JP5718141B2 - Dendritic elongation promoter - Google Patents

Description

  The present invention relates to an extension accelerator for dendrites of melanocytes.

  Melanocytes are migratory, migratory, dendritic cells from the neural crest, and are distributed in the basal layer and hair matrix in the skin. Melanocytes have organelles called melanosomes, and synthesize melanin within these melanosomes. Synthesized melanin matures and is donated to adjacent basal cells and spinous cells with the involvement of intermediate fibers. The donated basal cells gather melanosomes into nuclear information and form a square cap to protect DNA from ultraviolet rays.

Melanin synthesis is promoted by UV stimulation. Melanocytes that have been irradiated with ultraviolet rays produce α-MSH, and the stimulation thereof promotes the synthesis of melanin synthases, tyrosinase, TRP-1, and TRP-2 (Non-patent Document 1).
Dendritic elongation plays an important role in transporting melanosomes from melanocytes. Due to the extension of dendrites, the melanocyte motor proteins MyosinVa, Rab27, and melanophylline form a complex and are known to be excreted from Slp2-a at the tip of dendrites via F-actin. (Non-patent document 2). Α-MSH is known as a substance that promotes the extension of dendrites (Non-patent Document 3). In addition, it is known that the dendritic elongation action exists in plant extracts belonging to the genus Apiaceae (Patent Document 1). Furthermore, it is known that this action also exists in a xanthan derivative contained in a plant belonging to the genus Gentianaceae (Patent Document 2).

  Synthesized melanin is transported to keratinocytes via elongated dendrites. At this time, if dendrite elongation is hindered and transfer is hindered, it is said that melanocytes cause cell death, melanin production is hindered, and vitiligo is generated. The greatest cause of melanin transfer hindrance is inhibition of melanocyte dendrite elongation (see, for example, Patent Document 3). From these findings, it is considered that controlling the extension of dendrites is important for maintaining the health condition of human skin.

In recent years, various studies have been conducted on plant-derived compounds. The inventors have also filed a patent application by finding that the compound represented by the structure of the chemical formula (1) suppresses melanocyte production of melanocytes and brings a whitening effect (Patent Document 4).

JP 2005-1327845 A JP 2005-2056 A Japanese Unexamined Patent Publication No. 01-207225 JP 2011-46683 A

Yuji Yamaguchi, Vincent J. Hearing Biofactors. (2009); 35 (2): 193-199. Physiological factors that regulate skin pigmentation Kuroda, T.S. and Fukuda, M. Rab27A-binding protein Slp2-a is required for peripheral melanosome distribution and elongated cell shape in melanocytes.Nature Cell Biol. (2004) 6, 1195-1203 Yuji Yamaguchi, Vincent J. Hearing Biofactors. (2009); 35 (2): 193-199. Physiological factors that regulate skin pigmentation

  An object is to provide a dendrite elongation promoter and a melanin metabolism promoter.

The present invention has the following configuration.
(1) 2,2 ', 3,3'-Tetrahydro-5,5', 7,7'-tetrahydro-2,2'-bis (4-hydroxyphenyl) -3,3 represented by chemical formula (1) A dendrite elongation promoter containing '-bi [4H-benzopyran] -4,4'-dione as an active ingredient.

  In this patent specification, the compound of the chemical formula (1) used in the present invention may be referred to as kamejasmin for convenience.

  The present invention has the effect of extending the dendrites of melanocytes by administering the compound of the above chemical formula (1) to HIF. The extension of dendrites accelerates the transfer of melanin to melanocytes and promotes melanin metabolism. The transfer of melanin to the basal layer of Hif is promoted, and pigmentation due to sunburn etc. is quickly recovered. Also, melanin transfer to melanocytes is promoted to improve leukoplakia.

The graph which measured the change of the melanin amount inside and outside a cell when kamejasmin was administered to a human melanocyte. The graph which measured the ratio of the melanin which appears extracellularly when camejasmine is administered to a human epidermal melanocyte. The graph which shows the cell survival rate of the human epidermal melanocyte administered to a kamejasmin. The image which observed the extension of dendrite by kamejasmin administration. The graph which measured the extension of the dendrite of the melanocyte by the administration of kamejasmin. The image which observed the mode of the melanocyte of the tissue bottom in the culture | cultivation 15th day after administration of camejasmine to a three-dimensional skin model. Cell viability of the three-dimensional skin model when turtle jasmine is added. An image of the distribution of melanosomes in the tissue when camejasmine was added to a three-dimensional skin model. The arrows in the figure indicate the positions of melanin granules.

