JP6711506B2 - Topical skin agent and antibacterial agent - Google Patents

Description

The present invention relates to a skin external preparation and an antibacterial agent having antibacterial properties.
Further, the present invention relates to a skin-effective preparation that promotes gene expression of sorayacin, loricrin, and transglutaminase-1.

Conventionally, in the fields of skin external preparations, foods, etc., various fungicides and preservatives have been used in order to prevent contamination of products by microorganisms and to obtain storage stability of products. Further, in the fields of cosmetics, pharmaceuticals and the like, various fungicides and preservatives are used to prevent and improve skin diseases caused by microorganisms.
As a typical one, paraoxybenzoic acid ester is widely used. Paraoxybenzoic acid ester is also widely used in foods, but there is a history of allergies in the past, and problems with skin irritation have been pointed out.Therefore, the upper limit of blending is specified for blending into cosmetics. There is. In addition, benzoate, salicylate and dehydroacetate are also often used, but the problem of skin irritation has been pointed out like paraoxybenzoate.
Therefore, safe and highly antibacterial antibacterial agents have been developed. For example, mugwort extract and green tea extract (see Patent Document 1), various essential oils (see Patent Document 2), sorbic acid esters (see Patent Document 3). ), saponin (see Patent Documents 4 and 5) and the like are known, but the antibacterial properties are not sufficient, and development of safe and effective antibacterial agents is still desired.

The skin is located on the outermost side of the body and serves as a barrier against external stimuli such as bacteria. In the skin, keratinocytes change from basal cells to spiny cells, granule cells, and horny cells over about 4 weeks (keratinization), and in these cells, intercellular lipids and natural moisturizing factor (NMF) , And even achieves the barrier function by forming a cornified envelope. However, it is known that when the rate of keratinization decreases due to aging and the like, the skin becomes dull and the skin becomes rough. Therefore, keratinization accelerators that promote keratinization of the skin have been developed. For example, as a result of studying a substance capable of enhancing the expression of sorayacin with the expression level of sorayacin (=S100A7), which is a protein produced as a component of cornified envelope, as an index, butyric acid or its derivative, and Skin keratinization promoters containing a calcium ion supplying compound are known (Patent Documents 6 to 7).
Sorayacin (S100A7) was the first protein identified as a secreted protein overexpressed in psoriatic skin, and its overexpression was confirmed in many inflammatory diseases. Sorayacin (S100A7) has a function of killing bacteria on the surface of the body. Therefore, it has been revealed to be an antibacterial protein.

Loricrin is a major component of the peripheral zone (cornified cell envelope), and its expression begins in the granular layer and is gradually incorporated into the peripheral zone. It is one of the important constituent proteins of the peripheral zone, including the formation of natural moisturizing factors. Involves various stratum corneum functions and suppresses the formation of wrinkles, abnormal texture, and rough skin.

Transglutaminase-1 expresses proteins such as involucrin from the spinous layer to the granular layer during the differentiation process of keratinocytes, promotes cross-linking of these proteins, and is an insoluble cell membrane-like structure that wraps keratinocytes. To form a cornified envelope (hereinafter abbreviated as “CE”) that contributes to the stability of the cytoskeleton and structure of keratinocytes.
However, when the production amount of transglutaminase-1 in the epidermis is reduced due to various factors, CE formation becomes incomplete and keratinization is not normally performed. As a result, it is considered that the keratin barrier function and the skin moisturizing function are deteriorated, and skin symptoms such as rough skin and dry skin are exhibited.
From this, by increasing the production of transglutaminase-1 in the epidermis of keratinocytes, promoting the formation of CE and normalizing keratinization, the skin barrier function associated with external stimuli such as dryness and ultraviolet rays It is thought that it is possible to suppress deterioration and prevent and improve various skin symptoms such as dry skin and rough skin.

Japanese Patent Laid-Open No. 11-228325 JP, 2001-64163, A JP, 2000-256107, A JP-A-7-149608 JP 2001-39812 A JP, 2009-155269, A JP, 2010-105924, A

An object of the present invention is to obtain a skin external preparation and an antibacterial agent having an antibacterial property, and further to obtain a preparation effective for skin etc. which promotes gene expression of sorayacin, loricrin and transglutaminase-1.

