JP2002308791A - Ceramidase activity inhibitor, skin care preparation containing the activity inhibitor, cosmetic, and quasi-drug - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a ceramidase activity inhibitor capable of inhibiting decomposition of ceramide which is an intercellular lipid by inhibiting ceramidase activity in a horny layer, to provide a skin care preparation compounded from the inhibitor, having an effect of improving a depressed barrier function of the skin, excellent in safety, and excellent in a feeling when used, to provide a cosmetic, and to provide a quasi-drug. SOLUTION: This ceramidase activity inhibitor contains at least one kind of extract selected from Curcuma longa extract, Saxifraga stolonifera extract, Glechoma hederacea extract, and Sargassum fulvellum extract.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a ceramidase activity inhibitor which suppresses the decomposition of ceramide which is an intercellular lipid by suppressing ceramidase activity in the stratum corneum, and a skin external preparation containing the ceramidase activity inhibitor. Agents, cosmetics,
Related to quasi-drugs.

[0002]

2. Description of the Related Art As is well known, ceramide is abundantly present in the stratum corneum, which is the outermost part of the skin, and forms a lipid bilayer structure. It is known that it plays a central role in the skin barrier function that prevents invasion of allergens and chemical substances.

Hitherto, to improve the skin barrier function which has been reduced, the amount of ceramide in the stratum corneum is increased by applying the synthesized or extracted ceramide itself or activating ceramide synthesis, thereby improving the skin barrier function. Has been planned.

[0004]

However, as in the prior art, even if ceramide itself is applied, a lipid bilayer structure cannot be formed in the stratum corneum. In the case of rough skin due to social stress, etc., it has been reported that ceramide synthesis in the stratum corneum has already been activated. Therefore, the method of activating ceramide synthesis cannot sufficiently improve the skin barrier function. Was.

The present invention has been made to solve the above problems, and has an effect of improving a reduced skin barrier function, and is excellent in safety and usability, and has excellent skin feel, cosmetics, An object of the present invention is to provide a ceramidase activity inhibitor used as a quasi drug.

[0006]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve such problems, and as a result, ceramide extract, saxifrage extract, exotic plant extract and diatom extract were added to ceramide extract. It has been found that the activity of ceramidase, which is an enzyme that decomposes is specifically suppressed, and the skin barrier function is improved, and the present invention has been completed.

[0007] That is, the means for solving the above-mentioned problems of the present invention is that the ceramidase activity inhibitor contains at least one of a turmeric extract, a saxifrage extract, a rosewood extract, and a gypsophila extract. .

[0008] The present invention also provides a skin external preparation containing at least one of such turmeric extract, saxifraga extract, exotic plant extract, and gypsophila extract,
Cosmetics and quasi-drugs.

The amount of the turmeric extract, Saxifraga extract, genus carrageenum extract, and gypsophila extract in the skin preparation for external use, cosmetics, and quasi-drugs is not particularly limited. , 0.0001-20 based on total amount
%, More preferably 0.0005 to 5.0% by weight.

[0010] Skin external preparations, cosmetics and quasi-drugs containing the ceramidase activity inhibitor of the present invention include lotions,
It can be in the form of emulsions, creams, packs, ointments and the like.

When the external preparation for skin of the present invention is used as cosmetics, examples of the cosmetics include cleansing cosmetics, basic cosmetics, hair cosmetics, make-up cosmetics, soaps, facial cleansers and the like. Is done.

The external preparation for skin of the present invention includes a pigment, a preservative,
Surfactants, fragrances, pigments and the like can be appropriately blended.

[0013]

Embodiments of the present invention will be described below.

Example 1 This example is an example of a ceramidase activity inhibitor, and a ceramidase activity inhibition test was carried out using a turmeric extract as a ceramidase activity inhibitor.

(Extraction of Ceramidase) Ceramidase is
It was extracted according to the following conventional method.

The brain extracted from the mouse was homogenized with a buffer, and the supernatant obtained by centrifugation was subjected to ultrasonic treatment and 0.5%
Crude extraction was performed with a buffer containing sodium cholate (Merck).

The crude extract was subjected to dialysis, ammonium sulfate precipitation, and gel filtration to purify the ceramidase, which was stored frozen at -20 ° C. until the test was used.

(Preparation of Turmeric Extract) Turmeric extract is
Paste-like turmeric extract (Maruzen Pharmaceutical) with ultrapure water 1.0w
/ v%, and filtered to remove insolubles, 1.0w / v
% Solution was prepared.

