JP7724493B2 - Desmoglein reducer - Google Patents

Description

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

Increased desmosomal proteins are known to be one of the causes of deterioration of skin conditions, such as rough skin and desquamation of the stratum corneum caused by sunburn or dryness.
As a method for controlling the amount of desmosomal proteins, a method has been reported in which decomposition of desmosomal proteins accumulated in the stratum corneum with proteases is used to improve acne, dandruff, and desquamation (Patent Document 1).

Among desmosomal proteins, desmoglein in particular is known to be involved in adhesion between epidermal cells and keratinocytes (Patent Documents 2-4).

As a prior art, Patent Document 2 reports an agent for reducing desmoglein in stratum corneum cells, which contains astaxanthin.
Furthermore, Patent Document 4 reports an orally administered agent for inhibiting intercellular adhesion in the skin, which contains licorice extract as an active ingredient.

Patent Documents 5-7 report that extracts of Wild Thyme (Thymus serpyllum, hereafter referred to as "wild thyme") and extracts of Thymus plants in the Lamiaceae family have the effects of improving moisture retention, anti-inflammatory effects, preventing skin aging, and protecting against UV rays.

Special Publication No. 7-505383 JP 2016-37501 A Patent No. 4002635 Patent No. 4197194 Japanese Patent Application Laid-Open No. 2001-122729 Japanese Patent Application Laid-Open No. 2006-16337 JP 2009-256270 A

Fedoreyev SA et al, Planta Med 2005, 71, 5, 446-451

In light of the above-mentioned prior art, the objective of the present invention is to provide a novel technology for reducing desmogleins.

After extensive research, the inventors discovered that erythriquine has the effect of reducing desmoglein, leading to the completion of this invention.

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

In a preferred embodiment of the present invention, the desmoglein-reducing agent is used to inhibit intercellular adhesion of human epidermal keratinocytes.

In a preferred embodiment of the present invention, the desmoglein reducer is used to reduce desmoglein 1.

In a preferred embodiment of the present invention, the desmoglein reducer is used to prevent or improve sensitive skin.

In a preferred embodiment of the present invention, the desmoglein reducer is used to improve skin brightness.

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

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

In a preferred embodiment of the present invention, the desmoglein reducer is used to aid in the penetration of medicinal ingredients into the skin.

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

The present invention, which solves the above problem, is a composition containing the desmoglein reducer.

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

The present invention makes it possible to reduce the amount of desmoglein present on human skin.

FIG. 1 shows the experimental results of a test to confirm the desmoglein-reducing ability (control, 80% ethanol extract of wild thyme, and 60% methanol eluate). FIG. 1 shows the experimental results (isolated components 1 to 3) of a test to confirm the desmoglein-reducing ability. This figure shows the experimental results of a test to confirm the desmoglein-reducing ability (isolated components 4 to 6, component 7).

(1) Erythriquine The desmoglein-reducing agent of the present invention contains erythriquine as an active ingredient.

The structure of erythrichin ((2R)-3-(3,4-dihydroxyphenyl)-2-[4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2-naphthoyloxy]propanoic acid) is shown in Chemical Formula 1.

Erythrixine may be isolated from plant extracts or synthesized.

Erythrixine can be isolated from Erithrichium sericeum (Lehm.) A. DC. (Boraginaceae). An example of an isolation method is the method described in Non-Patent Document 1.

Erythriquine can also be obtained by the following method. First, 1 to 250 parts by weight of a solvent is added to 1 part by weight of the above-ground parts, roots, or dried material of wild thyme, and the mixture is soaked for several days at room temperature, or for several hours at temperatures near the boiling point. After soaking, erythriquine can be obtained by fractionation and purification using column chromatography packed with silica gel, synthetic adsorbents, octadecylsilylated silica gel, ion exchange resins, etc.

The extraction solvent is preferably a polar solvent, and suitable examples thereof include one or more selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butylene glycol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran.
Among these, preferred extraction solvents include ethanol and a mixed solvent of water and ethanol.

In addition, the desmoglein-reducing agent of the present invention can also use globoidnan A, which is not limited in its three-dimensional structure, as an active ingredient.

(2) Desmoglein-reducing agent The desmoglein-reducing agent of the present invention preferably targets desmoglein 1 for reduction.

The desmoglein-reducing agent of the present invention is preferably used for improving and/or preventing sensitive skin.
Here, "sensitive skin" in this specification refers to a skin type with reduced resistance (reduced skin irritation threshold) to external stimuli (dryness, ultraviolet rays, application of chemical compounds).

More specifically, "sensitive skin" in this specification refers to the skin types listed in (1) and/or (2).

