HK1149508B - Melanogenesis inhibitor and skin-whitening agent - Google Patents

Abstract

An object of the present invention is to provide melanogenesis inhibitor and skin-whitening agent. To solve the problem, there is provided melanogenesis inhibitor and skin-whitening agent containing compound which is represented by a chemical formula 1.

Description

Melanogenesis inhibitor and skin whitening agent

Technical Field

The present invention relates to a melanin production inhibitor and a whitening agent.

Background

The amount of ultraviolet rays reaching the epidermis tends to increase year by year due to air pollution and ozone layer destruction, and skin troubles such as skin spots, freckles, and blackened skin color due to ultraviolet rays are also increasing. Tyrosine present in the skin is oxidized by tyrosinase due to ultraviolet rays to produce melanin pigment, and when it is excessively produced, skin troubles such as speckle, freckle, blackened skin color, and the like are caused. As a method for suppressing the production of melanin pigment and preventing the formation of chloasma, freckles and darkening of skin color, a skin cosmetic composition containing arbutin (patent document 1: Japanese patent application laid-open No. 60-169906) and a glucoside of L-ascorbic acid and L-ascorbic acid as a derivative thereof (patent document 2: Japanese patent application laid-open No. 4-182412) has been proposed. Skin external preparations containing arbutin and whitening agents containing L-ascorbic acid and its derivatives such as glucose glycoside have insufficient whitening effect, or have safety problems when components exhibiting whitening effect are blended at a concentration at which the whitening effect is visible.

Further, the compound of the formula (1) is known as an antiviral component, but the effect of suppressing melanin production is not known (patent document 3: Japanese patent laid-open No. 8-311056).

Chemical formula (1)

Prior art documents

Patent document

[ patent document 1 ] Japanese patent laid-open No. Sho 60-016906

[ patent document 2 ] Japanese patent application laid-open No. H04-182412

[ patent document 3 ] Japanese patent application laid-open No. H08-311056

Disclosure of Invention

The present invention addresses the problem of providing an effective melanin production inhibitor and a whitening agent.

(carrying out the Process of the present invention)

In order to search for a compound having a melanin production-inhibiting function which is directly applicable to the human body in cosmetics and the like, a plurality of compounds expected to have a melanin production-inhibiting function are screened by a device for measuring a melanin production-inhibiting function, and a compound having a strong melanin production-inhibiting function is selected. In order to confirm safety of the compounds directly applicable to human bodies, a safety confirmation test was conducted to search for compounds having high melanin inhibitory activity and safety.

As a result, compounds having a melanin production-inhibiting function stronger than that of known compounds and having excellent safety were identified from among a plurality of compounds.

(Main constitution of the invention)

The specific compound is a compound of the following chemical formula (1), and the invention relates to a melanin production inhibitor and a whitening agent containing the compound.

Chemical formula (1)

The present invention provides a melanin production inhibitor and a whitening agent containing the compound of formula (1), which have a stronger melanin production-inhibiting effect than a whitening agent containing a conventional melanin production inhibitor, and which are excellent in safety while inhibiting the occurrence of speckle, freckle and skin color darkening.

Drawings

FIG. 1 is a graph comparing the IC50 value for melanin production inhibition and the IC50 value for cytotoxicity of the compounds of the present invention of formula (1) and positive control and comparative samples.

FIG. 2 is a graph showing a comparison of the safety factors of the compounds of the present invention represented by the formula (1) and positive controls and comparative samples.

FIG. 3 is a graph showing a comparison of images of the bottom surface of a melanotic part on day 5 of culture.

FIG. 4 is a graph comparing the melamine coverage of the bottom surface on day 5 of culture.

Detailed Description

The embodiments of the present invention will be described in more detail below.

