COMPOSITION FOR IMPROVING SKIN COMPRISING AMINO ACID COMPLEX AS ACTIVE INGREDIENT

Abstract

Provided is a composition for skin improvement, including an amino acid complex as an active ingredient. The composition according to an aspect including the amino acid complex as an active ingredient may proliferate cells, promote expression and synthesis of collagen, and have an effect on skin wrinkles and skin elasticity, and thus may be effectively used for overall skin improvements.

Claims

1. A cosmetic composition for skin improvement, comprising, as an active ingredient, an amino acid complex comprising glycine, L-alanine, L-proline, L-valine, L-leucine, L-lysine, serine, glutamic acid, aspartic acid, arginine, and histidine.

2. The cosmetic composition of claim 1, further comprising, as active ingredients, calcium pantothenate and a mineral component.

3. The cosmetic composition of claim 2, wherein the mineral component comprises, in a form of a single substance or a salt, at least three selected from the group consisting of calcium (Ca), magnesium (Mg), copper (Cu), and zinc (Zn).

4. The cosmetic composition of claim 3, wherein the mineral in a form of a salt comprises aspartate or gluconate of the mineral component.

5. The cosmetic composition of claim 1, wherein the cosmetic composition comprises, based on a total weight of the amino acid complex, glycine in an amount of about 20.0 wt% to about 35.0 wt%, L-alanine in an amount of about 15.0 wt% to about 30.0 wt%, L-proline in an amount of about 15.0 wt% to about 20.0 wt%, L-valine in an amount of about 10.0 wt% to about 25.0 wt%, L-leucine in an amount of about 1.0 wt% to about 5.0 wt%, L-lysine in an amount of about 1.0 wt% to about 5.0 wt%, serine in an amount of about 5.0 wt% to about 10.0 wt%, glutamic acid in an amount of about 5.0 wt% to about 10.0 wt%, aspartic acid in an amount of about 1.0 wt% to about 5.0 wt%, arginine in an amount of about 1.0 wt% to about 5.0 wt%, and histidine in an amount of about 1.0 wt% to about 5.0 wt%.

6. The cosmetic composition of claim 1, wherein the cosmetic composition comprises the amino acid complex in an amount of about 10.0 wt% to about 40.0 wt% based on a total weight of the cosmetic composition.

7. The cosmetic composition of claim 2, wherein the cosmetic composition comprises calcium pantothenate in an amount of about 0.1 wt% to about 5.0 wt% based on a total weight of the cosmetic composition.

8. The cosmetic composition of claim 2, wherein the cosmetic composition comprises the mineral component in an amount of about 0.1 wt% to about 5.0 wt% based on a total weight of the cosmetic composition.

9. The cosmetic composition of claim 1, wherein the skin improvement is at least one selected from the group consisting of improvement of skin barrier, prevention or improvement of skin damage, skin moisturizing, amelioration of skin wrinkles, inhibition of skin aging, improvement of skin elasticity, skin soothing, and skin regeneration.

10. A food composition for skin improvement, comprising, as an active ingredient, an amino acid complex comprising glycine, L-alanine, L-proline, L-valine, L-leucine, L-lysine, serine, glutamic acid, aspartic acid, arginine, and histidine.

11. The food composition of claim 10, further comprising, as active ingredients, calcium pantothenate and a mineral component.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0039] FIG. 1 is a graph confirming cytotoxicity of a composition according to an embodiment;

[0040] FIG. 2 is a graph showing an effect of a composition according to an embodiment on mRNA expression of collagen type I;

[0041] FIG. 3 is a graph showing an effect of a composition according to an embodiment on mRNA expression of collagen type III; and

[0042] FIG. 4 is a graph showing an effect of a composition according to an embodiment on inhibition of MMP-1 in cells damaged by ultraviolet rays.

DETAILED DESCRIPTION

[0043] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0044] Hereinafter, the present disclosure will be described in more detail through Examples below. However, these Examples are for illustrative purposes of one or more embodiments, and the scope of the present disclosure is not limited thereto.

Examples and Comparative Examples: Preparation of Amino Acid Complex

[0045] For the amino acid complex included in the composition of the present disclosure, amino acids shown in Table 1 were included to prepare each amino acid complex. Here, the amount was represented by wt%.

[0046] Amino acid complexes D to F were each prepared by excluding some natural amino acids.