As a result of diligent examination by the present inventors, the mantle of the pearl oyster, the mantle of the pearl oyster after infecting and stimulating the digestive tract or bacteria, the extract of the digestive tract or gill or a substance having an antibacterial property than these fractions The inventors have further found that these substances have an action of promoting the gene expression of sorayacin, loricrin, and transglutaminase-1.
In the present invention, pearl oysters (Pinctada fucata) or pearl oyster mantles of the related species, digestive tract or shellfish containing them are used as a starting material.
In addition, the fungus is administered to the shellfish of living pearl oysters or their related species. The administration method may be a method in which the fungus comes into contact with the shellfish by using an injection or a tube.
After administration, the animals are raised for 3 hours to 3 days. Even if the time is shorter than 3 hours, the effect of administering the bacteria does not appear, and the effect does not increase even after 3 days or more.
Further, the purpose can be achieved even if it is not administered to shellfish, if it is raised in seawater containing bacteria.
The number of bacteria to be administered varies depending on the method of administration, type of bacteria, rearing time, etc., but about 10 ² to 10 ¹⁵ are administered.
The type of the bacterium is not particularly limited, but a sufficient effect was observed with Vibrio parahaemolyticus.

An extract is obtained from the shell or specific region of any of the above pearl oysters or their relatives. Extract with a solution whose main component is water.
Although the pH varies depending on the site, an acidic aqueous solution tends to give a more effective extract. A pH of 0.5 to 7.0 is suitable.

Furthermore, it is also possible to fractionate.
The means used is not particularly limited. Examples include organic solvent precipitation, centrifugation, ultrafiltration membranes, high performance liquid chromatographs, column chromatographs, etc. Fractionation using one or more of these means to obtain more effective fractions It is also possible to fractionate the cost and effect.

These can be used as a skin external preparation, an antibacterial agent for foods, and a gene expression promoter for sorayacin, loricrin, and transglutaminase-1.
Of course, in addition to this, raw materials that can be used in foods and external preparations for skin can be blended as necessary.
The amount of these extracts incorporated into the preparation 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 solid content. is there.

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example-1
After collecting the pearl oysters and breeding them for 1 week, the following treatments were carried out for each 9 individuals.

H. A bacterial solution of 1×10 ¹² CFU/ml of Vibrio parahaemolyticus was injected into 0.1 ml shellfish per individual. (Hereinafter, it may be abbreviated as “high dose H” or “H”.)
M. A bacterial solution of 1×10 ⁸ CFU/ml of Vibrio parahaemolyticus was injected into 0.1 ml shellfish per individual. (Hereinafter, it may be abbreviated as "medium dose M" or "M".)
L. A bacterial solution of 1×10 ⁴ CFU/ml of Vibrio parahaemolyticus was injected into 0.1 ml shellfish per individual. (Hereinafter, it may be abbreviated as "low dose L" or "L".)
C. 0.8 ml of physiological saline was injected into 0.1 ml shellfish per individual. (Hereinafter, it may be abbreviated as “control C” or “C”.)
N. No processing (hereinafter sometimes abbreviated as “N”)

After the injection, the shellfish was divided into hemolymph, mucus, mantle, gill, digestive tract, and adductor muscle (scallop).
These were divided, the following solution was added and homogenized, followed by centrifugation (12000 rpm, 30 minutes) to obtain a supernatant.
・0.1% trifluoroacetic acid aqueous solution (pH=1.75) (hereinafter sometimes abbreviated as “TFA (pH=1.75)”)
0.01 M Tris-HCI (0.15 M NaCI) (pH=6.80) (hereinafter sometimes abbreviated as "Tris-HCI pH=6.80")

Antibacterial Confirmation Test The above supernatant was adjusted to a protein concentration of 5.0 mg/ml in a 96-well plate and then diluted 2-fold, 4-fold diluted, 8-fold diluted, 16-fold diluted, 32-fold diluted. , 64-fold dilution, 128-fold dilution, 256-fold dilution, Vibrio parahaemolyticus solution was added thereto, and the mixture was cultured at 37° C. for 72 hours.
Table 1 shows the results of extraction of gill, mantle and digestive tract with TFA (pH=1.75), FIG. 1 shows the results of mantle, and FIG. 2 shows the results of gill.
In the following, unless otherwise specified, the antibacterial property confirmation test was conducted using Vibrio parahaemolyticus.

