JP6716278B2 - Skin external preparation, wrinkle improvement promoter, sagging improvement promoter, matrix metalloproteinase gene expression inhibitor. - Google Patents

Description

The present invention relates to a preparation which suppresses the production of a matrix metalloprotease gene and is effective in improving wrinkles, sagging, etc.

Matrix metalloproteinase-1 (MMP-1) is known as an enzyme that decomposes collagen, which is a major constituent of the dermal extracellular matrix of skin.
It is considered that its expression is greatly increased by the irradiation of ultraviolet rays, contributes to the decrease/denaturation of collagen, and is a major factor such as the formation of wrinkles on the skin and the decrease in elasticity. Therefore, inhibiting the activity of MMP-1 is important for preventing and improving skin aging symptoms.
In addition, matrix metalloproteinases not only control the metabolism of dermal extracellular matrix, but are also deeply involved in cell infiltration and activation of physiologically active molecules such as cytokines, and their relationship with various diseases. It has been reported. For example, in the skin, transient excessive production of matrix metalloproteinases due to various cytokines, growth factors, cell interactions, etc., wounds, burns, ulcers, skin damage due to sunlight, blistering, moss, etc. It is known to be involved in various skin diseases such as scabies and granulation.
It is also effective in the prevention and treatment of arthritis and joint pain.
Furthermore, MMP-1 plays an important role in invasion and metastasis of cancer cells. For cancer cell invasion and metastasis, it is an important process to decompose extracellular matrix and invade tissues. Since cancer cells overexpress and secrete MMPs, they are deeply involved in invasion and metastasis of cancer cells. In particular, cancer cells that have passed through the basement membrane secrete MMP-1 to destroy the extracellular matrix mainly composed of type I, II, and III collagen, and reach the blood vessels or lymphatic vessels to reach the base of the blood vessels. It is known to destroy the membrane, invade into blood vessels, and metastasize to other organs.
Most of MMPs are produced as inactive precursors and are activated by other proteases such as MMPs. For example, since MMP-1 has a function of activating MMP-2 (Non-Patent Document 3), if the action of MMP-1 is suppressed, activation of MMP-2 is also inhibited, and as a result, The action of these MMP-2 can be suppressed. (Patent Document 1, Non-Patent Documents 1 to 3)

MMP-1 production inhibitor promoted in dermal fibroblasts by irradiation of ultraviolet rays, especially long wavelength ultraviolet rays (UVA), and contains one or more selected from compounds having sulfhydryl group and compounds having disulfide bond A type I matrix metalloprotease production inhibitor is disclosed. (Patent Document 2)
Matrix metalloprotease containing one or more selected from the group consisting of Touki and its related plants, scented plants, gardenia and its homologous plants, plant extracts of Dioscorea japonica, lipoic acid, its salts and its derivatives. Inhibitors are disclosed. (Patent Document 3)

A matrix metalloprotease inhibitor oral composition comprising a grape seed extract, which is used for preventing or treating periodontal disease, is disclosed. (Patent Document 4) Food for smokers or passive smokers for preventing or treating periodontal disease containing grape seed extract, vitamin C and vitamin E, which has a cell collagen synthesis promoting effect and a cell matrix metalloprotease inhibitory effect. Compositions or oral compositions are disclosed. (Patent Document 5)

(A) one or more selected from extracts containing natural polyphenols; and (B) one or more selected from the group consisting of vitamin C and its salts, vitamin E, vitamin A and beta carotene. Disclosed is a food composition for preventing or treating periodontal disease, which is particularly useful for smokers and has an inhibitory action on periodontal tissue cell-derived matrix metalloproteinase production by nicotine stimulation. (Patent Document 6)
Agents for inhibiting the approval of matrix metalloproteases or their precursors containing flavonoids derived from citrus are disclosed. (Patent Document 7)
However, some conventional matrix metalloprotease production inhibitors have insufficient inhibitory activity and some are unstable, and some of them are unsuitable for use as a material for cosmetics in terms of color and odor.

Fermented soymilk is also used as a raw material for cosmetics. (Patent Document 8)
However, there are concerns that allergic reactions may be caused by proteins with a large molecular weight contained in fermented soybeans, and some are unsuitable for use as a material for cosmetics.

