JP2016135766A - Kallikrein associated peptidase production-promoting agent, lekti production-promoting agent, slpi production-promoting agent, cornification normalizing agent, and corneodesmosome degradation normalizing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain formulations that promote productions of Kallikrein related peptidase, LEKTI, and SLPI, normalize Corneodesmosome degradation, and normalize keratinization, such as a horny layer thickening or inhibition of turnover delay.SOLUTION: By using one or more kinds of brown alga extracts selected from Eisenia arborea Areschoug extract and Fucus extract as an active ingredient, formulations that promote productions of kallikrein related peptidase, LEKTI, and SLPI, normalize Corneodesmosome degradation, and by extension normalize keratinization can be obtained.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a preparation that promotes the production of kallikrein-related peptidases, LEKTI, and SLPI, normalizes corneodesmosome degradation, and thus normalizes keratinization.

The epidermis begins with cell division of the basal layer and reaches the stratum corneum through the spinous and granular layers.
In stratum corneum, the water retention function by stratum corneum lipids such as ceramide is also an important factor, but the stratum corneum adheres to each other by the patchy distribution of corneodesmosomes, maintaining the lamellar structure and maintaining the integrity of the stratum corneum. I'm leaning.
Then, as new cells are gradually pushed out to the surface of the stratum corneum and moved to the upper stratum corneum, steroid sulfatase and lipase break down the stratum corneum intercellular lipids, and then the corneodesmosome is gradually broken down by the action of the protease. The adhesion becomes weak and eventually the stratum corneum peels off.
However, it is known that the barrier function of the epidermis is reduced due to external factors such as drying, ultraviolet rays, chemical substances, etc., and internal factors such as oxidative stress and aging, and these factors reduce the turnover of the epidermis. It is also known to do.
One of them is that the degradation of corneodesmosome is delayed, cell-cell adhesion is not weakened, and exfoliation and shedding of keratin may not be performed normally. (Chorn layer thickening)

Proteases that degrade corneodesmosome include kallikrein-related peptidase 5 and kallikrein-related peptidase 7. These serine proteases degrade desmoglein 1, corneodesmosine, desmocollin 1, and the like.
That is, when production of kallikrein-related peptidase 5 and kallikrein-related peptidase 7 decreases, exfoliation and shedding of keratin do not work.
Therefore, promoting production of kallikrein-related peptidase 5 and kallikrein-related peptidase 7 is an important factor for promoting skin turnover.
However, if the kallikrein-related peptidase 5 and the kallikrein-related peptidase 7 are excessively produced, abnormal desquamation may occur. Furthermore, it was found that kallikrein-related peptidase 7 degrades two types of lipid processing enzymes, β-glucocerebrosidase and acid sphingomyelinase.
In addition, kallikrein-related peptidase 5 and kallikrein-related peptidase 7 are balanced by activation and limited by LEKTI (Lympho-epithelial Kazal-type-related inhibitor) and SLPI (Secretory Leukocyte-Protease Inhibitor).
As described above, the production of kallikrein-related peptidase 5 and kallikrein-related peptidase 7 whose production has decreased due to aging, etc. is promoted, and the production of LEKTI and SLPI, which inhibit the production, simultaneously increases the production of corneodesmosomes. Normalizes, and thus normalizes keratinization. (Patent Document 1, Non-Patent Document 1)
In addition to skin keratinization, SLPI production promoters are applied to improve airway obstruction, to treat interstitial pneumonia, to prevent or treat retroviral infection, to treat post-burn scars, hypertrophic scars and keloids, etc. ing. (Patent Documents 2 to 5)
LEKTI production promoters are known to be effective for anti-inflammatory properties, Netherton syndrome, ichthyosis and the like.

Salamander (Eisenia arborea Areschoug) is a seaweed of brown algae, Laminariaceae, and genus Eisenia. Unlike alame, it has only primary side leaves, and secondary side leaves do not grow from the middle of the leaves. It is used as a foodstuff.
Furthermore, uses such as angiogenesis inhibitors, β-glucuronidase inhibitors, AGE production inhibitors, and aquaporin production-enhancing preparations are known. (See Patent Documents 6 to 9)

Hibamata is a seaweed that is classified into the brown alga Hibamata, and it is a seaweed that has been used for food in Europe for a long time and is rich in minerals. Among them, the content of iodine and zinc is high, and it is said to be useful for replenishing these components. Furthermore, mucus is a raw material for alginic acid esters necessary for food, cosmetics and pharmaceutical production. Furthermore, actions such as hyaluronidase inhibition, collagen gel contraction promotion, integrin expression promotion, and elastase inhibition are known. (Patent Documents 10 to 13)
In Europe, Fucus vesiculosus is mainly used and is also called Seaweed. Fucus evanescens is distributed in Japan.