Hereinafter, embodiments of the present invention will be described in more detail.
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'-bi [4H-1- benzopyran] -4,4'-dione, formula _is C _{30 H} 22 _{O 10.}
The compound of chemical formula (1) is a compound in which two molecules of flavanone are bonded to each other at the 3-position of the chromanone ring.

  A commercial item can be used for the compound of Chemical formula (1), for example, can be purchased from AnalytiCon.

  In using the dendrite elongation promoter and melanin metabolism promoter of the present invention, the agent of the present invention can be prepared by mixing with bases and additives of various cosmetics or external preparations.

  The dendrite elongation accelerator and melanin metabolism promoter of the present invention can be used in various known forms and applications such as cosmetics, creams, lotions, packs and the like. Further, the dendrite elongation promoter and melanin metabolism promoter of the present invention include skin external medicines and quasi drugs. Moreover, it is good also as an oral agent, an injection, and an oral medicine.

The dendrite elongation promoter and melanin metabolism promoter containing the compound of the chemical formula (1) of the present invention can be produced according to a formulation method that is usually used.
Examples of the dendritic elongation promoter and melanin metabolism promoter of the present invention include fats and oils such as vegetable oils, higher fatty acids, higher alcohols, silicones, anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. , Preservatives, sugars, sequestering agents, polymers such as water-soluble polymers, thickeners, powder components, UV absorbers, UV blockers, moisturizers such as hyaluronic acid, perfumes, pH adjusters Etc. can be contained. Vitamins, skin activators, blood circulation promoters, resident bacteria control agents, active oxygen scavengers, anti-inflammatory agents, other whitening agents, and other medicinal components such as bactericides, and physiologically active components can also be included.

  Examples of the oils and fats include liquid oils 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, triglycerin, glyceryl trioctanoate, and cocoa butter , Coconut oil, hydrogenated coconut oil, palm oil, palm kernel oil, molasses, mollusc kernel oil, hydrogenated oil, hydrogenated castor oil, beeswax, candelilla wax, cotton wax, nuta wax, lanolin, lanolin acetate, liquid lanolin And waxes such as sugarcane wax, liquid paraffin, squalene, squalane, and microcrystalline wax.

  Examples of higher fatty acids 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 cetostearyl alcohol, and branched chain alcohols such as monostearyl glycerin ether, lanolin alcohol, cholesterol, phytosterol, and octyldodecanol.

  Examples of the silicone include linear polysiloxanes such as dimethylpolysiloxane and methylphenylpolysiloxane, and cyclic polysiloxanes such as decamethylpolysiloxane.

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

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

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

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

  Examples of preservatives include methyl paraben and ethyl paraben.

  Examples of the sequestering agent include edetates such as disodium ethylenediaminetetraacetate, edetic acid, and sodium edetate.

  Examples of polymers include gum arabic, gum tragacanth, galactan, guar gum, carrageenan, pectin, agar, quince seed, dextran, pullulan, carboxymethyl starch, collagen, casein, gelatin, methylcellulose, methylhydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose Examples thereof include vinyl polymers such as sodium (CMC), sodium alginate, carboxyvinyl polymer (such as CARBOPOL), and bentonite.

  Examples of the thickener include carrageenan, gum tragacanth, quince seed, casein, dextrin, gelatin, CMC, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxyvinyl polymer, guar gum, xanthan gum and the like.

  Examples of the powder component include talc, kaolin, mica, silica, zeolite, polyethylene powder, polystyrene powder, cellulose powder, inorganic white pigment, inorganic red pigment, titanium oxide coated mica, titanium oxide coated talc, and colored titanium oxide coated mica. And organic pigments such as Red No. 201 and Red No. 202.

  Examples of the ultraviolet absorber include paraaminobenzoic acid, phenyl salicylate, isopropyl paramethoxycinnamate, octyl paramethoxycinnamate, 2,4-dihydroxybenzophenone, and the like.

