JPH0710740A - Melamine production-inhibiting agent - Google Patents

Abstract

PURPOSE:To obtain a melamine production-inhibiting agent expressing an excellent melamine production-inhibiting action and a moisture-retaining action, substantially not irritating skins, and useful also as a skin cosmetic ingredient. CONSTITUTION:The melamine production-inhibiting agent contains a fraction originated from the supernatant of the culture solution of a Bifidobacterium bacterium and having an estimated fraction mol.wt. of approximately 300-3000 by a high speed gel filtration method or its partial fraction as an active ingredient.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a melanin production inhibitor, and more specifically, it shows an excellent melanin production inhibitory action, and the action is reversible, and is excellent in safety. The present invention relates to an inhibitor and a skin cosmetic containing the same.

[0002]

BACKGROUND OF THE INVENTION Melanin, which is usually present in the skin, is an important medical and cosmetic factor that plays an important role in protecting the body from the effects of ultraviolet rays.

It is said that the synthesis of melanin is carried out in the pigment cells in the skin tissue by the action of tyrosinase, whereby tyrosine is changed into dopa and then dopaquinone, and then via 5,6-dihydroxyindole and the like.
When melanin is excessively synthesized, it exhibits a black color, while the uneven distribution of melanin on the skin forms spots, freckles, and the like, which are both serious cosmetic problems. The pigmentation of spots, freckles, etc. is exacerbated by ultraviolet irradiation. To promote the production of melanin in pigment cells induced by this ultraviolet irradiation,
Melanocyte stimulating hormone (α-MSH) is deeply involved.

Conventionally, hydroquinone, MBEH (monobenzyl ether of hydroquinone) and the like have been used for the purpose of obtaining fair skin, but these are for bleaching the skin by the action of denaturation and lethality of pigment cells. Therefore, the original physiological function of the skin is impaired, and irreversible vitiligo,
It has the drawback of causing side effects such as pigment abnormalities and rashes.

In order to correct the above-mentioned drawbacks, a substance which inhibits tyrosinase activity as a melanin production inhibitor is mainly searched for, and arbutin (JP-A-60-56912 and JP-A-61) as an inhibitor of tyrosinase activity.
-207316), kojic acid and its derivatives (JP-A-56-7776, JP-A-56-7727).
2, JP-A-59-122485, JP-A-6-
0-233071, etc.), vitamin C and its derivatives (JP-A-56-135411, JP-A-60-23).
7083, JP 61-286397),
Cysteine (Japanese Unexamined Patent Publication No. 61-239017), glutathione (Japanese Unexamined Patent Publication No. 61-189296), and other natural products derived from an agaraceae plant extract (Japanese Unexamined Patent Publication No.
No. 3-88333), many plant extracts and the like are disclosed. Recently, Smoto et al.
bacillus helveticus JCM11
It was found that the cyclic peptide having a molecular weight of 456 in 20 culture supernatants had tyrosinase inhibitory activity (1993 Journal of the Japanese Society of Agricultural Chemistry). Furthermore, as a substance that suppresses the production of tyrosinase itself and has an action of suppressing melanin production, organic acids and salts thereof (JP-A-2-193917,
JP-A-2-193918, JP-A-3-10681
No. 0) is disclosed.

[0006] However, it is said that these conventional melanin production inhibitors exert a melanin production inhibitory effect, such as the fact that some of them have an insufficient melanin production inhibitory effect in melanin-producing cells despite their high tyrosinase inhibitory activity. There were some difficulties and generally there were problems with stability and safety, which were not always satisfactory.

Therefore, the excellent melanin production inhibitory action is stably exhibited, and the action is reversible without impairing the physiological function of the skin, and the safety to the human body is also good, and environmental destruction is caused. It has been desired to develop a substance or composition derived from a natural product that does not invite it.