JP 2013-166768 A JP-A-6-298663 JP-A-7-82168 JP 2004-2455 A WO01 / 030389 JP 2006-022033 A JP 2006-045188 A JP 2008-214246 A JP 2013-087057 A Japanese Unexamined Patent Publication No. 09-0667266 Japanese Patent Laid-Open No. 10-072336 Japanese Patent Laid-Open No. 11-246428 JP 2000-053578 A Caubet C et al. J Invest Dermatol 122: 1235-1244,2004

  An object of the present invention is to obtain a preparation that promotes the production of kallikrein-related peptidase, LEKTI, and SLPI, normalizes corneodesmosome degradation, and normalizes keratinization such as suppression of stratum corneum thickening and turnover delay.

As a result of intensive studies by the present inventors, it has been found that the extract of sagarame and the extract of hibermata achieve the above-mentioned purpose.
Sagarame and hibamata are dried after necessary, and are extracted after processing such as chopping 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 water that has been subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, 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, 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.
In addition, when using the mixed solvent of the said water and a hydrophilic organic solvent, in the case of a lower alcohol, it is 1-20 mass parts with respect to 10 mass parts of water, and in the case of a lower aliphatic ketone, it is 10 mass parts of water. On the other hand, it is preferable to add 1 to 15 parts by mass. In the case of a 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. Furthermore, in order to raise extraction efficiency, you may stir or homogenize in an extraction solvent. The extraction temperature is suitably about 5 ° C. to 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 single 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 a plurality of times.
As a result of investigations by the present inventors, a substance that exerts the effect of the present invention is also extracted into water by 80% ethanol. Therefore, when purifying to some extent, after extracting with water, insoluble matters are removed, etc. It has also been found that further extraction may be performed by adding an amount to 5 times the amount of ethanol.
If necessary, purification treatment such as deodorization and decolorization may be further 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.
Further, this extract can be further purified using a synthetic adsorbent (Diaion HP20, Sephabies SP825, Amberlite XAD4, MCIgelCHP20P, etc.), dextran resin (Sephadex LH-20, etc.), ultrafiltration and the like. is there.

The preparation of the present invention exhibits drug efficacy for oral, injection, and external use, but is preferably used as a skin external preparation. Skin external preparations include skin cosmetics, external quasi-drugs, and medical skin external preparations.
The amount of these extracts in the preparation is 0.000001 to 10.0% by weight, preferably 0.00001 to 3.0% by weight, more preferably 0.00005 to 1.0% by weight, as a solid content. is there.

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

  As the surfactant, any of anionic, cationic, nonionic, natural, and synthetic surfactants can be used, but it is preferable to use a nonionic surfactant in consideration of irritation to the skin. 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 sorbite fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene glycol. , Polyoxyethylene polyoxypropylene alkyl ether, polyethylene glycol fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, alkylglycoside and the like.

  Examples of the oil component include fats and oils, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, essential oils, silicone oils, and the like. Examples of the fats and oils include soybean oil, nutka oil, jojoba oil, avocado oil, almond oil, olive oil, cacao oil, sesame oil, persic oil, castor oil, coconut oil, mink oil, beef fat, pork fat and the like, and these Hardened oil obtained by hydrogenation of natural fats and oils and synthetic triglycerides such as myristic acid glyceride and 2-ethylhexanoic acid triglyceride; waxes include, for example, carnauba wax, whale wax, beeswax, lanolin and the like; hydrocarbons For example, liquid paraffin, petrolatum, paraffin microcrystalline wax, ceresin, squalane, bristan etc .; higher fatty acids include, for example, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, Linolenic acid, lanolinic acid, isostearic acid, etc .; Examples of the class alcohols include lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, 2-hexyldecanol, etc .; examples of the esters include cetyl octanoate, triglyceride octanoate, myristyl lactate, cetyl lactate, Examples of essential oils include mint oil, jasmine, isopropyl myristate, myristyl myristate, octyldodecyl myristate, isopropyl palmitate, isopropyl adipate, butyl stearate, decyl oleate, cholesterol isostearate, POE sorbite fatty acid ester, etc. Oil, pepper brain oil, cypress oil, spruce oil, ryu oil, turpentine oil, cinnamon oil, bergamot oil, mandarin oil, ginger oil, pine , Lavender oil, bay oil, clove oil, hiba oil, rose oil, eucalyptus oil, lemon oil, thyme oil, peppermint oil, rose oil, sage oil, menthol, cineole, eugenol, citral, citronellal, borneol, linalool, geraniol, Camphor, thymol, spirantol, pinene, limonene, terpene compounds, etc .; examples of silicone oils include dimethylpolysiloxane. These oily components described above 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, petrolatum, paraffin microcrystalline wax, squalane, lauric acid, myristic acid, palmitic acid, linoleic acid, linolenic acid, isostearic acid , Cetyl alcohol, stearyl alcohol, oleyl alcohol, cholesterol, cetyl octanoate, triglyceride octanoate, isopropyl myristate, octyldodecyl myristate, cholesterol isostearate, POE sorbite fatty acid ester, peppermint oil, spruce oil, cinnamon oil, rose Oil, menthol, cineol, eugenol, citral, citronellal, geraniol, pinene, limonene, dimethylpolysiloxane It is preferable to use.