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

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

Examples of medicinal components include vitamin A oils such as vitamin A oil and retinol, vitamin B ₂ such as riboflavin, B ₆ such as pyridoxine hydrochloride, L-ascorbic acid, L-ascorbic acid phosphate, L- Vitamin Cs such as ascorbic acid monopalmitate, L-ascorbic acid dipalmitate, L-ascorbic acid-2-glucoside, pantothenic acids such as calcium pantothenate, vitamin Ds such as vitamin D ₂ and cholecalciferol Vitamins such as vitamin E such as α-tocopherol, tocopherol acetate, DL-α-tocopherol nicotinate;

  Other medicinal ingredients include whitening agents such as glutathione and yukinoshita extract, skin activators such as royal jelly and beech tree extract, blood circulation such as capsaicin, gingeron, cantalis tincture, ictamol, caffeine, tannic acid, and γ-oryzanol. Accelerators, glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives, anti-inflammatory agents such as azulene, amino acids such as arginine, serine, leucine and tryptophan, maltose sucrose condensates of resident bacteria control agents, lysozyme chloride and the like.

  In addition, chamomile extract, parsley extract, wine yeast extract, grapefruit extract, honeysuckle extract, rice extract, grape extract, hop extract, rice bran extract, loquat extract, buckwheat extract, yakuinin extract, assembly extract, merilot extract, birch extract, licorice extract, peony extract , Savonso extract, loofah extract, capsicum extract, lemon extract, gentian extract, perilla extract, aloe extract, rosemary extract, sage extract, thyme extract, tea extract, seaweed extract, cucumber extract, carrot extract, carrot extract, maronier extract, Examples include various extracts such as chammel extract and mulberry extract.

As an oral preparation, the compound of the chemical formula (1) is added as it is, or various nutritional components are added, or it is included in foods and drinks, so that nutritional supplements, functional foods, health foods, foods for specified health use or normal foods Can be used as a material. For example, suitable auxiliaries such as starch, lactose, maltose, vegetable oil powder, cocoa butter powder, stearic acid and the like can be added.
The effective dose of the compound of the chemical formula (1) as an oral agent is appropriately selected and determined according to the age, weight, symptom, administration route, administration schedule, formulation form, strength of activity of the material, etc. of the subject. For example, 1 to 5000 mg per day is preferable, and 10 to 1000 mg is particularly preferable. This may be divided into several times a day.

  Hereinafter, the present invention will be described in more detail with reference to examples, but it is needless to say that the present invention is not limited to such examples.

<Example 1>
Effect of turtle jasmine on melanin excretion (promoting melanin metabolism) from normal human skin melanocytes
[Method]
Normal human skin melanocyte PDL12 was used as the cell. Cells were seeded on a 24-well plate using 1 ml of 2 × 10 ⁵ cells / well medium. After 24 hours of cell attachment, 0.0 μg / ml of Medium 256 supplemented with α-MSH 100 nM, HMGS, and Penisilin streptmysin 1% was used. Kamejasmin (NP-007193 manufactured by AnalytiCon) was added to 0.4 μg / ml, 2.0 μg / ml, and 10.0 μg / ml and cultured for 48 hours. The DMSO concentration was adjusted to a final concentration of 0.5%.
After completion of the culture, the medium and cells were collected, and the MTT test was performed on 3 specimens in each test group. After collecting the medium, 2% SDS-PBS was added in the same amount as the collected medium to prepare a sample for measuring the amount of melanin. The cells were added with 2 ml of 1% SDS and 50% Medium 256 PBS, and the cells were pulverized with an ultrasonic crusher to prepare a sample for measuring the amount of melanin.
The amount of melanin was measured using a dot blot method. Using a nitrocellulose membrane (Hybond-C Extra), 100 μl of each melanin measurement sample was added with a dot blot apparatus (Bio-dot BioRad), and melanin was blotted on the membrane. The blotted melanin was subjected to image analysis based on the number of pixels using ImageJ, and the amount of melanin contained in the sample was measured. The value of the amount of melanin was determined as a relative value with the total amount of intracellular and extracellular melanin in the test group to which no chamejasmin was added being 100%.
Regarding the cell viability, the cell viability when no chamejasmin was added was taken as 100% from the MTT value, and the value of each test group was determined as a relative value.
In addition, as a result of analyzing the three-dimensional structure of kamejasmin, it was the structure of chemical formula (2).

