JP2014074015A - Aquaporin production promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a preparation which promotes production of aquaporin, particularly aquaporin 3 to increase freshness of a skin.SOLUTION: This invention provides an aquaporin production promoter including a protein hydrolysate derived from a pearl and/or a pearl layer as an active ingredient, the preparation promoting production of aquaporin, particularly aquaporin 3 to increase freshness of a skin.

Description

  The present invention relates to a preparation that promotes the production of aquaporin, particularly aquaporin 3, and keeps the skin healthy.

Aquaporin is a protein with pores present in the cell membrane, and is called a water channel that selectively permeates water molecules but does not permeate ions and other substances. Aquaporins are usually composed of four identical subunits, each monomer acting as a water channel. Water molecules pass through the pores of this channel. This water channel works to increase the cell membrane permeability of water. Systems that transport such water molecules are essential for many human cells, certain bacteria, and organic organisms such as plants.
In humans, the presence of 13 types of aquaporins (0 to 12) is known. In epidermal cells, aquaporin 3 is mainly present, and it is considered to play a role of taking in low molecular compounds such as glycerol and urea involved in water retention action in addition to water.

It is known that aquaporin 3 is present in the skin among aquaporins, and it has been reported that in mice lacking aquaporin 3, the water content and elasticity of the skin are significantly reduced. It has also been reported that wound healing is delayed in aquaporin 3-deficient mice. In this way, aquaporins are essential for maintaining the moisture and elasticity of the skin constantly, and have a significant impact on the ability to recover when wounded, such as rough skin. Thus, it has been considered that various skin problems can be improved.
Among natural products, saffron extract, royal jelly extract, butcher's bloom extract and the like are known as aquaporin 3 production promoters. (Patent Documents 1 to 3)

Pearls and nacreous protein hydrolysates have long been used in cosmetics as hydrolyzed conchiolin, and for example, effects such as histamine suppression and active oxygen suppression are known.
JP 2005-343882 A JP 2009-184955 A JP 2009-235044 Japanese Patent Application Laid-Open No. 06-211625 Japanese Patent Laid-Open No. 06-21640

  An object of the present invention is to obtain a preparation that promotes the production of aquaporins, particularly aquaporin 3, and keeps the skin healthy.

As a result of intensive studies by the present inventors, it has been found that a pearl and / or a protein hydrolyzate derived from a pearl layer achieves the above object.
The pearl and nacre used here will be described.
First, a pearl is a metabolite formed in a spherical or hemispherical shape (including some deformations) in a living pearl shell, and the main constituent of this visible portion is a pearl shell. This is the same quality as the pearl layer.
A pearl oyster is a shell with a nacre in its shell, but it refers to a specific old family of shells such as bivalves, gastropods, cephalopods, etc., for example, pearl oysters, white butterflies, black butterflies, black butterflies In addition to bivalves belonging to the genus Akoya, such as mussels, mussels, mussels, mussels, butterflies, kawashinjugai, limpets and the like can be mentioned.
Among these, pearl oysters, white butterflies, and black oysters are also cultivated and are preferable from the viewpoint of securing raw materials.
The pearl is left as it is or the nucleus is removed.
When using a shell with a nacreous layer, minerals such as tentacles, mollusks, arthropods, annelids, notochords, seaweeds, etc. attached to the shell, such as water, brushes, Remove with a spatula. (Of course, remove as much shellfish as possible)
Further, in some cases, the shell layer, the shell layer, and the ridge column layer are removed using a brush, a spatula, and a grinder. Also, since various methods are described in JP-A-62-120319, JP-A-2003-250489, JP-A-2011-72207, etc., any method may be selected.
As mentioned above, it deashes in order to remove a calcium carbonate etc. from one or both of the shell which has a pearl or a pearl layer. In order to perform this smoothly, a crushing and crushing process is added as necessary.
The crushing and crushing may be performed with a hammer, a rod, etc., or using a crusher or crusher such as a gyratory crusher, cone crusher, single roll crusher, double roll crusher, impact crusher, ball mill, hammer mill or rod mill. You may crush and grind | pulverize.

