JP6454481B2 - Melanin production inhibiting composition - Google Patents

Description

  The present invention is a collagen peptide obtained by decomposing a suppon-derived collagen with an enzyme, and has a melanin production inhibitory effect.

  The melanin inhibitor which suppresses the production | generation of melanin is utilized as cosmetics which have what is called a whitening effect. Patent Document 1 shows that a dipeptide represented by L-cysteinyl-L-tryptophan is a lipid peroxide degradation promoter and has a tyrosinase activity inhibitory effect. As is well known, melanin begins to form where tyrosinase acts on tyrosine. Therefore, by inhibiting the activity of tyrosinase, the production of melanin in skin cells can be suppressed, and it is considered that a whitening effect appears.

  Patent Document 2 discloses a collagen peptide having an average molecular weight of 200 to 1500 obtained by subjecting a collagen component-containing raw material to subcritical water treatment and having tyrosinase inhibitory activity.

Japanese Patent Laid-Open No. 06-345797 JP 2006-342107 A

  Cosmetics exhibiting a whitening effect have attracted particular attention in recent years. However, for whitening cosmetics, problems such as vitiligo have been pointed out. Whitening is considered to be possible by suppressing the synthesis of melanin in skin cells. And it is thought that the whitening effect is improved by suppressing the activity of tyrosinase, which is a key enzyme in the synthesis process of complex melanin.

  Screening of chemically synthesized products that suppress tyrosinase activity is still in progress. However, such a chemically synthesized product has a possibility of finding a highly effective substance, but is greatly concerned about side effects.

  Patent Documents 1 and 2 are both peptides (including polypeptides) derived from natural products. Therefore, although there are few concerns about side effects, we must consider the possibility of causing allergies.

The inventor of the present invention has found that an enzyme degradation product of collagen derived from suppon, which has been ingested for many years as a food having a high nourishing tonic effect in the first place and is also used as a supplement in recent years, has an inhibitory effect on melanin production. It came to complete. That is, the present onset Ming, humans, there is provided a melanogenesis inhibiting composition using a very small turtle derived collagen can cause allergic reactions.

More specifically, the composition for inhibiting melanin production according to the present invention is prepared by mixing collagen extracted from a suppon's foot or shell margin and papain or trypsin as a degrading enzyme, and incubating the degrading enzyme after incubation. And purified dialysis purified collagen peptide.

Since the composition for inhibiting melanin production according to the present invention is a degradation product of a suppon-derived collagen already used as a supplement, it has a very low possibility of causing an allergic reaction. In addition, many degrading enzymes cleave a specific position of an amino acid, and the same collagen peptide can always be obtained if the starting collagen is derived from the same substance.

In addition, since the melanin production-suppressing composition according to the present invention has a low possibility of causing an allergic reaction, it is not necessary to perform a separation step of separating only components that cause allergy after degrading collagen.

  Moreover, the low molecular component after decomposing | disassembling can be used as a component which has a moisturizing effect. That is, effective use of the suppon-derived collagen is possible.

It is a figure which shows the result of the light absorbency which shows the melanin inhibitory effect of the collagen peptide by trypsin. It is a figure which shows the result of the light absorbency which shows the melanin inhibitory effect of the collagen peptide by a papain. It is a photograph which shows the electrophoresis image of SDS-PAGE of a collagen peptide. It is a graph which shows the result of capillary electrophoresis of the collagen peptide by trypsin and papain. It is a graph which shows the result of CE of the collagen peptide by the trypsin after dialysis.

The melanin production inhibitory composition according to the present invention will be described below. In addition, the following description is the illustration about one embodiment and one Example of this invention, Comprising: This invention is not limited to the following description. The following embodiments can be modified without departing from the spirit of the present invention.

  The present invention is obtained by decomposing a suppon-derived collagen with degrading enzymes. The turtle (Pelodiscus sinensis) is a turtle classified as a genus Kyotosusupon in the reptile class. Suppons that can be used can be suitably used, such as Kyokuto Suppon, Florida Suppon, Toge Suppon, Mars Suppon, Lease Suppon, Orb Brief Suppon, Peacock Suppon, and the like.

  The site | part which can be utilized in a suppon is not specifically limited. However, the foot and the edge of the shell (the impeller) can be preferably used.

