KR101392194B1 - Cosmetic composition for improving anti-wrinkle effect comprising the extract of ascidian shell - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin wrinkles comprising an extract of Aspergillus oryzae as an active ingredient. The composition according to the present invention is effective for inhibiting collagenase activity associated with improvement of wrinkles of skin without skin irritation, It is excellent in regeneration activity and can be usefully used for improving the wrinkles of the skin.

Description

TECHNICAL FIELD The present invention relates to a cosmetic composition for improving skin wrinkles containing an extract of Aspergillus odoriferousum,

The present invention relates to a cosmetic composition for improving skin wrinkles containing an extract of the sea squirt, more specifically, a cosmetic composition containing a glycosaminoglycan extracted using a carotenoid extracted from a sea squirt or an additional protein hydrolyzing enzyme as an active ingredient And a cosmetic composition for improving skin wrinkles.

Aging is a functional, structural, and biochemical change that occurs throughout the whole body of cells and body tissues. Human skin has decreased hormone secretion over time and decreased function and activity of immune cells, resulting in intrinsic aging resulting from decreased biosynthesis of biocompatible proteins. In addition, dry skin, pollutants and ultraviolet light The skin is thinned, the wrinkles are increased, the elasticity is reduced, and aging phenomena such as spots, freckles and black blotch occur.

In particular, photooxidative skin damage by ultraviolet light is mediated by reactive oxygen species (ROS), leading to antioxidant depletion, lipid peroxidation, protein oxidation and oxidative damage of DNA. ROS refers to O2-, H2O2, ROO-, RO-, ROOH and HOCl as well as the highly reactive 1O2 and -OH. Of these, 10 and -OH are known to play an important role in skin photodamage. These ROS disrupt skin antioxidant defenses consisting of enzymatic and non-enzymatic antioxidants, oxidative deformation of biomolecules, damage to skin barrier and chain breaks such as collagen, hyaluronic acid, and wrinkles caused by abnormal cross-linking Accelerate skin aging.

Many functional cosmetics have been developed to reduce such aging of the skin, and in the functional cosmetics industry, various materials such as natural materials, microbial origins, and chemical compounds have been developed. Since 2003, there has been a market for products (food, beverage, and dietary supplements) consumed for cosmetics, and the development of dietary cosmetics has been activated, and efforts have been made to develop functional ingredients from marine life. Biological active substances extracted / purified from marine resources are very diverse, and they are invested because they can be a good resource for finding new substances because of various environmental conditions of marine environment, food organisms, and chemical diversity.

In recent years, due to the problems such as mad cow disease, avian flu and foot and mouth disease, the movement of vertebrate animals among countries is prohibited, so the reliability of raw materials obtained from aquatic products is increasing and the price is expected to rise. Thus, given the increasing public interest in marine products and the fact that the reliability of chemical synthetic substances added to these products is falling, it is expected that the natural ingredients obtained through the production of marine biotechnology will be used in the health and well- It seems to be able to bring a big advantage.

Therefore, the inventors of the present invention found that glycosaminoglycan obtained by extracting and purifying from the perennial skin which is always consumed, not obtained from dangerous materials such as vertebrate animals and birds during the development of functional cosmetic materials for anti-aging, and carotenoid pigments obtained by collagenase Inhibits the activity of the enzyme, promotes collagen production, and has cell regeneration activity, thereby improving skin wrinkles. Thus, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a cosmetic composition comprising a skin extract, which has collagenase inhibitory activity, collagen production activity and skin cell regeneration activity, and is excellent in skin wrinkle-reducing effect.

In order to achieve the above object, the present invention provides a cosmetic composition for improving skin wrinkles comprising an extract of Aspergillus oryzae as an active ingredient.

The cosmetic composition according to the present invention contains the glycosaminoglycan-containing extract or the carotenoid pigment-containing extract obtained from the skin of the axilla as an active ingredient, thereby being capable of inhibiting collagenase activity associated with skin wrinkle improvement without skin irritation, Production promotion, and cell regeneration activity, and thus can be usefully used for improving skin wrinkles.

Figure 1 shows the cytotoxicity of carotenoid pigment-containing extracts obtained from the sea squirt according to the present invention.

The cosmetic composition according to the present invention is characterized by containing an extract of A. cinerea exhibiting collagenase activity inhibition activity, collagen production promotion and cell regeneration activity, specifically, glycosaminoglycan or carotenoid pigment as an active ingredient.

