JP6846700B2 - Hyaluronan synthase gene expression promoter - Google Patents

Description

The present invention relates to a preparation that suppresses the decrease in skin elasticity and wrinkle formation, and prevents or improves arthritis.

It is known that a decrease in the amount of hyaluronic acid in the dermis is involved in aging phenomena such as a decrease in skin elasticity and wrinkle formation.
Hyaluronic acid is synthesized by hyaluronic acid synthase (hereinafter abbreviated as HAS). HAS1 mRNA, HAS2 mRNA, and HAS3 mRNA are known as enzymes involved in hyaluronic acid synthesis in vivo, but it is clear that hyaluronic acid synthesis is regulated mainly through the expression of HAS2 mRNA in fibroblasts. It has become.
Hyaluronic acid is known to be effective not only for reducing skin elasticity and wrinkle formation, but also for arthritis.
It has been reported that when degenerative arthritis occurs, the production of hyaluronic acid that lubricates the joints decreases, the destruction by protein enzymes increases, and the hyaluronic acid decreases in the joints. That is, as hyaluronic acid decreases in the joint, the joint cannot absorb or disperse the external impact, and the joint damage may become severe. From this, there is a method of injecting hyaluronic acid into the joint to alleviate arthritis, but ultimately, a method of increasing the synthesis of hyaluronic acid in the body is considered to be more effective.
Several substances are known to increase hyaluronic acid in the skin. (Patent Documents 1 and 2)
Collagen is widely used in external preparations for skin such as cosmetics, and collagen of shellfish and its hydrolyzate are also used in cosmetics. (Patent Documents 3 and 4)

Japanese Unexamined Patent Publication No. 2009-191039 Japanese Unexamined Patent Publication No. 2011-195493 Japanese Unexamined Patent Publication No. 2003-095854 Japanese Unexamined Patent Publication No. 2014-210766

An object of the present invention is a preparation for reducing skin elasticity, suppressing wrinkle formation, and preventing or ameliorating arthritis.

As a result of diligent studies by the present inventors, it was found that the collagen peptide is optimal for this purpose, and further fractionation revealed that the peptide of SEQ ID NO: 1 greatly promotes the expression of the hyaluronan synthase gene. It was.
Collagen is already known to be very useful for the skin, as mentioned above, but mammalian-derived collagen is something that some people don't want to use and can meet a variety of needs. As a result of diligent studies, it was found that collagen obtained from shellfish meat most closely matches the gist of the present invention. In addition, oysters, abalone, clams, etc. are used as food for shellfish, but in the case of pearl oysters, they are currently discarded as waste when pearl farming is completed. It is most effective to use it as a raw material for.
When pearl oysters are cultivated and the pearls are taken out, the pearl oysters are collected after removing the adductor muscles. Excluding the edible adductor muscle, the mantle contains a large amount of collagen, so if necessary, select the mantle from the shellfish.
The method for extracting collagen may be a known method. That is, for example, a method of adding water, stirring the mixture, and then separating the mixture with a centrifuge, or a method of allowing the mixture to stand and discarding the supernatant may be selected. In order to further remove impurities from this insoluble matter, salt solutions such as sodium chloride, potassium chloride, sodium acetate, sodium citrate, sodium hydrogen phosphate, etc., sodium hydroxide, potassium hydroxide, calcium hydroxide, aqueous ammonia, etc. A basic solution or a chelating agent such as ethylenediamine tetraacetate is added and stirred to collect insoluble matter. Collagen can be obtained by further removing impurities with a water-soluble organic solvent such as ethanol and then acid-extracting with acetic acid, citric acid, lactic acid, hydrochloric acid, phosphoric acid or the like. The concentration varies depending on the type of acid and various conditions, but the concentration is 0.01 to 2 mol. This solution is added and stirred for 2 to 48 hours to extract. The temperature is 30 ° C. or lower, preferably 2 to 10 ° C. In addition, after using an enzyme, extraction may be performed by a known method such as a method of extracting with an acid. Further, depending on the application, it may be better to further purify, and it is purified by using a hydrophilic organic solvent, a salting out method, or the like.
The collagen obtained as described above is hydrolyzed. The method of hydrolysis is to decompose by using proteolytic enzymes such as collagenase, papaine and pepsin, inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid, and organic acids such as acetic acid, lactic acid and succinic acid, alone or in combination of two or more. Remove unwanted enzymes and acids as needed. In addition, there is no problem even if a decomposition method such as an alkali other than an enzyme or an acid is used. One or a combination of these hydrolysis methods may be used to obtain the required molecular weight, but the required fraction may be obtained by using ultrafiltration, gel filtration, other chromatography and the like.
As a result of analyzing the results of analyzing the peptides obtained from these, it was found that the peptide of SEQ ID NO: 1 is very effective as a hyaluronan synthase gene expression promoter.

