KR101732639B1 - Novel Whitening compound and Whitening cosmetic composition comprising the same - Google Patents

Abstract

The present invention relates to a novel whitening compound and a whitening cosmetic composition containing the same, and more specifically to a whitening compound represented by the following general formula (1). The whitening compound according to the present invention is highly biocompatible, has no skin side effects, and has excellent skin whitening effect due to its ability to inhibit tyrosinase enzyme activity and melanin production.
≪ Formula 1 >
Azelaic-XY

Wherein X and Y are amino acids and at least two selected from Leu, Gly, Ala, Arg and Trp.

Description

[0001] The present invention relates to a novel whitening compound and a whitening cosmetic composition containing the same,

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

With the recent rise in the national income, the aging of the population and the advancement of women are increasing significantly. In addition, as the culture of LOHAS, well-being, etc. is emerging, the desire to maintain fine skin is increasing day by day. Among these, the demand of consumers for clear and clean skin is increasing, and accordingly, the technology development and discovery about the whitening-related material are attracting attention.

One of the most important factors in skin whitening is melanin formation in the skin's melanocyte (melanocyte).

Melanin is converted from tyrosine to dopa and dopaquinone by the action of a tyrosinase enzyme present in the melanin-forming cells and is produced via dopachrome. This melanin exists in the skin and protects the body from ultraviolet rays and has an important function to regulate hormone secretion in the body. However, excessive production of melanin is known to form spots and freckles, promotes skin aging, and plays an important role in skin cancer induction, so that research and development are actively conducted to prevent melanin overproduction.

Some of the substances known to be effective for skin whitening include vitamin C, extracts of Cabbage, kojic acid, arbutin, licorice extract, and hydroquinone. There is a case in which kojic acid is known as a carcinogenic substance and is prohibited to be used as a cosmetic ingredient except for the experiment. Hydroquinone is toxic to melanin-forming cells, and in vivo test results in permanent decolorization. It is also known that imports from cosmetics are prohibited in Korea.

Korean Patent Laid-Open Publication No. 10-2015-0079144 discloses that a high-concentrated azelaic acid is used as a whitening agent, but it has a disadvantage that the whitening effect of the whitening agent is not so high because the whitening agent has a poor ability to deliver skin.

Although the known chemical substances as described above have a whitening effect, there is a problem of toxicity or side effects to the skin, and thus there are limitations in their use. Recently, studies have been actively conducted to develop a biocompatible skin whitening ingredient which can minimize side effects to the skin. Since peptides are biocompatible and have very high activity, they are conducting research in the cosmetics and pharmaceuticals industry as the next generation of new materials. However, these peptides are difficult to maintain their efficacy because they are very easily degraded to protease, one of the most enzymes in human body.

It is an object of the present invention to provide a peptide composition which is safe for human body and has no side effects even when it is used for a long period of time, has improved stability by introducing azelaic acid into the peptide N-terminus and has excellent inhibitory activity on tyrosinase enzyme activity and melanin production To provide a novel whitening compound having excellent skin whitening effect and a whitening cosmetic composition containing the same.

In order to achieve the above object,

The present invention relates to a whitening compound represented by the following formula (1).

≪ Formula 1 >

Azelaic-X-Y

(Wherein X and Y are amino acids and at least two selected from Leu, Gly, Ala, Arg and Trp)

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 containing the whitening compound which can be safely applied to human body because of high biocompatibility while providing an excellent whitening effect by stably maintaining the whitening activity of the peptide in vivo.

1 shows HPLC analysis results of Azelaic-LG as a whitening compound of the present invention.
Fig. 2 shows MALDI-TOF analysis results of the whitening compound of the present invention, Azelaic-LG.
Fig. 3 shows the cell survival rate according to the cytotoxicity test of the whitening compound Azelaic-LG of the present invention.

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

The present invention provides a novel whitening compound into which a peptide having a whitening mechanism is introduced into a naturally occurring substance.