JP, 2014-221758, A JP-A-2002-47178 JP, 2000-319155, A JP 2000-191487 A JP, 2002-29953, A JP, 11-302142, A JP, 2000-80035, A JP, 2003-310154, A

FEBS letters, NO. 441, 137-140, 1998, Scott KA et al. Cancer Research Today (2) Cancer Cell Biology, Yoshimi Takai, Tohru Akiyama Editor/The University of Tokyo Press/2006 Exp. Dermatol. No. 6, 199-213, 1997, Kahri VM. et al.

An object of the present invention is to suppress MMP-1 gene expression, suppress skin wrinkles, prevent a decrease in elasticity and prevent skin tarmi. Furthermore, it is also effective for various skin diseases such as wounds, burns, ulcers, skin damage due to sunlight, blistering, lichen, granulation, etc., and is also effective for the prevention and treatment of arthritis and joint pain in addition to the skin. Is to obtain a safer formulation.

As a result of diligent studies by the present inventors, it was found that the protein hydrolyzate of the fermented soybean has an action of suppressing the expression of the MMP-1 gene and is highly safe.
Details are given below.

Soybean is subjected to appropriate treatment such as crushing, boiling, and dissolution, and then fermented by adding bacteria. As the bacterium used for fermentation, any koji mold, yeast, lactic acid bacterium, Bacillus subtilis, blue mold, etc. can be used as long as it is a bacterium species used for fermentation of food and the like.
However, fermentation with lactic acid bacteria was effective.

The obtained fermented soybean product is sterilized and heated or stirred to hydrolyze the protein. If washing, degreasing, crushing, or the like is performed as a pretreatment for protein hydrolysis, protein hydrolysis proceeds smoothly. Therefore, it is added if necessary.
For the protein hydrolysis, either acid hydrolysis or proteolytic enzyme is used, but some of them can be used in combination.
First, the method of acid hydrolysis will be described.
Examples of the acid used here include hydrochloric acid, nitric acid, sulfuric acid, boric acid, phosphoric acid, acetic acid, citric acid, etc., but the degree of decomposition, the concentration of the hydrolyzate, and further the use of the hydrolyzate Select one or more inorganic acids according to.

When the hydrolysis is completed, the mixture is cooled if necessary, and the hydroxide is gradually added with stirring to neutralize.
Examples of the hydroxide used here include lithium hydroxide, potassium hydroxide, sodium hydroxide, calcium hydroxide, strontium hydroxide, and barium hydroxide, but the degree of decomposition, the concentration of the hydrolyzate, Furthermore, one or more hydroxides are selected depending on the application of the hydrolyzate.
Neutralization causes precipitation, which is sometimes removed. The precipitate is removed by decantation, centrifugation, filtration, etc.
If necessary, hydrophilic organics such as methanol, ethanol, n-propyl alcohol, isopropyl alcohol, butyl alcohol, ethylene glycol, propylene glycol, diethylene glycol, glycerin, trimethylolpropane, 1.3 butylene glycol, acetone and methyl ethyl ketone. The solubilized neutralized salt can be precipitated by selecting and adding one or more solvents.

After allowing this to stand at room temperature or low temperature (0 to 10° C.), one or more arbitrary methods such as decantation, centrifugation, and filtration were selected to remove precipitates and suspended matters.
After that, when a highly volatile hydrophilic organic solvent such as ethanol or propanol is used as a part or all of the hydrophilic organic solvent, depending on the method of using the hydrolyzate, reduced pressure, heating, or a combination thereof may cause a hydrophilic property. It is also possible to remove part or all of the organic solvent.
Furthermore, it is possible to remove the water content and use it as a dried product. For example, decompression or heating, a combination thereof, freeze-drying or the like is performed.

The method using a protein hydrolase, as a protein hydrolase of plant origin, papain, bromelin, ficin, and the like, as an enzyme of animal origin, trypsin, chymotrypsin, kallikrein, pancreatin and the like, in particular, Trypsin and α-chymotrypsin are preferred, and they can be used alone or in combination of two or more. As the enzyme of microbial origin, proteolytic enzymes produced by Aspergillus, Bacillus subtilis, actinomycetes and the like can be used alone or in combination of two or more.
Select the amount and temperature of the fermented soybean that has been adjusted to a pH according to the type of enzyme, and after hydrolysis, add heating, centrifugation, filtration, etc. to inactivate or remove the enzyme. To do.