The compound of the following chemical formula (1) used in the present invention is 2, 2 ', 3, 3' -tetrahydro-5, 5 ', 7, 7' -tetrahydroxy-2, 2 '-bis (4-hydroxyphenyl) -3, 3' -bis [4H-1-benzo]-4, 4 ' -dione (2, 2 ', 3, 3 ' tetrahedron-5, 5 ', 7, 7 ' -tetrahydroxy-2, 2 ' -bis (4-hydroxyphenyl) -3, 3 ' -bi [ 4H-1-benzopyran)]-4, 4' -dione) having the compositional formula C₃₀H₂₂O₁₀。

Chemical formula (1)

The compound of formula (1) is a compound in which 2 molecules of flavanone are bound to each other at the 3-position of chroman-4-one ring. The 2-and 3-positions of the chroman-4-one ring are asymmetric carbons and can be any stereoisomer or mixture of stereoisomers.

The compound of formula (1) can be obtained commercially, for example, from AnalythiCon.

Further, it is known that the compound of formula (1) is contained in a Chinese medicinal herb, stellera chamaejasme, which is a pharmaceutical (patent document 3).

The melanin production inhibitor of the present invention can be used for exhibiting a whitening effect, and can be mixed with various bases and additives for whitening agents to be used as the whitening agent of the present invention.

The whitening agent of the present invention can be used in various known forms and applications, for example, as a whitening emulsion cosmetic, a whitening cream, a whitening lotion, a whitening oil cosmetic, a whitening mask, a whitening foundation, and the like. The whitening agent of the present invention includes a skin-whitening external preparation, a whitening external preparation, and a whitening cosmetic. The whitening agent of the present invention can also be used as an oral preparation, an injection, or an oral medicine for whitening.

The melanin production inhibitor and the whitening agent containing the compound of formula (1) of the present invention can be produced according to a generally used formulation method.

The melanin production inhibitor and the skin-whitening agent of the present invention may contain oils and fats such as vegetable oils, higher fatty acids, higher alcohols, silicones, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, preservatives, saccharides, and metal ion-blocking agents; such as water-soluble polymer, thickener, powder component, ultraviolet absorbent, and ultraviolet screening agent; such as humectant of hyaluronic acid, perfume, pH regulator, etc. It may also contain other effective components and physiological active components such as vitamins, skin activating agent, blood flow promoter, resident bacteria control agent, active oxygen scavenger, antiinflammatory agent, other whitening agent, and bactericide.

Examples of the oils and fats include liquid oils and fats such as camellia oil, evening primrose oil, macadamia nut oil, olive oil, rapeseed oil, corn oil, sesame oil, jojoba oil, germ oil, wheat germ oil, triglycerol, tricaprylin, and the like, solid oils and fats such as cocoa butter, coconut oil, hydrogenated coconut oil, palm kernel oil, wood wax kernel oil, hydrogenated oil, and hydrogenated castor oil, waxes such as beeswax, candelilla wax, cotton wax, bran wax, lanolin acetate, liquid lanolin, and sugar cane wax, and liquid paraffin, squalene, squalane, and microcrystalline wax.

Examples of the higher fatty acid include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and the like.

Examples of the higher alcohol include linear alcohols such as lauryl alcohol, stearyl alcohol, cetyl alcohol and stearyl alcohol, and branched alcohols such as stearyl glyceryl ether, lanolin alcohol, cholesterol, phytosterol and octyldodecanol.

Examples of the silicone include dimethylpolysiloxane such as a chain polysiloxane and methylphenylpolysiloxane, and decamethylpolysiloxane such as a cyclic polysiloxane.

Examples of the anionic surfactant include fatty acid salts such as sodium laurate, higher alkyl sulfate salts such as sodium lauryl sulfate, alkyl ether sulfate salts such as POE lauryl sulfate triethanolamine, N-acyl sarcosines, sulfosuccinates, and N-acyl amino acid salts.

Examples of the cationic surfactant include alkyltrimethylammonium salts such as octadecyltrimethylammonium chloride, benzalkonium chloride and benzethonium chloride.

Examples of the amphoteric surfactant include betaine surfactants such as alkyl betaine and amide betaine.

Examples of the nonionic surfactant include sorbitan fatty acid esters such as sorbitan monooleate, and hydrogenated castor oil derivatives.

Examples of the preservative include methyl paraben and ethyl paraben.

Examples of the metal ion-blocking agent include edetate salts such as disodium edetate, ethylenediaminetetraacetic acid and sodium edetate salt.

