KR100760875B1 - Melanin production inhibitory skin whitening cosmetic composition - Google Patents

Abstract

The present invention relates to a composition for inhibiting melanin production skin whitening, which will be described in more detail as a cosmetic composition containing gelatin peptides derived from fish for skin whitening such as inhibiting melanin production in the cell to improve blemishes, freckles, etc. The peptide compound has an amino acid sequence of Glycine-Proline-Hydroxyproline in the molecule, and is composed of 3 to 20 amino acids, which is obtained by enzymatic hydrolysis of fish gelatin, followed by molecular weight. The present invention relates to a cosmetic composition having excellent skin whitening effect from a peptide compound separated from hydrolyzate within the range of 4.5 kDa or less, and is commonly used as an ointment, cream, lotion, lotion, pack, powder, granule, tablet, capsule or patch. It may be prepared in a phosphorus formulation.

Description

 Composition for whitening skin by inhibiting melanin production {Compositions for whitening skin by inhibition of melanin synthesis}

The present invention relates to a cosmetic composition for skin whitening containing a gelatin peptide excellent in inhibiting melanin production, and more specifically, to a siamese ointment containing a gelatin peptide excellent in inhibiting skin melanocyte cell activity and melanin biosynthesis as an active ingredient, It is characterized by preparing a composition such as cream, lotion, lotion, the peptide compound according to the invention has an amino acid sequence of glycine-proline-hydroxyproline (Glycine- Proline-Hydroxyproline) in the molecule, 9-16 amino acids It is characterized in that, the production method is a peptide compound separated and purified from hydrolyzate having a molecular weight of 4.5 kDa or less after the enzyme-hydrolysis of fish gelatin.

Human skin color is determined by the concentration and distribution of melanin in the skin. In addition to genetic factors, it is also influenced by environmental or physiological conditions such as ultraviolet rays, fatigue and stress. Melanin is a type of amino acid tyrosine (tyrosinase) acts as a tyrosinase enzyme (tyrosinase) acts to convert to dopa (DOPA), dopaquinone (Dopaquinone) is produced through a non-enzymatic oxidation reaction. However, although the pathway by which melanin is made is known, it is still not clear what mechanisms induce melanin synthesis, the previous step in which tyrosinase works. When such synthesis of melanin occurs excessively in the skin, it may darken the skin tone, and may cause spots and freckles. Therefore, by inhibiting the synthesis of melanin pigment in the skin, not only can brighten the skin tone to realize skin whitening, but also improve skin hyperpigmentation such as spots, freckles, etc. caused by ultraviolet rays, hormones and genetic causes. have. Therefore, conventionally, a substance having an inhibitory activity against tyrosinase, such as hydroquinone, ascorbic acid, kojic acid, glutathione, and the like, is formulated into a cosmetic such as an ointment or an essence. Whitening was realized or skin hyperpigmentation such as blemishes and freckles was improved. However, hydroquinone exhibits a predetermined whitening effect, but has a problem in that skin irritation is severe and the amount of the compound is limited to a very small amount, and ascorbic acid is easily oxidized, resulting in problems such as discoloration and discoloration. There is this. In addition, thiol-based compounds such as glutathione and cysteine not only have a characteristic unpleasant odor, but also have problems with transdermal absorption, and glycosides and derivatives thereof have high polarity, making it difficult to use as a cosmetic ingredient. Placenta extract, on the other hand, has no irritation to the skin, but lacks a whitening effect.

The present invention has been obtained by hydrolyzing the fish gelatin in a special method to obtain a high antioxidant antioxidant gelatin hydrolyzate fraction and peptide (Korean Patent Publication No. 96-31582, 98-34825), continuing the study gelatin hydrolyzate fraction Among them, the peptide having a specific amino acid sequence was found to be particularly excellent in inhibiting melanogenesis and came to complete the present invention.

Therefore, an object of the present invention is to devise to improve the various problems as described above, inhibiting the production of excess melanin in skin cells to inhibit melanin production from fish for skin whitening such as blemishes, freckles, etc. To prepare a composition such as ointments, creams, lotions, lotions, sprays, ampoules, etc. containing an excellent safe gelatin peptide as an active ingredient, to prevent the accumulation of excessive melanin on the skin to provide a skin whitening have.

