KR20210090790A - Melanin production inhibitory skin whitening cosmetic composition - Google Patents

Abstract

The present invention relates to a composition for skin whitening that inhibits melanin production, and more specifically, to a cosmetic composition containing a gelatin peptide derived from fish for skin whitening such as alleviation of spots and freckles by inhibiting melanin production in cells. As such, the peptide compound has the amino acid sequence of glycine-proline-hydroxyproline in the molecule, and is characterized by consisting of 3 to 20 amino acids after enzymatic-hydrolysis of api gelatin. The present invention relates to a cosmetic composition with excellent skin whitening effect with a peptide compound separated and purified from hydrolyzate within the molecular weight range of 4.5 kDa or less, and can be prepared as conventional formulations such as ointment, cream, lotion, lotion, pack, powder, granule, tablet, capsule, or patch.

Description

Melanin production inhibitory skin whitening cosmetic composition {Melanin production inhibitory skin whitening cosmetic composition}

cosmetics

The present invention relates to a cosmetic composition for skin whitening containing a gelatin peptide having an excellent effect of inhibiting melanin production, and more particularly, an ointment, cream, and lotion containing a gelatin peptide excellent in inhibiting skin melanocyte cell activity and melanin biosynthesis as an active ingredient. , characterized in that it manufactures a composition such as a lotion.

The peptide compound according to the present invention has an amino acid sequence of Glycine-Proline-Hydroxyproline in the molecule, and is characterized in that it consists of 9 to 16 amino acids. It is a peptide compound separated and purified from hydrolysates with a molecular weight of 4.5 kDa or less after decomposition. Human skin color is determined by the concentration and distribution of melanin inside the skin. In addition to genetic factors, solar ultraviolet rays, fatigue, stress, etc. also influenced by the environmental or physiological conditions of

Melanin is produced through a non-enzymatic oxidation reaction after the enzyme tyrosinase acts on tyrosine, a kind of amino acid, to convert it into DOPA and dopaquinone.

However, although the pathway by which melanin is made is known, the mechanism that induces melanin synthesis, which is the previous step of tyrosinase action, is still not fully elucidated.

When this synthesis of melanin occurs excessively in the skin, it darkens the skin tone, and also causes spots, freckles, and the like.

Therefore, by inhibiting the synthesis of melanin in the skin, it is possible not only to realize skin whitening by brightening the skin tone, but also to improve skin hyperpigmentation such as spots and freckles caused by ultraviolet rays, hormones and genetic causes. can

Therefore, conventionally, a substance having inhibitory activity against tyrosinase, such as hydroquinone, ascorbic acid, kojic acid, and glutathione, is mixed with cosmetics such as ointments and essences to the skin. Whitening was realized or skin hyperpigmentation such as spots and freckles was improved.

However, hydroquinone exhibits a certain whitening effect, but has a problem that the amount of blending must be limited to a very small amount due to severe skin irritation, and ascorbic acid is easily oxidized, so cosmetics containing it have problems such as discoloration and discoloration. There are disadvantages.

In addition, thiol-based compounds such as glutathione and cysteine not only have a characteristic unpleasant odor, but also have a problem in transdermal absorption, and their glycosides and derivatives are also highly polar, making it difficult to use as a cosmetic ingredient. On the other hand, the placenta extract and the like do not irritate the skin, but the whitening effect is insufficient.

In the present invention, hydrolyzate fractions and peptides from fisheye gelatin with high antioxidant properties were obtained by hydrolyzing fisheye gelatin by a special method (Korean Patent Publication Nos. 96-31582, 98-34825), and the gelatin hydrolyzate fraction was continued through research. Among them, peptides having a specific amino acid sequence were found to be particularly excellent in inhibiting melanogenesis, leading to the completion of the present invention.

Therefore, the object of the present invention was devised to improve the various problems of the prior art, and the effect of inhibiting the production of melanin derived from fish for skin whitening such as improvement of spots and freckles by suppressing the production of excessive melanin in skin cells. The purpose of providing skin whitening by preventing excessive melanin accumulation in the skin by preparing compositions such as ointments, creams, lotions, lotions, sprays, and ampoules containing an excellent and safe gelatin peptide as an active ingredient There is this.

Animal testing and human skin testing.

The gelatin peptide compound is excellent in inhibiting the melanin biosynthesis of human melanocytes, so it is used for skin whitening such as skin, lotion, cream, foundation, essence, gel, pack, foam cleansing, cosmetics such as soap, and pharmaceuticals such as ointment for external use. When added to the composition, a strong skin whitening effect can be exhibited without any side effects.