Example-2
The extract of TFA (pH=1.75) from the gill of “High dose H” of Example-1 was analyzed with a column (2.6 cmφ×100 cmL) packed with Sephacryl S-200 in an amount of 0.002 MTris-HCI (0 A 0.03M NaCI) (pH=6.80) solution was eluted at an elution rate of 0.6 ml/min, and each fraction was divided into 5 ml.
With respect to each fraction, as antibacterial activity, the radius of the inhibition circle (mm), the absorbance at 280 nm, and the absorbance at 490 nm after color development by the phenol-sulfuric acid method were measured. The behavior of peak 1 (fraction numbers 42 to 48) and peak 2 (fraction numbers 86 to 90) was confirmed by electrophoresis.
Results are shown in FIG.

Example-3
Fraction Nos. 42 to 48 of Example-2 were put together, and a column (2.6 cmφ×100 cmL) packed with Sephadex G-50 was used to apply a 0.002M Tris-HCI (0.03M NaCI) (pH=6.80) solution. Was eluted at an elution rate of 0.4 ml/min, and each fraction was divided into 5 ml.
For each fraction, antibacterial activity, absorbance at 214 nm, and absorbance at 490 nm after color development by the phenol-sulfuric acid method were measured.
The antibacterial activity was measured by the radial diffusion method using Vibrio parahaemolyticus.
Further, the behavior of the substances of fraction numbers 22 to 37 (designated as AGPg) was confirmed by electrophoresis.
The result is shown in FIG.

Example-4
Bacteria were administered in the same manner as in Example-1, the following solution was added to the mantle, boiled for 5 minutes, allowed to cool, homogenized, and then centrifuged (12000 rpm, 30 minutes) to obtain a supernatant. ..
1.1.0% acetic acid aqueous solution (containing 0.2 mM PMSF)
2. 1.0% TFA aqueous solution (1M HCI, 5% formic acid, 1%NaCl, 0.2mMPMSF)
An experiment was performed in which the same treatment was performed except that "after boiling for 5 minutes and allowing to cool" was not performed.

The antibacterial activity of these samples is shown in FIGS.

Furthermore, after the extract of "high dose H" with 1.0% TFA was poured into a C18-E cartridge with 20 ml of purified water, a 0.05% trifluoroacetic acid aqueous solution (0.05% TFA may be abbreviated). Yes), and eluted with a 30% aqueous acetonitrile solution.
The peptide amount and antibacterial activity (inhibition circle) of this were measured.
The results are shown in Table 2.
The names of the eluted fractions were as follows.

SPE1 Fraction flowing out with purified water SPE2 Fraction flowing out with 0.05% TFA SPE3 Fraction flowing out with 30% acetonitrile aqueous solution

Example-5
The SPE2 obtained in Example-4 (fractions flowing out with 0.05% TFA) was applied to a TSKgel Amide-80 column (250 x 4.6 mm, TOSOH) column to obtain a linear gradient of 0 to 60% of 0.05% TFA. It was eluted at a rate of 0.45 ml/min over 70 minutes.

The results are shown in FIG. 7. The minimum inhibitory concentration and the minimum antibacterial concentration were measured for the fractions of peak Nos. 3, 6, 8, 10, and 11 shown in FIG. The results are shown in Table 3.

Further, the fractions of peaks Nos. 3, 6, 8, 10 and 11 were further fractionated with a gradient of acetonitrile containing C18 reverse phase column (4.6 mmφ×150 mmL Mightsil) 0.05% TFA, and peaks No. 3 to AMPm1 were peaked. AMPm2 was obtained from No6, AMPm3 was obtained from No8, AMPm4 was obtained from No10 and AMPm5 was obtained from No11.
For these fractions, the concentrations of peptide and sugar, the minimum inhibitory concentration and the minimum antibacterial concentration were measured. The results are shown in Table 4.