[0008]

DISCLOSURE OF THE INVENTION The inventors of the present invention have made earnest studies in view of such circumstances. As a result, a specific fraction obtained from the supernatant of a liquid culture of Bifidobacterium (genus Bifidobacterium), which has been widely used in foods, pharmaceuticals, etc. and has already been highly evaluated for its safety, produces excellent melanin production. It has been found that the inhibitory effect is stable, the effect is reversible, it is gentle to the skin, it has a moisturizing effect, and it can be widely mixed in cosmetics. Completed the invention.

That is, the present invention is derived from the culture supernatant of Bifidobacterium and has an estimated molecular weight cut-off of about 3 by the high speed gel filtration method.
The present invention provides a melanin production inhibitor containing a fraction of 00 to 3,000 or a fraction thereof as an active ingredient.

Furthermore, the present invention provides a skin cosmetic containing the fraction.

The bifidobacteria used in the present invention are obligately anaerobic hetero-lactic acid bacilli, and are classified into several tens of species according to their glycolytic properties. The Bifidobacterium strain that can be used in the present invention includes all of these, but is preferably human-derived Bifidobacterium longum (Bifidobact).
erium longum), Bifidobacterium breve (Bifidobacterium brev)
e), Bifidobacterium bifidum (Bifid
strains such as Obacterium bifidum) are used. Specific examples include Bifidobacterium longum ATCC15707 and Bifidobacterium longum No. 7. Bifidobacterium breve AT
CC15700, Bifidobacterium bifidum
ATCC11146 etc. are mentioned. These Bifidobacteria are widely used in fermented milk, lactic acid beverages, health foods, pharmaceuticals, feeds and the like, and are easily available.
The Applicant is the applicant of the Bifidobacterium longum (Bif
Idobacterium longum) No. 7 was deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology (FER
MP-13610).

[0012] In the present invention, the medium for culturing the bifidobacteria is not particularly limited as long as it is a medium in which the bifidobacteria can grow well, but preferably,
For example, a medium containing a hydrolyzate of milk protein, saccharides, yeast extract and the like can be mentioned. As a hydrolyzate of cow's milk protein, a hydrolyzate of skim milk, whey, casein or the like can be used, and as the saccharide, various sugars can be used, but lactose is sufficient.

The culture is carried out by a generally used anaerobic culture method. For example, a batch culture method by standing is preferable, and a neutralization culture method that allows further high-density culture is preferable. For example,
A lactic acid bacterium of the genus Bifidobacterium is inoculated into the medium (pH about 6.0) of the above composition, and the temperature is about 37 ° C while aeration with nitrogen gas.
Anaerobic culture is performed. During the culture, neutralize with sodium hydroxide or the like to maintain the pH of the medium at around 6.0. After culturing for about 12 hours, the maximum number of bacteria is reached, so culturing is stopped, the culture solution is centrifuged, and the culture supernatant is collected. The culture supernatant is preferably concentrated under reduced pressure about 3 to 5 times in order to stabilize the subsequent treatment. Next, organic acid salts and other low molecular weight substances contained in large amounts in the culture supernatant concentrate are removed.

Examples of the method for removing low molecular weight substances from the culture supernatant concentrate include gel filtration method, ultrafiltration method, dialysis method and the like, and these can be used alone or in appropriate combination.

When the gel filtration method is used, agar gel, dextran gel, polyacrylamide gel and the like can be used. For example, when using dextran gel, "S
Ephadex G-25 "(Pharmacia) can be used. The elution is performed using purified water, and the fraction from the void portion until the start of elution of the organic acid salt (sodium acetate, sodium lactate, etc.) is fractionated. An active substance that suppresses melanin production is contained in this fraction.

When the ultrafiltration method is used, a filtration membrane having a molecular weight cutoff of about 500 to 1,000 is used. The melanin production inhibitory substance of the present invention is contained in the ultrafiltration membrane non-permeable fraction. When the dialysis method is used, an electrodialysis method capable of rapidly dialyzing low-molecular dissociated ions in the culture supernatant is suitable. The non-dialysate contains the melanin production inhibitor of the present invention.

