JPH05194258A - Inhibitor of melanin pigment synthesis - Google Patents

Abstract

PURPOSE:To obtain an inhibitor of melanin pigment synthesis containing a fermented product of a lactic bacterium as an active ingredient, excellent in tyrosinase inhibiting activity and activity capable of inhibiting melanin pigment synthesis of cell by ultraviolet ray absorbing action, etc. CONSTITUTION:The objective inhibitor contains a product of lactic bacterium fermentation obtained by extracting or extracting and purifying ingredients obtained by culturing a lactic bacterium in an amount of >=0.2mg/ml as an active ingredient. The product of the lactic bacterium fermentation is a metal- containing tetracyclopeptide expressed by the formula (M is Zn, Cu, Mn, Mg, Ca or Fe) and can be used as a form of lotion, emulsion, cream, pack, etc. The lactic bacterium includes Lactobacillus helveti-cus JCM-1120.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a melanin pigment synthesis inhibitor.

[0002]

2. Description of the Related Art Generally, it is said that the occurrence of spots, freckles, etc. is caused by abnormal deposition of melanin pigment caused by ultraviolet rays and abnormal hormones that control the synthesis of melanin.

As an active ingredient of cosmetics for preventing spots, freckles and the like, ascorbic acid or cysteine has been proposed as a component for protecting the skin by absorbing ultraviolet rays, and stains and freckles in the body have been proposed. Hydroquinone preparations, arbutin, kojic acid, and the like are known as components that suppress the production itself. More recently, thiolurocanic acid and the like have been proposed as a component having both a melanin production suppressing action and an ultraviolet absorbing action.

On the other hand, it is desired that the component exhibiting the skin melanin pigment synthesis inhibitory effect is a substance which has sufficient safety and storage stability in addition to its effect. However, under the present circumstances, conventionally known melanin pigment synthesis inhibitors do not satisfy all of these effects at the same time. Specifically, for example, ascorbic acid as an active ingredient is easily discolored and odor easily due to containing water, and there is a problem in stability. Thiol compounds such as cysteine have a melanin pigment synthesis inhibitory action itself. Is not sufficient and is easily oxidized. Further, although the hydroquinone preparation is excellent in the melanin pigment synthesis inhibitory action, it has a problem in skin safety and the like.

Further, it has been known that metabolic components of lactic acid bacteria can be used as a cosmetic material, for example, Streptococcus zooepicus.
hyaluronic acid by dem-icus) is mass-produced. However, the lactic acid bacterium metabolic component merely utilizes a moisturizing action, and is not known at all to have a melanin pigment synthesis inhibitory action, and further known about a specific tetracyclopeptide of the present invention. The reality is that they do not.

[0006]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide melanin having excellent tyrosinase inhibitory activity, excellent melanin pigment synthesis inhibitory activity for cells such as ultraviolet absorption, and excellent safety and storage stability. It is to provide a pigment synthesis inhibitor.

[0007]

According to the present invention, there is provided a melanin pigment synthesis inhibitor containing a lactic acid bacterium fermentation product as an active ingredient.

The present invention will be described in more detail below.

In the melanin pigment synthesis inhibitor of the present invention, the lactic acid bacterium fermentation product used as an active ingredient may be one obtained by culturing a lactic acid bacterium by extraction or extraction / purification, and particularly ethyl acetate or the like. It is preferable to extract with an organic solvent and to use the concentrated residue of the extraction category as an active ingredient, and the property of the concentrated residue is a light yellowish brown, acidic viscous paste at room temperature, and a waxy solid at 5 ° C. Show. Further, the concentrated residue has a wide range of ultraviolet absorption ability, and is a component in which a decrease in melanin pigment synthesis inhibitory activity is not observed even after long-term storage. Further more preferably, the following structural formula 2 obtained by purifying the extract is
(In the formula, M represents Zn, Cu, Mn, Mg, Ca or Fe) and the like.