The naturally occurring substance is introduced into the N-terminus of the peptide having a whitening mechanism to the whitening compound to inhibit the degradation of the peptide in vivo, thereby increasing the stability of the peptide and providing continuous whitening activity.

In the present invention, the whitening compound may be represented by the following formula (1).

≪ Formula 1 >

Azelaic-X-Y

In Formula 1, X and Y are independently amino acids and dipeptide bonds are possible.

The amino acid is at least two selected from Leu, Gly, Alanine, Arg, and Trp, preferably leucine and glycine (Gly).

The compound of formula (1) has high biocompatibility including peptides and is designed to further enhance the efficacy of the peptide material by introducing azelaic acid into the N-terminus of the peptide.

Azelaic-X-Y is prepared through the coupling of a peptide with azelaic acid. It is a dipeptide form in which two amino acids are linked by an amide bond, and is a form that is a -COOH group at the C-terminal portion.

The compound of formula (1) is preferably a compound of formula (2).

(2)

In the present invention, the compound of Formula 2 is represented by Azelaic-LG.

The present invention provides a method for producing the whitening compound.

More specifically, the method for producing the whitening compound includes a peptide synthesis step, an organic substance introduction step, and a free step.

In the present invention, the peptide synthesis step produces a peptide by a solid phase synthesis method. Although the solid phase synthesis method is widely used in peptide synthesis, in the present invention, there is no particular limitation with respect to the synthesis conditions, but in order to maintain the OH group at the C-terminal of the amino acid, 2-CTC resin Tritic chloride resin) can be used (e. G., Scheme 1).

<Reaction Scheme 1>

(a) BOP, HOBt, DIEA (b) 20% piperidine (c) Fmoc-Leu (d) Azelaic acid

In the synthesis process, amine-protected amino acids and a support are reacted in a binding reagent with Fmoc (Fluorenylmethoxycarbonyl) to prevent side reactions. Next, the Fmoc (Fluorenylmethoxycarbonyl) protecting group is removed after washing and filtration. This synthesis process is repeatedly carried out by adding amino acid to form a desired peptide.

The binding reagent may be at least one of BOP {Benzotriazolyloxytris (dimethylamino) phosphonium Hexafluorophosphate}, HOBt (Hydroxybenzotriazole) and DIEA (Diisopropylethylamine) depending on the resin and reaction conditions, and the removal of the Fmoc (Fluorenylmethoxycarbonyl) Piperidine / NMP (N-Methyl-2-pyrrolidone) may be used.

In the present invention, the step of introducing the organic substance binds azelaic acid to the N-terminus of the peptide produced in the binding reagent. The carboxy group of the azelaic acid binds to the N-terminus of the peptide to form a whitening compound. The binding reagent is as mentioned above.

In the present invention, the free step is a step of separating the peptide bound to the resin after the introduction of the organic material, and the separation reagent is preferably Reagent K and Trifluoroacetic acid.

Reagent K is a mixed solution (82.5: 5: 5: 5: 2.5 (V / V)) of trifluoroacetic acid (TFA), thioanisole, water, phenol and ethanediol (EDT) The whitening compound obtained by the method can be further washed, purified and dried in a conventional manner.

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

The whitening cosmetic composition may contain the whitening compound in an effective amount, preferably 0.000001 to 30% by weight, and more preferably 0.001 to 10% by weight.

When the whitening compound is contained in an amount of less than 0.000001% by weight, skin whitening effect may be insignificant, and if it exceeds 30% by weight, the stability of the formulation may be lowered.

In the present invention, the formulation of the whitening cosmetic composition is not particularly limited and may be appropriately selected according to the purpose. For example, it can be applied to the skin such as external skin ointment, softening longevity, nutritional lotion, nutritional cream, massage cream, lotion, essence, eye cream, foundation, cleansing cream, cleansing foam, cleansing water, soap, pack, gel, spray and powder .