Since the concentrated liquid of the culture supernatant is usually turbid and contains a large amount of colloidal substances, these are previously removed by activated carbon treatment or ultrafiltration for the purpose of stabilizing the subsequent treatment steps. Preferably.

When the activated carbon treatment is used, either a batch method or a column method can be used. The appropriate amount of activated carbon varies depending on the type of activated carbon, the treatment method, and the treatment pH. For example, when treating with a batch method using powdered activated carbon manufactured by Wako Pure Chemical Industries, Ltd., the original culture supernatant liquid About 2% by weight
Add powdered activated carbon (about 10% by weight in 5 times concentrated solution),
Stirring is desirable.

In the case of the ultrafiltration membrane treatment, if the molecular weight cutoff of the separation membrane is 10,000 or more, particularly strict conditions are not required, and the treatment conditions are appropriately selected depending on the treatment amount of the culture supernatant. The operation method may be either batch filtration in which concentration and dilution are repeated or continuous filtration in which a solvent is added.

The estimated molecular weight cut off of the fraction thus obtained by the high speed gel filtration method is about 300 to 3,000.

From the fraction thus obtained, the melanin production inhibitor of the present invention containing the fraction as an active ingredient can be prepared.

The melanin production inhibitor of the present invention may be prepared according to a conventional method by concentrating or diluting the above-mentioned fractions as needed, and further adding any component within a range not impairing the effects of the present invention. it can.

When the above-mentioned fractions are mixed with various cosmetic bases, skin cosmetics intended for whitening can be obtained due to their melanin production-inhibiting action. The blending amount of the melanin production inhibitor in the cosmetic composition of the present invention varies depending on the form of the cosmetic composition, etc.
The dry weight is preferably 0.1 to 2% by weight, more preferably 1 to 2% by weight.

The skin cosmetics of the present invention include ordinary cosmetics,
Various optional ingredients used in quasi-drugs, pharmaceuticals, etc., such as oils, moisturizers, thickeners, preservatives, emulsifiers, pigments, powders, pH adjusters, medicinal ingredients, ultraviolet absorbers, antioxidants, and fragrances. Etc. can be blended appropriately.

The skin cosmetic of the present invention can be manufactured by a conventional method. Further, the skin cosmetics of the present invention are not limited to general skin cosmetics, but include quasi-drugs, external medicines, and the like, whose dosage form is cream,
Emulsion, lotion, foundation, pack, lotion, gel, solution, stick, etc. can be arbitrarily selected according to the purpose.

[0026]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. First, obtain the culture supernatant of Bifidobacterium by the following culture method,
The following fraction production examples were used. (Culturing method) To 3 kg of sodium casein, 100 kg of hot water is added and dissolved. 40 g of microbial-derived proteolytic enzyme powder is added and reacted at 50 ° C. for 4 hours to hydrolyze casein. Lactose (4 kg) and yeast extract (500 g) were added to this, and the pH was adjusted.
Adjust to 6.0. Heat at 121 ° C. for 10 minutes to perform sterilization and enzyme inactivation together. After cooling, Bifidobacterium longum is inoculated, and anaerobic culture is carried out at 37 ° C. with aeration of nitrogen gas. Sterilized during culture 10
% NaOH is added dropwise to adjust the pH to around 6.0. About 12
After culturing for a time, the mixture is cooled and centrifuged at 6,000 rpm to obtain a culture supernatant. The pH of this culture supernatant is adjusted to 10% Na
After adjusting to 7.0 with OH, it is concentrated under reduced pressure to obtain a 5 times concentrated solution.

Production Example 1 Activated carbon treatment was carried out for the purpose of removing turbidity of the culture supernatant. That is, 2 kg of activated carbon was added to 20 L of a 5-fold concentrated solution of the culture solution, the mixture was stirred at room temperature for 1 hour and allowed to stand, and the supernatant solution was filtered with a glass fiber filter paper to obtain a transparent filtrate. Gel filtration was then performed for desalting and removal of low molecular weight fractions.
That is, it was applied to a Sephadex G-25 gel filtration column (25 mm × 300 mm) manufactured by Pharmacia Co., and the mobile phase was fractionated by flowing purified water at a flow rate of 0.5 ml / min. Fractions before voiding of sodium acetate and sodium lactate from the void volume were collected. Table 1 shows the component analysis results of the obtained fractions.