TABLE-US-00001 Amino acid complex A B C D E F Glycine 24.2 26.1 26.3 31.6 28.1 25.9 L-Alanine 18.2 19.5 19.7 - 26.0 19.5 L-Proline 18.2 19.5 19.5 19.8 - 19.5 L-Valine 13.4 15.1 16.1 21.0 18.5 15.1 L-Leucine 3.4 4.0 4.0 5.0 4.8 - L-Lysine 2.6 3.7 3.7 2.6 2.6 - Serine 6.2 - 6.2 6.2 6.2 6.2 Glutamic acid 5.7 5.7 - 5.7 5.7 5.7 Aspartic acid 3.6 3.6 - 3.6 3.6 3.6 Arginine 2.8 2.8 2.8 2.8 2.8 2.8 Histidine 1.7 - 1.7 1.7 1.7 1.7 Sum 100.0 100.0 100.0 100.0 100.0 100.0

[0047] Compositions each including the amino acid complex prepared above, calcium pantothenate, and a mineral component were prepared as shown in Table 2.

TABLE-US-00002 Calcium pantothenate (wt%) Mineral component.sup.a(wt%) Amino acid complex (wt%) Example 1 0.7 0.6 A : 25.0 Example 2 0.7 0.6 B : 25.0 Example 3 0.7 0.6 C : 25.0 Comparative Example 1 0.7 0.6 D : 25.0 Comparative Example 2 0.7 0.6 E : 25.0 Comparative Example 3 0.7 0.6 F : 25.0 Comparative Example 4 0.7 0.6 A : 9.0 Comparative Example 5 0.7 0.6 - Comparative Example 6 0.7 - A : 25.0

[0048] Mineral component.sup.a: Complex of Zn-gluconate, Mg-gluconate, and Ca-gluconate at a weight ratio of 1:1:1

Experimental Example 1: Confirmation of Skin Cell Proliferation Effect

[0049] The effect of the composition including the amino acid complex, the calcium pantothenic acid salt, and the mineral component on proliferation of skin cells was confirmed.

[0050] In detail, human normal fibroblasts (available from Korean Cell Line Bank) were inoculated into a 96-well microplate to have a concentration of 1×10.sup.3 cells in each well, and cultured in DMEM (manufactured by Sigma company) 37° C. for 24 hours. Subsequently, the cells of experimental groups in which the prepared composition was included and a medium was replaced with fresh serum-free DMEM were further cultured for 36 hours, whereas the cells of a control group were further cultured for 36 hours without medium replacement. Next, 10 .Math.l of MTT solution (5 mg/ml, 3-(4,5-dimethylthiazole-2-yl)2,5-diphenyltetrazolium bromide) was added to each well, and the cells were cultured for 6 hours. After removing the medium, 100 .Math.l of DMSO solution was added to each well, and the cells were cultured by shaking for 20 minutes. Then, by using a microplate reader (Molecular Devices, USA), the absorbance of the supernatant was measured at 570 nm. The cell proliferation effect was calculated by substituting the measured value into Equation 1, and the results are shown in Table 3:

[00001]$\text{Cell proliferation effect}\left(\%\right)=$

[00002]$\frac{Absorbanceofexperimentalgroup-Absorbanceofcontrolgroup}{Absorbanceofcontrolgroup}\text{x 100}$

TABLE-US-00003 Entry Cell proliferation effect (%) Example 1 88.3 Example 2 81.2 Example 3 80.4 Comparative Example 67.9 Comparative Example 2 68.4 Comparative Example 3 69.2 Comparative Example 4 53.1 Comparative Example 5 33.4 Comparative Example 6 51.2

[0051] As shown in Table 3, it was found that the composition including the amino acid complex exhibited a significantly high effect on the proliferation of fibroblasts. In addition, it was confirmed that, as in cases of Examples 1 to 3, the compositions that essentially included glycine, L-alanine, L-proline, L-valine, L-leucine, and L-lysine exhibited better effects, whereas Comparative Examples 1 to 3 in which some of these amino acids were not included exhibited somewhat less effects. Meanwhile, as in case of Comparative Example 4, the effect was reduced when the amount of the amino acid complex was low. In addition, in case of Comparative Example 5 in which the amino acid complex was not included, the effect on the cell proliferation appeared to be low, and in case of Comparative Example 6 in which the amino acid complex was not used together with the mineral component, the effect on the cell proliferation was also low.

Experimental Example 2: Confirmation of Enhancement Effect on Collagen Synthesis

[0052] The enhancement effect of the composition including the amino acid complex, the calcium pantothenic acid salt, and the mineral component on collagen synthesis was confirmed.

