JP2022082369A - Desmoglein reducing agent - Google Patents

Abstract

To provide a novel desmoglein reducing agent.SOLUTION: Provided is a desmoglein reducing agent comprising salvianophosphate A as an active ingredient.SELECTED DRAWING: Figure 3

Description

The present invention relates to a desmoglein reducing agent and an external skin preparation containing a desmoglein reducing agent.

It is known that an increase in desmosome protein is one of the causes of deterioration of skin condition such as rough skin, desquamation of the stratum corneum caused by sunburn and dryness.
As a method for controlling the amount of desmosome protein, a method for decomposing the desmosome protein accumulated in the stratum corneum with a protease to improve acne, dandruff, and desquamation has been reported so far (Patent Document 1).

Here, among the desmosome proteins, it is known that desmoglein is particularly involved in the adhesion between epidermal cells and between keratinocytes (Patent Document 2-4).

As a prior art, Patent Document 2 reports a desmoglein reducing agent in astaxanthin-containing stratum corneum cells.
Further, Patent Document 4 reports an oral administration agent for suppressing cell-cell adhesion in the skin containing licorice extract as an active ingredient.

By the way, in Pat. It has been reported to have an anti-inflammatory effect, a skin aging prevention effect, and an ultraviolet protection effect.

Special Table No. 7-505383 Gazette Japanese Unexamined Patent Publication No. 2016-37501 Patent No. 4002635 Patent No. 4197194 Japanese Unexamined Patent Publication No. 2001-122729 Japanese Unexamined Patent Publication No. 2006-16337 Japanese Unexamined Patent Publication No. 2009-256270

Given the above-mentioned prior art, it is an object of the present invention to provide a novel technique for reducing desmoglein.

As a result of diligent research, the present inventors have found that salvianophosphate A has a desmoglein-reducing effect, and have completed the present invention.

The present invention that solves the above problems is a desmoglein reducing agent containing salvianophosphate A as an active ingredient.
In addition, in this specification, the concept of "desmoglein decrease" includes both the decrease of desmoglein and the suppression of increase of desmoglein.

In the preferred embodiment of the present invention, the desmoglein reducing agent is used for suppressing cell-cell adhesion of human epidermal keratinocytes.

In a preferred embodiment of the invention, the desmoglein reducing agent is used to reduce desmoglein 1.

In a preferred embodiment of the invention, the desmoglein reducing agent is used for the prevention or amelioration of sensitive skin.

In a preferred embodiment of the invention, the desmoglein reducing agent is used to improve the lightness of the skin.

In a preferred embodiment of the present invention, the desmoglein reducing agent is used to improve the transparency of the skin.

In a preferred embodiment of the invention, the desmoglein reducing agent is used for skin softening.

In the preferred embodiment of the present invention, the desmoglein reducing agent is used to assist the penetration of the medicinal ingredient into the skin.

In a preferred embodiment of the invention, the desmoglein reducing agent is used to improve and / or prevent exfoliation of the stratum corneum.

The present invention that solves the above-mentioned problems is a composition containing the above-mentioned desmoglein reducing agent.

In a preferred embodiment of the invention, the composition is a cosmetic or pharmaceutical product.

According to the present invention, desmoglein present on human skin can be reduced.

It is a figure which shows the experimental result (control, 80% ethanol extract of wild thyme, and 60% methanol elution part) of the desmoglein reduction ability confirmation test. It is a figure which shows the experimental result (isolated component 1 to 3) of the desmoglein reduction ability confirmation test. It is a figure which shows the experimental result (isolated component 4-6, component 7) of the desmoglein reduction ability confirmation test.

(1) Salvianophosphate A
The desmoglein reducing agent of the present invention contains salbianophosphate A as an active ingredient.

The structure of salvianolic acid A (Salvianolic acid A) is shown in Chemical formula 2.

Salvianophosphate A may be isolated from a plant extract or synthesized. Further, as salvianophosphate A, a commercially available product can be used.