[0028]

[Table 1]

Further, the above fractions exhibited the following properties. (1) Ultraviolet absorption spectrum Wide absorption is observed in the 250 to 350 nm region. (2) Solubility Up to 50% by weight is soluble in water, but insoluble in ethanol. (3) Properties It is a pale yellow powder with a slight peculiar odor. (4) pH 7.0. (5) Thermal stability Stable at 40 ° C. for 1 hour treatment (residual activity 109%), 90
Relatively stable at 1 ° C for 1 hour (remaining activity 60-100)
%). (6) Acid, alkali resistance It is relatively stable to 1 hour treatment at pH 1.0 and pH 11.0 (60% residual activity).

Production Example 2 Ultrafiltration treatment was performed for the purpose of removing the turbidity of the culture supernatant. That is, the culture supernatant was diluted with water so as to be a 3-fold concentrated solution, and filtered using an ultrafiltration membrane XM300 manufactured by AMICON (molecular cutoff of 300,000) to obtain a transparent permeate. The molecular weight cut-off size of the ultrafiltration membrane used is 10,000 to 10
Any number of 0,000 can be used. Gel filtration was then performed for desalting and removal of low molecular weight fractions. That is, this was applied to a Sephadex G-25 gel filtration column (25 mm x 850 mm) manufactured by Pharmacia, and the mobile phase was fractionated by flowing purified water at a flow rate of 0.5 ml / min. Fractions before voiding of sodium acetate and sodium lactate from the void volume were collected.

Production Example 3 Electrodialysis treatment was carried out for the purpose of desalting the culture supernatant. That is, an electrodialysis machine "DW-O type" manufactured by Asahi Glass Co., Ltd. was used to perform electrodialysis using an electrodialysis membrane "CSV / AMT" to obtain a desalted solution. Next, ultrafiltration treatment was performed for the purpose of removing turbidity. That is, the ultrafiltration membrane X manufactured by AMICON
Filtration was performed using M300 (molecular weight cutoff of 300,000) to obtain a transparent permeate. Then, ultrafiltration was performed to remove low molecular weight fractions. That is, the ultrafiltration membrane Y manufactured by AMICON
Filtration was performed using MI (molecular weight cutoff of 1000) to obtain a concentrated liquid fraction that did not pass through the filtration membrane. For ultrafiltration treatment, add purified water of the same volume as the treatment liquid to the concentrated liquid fraction,
Purification was performed.

Test Example 1 (1) TSK-GEL-G3000PW was prepared by dissolving 20 μl of each of the fractions obtained in Production Examples 1 to 3 in 45% acetonitrile (containing 0.1% trifluoroacetic acid).
_It was injected into _XL (7.8 mmφ × 300 mm, manufactured by Tosoh Corporation) and fractionated at the flow rate of 0.5 ml / min using the same moving bed. The fractionated molecular weight distribution was detected by the absorbance at 215 nm based on a molecular weight calibration curve prepared in advance using a standard peptide. The result is shown in FIG. As is clear from FIG. 1, it is clear that the fractions obtained in each production example all have a molecular weight cutoff in the range of about 300 to 3,000 under these conditions.

(2) The fraction obtained in Production Example 1 was analyzed by a reverse phase column. Production Example 1 for CAPCELL PAKC ₁₈
Was injected from 0.1% trifluoroacetic acid to 45%
Elution was performed with a linear concentration gradient (flow rate: 1.0 ml / min) to acetonitrile / 0.1% trifluoroacetic acid (column temperature 25 ° C). The elution fraction was detected by the absorbance at 215 nm. As a result, the existence of many peaks corresponding to peptides was found. Therefore, it is considered that the melanin production inhibitory activity of the active ingredient of the present invention exhibits high activity for the first time as an aggregate of a plurality of peptides.