First, the extract of A. sinensis according to the present invention can be prepared by the following process:

1) extracting the carotenoid pigment by adding an organic solvent such as acetone, ethyl acetate or ethanol to the ax skin;

2) concentrating the extract obtained by adding water and proteolytic enzyme to the skin of the axel from which the carotenoid is extracted;

3) treating the concentrate obtained in step 2) with acid and alkali to remove proteins and purifying the protein-removed filtrate; And

4) directly spray-drying the purified solution in step 3), or adding alcohol to the purified solution and lyophilizing the precipitate obtained by centrifugation to obtain a glycosaminoglycan-containing extract.

In step 1), the skin of the axilla can be used by being stored in a freeze-thaw (-40 ° C) after first washing, freezing, crushing and second washing. The carotenoid pigment-containing extract was obtained by extracting the carotenoid pigment using acetone as an extraction solvent, concentrating it after adding ether to dissolve it, and concentrating it under reduced pressure at 40 ° C or lower under a vacuum evaporator to remove the solvent. do. At this time, the acetone as the extraction solvent is used in an amount of 2 to 4 times the volume of the axilla skin.

In step 2), a hydrothermal extraction process is carried out at 90 to 125 ° C for 2 to 4 hours by using water as an extraction solvent, specifically, purified water at a content of 4 to 10 times as much as that of a sea squirt for the sea anthraquinone extracted with carotenoid pigment do. An enzyme selected from the group consisting of protamex, alcalase, flavorzyme and mixtures thereof is added to the hydrolyzed axilla and then the mixture is incubated at 50 to 65 ° C for 24 to 48 hours And hydrolyzed to obtain an enzyme hydrolyzate. At this time, the enzyme is used in an amount of 0.1 to 5.0% by weight (w / w) based on the rhizome skin weight. Subsequently, the extract and the enzyme hydrolyzate obtained from the hot water extraction step are concentrated under reduced pressure at 40 DEG C or less to obtain a concentrate having a brix adjusted to 7 to 15.

In step 3), the pH is adjusted to 4 to 5 by adding 2N HCl on the basis of 1,000 mL of the concentrate obtained in step 2), followed by heating at 60 to 90 ° C for about 20 minutes to precipitate the protein , The precipitate is removed by centrifugation, and the pH is adjusted to 6-7 by adding 2N NaOH to the supernatant.

The lyophilization process of step 4) comprises adding 95% alcohol to the pH-adjusted supernatant in an amount of 1 to 4 times as much as the supernatant, allowing the mixture to stand at 4 to room temperature for 12 to 24 hours to form a precipitate, And lyophilized to obtain a glycosaminoglycan-containing extract.

As described above, the carotenoid pigment-containing extract and the glycosaminoglycan-containing extract obtained from the sea squirt are excellent in collagenase activity inhibition activity, collagen production promotion and cell regeneration activity.

Accordingly, the cosmetic composition of the present invention containing the carotenoid pigment-containing extract and the glycosaminoglycan-containing extract obtained from the ax skin can be usefully used for improving skin wrinkles.

In addition, the composition of the present invention can be used variously in food additives, pharmaceutical compositions, and the like, and the use range of the composition is not particularly limited.

The cosmetic composition of the present invention may contain, as an active ingredient, an extract of carotenoid pigment-containing extract and a glycosaminoglycan-containing extract obtained from the mottle skin in an amount of 0.01 to 1.0% by weight based on the total weight of the composition. When the content of the extract is less than 0.01% by weight, it is difficult to exert the expected effect. When the content of the extract is more than 1.0% by weight, it is difficult to expect a synergistic effect by further use.

The cosmetic composition according to the present invention may contain, as an active ingredient, the ingredients commonly used in cosmetic compositions in addition to the above-mentioned extracts, and may contain conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, . ≪ / RTI >

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More preferably, it may be formulated into a formulation of a soft lotion (skin), a nutritional lotion (milk lotion), a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder .

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are intended to be illustrative of the present invention and are not intended to limit the present invention.

< Example  1> from the axilla Carotenoid  Preparation of pigment-containing extracts

After thawing the frozen-stored skull + shell (purchased from Tongyoung) at room temperature, 2 to 4 times the amount of acetone was added to 1 kg of the sample, and the mixture was allowed to stand at room temperature overnight. The dye component was then extracted and filtered three times, The carotenoid pigment was sufficiently eluted. The extract was filtered and concentrated at 40 ° C or lower using a rotary vacuum evaporator, and the residue was transferred to a separatory funnel. Ether was added thereto to dissolve the pigment components, and then the solvent was completely removed using a rotary vacuum evaporator to obtain a carotenoid dye- Containing extract. The total carotenoid content was 514 mg as measured by a UV / VIS spectrophotometer (Shimadzu UV-1700, Shimadzu Co., Kyoto, Japan) at 460 nm.