Salvianophosphate A can be isolated from red sage (Salvia miltiorrhiza) or wild thyme extract.
Add 1 to 250 parts by mass of the solvent to 1 part by mass of the plant or its dried product, and soak for several days at room temperature and for several hours at a temperature near the boiling point. After immersion, salbianophosphate A can be obtained by fractional purification by column chromatography or the like filled with silica gel, a synthetic adsorbent, octadecylsilylated silica gel, an ion exchange resin and the like.

As the extraction solvent, a polar solvent is preferable, and alcohols such as water, ethanol, isopropyl alcohol and butanol, and polyvalent alcohols such as 1,3-butylene glycol and polypropylene glycol are preferable. , One or more selected from ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran can be preferably exemplified.
Among them, ethanol and a mixed solvent of water and ethanol can be preferably mentioned as the extraction solvent.

(2) Desmoglein Reducing Agent The desmoglein reducing agent of the present invention preferably targets desmoglein 1 as a target for reduction.

The desmoglein reducing agent of the present invention is preferably used for improving and / or preventing sensitive skin.
Here, the term "sensitive skin" as used herein refers to a skin type having reduced resistance to external stimuli (dryness, ultraviolet rays, application of compounds) (decreased skin irritation threshold).

More specifically, "sensitive skin" in the present specification refers to the skin quality raised in (I) and / or (II).

(I) "Skin that reacts to substances such as external medicines, cosmetics, plants, ultraviolet rays, and metals and is prone to skin problems. Also, it is hypersensitive to allergic substances (pollen, fragrances, etc.) and irritating substances (alcohol, etc.). Skin "
(II) "Skin that is temporarily prone to skin problems due to irritants under conditions such as lack of sleep, overwork, menstruation, seasonal changes, and mental stress."

Further, "for improvement and / or prevention of sensitive skin" in the present specification means improvement of skin quality in which external stimulus resistance is reduced (skin stimulus threshold is lowered) and / or external stimulus resistance in skin quality. Refers to the prevention of a decrease in skin irritation (decrease in skin irritation threshold).

Further, the desmoglein reducing agent of the present invention is preferably used for suppressing cell-cell adhesion of human epidermal keratinocytes.
In the present specification, the term "cell adhesion" refers to abnormal stratum corneum adhesion function due to an increase in desmosome protein (protein group containing desmoglein) (progression of stratification of stratum corneum due to enhanced cell adhesion). (Refer to Japanese Patent No. 4197194 and Japanese Patent No. 4002635, if necessary).
Further, in the present specification, the concept of "inhibition of cell-cell adhesion" includes any of prevention or improvement of cell-cell adhesion.

Here, by suppressing the intercellular adhesion of human epidermal keratinocytes, the stratification of the stratum corneum is suppressed, and the effect of suppressing the exfoliation of the stratum corneum can be obtained.
That is, the desmoglein reducing agent of the present invention can be used for prevention or improvement of exfoliation of the stratum corneum.
In addition, in this specification, "weapon layer exfoliation" means exfoliation from the skin surface in a state where a plurality of stratum corneum are overlapped. Whether or not the skin is in a state where the stratum corneum is peeled off can be confirmed by collecting the stratum corneum by the tape stripping method, staining the collected stratum corneum, and observing it.

In addition, a large amount of desmoglein 1 is present in places where the lightness of the skin is low. Here, it is considered that the decrease in the lightness of the skin is due to the stratification of the stratum corneum.
In view of the above findings, by reducing desmoglein with the active ingredient of the present invention, it is possible to obtain an effect of improving the brightness of the skin.
That is, the active ingredient of the present invention can also be used as a skin brightness improving agent.
Here, in the present specification, "improvement of lightness of skin" means that the difference in lightness between normal skin and skin whose lightness is reduced due to the excessive presence of desmoglein is reduced.

Further, according to the desmoglein reducing agent of the present invention, a more transparent skin condition can be obtained. That is, the active ingredient of the present invention can also be used as a skin transparency improving agent.