(1) "Skin that reacts to substances such as topical medicines, cosmetics, plants, ultraviolet rays, and metals, and is prone to skin problems. Also, skin that is hypersensitive to allergens (pollen, fragrances, etc.) and irritants (alcohol, etc.)"
(2) "Skin that is temporarily more susceptible to skin problems due to lack of sleep, overwork, menstruation, seasonal changes, mental stress, etc."

In addition, in this specification, "for the improvement and/or prevention of sensitive skin" refers to the improvement of skin quality with reduced resistance to external stimuli (reduced skin irritation threshold) and/or the prevention of reduced resistance to external stimuli (reduced skin irritation threshold) in skin quality.

Furthermore, the desmoglein-reducing agent of the present invention is preferably used to inhibit intercellular adhesion of human epidermal keratinocytes.
In this specification, the term "intercellular adhesion" refers to abnormalities in stratum corneum adhesion (progression of stratum corneum stratification due to increased cell adhesion) caused by an increase in desmosomal proteins (a group of proteins including desmoglein) (see Patent Nos. 4,197,194 and 4,002,635 for details).
In addition, in this specification, the concept of "inhibiting intercellular adhesion" includes both prevention and improvement of intercellular adhesion.

Here, by inhibiting intercellular adhesion of human epidermal keratinocytes, stratification of the stratum corneum is inhibited, and an effect of inhibiting stratum corneum desquamation can be obtained.
That is, the desmoglein-reducing agent of the present invention can be used to prevent or improve stratum corneum desquamation.
As used herein, "stratum corneum delamination" refers to the peeling off of multiple layers of the stratum corneum from the skin surface. Whether or not the skin is in a state where stratum corneum delamination is possible can be confirmed by collecting a sample of the stratum corneum by tape stripping, staining the collected sample, and observing it.

Furthermore, areas of the skin with low brightness contain a large amount of desmoglein 1. Here, the decrease in skin brightness is thought to be due to stratification of the stratum corneum.
In view of the above findings, the active ingredient of the present invention can reduce desmoglein, thereby improving skin brightness.
That is, the active ingredient of the present invention can also be used as a skin lightening agent.
In this specification, "improving skin brightness" refers to reducing the difference in brightness between skin in a normal state and skin whose brightness has decreased due to the excessive presence of desmoglein.

Furthermore, the desmoglein-reducing agent of the present invention can achieve a more translucent skin condition. In other words, the active ingredient of the present invention can also be used as an agent for improving skin transparency.

Furthermore, areas of high skin hardness contain a large amount of desmoglein 1. Here, the cause of skin hardness is thought to be the stratification of the stratum corneum.
Based on the above findings, reducing desmoglein makes the skin softer.
In other words, the active ingredient of the present invention can reduce desmoglein, thereby providing a skin softening effect, and can also be used as a skin softener.

Here, softening the skin allows other medicinal ingredients to penetrate more efficiently, so by reducing desmoglein with the active ingredient of the present invention, it is possible to obtain the effect of allowing medicinal ingredients such as whitening agents to penetrate the skin more efficiently.
That is, the active ingredient of the present invention can also be used as an aid for the penetration of medicinal ingredients into the skin.

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

(3) Composition The present invention also relates to a composition containing the desmoglein-reducing agent described above.
In particular, the desmoglein-reducing agent of the present invention is preferably in the form of a composition for external use on the skin or an oral composition.

Suitable examples of compositions for topical use on the skin include cosmetics and pharmaceuticals.

In particular, it is preferable for the product to be in the form of a cosmetic product that can be used continuously, such as a lotion, emulsion, serum, cream, gel, sun care product, etc.

The concentration of erythriquinone in the entire composition for topical skin application is preferably 0.1 μg/μL or more, more preferably 0.5 μg/μL or more, even more preferably 1 μg/μL or more, particularly preferably 5 μg/μL or more, and most preferably 10 μg/μL or more.

When an oral composition is prepared, it is preferable to prepare a food composition containing the active ingredient of the present invention.
Specifically, it can be in the form of a supplement having the dosage form of a general food, tablet, granule, drink, or the like.

The concentration of erythrixine in the entire oral composition is preferably 0.1 μg/μL or more, more preferably 0.5 μg/μL or more, even more preferably 1 μg/μL or more, particularly preferably 5 μg/μL or more, and most preferably 10 μg/μL or more.

Test Example 1 Purification of Components in an 80% Methanol Extract of Wild Thyme A raw material of wild thyme (1.0 kg) was immersed in 80% ethanol to prepare an 80% ethanol extract of wild thyme.
The prepared 80% ethanol extract of wild thyme was passed through a Diaion HP20 column to obtain an unadsorbed/water eluted fraction, a 60% methanol eluted fraction, a methanol eluted fraction, and an acetone eluted fraction.
From the 60% methanol eluate, various chromatographic procedures were used to isolate luteolin 7-O-glucuronide (luteolin 7-O-glucuronide, isolated component 1, 87 mg), apigenin 7-O-glucuronide (apigenin 7-O-glucuronide, isolated component 2, 278 mg), erythrichin (eritrichin, isolated component 3, 44 mg), salvianolic acid A (salvianolic acid A, isolated component 4, 13 mg), luteolin 5-O-glucoside (luteolin 5-O-glucoside, isolated component 5, 607 mg), and diosmetin 5-O-glucoside (diosmetin 5-O-glucoside, isolated component 6, 124 mg).