< Example  2> from the axilla Glycosaminoglycan - Preparation of containing extracts

Purified water was added in an amount of 4 to 10 times to 1 kg of axel bark extracted with carotenoid pigment and subjected to hot extraction at 121 캜 for 2 to 4 hours. The alkaline enzyme was added to 1 kg of the hot-water extracted shell so as to have a concentration of 0.5 wt%, and hydrolyzed at 50 to 65 ° C for 24 to 48 hours according to the characteristics of each enzyme to obtain an enzyme hydrolyzate. The hydrothermal extract and enzyme hydrolyzate were collected and concentrated under reduced pressure at 40 캜 or lower to adjust the Bricks to 7 to 15. 1,000 mL of the concentrate was treated with 2N HCl to adjust the pH to 4 to 5 and then heated at 80 DEG C for about 20 minutes to precipitate the protein and remove the precipitate by centrifugation. The supernatant was treated with 2N NaOH to adjust the pH to 6-7. The pH-adjusted supernatant was immediately spray-dried or added with 1 to 4 times 95% alcohol and allowed to stand at 4 ° C to room temperature for 16 hours to form a precipitate. The precipitate was centrifuged and lyophilized to obtain 35 g of glycosaminoglycan- .

< Experimental Example  1> Carotenoid  Of pigment-containing extracts DPPH (1,1- 피덴 -2- picrylhydrazyl ) Through the reaction Radical  Measurement of scavenging activity

The extract of carotenoid pigment-containing extract obtained from Example 1 was diluted with ethanol to a concentration of 0.1 mg / mL, 1 mg / mL, 10 mg / mL, and 100 mg / mL to prepare a sample solution. To a 96-well plate test tube, 100 μL of the sample solution and 100 μL of ethanol were added, and 50 μL of a 0.5 mM DPPH / ethanol solution was added. After standing at room temperature for 30 minutes at 25 ° C, the absorbance was measured at 517 nm. For the control group, 100 μL of deionized water was used instead of the sample. For the blank test, 150 μL of ethanol was used instead of DPPH and distilled water. In addition, 100 μL of the sample solution and 150 μL of ethanol were mixed to measure the absorbance, and then the absorbance of the sample was corrected. As a positive control, ascorbic acid was diluted to a concentration of 0.01 mg / mL, 0.1 mg / mL, 1 mg / mL, and 10 mg / mL, and the absorbance was measured by the same method. The radical scavenging activity was calculated according to the following formula and is shown in Table 1.

Radical scavenging activity of carotenoid pigment extract (ASPE) Sample Scavenging activity (%) Control group 100.0 ± 1.9 Ascorbic acid 0.01 mg / mL
0.1 mg / mL
1 mg / mL
10 mg / mL 41.6 ± 6.0
56.6 ± 2.4
93.5 ± 0.4
94.6 ± 0.8 ASPE 0.1 mg / mL
ASPE 1 mg / mL
ASPE 10 mg / mL
ASPE 100 mg / mL 33.2 ± 2.1
35.9 ± 2.0
41.3 ± 3.9
47.2 ± 4.6

As shown in Table 1, ascorbic acid, a positive control, exhibited more than 90% of the radical scavenging activity at a concentration of 1 mg / mL, and the concentration (IC 50) at which the radical was inhibited by 50% was 0.023 mg / mL. The radical scavenging activity was slightly increased with increasing concentration of carotenoid pigment - containing extract from the skeleton skin, and showed a radical scavenging activity of 47.2% at 100 mg / mL of high concentration

< Experimental Example  2> Glycosaminoglycan - containing extract DPPH  Through the reaction Radical  Measurement of scavenging activity

The radical scavenging activity of the glycosaminoglycan-containing extract of the sea squirt skin obtained in Example 2 was measured by the same method as Experimental Example 1, and the results are shown in Table 2.

Radical scavenging activity of glycansaminoglycan extract (AGE) Sample Scavenging activity (%) Control group 100.0 ± 1.9 Ascorbic acid 0.01 mg / mL
0.1 mg / ml
1 mg / mL
10 mg / mL 41.6 ± 6.0
56.6 ± 2.4
93.5 ± 0.4
94.6 ± 0.8 AGE 1 mg / mL
5 mg / mL
25 mg / mL
50 mg / mL 13.3 ± 4.2
15.4 ± 4.5
29.5 ± 3.4
92.8 ± 5.5

As can be seen in Table 2, the activity of 93.5% at 1 mg / mL of ascorbic acid, while the activity of radical scavenging increased with increasing concentration of glycosaminoglycan-containing extract, At a high concentration of 92.8% of the radical scavenging activity. Although the concentration of the glycosaminoglycan - containing extract of the skeleton skin is different from that of ascorbic acid, it has a high radical scavenging activity and has an antioxidative effect considering that ascorbic acid is a single component.