Examples of the polymer include ethylene polymers such as gum arabic, tragacanth gum, galactan, guar gum, carrageenan, pectin, agar, quince seed, dextran, pullulan, carboxymethyl starch, collagen, casein, gelatin, methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose (CMC), sodium alginate, and CARBOPOL (CARBOPOL), and bentonite.

Examples of the thickener include carrageenan, tragacanth gum, quince seed, casein, dextrin, gelatin, sodium carboxymethylcellulose (CMC), hydroxyethyl cellulose, hydroxypropyl cellulose, carbopol, guar gum, and xanthan gum.

Examples of the powder component include talc, kaolin, mica, silica, zeolite, polyethylene powder, polystyrene powder, cellulose powder, inorganic white pigment, inorganic red pigment, mica titanium pearl pigment, talc coated with titanium oxide, pearl pigment such as coloring mica titanium pearl pigment, and organic pigment such as red 201 and red 202.

Examples of the ultraviolet absorber include p-aminobenzoic acid, phenyl salicylate, isopropyl p-methoxycinnamate, octyl p-methoxycinnamate, and 2, 4-dihydroxybenzophenone.

Examples of the ultraviolet shielding agent include titanium oxide, talc, carmine, bentonite, kaolin, and zinc oxide.

Examples of the humectant include polyethylene glycol, propylene glycol, dipropylene glycol, 1, 3-butylene glycol, glycerin, diglycerin, polyglycerol, xylitol, maltitol, maltose, sorbitol, glucose, fructose, sucrose, lactose, sodium chondroitin sulfate, sodium hyaluronate, sodium lactate, pyrrolidone carboxylic acid, and cyclodextrin.

Examples of the pharmaceutically active ingredient include vitamin A compounds such as vitamin A oil and retinol, and vitamin B compounds such as riboflavin₂Class B of pyridoxine hydrochloride₆Vitamins C such as L-ascorbic acid, L-ascorbic acid phosphate, L-ascorbic acid monopalmitate, L-ascorbic acid dipalmitate, L-ascorbic acid 2-glucoside, pantothenic acid such as calcium pantothenate, vitamin D₂Vitamin D₃And the like; vitamins such as vitamin E including α -tocopherol and vitamin E, DL- α -tocopherol nicotinate.

Examples of the other active ingredients include skin whitening agents such as glutathione and saxifrage extract, skin activating agents such as royal jelly and beech extract, blood flow promoters such as capsaicin, zingerone, cantharidin, ichthammol, caffeine, tannic acid and γ -oryzanol, anti-inflammatory agents such as glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives and azulene, amino acids such as arginine, serine, leucine and tryptophan, maltose-sucrose condensation products which are resident bacteria control agents, lysozyme chloride, and the like.

Further, various extracts such as chamomile extract, parsley extract, wine yeast extract, grapefruit extract, honeysuckle extract, rice extract, grape extract, hop extract, rice bran extract, loquat extract, cork tree extract, coix seed extract, swertia herb extract, sweet clover extract, birch extract, licorice extract, peony extract, soapwort extract, luffa extract, capsicum extract, lemon extract, gentian extract, perilla extract, aloe extract, rosemary extract, sage extract, thyme extract, tea extract, seaweed extract, cucumber extract, clove extract, carrot extract, aesculus hippocastanum extract, witch hazel extract, mulberry extract, and the like can be exemplified.

The whitening oral preparation can be prepared by directly using the compound of formula (1), or adding various nutritional ingredients to foods and beverages, or can be prepared into foods and beverages as materials for nutritional supplement food, functional food, health food, specific health food, or general food. For example, suitable auxiliaries such as starch, lactose, maltose, vegetable fat powder, cacao butter powder and stearic acid may be added.

The effective dose of the compound of formula (1) as an oral preparation for whitening skin is appropriately selected depending on the age, body weight, symptoms, administration route, administration schedule, preparation form, and the intensity of material activity of the subject, and is, for example, preferably 1 to 5000mg, and particularly preferably 10 to 1000mg per 1 day. It can also be administered in several times on 1 day.