[0010]

[Chemical 2]

The lactic acid bacterium is not particularly limited as long as it can grow in a general medium, but is preferably Lactobacillus helveticus JCM-1120 (Lactobacil).
lushelveti-cus JCM-1120) and other Lactobacillus helveticus, Lactobacillus fermentum JCM-1173 (La
Lactobacillus such as ctobacillus fermentum JCM-1173)
Fermentum, Lactobacillus confusus JCM-1
093 (Lactobacillus confusus JCM-1093) and other Lactobacillus confusus, Lactobacillus reuteri JCM-11
12 (Lactobacillus reuteri JCM-1112) and other Lactobacillus reuteri, Lactobacillus buchneri JCM-1115 (Lacto
Lactobacillus buchneri such as bacillus buchneri JCM-1115) can be preferably mentioned, and Lactobacillus helveticus JCM-1120 and Lactobacillus fermentum JCM-1173 are most preferable.

In the present invention, in order to prepare the lactic acid bacterium fermentation product, the lactic acid bacterium is inoculated into the medium and cultivated to obtain a lactic acid bacterium culture solution, which is obtained by a method of extracting the active ingredient or a method of extracting and purifying the active ingredient. it can. The medium, for example, as carbon source, assimilable carbon source, specifically, glucose, sucrose, molasses, and the like, and as the nitrogen source, yeast extract, peptone, meat extract, casein hydrolyzate, etc. Can be mentioned. As other components, inorganic salts that promote the growth of lactic acid bacteria, vitamins, amino acids, known defoaming agents, surfactants and the like can also be added, and specific media include, for example, Brigg's medium and skim milk-tryptone. A medium or the like can be preferably used. Various culture conditions can be selected according to the properties of the lactic acid bacterium to be used, and specifically, the culture temperature can be 25 to 40 ° C. and the culture time can be 12 to 48 hours under aerobic or anaerobic conditions. At this time, the amount of the lactic acid bacterium starter inoculated to the medium is preferably 1 to 5% by weight.
Then, the cultivated lactic acid bacterium culture fluid is separated by centrifugation or filtration to separate the culture supernatant fluid, and the obtained culture supernatant fluid is subjected to extraction treatment with a non-water-soluble organic solvent such as ethyl acetate or 1-butanol. The active ingredient can be obtained by a method of re-dissolving in water, a buffer such as 0.2 M sodium phosphate buffer, or the like.

In the lactic acid bacterium fermentation product used in the present invention, a redissolved solution obtained by extraction from the lactic acid bacterium culture solution can be used as an active ingredient, but it is further purified and, for example, tetracyclo represented by the structural formula 2 above. A peptide or the like can also be used as an active ingredient. In order to prepare the tetracyclopeptide, for example, a solution obtained by the same method as the re-dissolved solution that can be used as an active ingredient is treated with an ion exchange resin such as a trade name "Amberlite XAD" (manufactured by Organo), A combination of known purification methods such as reverse phase resin, column chromatography using silica gel, gel filtration,
Alternatively, it can be obtained by repeating further purification.

In the present invention, the minimum effective concentration of the lactic acid bacterium fermentation product as an active ingredient is preferably 0.2 mg / ml or more, and particularly preferably 0.8 mg / ml or more.

In the melanin pigment synthesis inhibitor of the present invention, in addition to the lactic acid bacterium fermentation product which is the above-mentioned active ingredient, known vitamins such as vitamin A, vitamin B ₆ and vitamin H, ascorbic acid and cystine are known. Antioxidants, white petrolatum, paraffin, squalane, and other cosmetic raw materials may also be included.

The melanin pigment synthesis inhibitor of the present invention can be used in the form of lotions, emulsions, creams, packs and the like.

[0017]

EFFECT OF THE INVENTION The melanin pigment synthesis inhibitor of the present invention has excellent tyrosinase inhibitory activity, excellent melanin pigment synthesis inhibitory activity of cells such as UV absorption, and also has excellent safety and storage stability. As cosmetic materials,
It is useful for lotions, emulsions, creams, packs and the like.