< Experimental Example  3> Carotenoid  Pericyanide of the pigment-containing extract ( ferricyanide  Evaluation of reducing power by

The extract of the carotenoid pigment-containing extract obtained from Example 1 was diluted with ethanol to a concentration of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, and 10 mg / mL to prepare a sample solution. 0.3 mL of the sample solution was placed in a test tube and 0.7 mL of 50 mM phosphate buffer solution (pH 6.6) and 0.5 mL of 1% potassium ferricyanide solution were added thereto, followed by reaction at 50 ° C. for 20 minutes. To remove proteins, 0.5 mL of 10% TCA solution was added and centrifuged at 2500 x g for 10 minutes. Take 0.5 mL of the supernatant and add 0.5 mL of deionized water and 0.1% FeCl ₃ 0.1 mL was added and the absorbance was measured at 700 nm after 10 minutes. Absorbance was measured using ascorbic acid as a comparative control at concentrations of 0.01, 0.1, 1.0, and 10 mg / mL. The blank test was performed using the same amount of distilled water instead of the sample. The absorbance results are shown in Table 3.

Reduction of carotenoid pigment extract (ASPE) Sample Absorbance (nm) Ascorbic acid 0.01 mg / mL
0.1 mg / mL
1 mg / mL
10 mg / mL 0.162 ± 0.003
0.430 ± 0.000
0.536 ± 0.002
1.584 ± 0.007 ASPE 0.1 mg / mL
ASPE 0.5 mg / mL
ASPE 1 mg / mL
ASPE 10 mg / mL 0.009 ± 0.001
0.049 ± 0.003
0.203 ± 0.008
1.025 + 0.055

As can be seen in Table 3, the reducing power was greatly increased with increasing concentration of carotenoid extract. Ascorbic acid increased more than 3 times compared to 0.01 mg / mL absorbance at 1 mg / mL and increased nearly 10 fold at 10 mg / mL concentration.

< Experimental Example  4> Glycosaminoglycan - containing extract On ferricyanide  Evaluation of reducing power by

To evaluate the reducing power of the glycosaminoglycan-containing extract of the sea squirt skin obtained in Example 2, the absorbance was measured at the concentrations of 1, 5, 25 and 50 mg / mL in the same manner as in Experimental Example 3, The results are shown in Table 4.

Reducing power of glycansaminoglycan extract (AGE) Sample Absorbance Ascorbic acid 0.01 mg / mL
0.1 mg / ml
1 mg / mL
10 mg / mL 0.162 ± 0.003
0.430 ± 0.000
0.536 ± 0.002
1.584 ± 0.007 AGE 1 mg / mL
5 mg / mL
25 mg / mL
50 mg / mL 0.003 0.005
0.001 0.002
0.017 ± 0.001
0.156 ± 0.002

As can be seen in Table 4, the reducing power of the glycosaminoglycan-containing extract was remarkably increased at 25 mg / mL, and the reducing power was increased about 10 times at 25 mg / mL at 50 mg / mL gave. Ascorbic acid used as a control group showed activity even at a low concentration of 0.01 mg / mL, and abruptly decreased with increasing concentration.

< Experimental Example  5> Carotenoid  Cytotoxicity measurement of pigment-containing extracts

The cytotoxicity of the carotenoid pigment-containing extract of the axilla skin obtained in Example 1 was confirmed by the RAW 264.7 macrophage line. Specifically, cells were removed from a RAW 264.7 macrophage cell line cultured in a cell culture flask using a 10 mL pipette, and the cells were cultured in DMEM medium containing 10% FBS (fetal bovine serum, Gibco) and penicillin / streptomycin Co., USA). RAW 264.7 macrophages were dispensed into 96-well plates at a concentration of 1 x 10 ⁴ cells / well, and CO ₂ adjusted to 35 ° C, 95% humidity, 5% CO ₂ And cultured in an incubator for 24 hours. The medium was changed every 2 to 3 days. The concentrations of the samples were adjusted to 25, 50, 100 and 200 μg / mL in the new medium, and the cells were treated for 24 hours. After the MTS reagent was treated for 1 hour, the microplate reader (Perkin Elmer 1420, VICTOR ^TM X Multilabel Plate Readers, Waltham, Mass., USA) at 490 nm. The survival rate of the cells was expressed as the absorbance of the sample treated group against the untreated control sample, and the results are shown in Fig.