[0053] In detail, human normal fibroblasts (available from Korean Cell Line Bank) were inoculated into a 96-well microplate to have a concentration of 2×10.sup.3 cells in each well, and cultured in DMEM at 37° C. for 24 hours. Subsequently, the cells of experimental groups in which the prepared composition was included and a medium was replaced with fresh serum-free DMEM were further cultured for 48 hours, whereas the cells of a control group was further cultured for 48 hours without medium replacement. After the culture, the supernatant of each well was collected to measure the amount of procollagen type I C-peptide (PICP) by using a kit (manufactured by Takara, Japan), so as to measure newly synthesized collagen. The measured amount was converted to ng/2×10.sup.4 cells, and the results are shown in Table 4.

TABLE-US-00004 Entry Collagen synthesis amount (ng/2×10.sup.4) Example 1 301 Example 2 250 Example 3 254 Comparative Example 1 201 Comparative Example 2 198 Comparative Example 3 196 Comparative Example 4 131 Comparative Example 5 96 Comparative Example 6 145

[0054] As shown in Table 4, the composition including the amino acid complex showed a very high synthesis amount of collagen. In addition, it was confirmed that, as in cases of Examples 1 to 3, the cosmetic compositions that essentially included glycine, L-alanine, L-proline, L-valine, L-leucine, and L-lysine exhibited better effects on the collagen synthesis, whereas Comparative Examples 1 to 3 in which some of these amino acids were not included exhibited somewhat less effects. Meanwhile, as in case of Comparative Example 4, the effect was reduced when the amount of the amino acid complex was low. In addition, in case of Comparative Example 5 in which the amino acid complex was not included, the effect on the collagen synthesis appeared to be low, and in case of Comparative Example 6 in which the amino acid complex was not used together with the mineral component, the effect on the collagen synthesis was also low.

Experimental Example 3: Confirmation of Cytotoxicity

[0055] The cytotoxicity of the composition of Example 1 including the amino acid complex, the calcium pantothenic acid salt, and the mineral component was confirmed.

[0056] In detail, the human dermal fibroblasts (CCD-1064Sk, ATCC® CRL-2076™) were subjected to toxicity evaluation by using Iscove’s modified Dulbecco’s medium (IMDM, Welgene, Korea). The human dermal fibroblasts were inoculated into a 24-well plate, cultured for 24 hours, and then replaced with fresh serum-free IMDM. The composition of Example 1 was treated at different concentrations and cultured for 24 hours. After the culture was completed, the medium was replaced with a 10-fold diluted medium with a 2.5 mg/ml MTT solution, and the cells were allowed for a reaction for 4 hours. Then, the supernatant was removed, and 1 ml of DMSO was added to dissolve the MTT-formazan crystals produced, so as to measure absorbance at 570 nm by using an ELISA reader.

[0057] As a result, it was confirmed that the composition of Example 1 had no cytotoxicity as shown in Table 5 and FIG. 1.

TABLE-US-00005 Sample Concentration Mean Standard deviation p-value Control group - 100.00 3.76 1.000 Example 1 (%) 0.1 99.32 3.10 0.821 0.5 100.68 4.02 0.841 1 99.44 0.93 0.815 2 101.73 1.18 0.490 3 99.91 1.08 0.970

Experimental Example 4: Confirmation of Gene Expression of Collagen Type I

[0058] The effect of the composition of Example 1 including the amino acid complex, the calcium pantothenic acid salt, and the mineral component on gene expression of collagen type I was confirmed.

[0059] In detail, vitamin C was used as a positive control group, and the composition of Example 1 was treated at different concentrations, so as to confirm the gene expression level of collagen type I. Human dermal fibroblasts (CCD-1064Sk, ATCC® CRL-2076™) were inoculated into a 60 mm dish using IMDM (Welgene, Korea), cultured for 24 hours, and then replaced with fresh serum-free IMDM. The composition of Example 1 was treated at different concentrations and cultured for 24 hours at 37° C. in a 5 % CO.sub.2 incubator. After removal of the medium, the cells were collected by using QIAzol™ Lysis Reagent (Qiagen), and RNA thereof was isolated according to the protocol of the manufacturer. After quantifying the isolated RNA, cDNA was synthesized with 1 .Math.g of RNA, followed by Real-time PCR (Applied Biosystems, 7500 FAST Real-Time PCR System).

[0060] As a result, as shown in Table 6 and FIG. 2, it was confirmed that the composition of Example 1 increased the gene expression of collagen type I to a similar gene expression level with the positive control group.