The protein hydrolyzate of fermented soybean obtained by the above-mentioned operation or a solution thereof can be used for medicines, foods, and cosmetics. In particular, when it is used as an external preparation for skin such as cosmetics, it is particularly effective because it does not cause allergies due to a low salt concentration or a protein having a large molecular weight.

In addition to these hydrolyzates, raw materials that can be used in medicines, foods, and cosmetics can be used to prepare formulations, or these can be used by adding them to existing medicines, foods, and cosmetics. For example, the raw materials that can be used
Avocado oil, almond oil, fennel oil, sesame oil, olive oil, orange oil, orange lafer oil, sesame oil, cacao butter, chamomile oil, carrot oil, cucumber oil, beef tallow fatty acid, kukui nut oil, safflower oil, shea butter, liquid shea Fat, soybean oil, camellia oil, corn oil, rapeseed oil, persic oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, mokuro, palm oil, beef tallow, lard , Squalene, squalane, pristane or various oils and fats such as hydrogenated products (hardened oil etc.) of these oils and fats.
Waxes such as beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax and rice wax.
Mineral oils such as liquid paraffin, petrolatum, paraffin, ozokeride, ceresin, and microcristan wax.

Fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, and lanolin fatty acid.
Alcohols such as ethanol, isopyranol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, phenoxyethanol, 2-hexyldecanol, isostearyl alcohol, 2-octyldodecanol.
Ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene glycol, polypropylene glycol, 1,3-butylene glycol, pentyl glycol, glycerin, Polyhydric alcohols such as pentaerythritol, threitol, arabitol, xylitol, galactitol, sorbitol, lactitol and maltitol.

Isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate, diethyl phthalate, phthalate Esters such as dibutyl acid, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, and propylene glycol dioleate.
Metal soaps such as aluminum stearate, magnesium stearate, zinc stearate, calcium stearate and zinc palmitate.
Arabic gum, guaiac butter, karaya gum, tragacanth gum, quince seed, agar, casein, lactose, fructose, sucrose or its ester, trehalose or its derivative, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, ethylene oxide. Alkylene (C2-C4) oxide-added hydroxyalkyl (C2-C4) chitin or chitosan, low molecular weight chitin or chitosan, chitosan salt, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or a salt thereof, Hyaluronic acid or its salt, chondroitin sulfate or its salt, heparin, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, carboxyethyl cellulose sodium, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone. , Polyvinylmethacrylate, polyacrylic acid salts, polyalkylene oxides such as polyethylene oxide and polypropylene oxide or cross-linked polymers thereof, carboxyvinyl polymers, gums such as polyethyleneimine, sugars and water-soluble polymer compounds.
Alkyl carboxylate, alkyl sulfonate, alkyl sulfate ester salt, alkyl phosphate ester salt, alkyl amine salt, alkyl quaternary ammonium salt, carboxylic acid type amphoteric surfactant, sulfate ester type amphoteric surfactant, sulfonic acid type Amphoteric surfactant, phosphate ester type amphoteric surfactant, ether type nonionic surfactant, ether ester type nonionic surfactant, ester type nonionic surfactant, block polymer type nonionic surfactant, nitrogen-containing surfactant Type anionic surfactants such as nonionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants.

Retinol, retinal, dehydroretinal, carotene, lycopene, thiamine hydrochloride, thiamine sulfate, riboflavin, pyridoxine, cyanocobalamin, folic acid, nicotinic acid, pantothenic acid, biotin, choline, inositol, vitamin C or its derivative, ergocalci Ferrol, cholecalciferol, dihydrotaxosterol, vitamin E or its derivative, various vitamins such as ubiquinones or its derivative.
Valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, ornithine, histidine, etc. , Various amino acids such as their sulfates, phosphates, nitrates, citrates, or amino acid derivatives such as pyrrolidonecarboxylic acid.