As can be seen in FIG. 1, 60% of the cells died when the concentration of the carotenoid pigment-containing extract was 100 μg / mL, and no significant cytotoxicity was observed at the low concentration of 10 μg / mL. Therefore, subsequent anti-inflammatory efficacy experiments were performed at concentrations below 10 μg / mL.

< Experimental Example  6> Carotenoid  Of pigment-containing extracts NO  Inhibitory force measurement

In order to confirm the anti-inflammatory effect of the carotenoid pigment-containing extract of the axilla skin obtained in Example 1, the NO formation inhibitory effect was measured by the griess method through the RAW 264.7 macrophage line (ATCC Accession Number: CRL-2278) Respectively.

Specifically, the RAW 264.7 macrophage cell line was dispensed into a 96-well plate, and the sample was treated when the cells had grown to about 70 to 80% on the bottom. LPS (lipopolysaccharide) (Sigma Co., St. Louis, Mo., USA) was added to 1% FBS medium to a final concentration of 100 ng / mL to induce inflammation . The positive control group was treated with LPS alone, and the test group was treated with the LPS-containing medium prepared for each concentration and cultured for 24 hours. 100 μL of the culture supernatant was taken into a new 96-well plate, and 100 μL of the grease reagent was added and reacted at room temperature for 10 minutes. The grease reagent was prepared by dissolving 1% sulfanilamide and 0.1% naphthylenediamine in 2.5% phosphoric acid. Then, absorbance at 570 nm was measured with a microplate reader to compare NO formation amounts. The results are shown in Table 5.

Effect of Carotenoid Dye Extract (ASPE) on the NO Production Sample NO formation (%) Control group 38.3 ± 2.1  LPS 100 ng / mL 100.0 + - 3.7 ASPE 0.01 μg / mL + LPS 100 ng / mL
ASPE 0.1 μg / mL + LPS 100 ng / mL
ASPE 1 μg / mL + LPS 100 ng / mL
ASPE 5 μg / mL + LPS 100 ng / mL
ASPE 10 μg / mL + LPS 100 ng / mL 98.5 ± 3.8
98.0 ± 6.0
98.0 ± 4.4
99.0 + - 6.0
95.0 ± 7.2

As shown in Table 5, the amount of NO produced by the LPS-treated cells (positive control group) was about three times higher than that of untreated cells (control group). According to the present invention, the carotenoid pigment-containing extract obtained from the sea squirt showed inhibitory activity of about 5% at a concentration of 10 μg / mL, but no significant amount of change was observed at all concentrations.

< Experimental Example  7> Carotenoid  Of pigment-containing extracts COX -2 activity measurement

COX-2 is a substance that is produced by infection, wound or stress in macrophages and induces a large amount of prostaglandin production to induce inflammation. Therefore, in this experiment, the amount of COX-2 produced was measured to confirm the anti-inflammatory effect.

First, the RAW264.7 macrophage cell line was divided into 96-well plates and cultured at 37 ° C in 5% CO ₂ The cells were cultured in an incubator, and then cultured until the cells were grown to the bottom of 70%. After washing with PBS, the macrophage-activated LPS in DMEM medium supplemented with 1% FBS and the extract of the carotenoid pigment-containing extract obtained in Example 1 were added to a final concentration of 0.1, 1, 5 and 10 / mL And cultured in a 5% CO 2 incubator at 37 ° C for 24 hours.

The COX-2 assay kit (Cat. No. 760151; Cayman Chemical Company, USA) was used to dilute the assay buffer contained in the reagent 10 times, and the heme was diluted Lt; / RTI &gt; buffer. The COX standard solution was prepared by thawing on ice, and the arachidonic acid was prepared by mixing with the same amount of KOH and diluting 10 times with distilled water.

The cultured cells were collected by a screen, centrifuged at 1000 x g to 2000 x g for 10 minutes, and homogenized by adding cold buffer solution (0.1 M tris-HCl, pH 7.8 1 mM EDTA). After centrifugation at 10,000 x g for 15 minutes at 4 ° C, 150 μL of supernatant was added to the EP tube. The mixture was heated in boiling water for 5 minutes, centrifuged at 8,000 × g for 1 minute, and the supernatant was used for the experiment. The amount of COX-2 produced was measured by ELISA kit reagent.

The reagents and samples were added to the 96-well plate as follows, and the plates were carefully shaken for a few seconds and incubated at 25 ° C for 5 minutes. To this, 20 μL of a colorimetric substrate and 20 μL of arachidonic acid were added and mixed carefully. Incubation was carried out at 25 ° C for 5 minutes and absorbance was measured at 590 nm.