In addition to the peptide of SEQ ID NO: 1 or the peptide containing the peptide of SEQ ID NO: 1, a preparation may be prepared together with raw materials that can be used for pharmaceuticals, foods, and cosmetics, or these may be added to existing pharmaceuticals, foods, and cosmetics for use. You can also.
To give an example of available raw materials
Avocado oil, almond oil, uikyo oil, sesame oil, olive oil, orange oil, orange raffer oil, sesame oil, coconut oil, chamomile oil, carrot oil, cucumber oil, beef fat fatty acid, kukui nut oil, saflower 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, mokurou, palm oil, beef fat, pork fat , Squalene, Squalane, Prystan or various fats and oils such as hydrogenated additives (hardened oils, etc.) of these fats and oils.
Waxes such as beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, cellac wax, and rice wax.
Mineral oils such as liquid paraffin, petrolatum, paraffin, ozokelide, selecin, and microkristang wax.
Fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, bechenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, and lanolin fatty acids.
Alcohols such as ethanol, isopyropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, phenoxyethanol, 2-hexyldecanol, isostearyl alcohol, 2-octyldodecanol and the like.
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, tretol, arabitol, xylitol, galactitol, sorbitol, lactitol, and multitol.

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 acid, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, and propylene glycol dioleate.
Metallic soaps such as aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, and zinc palmitate.
Arabian gum, guayaku fat, karaya rubber, tragant rubber, quinseed, agar, casein, lactose, fructose, sucrose or its ester, trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, ethylene oxide 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, etc. Hyaluronic acid or its salt, chondroitin sulfate or its salt, heparin, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, sodium carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone , Polyalkylene methacrylate, polyacrylate, polyalkylene oxide such as polyethylene oxide and polypropylene oxide or a crosslinked polymer thereof, gum substance such as carboxyvinyl polymer and polyethyleneimine, saccharide or water-soluble polymer compound.

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 Anionic surfactants such as type nonionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants.

Retinol, retinal, dehydroretinal, carotene, lycopene, thiamine hydrochloride, thiamine sulfate, riboflavin, pyridoxin, cyanocobalamine, folic acids, nicotinic acids, pantothenic acids, biotins, choline, inositols, vitamin C or its derivatives, ergocalciferol Various vitamins such as ferrol, choleciferol, dihydrotachysterol, vitamin E or its derivatives, ubiquinones or its derivatives.
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. , Their sulfates, phosphates, nitrates, citrates, or various amino acids such as amino acid derivatives such as pyrrolidone carboxylic acid.

Red bud Kashiwa, Jihuang, Kidori, Jidori, Ichiyakuso, Ginkgo, Woolong tea, Uikyo, Ukon, Summer dead grass, Chenpi, Ezokogi, Sekimeiko, Ouren, White ginseng, Panax japonicus, Okura, Panax japonicus or Panax japonicus , Orange, chamomile or roma chamomile, camcam, galana, chenpi, gyoryu, goose berry, clara, cranberry, grapefruit, clove, kobushi, mokuren, reisen, cherry, sardine, ginseng, magnolia obovata, sansho, sandscon, Shikunshi, Shiso Shiso, Shiso, Shakuyaku, Watermelon, Magnolia obovata, Celoli, Senkyu, Senburi, Daiou, Tachijakousou, Chikusetsu carrot, Tengusa, Jincha, Tendaiuyaku, Tokusa, Dokudami, Tochu, Toneriko , Novara, Hatomugi, Rose, Ryomi, Biwa, Blackberry, Prune, Magnolia obovata, Bowfu (windproof), Magnolia obovata, Bodaiju, Button, Hope, Hohoba, Maou, Maca, Macademia nuts, Mulberry bark, Carrot, Mannentake, Ginseng , Mirobaran, Mukuroji, Murasaki, Ginseng, Mematsuyoigusa, Melissa, Merilot, Mokko, Yakon, Yukari, Yukinoshita, Yukka, Yuzu, Yuri, Yoroigusa, Yomogi, Lavender, Lemon, Lemongrass, Rosemary, Jiyoku, Chlorella, Chlorella, Various extracts of plants such as konbu, wakame, giant kelp, hijiki, hibamata, ginseng, asakusanori, susabinori, spirulina, ishige, iroro, and owl, seaweed, and algae.