Test Example 2 (Measurement of Melanin Production Inhibitory Activity) Using the respective fractions obtained in Production Examples 1 to 3 as samples, the melanin production inhibitory activity of these samples was measured using B16 mouse melanoma cells. B16 mouse melanoma cells in 96-well plate containing 10% FCS in DME
One well of 500 cells was seeded in the M medium and cultured at 37 ° C. for 4 hours to attach the cells. Thereafter, the medium was removed, and the cells were cultured at 37 ° C. for 4 to 5 days in a medium to which samples of various concentrations were added. In addition,
Cell culture is 5% in a CO ₂ incubator at 37 ° C.
It was carried out under the condition of CO ₂ -95% air. The number of viable cells is MT
T method (CHEMICON INTERNATIONAL
INC. Material Colorimetric (MTT)
Assay for Cell Survival an
(d Proliferation), the absorbance at 570 nm was measured with an auto reader at 630 nm as a background, and the value was used as a relative value representing the number of viable cells. The amount of melanin produced was determined by dissolving the cells in 0.2N NaOH and measuring the absorbance at 490 nm with an auto reader, and taking that value as the relative value representing the amount of melanin produced. The melanin production amount of the number of viable cells is shown by the following formula.

[0035]

[Equation 1]

2, 3, and 4 show the amount of melanin produced per living cell with respect to the concentration of each sample obtained in Production Examples 1 to 3. The fractions obtained in Production Examples 1 to 3 were concentrated to about 50, 50 and 25 times the culture supernatant, respectively. It can be seen that the addition of any of the fractions markedly decreases the amount of melanin produced as the sample concentration increases.

Test Example 3 (Suppression of melanin production in cells and its reversibility test) Two systems were tested, one containing α-MSH (final concentration 0.2 μM) in the medium and the other without. The fraction obtained in Production Example 1 (about 2% corresponding to the original culture supernatant)
5 times concentrated solutions) having the concentrations shown in Tables 2 and 3 below were prepared and provided as samples.

(1) Melanin production inhibition test B16 mouse melanoma cells were inoculated into each well of a 6-well plate in an amount of 1.6 × 10 ⁴ cells (about 1.8 × 10 ³ cells / cm ² ), and the medium (10% FCS) was inoculated. In DMEM medium containing 4)
After culturing for a period of time, the cells were attached to the wells. Then, the above-mentioned cultured cells are exchanged with a sample-free medium (sample-free medium) and a medium containing each concentration of the sample (sample-containing medium).
Cultivation was carried out for a day (cell density was about 80 to 90% saturation). Culturing was performed using 3 wells for each concentration of one sample. On the 5th day of culture, the cells were washed with PBS to give 0.125
% Trypsin solution was used to detach the cells, and the number of viable cells after trypan blue staining was measured for a portion of the cells, and a portion of the cultured cells in sample-free medium and sample-containing medium (system with the highest sample concentration) Reserved for cell passage for each well. The remaining cells were collected in a glass tube by centrifugation and stored frozen.

(2) Reversibility test of melanin production inhibitory effect The cells secured for cell passage in (1) were seeded in a medium at the above cell density in each well, and after 4 hours, each medium was replaced. That is, cultured cells (3) in the sample-free medium of (1)
Wells) were further cultured in the same medium, and cells (6 wells) cultured in the sample-containing medium in (1) were used in the sample-free medium (3).
Wells) and (1) in the same concentration as the sample-containing medium (3 wells), and after culturing for 5 days, the number of cells was measured according to the above method, and the remaining cells were collected in a glass tube, and if necessary, It was stored frozen.

