JP2021169532A - Transglutaminase production promoter - Google Patents

Abstract

To provide formulations effective for the alleviation of dry skin and atopic dermatitis symptoms as well as the prevention and treatment of ichthyosis, by promoting transglutaminase production and normalizing epidermal corneocyte.SOLUTION: A formulation promoting transglutaminase production can be obtained by compounding as an active ingredient one or more extracts selected from Eisenia arborea Areschoug extract, Artemisia princeps extract, Rose Fruit extract, and Panax ginseng extract.SELECTED DRAWING: Figure 1

Description

The present invention relates to a transglutaminase production promoter.

Transglutaminase is distributed in animal organs including humans. It is a protein with a molecular weight of about 30,000 to 100,000.
Its main action is to catalyze the reaction between free glutamate residues present in the outermost layer of the skin and lysine residues, and to form crosslinks consisting of ε-lysine (γ-glutamyl) bonds, thereby densifying the surface structure. That is. There are three reaction modes depending on the composition of the reaction system. In either case, the γ-carboxyamide group of the glutamate residue in the peptide chain serves as the acyl donor, but in the presence of the amine compound, the amino group serves as the acyl acceptor and the addition reaction of the amine compound to the peptide. Occurs. When the ε-amino group of the lysine residue in the peptide reacts as an acyl acceptor, a crosslink between the peptide chains is formed by the ε-lysine-isoptide bond.
In the above reaction, hydrophobic proteins such as involucrin and loricrin are covalently cross-linked during the keratosis process to become insoluble cornified envelope (cornified envelope), which is a backing protein of the cell membrane and stabilizes the stratum corneum. Contribute to the conversion. Deficiency in the activity of transglutaminase, which plays the most important role in this formation, becomes a serious disease such as ichthyosis that loses the skin barrier. In recent years, it has become clear that ceramide alone cannot exert a sufficient barrier effect, and that cross-linking by the enzyme is important for enhancing the keratin barrier.
Therefore, promotion of transglutaminase expression and enhancement of activity are effective in normalizing epidermal keratinocytes, reducing dry skin and atopic dermatitis, and preventing and treating ichthyosis.
As a transglutaminase production promoter, external preparations such as evening primrose extract, calcium pantotein sulfonate and calcium gluconate have been proposed. (Patent Documents 1 and 2)
However, there is a need for further transglutaminase production promoters.

Sagarame (Eisenia arborea Areschoug) is a seaweed of brown algae, kelp family (Laminariaceae), and genus Arame (Eisenia). It is used as an ingredient.
Further, applications such as an angiogenesis inhibitor, β-glucuronidase inhibitor, AGE production inhibitor, and aquaporin production-enhancing preparation are known. (See Patent Documents 3 to 6)
Mugwort (scientific name: Artemisia indica or Artemisia princeps), wormwood (Artemisia absinthium), wormwood (Artemisia mongolia) is a dried leaf or whole plant, and is called a herbal medicine (moxa leaf) that is effective for blood stoppage, pain relief, diarrhea prevention, etc. ) Has been used. Further, it is used as a raw material for cosmetics effective in moisturizing, hygroscopicity, claudin production promoting agent, occludin production promoting, etc., and also as an anti-itch external application, a blood viscosity lowering agent, and a glycation inhibitor. (Patent Documents 7 to 11)

Agetsu is a fruit of Rosa multiflora, which has been known as a Chinese herbal medicine for a long time and has been used as a laxative and a diuretic.
It is also known as a hair cosmetic, an external skin agent for preventing dullness, an elastase inhibitor, and a ceramide production promoter. (Patent Documents 12 to 15)
Panax ginseng is also called ginseng (Korean ginseng), ginseng (Korean ginseng), or simply ginseng.
It is a herbal medicine that has been used for a long time and is also described in "God Emperor Honsokyo".
Further, applications such as a ceramide production promoter, a hair restorer, a glutathione production promoter, an anti-inflammatory agent, an anti-aging agent, an anti-obesity agent, and an elastin production promoter are known. (Patent Documents 16 to 20)

Japanese Unexamined Patent Publication No. 2004-91376 Japanese Unexamined Patent Publication No. 11-100320 Japanese Unexamined Patent Publication No. 2006-022033 Japanese Unexamined Patent Publication No. 2006-04518 Japanese Unexamined Patent Publication No. 2008-21246 Japanese Unexamined Patent Publication No. 2013-087057 Japanese Unexamined Patent Publication No. 05-271047 Japanese Unexamined Patent Publication No. 06-172196 Japanese Unexamined Patent Publication No. 06-21179 Japanese Unexamined Patent Publication No. 2004-250445 JP-A-2009-256244 Japanese Unexamined Patent Publication No. 11-005719 Japanese Unexamined Patent Publication No. 11-335236 Japanese Unexamined Patent Publication No. 2000-119189 Japanese Unexamined Patent Publication No. 2000-169359 Japanese Unexamined Patent Publication No. 2000-169359 Japanese Unexamined Patent Publication No. 2003-238365 Japanese Unexamined Patent Publication No. 2009-132662 Japanese Unexamined Patent Publication No. 2010-155787 Japanese Unexamined Patent Publication No. 2012-056933

An object of the present invention is to promote the production of transglutaminase, normalize epidermal keratinocytes, reduce dry skin and atopic dermatitis, and obtain an external skin preparation effective for the prevention and treatment of ichthyosis.