The following components can be further added to the preparation of the present invention, but the components are not limited thereto.
Colors: Yellow color No. 4, Blue No. 1 and Yellow No. 202 are recognized as food additives such as tar color dyes I and II, chlorophyll, riboflavin, crocin, safflower, anthraquinone, etc. Natural pigments.
Vitamins; vitamin A, vitamin C, vitamin D, vitamin E, etc.
Others: bactericides, preservatives, other ingredients necessary for formulation.

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

EXAMPLES Next, an Example is given and this invention is demonstrated in detail.

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 and freeze-dry it did.

Example 2
After adding 2 liters of 50% (V / V) aqueous ethanol solution to 50 g of Fucus vesiculosus (dried product, shredded product), extracting it for 24 hours with occasional stirring, filtering (No5C) and evaporating. This was lyophilized.

Confirmation test HuMedia- cultivated human foreskin-derived epidermis cells (Kurabo) in the 2nd passage in HuMedia-KG2 medium (without phenol red) so as to be 50-70% confluent, and the calcium concentration was changed to 1.8 mM the day before. Examples were added to KG2 medium and cultured in a 37 ° C., 5% CO 2 incubator for 2 days.

<Extraction of RNA>
Total RNA was extracted from the cells after exfoliation with trypsin / EDTA and then using 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>
Using 2.5 μg of total RNA, RT reaction was performed according to Toyobo recommended protocol (TOYOBO BIOCHEMICALS FOR LIFE SCIENCE 2008/2009, page 1-42) using MMLV Reverse Transcriptase RNase H- (Toyobo).
Real-time PCR was performed as follows using Applied Biosystems 7500 Real-Time PCR System. Using the SYBR Green method (THUNDERBIRD SYBR qPCR Mix, Toyobo Co., Ltd.) and the 7500 Real-Time PCR System operation manual (Applied Biosystems), gene expression comparison was performed by the Comparative CT (ΔΔCT) method (n = 3). GAPDH was used as an internal standard.
The target genes are kallikrein-related peptidase 5, kallikrein-related peptidase 7, LEKTI, and SLPI.

The result of the confirmation test is shown in FIG.
In both Example 1 and Example 2, the gene expression levels of kallikrein-related peptidase 5, kallikrein-related peptidase 7, LEKTI, and SLPI were greatly promoted as compared to the control.

Moreover, the external preparation which mix | blended the Example was created, and as a result of actually using it, it was effective in promotion of stratum corneum thickening and turnover.

In Example 1 (acting concentration 0.05%) and Example 2 (acting concentration 0.1%), changes in the production amount of genes for kallikrein-related peptidase 5, kallikrein-related peptidase 7, LEKTI, and SLPI were observed. The vertical axis represents the gene expression level when the gene expression level when no example is added is 1.

Claims (5)

  A kallikrein-related peptidase production promoter containing, as an active ingredient, one or more extracts of brown algae selected from an extract of sagarame and an extract of hibermata.   A LEKTI production promoter containing, as an active ingredient, one or more extracts of brown algae selected from extracts of sagarame and Hibamata.   A SLPI production promoter containing, as an active ingredient, one or more extracts of brown algae selected from an extract of sagarame and an extract of hibermata.   A corneodesmosome degradation normalizing agent containing, as an active ingredient, one or more extracts of brown algae selected from extracts of sagarame and Hibamata.   A keratinization normalizing agent containing, as an active ingredient, one or more extracts of brown algae selected from an extract of sagarame and an extract of hibermata.