In addition, a large amount of desmoglein 1 is present in places where the hardness of the skin is high. Here, the factor of the hardness of the skin is considered to be due to the stratification of the stratum corneum.
Based on the above findings, reducing desmoglein softens the skin.
That is, by reducing desmoglein with the active ingredient of the present invention, the effect of softening the skin can be obtained. That is, the active ingredient of the present invention can also be used as a skin softener.

Here, by softening the skin, it is possible to more efficiently permeate other drug components. Therefore, by reducing desmoglein with the active ingredient of the present invention, it is possible to obtain the effect of more efficiently permeating a medicinal ingredient such as a whitening agent into the skin.
That is, the active ingredient of the present invention can also be used as an auxiliary agent for permeation of medicinal ingredients into the skin.

In particular, the active ingredient of the present invention is preferably used as a whitening ingredient permeation aid for use in combination with a whitening agent.

(3) Composition The present invention relates to a composition containing the above-mentioned desmoglein reducing agent.
Above all, it is preferable to use the form of an external composition for skin or an oral composition.

As the composition for external use on the skin, cosmetics, pharmaceuticals and the like can be preferably exemplified.

Above all, it is preferably in the form of a cosmetic that can be used continuously, and for example, it can be in the form of a lotion, a milky lotion, a beauty essence, a cream, a gel, a sun care product, or the like.

The concentration of salvianophosphate A with respect to the total amount of the external composition for skin is preferably 3 μg / μL or more, more preferably 5 μg / μL or more, still more preferably 8 μg / μL or more, and particularly preferably 10 μg / μL or more. Is.

In the case of an oral composition, it is preferable to use a food composition containing the active ingredient of the present invention.
Specifically, it can be in the form of a supplement having a dosage form such as a general food, a tablet, a granule, or a drink.

The concentration of salvianophosphate A with respect to the entire oral composition is preferably 3 μg / μL or more, more preferably 5 μg / μL or more, still more preferably 8 μg / μL or more, and particularly preferably 10 μg / μL or more. Is.

[Test Example 1] Purification of components in wild thyme 80% ethanol extract A wild thyme raw material (1.0 kg) was immersed in 80% ethanol to prepare a wild thyme 80% ethanol extract.
The prepared 80% ethanol extract of wild thyme was used to obtain an unadsorbed / water elution part, a 60% methanol elution part, a methanol elution part, and an acetone elution part using a Diaion HP20 column.
By using various chromatographies from the 60% elution part, luteolin 7-O-glucuronide (isolated component 1,87 mg), apigenin 7-O-glucuronide (apigenin 7-O-glucuronide) , Isolated component 2,278 mg), eritricin (isolated component 3,44 mg), salvianolic acid A (isolated component 4, 13 mg), luteolin 5-O-glucoside (luteolin 5) -O-glucoside, isolated component 5,607 mg), diosmetin 5-O-glucoside (diosmetin 5-O-glucoside, isolated component 6,124 mg) were isolated.

[Test Example 2] Desmoglein reduction ability confirmation test of wild thyme fraction <Preparation of test sample and control sample>
80% ethanol extract prepared from wild thyme raw material, 60% methanol elution part, isolated 6 kinds of components and rosmarinic acid (Rosmarinic acid, manufactured by Wako Pure Chemical Industries, Ltd., component 7) were used as test samples.

The wild thyme 80% ethanol extract and the wild thyme 60% methanol eluate were prepared by DMSO so that the concentration was 6 μg / μL. 100 μL (final concentration 30 μg / mL) was added to 20 mL of PBS dispensed into a 50 mL tube, and the mixture was stirred with vortex to obtain a test sample.
The isolated components 1 to 6 and the component 7 were prepared by DMSO so that the concentration was 2 μg / μL. 10 μL (final concentration 1 μg / mL, low concentration sample) or 100 μL (final concentration 10 μg / mL, high concentration sample) was added to 20 mL of PBS dispensed into a 50 mL tube, and the mixture was stirred with vortex.
For low-concentration samples, 90 μL of DMSO was added and used as a test sample.
In addition, 100 μL of DMSO (final concentration 0.5%) was prepared as a control sample.