FIG. 8 shows the result of electrophoresis.

Confirmation test After the second passage, human foreskin-derived epidermal cells (Kurabo) were cultured in HuMedia-KG2 medium (phenol red-free) so as to be 50-70% confluent, and then HuMedia-in which the calcium concentration was changed to 1.8 mM the day before. The production example was added to the KG2 medium, and the cells were cultured at 37° C. in a 5% CO 2 incubator for 2 days.

<RNA extraction>
Extraction of Total RNA from the cells was carried out by using SV Total RNA Isolation System (Promega) after stripping with trypsin/EDTA, according to the attached manual of Promega (Japanese Protocol No TM048J June 2001). The RNA concentration was calculated using NanoDrop1000 (Thermo SCIENTIFIC).

<RT reaction and real-time PCR>
Using 2.5 μg of total RNA, MMLV Reverse Transcriptase RNase H- (Toyobo Co., Ltd.) was used to perform an RT reaction according to the recommended protocol (TOYOBO BIOCHEMICALS FOR LIFE SCIENCE 2008/2009).
Real-time PCR was carried out as follows using Applied Biosystems 7500 Real-time PCR System. 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, the comparative CT (ΔΔCT) method (n=3) was used to compare the gene expression. GAPDH was used as an internal standard.
As the primer used, the primer described in JP2013-166710A was used.

FIG. 9 shows the results of gene expression experiments of solayacin, loricrin, and transglutaminase-1 for AMPm4, AMPm5, and AGPg.

It is a result of the antibacterial activity of the mantle of Example-1, and the transparent part of the 96-well plate is a sample in which the growth of bacteria was inhibited. The left half is extracted with 0.1% trifluoroacetic acid aqueous solution (pH=1.75), and the right half is extracted with 0.01 MTris-HCI (0.15M NaCI) (pH=6.80). For the upper HMLCN, see paragraph No. 0013, and the dilution ratio is 2-fold dilution, 4-fold dilution, 8-fold dilution, etc. based on the protein concentration of 5.0 mg/ml. The mantle extracted with 0.1% trifluoroacetic acid aqueous solution (pH=1.75) showed a high antibacterial activity regardless of the presence or absence of administration of bacteria and the concentration thereof. It is a result of the antibacterial activity of the gill of Example-1, and the transparent part of the 96-well plate is a sample in which the growth of bacteria was inhibited. The left half is extracted with 0.01 MTris-HCI (0.15M NaCI) (pH=6.80), and the right half is extracted with 0.1% trifluoroacetic acid aqueous solution (pH=1.75). For the upper HMLCN, see paragraph No. 0013, and the dilution ratio is 2-fold dilution, 4-fold dilution, 8-fold dilution, etc. based on the protein concentration of 5.0 mg/ml. The gill had an antibacterial activity increased depending on the concentration of administration of the bacteria in the gill extracted with 0.1% trifluoroacetic acid aqueous solution (pH=1.75). It is a test result of Example-2. For each fraction, the antibacterial activity is the result of the radius of the inhibition circle (mm) on the right scale, the absorbance at 280 nm, and the absorbance at 490 nm after color development by the phenol-sulfuric acid method (all on the left scale). The electrophoresis on the right was performed on peak 1 (fractions no. 42-48) and peak 2 (fractions no. 86-90). It is a test result of Example-3. For each fraction, the absorbance at 214 nm (solid line) and the absorbance at 490 nm after color development by the phenol-sulfuric acid method (dashed line) were measured. For the absorbance, the scale on the right side was used. The antibacterial activity was collectively measured as AGPg. The electrophoresis on the right is for AGPg. It is a test result of extraction with the 1.0% acetic acid aqueous solution of Example-4. It is a test result of extraction with a 1.0% TFA aqueous solution of Example-4. It is a test result of Example-5. As a whole, SPE2 (fractions flowing out with 0.05% TFA) was applied to a TSKgel Amide-80 column (250 × 4.6 mm, TOSOH) column for more than 70 minutes with a 0-60% linear gradient of 0.05% TFA. It is a graph of the absorbance at 214 nm as a result of chromatography eluted at a rate of 0.45 ml/min. Further, the five small frames are the results of the C18 reverse phase column for the fractions of peak Nos. 3, 6, 8, 10, and 11, and are graphs of the absorbance at 214 nm. It is a test result of Example-5. The results of AMPm1 to AMPm5 and SPE2 before fractionation are shown. The result of the gene expression test of solayacin, loricrin, and transglutaminase-1 of AMPm4, AMPm5, and AGPg is shown.