COX standard wells: 150 μL of assay buffer + 10 μL of hem + 10 μL of standard solution

Background well: 120 μL of assay buffer + 10 μL of hem + 40 μL of inactivated sample

Sample well: 120 μL of black buffer solution + 10 μL of heme + 40 μL of sample (triple)

Inhibitor wells: 110 μL of black buffer solution + 10 μL of hem + 40 μL of sample (triple) + COX-1 inhibitor

The activity of COX-2 was calculated according to the following formula, and the results are shown in Table 6.

Production of COX-2 according to content of carotenoid pigment extract (ASPE) Sample Production of COX-2 (nmol / min / mL) Control group 6.97 + - 0.30  LPS 100 ng / mL 8.30 ± 2.31 ASPE 0.01 μg / mL + LPS 100 ng / mL
ASPE 0.1 μg / mL + LPS 100 ng / mL
ASPE 1 μg / mL + LPS 100 ng / mL
ASPE 5 μg / mL + LPS 100 ng / mL 8.12 ± 0.39
8.34 + - 0.40
8.11 + - 0.34
8.25 + - 0.10

As shown in Table 6, the amount of COX-2 produced was 6.97 ± 0.30 nmol / min / mL in the control group and 8.30 ± 2.30 nmol / min / mL in the LPS-treated group, , But no significant increase in production was observed in the comparison of the test groups treated with LPS and LPS.

< Experimental Example  8> Glycosaminoglycan - containing extract Collagenase  activation Inferior  Measure

The skin dermis consists mainly of collagen, and collagenase, which is an enzyme that breaks down collagen by ultraviolet rays or aging, forms wrinkles of the skin, and skin elasticity is lowered. The activity of such collagenase can be inhibited and the generation of wrinkles can be delayed. Thus, the inhibitory effect of the collagenase on the glycosaminoglycan-containing extract of the axal skin obtained in Example 2 was tested with a kit reagent prepared by Cayman Co., USA.

First, DG gelatin solution (1 mg / mL) was prepared by adding 1.0 mL of DDW to a vial containing 1 mg of DG gelatin. Then, 18 mL of DDW was added to 2 mL of the reaction buffer solution, and the reaction buffer solution was diluted. Next, the collagenase enzyme reagent was prepared and diluted with the working buffer solution to a final concentration of 0.2 U / mL. The glycosaminoglycan-containing extract to be used in the experiment was dissolved in a reaction buffer to prepare sample solutions such that the concentrations were 1, 10, 25 and 50 μg / mL. The prepared sample solution was prepared for repeated experiment three times in a 96-well plate in 90 μL increments. 10 μL of DG gelatin was added to the sample, 100 μL of the reaction buffer solution was added to the negative control, and 100 μL of the enzyme reagent was added to the positive control. The fluorescence intensity was measured at 495 nm / 515 nm after leaving for 1 to 2 hours at room temperature in the state of blocking light, and the results are shown in Table 7.

Inhibition of collagenase activity of glycansaminoglycan extract (AGE) Sample Collagenase activity inhibition (%) Control group 100.0 + - 5.7 Adenosine 100.0 + - 5.7 Retinyl acetate 0.1 mg / mL
10 mg / mL 24.5 ± 2.9
42.0 ± 1.4 AGE 1.0 mg / mL
10 mg / mL
25 mg / mL
50 mg / mL 44.9 ± 2.6
59.9 ± 1.9
71.1 ± 0.4
75.2 ± 5.9

As can be seen in Table 7, retinyl acetate showed about 42.0% inhibition at a concentration of 10 mg / mL, while the extract of the skeletal glycosaminoglycan-containing extract showed a much lower concentration of 0.5 mg / mL And 44.9%, respectively.

< Experimental Example  9> Glycosaminoglycan - Effect of collagen production on extract

Collagen, the main protein that makes up the skin, is synthesized in fibroblasts in the dermis of skin tissue. First, procollagen is synthesized and active collagen is formed by decomposition of C-peptide bound to procollagen. By measuring the content of C-peptide by hydrolyzing the skin homogenate, the activated collagen content can be measured.

First, fibroblast (CCD 986sk) (ATCC, Seoul, Korea), a human dermal-derived dermis cell, was cultured in an IMDM medium (Carlsbad, CA, USA) containing 10% FBS and cultured every 2 to 3 days The badge was exchanged. Fibroblast CCD 986sk was cultured on a 96-well plate in a medium containing 10% FBS. When the cells grew by about 70%, glycosaminoglycans of the skeleton skin obtained in Example 2 with FBS free medium were treated with 1 , 10, 50 and 100 μg / mL, and cultured for 24 hours. The culture supernatant was taken and the amount of C-peptide produced was measured using a procollagen type 1 C-peptide EIA kit (Takara Bio Inc., Japan), and the results are shown in Table 8.

