihydroguaiaretic acid . Furthermore, as shown in FIG. 2b, the melanin production promoting action by nordihydroguaiaretic acid reached about 6.9 times as compared with the control addition of DEMSO at the seventh addition.

  As shown in FIGS. 2a and 2b, it was revealed that nordihydroguaiaretic acid increases the amount of melanin produced by human skin-derived melanocyte cells (HMVII).

Effect of lignan on tyrosinase activity in HMVII cells The reaction that becomes the rate-limiting step in the melanin production process in the cell is an enzyme-catalyzed reaction from tyrosine to dopa and dopaquinone by tyrosinase. In addition, it is speculated that there is some relationship between the promotion of melanin production by lignans and tyrosinase activity. Measured.

After washing about 1 × 10 ⁶ human skin-derived melanocyte cells (HMVII) with a phosphate buffer, 45 μl of 1% Triton X-100-containing phosphate buffer (pH 7.4) was added to lyse the cells. 5 μl of 20 mM L-dopa was added to this cell lysate and reacted at 37 ° C. for 60 minutes. The absorbance (475 nm) of this reaction solution was measured by SPECTRAmax 250 microplate reader (Molecular Device Co. USA). The enzyme activity was determined using a tyrosinase protein (derived from mushrooms) having a known enzyme activity as a control. The results are shown in FIGS. 3a and 3b.

  As a result, it was found that after adding nordihydroguaiaretic acid (final concentrations 1 μM, 5 μM, 10 μM, 20 μM), tyrosinase activity increased significantly with time (FIG. 3 a). Tyrosinase activity increased depending on the amount of nordihydroguaiaretic acid added (FIG. 3b). From this result, it was found that the increase in the amount of intracellular melanin by nordihydroguaiaretic acid was due to the increase in tyrosinase activity in the cells.

Effect of lignans on in-gel tyrosinase activity Furthermore, tyrosinase activity staining was performed as follows in an electrophoresis gel using L-dopa as a substrate.
1 × 10 ⁶ HMVII cells were treated with 100 μl of cell disruption solution (1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 μg / ml aprotin, 10 μg / ml leupeptin, 0.1 M phosphate buffer (pH 6.8). )) Was added and sonicated to prepare a cell lysate. Using this crushed liquid, sodium dodecyl sulfate (SDS) -10% polyacrylamide gel electrophoresis was performed. After electrophoresis, the polyacrylamide gel was washed with 200 ml of 0.1 M phosphate buffer (pH 6.8). The washed gel was soaked in 200 ml of 0.1 M phosphate buffer (pH 6.8) containing 5 mM L-dopa and gently shaken at 37 ° C. for 30 minutes to try tyrosinase activity staining.
As a result, as shown in FIG. 4a, after adding nordihydroguaiaretic acid, a single band with positive tyrosinase activity staining that is recognized at a position corresponding to the molecular weight of tyrosinase protein as time passes 1, 3, 5 and 7 days. The strength of has increased significantly.
In addition, as shown in FIG. 4b, the intensity of the band of tyrosinase activity staining was dependent on the concentration of nordihydroguaiaretic acid.

Effect of lignans on tyrosinase protein expression in HMVII cells It was speculated that the increase in tyrosinase activity by nordihydroguaiaretic acid shown above was due to an increase in intracellular tyrosinase protein. Therefore, the amount of tyrosinase protein after addition of nordihydroguaiaretic acid was observed by an immunoblotting method. The experiment was performed as follows.

1 × 10 ⁶ HMVII cells with 100 μl of cell disruption solution (1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 μg / ml aprotin, 10 μg / ml leupebutine, 0.1 M phosphate buffer (ph 6.8)) And sonicated to prepare a cell lysate. Sodium dodecyl sulfate (final concentration 1%) and 2-mercaptoethanol (final concentration 5%) were added to the crushed liquid, and heat treatment was performed at 95 ° C. for 5 minutes. Using this solution as a sample, sodium dodecyl sulfate (SDS) -10% polyacrylamide gel electrophoresis was performed. After electrophoresis, the protein in the polyacrylamide gel was transferred to a nitrocellulose membrane. The nitrocellulose membrane was immersed in a TBS buffer (tris-buffered saline) containing 5% non-fat dry milk and 0.1% Tween and shaken for 1 hour. Next, the nitrocellulose membrane was reacted with an anti-human tyrosinase-mouse antibody diluted at room temperature for 1 hour. After washing 5 times with 0.1% Tween-containing TBS buffer, the nitrocellulose membrane was reacted with horseradish peroxidase-labeled anti-mouse immunoglobulin antibody for 1 hour at room temperature. The band of tyrosinase protein was detected by chemiluminescence method. The results are shown in FIGS. 5a and 5b.

  As a result, the concentration of the tyrosinase protein band observed by the immunoblotting method increased depending on the action time (FIG. 5a) and concentration (FIG. 5b) of nordihydroguaiaretic acid. This revealed that nordihydroguaiaretic acid promotes the synthesis of tyrosinase protein.

  As shown in FIGS. 3 to 5, nordihydroguaiaretic acid was observed to promote tyrosinase activity of human skin-derived melanocyte cells (HMVII).

Formulation Example The following is a formulation example of the hair cosmetic composition for improving gray hair according to the present invention and a preventive and / or therapeutic agent for acquired depigmentation (white spot).

Formulation Example 1: Hair cosmetic for improving white hair A hair cosmetic for improving white hair was prepared according to the formulation using the ingredients shown in Table 1 below. That is, each compounding component was stirred and solubilized at room temperature to obtain a hair cosmetic for improving gray hair.

Formulation Example 2: Agent for Preventing and / or Treating Acquired Depigmentation (Vitiligo) According to the formulation shown below, using the ingredients shown in Table 2 below, and / or preventing acquired depigmentation (white spot) A therapeutic agent was made. That is, each compounding component was weighed in a kneader and kneaded well to obtain a preventive and / or therapeutic agent for acquired depigmentation (white spot).

Test Example 1: Acquired depigmentation (white spot) improvement effect Patients with 10 melanocytes that have not disappeared at the initial lesion (10 patients) were mixed with petrolatum 15% nordihydroguaiaretic acid for 1 day in the white spot. The external application was performed twice. The application amount was limited to 2-3 g at a time depending on the surface area of the vitiligo portion. When 3 months passed after the external application, the degree of pigmentation in the vitiligo portion was visually evaluated. The standard of visual evaluation is `` improvement '' when the boundary between vitiligo and normal skin is unclear, `` no change '' when the boundary can be clearly recognized, `` between `` improvement '' and `` no change '' Somewhat improved. As a result, as shown in Table 3, skin pigmentation was observed in 80% of cases, and improvement of vitiligo was confirmed.

Diagram showing the effect of lignans on HMVII cells Diagram showing the effect of lignans on the melanin content of HMVII cells Diagram showing the effect of lignans on tyrosinase activity in HMVII cells Diagram showing the effect of lignans on in-gel tyrosinase activity Diagram showing the effect of lignans on tyrosinase protein expression in HMVII cells

Claims (1)

A preventive and / or therapeutic agent for acquired depigmentation (white spot) comprising a melanin production promoter composed of nordihydroguaiaretic acid represented by the following general formula (II) as an active ingredient .