TABLE-US-00006 Sample Concentration Mean Standard deviation p-value Control group - 100.0 0.00 1.000 Vitamin C (.Math.g/mℓ) 100 134.2 1.97 0.000 Example 1 (%) 1 96.7 7.41 0.484 2 123.1 4.32 0.001 3 127.5 0.41 0.000

Experimental Example 5: Confirmation of Gene Expression of Collagen type III

[0061] The effect of the composition of Example 1 including the amino acid complex, the calcium pantothenic acid salt, and the mineral component on gene expression of collagen type III was confirmed.

[0062] In detail, vitamin C was used as a positive control group, and the composition of Example 1 was treated at different concentrations, so as to confirm the gene expression level of collagen type III. Human dermal fibroblasts (CCD-1064Sk, ATCC® CRL-2076™) were inoculated into a 60 mm dish using IMDM (Welgene, Korea), cultured for 24 hours, and then replaced with fresh serum-free IMDM. The composition of Example 1 was treated at different concentrations and cultured for 24 hours at 37° C. in a 5 % CO.sub.2 incubator. After removal of the medium, the cells were collected by using QIAzol™ Lysis Reagent (Qiagen), and RNA thereof was isolated according to the protocol of the manufacturer. After quantifying the isolated RNA, cDNA was synthesized with 1 .Math.g of RNA, followed by Real-time PCR (Applied Biosystems, 7500 FAST Real-Time PCR System).

[0063] As a result, as shown in Table 7 and FIG. 3, it was confirmed that the composition of Example 1 increased the gene expression of collagen type III to a similar gene expression level with the positive control group.

TABLE-US-00007 Sample Concentration Mean Standard deviation p-value Control group - 100.0 0.00 1.000 Vitamin C (.Math.g/mℓ) 100 135.6 2.68 0.000 1(%) 1 91.1 4.17 0.021 2 125.4 8.17 0.006 3 130.5 8.62 0.004

Experimental Example 6: Confirmation of Inhibition of MMP-1 Gene expression

[0064] The inhibitory effect of the composition of Example 1 including the amino acid complex, the calcium pantothenic acid salt, and the mineral component on gene expression of MMP-1 was confirmed.

[0065] In detail, as a positive control group, cells damaged by ultraviolet A (UVA) irradiation were treated with EGCG, and the composition of Example 1 was treated at different concentrations, so as to confirm the gene expression level of MMP-1. Human dermal fibroblasts (CCD-1064Sk, ATCC® CRL-2076™) were inoculated into a 60-mm dish using IMDM (Welgene, Korea) and cultured for 24 hours, followed by UVA irradiation. Afterwards, the medium was replaced with fresh serum-free IMDM, and samples were each treated at different concentrations and cultured for 24 hours at 37° C. in a 5 % CO.sub.2 incubator. After removal of the medium, the cells were collected by using QIAzol™ Lysis Reagent (Qiagen), and RNA thereof was isolated according to the protocol of the manufacturer. After quantifying the isolated RNA, cDNA was synthesized with 1 .Math.g of RNA, followed by Real-time PCR (Applied Biosystems, 7500 FAST Real-Time PCR System).

[0066] As a result, as shown in Table 8 and FIG. 4, it was confirmed that the composition of Example 1 reduced the gene expression of MMP-1 in a concentration-dependent manner.

TABLE-US-00008 Sample Concentration Mean Standard deviation p-value Control group - 50.5 6.13 0.000 UVA - 100.0 0.00 1.000 UVA+EGCG (.Math.g/mℓ) 1 56.7 1.27 0.000 1(%) 1 86.9 7.10 0.033 2 73.1 3.30 0.000 3 70.4 1.94 0.000

Preparation Examples: Preparation of Cosmetic Composition

[0067] In order to conduct a clinical test for effects of a cosmetics including all of the amino acid complex, the calcium pantothenate, and the mineral component on the amelioration of skin wrinkles and improvement of elasticity, cosmetic compositions were prepared with the components and amounts as shown in Table 9.

[0068] A cosmetic composition not including the amino acid complex was prepared in Comparative Preparation Example 1. Specific components and amounts are as shown in Table 9.