The present invention is a cosmetic composition containing a fish-derived gelatin peptide for skin whitening such as improvement of spots and freckles by inhibiting melanin production in cells. The peptide compound according to the present invention has the amino acid sequence of Glycine-Proline-Hydroxyproline in the molecule, and is characterized in that it consists of 9 to 16 amino acids, which enzymatically hydrolyzes API gelatin. Then, it relates to a cosmetic composition with excellent skin whitening effect with a peptide compound separated and purified from hydrolyzate within the molecular weight range of 1.5 - 4.5 kDa and ∼1 kDa, including ointments, creams, lotions, lotions, packs, powders, granules, tablets, It can be prepared in conventional dosage forms such as capsules or patches.

The configuration of the present invention will be described through reference examples, experimental examples, examples and comparative examples, and effect test examples as follows.

[Reference Example] Extraction method of gelatin peptide compound

The antioxidant peptide according to the present invention is obtained from api gelatin. That is, after extracting gelatin from apis, it can be hydrolyzed to obtain a mixture of peptides of various molecular weight ranges and separated therefrom. In a specific example of the present invention, gelatin is obtained from apis 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 gelatin hydrolysis method described in Korean Patent Laid-Open No. 96-31582 are also described in detail herein. In particular, in the present invention, gelatin is extracted from the skins of tuna, pollack, mint or squid by hot water extraction, and the gelatin is hydrolyzed with enzymes using a three-stage membrane reactor apparatus, and then peptides with excellent melanogenesis inhibitory activity are isolated therefrom. Purified. Separation and purification of a peptide having excellent antioxidant activity from a hydrolyzate of gelatin may be carried out according to a conventional method for separating and purifying peptides.

The present invention was carried out using a series of chromatography 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. It was possible to isolate a specific peptide having particularly excellent inhibitory activity and to identify its amino acid sequence. 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. The characteristic sequence of glycine-proline-hydroxyproline included in the molecule may be repeated once or twice or more in the molecule. Among them, it has glycine at the C-terminus and consists of 3 to 20 amino acids. In the present invention, fish skin can be used as a raw material for gelatin, and fish skin is particularly advantageously used.

Hereinafter, various materials and test methods 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. At a concentration where the content of the gelatin peptide compound is less than 0.0001% by weight, efficacy and effect on the inhibition of melanogenesis cannot be expected, and when it exceeds 10% by weight, it is inappropriate because of deterioration of product safety. The pharmaceutical containing the starfish extract of the present invention can be prepared in any form for the efficacy against the melanogenesis inhibitory effect, and these forms include ointments, creams, lotions, lotions, packs, powders, granules, tablets, A capsule preparation or a patch preparation is mentioned.

The present invention will be described in more detail through the following examples and comparative examples. However, the examples are intended to illustrate the present invention and are not limited thereto, 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 (Rutin), taxifolin (Taxifolin), campferol (Kaempferol) ), Myricetin, Curcumin, Resveratrol, Arecoline, Apigenin, Wogonin, Luteolin and Tectorigenin It may further contain one or more active oxygen production inhibitors selected from the group consisting of. In the present invention, products from Sigma, which have already been purified, were purchased and one or more of them were selected and used.

If the content of the active oxygen production inhibitor is too small, 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 excellent in inhibiting melanin biosynthesis of skin melanocytes as an active ingredient.

The gelatin peptide compound according to the present invention can be prepared in any form to promote skin whitening by inhibiting skin melanocyte melanin biosynthesis.

This type, for example, can be applied to all skin cosmetic compositions targeting the back skin. These cosmetic compositions for skin can be prepared according to a conventional method in the art.

As a result of preparing the composition of the present invention and evaluating the effect of promoting skin whitening in humans, it was found that there was a statistically significant effect.

Hereinafter, the present invention will be described in more detail through examples. However, these Examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto. In this example, the gelatin peptide compound was tested to prepare a cosmetic composition for skin whitening, but this is also an example of the present invention, and 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 compound on melanocyte cell growth