This is diluted with ultrapure water to obtain 0.01, 0.1, 0.5
And 1.0 w / v%.

(Measurement of ceramidase activity) After the turmeric extract was allowed to act on the ceramidase extract, the substrate C16
A suspension of ceramide (N-palmitoyl-dl-sphingosine: Funakoshi) was added, and an enzyme reaction was performed at 37 ° C.

An ethanol solution of NBD-Cl (4-chloro-7-nitrobenzofurazan: Dojindo) was added to the enzyme reaction solution, and sphingosine released in the enzyme reaction solution was removed.
An NBD induction reaction was performed at 55 ° C.

After the reaction was stopped by adding methanol to the reaction solution for deriving NBD, the HPLC was connected to a fluorescence detector.
Wavelength of sphingosine labeled with NBD at 469n
m, the fluorescence intensity at a fluorescence wavelength of 530 nm was measured.

To prepare a calibration curve, d-sphingosine (BIOMO) diluted sequentially with a 40 mg / mL aqueous solution of sodium cholate was used.
L) was used to induce NBD, and the fluorescence intensity was measured by HPLC.

FIG. 1 shows the test results.

FIG. 1 shows the amount of sphingosine released from ceramide by the action of ceramidase.

As shown in FIG. 1, the amount of sphingosine released by the action of the turmeric extract on the ceramidase is reduced in a concentration-dependent manner in the turmeric extract, and the release of sphingosine from the ceramide is controlled by the control of the ceramide. It was significantly suppressed compared to pure water.

Therefore, it is recognized that the turmeric extract has an action of suppressing ceramidase activity.

Example 2 This example is another example of a ceramidase activity inhibitor. A ceramidase activity inhibition test was carried out using a saxifrage extract as a ceramidase activity inhibitor.

(Extraction of ceramidase) The extraction of ceramidase was performed in the same manner as in Example 1.

(Preparation of Saxifraga stolonifera extract) The Saxifraga extract was prepared by drying dried Saxifraga stolonifera.
This is an extract obtained by immersing Meerburg root in ultrapure water.

The Saxifrage extract was filtered, dried and dried.
mg / mL dissolved in 50% ethanol
Diluted with ultrapure water to 0.1, 1, 5 and 10 v / v%.

(Measurement of ceramidase activity) The ceramidase activity was measured in the same manner as in Example 1.

FIG. 2 shows the test results of Example 2.

As shown in FIG. 2, the amount of sphingosine released by the action of the sacrificial extract on the ceramidase was reduced in a concentration-dependent manner, and the release of sphingosine from the ceramide was controlled by 5%. % Ethanol was significantly suppressed.

Accordingly, it is recognized that the Saxifraga extract has an action of suppressing ceramidase activity.

Example 3 This example is another example of a ceramidase activity inhibitor. A ceramidase activity inhibitory test was carried out using a rosewood extract as a ceramidase activity inhibitor.

(Extraction of ceramidase) The extraction of ceramidase was performed in the same manner as in Examples 1 and 2.

(Preparation of a Rosewood extract) A Rosewood extract was prepared by diluting the Rosewood extract-J (Maruzen Pharmaceutical) with ultrapure water to 0.01, 0.1, 1.0, and 5.0 v / v%.

(Measurement of ceramidase activity) The measurement of ceramidase activity was carried out in the same manner as in Examples 1 and 2.

FIG. 3 shows the test results of the third embodiment.

As shown in FIG. 3, the amount of sphingosine released by the action of the dextrin extract on the ceramidase was reduced depending on the concentration of the extract, and the release of sphingosine from the ceramide was controlled by 5%. % Ethanol was significantly suppressed.

Therefore, it is recognized that the extract of Hemerocallopus L. has the effect of suppressing ceramidase activity.

Example 4 This example is another example of a ceramidase activity inhibitor. A ceramidase activity inhibition test was carried out using diatom extract as a ceramidase activity inhibitor.

(Extraction of ceramidase) The extraction of ceramidase was performed in the same manner as in Examples 1 to 3.

(Preparation of diatom extract) The diatom extract was prepared by diluting a diatom extract (Maruzen Pharmaceutical) with ultrapure water to obtain 0.1%.
01, 0.1, 1.0, and 5.0 v / v%.

(Measurement of ceramidase activity) The measurement of ceramidase activity was performed in the same manner as in Examples 1 to 3.