In addition, the cosmetic composition of the present invention may contain, in addition to the components described above, one kind selected from oil, water, surfactant, moisturizer, ethanol, thickener, chelating agent, coloring agent, preservative, The above additives may be appropriately used in combination as needed.

Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples. This is for the purpose of illustrating the present invention, and thus the scope of the present invention is not limited thereto.

< Manufacturing example  1> Azelaic -LG Manufacturing

1 g of 2-CTC resin (2-chlorotritic chloride resin) (substitution ratio: 1 mmol / g) was added to the reactor and swelled in a solvent of N-methylpyrrolidone (NMP) Piperidine / N-methylpyrrolidone is added and the reaction is carried out for 30 minutes to remove the Fmoc protecting group of the resin. After removing the solution, 20% piperidine / N-methylpyrrolidone is added to the reactor containing the resin and reacted for 1 hour. After removing the solution again, the resin was washed three times each with N-methylpyrrolidone, methanol and dichloromethane (DCM), and finally washed again with N-methylpyrrolidone to remove the excess reaction solution do.

The reactor was charged with 297 mg of Fmoc-Gly (2 equivalents of substituent), 442 mg of BOP reagent (equivalent to amino acid reagent), 135 mg of 1-hydroxybenzotriazole (HOBt) (equivalent to amino acid reagent) 142 mg of diisopropylethylamine (DIEA) (1.1 equivalents of amino acid reagent) was added, and 10 mL of N-methylpyrrolidone was added, followed by reaction for 3 hours.

The solution in the reactor was removed and the resin was washed three times with N-methylpyrrolidone, methanol and dichloromethane, respectively, and finally washed again with N-methylpyrrolidone. 20% piperidine / N-methylpyrrolidone was added to the washed resin, and the same reaction as described above was carried out to remove the Fmoc protecting group. Subsequently, Fmoc-Leu was introduced into the resin in the same manner.

Azelaic acid 180 mg (2 equivalents of substituent), 442 mg of BOP reagent (the same amount as the amino acid reagent), 1-hydroxy-3-hydroxypentanoic acid 135 mg of benzotriazole (equivalent to the amino acid reagent) and 142 mg of diisopropylethylamine (1.1 equivalent of the amino acid reagent) were added, and 10 mL of N-methylpyrrolidone was added, followed by reaction for 24 hours. The reaction was terminated using Kaiser's ninhydrin test.

As described above, Azelaic-Leu-Gly prepared on 2-CTC resin can be synthesized by a known method using Reagent K including Trifluoroacetic acid (Synthetic Peptides: A User's Guide (GA Grant, ed.), WH Freeman and Company, New York, 1992). The separated peptide derivative was precipitated with ether and then separated from the solution by centrifugation at 0 ° C. The separated peptide derivative was subjected to centrifugal separation by adding 20 mL of cold ether under the same conditions and repeated three times to remove impurities. The centrifuged peptide derivative was lyophilized for 24 hours to obtain about 200 mg of a final product having a purity of 97.0%.

The presence of the final product was confirmed by HPLC analysis and MALDI-TOF (Matrix-assisted laser desorption ionization time of flight mass spectrometry) analysis. The results are shown in Fig. 1 and Fig.

< Experimental Example  1> Tyrosinase  Experiment of inhibition of activity

When tyrosinase becomes active due to ultraviolet rays or the surrounding environment and internal hormone, tyrosine or dioxyphenylalanine (DOPA) undergoes the pathway of dopachrome formation, and melanin Polymer.

The degree of inhibition of melanin pigmentation was measured by measuring the production of dopa chrome, an intermediate production step by tyrosinase, in this experiment.

Tyrosinase was isolated and purified from mushroom and purchased from Sigma.