 The present invention is a cosmetic composition containing gelatin peptides derived from fish for skin whitening such as blemishes, freckles, etc. by inhibiting the production of melanin in the cells. The peptide compound according to the present invention has an amino acid sequence of Glycine-Proline-Hydroxyproline in the molecule, and is characterized by consisting of 9 to 16 amino acids, which is enzyme-hydrolyzed from fish gelatin. The present invention relates to a cosmetic composition having excellent skin whitening effect with a peptide compound separated and purified from a hydrolyzate in the range of 1.5 to 4.5 kDa and a molecular weight of 1 to 1 kDa, and includes an ointment, cream, lotion, lotion, pack, powder, granule, tablet, and capsule. Or in conventional formulations such as patching agents.

Referring to the configuration of the present invention, reference examples, experimental examples, examples and comparative examples, effect test examples are as follows.

Reference Example Extraction Method of Gelatin Peptide Compound

The antioxidant peptides according to the invention are obtained from fish gelatin. That is, the gelatine may be extracted from the skin, and then hydrolyzed to obtain a peptide mixture of various molecular weight ranges, and then separated therefrom. In a specific example of the present invention, gelatin is obtained from the skin and hydrolyzed. Although the method described in Korean Patent Publication No. 96-31582 by the applicant was used, the present invention is not limited to this specific method. The gelatin extraction method and the hydrolysis method of gelatin described in Korean Patent Publication No. 96-31582 are also described in detail herein. In particular, in the present invention, gelatin is extracted from the skin of tuna, pollock, minka or squid by hot water extraction, and the gelatine is hydrolyzed with an enzyme using a three-stage membrane reactor, and then a peptide having excellent melanogenesis inhibitory activity is separated therefrom. Purified. Separation and purification of peptides having excellent antioxidant activity from gelatin hydrolyzate can be carried out according to conventional peptide separation and purification methods.

The present invention was carried out using a series of chromatographic techniques, specifically, melanogenesis by measuring the melanogenesis inhibitory activity for each fraction at each step while sequentially performing gel filtration, ion exchange and high performance liquid chromatography. The specific peptide which was especially excellent in inhibitory activity was isolate | separated, and the amino acid sequence was identified. The antioxidant peptide provided according to the present invention has an amino acid sequence of Glycine-Proline-Hydroxyproline in the molecule and consists of 3 to 20 amino acids. Characteristic sequences of Glycine-Proline-Hydroxyproline included in a molecule may be present one or more times in the molecule. Among them, in particular the glycine at the C-terminus, consisting of 3 to 20 amino acids. In the present invention, skin can be used as a raw material of gelatin, and in particular, tuna skin is advantageously used. Hereinafter, various materials, test methods, and the like used in the present invention will be described in detail.

The content of the gelatin peptide compound used in the present invention is 0.0001 to 10% by weight, preferably 0.01 to 5% by weight. When the content of the gelatin peptide compound is less than 0.0001% by weight, the efficacy and effect of inhibiting melanogenesis cannot be expected. If the content of the gelatin peptide compound exceeds 10% by weight, it is unsuitable for lowering the safety of the product. The drug containing the starfish extract of the present invention can be prepared in any form of the drug for the effect on the melanogenesis inhibitory effect, these forms ointment, cream, lotion, lotion, pack, powder, granules, tablets, capsules Or a patch agent.

The present invention will be described in more detail with reference to the following examples and comparative examples. However, the examples are not intended to limit the present invention only to illustrate the invention, and various modifications are possible.

The composition of the present invention for the purpose of improving the melanogenesis inhibitory effect, tocopherol (Vitamin E), ascorbic acid (Vitamin C), quercetin (Quercetin), Rutin, Taxifolin (Taxifolin), Camperol (Kaempferol) ), Myricetin, Curcumin, Resveratrol, Arecoline, Apigenin, Wogonin, Luteolin and Tectorigenin It may further contain one or more active oxygen production inhibitor selected from the group consisting of). In the present invention, the already purified Sigma (Sigma) company purchased and used at least one of them.