After skin tissue is harvested during skin transplantation, the skin tissue is cut into fine sections and then phosphate buffered solution containing antibiotics (50mg/ml gentamicin, 50mg/mlamphotericin, 50U/ml penicillin, 50㎍/streptomycin). After washing with and treating with 0.25% trypsin / EDTA for 5 minutes, melanocyte selective medium, M2 medium purchased from Promo Cell, 5% carbon dioxide and 37 ° C in an incubator for 15 days. , Melanocyte cells were again removed with 0.25% Trypsin/EDTA, counted with a hemocytometer, and 1 × 10 cells per well of a 24-well plate containing M2 medium. rushed to get in. After culturing in a 37°C incubator with 5% carbon dioxide and 100% humidity for 1 day so that the cells can adhere, saline was added to the control group and 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005 and 0.001 gelatin peptide compounds were added to the experimental group, respectively. It was added at a test concentration of % by weight and cultured for 7 days. After each time elapsed, 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 was added to each It was added to the well and incubated for 4 hours. After incubation, the medium was removed and 200 μl of dimethyl sulfoxide (DMSO, dimethyl sulfoxide) was added to dissolve formazan crystals formed, and then transferred to a 96-well plate to check 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 set to 100%, and the cell activity effect was expressed as a percentage compared to the experimental group.

(See Fig. 1. Cell activity increase according to the content of the gelatin peptide compound. Refer to the comparison table)

As shown in FIG. 1, the gelatin peptide compound did not have any detrimental effect on the cytotoxicity of melanocytes.

[Experimental Example 2] Melanin biosynthesis inhibitory effect of gelatin peptide compound in skin melanocytes

After skin grafting, the skin tissue is cut into fine sections and washed with a phosphate buffer solution (PhosphateBufferedSaline) containing antibiotics (50mg/ml gentamicin, 50mg/mlamphotericin, 50U/ml penicillin, 50㎍/streptomycin). and 0.25% Trypsin/EDTA for 5 minutes and cultured in M2 medium (Promocell), which is a melanocyte selective medium, for 15 days in an incubator under 5% CO2 and 37°C conditions, then melanocyte cells were again re-incubated with 0.25% Trypsin/EDTA Count the cells with a hemocytometer, and inoculate 1×10 per well into 24-wells containing M2 medium (Promocell), and then incubate at 37°C until the cells adhere to the well at least 80%d. After culturing for 1 day, dopa, a precursor of melanin pigment, was added to 0.08 mM, and then gelatin peptide compounds were added at test concentrations of 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005 and 0.001 wt%, respectively, and cultured for 3 days. Saline was added to the control group. After removing the medium, the cells are washed with a phosphate buffer solution (PBS, PhosphateBufferedSaline) and treated with trypsin to recover the cells. After measuring the number of cells using a hematocytometer, the recovered cells are centrifuged at 5,000 to 10,000 for 10 minutes, and then the supernatant is removed to obtain a precipitate. Add 100 μl of 1M sodium hydroxide solution and obtain intracellular melanin in a thermostat at 60°C. The absorbance was measured at 490 with a microplate with this solution, and the melanin content per cell count was expressed as a percentage by comparing the control group not treated with the gelatin peptide compound and the experimental group.

(See Figure 2. Melanin biosynthesis promotion comparison table according to the content of each gelatin peptide compound)

As shown in FIG. 2, it was found that melanin biosynthesis of melanocytes was inhibited as the content of the gelatin peptide compound increased.

[Experimental Example 3] Whitening effect of gelatin peptide compound in animals

After dividing the brownish guinea pig weighing around 400g into 7 test groups and 1 control group of 10 each, the back area was depilated, and two 3x3cm square material application sites were marked on the skin. At this time, one site is a site for application of the gelatin peptide compound of the present invention, and one site is a control site. It was applied once or twice a day, and this was continued for 30 days. For comparison, a control (propylene glycol: ethanol: purified water=5:3:2 mixed solvent) was applied to the control site with a cotton swab, and the experimental group was prepared by dissolving 0.01 to 1% by weight of the starfish extract in the solvent. 0.1ml was applied to the test group application site twice daily with a cotton swab. The minimum amount of erythema was irradiated once a day, a total of 3 times. After 1 month, the criteria for determining pigmentation were 0 to 25% (1 point), 26 to 50% (2 points), 51 to 75% ( 3 points) and 76-100% (1 point), the number of animals was determined in 4 stages, and the results are described in FIG.

As can be seen from the results of FIG. 3 , it was confirmed that the gelatin peptide compound had an inhibitory effect on the melanin production of the brownish guinea pig induced by UV A, and when treated with ascorbic acid, a synergistic effect on the whitening effect than when used alone. appeared to have

[Example 1 to Comparative Example 1] Preparation of ointment

Gelatin peptide compound is 0.01%, diethyl sebacate 8.0%, braze 5.0%, polyoxyethylene oleyl-ether phosphate (EO4) 6.0%, appropriate amount of paraoxybenzoic acid ester, and the remaining amount of vaseline. Prepare according to a conventional ointment manufacturing method. . In the comparative example, the gelatin peptide compound was excluded from this composition.