After adding 50 μL of a sample at a concentration of 100 μM and 50 μM into a 96-well plate, 50 μL of a 2400 units / mL tyrosinase solution and 100 μL of a 0.3 mg / mL L-tyrosine solution were added in this order. For the blank, the same amount of the solvent in which the sample was dissolved was added instead of the sample. After incubation at 37 ° C for 10 minutes, the absorbance was measured at 475 nm using an ELISA reader. Arbutin was used as a positive control.

The inhibitory rate of tyrosinase activity of each sample liquid was calculated according to the following equation (1), and the experimental results are shown in Table 1 below.

(1)

(%) = 100- (B-B ') / (A-A') X 100

A: Absorbance after reaction of the blank

B: Absorbance after reaction of the sample solution

A 'and B': Absorbance measured by replacing with buffer instead of tyrosine

Inhibition rate of tyrosinase activity (%) Azelaic-LG 100 μM 28.7 Azelaic-LG 50 μM 22.5 Azelaic-AR 100 μM 25.4 Azelaic-AR 50 μM 20.6 Azelaic-RW 100 μM 26.5 Azelaic-RW 50 μM 21.8 Azelaic acid 100 μM 23.2 Azelaic acid 50 μM 18.5 Arbutin 100 μM 14.3 Arbutin 50 μM 9.3

As shown in Table 1, the tyrosinase inhibitory effect of Azelaic-LG was superior to the comparative substances arbutin and azelaic acid. Based on this, it was expected that the whitening compound of the present invention effectively inhibited the activity of tyrosinase, thereby being effective for whitening of the skin.

< Experimental Example  2> Melanin formation inhibitory effect experiment

B16F1 melanoma cells were used to measure the inhibitory effect of melanin formation. B16F1 melanoma cells were inoculated into 6-well plates at a density of 1 x 10 &lt; ⁵ &gt; cells in DMEM supplemented with 10% FBS and incubated at 37 ° C in 5% CO ₂ for one day. After replacing with new DMEM, samples were treated at 1, 10, and 50 μM, respectively, and cultured for 3 days. After 3 days, the cells were washed with PBS (phosphate buffered saline) and treated with trypsin. The recovered cells were separated by centrifugation, the supernatant was discarded, and 0.2 ml of 1 N NaOH + 10% DMSO solution was added to obtain intracellular melanin. The collected cells were transferred to a 96-well plate and absorbance was measured at 490 nm. The amount of melanin in the cells was determined by preparing a standard curve using synthetic melanin as a standard substance, and the amount of protein was quantified by measuring the absorbance using a protein assay reagent. The amount of melanin produced was determined as the amount of melanin for a certain protein. As a positive control group, niacinamide was used at a concentration of 50 μM, and a negative control group was used. The experimental results of the inhibitory effect on the melanin formation in the negative control group are shown in Table 2.

Melanin production inhibition rate (%) Azelaic-LG 1 μM 18.9 Azelaic-LG 10 μM 21.3 Azelaic-LG 50 μM 25.1 Niacinamide 50 μM 10.4

From the above results, it can be confirmed that the effect of inhibiting melanin formation of Azelaic-LG is excellent, and based on this, the whitening compound of the present invention effectively inhibits melanin production, thereby skin whitening can be expected.

< Experimental Example  3> Cytotoxicity experiment

The degree of cytotoxicity was measured using B16F1 melanoma cells. B16F1 melanoma cells were inoculated into 6-well plates at a density of 1 x 10 &lt; ⁵ &gt; cells in DMEM supplemented with 10% FBS and incubated at 37 ° C in 5% CO ₂ for one day. After replacing with new DMEM, samples were treated at 1, 10, 50, and 100 μM for 3 days. After 3 days, the medium was treated with MTT reagent at a concentration of 5 mg / mL, and then cultured at 37 ° C in 5% CO ₂ for 4 hours. The medium was removed and 2 mL of DMSO reagent was added to dissolve the purple crystals and the absorbance was measured at 540 nm.