FIG. 4 shows the test results of Example 4.

As shown in FIG. 4, the amount of sphingosine released by the action of diatom extract on ceramidase was reduced in a concentration-dependent manner on the diatom extract, and the release of sphingosine from ceramide was controlled by 2.5%.
% Ethanol was significantly suppressed.

Accordingly, it is recognized that the diatom extract has an action of suppressing ceramidase activity.

(Examples 5 to 8 and Comparative Examples 1 and 2) In the present example, turmeric extract, saxifrage extract, genus carrageensis extract, and gypsophila extract, which are ceramidase activity inhibitors shown in the above examples 1 to 4, were used. The improvement effect of the liquid on the skin barrier function was tested.

(Preparation of turmeric extract)
Turmeric extract in paste form (Maruzen Pharmaceutical), 5.0w / v%
Example 5 was prepared by preparing a dissolved 10% ethanol solution and filtering insolubles.

(Preparation of Saxifraga extract) A Saxifraga extract was extracted in the same manner as in Example 2.

This extract of Saxifraga was dissolved at 5 v / v%.
A 10% ethanol solution was prepared, and this was designated as Example 6.

(Preparation of Hemerocarpus extract) The Hemerocarpus extract was prepared by using Hemerocarpus extract J (Maruzen Pharmaceutical).
Example 7 A 10% ethanol solution in which v / v% was dissolved was prepared and
And

(Preparation of diatom extract) The diatom extract was prepared by dissolving 5 v / v% of diatom extract (Maruzen Pharmaceutical).
Example 8 was prepared as a% ethanol solution.

(Test method) BALB / c with back shaved
Male mice, 7 weeks old, reared in 40 / cage for 10 days,
Overcrowding stress was applied.

During overstress, Examples 5-8 were applied daily to the shaved back.

After releasing from the overcrowding stress, the amount of transepidermal water evaporation of the back skin, the amount of ceramide per corneal area, and the amount of ceramide per total lipid in the stratum corneum were measured. It was created and the condition of the tissue was observed.

The same measurement and observation were carried out as in Comparative Example 1 except that a non-stress load, which was bred in cages of 5 animals per cage and not subjected to overcrowding stress, was used as Comparative Example 1.

The case where only the stress was applied and no application was performed was set as Comparative Example 2, and the case where 10% ethanol as the base of Examples 5 to 8 was applied while applying the stress was set as Comparative Example 3.

(Measurement of Barrier Function) The transepidermal water loss of the back skin of Examples 5 to 8 and Comparative Examples 1 to 3 was measured using a Tewa meter.
It was measured by TM210 (COURAGE + KHAZAKA Electronic GmbH), and the difference between the measured values of Comparative Example 1 and Comparative Example 2 was converted to 100, and Examples 5 to 8 and Comparative Example 3 were shown by the recovery rate. FIG. 5 shows the measurement results of the recovery rate of the barrier function.

As is clear from FIG. 5, the mouse skin to which Examples 5 to 8 have been applied has recovered the barrier function in all the Examples compared to the case where the base of Comparative Example 1 was applied. I understand. This indicates that application of Examples 5 to 8 to the skin improved the skin barrier function.

(Measurement of amount of ceramide in stratum corneum)
8 and the horny layer were collected from the back skin of Comparative Examples 1 to 3 using a cyanoacrylate resin, and hexane: ethanol (95: 5) was used.
The lipid was extracted with the solution.

The extracted lipids were separated by thin-layer chromatography and after coloring, the density of each spot appeared was quantified, and the amount of ceramide per area of the collected stratum corneum and the ratio of the amount of ceramide to the total amount of extracted lipid were determined. Indicated. FIG. 6 shows the amount of ceramide per area of the stratum corneum, and FIG. 7 shows the ratio of the amount of ceramide to the total amount of lipid in the stratum corneum.

As is clear from FIG.
It was found that the amount of ceramide per unit area of the horny layer was increased in the mouse skin in which the base was applied in all the examples, as compared with the case where the base of Comparative Example 3 was applied.

This indicates that the reduction of the amount of ceramide in the stratum corneum due to stress was suppressed by applying Examples 5 to 8 to the skin.

Further, in FIG. 7 as well, the mouse skin to which Examples 5 to 8 were applied showed that the ceramide relative to the total lipid content in the stratum corneum in all the Examples compared to the case where the base of Comparative Example 3 was applied. It can be seen that the proportion of the amount is increasing or increasing.