[result]
The measurement results of intracellular and extracellular melanin are shown in Table 1 and FIG.

The total amount of melanin, which is the sum of the amount of intracellular melanin and the amount of melanin in the medium, was not changed by the addition of kamejasmin as clearly shown in Table 1 and FIG. However, the intracellular melanin content decreased in a concentration-dependent manner, and the melanin content in the medium increased in a concentration-dependent manner. As shown in FIG. 2, the amount of melanin in the medium is significant in the groups to which 2.0 μg / ml and 10.0 μg / ml of kamejasmin are added, compared to the amount of melanin in the group to which no chamejasmin is added. The amount of melanin increased. Further, it was confirmed that the extracellular melanin did not appear in the culture medium due to cell death as shown in FIG.
In melanocytes, it is known that melanin is produced intracellularly and excreted outside the cell through the dendrite of the cell. The fact that there are more melanins outside the cells than in the cells has confirmed that kamejasmin has the action of promoting the excretion of melanin out of the cells. Melanin can cause stains if it accumulates in cells. The agent of the present invention has an effect of improving such spots.

<Example 2>
Morphological changes and dendritic elongation of turtle jasmine
[Measuring method]
Normal human skin melanocyte PDL12 was used as the cell. Cells were seeded at 2 × 10 ⁵ cells / well in a 24-well plate, seeded with 1 ml of Medium256 supplemented with HMGS and Penisilin streptmysin 1%, and after 24 hours of cell attachment, α-MSH100nM and kamejasmin were added. The cells were cultured for 48 hours. The concentrations of kamejasmin were 0.0 μg / ml, 0.4 μg / ml, 2.0 μg / ml, 10.0 μg / ml, and the DMSO concentration was 0.5%. After completion of the culture, photographing under a microscope was performed to confirm the state of dendrite. In addition, the length of the dendrite was measured at random for 15 cells under the respective conditions of adding kamejasmin. For the measurement of dendrite length, the total length of dendrites from one cell was taken as the dendrite length for each cell. The length of dendrites was analyzed by analysis of variance, and compared with Dunnett's test when no chamejasmin was added.

[result]
An image obtained by observing dendrite elongation is shown in FIG. As is clear from FIG. 4, melanocytes increased the number of dendrites by adding kamejasmin, and elongated. Dendrites were most elongated at a camejasmin concentration of 10 μg / ml, and the control ratio was 201.8% as shown in the graph of FIG. From this, it was confirmed that kamejasmin has an action of extending the dendrites of melanocytes.
It is known that the extension effect of dendrite is caused by α-MSH produced by stimuli such as ultraviolet rays. α-MSH is known to promote melanin production, but it was confirmed that kamejasmin extends dendrites without promoting melanin production.

<Example 3>
Evaluation by 3D skin model
[Method]
The three-dimensional skin model is an in vitro test system that reproduces the structure of Hif. It has been confirmed that the results of this test system can be deduced to the skin.
The three-dimensional skin model was performed using MEL-300A (Lot # 10892, MatTek Corp.). In the 3D skin model, keratinization was started according to the instructions recommended by MEL-300. The medium was EPI-100-LLMM maintenance medium (Lot # 302410PND). As a sample, a kamejasmin solution and a negative control were used. The kamejasmin-dissolved sample solution was prepared by dissolving kamejasmin (NP-010092 AnalytiCon Discovery Co. Ltd.) with dimethyl sulfoxide (049-07213 Wako Pure Chemical Industries, Ltd.) and adjusting the concentration of dimethyl sulfoxide to 0.5%. It was obtained by the addition adjustment ^- PBS. The final concentrations of the kamejasmin-dissolved sample solution were 0.4 μg / ml, 2.0 μg / ml, 10 μg / ml, and 50 μg / ml. Negative controls dimethylsulfoxide concentration D-PBS such that 0.5% ^- was obtained by the addition adjustment. The final concentration of the negative control solution was 0.05% by weight.
The culture and sample addition conditions were as follows. The prepared sample solution was directly administered to 4 cups of 50 μl each in a MEL-300 skin model cup. The culture was performed by stacking two Stelile Washers (MatTek Corp.) at the center of each 6-well plate, adding 5 ml each of EIL-LMM maintenance medium, and placing a skin model cup thereon. The culture medium and sample were performed every 2 days and cultured for 15 days.
At the end of the culture, cell viability was measured by MTT test for 3 samples under each test condition. The MTT test was conducted according to the instruction manual of MatTek.
The melanocytes on the bottom of the tissue were observed 15 days after the start of the culture. For observation, the skin model cup was photographed with a CCD camera using an inverted microscope. The magnification at the time of observation was set to 100 times that of X10X10. An observation image of the tissue bottom is shown in FIG.
The three-dimensional skin model D-PBS ^- washed with, after 24 hours fixation in 10% neutral buffered formalin, paraffin embedded, were stained by HE staining after cut sections. After staining, the distribution of melanosomes was observed. An image confirming the distribution of melanosomes is shown in FIG.

