KR20130046702A - Novel whitening compound and whitening cosmetic composition comprising the same - Google Patents

Abstract

PURPOSE: A novel whitening compound and a cosmetic composition containing the same for skin whitening are provided to suppress tyrosinase expression and to stably maintain whitening activity of a peptide. CONSTITUTION: A novel compound for skin whitening is denoted by chemical formula 1. A cosmetic composition for skin whitening contains 0.001-30 wt% of the compound. The compound enhances stability of a peptide by introducing a natural product-derived material to N-terminal of the peptide with a whitening mechanism, and suppressing decomposition of the peptide in vivo.

Description

Novel whitening compound and whitening cosmetic composition comprising the same

The present invention relates to a novel whitening compound that can be utilized as a whitening material and a cosmetic composition for whitening comprising the same.

With the recent increase in national income, the aging of the population and the entry of women have increased significantly. In addition, as the culture of LOHAS and well-being rises, the desire to maintain fine skin is increasing day by day. Among these, consumer demand for clear and clean skin is increasing, and accordingly, technology development and discovery regarding whitening-related materials are attracting attention.

In particular, recently, a new mechanism has been studied and approached from one of the existing whitening mechanisms of inhibition of tyrosinase. This is to overcome this with a new approach as the limitations of previously commercialized whitening materials are gradually revealed. This new approach reduces the effects of ultraviolet light, cytokines and hormones that promote melanin formation, pre-melanin production that regulates tyrosinase transcription, melanin production that inhibits tyrosinase activity, and the resulting melanin Materials that regulate a number of different mechanisms have been developed, including those that regulate the delivery stage of melanin, which is delivered to keratinocytes in the form of moths. Research into the use of peptides underlying the materials involved in each step of these materials is being activated (US 5731408, US 6054556, US 7084111, US 7582610, WO 2009010356A1).

In addition, since the peptide is biocompatible and has a very high activity, many researches have been conducted in the cosmetic and pharmaceutical industries as the next-generation new material. However, since the peptide has a property of being easily degraded by protease, which is one of the most enzymes in the human body, it is difficult to maintain its efficacy. In order to solve this problem, a method of slowing down the degradation by modifying a part of the peptide has been widely used. In general, a form in which an acetyl group is introduced at the N-terminus has been proposed ( Pharm . Res . , 10 , 1268, (1993). Int . J. Cancer , 83 , 326, (1999).

The present invention, by applying a peptide containing an antagonist to the Melanocortin receptor 1 (MC1R) involved in the expression of tyrosinase, not only inhibits the expression of tyrosinase more effectively, but also can stably maintain the whitening activity of the peptide. It is to provide a novel whitening compound and a whitening cosmetic composition comprising the same.

According to an aspect of the present invention,

It relates to a whitening compound represented by the following formula (1).

[Formula 1]

(In Formula 1, R ₁ and R ₂ are the same as or different from each other, H or OH, X is a peptide consisting of 2 to 4 amino acids, the amino acid is one of Arg, Lys, Ala, Pro and Val) Y is the C-terminal portion of the peptide and is OH or NH _2. )

Another aspect of the present invention relates to a whitening cosmetic composition comprising the whitening compound as an active ingredient.

The present invention can provide a whitening compound and a whitening cosmetic composition comprising the same, which can be stably applied to the human body with high biocompatibility while stably sustaining the whitening activity of the peptide in vivo and providing excellent whitening effect. .