If the content of the active oxygen generation inhibitor is too small, the efficacy cannot be expected, and if too much, the safety of the product is lowered, so the appropriate content is 0.001 to 5% by weight, preferably 0.01 to 3% by weight.

The composition of the present invention relates to a composition having a skin whitening promoting effect containing a gelatin peptide compound having an excellent effect of inhibiting melanin biosynthesis of skin melanocytes as an active ingredient.

The gelatin peptide compound according to the present invention may be prepared in any form for promoting skin whitening by inhibiting skin melanocyte melanin biosynthesis. In this form, for example, it is applicable to all skin cosmetic compositions for the skin. These cosmetic compositions for skin can be prepared according to methods conventional in the art.

When the composition of the present invention was prepared and evaluated for skin whitening promoting effect in humans, it was found to have a statistically significant effect.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only illustrative of the present invention, and the scope of the present invention is not limited to these. In this embodiment, the gelatin peptide compound has been tested to prepare a cosmetic composition for skin whitening, but this is also merely an example of the present invention, it will be apparent that the technical scope of the present invention is not limited only to these examples.

Experimental Example 1 Effect of Gelatin Peptide Compounds on Melanosite Cell Growth

When skin grafts are taken, skin tissues are cut into fine sections and phosphate buffered saline containing antibiotics (50mg / ml gentamicin, 50mg / ml amphotericin, 50U / ml penicillin, 50㎍ / streptomycin). After 5 minutes of treatment with 0.25% Trypsin / EDTA for 5 minutes and incubated for 15 days in an incubator at 5% carbon dioxide and 37 ℃ conditions in M2 medium purchased from the promocel company, melanocyte selection medium Melanocite cells were removed again with 0.25% Trypsin / EDTA and counted with a hemocytometer to count 1 × 10 ⁴ cells in each well of a 24-well plate containing M2 medium. Was busy to enter. The cells were incubated in a 37% incubator at 5% carbon dioxide and 100% humidity for one day, and then saline was added to the control group, and gelatin peptide compounds were added to the experimental group, respectively, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005 and It was added at a test concentration of 0.001% by weight and incubated for 7 days. After each time, 300 μl of MTT (3- (4,5-dimethyl- 2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide, Sigma Chemical Co. USA) solution dissolved in physiological saline solution It was added to the wells and incubated for 4 hours. After incubation, the medium was removed and 200 μl of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals, which were then transferred onto a 96-well plate for cell activity. Absorbance was measured at 540 nm with a microplate reader (GENios, TECAN Co., USA). In the group not treated with the gelatin peptide compound, the cell activity of the control group was 100%, and the cell activity effect was expressed in% compared with the experimental group.

Table 1. Comparison of increase in cell activity according to the content of gelatin peptide compound

 As shown in Table 1 above, the gelatin peptide compound was found to have no detrimental effect on the cytotoxicity of melanocytes.

Experimental Example 2 Inhibitory Effect of Gelatin Peptide Compounds on Melanin Biosynthesis in Skin Melanosite

After skin graft, skin tissue was collected and cut into fine sections, followed by phosphate buffered saline containing antibiotics (50mg / ml gentamicin, 50mg / ml amphotericin, 50U / ml penicillin, 50㎍ / streptomycin). After washing and incubating with 0.25% Trypsin / EDTA for 5 minutes and incubating for 15 days in an incubator at 37 ° C. with 5% CO2 in melanocyte selective medium M2 medium (Promocell), the melanocytes were again 0.25% Trypsin / Remove the cells with EDTA, count the cells with a hemocytometer, inoculate 1 × 10 ⁴ cells per well in 24-well containing M2 medium (Promocell), and incubate at 37 ° C until more than 80% d of the pore well is attached. . After 1 day of incubation, dopa, a precursor of melanin pigment, was added to 0.08 mM, and then the gelatin peptide compound was added at test concentrations of 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005 and 0.001% by weight, respectively, and cultured for 3 days. Saline was added to the control group. The cells from which the medium has been removed are washed with phosphate buffered solution (PBS) and treated with trypsin to recover the cells. The recovered cells were measured by using a hematocytometer, and then centrifuged at 5,000 to 10,000 for 10 minutes, and then the supernatant was removed to obtain a precipitate. 100 μl of 1 M sodium hydroxide solution was added to obtain intracellular melanin in a 60 ° C. thermostat. Using this solution, the absorbance was measured at 490 using a microplate, and the melanin content per cell number was expressed in% compared to the control group without treatment with the gelatin peptide compound.