[0018]

EXAMPLES The present invention will be specifically described below based on examples, but the present invention is not limited thereto.

[0019]

Example 1 (1) Preparation of Medium A Briggs agar medium having the composition shown in Table 1 below was prepared, adjusted to pH 6.8 with a 1N sodium hydroxide solution, and further sterilized under high pressure to obtain a medium. ..

[0020]

[Table 1]

(2) Culture of lactic acid bacteria Preserved strains of lactic acid bacteria (suspension of lactic acid bacteria containing 30% by weight of glycerol (bacteria number about 10 ⁸ / ml), stored at −80 ° C.) 2 strains (Lactobacillus helveticus JCM-1120, lacto Bacillus fermentum JCM-1173) was inoculated into the Briggs agar medium prepared in (1) above with 1 platinum loop, and the mixture was incubated at 37 ° C. under anaerobic conditions.
It was cultured for 24 hours. After culturing, a colony was scraped from each agar medium, inoculated into an L-shaped tube of 10 ml of Briggs medium, and cultured by shaking for 18 hours. Scaled up in sequence and finally 3 with a 60 liter jar fermenter
After stirring and culturing at 7 ° C. for 48 hours, 40 kg of a lactic acid bacterium culture solution was obtained.

(3) Preparation of Lactic Acid Bacterial Fermentation Product 40 kg of each culture solution prepared in (2) was centrifuged, and 400 g of 6N hydrochloric acid was added to the supernatant after sterilization to give a pH of 3.
It was adjusted to 0 and the extraction was repeated twice with 20 kg of ethyl acetate. The extraction operation was carried out by collecting the upper layer (ethyl acetate layer) which was separated by standing after 30 minutes of stirring. The ethyl acetate layer was concentrated under vacuum (40 ° C. or less, vacuum degree 76 mmHg) to distill off the ethyl acetate, and the fermentation product of lactic acid bacteria as an active ingredient was obtained as the rest of 20 g.

Next, the following two tests were carried out on the obtained two types of fermentation products of lactic acid bacteria in order to measure the melanin pigment synthesis inhibitory action and safety.

Tyrosinase activity inhibition test (absorbance
Method) 0.1 ml of mushroom-derived tyrosinase (300 unit / ml, Sigma) and 0.1 mM 3,4 as a substrate
-Dihydroxyphenylalanine (L-DOPA) 1
ml and the fermentation product of lactic acid bacteria with 0.2M phosphate buffer for 10
0.1m of sample solution diluted to a concentration of mg / ml
1 was mixed and the reaction was started at 25 ° C. At this time, the production amount of dopachrome was measured for 5 minutes by increasing the absorbance at 475 nm. 0.2M as a control
The same procedure was performed using sodium phosphate buffer. The results are shown in Figure 1. From the results in Figure 1, Lactobacillus helveticus JCM-1120, Lactobacillus fermentum JCM-1
A strong inhibition of tyrosinase activity was observed in 173 fermentation products.

In a similar manner, the relationship between the concentration of the fermentation product of Lactobacillus helveticus JCM-1120 and the effect was examined. The results are shown in Table 2.

[0026]

[Table 2]

From the results of Table 2, the minimum effective concentration of the fermentation product of Lactobacillus helveticus JCM-1120 is 0.2.
It was mg / ml. The inhibitory activity of 10% or more is
Obtained above 0.8 mg / ml. (The concentration showing 2 or more activities of the control was defined as the minimum effective concentration.) Melanin synthetic oxygen activity inhibitory test ( ¹⁴ C-tyrosine
Method) Mouse melanoma cell line clone M-3 (ATCC N
O. CCL-53.1) cell extract was used as enzyme solution (melanin synthase group), and the substrate was 500 mCi / mmol.
¹⁴ C-tyrosine (manufactured by NEN Research Products) was used.