  The method for extracting collagen is not particularly limited. The basic enzyme extraction method, acid extraction method, and hot water extraction method can be suitably used, and a method in which these are modified may be used.

  As the degrading enzyme, a hydrolase can be preferably used. If it is a hydrolase, there will be no restriction | limiting in particular. The inventor has confirmed that hydrolyzing enzymes such as trypsin and papain can be suitably used.

The composition for inhibiting melanin production according to the present invention is obtained by mixing and incubating suppon-derived collagen and a degrading enzyme, and then inactivating the decomposing enzyme. Incubation may be said to be a step of degrading the suppon-derived collagen with a degrading enzyme. Moreover, since the degradation enzyme may cleave a non-specific site after the substrate is reduced as it is, after degrading for a certain period of time, it is deactivated to stop the degradation. The deactivation can be deactivated by performing a heat treatment at 100 ° C. for about 10 minutes. The melanin production inhibiting composition according to the present invention may contain a deactivated degrading enzyme.

  Moreover, you may remove the digest fragment | piece and salt which were decomposed | disassembled non-specifically and were made too low molecular weight. This can be performed by a process of dialysis of the obtained collagen peptide against a solvent such as water or filtration with a filter. The collagen peptide from which unnecessary substances are removed after deactivating the degrading enzyme is referred to as “purified collagen peptide”.

  In particular, when collagen is decomposed, a low molecular compound (amines) having an amino group and a low molecular compound (organic acids) having an acidic group such as a carboxyl group are generated. Since this has a relatively strong odor and an unpleasant odor, it is not preferable as a whitening agent (cosmetics). Dialysis against collagen peptides significantly removes amines and organic acids. That is, the obtained collagen peptide can obtain a collagen peptide that does not have an irritating odor such as an amine odor, that is, an unpleasant odor.

Example 1
<Extraction of suppon-derived collagen>
10 g of the emperor portion of Kyokuto suppon cultivated at Shizuoka Prefectural Yaizu Suisan High School was immersed in 100 mL of 0.1N hydrochloric acid (HCl) solution and stirred for 24 hours. Thereafter, centrifugation was performed at 7000 rpm for 20 minutes to collect the precipitate.

  100 mL of 0.1N sodium hydroxide (NaOH) was added to the resulting precipitate and stirred for 24 hours. Thereafter, the mixture was centrifuged at 7000 rpm for 20 minutes to collect the precipitate. This treatment was performed three times.

  To about 10 g of the obtained precipitate, 100 mL of a 0.03 M citric acid solution was added and incubated at 37 ° C. for 24 hours. Then, it centrifuged at 7000 rpm for 20 minutes, collect | recovered supernatant solutions, and obtained the suppon origin collagen solution.

<Decomposition of suppon-derived collagen>
The suppon-derived collagen obtained above was degraded using a degrading enzyme in the following procedure.

100 mg of suppon-derived collagen was suspended in an ammonium hydrogen carbonate (NH ₄ HCO ₃ ) solution (final concentration 100 mM) in a 100 mL Meyer flask. Here, 100 mg of suppon-derived collagen means that 100 mg of collagen dissolved in a 0.03M citric acid solution at about 2 mg / mL was used.

  A bovine-derived trypsin (manufactured by SIGMA-ALDRICH) solution (10 mg / mL × 100 μL = 1 mg) was added and incubated at 37 ° C. with shaking. Five days after the start of incubation, the enzyme activity was deactivated by heating at 100 ° C. for 10 minutes. A collagen peptide is contained in the solution in which the enzyme activity is deactivated. This solution may be referred to as a collagen peptide solution.

  It was confirmed by SDS-PAGE and CE whether or not it could be degraded by a degrading enzyme. In SDS-PAGE, the measurement was also performed on collagen peptides at 24 hours, 48 hours, and 72 hours of incubation. Further, the collagen peptide solution 5 days after the incubation was dialyzed against distilled water using a dialysis membrane of MWCO 100 to 500 to remove low-molecular digest fragments and salts. This is a purified collagen peptide solution containing a purified collagen peptide.

<SDS-PAGE>
SDS-PAGE (Sodium Dodecyl Sulfate-Polyacylamide Gel Electrophoresis) was performed by the following procedure.