Red sprout, Jihuang, Kidori, Jiran, one medicinal herb, ginkgo, oolong tea, fennel, turmeric, summer hay, citrus peel, eleuthero, kumiko, oren, white jelly, sagebrush, okra, ginseng or carrot ginseng (carrot), olive, oregano , Orange, chamomile or Roman chamomile, camu camu, guarana, magnolia, gyoryu, gooseberry, clara, cranberry, grapefruit, clove, kobushi, magnolia, gaisen, sakura, sashima, sanshini ginseng, wild mushroom, sansho, sandscon, Shikunshi, perilla perilla, shiso, peony, honeysuckle, horse chestnut, celery, senkyu, senburi, rhubarb, pearl oyster, pearl millet, ginseng, ginseng, beetle tea, tendaiyaku, horse mackerel, dokudami, eucommia, nematode, five times eel, jujube, jujube, jujube, jujube , Nobara, pearl barley, rose, hishimi, loquat, blackberry, prunes, horn, bow hu (windbreak), honoki, bodaiju, button, hops, jojoba, maou, maca, macadamia nuts, mulberry bark, bitter ginger, ganoderma lucidum, currant fruit , Myrobalan, swordfish, purple, maternity herb, evening primrose, melissa, merlot, mokko, yacon, eucalyptus, yukinoshita, yucca, yuzu, lily, yorogusa, mugwort, lavender, lemon, lemongrass, rosemary, earthworm, chlorella, chlorella, Various extracts of plants, seaweed, algae such as kelp, seaweed, giant kelp, hijiki, hibamatata, giraffe, asakusanori, susabinori, spirulina, ishige, iroro, fukuronori.

Placenta extract of horse or pig, silk protein and its degradation products or their derivatives, milk, casein and its degradation products or their derivatives, skim milk powder and its degradation products or their derivatives, lactoferrin or its degradation products, chicken egg components , Substances derived from animal sources such as fish meat degradation products and nucleic acid-related substances (ribonucleic acid, deoxyribonucleic acid).
Marine components Deep sea water, seawater salt, dried seawater, inorganic salts obtained from the Dead Sea or the Atlantic Ocean or the Pacific Ocean, marine products such as sea mud.
Yeast extract, bacterial metabolite, bacterial extract, mold or actinomycete metabolite, mold or actinomycete extract, natto metabolite, natto extract, rice fermented extract, rice bran (red bran, white bran) fermented extract, Fungal substances such as lactic acid fermented raw milk or skim milk, fermented lactic acid bacteria of legumes, fermented lactic acid bacteria of coconut plants.
Α-Hydroxy acids such as glycolic acid, citric acid, malic acid, tartaric acid and lactic acid.
Silica anhydride, magnesium silicate, talc, kaolin, bentonite, mica, titanium mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, calcium carbonate, magnesium carbonate, yellow iron oxide, red iron oxide, black iron oxide Inorganic pigments such as red, yellow oxide, chromium oxide, chromium hydroxide, carbon black, and calamine.
UV absorbers or blockers such as benzophenone derivatives, para-aminobenzoic acid derivatives, methoxycinnamic acid derivatives, salicylic acid derivatives, and urocanic acid derivatives.

Para-aminobenzoic acid derivative, salicylic acid derivative, anthranilic acid derivative, coumarin derivative, amino acid compound, benzotriazole derivative, hydroquinone or its derivative, nicotinic acid derivative, kojic acid or its derivative, oxybenzone, benzophenone, arbutin, guaiazulene, shikonin, baicalin , Whitening agents such as baicalein, berberine, placenta extract, ellagic acid, and rucinol.
Ictamol, indomethacin, kaolin, salicylic acid, sodium salicylate, methyl salicylate, acetylsalicylic acid, diphenhydramine hydrochloride, d-camphor, dl-camphor, hydrocortisone, guaiazulene, camazulene, chlorpheniramine maleate, glycyrrhizic acid or its salt, glycyrrhetinic acid or its salt. Salt anti-inflammatory agent.
Antibacterial/sterilizing/disinfecting agents such as acrinol, sulfur, calcium gluconate, chlorhexidine gluconate, and triclosan.
Musk, Civet, Castorium, Ambergris, Ylang Ylang Essential Oil, Iris Essential Oil, Fennel Essential Oil, Orange Essential Oil, Cardamom Essential Oil, Cinnamon Essential Oil, Geranium Essential Oil, Copaiba Balsam Essential Oil, Cedarwood Essential Oil, Jasmine Essential Oil, Rose Essential Oil, Bergamot Essential Oil, Lavender Essential Oil, Animal and vegetable flavors such as lemongrass essential oil, lemon essential oil, rosemary essential oil, and other synthetic flavors.
Photosensitizer No. 101, photosensitizer No. 201, photosensitizer No. 401, photosensitizer No. 301, hinokitiol, pantothenic acid or its derivatives, allantoin, pentadecanoic acid glyceride, urea, guanidine and other various agents.
Other hormones, sequestering agents, pH adjusting agents, etc. may be mentioned.