As a result of diligent studies by the present inventors, it was found that the extract of sagarame, the extract of mugwort, the extract of ages, and the extract of Panax ginseng achieve the above objectives.
Sagarame, mugwort, ages, and Panax ginseng are extracted after being dried as necessary and then subjected to treatments such as shredding and crushing in consideration of extraction efficiency.
Drying may be performed in the sun or using a commonly used dryer.
As the solvent used for the extraction, water, a hydrophilic organic solvent, or a mixture thereof is used.
Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.
Examples of the hydrophilic organic solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol and propylene glycol. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycerin, and a mixed solvent of these hydrophilic organic solvents and water can be used.
When a mixed solvent of the water and the hydrophilic organic solvent is used, 1 to 20 parts by mass with respect to 10 parts by mass of water in the case of lower alcohol, and 10 parts by mass of water in the case of lower aliphatic ketone. On the other hand, it is preferable to add 1 to 15 parts by mass. In the case of polyhydric alcohol, it is preferable to add 1 to 20 parts by mass with respect to 10 parts by mass of water.

The amount of the organic solvent used for extraction is preferably about 5 to 100 times, more preferably about 10 to 50 times the amount of the plant as a raw material. In order to further increase the extraction efficiency, stirring or homogenization may be performed in the extraction solvent. It is appropriate that the extraction temperature is from about 5 ° C. to a temperature equal to or lower than the boiling point of the extraction solvent. The extraction time varies depending on the type of extraction solvent and the extraction temperature, but it is appropriate to set it to about 1 hour to 14 days.
The extraction operation is not limited to a one-time operation. A fresh solvent can be added again to the residue after extraction to perform an extraction operation, or the extraction solvent can be brought into contact with the extraction raw material multiple times.
As a result of the examination by the present inventors, 80% of the substance exhibiting the effect of the present invention is extracted with ethanol in water. Therefore, when purifying to some extent, after extracting with water, the insoluble matter should be removed, etc. It was also found that further extraction should be performed by adding an amount to 5 times the amount of ethanol.
If necessary, further purification treatment such as deodorization and decolorization may be added within a range that does not affect the effect, and the concentration can be achieved by a vacuum concentrator such as an evaporator or solvent removal by heating.
It is also possible to further purify this extract using a synthetic adsorbent (Diaion HP20, Sephadex SP825, Amberlite XAD4, MCIgelCHP20P, etc.), dextran resin (Sephadex LH-20, etc.), ultrafiltration, etc. be.

Although the pharmaceutical product of the present invention exhibits medicinal effects by oral, injectable, or external use, it is preferably used as a skin external preparation. External dermatological agents include skin cosmetics, quasi-drugs for external use, and external dermatological agents for medical use.
The amount of these extracts to be added to the formulation is 0.000001 to 10.0% by weight, preferably 0.00001 to 3.0% by weight, and more preferably 0.00005 to 1.0% by weight as a solid content. be.

In addition to the above ingredients, the formulations of the present invention include surfactants, oily ingredients, moisturizers, polymer compounds, ultraviolet absorbers, anti-inflammatory agents, and bactericides used in various pharmaceutical and cosmetic formulations. Antioxidants, metal ion blockers, preservatives, vitamins, pigments, fragrances, water and the like can be blended.

As the above-mentioned surfactant, any of anionic, cationic, nonionic, natural and synthetic surfactants can be used, but it is preferable to use nonionic surfactants in consideration of skin irritation. Examples of the nonionic surfactant include glycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbit fatty acid ester, polyoxyethylene alkyl ether, and polyoxyethylene polyoxypropylene glycol. , Polyoxyethylene polyoxypropylene alkyl ether, polyethylene glycol fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, alkyl glycoside and the like.