  A gel for electrophoresis was prepared according to a conventional method. Specifically, it was as follows. Separation gels and concentration gels were prepared according to the composition in Table 1 using water, acrylamide, Tris, APS (ammonium peroxodisulfate), TEMED (tetramethylethylenediamine).

  The concentration of the sample to be electrophoresed (collagen peptide) was adjusted with PBS so that the amount was adjusted to about 5 to 10 μg. The sample was mixed with Laemmili Sample Buffer (containing Tris, Glycerol, SDS, Pyronin Y, β-mercaptoethanol) at a ratio of 1: 1 and denatured by heating at 95 ° C. for 10 minutes.

  Samples and molecular weight markers were applied to each lane. A constant current was applied at 20 mA per gel. When the dye (Pyronin Y) in the sample buffer came to the lower side of the gel, the electrophoresis was terminated. When the electrophoresis was completed, it was stained with CBB (Coomassie Brilliant Blue).

<Capillary electrophoresis>
The collagen peptide was subjected to capillary electrophoresis (CE). Fused silica was used for the capillary. As the electrophoresis solution, a solution obtained by adding 20 mM SDS to 50 mM borate buffer (pH 10.5) was used. The drop method was used for sample introduction. The applied voltage was 20 kV, and detection was performed with UV absorption of 200 to 300 nm. CE was also performed on purified collagen peptides dialyzed against trypsin collagen peptides.

The temperature at which electrophoresis was performed was 25 ° C. The denaturing buffer was pH 6.8 with 1M Tris at 1.25 mL, glycerol at 2.0 mL and SDS at 0.4 mL until water totaled to 10 mL. The sample was mixed with sodium cinnamate (C ₆ H ₅ CH = CHCOONa) immediately before the electrophoresis.

<Confirmation of melanin production inhibitory effect>
The collagen peptide solution (1 mg / ml) was diluted 10-fold with phosphate buffered saline (hereinafter referred to as “PBS”) and then sterilized by filtration through a 0.2 μm filter.

B16F1 cells (mouse malignant melanoma) were dispensed at 2 × 10 ⁵ cells / 60 mm dish. 100 μL or 50 μL of a 0.1 mg / mL collagen peptide solution was added to 5 mL of a medium in which 10% FBS (Fetal bovine serum) was added to RMI1640 medium manufactured by Wako.

  After culturing B16F1 cells for 24 hours, a sterilized collagen peptide is administered. As a control, PBS used for dilution was added to B16F1 cells. In addition, collagen equivalent to the maximum dose of peptide (collagen not degraded by enzyme) was also prepared as a control.

After administration of the collagen peptide, further cultivation was performed 48 hours later. Thereafter, the cells adhering to the culture dish were detached with trypsin, and 1 × 10 ⁶ cells were collected.

  The collected cells were washed with 5 mL of PBS and suspended in 500 μl of 0.85 M potassium hydroxide (KOH), 10% dimethyl sulfoxide (DMSO) solution. Next, it incubated at 80 degreeC for 30 minutes, and dissolved melanin in a cell. A solution in which melanin is dissolved is called a cellular melanin solution.

  200 μl of the cell melanin solution was transferred to a 96-well plate, and the absorbance at 405 nm was measured.

(Example 2)
Except for the following points, the same procedure was repeated except that papain (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of trypsin in Example 1, and the absorbance at 405 nm of the cell melanin solution was measured.
(1) Only 100 μL of collagen peptide was added to the medium of B16F1 cells (mouse malignant melanoma).
(2) CE was performed only for the collagen peptide, not for the purified collagen peptide.

<Result>
The absorbance results are shown in FIG. 1 (trypsin) and FIG. 2 (papain). 1 and 2, the vertical axis represents the ratio (%) when the absorbance of a control (referred to as control) when PBS is added is defined as 100. FIG. The horizontal axis represents the type of each sample.

  FIG. 1 shows the results when the degrading enzyme is trypsin. Referring to FIG. 1, it can be said that melanin production was suppressed by administering the collagen peptide as compared to control (PBS). Specifically, when the collagen peptide was added at 1 μg / mL, about 20% of the melanin production was suppressed at 2 μg / mL. In addition, the absorbance of the collagen not degraded by the enzyme was also reduced by about 20% with respect to the control.