1 shows the results of HPLC analysis of caffeic-KVAR-NH ₂ as a whitening compound according to an embodiment of the present invention.
Figure 2 shows the MALDI-Tof analysis of the whitening compound caffeic-KVAR-NH ₂ according to an embodiment of the present invention.
Figure 3 shows the results of HPLC analysis of caffeic-VAR-NH ₂ as a whitening compound according to an embodiment of the present invention.
Figure 4 shows the results of MALDI-Tof analysis of caffeic-VAR-NH ₂ as a whitening compound according to an embodiment of the present invention.
5 shows the results of HPLC analysis of caffeic-AR-NH ₂ as a whitening compound according to an embodiment of the present invention.
Figure 6 shows the results of MALDI-Tof analysis of caffeic-AR-NH ₂ as a whitening compound according to an embodiment of the present invention.
7 shows the results of HPLC analysis of caffeic-KVAR-OH, a compound for whitening according to an embodiment of the present invention.
Figure 8 shows the MALDI-Tof analysis of the caffeic-KVAR-OH whitening compound according to an embodiment of the present invention.
9 shows the results of HPLC analysis of caffeic-VAR-OH, a compound for whitening according to an embodiment of the present invention.
Figure 10 shows the MALDI-Tof analysis of the caffeic-VAR-OH whitening compound according to an embodiment of the present invention.
11 shows the results of HPLC analysis of caffeic-AR-OH, a compound for whitening according to an embodiment of the present invention.
12 shows the results of MALDI-Tof analysis of caffeic-AR-OH, a whitening compound according to an embodiment of the present invention.
FIG. 13 shows the results of HPLC analysis of caffeic-VARP-OH, a compound for whitening according to an embodiment of the present invention. FIG.
Figure 14 shows the MALDI-Tof analysis of the whitening compound caffeic-VARP-OH according to an embodiment of the present invention.
15 shows the results of HPLC analysis of caffeic-RP-OH, a compound for whitening according to one embodiment of the present invention.
16 shows the results of MALDI-Tof analysis of caffeic-RP-OH, a compound for whitening according to an embodiment of the present invention.
17 shows the results of HPLC analysis of caffeic-KV-OH, a compound for whitening according to one embodiment of the present invention.
18 shows the results of MALDI-Tof analysis of caffeic-KV-OH, a whitening compound according to an embodiment of the present invention.
19 shows the results of HPLC analysis of coumaric-KVAR-NH ₂ as a whitening compound according to an embodiment of the present invention.
20 shows the results of MALDI-Tof analysis of coumaric-KVAR-NH ₂ , a whitening compound according to an embodiment of the present invention.
21 shows the results of HPLC analysis of coumaric-AR-NH ₂ as a whitening compound according to an embodiment of the present invention.
22 shows the results of MALDI-Tof analysis of coumaric-AR-NH ₂ which is a whitening compound according to an embodiment of the present invention.
FIG. 23 shows the results of HPLC analysis of coumaric-VARP-NH ₂ as a whitening compound according to an embodiment of the present invention. FIG.
FIG. 24 shows MALDI-Tof analysis results of coumaric-VARP-NH ₂ which is a whitening compound according to an embodiment of the present invention.
25 shows the results of HPLC analysis of coumaric-RP-NH ₂ as a whitening compound according to an embodiment of the present invention.
Figure 26 shows the MALDI-Tof analysis of the whitening compound coumaric-RP-NH ₂ according to an embodiment of the present invention.
27 shows the results of HPLC analysis of coumaric-KV-NH ₂ as a whitening compound according to an embodiment of the present invention.
28 shows the results of MALDI-Tof analysis of coumaric-KV-NH ₂ which is a whitening compound according to an embodiment of the present invention.
29 shows the results of HPLC analysis of coumaric-KVAR-OH, a compound for whitening according to an embodiment of the present invention.
30 shows the results of MALDI-Tof analysis of coumaric-KVAR-OH, a compound for whitening according to an embodiment of the present invention.
FIG. 31 shows the results of HPLC analysis of coumaric-VAR-OH as a whitening compound according to an embodiment of the present invention. FIG.
32 shows the results of MALDI-Tof analysis of coumaric-VAR-OH, a compound for whitening according to an embodiment of the present invention.
FIG. 33 shows the results of HPLC analysis of coumaric-AR-OH as a whitening compound according to an embodiment of the present invention. FIG.
Figure 34 shows the MALDI-Tof analysis of the whitening compound coumaric-AR-OH according to an embodiment of the present invention.
35 shows the results of HPLC analysis of coumaric-RP-OH, a compound for whitening according to an embodiment of the present invention.
36 shows the results of MALDI-Tof analysis of coumaric-RP-OH, a compound for whitening according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention provides a novel whitening compound in which a peptide having a whitening mechanism is introduced into a naturally derived material.

The whitening compound may introduce a naturally-derived material into the N-terminus of the peptide having a whitening mechanism to inhibit degradation of the peptide in vivo, thereby enhancing stability of the peptide and providing continuous whitening activity. The whitening compound may be represented by the following Chemical Formula 1.

[Formula 1]

In Formula 1, R ₁ and R ₂ are the same as or different from each other, H or OH, X is a peptide consisting of 2 to 4 amino acids, the amino acid is one or more of Arg, Lys, Ala, Pro and Val And Y is the C-terminal portion of the peptide and is OH or NH ₂ .

Preferably in formula 1 R ₁ And R ₂ is OH the caffeic acid (caffeic acid), or R ₁ is H and R ₂ is OH, and in Kumar acid compounds for whitening the N- terminus of the carboxyl group and the peptide bond of (coumaric acid), of formula (II) to And represented by the formula (3). Kumaric acid and caffeic acid are naturally derived substances having a whitening activity, have high biocompatibility, and may enhance stability of the peptide in vivo. In addition, since the peptide shows whitening activity after degradation, it can provide a continuous whitening function.