As a negative control, a reaction solution to which no sample was added was used. The cell viability by each sample solution was calculated according to the following equation (2), and the measurement results are shown in FIG.

(2)

Cell viability (%) = A / B X 100

A: Absorbance of the sample

B: absorbance of negative control group

From the results shown in FIG. 3, it was confirmed that Azelaic-LG had almost no cytotoxicity at a concentration of 100 μM or less.

< Experimental Example  4> Skin stimulation experiment

A human patch test was performed to evaluate the degree of irritation to the skin. The skin patch test was carried out on 20 normal subjects aged 20 years or older. The samples were made into 0.001%, 0.01%, 0.1%, 1%, and 10% aqueous solutions and then in the Finn chamber for 48 hours. And then the first reading was performed after the removal, and the second reading was performed after 72 hours to determine the skin reaction result. The evaluation criteria and the stimulation index are shown in Table 3 below and the results are shown in Table 4.

reaction weight Evaluation standard - 0.0 No reaction ± 0.5 Suspicious Positive Response + 1.0 Weakly positive reaction ++ 2.0 Strong positive reaction +++ 3.0 Polar positive reaction

(3)

Skin irritation index = Σ (weight X response factor) / (maximum weight X total subjects) X 100

Skin irritation index Degree of stimulation Azelaic-LG 0.001% 0.0 No stimulation Azelaic-LG 0.01% 0.0 No stimulation Azelaic-LG 0.1% 0.0 No stimulation Azelaic-LG 1% 0.0 No stimulation Azelaic-LG 10% 0.4 No stimulation

As a result, Azelaic-LG showed no irritation to the skin under 10%, and it is considered that there is no irritation to the skin when it is used as a raw material for cosmetics.

< Experimental Example  5> Skin whitening  Clinical trial

The cream compositions of Example 1 and Comparative Examples 1 and 2 were prepared in the composition shown in Table 5 below (unit: wt%).

Ingredients Content (% by weight) Example 1 Comparative Example 1 Comparative Example 2 Azelaic-LG 0.01 - - Azelaic-acid - - 0.01 glycerin 5.0 5.0 5.0 Stearic acid 8.0 8.0 8.0 Squalane 5.0 5.0 5.0 Self emulsifying glycerin monostearate 2.5 2.5 2.5 Polyoxyethylene sorbitan monostearate 1.5 1.5 1.5 Propylene glycol 4.0 4.0 4.0 Stearyl glycyrrhetinate 0.2 0.2 0.2 Vaseline 2.0 2.0 2.0 Antioxidant Suitable amount Suitable amount Suitable amount Spices Suitable amount Suitable amount Suitable amount antiseptic Suitable amount Suitable amount Suitable amount Purified water Balance Balance Balance

Clinical tests were carried out to confirm the skin whitening effect of the cream prepared in Example 1 and Comparative Examples 1 and 2. 30 women in their 20s and 40s were divided into 3 groups of 10 persons in Example 1 and Comparative Examples 1 and 2 and used for 4 weeks after morning washing and evening washing after twice daily.

The skin melanin index before and after use was measured using Mexameter® MX 18 (CK Electronic GmbH, Cologne, Germany), and the value was calculated as ΔM.I. Values were calculated according to Equation (4) and the effects were compared. The results are shown in Table 6 below.

(4)

? MI. = M.I. Value - before use M.I. value

? MI. Example 1 -13.03 Comparative Example 1 -10.19 Comparative Example 2 -11.54

As a result of the experiment, it was confirmed that when Example 1 containing Azelaic-LG was used for 4 weeks, skin whitening effect was higher than Comparative Examples 1 and 2.

< Experimental Example  6> Light stability test

Photostability tests were carried out to confirm the presence of photodegradation. In the light stability test, the sample was diluted to a concentration of 0.5%, stored for one week under external light conditions, and the peak area was determined by HPLC analysis. The content% of the sample was calculated according to the following equation (5), and the experimental results are shown in Table 7 below.