 Table 2. Comparative Table for Promoting Melanin Biosynthesis According to Gelatin Peptide Compound Contents

 As shown in Table 2, the melanin biosynthesis of melanocytes was suppressed as the gelatin peptide compound content increased.

Experimental Example 3 Whitening Effect of Gelatin Peptide Compound in Animals

 10 Brownish guinea pigs weighing about 400g were divided into 7 test groups and 1 control group, each with hair loss at the back, and the skin was marked with two 3 × 3 cm square material spots. At this time, one place is to apply the gelatin peptide compound of the present invention, and one place is the control site. Apply once or twice a day, and continue for 30 days. For comparison, a control group (propylene solvent: ethanol: purified water = 5: 3: 2 mixed solvent) with no cumulative stimulus was applied to the control area with a cotton swab, and the experimental group dissolved starfish extract in a solvent at 0.01 to 1% by weight. 0.1ml was applied to the experimental group application site twice with a cotton swab every day. Ultraviolet rays of the minimum amount of erythema were irradiated three times a day. After 1 month, the criterion for pigmentation was 0-25% (1 point), 26-50% (2 points), 51-75% (pigment area) of the treated groups of each compound compared to the control group (4 points). 3 points), 76 ~ 100% (1 point) was determined by the number of animals in four stages, the results are shown in Table 3.

Table 3. Comparison of Whitening Effect of Gelatin Peptide Compounds in Animals

As can be seen from the results of Table 3, it was confirmed that the gelatine peptide compound had an inhibitory effect on melanin production of brownish guinea pigs induced by UVA, and a synergistic effect on the whitening effect when treated with ascorbic acid alone. Appeared to be.

Example 1 to Comparative Example 1 Preparation of Ointment

Gelatin peptide compound is prepared according to the conventional ointment manufacturing method in 0.01%, diethyl sebacate 8.0%, light lead 5.0%, polyoxyethylene oleyl-ether phosphate (EO 4) 6.0%, a suitable amount of paraoxybenzoic acid ester, residual amount of petrolatum. do. Comparative Example excludes gelatin peptide compounds from this composition.

Example 2 to Comparative Example 2 Preparation of Soft Cosmetics

Gelatin Peptide Compound 0.1%, Glycerin 2.0%, Hyaluronic Acid 1.0%, Polyoxyethylene Oleyl Ether (EO20) 0.1%, Polyoxyethylene Cured Castor Oil (EO40) 0.1%, Paraoxybenzoic Acid Ester Amount, Flavor Amount, Pigment Amount In the remaining amount of purified water, it is prepared according to a conventional flexible cosmetic preparation method. Comparative Example excludes gelatin peptide compounds from this composition.

Example 3 to Comparative Example 3 Preparation of Nutrients

Gelatin Peptide Compound 1%, Cetanol 1.0%, Beeswax 0.5%, Vaseline 2.0%, Squalane 6.0%, Ethanol 3.0%, 1,3-Butylene Glycol 4.0%, Polysorbate 60 1.0%, Solbitan Sesquioleate 0.3%, carboxy vinyl polymer 0.3%, triethanolamine 0.3%, the amount of paraoxybenzoic acid ester, the amount of perfume, the amount of pigment, the amount of purified water is prepared according to the conventional nutrient emulsion production method. Comparative Example excludes gelatin peptide compounds from this composition.

