JP3129646B2 - Ceramide synthesis promoter - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a ceramide synthesis accelerator which is expected to improve rough skin and various skin diseases by activating ceramide synthesis of epidermal cells themselves in the skin surface layer and improving the skin barrier function. About.

[0002]

2. Description of the Related Art It has recently been known that ceramide, which is a kind of lipid, has an important physiological role although its quantity is smaller than that of glycerolipid which occupies most of the body. Biodistribution in mammals, including humans, is also a physiologically important place, but it is known that it is accumulated in the brain, liver, skin and the like.

[0003] In the skin, ceramide is accumulated especially in the stratum corneum of the epidermis, which is synthesized and secreted by epidermal cells and is a main component of intercellular lipids forming a unique lamellar structure between cells ( Lukas Landmann: Anat Embryol, 1
78, pages 1-3, 1988). The stratum corneum has a series of physiological roles, including the ability to moisturize the skin and physical protection of the living body, so-called barrier functions, but intercellular lipids are the substance of this barrier function and are the most important in life support (Genji Imokawa: Journal of Cosmetic Society, Vol. 15 (4), pp. 250-253, 1991)
Year). In this sense, it can be said that skin ceramide is one of the important substances in biological defense.

[0004] For rough and dry skin and various skin diseases,
It has been reported that the healthy formation of the stratum corneum is hindered and the barrier function is reduced. Specific examples include a decrease in the turnover of the epidermal layer due to aging of the skin surface, and rough or dry skin caused by external factors such as light, temperature, and weather conditions. This is thought to be caused by a decrease in the barrier function, and a decrease in the moisturizing capacity of the skin and an increase in the amount of water transpiration (Shuichi Akasaki et al .: Nisshin Gakkai, Vol. 98 ( 1
No.), pp. 41-51, 1988).

[0005] Among skin diseases, in atopic dermatitis, the barrier function is deteriorated or degraded not only in the inflamed part but also in the non-inflamed part of the patient, and ceramide in the patient's skin, in general or in a specific type, is reduced. A decrease in the content has been reported (Shin Kawashima: Journal of Cosmetic Society, Vol. 15, No. 4, 261-262).
1991). In addition, changes in the amount of ceramide in the skin of patients have been reported in psoriasis (Stefania.M: Arch De
rmatol. 130, 452-456, 1994),
Again, this variation is thought to be related to barrier collapse.

[0006] With respect to skin diseases and insufficiencies caused by such deterioration or breakdown of the skin barrier function, administration of a moisturizer has been conventionally used to prevent the skin from becoming dry and moist, and to prevent eczema caused by anti-inflammatory agents. Control has been attempted. However, these methods have a temporary effect because they replenish some of the water or moisturizing components on the keratinous surface, and cannot provide sufficient moisture inside the skin continuously (Takemura Toshiyuki: Pharmacia, vol. 28, p. 1, 1992
Years), even if the temporary inflammation was suppressed, there were often problems with the persistence of the effects and side effects.

[0007] On the other hand, recently, attempts have been made to improve the skin by externally supplementing ceramide, which is a main component of the barrier, and it has been reported that it is effective for rough skin and atopic dermatitis (Yuko Higaki et al .: Allergy Clinic, 13 (12), 26-28, 1993). However, this method seems to have an early effect, but it is inadequate in the persistence of the effect as well as the moisturizers that have been used in the past, and the difference in transdermal absorption due to the condition of the skin. However, there is a disadvantage that the effect is not sufficiently exhibited.

On the other hand, nicotinic acid and nicotinamide are one of vitamins widely present in animal and plant tissues, and have been conventionally used as growth factors in culturing yeasts and other bacteria. However, it is not known that these bacterial cultures promote ceramide synthesis of epidermal cells.

[0009]

In view of such circumstances, the inventors of the present invention intend to activate ceramide synthesis of epidermal cells themselves within the skin surface layer and improve the skin barrier function, and to improve the skin barrier function. As a result of diligent examination of the search for nicotinic acid and / or nicotinamide, they found that a bacterial culture containing nicotinic acid and / or nicotinamide had an effective ceramide synthesis accelerating action, and completed the present invention. However, it is an object of the present invention to provide a ceramide synthesis promoter that is expected to improve rough skin and various skin diseases by activating ceramide synthesis of epidermal cells themselves in the skin surface layer and improving the skin barrier function.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to provide a lactic acid bacteria culture containing nicotinic acid and / or nicotinamide .
Of ceramide synthesis using yeast or yeast culture as an active ingredient
Thus it is achieved agents.

[0011]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The configuration of the present invention will be described below in detail.

Lactic acid bacteria culture or yeast containing nicotinic acid and / or nicotinamide used in the present invention
The culture, for example, lactic acid bacteria culture cultured in a medium containing nicotinic acid and / or nicotinamide include yeast fungus culture product.