Claims (9)

An antibacterial agent containing, as an active ingredient , an extract obtained by extracting the mantle of the pearl oyster with an acidic aqueous solution . An antibacterial agent containing, as an active ingredient , an extract obtained by extracting the gill of pearl oyster after administration of the bacterium with an acidic aqueous solution . The antibacterial agent according to claim 2, wherein the infected bacterium is Vibrio parahaemolyticus. The extract obtained by extracting the pearl oyster mantle with an acidic aqueous solution was used to flow 20 mL of purified water using a C18-E cartridge, and then the fraction eluted with 0.05% TFA was added to 0.05% using a TSKgel Amide-80 column. Solayacin containing, as an active ingredient, peak numbers 10 and/or 11 among fractions eluted with a linear gradient of% TFA, and fractions eluted with a gradient of acetonitrile containing 0.05% TFA using a C18 reverse phase column. Gene expression promoter . The extract obtained by extracting the gills of the pearl oyster after administration of Vibrio parahaemolyticus with an acidic aqueous solution was fractionated with Tris-HCl solution at a fraction of 5 mL using a column packed with Sephacryl S-200, and the fraction number 42 to 48 are collected together, and this is fractionated with a Tris-HCl solution in a fraction of 5 mL using a column packed with Sephadex G-50, and a sorayasin gene expression promoter containing fractions 22 to 37 as an active ingredient. .. The extract obtained by extracting the pearl oyster mantle with an acidic aqueous solution was used to flow 20 mL of purified water using a C18-E cartridge, and then the fraction eluted with 0.05% TFA was added to 0.05% using a TSKgel Amide-80 column. % Of the fractions eluted with a linear gradient of% TFA, and peak number 10 and/or 11 was eluted with a gradient of acetonitrile containing 0.05% TFA using a C18 reverse phase column. Gene expression promoter (excluding applications for suppressing wrinkle formation). The extract obtained by extracting the gills of the pearl oyster after administration of Vibrio parahaemolyticus with an acidic aqueous solution was fractionated with Tris-HCl solution at a fraction of 5 mL using a column packed with Sephacryl S-200, and the fraction number 42 to 48 are collected together, and this is fractionated with Tris-HCl solution in a fraction of 5 mL using a column packed with Sephadex G-50, and a loricrin gene expression promoter containing fractions 22 to 37 as an active ingredient. (However, excluding wrinkle formation suppression applications). The extract obtained by extracting the pearl oyster mantle with an acidic aqueous solution was used to flow 20 mL of purified water using a C18-E cartridge, and then the fraction eluted with 0.05% TFA was added to a fraction using a TSKgel Amide-80 column. Peak number 10 and/or 11 out of the fractions eluted with a linear gradient of% TFA is the trans fraction containing the fraction eluted with a gradient of acetonitrile containing 0.05% TFA as an active ingredient using a C18 reverse phase column. A glutaminase-1 gene expression promoter. The extract obtained by extracting the gills of the pearl oyster after administration of Vibrio parahaemolyticus with an acidic aqueous solution was fractionated with Tris-HCl solution at a fraction of 5 mL using a column packed with Sephacryl S-200, and the fraction number 42 to 48 were collected together, and this was fractionated with a Tris-HCl solution in a fraction of 5 mL using a column packed with Sephadex G-50, and the transglutaminase-1 gene containing fractions 22 to 37 as an active ingredient. Expression promoter.