This suggests that the decrease in the amount of ceramide in the stratum corneum due to stress is not due to the decrease in intercellular lipid, but to the decrease in ceramide itself.

(Observation of Tissue Condition) The back skin removed from Examples 5 to 8 and Comparative Examples 1 to 3 was fixed in a 20% neutral buffer of formalin, and a pathological tissue specimen stained with hematoxylin and eosin was prepared. And observed under a microscope.

In the observation of the tissues, the degree of epidermal layer thickening, basal cell spindle formation, stratum corneum stratification, and dyskeratinism are observed in +++ to 80% of the cases where the degree of remarkable observation is observed. The case was represented by +, the case observed in the-part was represented by ±, and the case not observed was represented by-. Table 1 shows the observation results of the tissue state.

[0071]

[Table 1] From the observation of the tissue state shown in Table 1, it can be seen that abnormalities in the keratinization in the epidermis layer were suppressed in the skin to which Examples 5 to 8 were applied, as compared with Comparative Examples 2 and 3. This indicates that by applying Examples 5 to 8, abnormal keratinization caused by stress was improved and a more normal state was maintained.

Turmeric extraction was performed based on the results of the above-described measurement of the barrier function, the measurement results of the amount of ceramide per area of the stratum corneum, the measurement results of the ratio of the amount of ceramide to the total lipid amount in the stratum corneum, and the observation results of the tissue state. It was found that the liquid, the saxifrage extract, the vegetative extract, and the gypsophila extract have the effect of suppressing the decrease in ceramide due to stress and maintaining the skin barrier function in a more normal state.

(Test for Improvement of Skin Roughness) In this example, a formulation was prepared in which the above-mentioned ceramidase activity inhibitor, a turmeric extract, a saxifrage extract, a rosewood extract, and a gypsophila extract, were incorporated into a cream as an example of a cosmetic. This is an example, and a cream roughening improvement test was performed on the cream.

The preparation of the cream was carried out as follows.

Squalene, cetyl isooctanoate,
After heating and dissolving the microcrystalline wax, the clay mineral and POE glycerol triisostearate were added, the temperature was adjusted to 70 ° C., and the mixture was uniformly dispersed and dissolved to obtain an oily gel.

Each of the turmeric extract, Saxifraga extract, Rosewood extract, and Gypsophila extract was dissolved in purified water of a predetermined concentration, adjusted to 70 ° C., and slowly added to the oily gel with sufficient stirring. .

After homogeneously mixing with a homomixer,
After filtration, the mixture was cooled to 30 ° C. to obtain a cream. The compositions and compounding ratios of the obtained creams of Examples 9 to 12 are as follows.

Example 9 Composition Mixing ratio (% by weight) Squalene 20% Cetyl isooctanoate 8.5% Microcrystalline wax 1% Clay mineral 1.3% POE glycerol triisostearate 0.2% Turmeric extract 0.1% Water balance

Example 10 Composition Mixing ratio (% by weight) Squalene 20% Cetyl isooctanoate 8.5% Microcrystalline wax 1% Clay mineral 1.3% POE glycerol triisostearate 0.2% Saxifrage extract 0.1% Water remaining amount

Example 11 Composition Compounding ratio (% by weight) Squalene 20% Cetyl isooctanoate 8.5% Microcrystalline wax 1% Clay mineral 1.3% POE glycerol triisostearate 0.2%

Example 12 Composition Blending ratio (% by weight) Squalene 20% Cetyl isooctanoate 8.5% Microcrystalline wax 1% Clay mineral 1.3% POE glycerol triisostearate 0.2% Kaisou extract 0.1% Water remaining amount

Using the creams of Examples 9 to 12 thus prepared, a practical test for rough skin was carried out.

The test method is as follows.

For 30 female subjects (25 to 45 years old) complaining of rough or dry skin, the sample and the cream base were applied twice a day to the affected area twice a day for one month.

The evaluation was based on the number of persons who answered that the degree of improvement of rough and dry skin was better in the sample application site than in the cream base application site.

On the other hand, a cream containing no turmeric extract or the like was subjected to the same test as Comparative Example 4.

Table 2 shows the test results.

[0088]

[Table 2]

As is clear from Table 2, in Examples 9 to 12, the number of persons who answered that they felt moist was about twice or more as compared with Comparative Example 4.