From these, various selections are made and used in any form. Examples of forms include capsules, powders, granules, solids, liquids, gels, bubbles, emulsions, creams, ointments, sheets and the like.

Example 1
To 200 g of soybean, an equal amount of water was added, and the mixture was immersed at room temperature for 12 hours, mashed with a mortar, and boiled for 5 minutes.
After cooling, the filtrate from which solids were removed by filtration was sterilized under high pressure, and then lactic acid bacteria were inoculated and cultured at 37° C. for 5 days.
The culture solution was sterilized under high pressure to obtain 300 g of a fermented soybean product.
300 g of the fermented soybean product was placed in a flask equipped with a condenser, 30 g of sulfuric acid was added, and the mixture was heated at 110° C. for 24 hours.
After cooling, 20 g of calcium hydroxide was gradually added and stirred for 1 hour.
Further, calcium hydroxide was gradually added to adjust the pH to 7.5.
This liquid was centrifuged (3000 rpm×15 minutes) to obtain a supernatant. To this was added twice the amount of ethanol and the mixture was stirred.
After standing at 4° C. for 14 days, it was filtered to obtain a filtrate.
This was evaporated and then freeze-dried.

The following experiment was conducted for Example 1.
Human normal fibroblasts (Kurabo) were seeded on a 6-well plate, and cultured in DMEM medium containing 10% FBS for 3 days as a preliminary culture. After culturing, an example was added and the cells were cultivated for 3 hours. As a control, the test was also conducted in a system in which the example was not added.

Extraction of RNA The extraction of Total RNA from the cells was adjusted according to the attached manual using PureLink (registered trademark) RNA Mini Kit (Life Technologies Japan). The RNA concentration was calculated using NanoDop1000 (Thermo Fisher scientific).

RT reaction and real-time PCR
Using the extracted Total RNA, RT reaction was performed according to the attached manual using High-Capacity RNA-to-cDNA Kit (Life Technologies Japan).
Real-time PCR was performed using 7500 Real-Time PCR System (Life Technologies Japan).
In detail, the above-mentioned RT product, MMP-1 and TaqMan (registered trademark) primer of GAPDH (Life Technologies Japan), TaqMan (registered trademark) Gene Expression Master Mix (Life Technologies Japan) were used as endogenous controls. Then, the gene expression levels were compared by the ΔΔCT method.

The results are shown in Figure 1.

In addition, as a result of preparing an external preparation containing the examples and actually using it, a preparation was obtained in which wrinkles on the skin were suppressed, elasticity was prevented from decreasing, and skin tarmi was prevented.

It is a figure which shows the result of an Example.

Claims (5)

A skin external preparation containing a protein hydrolyzate of a fermented soybean by a lactic acid bacterium as an active ingredient. The external preparation for skin containing the protein hydrolyzate as an active ingredient according to claim 1 , wherein the fermented soybean is a fermented product of a hot water extract. Wrinkle reduction promoter having for an active ingredient one of the protein hydrolyzate according to claim 1 or 2. Sagging improved promoter having for an active ingredient one of the protein hydrolyzate according to claim 1 or 2. Matrix metalloproteinase 1 gene expression inhibitor comprising as an active ingredient one of the protein hydrolyzate according to claim 1 or 2.