Collagen production of glycansaminoglycan extract (AGE) of axillae Sample C-peptide production (μg / mL) C-peptide production ratio (%) Control group 24.3 ± 1.8 100.0 + - 7.3 AGE 5 mg / mL
10 mg / mL
50 mg / mL
100 mg / mL 26.7 ± 1.1
27.0 ± 1.3
27.6 ± 1.5
28.5 ± 1.3 109.8 ± 4.5
111.2 ± 5.5
113.9 ± 6.4
117.3 ± 5.3

As shown in Table 8, the amount of C-peptide produced in the control group was 24.3 ± 1.8 μg / mL (100.0 ± 7.3%), and the amount of the glycosaminoglycan-containing extract of the skeleton skin And at the high concentration of 100 mg / mL, it increased by 17% compared to the control group.

< Experimental Example  10> Glycosaminoglycan - Effect of collagen production on extract

The proliferation rate of the fibroblast CCD 986sk, a human skin-derived dermis cell, was measured in order to examine the regenerating ability of the skin cell of the glycocarcinoglycan-containing extract of the sea squirt skin obtained in Example 2.

First, the fibroblast CCD 986sk was cultured in IMDM medium (USA) containing 10% FBS and the medium was changed every 2 to 3 days during the culture. Fibroblast CCD 986sk was diluted to 2000 μL / well on a 96-well plate and cultured for 24 hours in 10% FBS culture medium. FBS free medium was added to the final concentration of 1, 10, 50, 100 ug / And then cultured for 24 hours and 72 hours. Cell proliferation was measured by treatment with MTS reagent, and the results are shown in Table 9.

Cell regeneration activity of glycansaminoglycan extract (AGE) Sample Cell regeneration activity (%) 24 hour culture 72 hours culture Control group 100.0 + - 4.6 100.0 + - 2.9 AGE 1 μg / mL
10 μg / mL
50 μg / mL
100 μg / mL 109.8 ± 6.7
106.5 ± 6.7
112.3 ± 5.5 ^*
115.1 ± 3.7 ^** 122.6 ± 5.6 ^**
119.8 ± 4.2 ^**
127.1 ± 4.7 ^**
128.9 ± 5.0 ^**

As can be seen in Table 9, the proliferation rate of the extract of glycansaminoglycan-containing extracts of the sea squirt increased with increasing concentration, especially at a concentration of 50 μg / mL and a concentration of 100 μg / mL Respectively. The growth rate of CCD 986sk cell line was slow, indicating that the significant increase in the culture for 3 days is highly expected.

As such, the glycosaminoglycans of the axilla inhibited the activity of collagenase and showed a functional effect of promoting the synthesis of collagen. It is also considered to be a substance which not only improves the regeneration ability of dermal fibroblasts but also delays skin aging.

< Experimental Example  11> Glycosaminoglycan Of cosmetics containing Collagenase  activation Low performance  Measure

The collagenase inhibitory activity of cosmetics containing glycosaminoglycan - containing extracts obtained from the sea squirt skin and cosmetics containing no glycosaminoglycan - containing extracts was compared. The sample was diluted to a final concentration of 0.1% with the glycosaminoglycan-containing extract obtained in Example 2 to prepare a cosmetic formulation by a conventional method, and then the same experiment as in Experiment 8 was conducted. Are shown in Table 10 below.

Cosmetics containing an astringent skin glycosaminoglycan extract (AGE)
Inhibition of collagenase activity Sample Collagenase activity inhibition (%) Control group 100.0 + - 5.7 Skin control
AGE-containing skins 93.5 ± 1.1
94.6 ± 0.8 Essence control group
AGE Containing Essence 66.5 ± 2.5
80.1 ± 4.9

As can be seen in Table 10, in the case of the skin, both the sample containing no glycosaminoglycan and the cosmetic containing the same exhibited the activity of over 90% of collagenase activity, and the sample containing glycosaminoglycan was 94.6 %, Indicating a slightly higher activity (93.5%) than the glycosaminoglycan-free skin. In the case of the essence, the cosmetic containing the glycosaminoglycan-containing extract showed an inhibitory activity of 80.1%, showing higher activity than the non-containing essence (66.5%). In fact, it was confirmed that even when the content of glycosaminoglycan in the skin of the sea squirt contained in cosmetics is 1%, it shows activity even at a low content.