[Formula 2]

(3)

(In Formulas 2 and 3, X and Y are as mentioned above.)

The whitening compound is a peptide containing an antagonist against MC1R by shortening or altering the sequence of the Agouti signal protein, known as an antagonist of MC1R (-Lys-Val-Ala-Arg-), or by replacing it with a similar amino acid. It is prepared by applying. Since the peptide is the basis for constituting the human body, the peptide may exhibit not only high biocompatibility but also excellent activity. In addition, the peptide may act as an antagonist to MC1R to more effectively inhibit the expression of tyrosinase can provide excellent whitening efficacy. The peptides are represented by X in Formulas 1 to 3, wherein Y is introduced at the C-terminus of the peptide.

Preferably X is a peptide comprising the Ala-Alg sequence. Y is NH ₂ , which means that the C-terminus of the peptide is converted into the structure of -CONH ₂ .

The present invention provides a method for preparing the compound for whitening. More specifically, the preparation method includes a peptide synthesis step, an organic material introduction step and a free step.

The peptide synthesis step is to prepare a peptide by a solid phase synthesis method. The solid phase synthesis method is a widely used method for peptide synthesis, and the present invention is not particularly limited in terms of the synthesis conditions, but preferably in order to maintain the OH group at the C-terminus of the amino acid 2-CTC resin (2- Chlorotrityl chloride resin (Scheme 1), and polystyrene-based resin having a link amide linker introduced to form -CONH ₂ by introducing NH ₂ to the C-terminus of the amino acid. 2) can be used.

For example, referring to Scheme 1 and Scheme 2, R represents various side chains of amino acids, and X is a peptide consisting of 2-4 amino acids. The synthesis process reacts the amino acid protected with amine with Fmoc in the binding reagent to prevent side reactions. Next, the Fmoc protecting group is removed after washing and filtration. This synthesis process is repeated by adding amino acids to form the desired peptide.

The binding reagent may be applied to at least one of BOP, HOBt and DIEA depending on the resin and reaction conditions, and the removal of the Fmoc protecting group may use 20% piperidine / NMP.

The introduction step of the organic material binds a naturally derived material to the N-terminus of the prepared peptide in the binding reagent. For example, the naturally-derived material may be caffeic acid, coumaric acid, or the like, and the carboxyl groups of the caffeic acid and coumaric acid and the N-terminus of the peptide combine to form a whitening compound. The binding reagent is as mentioned above.

The free step separates the peptide bound to the resin by a free reaction using trifluoroacetic acid (reagent K, Trifuloroacetic acid) after the introduction of the organic material.

The whitening compound obtained by the above method may be further washed, purified and dried in a conventionally known manner.

[Reaction Scheme 1]

[Reaction Scheme 2]

The present invention provides a whitening cosmetic composition comprising the whitening compound as an active ingredient.

The cosmetic composition for whitening may include the compound for whitening in an effective amount, more specifically, may comprise 0.001 to 30% by weight. If the whitening compound is included in the above range it can exhibit the stability and excellent whitening effect of the formulation.

The cosmetic composition for whitening may be configured by selecting a cosmetically acceptable carrier within a range that does not reduce the effect of the present invention, more specifically, a solvent, a viscosity modifier, a UV absorber, a foaming agent, a cleaning agent, a pigment, a moisturizing agent Preservatives, flavors, pigments, oils, gelling agents, solubilizers, emulsions, surfactants, alcohols, chelating agents, water, oils, inorganic powders and the like.

The cosmetic composition for whitening may be prepared in any formulation commonly prepared in the art, for example, skin ointment, softening lotion, nutrition lotion, nutrition cream, massage cream, lotion, essence, eye cream, foundation , Cleansing cream, cleansing foam, cleansing water, soap, pack, gel, spray, powder and the like.

Hereinafter, the present invention will be described in more detail by way of examples. The following examples are only for the description of the present invention, and thus the scope of the present invention is not limited.

Example 1 Preparation of caffeic acid-Lys-Val-Ala-Arg-NH ₂

1 g of Rink amide (substitution rate: 0.5 mmol / g) was added to the reactor, swollen in NMP (n-Methylpyrrolidone) solvent for 2 hours, and then 20% piperidine / NMP was added for 30 minutes. In reaction to remove the Fmoc protecting group of the resin. After the solution was removed, 20% piperidine / NMP was added back to the reactor containing the resin and allowed to react for 1 hour. After the solution was removed again, the resin was washed three times with NMP, MeOH and DCM (Dichloromethane), respectively, and finally with NMP again to remove excess reaction solution.