The composition of the enzyme reaction solution was as follows: 20 μl of 500 mM phosphate buffer (pH 7.4), 20 μl of 5 mg / ml bovine serum albumin, 50 μM 10 μl of 3,4-dihydroxyphenylalanine (L-DOPA), substrate ¹⁴ C-tyrosine. 10 μl and 10 μl of enzyme solution were used.

10 μl each of the sample solutions having the respective concentrations shown in Table 3 were added to the enzyme reaction solution and reacted at 37 ° C. for 60 minutes. After the reaction, filter paper (brand name "Whatman 3MM filter",
The reaction solution was dipped in Whatman Inc.), air-dried and then washed by the following method. After washing once with a 0.1N hydrochloric acid solution containing 0.1% tyrosine, further washing twice with a 0.1N hydrochloric acid solution,
Next, it was washed 3 times with ethanol and once with acetone, and the ¹⁴ C-labeled acid-insoluble fraction (melanin fraction) remained on the filter paper.
Liquid scintillation counter (model LSC-90
0, manufactured by Aloka Co., Ltd.).

Lactobacillus helveticus JCM-1120
Table 3 shows the results when the fermentation product of No. 1 was used as a sample. As a result, it was found that the melanin pigment synthesis inhibitory activity depends on the concentration of the fermentation product.

[0031]

[Table 3]

Inhibitory action on melanin pigment synthetic ability in cells 75 cm ² of mouse melanoma cell line clone M-3 as melanin-synthesizing cells was used in F-10 medium containing 2.5% fetal bovine serum and 15% horse serum. The cells were cultured in the cell culture flask for 48 hours. Then, the cells were detached with 0.25% trypsin, and the cells were washed with 3 × 10 ⁷ cells /
ml was resuspended in serum-free F-10 medium. To each of the obtained cell suspensions, a sample solution (a fermentation product of Lactobacillus helveticus JCM-1120) of each concentration shown in Table 4 was added so as to be 1/10 of the volume, and the solution was added at 37 ° C for 6 hours.
The reaction was allowed for 0 minutes. After the reaction, phosphate buffered saline (P
The cells were collected by washing 3 times with (BS) and 0.5% trade name "Triton X-100" (manufactured by Wako Pure Chemical Industries, Ltd.).
10 mM sodium phosphate buffer (pH 6.8) containing
The cells were treated with and homogenized. Then 5000
After centrifugation at 5 rpm for 5 minutes, the supernatant was used to measure the ability to suppress melanin synthesis according to the ¹⁴ C-tyrosine method. The results are shown in Table 4.

[0033]

[Table 4]

Cytotoxicity test Each of mouse melanoma cell line clone M-3 and rabbit corneal cell line SIRC was added to F-12 medium containing 10% fetal bovine serum and incubated at 37 ° C. in a 25 cm ² cell culture flask. Cultured (1 x 10 ⁶ CELLS
/ Ml). After 24 hours, the fermentation product of Lactobacillus helveticus JCM-1120 or arbutin was added so that the final concentrations were 0 to 8 mg / ml and 0 to 10 mM, respectively, and the cells were further cultured for 24 hours. These steps were performed while changing the medium every day. On the 4th day after cell inoculation, the survival rate of the cells was measured by the dye exclusion ability of trypan blue. The results are shown in Figure 2. As a result, the fermentation product of lactic acid bacteria was 8 mg / ml.
No cytotoxicity was observed to a degree. On the other hand, arbutin, which is an active ingredient of conventionally known cosmetic materials, decreased to a survival rate of about 20% at 1 mM.

Storage stability of residual action of tyrosinase activity inhibition
Sex test The fermentation product of Lactobacillus helveticus JCM-1120 was adjusted to 10 mg / ml and stored in a solution state at 37 ° C. for 4 weeks in the dark. Sampling was performed every week, and the residual activity of tyrosinase inhibition was measured by the ¹⁴ C-tyrosine method.
As a result, it was found that the tyrosinase inhibitory activity was not decreased even after 4 weeks and the storage stability was excellent.