(3) Evaluation of melanin production inhibitory activity of the sample The cryopreserved cells were thawed, the color tone of the cells was visually observed, and the whitening degree of the cells was evaluated according to the following 5 grades. + Indicates whiteness. 4+: White, 3+: White to gray, 2+: Gray, 1+ Gray to black,-: Black. After that, 0.2N the cells
It was dissolved in NaOH (use 0.5-1.0 ml depending on the number of cells) and the absorbance at 475 nm was measured. Separately, the total amount of melanin contained in the cells was obtained based on the calibration curve prepared with the melanin solution, and the amount of melanin per cell was determined by α-MSH.
Calculations were made for the system containing no and the system containing.
The results are shown in Table 2 (no α-MSH added) and Table 3 (α-MSH added).
Addition).

[0041]

[Table 2]

[0042]

[Table 3]

As is clear from the results shown in Tables 2 and 3, in both the system in which α-MSH was not added to the medium and the system in which α-MSH was added, the amount of melanin per cell was decreased at the concentration of addition of the sample that did not inhibit cell growth. And whitening of cells was observed. When cells that had been whitened by adding the sample were subcultured without adding the sample, an increase in the amount of melanin per cell and a blackening of the cells were observed. The above results show that the fraction also suppresses melanin production promoted by α-MSH, and further, this action does not destroy the melanin production apparatus of melanocytes, and the action is It is shown to be reversible.

Test Example 4 (Primary Skin Irritation Test) Using the fractions obtained in Production Example 1, a skin primary irritation test for Japanese white rabbits (male, 2 to 2.5 kg) was conducted according to the Draize method. Under the test conditions of (Conditions) Dose: 0.25 ml of the test substance (about 100 times concentrated liquid equivalent to the original culture supernatant, about 450 mg per 1 ml of solid content) was used on a 1.5 × 2.0 cm lint cloth. Exposure time: closed application, 24 hours. Fixation of animals during exposure: Fix the animals in the neck for 24 hours. Cleaning after application: Wipe gently with water. Number of animals used: 6 Rubbed and normal skin: 5 each (4 were normal on one side, one was rubbed on one side, and the other 2 were normal and scratched skin respectively) Evaluation: Change of skin immediately after removal of application, 24, 48 and 72 hours later Observe, record and evaluate. Judgment: Performed by the Draize method. That is, the erythema score and the edema score at 24 hours and 72 hours were added to calculate an average value, and the primary irritation index (PII, Primary) was calculated.
It was classified as the Irritation Index) according to the following criteria. (Classification Criteria) P. I. I <2: Almost no irritation. 2 ≦ P. I. I <6: Slightly irritating. 6 ≦ P. I. I ≦ 8: There is irritation. The results are shown in Table 4.

[0045]

[Table 4]

As is clear from the results shown in Table 4, the above fraction (Production Example 1) has extremely low skin irritation. When the fractions obtained in Production Examples 2 and 3 were tested in the same manner, they also showed extremely low skin irritation.

Test Example 5 (Measurement of Tyrosinase Inhibitory Activity) The fraction obtained in Production Example 1 (about 25-fold concentrated solution corresponding to the original culture supernatant) was used as a sample, and its tyrosinase inhibitory activity was measured as follows (2), ( It measured by the method of 3). (1) Preparation of Tyrosinase Crude Enzyme Solution from B16 Mouse Melanoma Cells About 4 × 10 ⁷ B16 mouse melanoma cells cultured and grown in a medium were scraped directly from a petri dish using a rubber policeman, and 10 ml of 10 mM Na-phosphate buffer was used. The mixture was triturated with a homogenizer in the liquid (pH 7.4). 1 x 10
^The product was centrifuged at ⁴ G for 20 minutes, and the supernatant was sufficiently dialyzed against the above buffer solution to give a crude tyrosinase enzyme solution. For the measurement, the enzyme solution was appropriately diluted with the above buffer solution.