(5)

Content of sample% = A / B X 100

A: Peak area of sample after external light test

B: Peak area of sample before external light test

Initial content% Azelaic-LG 93.6 Coumaric-LG 34

From the above results, it was confirmed that Azelaic-LG is superior in light stability effect to Coumaric-LG.

Based on the results of the above Experimental Examples, various formulation examples using Azelaic-LG are prepared as shown in Tables 8 to 12 below. However, it should be apparent to those skilled in the art that these formulation examples are for illustrating the present invention and that the formulation of the present invention is not limited to these formulation examples.

&Lt; Formulation Example 1 > Soft skin length (skin toner)

Ingredients Content (% by weight) Azelaic-LG 0.01 glycerin 5.0 1,3-butylene glycol 3.0 ethanol 5.0 Polyoxyethylene nonylphenyl ether 0.5 Spices Suitable amount antiseptic Suitable amount Purified water Balance

&Lt; Formulation Example 2 >

Ingredients Content (% by weight) Azelaic-LG 0.01 glycerin 3.0 Citric acid 0.1 ethanol 10.0 Polyoxyethylene oleyl ether 1.0 Sorbitol 2.0 Spices Suitable amount antiseptic Suitable amount Purified water Balance

Formulation Example 3 Lotion (emulsion)

Ingredients Content (% by weight) Azelaic-LG 0.01 glycerin 5.0 1,3-butylene glycol 8.0 Squalane 10.0 Polyoxyethylene sorbitan monooleate 2.0 Triethanolamine 1.5 Glyceryl stearate 0.5 Stearyl glycyrrhetinate 0.2 Carboxyvinyl polymer 0.1 Arginine 0.1 Spices Suitable amount antiseptic Suitable amount Purified water Balance

&Lt; Formulation Example 4 >

Ingredients Content (% by weight) Azelaic-LG 0.01 Sorbitol 8.0 Polyoxyethylene glycol 1500 6.0 ethanol 5.0 glycerin 3.0 1,3-butylene glycol 3.0 Polyoxyethylene oleyl alcohol ether 1.0 olive oil 0.3 Hyaluronic acid 0.2 Antioxidant Suitable amount Spices Suitable amount antiseptic Suitable amount Purified water Balance

&Lt; Formulation Example 5 >

Ingredients Weight (% by weight) Azelaic-LG 0.01 ethanol 10.0 glycerin 4.0 Propylene glycol 4.0 Polyoxyethylene hardened castor oil 0.1 Carboxy polymer 0.3 Triethanolamine 0.3 Antioxidant Suitable amount Spices Suitable amount antiseptic Suitable amount Purified water Balance

In the present invention, as shown in Tables 8 to 12, the novel whitening compounds prepared by using various formulations using Azelaic-LG inhibit the degradation of peptides in vivo by introducing a naturally occurring substance into the N-terminus of a peptide having a whitening mechanism To increase the stability of the peptide, to provide continuous whitening activity, and to exhibit excellent whitening efficacy.

Claims (6)

(1). &Lt; / RTI >

&Lt; Formula 1 &gt;
Azelaic-XY

Wherein X and Y are amino acids and at least two selected from Leu, Gly, Ala, Arg and Trp.
The method according to claim 1,
Wherein said Azelaic-XY is represented by the following formula (2), wherein X is Leu and Y is Gly.

(2)

A cosmetic composition for whitening, which comprises the whitening compound of any one of claims 1 and 2 as an active ingredient.
The method of claim 3,
Wherein the whitening compound is contained in an amount of 0.000001 to 30% by weight based on the total composition.
delete The method of claim 3,
The formulation of the whitening cosmetic composition may be at least one selected from the group consisting of external ointment for skin, softening longevity, convergent lotion, nutritional lotion, nutritional cream, massage cream, lotion, essence, eye cream, foundation, cleansing cream, cleansing foam, cleansing water, , A spray, and a powder.

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