<Preparation process of stratum corneum cell sample>
The fourth layer of the stratum corneum on the outside of the human forearm was collected by the tape stripping method, and the cellophane tape to which the stratum corneum was attached was cut into squares of about 4 mm and attached to a slide glass. After the slide glass was immersed in xylene overnight, the immersion in xylene for 10 minutes was repeated twice, and the slide glass was air-dried until there were no water droplets on the slide glass.

<Immunostaining test>
After air-drying, the slide glass was immersed in the PBS containing the control sample or the PBS containing the test sample for 24 hours. After 24 hours, the slide glass was rinsed with PBS and immersed in 4% PFA for 15 minutes to fix the stratum corneum cells.
The slide glass was then rinsed with PBS and immersed in 0.5% Triton X-100 / PBS for 5 minutes at room temperature for permeabilization. Furthermore, after rinsing with PBS, blocking was performed at room temperature for 1 hour with 20% Block Ace (KAC). Then, the reaction with the primary antibody (anti-Dessmoglein 1 mouse monoclonal, DSG1-P23; PROGEN Biotechnik GmbH) was carried out at room temperature for 2 hours.

After repeating the immersion in PBS for 5 minutes three times, the reaction with a secondary antibody (Goat anti-mouse IgG (H + L) Cross-Adsorbed Sequence Antibody, Alexa Fluor 488; Invitrogen) was carried out at room temperature for 1 hour in a shaded state. .. Immersion in PBS for 5 minutes was repeated 3 times, then rinsed twice with distilled water and the sample was encapsulated.

Then, four analysis images were taken for each sample using an all-in-one fluorescence microscope BZ-X810 (KEYENCE). For the four analysis images, the area of the fluorescent portion (stained desmoglein 1) was calculated with the area of the entire analysis region as 100%, and the average value of the area of the fluorescent portion was calculated. The results are shown in FIGS. 1, 2 and 3.

In addition, the desmoglein 1 amount reduction rate (Dsg-1 reduction rate) of each test sample with respect to the control sample was calculated by the following formula. The results are shown in Table 1.

(Calculation formula for the rate of decrease in 1 amount of desmoglein)
Desmoglein 1 amount reduction rate (%) = (1-A / B) x 100
A: Dsg-1 amount (%) per area of the entire analysis image when the test sample is added.
B: Dsg-1 amount (%) per area of the entire analysis image of the control sample

A significant Dsg-1 reducing effect was observed in the wild thyme 80% ethanol extract and the wild thyme 60% methanol eluate, as well as the isolated component 3 (erytrikin) and the isolated component 4 (salbianophosphate A).

The present invention can be applied to cosmetics and pharmaceuticals that improve various skin conditions.

Claims (11)

A desmoglein reducing agent containing salvianophosphate A as an active ingredient. The desmoglein reducing agent according to claim 1, for suppressing cell-cell adhesion of human epidermal keratinocytes. The desmoglein reducing agent according to claim 1 or 2, wherein the target of reduction is desmoglein 1. The desmoglein reducing agent according to any one of claims 1 to 3, for improving and / or preventing sensitive skin. The desmoglein reducing agent according to any one of claims 1 to 3, for improving the lightness of the skin. The desmoglein reducing agent according to any one of claims 1 to 3, for improving the transparency of the skin. The desmoglein reducing agent according to any one of claims 1 to 3, for softening the skin. The desmoglein reducing agent according to any one of claims 1 to 3, for assisting the penetration of a medicinal ingredient into the skin. The desmoglein reducing agent according to any one of claims 1 to 3, for improving and / or preventing exfoliation of the stratum corneum. A composition comprising the desmoglein reducing agent according to any one of claims 1 to 9. The composition according to claim 10, which is a cosmetic or a pharmaceutical product.

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