Example 4 to Comparative Example 4 Preparation of Cream

Gelatin Peptide Compound 0.01%, Ascorbic Acid 0.01%, Stearic Acid 2.0%, Nong glycerin 1.0%, Squalane 9.0%, 1,3-butylene glycol 6.0%, Polysorbate 60 1.5%, Polyethylene glycol 1000 4.0%, Curing Lanolin 4.0%, octyl dodecanol 10.0%, sorbitan stearate 0.8%, triethanolamine 0.5%, the amount of paraoxybenzoic acid ester, the amount of perfume, the amount of pigment, the amount of purified water according to the conventional cream preparation method. Comparative Example excludes gelatin peptide compounds from this composition.

Example 5 to Comparative Example 5 Preparation of Essence

Cyclomethicone 15.0%, Caprylic Capric Triglyceride 3.0%, Mineral Oil 3.0%, Beeswax 1.0%, Cetyl Dimethicone Copolyol 3.0%, Gelatin Peptide Compound 0.1%, 0.01%, Ascorbic Acid, Glycerin 5.0%, Sulfuric Acid Magnesium 3.0%, paraoxybenzoic acid ester amount, perfume amount, pigment amount, purified water residual amount is prepared according to a conventional essence production method. Comparative Example excludes gelatin peptide compounds from this composition.

Experimental Example 4 Whitening Effect of Comparative Examples and Examples and 1 to 5 in Humans

Example for healthy women and men in their 40s and 50s who suffer from melanin on the skin, 100 randomly selected total 10 persons per each composition of the present invention, the face is divided into two vertically, Examples 1 to 5 on the left cheek The present invention composition of the right side, the control of Comparative Examples 1 to 5 was applied twice a day, morning and evening, for two months. Determination of the whitening effect was to evaluate the degree of whitening effect through the following evaluation score by subjective evaluation of the individual after using the composition of the present invention. The results are shown in Table 7 below.

Table 7. Evaluation table for the effect of suppressing white development and promoting black hair in human

(Score)

3: The whitening effect is more than 75% better than the control group

2: Whitening effect is 50 ~ 75% superior to the control group

1: Whitening effect is 25 ~ 50% better than the control group

0: whitening effect is 25% or less than the control group.

When the composition of the present invention is used in cosmetic compositions such as ointment, softening water, nutrient emulsion, cream or essence, as shown in the above experimental results, all of the cosmetic compositions of Examples 1 to 5 containing a gelatin peptide compound Compared with Comparative Examples 1 to 5, the whitening effect was excellent, and the whitening effect was increased with increasing content of the gelatin peptide compound and when used with ascorbic acid.

As described above, although the present invention has been described with reference to the above embodiments, it is not necessarily limited thereto, and various modifications may be made without departing from the scope and spirit of the present invention.

As shown above, the gelatine peptide compound is excellent in inhibiting melanin biosynthesis of human melanocytes, such as skins, lotions, creams, foundations, essences, gels, packs, foam cleansing, cosmetics such as soaps, and cosmetics such as skin ointments. When added to such a composition for skin whitening, it can exhibit a powerful skin whitening effect without any side effects.

Claims (6)

 In the composition for inhibiting melanin production, A composition for skin whitening having an amino acid sequence of glycine-proline-hydroxyproline and containing a gelatin peptide compound consisting of 3 to 20 amino acids. delete The composition of claim 1, wherein the gelatin peptide compound has glycine (Glycine) at the carbon chain (C) -terminus and consists of 3 to 20 amino acids. The composition for skin whitening according to claim 1, wherein the gelatin peptide compound contains 0.001 to 10% by weight based on the total weight of the composition. The composition of claim 1, wherein the composition for skin whitening is Quercetin, Rutin, Taxifolin, Kaempferol, Myricetin, Curcumin, Resveratrol At least one active oxygen-producing inhibitor selected from the group consisting of arecoline, apigenin, apigenin, wogonin, luteolin and tetorigenin Skin whitening composition, characterized in that it further comprises 0.001 to 5% by weight. The composition of claim 1, wherein the composition for skin whitening comprises skin, lotion, cream, foundation, essence, gel, pack, foam cleansing, cosmetic such as soap, pharmaceutical ointment such as external skin ointment, cream, lotion, cosmetic composition Skin whitening composition, characterized in that.