[Examples 13 to 16] In this example, the above-mentioned ceramidase activity inhibitor, a turmeric extract, a saxifrage extract, a rosewood extract, and a gypsophila extract, were mixed into a lotion as an example of cosmetics. This is a formulation example in which the rough skin lotion was tested for practical use.

Preparation of the lotion was carried out as follows.

A lotion base containing the surfactant POE (20) oleyl alcohol ether, the thickener methylcellulose, quince seed and ethanol was prepared, and a predetermined concentration of turmeric extract and the like were added.

The compositions and compounding ratios of the obtained lotions of Examples 13 to 16 are as follows.

Example 13 Composition Mixing ratio (% by weight) POE (20) Oleyl alcohol Ether squalene 0.5% Methyl cellulose 0.2% Quince seed 0.1% Ethanol 10% Turmeric extract 0.1% Water Remaining amount

Example 14 Composition Compounding ratio (% by weight) POE (20) Oleyl alcohol Ether squalene 0.5% Methyl cellulose 0.2% Quince seed 0.1% Ethanol 10% Saxifrage extract 0.1% Water remaining amount

Example 15 Composition Mixing ratio (% by weight) POE (20) Oleyl alcohol Ether squalene 0.5% Methylcellulose 0.2% Quince seed 0.1% Ethanol 10% Butterfly extract 0.1% Water balance

Example 16 Composition Mixing ratio (% by weight) POE (20) Oleyl alcohol Ether squalene 0.5% Methylcellulose 0.2% Quince seed 0.1% Ethanol 10% Kiso extract 0.1% Water Remaining amount

Using the lotions of Examples 13 to 16 prepared as described above, a practical test for rough skin was conducted. The test method is as follows.

For 30 female subjects (25 to 45 years old) complaining of rough and dry skin, the affected part was applied twice a day twice a day with a sample and a lotion base for one month.

The evaluation was made based on the number of persons who answered that the degree of improvement of rough and dry skin was better at the site where the sample was applied than at the site where the lotion base was applied.

On the other hand, the same test as Comparative Example 5 was conducted on a lotion not containing a turmeric extract or the like.

Table 3 shows the test results.

[0103]

[Table 3]

As is clear from Table 3, in Examples 13 to 16, the number of persons who answered that they felt moist was about 2 to 3 times as large as that in Comparative Example 5.

[Other Embodiments] In the above embodiment,
Although the case where only one of the turmeric extract, the Saxifraga extract, the rosewood extract, and the gypsophila extract is used has been described, it is also possible to mix and use two or more kinds.

[0106]

As described above, according to the present invention, it is possible to provide useful skin cosmetics, quasi-drugs, and skin external preparations having a remarkable skin roughness improving effect and excellent in usability. It became.

[Brief description of the drawings]

FIG. 1 is a graph showing the correlation between the concentration of a turmeric extract that acts on ceramidase and the amount of sphingosine released from ceramide.

FIG. 2 is a graph showing the correlation between the concentration of a saxifrage extract that acts on ceramidase and the amount of sphingosine released from ceramide.

FIG. 3 is a graph showing the correlation between the concentration of a rosewood extract that acts on ceramidase and the amount of sphingosine released from ceramide.

FIG. 4 is a graph showing a correlation between the concentration of an extract of diatoms that act on ceramidase and the amount of sphingosine released from ceramide.

FIG. 5 is a graph showing a recovery rate of a barrier function.

FIG. 6 is a graph showing the amount of ceramide per stratum corneum area.

FIG. 7 is a graph showing the amount of ceramide per total lipid amount.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 43/00 111 A61P 43/00 111 (72) Inventor Kazuhiko Hamada Kamigamo Toyodacho, Kita-ku, Kyoto-shi, Kyoto 64-5 F term (reference) 4C083 AA111 AA112 AB442 AC022 AC102 AC182 AC352 AC422 AD262 AD352 CC04 CC05 EE12 4C088 AB11 AB66 AB81 AC11 BA08 CA05 NA14 ZA89 ZC20

Claims (4)

[Claims] 1. A ceramidase activity inhibitor comprising at least one of a turmeric extract, a saxifrage extract, a rosewood extract, and a gypsophila extract. 2. An external preparation for skin, comprising the ceramidase activity inhibitor according to claim 1. 3. A cosmetic comprising the ceramidase activity inhibitor according to claim 1. 4. A quasi-drug containing the ceramidase activity inhibitor according to claim 1.