As lactic acid bacteria, for example, Streptococcus
thermophilus, Lactobacillus bulugaricus, Lactoco
ccus lactis, etc., as the yeast bacteria, for example Saccharomy
cescerevisiae, Endomyces magnusii and the like.

In obtaining the ceramide synthesis promoter of the present invention, the amount of nicotinic acid or nicotinamide added to the culture system of these bacteria is 0.0001 to 1% by weight (hereinafter referred to as%).
Is preferable).

The use form of the ceramide synthesis promoter of the present invention includes, in addition to additives to cultured cells, external preparations for skin.
For example, ointments, creams, lotions and the like can be mentioned.
The base of the external preparation may be a known external base and is not particularly limited.

When the bacterial culture which is the ceramide synthesis promoter of the present invention is added to a cultured epidermal cell line to promote ceramide synthesis, the amount added is preferably 0.001 to 10%. 1
The content of 0% or less is preferable in that the stimulation to epidermal cells is small.

The amount of the ceramide synthesis promoter of the present invention to be added to the external preparation for skin is sufficient because it promotes ceramide synthesis sufficiently and hardly gives out color or odor of the culture. The content is preferably from 0.01 to 20%, particularly preferably from 0.1 to 10%.

[0018]

The present invention will be described in detail below with reference to examples and comparative examples.

Examples 1 to 6, Comparative Examples 1 to 3 (Lactic acid bacterium culture) Purified water was added to the compositions shown in Table 1 to make up to 100 ml.
A medium was prepared by autoclaving at 21 ° C. for 20 minutes (Skim milk was manufactured by Difco, and glucose, nicotinic acid and nicotinamide were manufactured by Kanto Kagaku). Lactococcus lact was pre-cultured in the same medium at 37 ° C for 24 hours.
is (IFO 12007), Streptococcus thermophilus (AT
CC 19254) and Lactobacillus bulgaricus (ATCC 1
1842). After static culture at 37 ° C for 24 hours,
The lactic acid bacteria culture obtained by removing the cells by centrifugation and treating the culture supernatant at 80 ° C. for 30 minutes was used as the ceramide synthesis promoter of the present invention (Examples 1 to 6).

As comparative examples, lactic acid bacteria cultures were obtained in the same manner as in Examples 1 to 6, except that neither nicotinic acid nor nicotinamide was used (Comparative Examples 1 to 3).

[0021]

[Table 1]

Examples 7 and 8, Comparative Example 4 (Yeast culture) Purified water was added to the compositions shown in Table 2 to make up to 100 ml.
A medium was prepared by autoclaving at 21 ° C. for 20 minutes (malt extract broth was from Difco and nicotinic acid was from Kanto Kagaku). Add 28 ° C, 48
Saccharomyces cerevisiae (IFO 237
5) was inoculated at 1%. After shaking culture at 28 ° C for 48 hours,
The yeast culture obtained by removing the cells by centrifugation and treating the culture supernatant at 80 ° C. for 30 minutes was used as the ceramide synthesis promoter of the present invention (Examples 7 to 8).

As a comparative example, a lactic acid bacteria culture was obtained in the same manner as in Examples 7 and 8, except that neither nicotinic acid nor nicotinamide was used (Comparative Example 4).

[0024]

[Table 2]

Hereinafter, a ceramide synthesis promotion test and a skin barrier recovery test using the ceramide synthesis promoters of Examples 1 to 8 and the bacterial cultures obtained in Comparative Examples 1 to 4 will be described.

Test Example 1 Ceramide synthesis promotion test (1) Method (a) Cultured epidermal cells Normal human epidermal cells are commercially available (Cascad)
e Biologic).

(B) Medium for Cell Culture The medium used was an MCDB153 medium supplemented with BPE (bovine pituitary gland) as a growth factor.

(C) Preparation of Hepes buffer 7.15 g of Hepes, 1.8 g of glucose, 0.22 g of potassium chloride, 7.7 g of sodium chloride and 0.27 g of disodium hydrogenphosphate dodecahydrate are dissolved in purified water. Then, the pH was adjusted to 7.4 with a 1N aqueous solution of sodium hydroxide, and the volume was increased to 1 liter.

(D) Cell culture The number of normal human epidermal cells was counted in MCDB153 medium to 1
× 10 ⁴ cells / ml, inoculated in 4 ml portions on a 60 mm collagen-coated plate (Falcon) and 95%
In an atmosphere of air (V / V) -5% (V / V) carbon dioxide,
The cells were cultured at 37 ° C. for 5 days.

The culture supernatant was removed by suction, and the ceramide synthesis promoter of Examples 1 to 8 and the bacterial culture of Comparative Examples 1 to 4 were mixed with 1
4% of MCDB153 medium to which the weight% was added was added to each dish. In addition, Hepes buffer solution was added as a control. 95% air (V /
V) -6% at 37 ° C in an atmosphere of -5% (V / V) carbon dioxide gas.
The culture was allowed to stand for a day.