[0036]

Example 2 100 ml of the same medium as in Example 1 was added to 50
Put it in a Erlenmeyer flask with a 0 ml volume silicon stopper, 121
After sterilization at 20 ° C. for 20 minutes, 1 platinum loop of Lactobacillus helveticus JCM-1120 (bacteria number: about 10 ⁸ / ml) grown on an agar medium was inoculated and shake-cultured at 37 ° C. for 2 days under anaerobic conditions. Sequential scale-up was carried out, and finally 10 liters of the culture solution of lactic acid bacteria was collected, and then the culture supernatant liquid free of the bacterial cells was separated by centrifugation and extracted with the same amount of ethyl acetate. The extraction treatment was carried out by recovering the upper layer (ethyl acetate layer) which was left standing and separated after 30 minutes of stirring. Next, the ethyl acetate layer was evaporated to dryness under reduced pressure to obtain 4 g of a distillation residue, 300 ml of distilled water was added to the distillation residue, and 1000 ml of an ion exchange resin (trade name "Amberlite XAD-7", manufactured by Organo). After being adsorbed on, the elution treatment was carried out with 2000 ml of ethyl alcohol. Next, ethanol was distilled off with a rotary evaporator to obtain an oily fraction, which was then suspended in a solution of chloroform: methanol = 8: 2, and a silica gel column (trade name "Wakogel C-200", manufactured by Wako Pure Chemical Industries, Ltd., Fractionation was performed at 2.5 × 40 cm). After collecting the active fraction and distilling off the solvent, it was dissolved in distilled water,
Reversed phase resin column (trade name "Cosmo Seal 75C ₁₈ -OP
N ", manufactured by Nacalai Tesque, Inc.). Next, elution was performed by sequentially increasing the methanol concentration, the active fraction was collected, and the solvent was distilled off. 4 ml of residue 90
It was dissolved in% methanol and purified by the following high performance liquid chromatography. The high performance liquid chromatography process is performed using a gel filtration column (trade name “Asahi Pack GS-32
0 ", manufactured by Asahi Kasei Co., Ltd.), the methanol was distilled off by vacuum distillation, and the residue was redissolved in 1 ml of distilled water. The reverse phase column (trade name" Capsule Pack SG-120 ", Ltd.) Shiseido Co., Ltd.), and the active fraction was collected with an acetonitrile gradient and dried under reduced pressure to obtain a pure cyclopeptide represented by the following structural formula 3. The physicochemical properties of the obtained tetracyclopeptide were as follows.

[0037]

[Chemical 3]

Physicochemical properties Appearance: White powder FAB mass spectrum: m / z 457 (M + H) +, 5
21 (M + Zn) + Molecular formula: C ₂₄ H ₃₂ N ₄ O ₅ Molecular weight: 456 UV absorption spectrum: A peak exists at 274 nm when measured with an aqueous solution. ¹ H-NMR spectrum: The spectrum result measured at 400 MHz in heavy water is shown in FIG. ¹³ C-NMR spectrum: The spectrum result measured at 100 MHz in heavy water is shown in FIG. Distinction between acidic, medieval, and basic: weakly acidic substances Constituent amino acids: tyrosine, valine, proline (2 molecules)
Next, the following tests were carried out on the obtained tetracyclopeptide, which is a fermentation product of lactic acid bacteria, in order to measure the melanin pigment synthesis inhibitory action.

Tyrosinase activity inhibitory action test (absorbance
Method) 0.1 ml of mushroom-derived tyrosinase (300 unit / ml, Sigma) and 0.1 mM 3,4 as a substrate
-Dihydroxyphenylalanine (L-DOPA) 1
ml and a lactic acid bacterium fermentation product were dissolved in a 0.2 M phosphate buffer solution to a concentration shown in FIG.
The reaction was started at ° C. At this time, the production amount of dopachrome was measured for 5 minutes by increasing the absorbance at 475 nm. As a control, the same procedure was performed using 0.2 M sodium phosphate buffer. Results are shown in FIG. Figure 5
From these results, strong inhibition of tyrosinase activity was observed in the tetracyclopeptide.