Examples of the oily component include fats and oils, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, essential oils, silicone oils and the like. Examples of fats and oils include soybean oil, nuka oil, jojoba oil, avocado oil, almond oil, olive oil, cacao oil, sesame oil, persic oil, castor oil, palm oil, minced oil, beef fat, pork fat and other natural fats and oils. Hardened oil obtained by hydrogenating natural fats and oils from Japan and synthetic triglycerides such as myristic acid glyceride and 2-ethylhexanoic acid triglyceride; Examples of waxes include carnauba wax, whale wax, honeydew, lanolin and the like; hydrocarbons Examples include liquid paraffin, vaseline, paraffin microcrystalin wax, selecin, squalane, bristan, etc .; higher fatty acids include, for example, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, etc. Linolenic acid, lanolinic acid, isostearic acid and the like; higher alcohols include, for example, lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, 2-hexyldecanol and the like; esters include, for example, cetyl octanate. , Octanoic triglyceride, myristyl lactate, cetyl lactate, isopropyl myristate, myristyl myristate, octyldodecyl myristate, isopropyl palmitate, isopropyl adipate, butyl stearate, decyl oleate, cholesterol isostearate, POE sorbit fatty acid ester, etc. The essential oils include, for example, peppermint oil, jasmine oil, show brain oil, hinoki oil, peppermint oil, ryu oil, terepine oil, coconut skin oil, bergamot oil, orange oil, shove oil, pine oil, lavender oil, and bay oil. , Clove oil, hiba oil, rose oil, eucalyptus oil, lemon oil, thyme oil, peppermint oil, rose oil, sage oil, menthol, cineole, eugenol, citral, citroneral, borneol, linalol, geraniol, camphor, timol, spirantol, Pinen, limonene, terpene-based compounds and the like; Examples of silicone oils include dimethylpolysiloxane and the like. These above-mentioned oily components can be used alone or in combination of two or more. In the present invention, among these, myristic acid glyceride, 2-ethylhexanoic acid triglyceride, lanolin, liquid paraffin, vaseline, paraffin microcrystalin wax, squalane, lauric acid, myristic acid, palmitic acid, linoleic acid, linolenic acid, isostearic acid. , Cetyl alcohol, stearyl alcohol, oleyl alcohol, cholesterol, cetyl octanoate, triglyceride octanate, isopropyl myristyrene, octyldodecyl myristate, cholesterol isostearate, POE sorbit fatty acid ester, peppermint oil, peppermint oil, coconut oil, rose It is preferable to use oil, menthol, cineol, eugenol, citral, citroneral, geraniol, pinen, limonene and dimethylpolysiloxane.

The following components can be further added to the formulation of the present invention, but the components are not limited thereto.
Pigments: Tar dyes specified in the Ordinance of the Ministry of Health and Welfare such as Yellow No. 4, Blue No. 1, Yellow No. 202, etc. Approved as food additives such as pigments in Appendix I and II, chlorophyll, riboflavin, crocin, safflower, anthraquinone, etc. Natural pigments, etc.
Vitamins; vitamin A, vitamin C, vitamin D, vitamin E, etc.
Others; bactericides, preservatives, other ingredients necessary for formulation, etc.

The pharmaceutical product of the present invention can be produced according to a conventional method by adding the optional ingredient to the essential ingredient as necessary.

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

Example 1
Add 2 liters of 50% (V / V) ethanol aqueous solution to 50 g of sagarame (dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), evaporate this, and then freeze-dry. bottom.

Example 2
Add 2 liters of 50% (V / V) ethanol aqueous solution to 30 g of mugwort leaves (dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), and evaporate this. It was lyophilized.

Example 3
Add 2 liters of 50% (V / V) ethanol aqueous solution to 30 g of Agetsu (dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), evaporate this, and then freeze-dry. bottom.

Example 4
Add 2 liters of 20% (V / V) ethanol aqueous solution to 50 g of Panax ginseng (root, dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), and evaporate this. It was lyophilized.

Confirmation test After culturing the second-generation human foreskin-derived epidermal cells (Kurabou) in HuMedia-KG2 medium (without phenol red) so as to be 50-70% confluent, the calcium concentration was changed to 1.8 mM the day before. Examples were added to KG2 medium and cultured at 37 ° C. in a 5% CO2 incubator for 2 days.

<RNA extraction>
Extraction of Total RNA from cells was prepared using trypsin / EDTA and then using the illustra RNA Mini RNA Isolation Kit (GE Healthcare) according to the attached manual of GE Healthcare. The RNA concentration was calculated using NanoDrop1000 (Thermo SCIENTIFIC).

<RT reaction and real-time PCR>
An RT reaction was performed using 2.5 μg of Total RNA and MMLV Reverse Transcriptase RNase H- (Toyobo) according to the Toyobo recommended protocol (TOYOBO BIOCHEMICALS FOR LIFE SCIEENCE 2008/2009, page 1-42).
Real-time PCR was performed using Applied Biosystems 7500 real-time PCR System as follows. Gene expression by Comparative CT (△△ CT) method (n = 3) using the SYBR Green method (THUNDERBIRD SYBR qPCR Mix, Toyobo Co., Ltd.) and using the operation manual (Applied Biosystems) of the 7500 real-time PCR system. GAPDH was used as the internal standard.
The target gene is transglutaminase 1.

The result of the confirmation test is shown in FIG.

In addition, as a result of preparing an external preparation containing the examples and actually using it, improvement was observed in dry skin and atopic dermatitis.

The vertical axis is the gene expression level when the gene expression level when the example is not added is 1.

Claims (3)

A transglutaminase production promoter containing one or more extracts selected from the extract of agetsu, the extract of mugwort, and the extract of Panax ginseng as an active ingredient. The transglutaminase production promoter according to claim 1, wherein the mugwort used in the mugwort extract has not been fermented. The transglutaminase production promoter according to claim 1 or 2, wherein the site of Panax ginseng used in the extract of Panax ginseng is a root.

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