The amount of the compound of formula (1) to be blended in the whitening agent is preferably 0.00001 to 80% by mass, but is not limited thereto, and may be 0.0001 to 80% by mass.

Examples

[ test for inhibiting melanin production ]

For the compound of chemical formula (1), the melanin production-inhibiting activity was determined by the following assay.

The compound of formula (1) was used as a sample, arbutin was used as a positive control, hydroxytyrosol, hydroquinone, kojic acid, and ascorbic acid 2-glycoside were used as comparative samples, and 10mg/ml of each was prepared from dimethyl sulfoxide.

The melanin production-inhibiting activity was determined by the following method.

A mouse-derived B16 melanoma F10 was inoculated to 20% and the resulting mixture was inoculated with [ NLE4, D-PHE7] alpha-MSH (SIGMA-ALDRICH Co., Ltd.), and each sample of a compound of formula (1), arbutin, hydroxytyrosol, hydroquinone, kojic acid, and ascorbic acid 2-glycoside so that the concentration of the sample in the culture medium became 1.56. mu.M to 400. mu.M. Each sample was added, cultured for 96 hours, the culture medium was removed, and the cells were washed with PBS. The amount of melanin production by the cells was determined by colorimetric quantification. The melanin production amount when each sample was added at each concentration was calculated by taking the melanin production amount when only [ NLE4, D-PHE7] alpha-MSH was treated as 100%.

The melanin production (%) was determined by the following equation, using the quantitative value obtained when only [ NLE4, D-PHE7] alpha-MSH was treated As A and the quantitative values obtained when the samples were added at the respective concentrations As As.

Melanin production (%) ═ As/Ax 100

The results are shown in Table 1. IC50 (concentration of the sample in the culture medium when the amount of melanin production was 50%) that inhibited the production of melanin was determined from the measured values of the amount of melanin production at each sample concentration. IC50 for the inhibition of melanin production is plotted in fig. 1. In addition, in the case of the system in which kojic acid and ascorbic acid 2-glycoside were added, since melanin production could not be inhibited in the concentration range of the experimental system, IC50 for inhibiting melanin production could not be obtained.

TABLE 1

[ cell survival assay (MTT assay) ]

The compound of formula (1) was used as a sample, arbutin was used as a positive control, hydroxytyrosol, hydroquinone, kojic acid, and ascorbic acid 2-glycoside were used as comparative samples, and 10mg/ml of each was prepared from dimethyl sulfoxide.

Cell viability was determined by MTT assay. A mouse-derived B16 melanoma F10 was inoculated to 20% and the resulting mixture was inoculated with [ NLE4, D-PHE7] alpha-MSH (SIGMA-ALDRICH Co., Ltd.), and each sample of a compound of formula (1), arbutin, hydroxytyrosol, hydroquinone, kojic acid, and ascorbic acid 2-glycoside so that the concentration of the sample in the culture medium became 1.56. mu.M to 400. mu.M. After 96 hours of culture, the cells were treated according to a usual experimental method of the MTT assay, and the cell viability was measured. The cell viability when each sample was added at each concentration was calculated by taking the cell viability when only [ NLE4, D-PHE7] alpha-MSH was treated as 100%. The cell survival rate (%) was determined by the following equation, where the quantitative value obtained by treating only [ NLE4, D-PHE7] α -MSH was B, and the quantitative value obtained by adding the sample at each concentration was Bs.

(Bs/bx 100) cell survival rate [% ]

The results are shown in Table 2. The IC50 of cytotoxicity (concentration of the sample in the culture medium at 50% cell viability) was determined from the measurement values of cell viability at each sample concentration. The cytotoxic IC50 is plotted in figure 1. In addition, in the case of the system to which ascorbic acid 2-glycoside was added, the cell viability was not reduced in the concentration range of the experimental system, and therefore, IC50 indicating cytotoxicity could not be obtained.

TABLE 2

[ safety factor ]

The safety factor was determined by dividing the cytotoxic IC50 by the IC50 value that inhibited melanin production. The greater the safety factor, the more potent it is at low cytotoxicity to inhibit melanin production. The compound of formula (1) has a higher safety factor than arbutin and is excellent as a whitening agent. The safety factor is shown in table 3, plotted as shown in fig. 2. In addition, as for kojic acid and ascorbic acid 2-glycoside, IC50 inhibiting melanin production and cytotoxicity could not be obtained, and therefore, the safety factor could not be obtained.