[Test Example 2] Test to confirm the desmoglein-reducing ability of wild thyme fractions <Preparation of test samples and control samples>
The test samples were an 80% ethanol extract prepared from wild thyme raw material, a 60% methanol eluate, six isolated components, and rosmarinic acid (component 7, manufactured by Wako Pure Chemical Industries, Ltd.).

The 80% ethanol extract and 60% methanol eluate of wild thyme were adjusted with DMSO to a concentration of 6 μg/μL, and 100 μL of the prepared solution (final concentration: 30 μg/mL) was added to 20 mL of PBS dispensed into a 50 mL tube and then vortexed to obtain test samples.
Isolated components 1 to 6 and component 7 were prepared in DMSO to a concentration of 2 μg/μL. 10 μL (final concentration 1 μg/mL, low-concentration sample) or 100 μL (final concentration 10 μg/mL, high-concentration sample) of the prepared solution was added to 20 mL of PBS dispensed into a 50 mL tube, and the mixture was stirred with a vortex mixer.
For the low concentration sample, 90 μL of DMSO was added, and this was used as the test sample.
As a control sample, 100 μL of DMSO (final concentration: 0.5%) was prepared.

<Steps for preparing stratum corneum cell samples>
The fourth layer of the stratum corneum was collected from the outer surface of a human forearm using tape stripping. The cellophane tape containing the stratum corneum was cut into approximately 4 mm squares and attached to a glass slide. The glass slide was immersed in xylene overnight, then immersed in xylene for 10 minutes twice, and air-dried until all water droplets had disappeared.

<Immunostaining test>
After air drying, the slides were immersed in PBS containing a control sample or PBS containing a test sample for 24 hours, after which they were rinsed with PBS and immersed in 4% PFA for 15 minutes to fix the keratinocytes.
The slides were then rinsed with PBS and immersed in 0.5% Triton X-100/PBS at room temperature for 5 minutes for permeabilization. After further rinsing with PBS, they were blocked with 20% Block Ace (KAC) at room temperature for 1 hour. Subsequently, they were incubated with a primary antibody (anti-Desmoglein 1 mouse monoclonal, DSG1-P23; PROGEN Biotechnik GmbH) for 2 hours at room temperature.

After immersion in PBS for 5 minutes three times, the sections were incubated with a secondary antibody (Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488; Invitrogen) for 1 hour at room temperature in the dark. After immersion in PBS for 5 minutes three times, the sections were rinsed twice with distilled water and then mounted.

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

The desmoglein 1 reduction rate (Dsg-1 reduction rate) for each test sample relative to the control sample was calculated using the following formula. The results are shown in Table 1.

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

As shown in Table 1 and Figures 1 to 3, the 80% ethanol extract of wild thyme, the 60% methanol extract of wild thyme, as well as isolated component 3 (erythrixine) and isolated component 4 (salvianolic acid A) were found to have a significant Dsg-1 reducing effect.
Erythriquine was found to have a significant Dsg-1 reducing effect even at a low concentration of 1 μg/mL.

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

Claims (11)

A desmoglein reducer whose active ingredient is erythriquine. The desmoglein-reducing agent according to claim 1 for inhibiting intercellular adhesion of human epidermal keratinocytes. The desmoglein reducer according to claim 1 or 2, wherein the target to be reduced is desmoglein 1. A desmoglein-reducing agent according to any one of claims 1 to 3 for the improvement and/or prevention of sensitive skin. A desmoglein-reducing agent according to any one of claims 1 to 3 for improving skin brightness. A desmoglein-reducing agent according to any one of claims 1 to 3 for improving skin clarity. A desmoglein-reducing agent according to any one of claims 1 to 3 for softening the skin. The desmoglein reducer according to any one of claims 1 to 3, for aiding the penetration of medicinal ingredients into the skin. The desmoglein-reducing agent according to any one of claims 1 to 3 for improving and/or preventing stratum corneum desquamation. A composition for reducing desmoglein , which contains the desmoglein reducer according to any one of claims 1 to 3 and contains erythriquinone as an active ingredient, and which is intended to improve stratum corneum stratification, decreased skin brightness, and/or increased skin hardness caused by increased desmoglein. The desmoglein-reducing composition according to claim 10, which is a cosmetic or pharmaceutical product.