Test for inhibiting melanin synthesis oxygen activity ( ¹⁴ C
-Tyrosine method) Mouse melanoma cell line clone M-3 (ATCC NO.CCL-5
The cell extract of 3.1) was used as an enzyme solution (melanin synthase group), and 500 mCi / mmol ¹⁴ C-tyrosine (manufactured by NEN Research Products) was used as a substrate.

The composition of the enzyme reaction solution was as follows: 20 μl of 500 mM phosphate buffer (pH 7.4), 20 μl of 5 mg / ml bovine serum albumin, 50 μM 10 μl of 3,4-dihydroxyphenylalanine (L-DOPA), substrate ¹⁴ C-tyrosine. 10 μl and 10 μl of enzyme solution were prepared.

10 μl of each concentration of the sample solution shown in FIG. 6 was added to the enzyme reaction solution and reacted at 37 ° C. for 60 minutes. After the reaction, filter paper (brand name "Whatman 3MM filter",
The reaction solution was dipped in Whatman Inc.), air-dried and then washed by the following method. After washing once with a 0.1N hydrochloric acid solution containing 0.1% tyrosine, further washing twice with a 0.1N hydrochloric acid solution,
Next, it was washed 3 times with ethanol and once with acetone, and the ¹⁴ C-labeled acid-insoluble fraction (melanin fraction) remained on the filter paper.
Liquid scintillation counter (model LSC-90
0, manufactured by Aloka Co., Ltd.). The sample solutions used were distilled water as a control, arbutin and kojic acid sample solutions as existing melanin pigment synthesis inhibitors, and the tetracyclopeptide. The result is shown in FIG. 6. From FIG. 6, the sample solution using the tetracyclopeptide was
It showed remarkable melanin pigment synthesis inhibitory activity in a concentration-dependent manner. When converted from the concentration showing 50% inhibitory activity of each sample solution, the sample solution containing the tetracyclopeptide exhibited melanin pigment synthesis inhibitory activity about 5 times that of kojic acid and about 100 times that of arbutin.

Inhibition of melanin pigment synthesizing ability in cells The melanin synthesis-inhibiting ability was measured in the same manner as in Example 1 except that the sample solutions (the above-mentioned tetracyclopeptides) having the respective concentrations shown in Table 5 were added. The results are shown in Table 5.

[0044]

[Table 5]

[Brief description of drawings]

FIG. 1 is a chart showing the increase in absorbance over time of a control and two types of fermentation products of lactic acid bacteria in the tyrosinase activity inhibitory activity test of Example 1.

FIG. 2 is a chart showing the survival rate of cells at various concentrations of a lactic acid bacterium fermentation product and arbutin in the cytotoxicity test of Example 1.

FIG. 3 shows the tetracyclopeptide prepared in Example 2.
It is a chart which shows a ¹ H-NMR spectrum.

FIG. 4 shows the tetracyclopeptide prepared in Example 2.
It is a chart which shows a ¹³ C-NMR spectrum.

FIG. 5 is a chart showing the increase in absorbance over time of the control and tetracyclopeptide in the tyrosinase activity inhibitory action test of Example 2.

FIG. 6 is a chart showing the amount of melanin synthesis at each concentration of control, tetracyclopeptide, arbutin and kojic acid in the melanin synthase activity inhibitory action test of Example 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location // C12N 9/99 C12P 21/04 8214-4B (C12P 21/04 C12R 1: 225)

Claims (2)

[Claims] 1. A melanin pigment synthesis inhibitor containing a lactic acid bacterium fermentation product as an active ingredient. 2. The lactic acid bacterium fermentation product has the following structural formula 1.
The melanin pigment synthesis inhibitor according to claim 1, which is a metal-containing tetracyclopeptide represented by the formula (M represents Zn, Cu, Mn, Mg, Ca, or Fe). [Chemical 1]