TABLE 3

	A compound of formula (1)	Arbutin	Hydroxytyrosol	Hydroquinone
					Factor of safety	39.4	11.8	1.3	0.8

From the above results, it is understood that the compound of formula (1) in the present invention exhibits the effect of suppressing melanin production in a concentration range of 1.5625 to 25 μ M, since the melanin production is 53 to 23% and is significantly less than the other 5 compounds. In addition, the melanin production inhibiting effect is remarkable compared with arbutin, hydroxytyrosol, kojic acid and ascorbic acid 2-glucoside at a concentration of 50-100 mu M. Further, the compound of formula (1) showed melanin production-inhibiting effect even at a lower concentration than the other 5 compounds because IC50(μ M) for melanin production-inhibiting effect was 1.8 μ M, which was significantly smaller than the other 5 compounds. Further, regarding the cytotoxicity IC50, the compound of formula (1) in the present invention has the same or higher value than hydroxytyrosol and hydroquinone, and has low toxicity.

From the above data, it is shown that the safety factor of the compound of formula (1) of the present invention is 39.4, the safety is about 4 times higher than that of arbutin 11.8, and the safety is more excellent than that of the compound of hydroxytyrosol and hydroquinone having a value of about 1, as shown in table 3 and fig. 2, in which the cytotoxicity per 1 unit IC50 of the IC50 value for inhibiting melanin production is obtained.

From the above results, it is understood that the compound of formula (1) of the present invention, when contained in a melanin production inhibitor and a whitening agent, has a stronger melanin production-inhibiting effect than other compounds having a melanin production-inhibiting effect, and is a more safe cosmetic that exhibits a whitening effect.

[ melanin production inhibition test Using three-dimensional skin model ]

For the compound of chemical formula (1), the melanin production-inhibiting effect in the three-dimensional skin model was observed by the following test.

(preparation of sample solution)

The compound of the formula (1) was prepared with dimethylsulfoxide (049-. The concentration of the compound of formula (1) in the sample solution was 0.05 mass%. Furthermore, a solution containing no compound of formula (1) and dimethyl sulfoxide added at a concentration of 0.5% by mass in D-PBS-was used as a control.

(culture of three-dimensional skin model and addition of sample solution)

Three-dimensional skin model MEL-300A (Lot #10892, MatTek Corp.) was used. MEL-300A is a three-dimensional model of human skin consisting of normal human epidermal keratinocytes containing normal human epidermal melanocytes. The three-dimensional skin model was cultured (cornified) starting according to the kit instructions. Media EPI-100-LLMM maintenance medium (Lot #302410PND, MatTek Corp.) was used. Culturing was performed by overlapping Stelile Washers (MatTek Corp.) every 2 cells at the center of a 6-well plate, adding 5ml of each of the EPI-100-LLMM maintenance medium, and placing a skin model Cup (Cup) thereon.

The prepared sample solutions were applied to 50. mu.l of each Cup (Cup) surface of a MEL-300A three-dimensional skin model, and used as a model for cosmetics. The medium was replaced with a new medium after 2 days and 4 days, and the culture was carried out for 5 days (n-3). Sample solution after 2 days and 4 days, the old sample solution was removed by suction, and after washing with D-PBS, the sample solution was replaced with a new one to be used as a model for cosmetic.

(Observation of the bottom surface of three-dimensional skin model)

After 5 days from the start of the culture, the bottom surface of the three-dimensional skin model was observed for the pigmented melanocytes. The three-dimensional skin model Cup (Cup) during culture was observed by imaging with a CCD camera using an inverted microscope. The magnification in observation was 100 times. Fig. 3a shows a bottom image of the three-dimensional skin model to which the control was applied, and fig. 3b shows a bottom image of the three-dimensional skin model to which the sample solution of the compound of chemical formula (1) was applied. When a sample solution of the compound of formula (1) was administered, the melanocytes were light in color and the deposited melanin was less than that of the control.