648 mg of Fmoc-Arg (Pbf) -OH (2 equivalents of substituent), 442 mg of BOP reagent (equivalent to amino acid reagent), 135 mg of HOBt (equivalent to amino acid reagent), 142 mg of DIEA (1.1 equivalent of amino acid reagent) were added to the reactor. After adding 10ml, it is reacted for 3 hours. Termination of the reaction was confirmed using kaiser's ninhydrin test ( Anal. Biochem. , 1981, 117, 147). After removing all the solutions in the reactor, the resin was washed three times with NMP, MeOH and DCM, respectively, and finally with NMP again. After the washing was completed, 20% piperidine / NMP was added to the resin and the Fmoc protecting group was removed by the same reaction as described above. Subsequently, Fmoc-Ala-OH, Fmoc-Val-OH and Fmoc-Lys (Boc) -OH are introduced into the resin in the same manner.

Lys-Val-Ala-Arg-injected resin contains caffeic acid (180 mg, 2 equivalents of substituent), BOP reagent 442 mg (equivalent to amino acid reagent), HOBt 135 mg (hydroxybenzotriazol, equivalent to amino acid reagent), DIEA 142 mg (N , N-Diisopropylethylamine, 1.1 equivalents of amino acid reagent) is added and 10 ml of NMP is added, followed by reaction for 24 hours. Termination of the reaction was confirmed using the kaiser's ninhydrin test.

Caffeic acid-Lys-Val-Ala-Arg-NH ₂ prepared on Rink amide resin as described above using reagent K (Trifuloroacetic acid) (Synthetic Peptides: A User's Guide (GA Grant, ed.), WH Freeman and Company, New York, 1992). The isolated peptide derivative was precipitated using ether and then separated from the solution by centrifugation at 0 ° C. The isolated peptide derivative was added to 20 ml of ether under the same conditions and centrifuged three times to remove impurities. The final product was lyophilized for 24 hours lyophilized peptide derivative to give 323 mg.

The presence of the final product was confirmed using HPLC analysis and MALDI-Tof (Matrix-assisted laser desorption ionization time of flight mass spectroscopy) analysis. The results are shown in FIGS. 1-2 and Table 2.

<Example 2-3>

Compounds were synthesized in the same manner as in Example 1, except that the amino acid sequences shown in Table 1 were used. The presence of the final product was confirmed using HPLC analysis and MALDI-Tof analysis. The results are shown in FIGS. 3 to 6 and Table 2.

<Example 4-9>

Using 2-CTC-resin (2-chlorotrityl chloride), the compound was synthesized in the same manner as in Example 1 except that the amino acid sequence shown in Table 1 was synthesized. The presence of the final product was confirmed using HPLC analysis and MALDI-Tof analysis. The results are shown in Figures 7 to 18 and Table 2.

<Example 10-14>

A compound was synthesized in the same manner as in Example 1 except that the amino acid sequence shown in Table 1 was synthesized and coumaric acid was introduced into the peptide. The presence of the final product was confirmed using HPLC analysis and MALDI-Tof analysis. The results are shown in FIGS. 19 to 28 and Table 2.

&Lt; Examples 15-18 >

Compounds were synthesized in the same manner as in Example 4-9, except that the amino acid sequences shown in Table 1 were synthesized and Coumaric acid was introduced into the peptide. The presence of the final product was confirmed using HPLC analysis and MALDI-Tof analysis. The results are shown in FIGS. 29 to 36 and Table 2.

Example compound M.W. water One caffeic-KVAR-NH ₂ 632 96.647% 2 caffeic-VAR-NH ₂ 505.5 97.672% 3 caffeic-AR-NH ₂ 406.9 97.074% 4 caffeic-KVAR-OH 634.7 97.552% 5 caffeic-VAR-OH 506.7 97.334% 6 caffeic-AR-OH 407.9 96.549% 7 caffeic-VARP-OH 603.5 96.900% 8 caffeic-RP-OH 433.4 96.682% 9 caffeic-KV-OH 407.4 97.437% 10 coumaric-KVAR-NH ₂ 616.2 99.451% 11 coumaric-AR-NH ₂ 390.4 98.072% 12 coumaric-VARP-NH ₂ 586.5 99.116% 13 coumaric-RP-NH ₂ 416.1 98.792% 14 coumaric-KV-NH ₂ 390.4 95.184% 15 coumaric-KVAR-OH 618.2 97.253% 16 coumaric-VAR-OH 490.7 99.448% 17 coumaric-AR-OH 391.3 98.929% 18 coumaric-RP-OH 417.6 98.417%