Horse or pig placenta extract, silk protein and its degradation products or derivatives thereof, 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 , Fish meat decomposition products, nucleic acid-related substances (ribonucleic acid, deoxyribonucleic acid) and other animal-based raw material-derived substances.
Marine components Deep sea water, sea salt, dried sea water, inorganic salts obtained from the Dead Sea or the Atlantic or Pacific Ocean, sea mud and other marine products.
Yeast bacterium extract, bacterial metabolite, bacterial extract, mold or lactic acid bacterium metabolite, mold or lactic acid bacterium extract, natto bacterium metabolite, natto extract, rice fermented extract, rice bran (red bran, white bran) fermented extract, Bacterial-derived substances such as lactic acid fermented products of raw milk or non-fat dry milk, lactic acid bacteria fermented products of legumes, and lactic acid bacteria fermented products of coconut palm 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, mica titanium, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, calcium carbonate, magnesium carbonate, yellow iron oxide, red iron oxide, black iron oxide , Gunjo, Chromium Oxide, Chromium Hydroxide, Carbon Black, Caramin and other inorganic pigments.
Ultraviolet absorbing or blocking agents such as benzophenone derivatives, para-aminobenzoic acid derivatives, methoxycinnamic acid derivatives, salicylic acid derivatives, and urocanic acid induction.

Paraaminobenzoic acid derivative, sulcylic acid derivative, anthranic acid derivative, coumarin derivative, amino acid compound, benzotriazole derivative, hydroquinone or its derivative, nicotinic acid derivative, kodiic acid or its derivative, oxybenzone, benzophenone, albutin, guaiazulene, ciconin, baicalein , Baicalein, velverine, placenta extract, ellagic acid, lucinol and other whitening agents.
Ictamol, indomethacin, kaolin, salicylic acid, sodium salicylate, methyl salicylate, acetylsalicylic acid, diphenhydramine hydrochloride, d-camphor, dl-camphor, hydrocortisone, guaiazulene, chamazulene, chlorpheniramine maleate, glycyrrhizinic acid or its salts, glycyrrhetinic acid or its salts. Salt anti-inflammatory agent.
Antibacterial, bactericidal and disinfectants such as acrinol, sulfur, calcium gluconate, chlorhexidine gluconate and triclosan.
Jaco, Civet, Castorium, Ambergris, Iran Iran Essential Oil, Iris Essential Oil, Wikyo Essential Oil, Orange Essential Oil, Cardamon 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 Animal and vegetable fragrances such as lemongrass essential oil, lemon essential oil, rosemary essential oil, and other synthetic fragrances.
Various chemicals such as photosensitizer 101, photosensitizer 201, photosensitizer 401, photosensitizer 301, hinokitiol, pantothenic acid or a derivative thereof, allantoin, pentadecanoic acid glyceride, urea, and guanidine.
Other examples include hormones, sequestrants, pH regulators and the like.

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.

Next, the present invention will be described in detail with reference to examples.

Example 1. Extraction of atelocollagen The mantle was collected from raw pearl oysters, frozen and stored, and thawed in cold water at appropriate times for use in the following.
A 1M aqueous NaCl solution was added to the mantle for homogenization. This was centrifuged to obtain a precipitate. Next, this precipitate was dispersed in water and then centrifuged to obtain a precipitate. By repeating this, water-soluble components such as salts were removed.
This precipitate was stirred in a dilute aqueous citric acid solution for about 1 hour, and then adjusted to around pH 2.5 with citric acid if necessary. Pepsin having a weight ratio of about 1.0% based on the weight of the mantle was added and enzymatically decomposed for 15 hours or more under the condition of 10 ° C. or lower to atelolate collagen.
This was centrifuged and the supernatant was collected. A concentrated NaOH aqueous solution was added to the obtained supernatant to adjust the pH to 10, and the mixture was stirred overnight under the condition of 10 ° C. or lower to inactivate pepsin.
Next, citric acid was added to this solution to adjust the pH to 2.5 again, and then NaCl was gradually added for salting out. This was centrifuged to obtain a precipitate containing atelocollagen.
This precipitate was dissolved again in a dilute aqueous citric acid solution, and then the insoluble precipitate was removed by centrifugation or the like. This was salted out by the same operation as above to obtain a precipitate.
After dispersing this precipitate in hydrous ethanol, the precipitate composed of atelocollagen was recovered by centrifugation.