(observation of the bottom surface of the three-dimensional skin model after image processing and measurement of the melanin covered area)

To quantitatively compare the melanotic regions, the melanin covered area of the bottom surface of the three-dimensional skin model was quantified by image analysis. The image of the bottom surface of the three-dimensional skin model is converted into a gray mode through computer software (Adobe photoshop), and background values of all the images are unified by using 256 gradations of background gradations in the image. Thereafter, the Image was binarized using 180 steps as the steps of the portion stained with melanin as a threshold value, and the black portion was digitized as the melanin covered area of the bottom surface of the three-dimensional skin model using the Analyze PartiCle function of the Image analysis computer software (Image J).

FIG. 3c shows the binarized image of the melanised area to which the control was applied, and FIG. 3d shows the binarized image of the melanised area to which the sample solution of the compound of formula (1) was applied.

The comparison of the melanin covered areas of the bottom surfaces of the three-dimensional skin models was performed by percentage with the total of the melanin covered areas of the bottom surfaces of the control and control regions as 100%, and was statistically processed using Student-T-test. The results are shown in fig. 4 (comparison of the coverage area of melanin on the bottom surface when the sample solution (0.05% of the compound of formula (1)) and the control (0% of the compound of formula (1)) were administered (Mean ═ s.d.n ═ 3)).

The sum of the melanin covered areas on the bottom surface of the three-dimensional skin model to which the sample solution of the compound of chemical formula (1) was administered was 48% of that of the control.

According to the test for suppressing melanin production using the three-dimensional skin model, it was confirmed that the compound of formula (1) not only reduces the amount of melanin production but also has the effect of reducing the area of the melanin-deposited part, that is, the area of melanin coverage, that is, the area where speckle, freckle and blackened skin color are generated, is reduced or becomes lighter even by visual observation, and thus it was also confirmed that the whitening effect is exhibited when the compound of formula (1) is blended in a cosmetic.

The following shows a formulation example of the present invention. The production was carried out according to the conventional method.

Prescription example 1 whitening lotion

Ingredient mixing amount (% by mass)

1. Glycerol 10

2, 1, 3-butanediol 5

3. Glucose 2

4. Ethanol 5

5. Carboxyvinyl 0.02

6. Dipotassium glycyrrhizinate 0.1

7. Sodium hyaluronate 0.001

8. Compound of formula (1) 0.1

9. Citric acid 0.05

10. Sodium citrate 0.1

11. 0.01 part of potassium hydroxide

12. Pure water surplus

Prescription example 2 whitening cream

Ingredient mixing amount (% by mass)

1. Stearyl alcohol 6

2. Stearic acid 2

3. Squalane 10

4. Octyl dodecanol 5

5. Olive oil 5

6.1, 3-butanediol 8

7. Polyethylene glycol 15004

POE (25) Hexadecyl Ether 3

9. Glyceryl monostearate 2

10. Compound of formula (1) 0.1

11. Pure water surplus

Prescription example 3 whitening mask

Ingredient mixing amount (% by mass)

1. Polyvinyl alcohol 15

2. Carboxymethyl cellulose 5

3, 1, 3-butanediol 5

4. Ethanol 12

5. Compound of formula (1) 0.05

POE oleyl alcohol Ether 0.5

7. Citric acid 0.02

8. Sodium citrate 0.04

9. Pure water surplus

Formulation example 4 whitening tablet

Ingredient mixing amount (% by mass)

1. Compound 63 of formula (1)

2. Lactose 24

3. Corn starch 12

4. Guar gum 1

Formulation example 5 whitening beverage

Ingredient mixing amount (% by mass)

1. Compound 10 of formula (1)

2. Fructose glucose liquid sugar 15

3. Citric acid 10

4. Vitamin C5

5. Fragrance 1

6. Pigment 1

7. Pure water surplus

Claims (2)

1. Use of a compound of the following chemical formula (1) as an active ingredient in the preparation of a melanin production inhibitorChemical formula (1).

2. Application of compound containing the following chemical formula (1) in preparing whitening agentChemical formula (1).