TABLE-US-00009 Component Amount (wt%) Preparation Example 1 Preparation Example 2 Preparation Example 3 Comparative Preparation Example 1 Vaseline 7.0 7.0 7.0 7.0 Liquid paraffin 5.0 5.0 5.0 5.0 Wax 2.0 2.0 2.0 2.0 Polysorbate-60 2.0 2.0 2.0 2.0 Sorbitan sesquioleate 2.5 2.5 2.5 2.5 Squalane 3.0 3.0 3.0 3.0 Propylene glycol 6.0 6.0 6.0 6.0 Glycerin 4.0 4.0 4.0 4.0 Triethanolamine 0.5 0.5 0.5 0.5 Carboxyvinyl polymer 0.5 0.5 0.5 0.5 Tocopherol acetate 0.1 0.1 0.1 0.1 Purified water To 100 To 100 To 100 To 100 Calcium pantothenate 0.7 0.7 0.7 0.7 Mineral component.sup.a 0.6 0.6 0.6 0.6 Amino acid complex A 25.0 - - - Amino acid complex B - 25.0 - - Amino acid complex F - - 25.0 - Fragrance, preservative Trace amount Trace amount Trace amount Trace amount

[0069] Mineral component.sup.a: Complex of Zn-gluconate, Mg-gluconate, and Ca-gluconate at a weight ratio of 1:1:1

Experimental Example 7: Confirmation of Effect of Skin Wrinkle Amelioration

[0070] In order to examine effects of the cosmetic compositions of Preparation Examples 1 to 3 and Comparative Preparation Example 1 on the amelioration of skin wrinkles, skin wrinkles were measured from 40 healthy women aged between 35 to 50 as follows.

[0071] First, the subject women were divided into four groups, and Group A (10 people), Group B (10 people), Group C (10 people), and Group D (10 people) were treated with the formulations of Preparation Examples 1 to 3 and Comparative Preparation Example 1, respectively, by applying the formulations onto the face twice a day (in the morning and night) for 3 months. After 3 months, the degree of amelioration of wrinkles was evaluated through a questionnaire of the subject women and image analysis on the wrinkles.

[0072] The questionnaire of the subject women was determined as 4 stages of no improvement, slight improvement, moderate improvement, and significant improvement compared to the condition before use, and the results are shown in Table 10.

[0073] For the evaluation of the wrinkles by image analysis, a replica was harvested from an area underneath the eye before the experiments began, and a replica immediately after the end of the experiment was harvested from the same area underneath the eye, so as to measure density of the wrinkles by two-dimensional analysis of the wrinkles through image analysis. The measurement results of the wrinkle density by image analysis were indicated by the reduction rate of the wrinkle density before use, and the results are shown in Table 11.

TABLE-US-00010 Sample No improvement (people) Slight improvement (people) Moderate improvement (people) Significant improvement (people) Preparation Example 1 0 1 2 7 Preparation Example 2 0 2 3 5 Preparation Example 3 2 1 3 4 Comparative Preparation Example 1 4 4 1 1

TABLE-US-00011 Sample Wrinkle density reduction rate Preparation Example 1 55 Preparation Example 2 49 Preparation Example 3 43 Comparative Preparation Example 1 15

[0074] As shown in the results of Tables 10 and 11, it was found that the cosmetic composition including the amino acid complex exhibited very excellent effects on the amelioration of skin wrinkles compared to the composition not including the amino acid complex.

Experimental Example 8: Confirmation of Effect on Skin Elasticity improvement

[0075] In order to examine the enhancement effects of the cosmetic compositions of Preparation Examples 1 to 3 and Comparative Preparation Example 1 on skin elasticity, measurements were carried out as follows.

[0076] First, 40 healthy women over 20 years old (average age 37) were divided into four groups, and Group A (10 people), Group B (10 people), Group C (10 people), and Group D (10 people) were treated with the formulations of Preparation Examples 1 to 3 and Comparative Preparation Example 1, respectively, by applying the formulations around the eyes twice a day (in the morning and night) for 12 weeks under the conditions of a temperature in a range of about 24° C. to about 26° C. and a humidity of 75 %. Then, the skin elasticity was measured by using a skin elasticity measuring device (Cutometer MPA580, Conrage+Khazaka company, Germany). The test results were calculated as R8 value (R8 (at 12 weeks)-R8 (at 0 week) of the cutometer MPA580, and the results are shown in Table 12. Here, the R8 value represents the property of skin viscoelasticity.

TABLE-US-00012 Sample Skin elasticity effect Preparation Example 1 0.79 Preparation Example 2 0.69 Preparation Example 3 0.51 Comparative Preparation Example 1 0.29

[0077] As shown in the results of Table 12, it was confirmed that the cosmetic composition including the amino acid complex significantly increased the enhancement effect on skin elasticity compared to the composition not including the amino acid complex.

[0078] The composition according to an aspect including the amino acid complex as an active ingredient may proliferate cells, promote expression and synthesis of collagen, and have an effect on skin wrinkles and skin elasticity, and thus may be effectively used for overall skin improvements.

[0079] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope as defined by the following claims.