2. Peptidization of Atelocollagen To 1 mg of the collagen obtained above, 100 μl of 0.1% sodium dodecyl sulfate, 1 mM tris (2-carboxyethyl) phosphine, and 100 mM sodium hydrogen carbonate were added, and then the mixture was heated at 100 ° C. for 10 minutes. To 10 μl of this solution, 2 μl of 100 mM iodoacetamide was added and left at 37 ° C. for 1 hour, then 6 μl of 30% acrylamide, 1 μl of 10% ammonium persulfate and 1 μl of tetramethylethylenediamine were added, and the mixture was left at room temperature for 20 minutes. Gelled and finely chopped. This gel was washed by adding 10% acetic acid: methanol (= 1: 1), mixing for 10 minutes, and then discarding the supernatant. After repeating this washing operation three times, 100 mM ammonium hydrogen carbonate was added and mixed for 10 minutes. After removing the supernatant, acetonitrile was added to the gel to dehydrate the gel, and the gel was dried by removing acetonitrile. To this, 1 μl of 0.5 mg / ml trypsin proteomics grade (Sigma-Aldrich) was added, absorbed into a gel for 5 minutes, and then 40 μl of 100 mM ammonium hydrogen carbonate was added and reacted at 37 ° C. overnight. After collecting the supernatant, the peptide was extracted again with 50 μl of 50% acetonitrile and 0.1% trifluoroacetic acid, and combined with the previously collected supernatant to prepare a peptide fragment.

3. 3. Determination of Peptide Amino Acid Sequence The atelocollagen peptide fragment obtained above was analyzed using a liquid chromatography-mass spectrometer. A reverse phase column Cadenza CD-C18 (Intakt, φ2 mmx15 cm) was used for the analysis, and the analysis was performed at a flow velocity of 0.2 ml / min. After equilibration with 0.1% formic acid and 5% acetonitrile, the peptide was eluted with a concentration gradient of acetonitrile. At the same time, each peptide was subjected to a tandem mass spectrometer (MS / MS) to analyze the amino acid sequence, and the obtained sequence was subjected to X! Tandem analysis using the gene model of Akoyagai genome ver. 2.0 as a database to perform X! Tandem analysis of each peptide. The amino acid sequence was finalized.
As a result of synthesizing the obtained peptides having several amino acid sequences and conducting a confirmation test, it was found that the peptide of SEQ ID NO: 1 greatly increased the expression of the HAS2 gene at a low concentration.
The amino acid sequence determined by SEQ ID NO: 1 is Gln-Pro-Gln-Gly-Gln-Gln-Gly-Glu-Leu-Gly-Asp-Pro-Gly-Met-Hyp-Gly-Ala-Hyp-Gly-. Pro-Gln-Gly-Ala-Arg, but replaced Hyp with Pro, Gln-Pro-Gln-Gly-Gln-Gln-Gly-Glu-Leu-Gly-Asp-Pro-Gly-Met-Pro-Gly The following confirmation tests were performed using −Ala-Pro-Gly-Pro-Gln-Gly-Ala-Arg.

Confirmation test In the test, human fibroblasts were used, cultured to semiconfluent, replaced with a medium containing the test product, and cultured for 3 days. After RNA was extracted, cDNA was obtained by reverse transcription, and the expression level of the HAS2 gene was measured using a real-time PCR system (Applied Biosystems). GAPDH was used as the endogenous control.
The result is shown in FIG.

The peptide of SEQ ID NO: 1 can be used as it is as an external preparation, but it is also possible to prepare an external preparation by blending it with other raw materials.

The result of the HAS2 gene expression test of SEQ ID NO: 1 is shown. The gene expression level was shown with the control not containing SEQ ID NO: 1 as 1.

Claims (1)

SEQ ID NO: 1 as an active ingredient, comprising a peptide of hyaluronic acid synthase gene expression promoters described in the sequence listing.

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