(2) Measurement of melanin production inhibitory activity This activity was measured by the method of Hearing (V. J. He.
aring and T. M. Ekel, Bioche
m. J. 157 , 549 (1976)). Tyrosinase crude enzyme solution, 0.1 M Na-phosphate buffer (pH 7.4), 0.1 mg bovine albumin, 50 μM [U
- ¹⁴ C] tyrosine (sp.act.25mCi / mmol, 0.
(125 μCi / ml), 5 μM DOPA and the sample of Production Example 1 to make a total volume of 50 μl, and after mixing, the mixture was reacted at 37 ° C. for 60 minutes. After the reaction, the whole amount was adsorbed on a 2.4 cm Whatman 3MM filter paper, washed several times with 0.1N HCl-0.1% tyrosine solution, then washed with ethanol and acetone and dried, and the isotope incorporated in the acid-insoluble fraction. Quantities were measured using a liquid scintillation spectrometer.
The melanin production inhibitory activity of the sample can be determined as a difference from the uptake amount in the sample-free reaction system.

FIG. 5 shows the relationship between the effective concentration and the tyrosinase (melanin production) inhibitory activity in the test using the cultured cells of the above fractions. From this, it can be seen that the tyrosinase inhibitory activity due to the presence of the fraction is relatively small.

(3) Measurement of dopa oxydase inhibitory activity In a 1 ml cuvette, tyrosinase crude enzyme solution, 0.1M was added.
Na-phosphate buffer (pH 7.4), 0.5 mM DOPA and fractions of each concentration were added to make the total volume 1 ml and reacted at 37 ° C. The formation of melanin and its precursors was
The absorbance change at 5 nm was monitored over time. The inhibition of the activity by the above fractions can be determined as the difference from the absorbance in the sample-free reaction system.

FIG. 6 shows the relationship between the measuring time and the inhibitory activity. From this, it can be seen that inhibition of dopaoxydase activity by the above fractions is extremely small.

Test Example 6 (Hygroscopicity and Moisture Retention Test) The hygroscopicity and moisturizing effect of the fraction (freeze-dried product) obtained in Production Example 1 were evaluated. As a control product, potassium hyaluronate (Wako Pure Chemical Industries, Ltd., made from chicken cob) was used. (1) Hygroscopicity test Approximately 200 mg of each sample was weighed into a weighing bottle, left for 18 hours under reduced pressure (room temperature) in a dryer containing phosphorus pentoxide, and dried to be used for the test. These dried samples were allowed to stand in a thermo-hygrostat having relative humidity of 34% and 74% (temperature 20 ° C.), and the weight was measured over time. The weight increase rate (%) obtained by the following equation was used as an index of the moisture absorption capacity.

[0053]

[Equation 2]

(2) Moisture retention test A sample absorbed under high humidity (temperature 20 ° C., relative humidity 74%) is allowed to stand in a constant temperature and humidity tank at 20 ° C. and 40% relative humidity, and then in a desiccator (room temperature), The weight was measured over time. Moisture retention rate (%) calculated by the following formula as an index of moisture retention
Was used.

[0055]

[Equation 3]

7 and 8 show the time variation (hygroscopicity) of the moisture absorption weight increase rate of each sample under the conditions of relative humidity of 34% and 74% (temperature 20 ° C.). In addition, the moisture retention of each sample,
Using each sample that was made to absorb moisture under high humidity, it was represented by the time variation of the residual moisture rate under that low humidity (FIG. 9). The rate of increase in hygroscopic weight (hygroscopicity) of the component of Production Example 1 was slightly lower than that of hyaluronic acid, but exhibited almost the same behavior. The residual water content of the components of Production Example 1 is calculated to be about 10% of the weight of the sample despite being left in the desiccator. A substance that has both high hygroscopicity and moisturizing property may give "stickiness" to the skin at high humidity. Therefore, it is considered that an ideal moisturizing agent is not very hygroscopic and has excellent moisturizing property. . The desirable water content in the human stratum corneum is 10% or more. Therefore, the product of the present invention, which retains about 10% of water even under low humidity, has a desirable moisturizing effect of improving the water retention in the stratum corneum and further suppressing the release of water in a low humidity environment such as winter. Was found to have. This effect is the same in Production Examples 2 and 3, and is comparable to hyaluronic acid, which is currently considered to be an excellent moisturizing substance.