Amino Acid Reagents Used in the Synthesis of Compounds in Table 1

1) Fmoc-Lys (Boc) -OH

2) Fmoc-Arg (Pbf) -OH

3) Fmoc-Ala-OH

4) Fmoc-Val-OH

5) Fmoc-Pro-OH

&Lt; Test Example 1 >

The melanogenesis inhibitory effect using B16-F1 melanoma cells was measured. B16-F1 melanoma cells were inoculated into 6-well plates in Dulbecco's modified Eagle's medium (DMEM) with 10% Fetal Bovine Serum (FBS) at a density of 3 × 10 ⁵ cells and at 5% CO ₂ , 37 ° C. for ₁ day. Incubated. Thereafter, after replacing with fresh DMEM, the compounds of Examples 1 to 18 were treated at 50 uM concentration and incubated for 2 days. After 2 days, the cells from which the medium was removed were washed with PBS (phosphate buffered saline) and treated with trypsin. The recovered cells were separated by centrifugation, and the supernatant was discarded. The cells were treated with 0.2 ml of 1N NaOH + 10% DMSO solution to obtain intracellular melanin. The collected cells were transferred to a 96-well plate, and the absorbance was measured at 490 nm. A standard curve was prepared using synthetic melanin as a standard material to determine the intracellular melanin amount, and the amount of protein was measured by using a protein assay reagent. Quantification through The amount of melanin produced was determined by the amount of melanin for a certain protein. The control group was used kojiic acid (Kojic acid) of 50uM concentration, the negative control group was used as the reaction solution without the sample. The experimental results of the melanin production inhibitory effect on the negative control group are shown in Table 2 and Table 3.

Example compound Inhibition% One caffeic-KVAR-NH ₂ 36% 2 caffeic-VAR-NH ₂ 26% 3 caffeic-AR-NH ₂ 28% 4 caffeic-KVAR-OH 16.6% 5 caffeic-VAR-OH 72% 6 caffeic-AR-OH 61% 7 caffeic-VARP-OH 19.5% 8 caffeic-RP-OH 32% 9 caffeic-KV-OH 20.8% 10 coumaric-KVAR-NH ₂ 36% 11 coumaric-AR-NH ₂ 68% 12 coumaric-VARP-NH ₂ 42% 13 coumaric-RP-NH ₂ 22% 14 coumaric-KV-NH ₂ 32% 15 coumaric-KVAR-OH 29.4% 16 coumaric-VAR-OH 43% 17 coumaric-AR-OH 66% 18 coumaric-RP-OH 24%

Control group matter Inhibition% One coumaric acid 23.2% 2 caffeic acid 15.6% 3 Kojic acid 13.8%

As shown in Table 2, specific compounds of the whitening compound in which the peptide is bound to coumaric acid or caffeic acid can be confirmed to more effectively inhibit melanin production. In particular, Examples 5, 6, 11, 17 showed an excellent melanin formation inhibitory effect corresponding to 4 to 5 times the kojic acid. The amino acid sequences of the compounds of Examples 5, 6, 11, and 17 commonly include Ala-Arg.

The novel whitening compound according to the present invention stably sustains the whitening activity of the peptide by introducing a naturally occurring substance into the peptide having a whitening mechanism, and exhibits superior whitening efficacy when applying a naturally-derived substance having a whitening effect. Can be.

Claims (6)

A compound for whitening, characterized in that represented by the following formula (1).
[Formula 1]

(In Formula 1, R ₁ and R ₂ are the same as or different from each other, H or OH, X is a peptide consisting of 2 to 4 amino acids, the amino acid is one of Arg, Lys, Ala, Pro and Val) And Y is the C-terminal portion of the peptide and is OH or NH _2. )
The method of claim 1,
In Formula 1, R &lt; 1 _&gt; And R ₂ is OH, characterized in that represented by the formula (2) Whitening compound.
(2)

The method of claim 1,
In Formula 1, R ₁ is H and R ₂ is OH, characterized in that represented by the formula (3) Whitening compound.
(3)

The method of claim 1,
X is a compound for whitening, characterized in that the peptide comprising the Ala-Arg sequence.
A whitening cosmetic composition comprising a compound for whitening according to any one of claims 1 to 4 as an active ingredient.
The method of claim 5,
The whitening compound is a cosmetic composition for whitening, characterized in that contained in 0.001 to 30% by weight relative to the composition.

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