[Example 2 to Comparative Example 2] Preparation of softening lotion

Gelatin peptide compound 0.1%, glycerin 2.0%, hyaluronic acid 1.0%, polyoxyethylene oleyl ether (EO20) 0.1%, polyoxyethylene hydrogenated castor oil (EO40) 0.1%, paraoxybenzoic acid ester appropriate amount, fragrance appropriate amount, dye appropriate amount , It is prepared according to the usual softening lotion manufacturing method with the remaining amount of purified water. In the comparative example, the gelatin peptide compound was excluded from this composition.

[Example 3 to Comparative Example 3] Preparation of nutrient emulsion

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%, sorbitan sesquioleate 0.3%, carboxyvinyl polymer 0.3%, triethanolamine 0.3%, paraoxybenzoic acid ester, fragrance, dye, and remaining amount of purified water. In the comparative example, the gelatin peptide compound was excluded 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%, concentrated glycerin 1.0%, squalane 9.0%, 1,3-butylene glycol 6.0%, polysorbate 60 1.5%, polyethylene glycol 1000 4.0%, hydrogenated lanolin 4.0%, octyl dodecanol 10.0%, sorbitan stearate 0.8%, triethanolamine 0.5%, paraoxybenzoic acid ester, fragrance, pigment, and purified water. In the comparative example, the gelatin peptide compound was excluded 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%, cetyldimethicone copolyol 3.0%, gelatin peptide compound 0.1%, 0.01%, ascorbic acid, glycerin 5.0%, sulfuric acid Magnesium 3.0%, paraoxybenzoic acid ester, fragrance, pigment, and purified water are prepared according to a conventional essence preparation method. In the comparative example, the gelatin peptide compound was excluded from this composition.

[Experimental Example 4] Whitening effect of Comparative Examples and Examples and 1 to 5 in humans

For healthy women and men in their 40s to 50s who are worried about melanin in their skin, a total of 100 people were randomly selected for each composition of the present invention in each of the Examples, and the face was vertically divided into two, and Examples 1 to 5 on the left cheek of the composition of the present invention, the control group of Comparative Examples 1 to 5 was applied to the right cheek twice a day, morning and evening, for 2 months. To determine the whitening effect, after using the composition of the present invention, the degree of the whitening effect was evaluated through the following evaluation score as a subjective evaluation of each subject. The results are shown in FIG. 7 below.

Evaluation result (refer to the evaluation table of the effect of suppressing leukogenesis and promoting black hair in humans in FIG. 4)

3: Whitening effect is more than 75% superior to that of the control group

2: Whitening effect is more than 50~75% superior to that of the control group

1: Whitening effect is more than 25~50% superior to that of the control group

0: The whitening effect is 25% or less compared to the control.

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

As described above, although the present invention has been described only with respect to the above embodiments, it is not necessarily limited thereto, and various modifications are possible within the scope and spirit of the present invention.

Description of the evaluation table for the suppression of leukogenesis and the promotion of black hair in humans.
3: Whitening effect is more than 75% superior to that of the control group
2: Whitening effect is more than 50~75% superior to that of the control group
1: Whitening effect is more than 25~50% superior to that of the control group
0: The whitening effect is 25% or less compared to the control.

Claims (5)

In the melanin production inhibition cosmetic composition,
A composition for skin whitening, characterized in that it contains a gelatin peptide compound having the amino acid sequence of Glycine-Proline-Hydroxyproline and consisting of 3 to 20 amino acids. The composition for skin whitening according to claim 1, wherein the gelatin peptide compound has 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 is contained in an amount of 0.001 to 10% by weight based on the total weight of the composition. According to claim 1, wherein the skin whitening composition is Quercetin (Quercetin), Rutin (Rutin), Taxifolin (Taxifolin), Campferol (Kaempferol), Myricetin (Myricetin), Curcumin (Curcumin), Resveratrol (Resveratrol) , Arecoline, apigenin, wogonin, luteolin, and one or more active oxygen production inhibitors selected from the group consisting of tectorigenin in the total weight of the composition The composition for skin whitening, characterized in that it further contains 0.001 to 5% by weight with respect to the. According to claim 1, wherein the composition for skin whitening is skin, lotion, cream, foundation, essence, gel, pack, foam cleansing, cosmetics such as soap, pharmaceutical ointments such as external skin ointments, creams, lotions, and cosmetic compositions A composition for skin whitening, comprising:

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