On the sixth day, 0.5 μCi of [ ¹⁴ C] -serine (American Radiolabeled Ch) was used.
(manufactured by Chemicals Inc.) was added to the medium, and the culture was further performed for 2 days. After the culture, the cells were treated as follows.

(E) Extraction of lipid The medium supernatant was removed by suction, washed twice with 5 ml of Hepes buffer, and the cells were scraped off from the dish with a cell scraper (Sumitomo Bakelite). 1.6 ml of this
In Hepes buffer, 4 ml of methanol and 2
Add ml of chloroform and mix. After allowing to stand at room temperature for 20 minutes, a 1.6 ml chloroform layer was taken to obtain a lipid fraction. Remove chloroform by centrifugation and remove 1 ml
Redissolved in benzene.

(F) Isolation of Ceramide Fraction Using Iatrobeads Column A lipid sample dissolved in benzene was subjected to Iatrobeads 100 β
The solution was applied to a column packed with 1, washed with a benzene-ethyl acetate (4: 1) solution, and eluted with 1 ml of ethyl acetate to obtain a ceramide fraction.

(G) Measurement of radioactivity of [ ¹⁴ C] -labeled ceramide The radioactivity incorporated in the ceramide fraction was measured with a liquid scintillation counter.

(2) Results The results are shown in FIG. As is clear from FIG. 1, the ceramide synthesis promoting agent of the present invention (Examples 1 to 8) comprising a bacterial culture cultured in a medium to which nicotinic acid and / or nicotinamide was added showed an effect of promoting ceramide synthesis. .

Test Example 2 Skin Barrier Recovery Test (1) Method As a test animal, a Skh: hr hairless mouse male (Japan SLC), 6 weeks old, was purchased and reared for 2 weeks.
The experiment was started with 5 animals per group.

Rough skin is made of retinoic acid (vitamin A acid: al
20 μg of l-transretinoic acid (SIGMA) was dissolved in ethanol and applied to the buttocks of a mouse once a day (in the morning) for 3 days.

Ceramide synthesis accelerator 2 of Examples 1, 3 and 5
lyophilized, dissolved in the same amount of 50% (V / V) ethanol, and from the afternoon of the day on which retinoic acid was applied,
Similarly, 100 μl was applied once a day (afternoon) for 3 days. In addition, only 50% (V / V) ethanol was applied as a control.

Three days after the application of retinoic acid, the transepidermal water loss (TEWL) was measured and expressed as the water loss (mg / cm ² / min). TEWL is an index for measuring the skin barrier function.
It rises when the barrier function is destroyed and falls when it recovers. The measurement of TEWL was performed using AUM-3 manufactured by Forsion.

(2) Results The results are shown in FIG. As is clear from FIG. 2, the TEWL of the ceramide synthesis accelerator applied group of Examples 1, 3, and 5 three days after application of retinoic acid was lower than that of the control, and the application of the ceramide synthesis accelerator of Examples 1, 3, and 5 was performed. As a result, it was found that retinoic acid restored damage to the skin barrier function.

Hereinafter, application examples of the ceramide synthesis accelerator of the present invention will be described.

Application Examples 1 to 3 (Skin Cream) A skin cream was prepared by blending the ceramide synthesis accelerators of Examples 1, 3 and 5 with the compositions shown in Table 3 (Application Example 1).
~ 3).

(1) Composition

[Table 3]

(2) Preparation Method The components (A) and (B) were each heated and dissolved at 80 ° C., mixed, cooled with stirring, and cooled to 30 ° C. to prepare a skin cream.

Application Examples 4 to 6 (Lotions) The ceramide synthesis accelerators of Examples 1, 3 and 5 were blended in the composition shown in Table 4 to prepare lotions (Application Examples 4 to 6).

(1) Composition

[Table 4]

(2) Preparation method The components (A) and (B) are mixed and dissolved, respectively.
(B) was added to (A), and mixed and stirred to prepare a lotion.

[0048]

As described above, according to the present invention, ceramide synthesis which is expected to improve rough skin and various skin diseases by activating ceramide synthesis of epidermal cells themselves in the skin surface layer and improving the skin barrier function. Clearly, an accelerator can be provided.

[Brief description of the drawings]

FIG. 1 is a graph showing the ceramide synthesis promoting effects of the ceramide synthesis promoters of Examples 1 to 8.

FIG. 2 is a graph showing the effect of improving the rough skin by the ceramide synthesis accelerators of Examples 1, 3, and 5.

Continuation of the front page (51) Int.Cl. ⁷ identification code FI A61P 17/16 A61P 17/16 (56) References JP-A-59-137405 (JP, A) JP-A-6-293649 (JP, A) JP-A-8-217658 (JP, A) JP-A-10-259135 (JP, A) JP-A-6-40881 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35/72 A61K 7/00 A61K 7/48

Claims (1)

(57) [Claims] [Claim 1] A ceramide synthesis promoter containing nicotinic acid and / or nicotinamide, comprising a lactic acid bacteria culture or a yeast culture as an active ingredient.