Example 1 (Preparation of Vanishing Cream) Using the fraction obtained in Production Example 1 (about 50 times concentrated solution corresponding to the original culture supernatant, about 225 mg per 1 ml of solid content), the vanishing composition is shown below. A cream (O / W type) was prepared by the following production method.

(Composition) (wt%) Solution A Stearic acid 10.0 Stearyl alcohol 4.0 Butyl stearate 8.0 Glycerin monostearate 2.0 Methylparaben 0.048 Dibutylhydroxytoluene 0.01 Solution B Propylene glycol 10 0.0 Glycerin 4.0 Potassium hydroxide 0.4 Purified water Remaining fraction (Production Example 1) 5.0 Methylparaben 0.152

(Production Method) 1) Solution A is heated and dissolved in a water bath at 70 ° C. 2) While heating and stirring in a water bath at 70 ° C., potassium hydroxide and methylparaben are first added in this order to purified water to sufficiently dissolve them, and propylene glycol and glycerin and finally a fraction are added thereto. 3) While stirring Solution B in a 70 ° C. water bath, Solution A heated and dissolved therein is gradually added. 4) After emulsifying solutions A and B, set the water temperature to 3 with stirring.
Slowly lower to 0 ° C. 5) Cool to obtain a stable creamy vanishing cream.

Example 2 (Preparation of Vanishing Cream) A vanishing cream was obtained in the same manner as in Example 1 except that the content of the fraction (Production Example 1) was 10% by weight.

[0061]

The melanin production inhibitor of the present invention has an excellent melanin production inhibitory action, an excellent moisturizing action, and almost no skin irritation. Moreover, since the above-mentioned melanin production inhibitory action is reversible without damaging living tissues, the agent of the present invention is useful as a skin cosmetic ingredient.

[Brief description of drawings]

FIG. 1 is a high-speed gel filtration method for each fraction obtained in Production Examples 1 to 3 (eluent: acetonitrile, marker: peptides).
It is a figure which shows the molecular weight cut off distribution by.

FIG. 2 is a drawing showing the relationship between the concentration of the fraction obtained in Production Example 1, the number of living cells, and the amount of melanin produced per living cell.

FIG. 3 is a drawing showing the relationship between the concentration of the fraction obtained in Production Example 2, the number of living cells, and the amount of melanin produced per living cell.

FIG. 4 is a drawing showing the relationship between the concentration of the fraction obtained in Production Example 3, the number of living cells, and the amount of melanin produced per living cell.

FIG. 5 is a drawing showing the relationship between the concentration of the fraction obtained in Production Example 1 and tyrosinase (melanin production) inhibitory activity.

FIG. 6 is a drawing showing changes over time in dopa oxidase activity depending on the concentration of the fraction obtained in Production Example 1.

FIG. 7 is a drawing showing the hygroscopicity of the fraction obtained in Production Example 1 at a relative humidity of 34% (temperature 20 ° C.).

FIG. 8 is a drawing showing the hygroscopicity of the fraction obtained in Production Example 1 at a relative humidity of 74% (temperature 20 ° C.).

FIG. 9: The fraction obtained in Production Example 1 was allowed to stand at 74% relative humidity (temperature 20 ° C.) for 24 hours, then at 40% relative humidity (temperature 20 ° C.) for 24 hours, and then in a desiccator (room temperature). 7) is a drawing showing the moisturizing property when left to stand.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical display location A61K 35/74 ADS G 7431-4C AED G 7431-4C C12N 9/99 9152-4B (72) Invention Yoshiyoshi Yoshiyama, 540 Narita, Odawara City, Kanagawa Prefecture, Meiji Dairy Health Science Research Institute

Claims (2)

[Claims] 1. A melanin production inhibitor comprising a fraction derived from a culture supernatant of Bifidobacterium and having an estimated cutoff molecular weight of about 300 to 3,000 by a high speed gel filtration method or a fraction thereof as an active ingredient. 2. A skin cosmetic containing the fraction according to claim 1.