[result]
As shown in the graph of FIG. 7, as a result of the assay using a three-dimensional skin model, no cytotoxicity was observed at a camejasmin concentration of 50 μg / ml or less. As shown in FIG. 6, the melanocyte morphological change in the three-dimensional skin model was extended by dendrite by the addition of kamejasmin, similar to the results obtained with monolayer cells. In addition, the melanin produced in the melanocytes is transferred to the keratinocytes by culturing using the three-dimensional skin model, and the image of FIG. 8 is observed to clearly transfer melanin to the keratinocytes as compared with the control. I was able to confirm. The arrow in the figure is the melanin granule, and it moves from the bottom of the three-dimensional skin model to the culture surface (basal layer to stratum corneum) as the kamejasmin concentration increases. From this, it was confirmed that kamejasmin has an action of promoting metabolism of melanin in the skin.

Below, the formulation example of this invention is shown. Each was produced according to a conventional method.
Formulation Example 1
Cosmetic lotion ingredients Amount (% by mass)
1. Glycerin 10
2. 1,3-butylene glycol 5
3. Glucose 2
4). Ethanol 5
5. Carboxyvinyl polymer 0.02
6). Dipotassium glycyrrhizinate 0.1
7). Sodium hyaluronate 0.001
8). Compound of chemical formula (2) 0.1
9. Citric acid 0.05
Ten. Sodium citrate 0.1
11． Potassium hydroxide 0.01
12． Purified water residue

Formulation Example 2 Beauty Cream Ingredient Amount (% by mass)
1. Stearyl alcohol 6
2. Stearic acid 2
3. Squalane 10
4). Octild decanol 5
5. Olive oil 5
6. 1,3-Butylene glycol 8
7). Polyethylene glycol 1500 4
8). POE (25) cetyl alcohol ether 3
9. Glyceryl monostearate 2
Ten. Compound of chemical formula (2) 0.1
11． Purified water residue

Formulation Example 3 Cosmetic Pack Ingredient Amount (% by mass)
1. Polyvinyl alcohol 15
2. Carboxymethylcellulose 5
3. 1,3-butylene glycol 5
4). Ethanol 12
5. Compound of chemical formula (2) 0.05
6). POE oleyl alcohol ether 0.5
7). Citric acid 0.02
8). Sodium citrate 0.04
9. Purified water residue

Formulation Example 4 Cosmetic Tablet Ingredient Amount (mass%)
1. Compound of chemical formula (2) 63
2. Lactose 24
3. Cornstarch 12
4). Guar gum 1

Formulation Example 5 Beauty Beverage Ingredient Amount (% by mass)
1. Compound of chemical formula (2) 10
2. Fructose butter sugar liquid sugar 15
3. Citric acid 10
4). Vitamin C 5
5. Fragrance 1
6). Dye 1
7). Purified water residue

Claims (1)

2,2 ', 3,3'-Tetrahydro-5,5', 7,7'-tetrahydro-2,2'-bis (4-hydroxyphenyl) -3,3'-bi represented by chemical formula (1) A dendrite elongation promoter containing [4H-benzopyran] -4,4'-dione as an active ingredient.