Decalcification is usually performed using an acid. Any acid can be used as long as it reacts with calcium and dissolves in water. For example, inorganic acids and organic acids such as hydrochloric acid, nitric acid, lactic acid, acetic acid, and glycolic acid can be mentioned. Of these, hydrochloric acid is often selected from the viewpoint of cost. When hydrochloric acid is used, it foams. If necessary, add water and stir while gradually adding hydrochloric acid. Agitation is selected according to the amount of water added, the degree of shell crushing, the reaction temperature, the degree of firing, and the like.
The reaction time varies depending on the degree of pulverization, reaction temperature and the like, but is preferably about 10 hours to 10 days.
The amount of hydrochloric acid to be added may be such that calcium carbonate dissolves. However, when acid is used in the protein hydrolysis step in the subsequent step, the acid concentration during hydrolysis changes, so the amount of hydrochloric acid dissolves calcium carbonate. Slightly more than necessary and decalcify well.
Insoluble matters are collected by centrifugation, filtration, and the like. Depending on the application, when performing the desalting step described later, depending on the desalting method, it is necessary to remove the salt sufficiently in this step, so add water to the insoluble matter, stir, then insoluble by centrifugation, filtration, etc. The process of collecting things may be repeated multiple times.

The collected insoluble matter is hydrolyzed. Hydrolysis is carried out using acid, alkali, proteolytic enzyme, etc., but pearls or nacreous proteins cannot be decomposed by ordinary proteolytic enzymes, so it is necessary to devise the type of enzyme and the decomposition conditions.
Examples of the acid include sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, sulfurous acid, oxalic acid, citric acid, acetic acid, formic acid, and lactic acid. Examples of the alkali include sodium hydroxide and potassium hydroxide. The protein is hydrolyzed using one or more of them, and further in combination with a protease. Of these, hydrochloric acid and sulfuric acid are often used.
Hydrolysis conditions are selected according to the type of acid, alkali, and enzyme, but when hydrochloric acid or sulfuric acid is used, 1 to 10 mol, room temperature to 120 ° C., about 1 hour to 10 days is good, and pressure is also applied. May be.
After the hydrolysis, neutralization is performed as necessary when acid or alkali is used, and deactivation such as heating is performed as necessary when an enzyme is used. Further, depending on the application, the salt is completely or partially desalted.
Then, a decolorization process and a drying process are added as needed, and a pearl and / or a pearl layer origin protein hydrolyzate are obtained.
Further, the methods disclosed in JP-A-62-2223104, JP-A-02-075597, and JP-A-05-043444 are also helpful.

  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.

  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, and can be in the form of cream, emulsion, lotion or the like.

Next, an Example (manufacturing example) is given and this invention is demonstrated in detail.

Production Example 1
Deposits were removed from the shellfish and shells with water and a brush, and 450 kg of water was added to 50 kg of pearl oyster shells crushed to about 1 to 5 mm, and 100 kg of hydrochloric acid was gradually added. Further, 100 kg of water was added and stirred for 3 days. Hydrochloric acid 10 kg was gradually added. Further, 200 kg of water was added and stirred for 2 days. While stirring, 3 kg of sodium hydroxide was gradually added.
After adjusting the pH to 25 with sodium hydroxide 25%, 200 kg of water was added. The precipitate was collected with a sharp press centrifuge.
The precipitate was crushed, 20 kg of 0.1 molar hydrochloric acid was added, and the mixture was slowly stirred for 24 days. To this was added 1000 kg of water with stirring, and the precipitate was collected with a sharp press centrifuge.
The precipitate was crushed and 1000 kg of water was added to this with stirring, and then the precipitate was collected with a sharp press centrifuge.
To 200 g of this precipitate, 3 kg of 10-fold diluted sulfuric acid was added and hydrolyzed in an oil bath at 110 ° C. for 24 hours.
After allowing to cool, the pH was adjusted to 3.0 while gradually adding barium hydroxide, and then centrifuged (6000 rpm, 15 minutes), and Amberlite IRA910 was gradually added to the supernatant until the pH reached 7.0. This was filtered, 10 g of activated carbon was added to the filtrate, and the mixture was stirred for 30 minutes.
This was filtered and lyophilized.

Production Example 2
Deposits were removed from the shellfish and shells with water and a brush, and 45 kg of water was added to 5 kg of black mussel shells crushed to about 1 to 5 mm, and 10 kg of hydrochloric acid was gradually added. Further, 10 kg of water was added and stirred for 3 days. 1 kg of hydrochloric acid was gradually added. Further, 200 kg of water was added and stirred for 2 days. While stirring, 300 g of sodium hydroxide was gradually added.
500 g of 10% sulfuric acid was added and hydrolyzed in an oil bath at 110 ° C. for 24 hours.
After allowing to cool, the pH was adjusted to 7.0 while gradually adding sodium hydroxide, and then centrifuged (6000 rpm, 15 minutes) to obtain a supernatant. Two times the amount of ethanol was added thereto, and the mixture was slowly stirred and allowed to stand at 4 ° C. for 1 week.
This was filtered and lyophilized.