  A color sample is shown on the graph of FIG. This is an example of the appearance when the gradation is reduced by 20% and 40% using image data having 256 gradations. The control gradation (reference numeral 10) was 128. When collagen alone is used (symbol 12) and when tryptic collagen peptide is added at 1 μg / mL (symbol 14), there is little difference in the effect on the control.

  When 2 μg / mL of collagen peptide by trypsin is added (symbol 16), the density can be seen even by comparing the color samples. Thus, it is considered that a whitening effect can be expected if there is a change in absorbance of 50% or more with respect to at least the control.

  In addition, as shown in FIG. 1, the effect of inhibiting melanin production was shown in inverse proportion by increasing the amount of collagen peptide added. This is an easy-to-use characteristic in that the intake for the whitening effect can be adjusted when used on the human body.

  FIG. 2 shows the results when the degrading enzyme is papain. Referring to FIG. 2, when 2 μg / mL of collagen peptide having papain as a degrading enzyme was added to the control, about 60% of melanin production suppression was confirmed with respect to the control.

  A color sample is shown on the graph of FIG. It can be recognized that when the gradation is changed by 60% with respect to the control (reference numeral 10) (reference numeral 18), it becomes clear white.

  FIG. 3 shows the results of SDS-PAGE of purified collagen peptides enzymatically degraded with trypsin and papain. The results of changing the decomposition time to 24 hours, 48 hours, and 72 hours are shown side by side. The first, second, and third lanes are electrophoretic images of collagen peptides by papain after 24, 48, and 72 hours. The fourth, fifth, and sixth lanes are the migration images of collagen peptides with trypsin after 24, 48, and 72 hours. The zero lane is a molecular weight marker.

  A plurality of distinct electrophoretic images were confirmed for the collagen peptide enzymatically decomposed with papain, ranging from 15 to 43 kDa. Moreover, about the decomposition | disassembly time, the electrophoretic image of 48 hours and 72 hours was the same. Therefore, it was considered that the enzyme was almost completely degraded by degradation for at least 48 hours.

  On the other hand, with trypsin, no electrophoretic image was confirmed after 24 hours. From this, it was considered that a small collagen peptide of 3.5 kDa or less was produced when digested with trypsin.

FIG. 4 shows the result of CE. FIG. 4 (a) shows a case where the enzyme is decomposed with trypsin, and FIG. 4 (b) shows a case where the enzyme is decomposed with papain. In each case, the horizontal axis represents the migration time (minutes), and the vertical axis represents the absorbance. Compared with the peak of sodium cinnamate (C ₆ H ₅ CH = CHCOONa), which is a reference peak, when the enzyme is degraded with papain, a peptide with a large molecular weight is present in a rich manner. When the enzyme is digested with trypsin It can be seen that peptides having a small molecular weight are present in a rich manner. This is consistent with the results of SDS-PAGE (FIG. 3).

  FIG. 5 shows the results of CE when a collagen peptide using trypsin is dialyzed into a purified collagen peptide. The horizontal and vertical axes represent the migration time (minutes) and absorbance. In this case, the purified collagen peptide is a solution remaining after dialysis. That is, a substance having a pore size smaller than that of the membrane used in dialysis is a removed collagen peptide. Compared to FIG. 4A, the position of the peak and the distribution ratio were hardly changed.

  It has been confirmed that this purified collagen peptide also has an inhibitory effect on melanin production. Therefore, it is considered that the melanin production inhibitory effect is not due to the contribution of low-molecular substances removed by dialysis but to the collagen peptide remaining after dialysis. The purified collagen peptide after dialysis did not have an unpleasant odor such as an amine odor that had been present after the incubation.

The melanin production inhibiting composition according to the present invention can be suitably used as a raw material for cosmetics having a whitening effect.

Claims (2)

Melanin production-suppressing composition comprising a purified collagen peptide that is prepared by mixing and incubating papain or trypsin as a degrading enzyme with collagen extracted from the leg or shell edge of a suppon , incubating, deactivating the degrading enzyme, and performing dialysis .   The composition for inhibiting melanin production according to claim 1, which does not have an amine odor.