Confirmation test 1 DNA microarray analysis In the test method, 0.5% of Production Example 1 was allowed to act on epidermal keratinocytes, 48 hours later, RNA was extracted together with the control in the same manner as above (RealTime PCR), and Quick Amp was used from 3 μg of each RNA. Cy3 and Cy5 labeled probes were prepared using Labeling Kit, two-color (Agilent Technologies). After purification of the labeled probe using a Qiagen RNeasy mini kit (Qiagen), it was performed using a Gene Expression Hybridization kit (Agilent Technologies) at a Whole Human Genome slide (4 × 44 K, at an electron temperature of 60 ° C., at 17 ° C., at 17 ° C., at a temperature of 17 ° C., at a temperature of 17 ° C. After washing, scanning was performed using an Agilent DNA microarray scanner (Agilent Technologies), and data processing was performed using Feature Extraction software.

  The test results were as Log Ratio, aquaporin 3 was 0.469, aquaporin 6 was 0.816, and aquaporin 9 was 0.376.

Confirmation test 2
The second passage human foreskin-derived epidermis cells (Kurabo) were cultured in HuMedia-KG2 medium (without phenol red) so as to be 50-70% confluent, and then the media concentration was changed to 1.8 mM on the previous day, HuMedia-KG2 medium. Then, the preparation example was added, and the cells were cultured at 37 ° C. in a 5% CO 2 incubator for 2 days.

<Extraction of RNA>
Total RNA was extracted from the cells after peeling with trypsin / EDTA and using SV Total RNA Isolation System (Promega) according to the manual attached to Promega (prepared in Japanese protocol NoTM048J, June 2001). 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.

<Primer used>
AQP3: A set of a forward primer is AGAGCCCCCTTCAGGATTTT (SEQ ID NO: 1) and a reverse primer is TTCCCTTGCCCTGAATATCTG (SEQ ID NO: 2) Set with the base sequence of TTGATTTTGGAGGGATCTCG (SEQ ID NO: 4)

The result of the confirmation test 2 is shown in FIG.
From the results, it was found that Production Example 1 increased the gene expression level of aquaporin 3 by about 4.0 times when acting at 0.5%.
Next, FIG. 2 shows the results of an experiment conducted with Production Example 2 acting at 0.5%.
From the results, it was found that Production Example 2 increased the gene expression level of aquaporin 3 by about 2.2 times when acting at 0.5%.

As described above, Production Example 1 derived from the pearl oyster was shown to promote the expression of each gene of aquaporins 3, 6, and 9 by DNA microarray analysis.
Furthermore, when the expression of the aquaporin 3 gene was confirmed by a quantitative mRNA gene expression test, it was confirmed that the gene expression was promoted.
Moreover, it confirmed that the expression of the gene of aquaporin 3 was promoted also in the manufacture example 2 derived from the black mussel.

  In addition, as a result of preparing and actually using an external preparation blended with production examples, it was found that the production of aquaporin, particularly aquaporin 3, was promoted, and fresh skin was obtained.

It is a figure which shows the gene expression level change of aquaporin 3 by the result of the confirmation test 2 in final concentration 0.5% of manufacture example 1. FIG. The vertical axis represents the gene expression level when the gene expression level is 1 when no production example is added. It is a figure which shows the gene expression level change of aquaporin 3 by the result of the confirmation test 2 in final concentration 0.5% of manufacture example 2. FIG. The vertical axis represents the gene expression level when the gene expression level is 1 when no production example is added.

Claims (4)

  The aquaporin production promoter which uses a pearl and / or a protein hydrolyzate derived from a pearl layer as an active ingredient.   Aquaporin 3 production promoter which uses a pearl and / or a protein hydrolyzate derived from a pearl layer as an active ingredient.   The aquaporin production promoter of Claim 1 which uses as an active ingredient the pearl and / or pearl layer origin protein hydrolyzate which are derived from the bivalve of the pearl genus.   The aquaporin 3 production promoter of Claim 2 which uses the pearl and / or nacreous-derived protein hydrolyzate derived from the bivalve of the genus Akoyaga as an active ingredient.