< Experimental Example  12> Glycosaminoglycan - Collagen in cosmetics containing extracts containing Generation  Measure

The collagen production ability of the cosmetic product containing the glycosaminoglycan-containing extract obtained from the sea squirt skin and the cosmetic product containing no glycosaminoglycan-containing extract was compared. The sample was prepared by diluting the glycosaminoglycan-containing extract obtained in Example 2 to a final concentration of 0.1% to prepare a cosmetic formulation by a conventional method, and then treating the skin fibroblasts for 72 hours to produce The content of procollagene was measured and the results are shown in Table 11 below.

Procollagen production of cosmetics containing skimmed peel glycosaminoglycan extract (AGE) Sample Production of procollagen (μg / mL) Procollagen production rate (%) Control group 18.7 ± 1.0 100.0 + - 5.4 Skin control
AGE-containing skins 20.0 ± 0.7
20.9 ± 1.1 107.0 + - 3.6
111.7 ± 6.0 Essence control group
AGE Containing Essence 19.5 ± 0.8
20.6 ± 0.5 104.1 ± 4.5
110.2 ± 2.5

As can be seen in Table 11, the amount of procollagen produced was increased in cosmetics containing glycosaminoglycan-containing extracts of all axillae compared to the control. That is, in the case of the skins containing no glycosaminoglycan, the amount of the produced product was 20.0 ± 0.7 μg / mL, but the amount of the product was slightly increased, but there was no significant difference. However, in the case of the skin containing glycosaminoglycan, the amount of the glycosaminoglycan was significantly increased to 20.9 ± 1.1 μg / mL.

< Experimental Example  13> Glycosaminoglycan Of fibroblasts in cosmetics containing Proliferative ability  Measure

The fibroblast proliferative capacity of the cosmetic products containing the glycosaminoglycan - containing extract obtained from the sea squirt skin and the cosmetic products not containing the glycosaminoglycan - containing extracts were compared. As a sample, a cosmetic formulation was prepared using the glycosaminoglycan-containing extract obtained in Example 2, and the skin fibroblasts were treated for 72 hours and then treated with MTS reagent to measure the degree of cell proliferation And the results are shown in Table 12 below.

Fibroblast proliferative activity of cosmetics containing skeletal glycosaminoglycan extract (AGE) Sample Cell proliferation (%) Control group 100.0 + - 5.7 Skin control
AGE-containing skins 101.6 ± 3.4
104.3 ± 3.1 ^* Essence control group
AGE Containing Essence 103.1 ± 1.7 ^*
107.1 ± 3.0 ^**

As can be seen in Table 12, significant cellular proliferative potential was observed in cells containing the glycosaminoglycan-containing extract of the axilla. In the case of the extract, the extracts containing the glycosaminoglycan - containing extracts of the mottle skin were 103.1 ± 1.7% and 107.1 ± 3.0%, respectively, and the extracts containing the glycosaminoglycan - , It was confirmed that the activity increase was about 4.0%. Thus, the glycosaminoglycan-containing extract of the skinshell showed the ability of the fibroblast to proliferate not only in the raw material but also in the cosmetics containing it.

As described above, the glycosaminoglycan-containing extract obtained from the skins showed wrinkle-improving effect in the measurement of the activity of the raw material as well as the prototype containing the raw material. Therefore, the glycosaminoglycan-containing extract of the skinshells showed a high moisture-retaining effect as well as an excellent wrinkle-improving effect by containing a mucopolysaccharide exhibiting a high viscosity, thus showing the possibility of developing cosmetic raw materials using marine resources.

Claims (9)

It contains an extract of Aspergillus oryzae as an active ingredient,
The cosmetic composition for improving skin wrinkles is characterized in that the sea squam skin extract contains a carotenoid pigment component or a glycosaminoglycan component. delete The method according to claim 1,
Wherein the skin extract is contained in an amount of 0.01 to 1.0% by weight based on the total weight of the composition. A method for producing a cosmetic composition for improving skin wrinkles containing an extract of Aspergillus oryzae as an active ingredient,
1) extracting the carotenoid pigment by adding an organic solvent to the skin of the axilla;
2) concentrating the extract obtained by adding water and proteolytic enzyme to the skin of the axel from which the carotenoid is extracted;
3) treating the concentrate obtained in step 2) with acid and alkali to remove proteins and purifying the protein-removed filtrate; And
4) A process comprising directly spray-drying the purified solution in step 3), or adding alcohol to the purified solution and lyophilizing the precipitate obtained by centrifugation to obtain a glycosaminoglycan-containing extract Wherein the skin extract is obtained by a method comprising the steps of: 5. The method of claim 4,
Wherein the organic solvent used in step 1) is acetone, ethyl acetate or ethanol. 5. The method of claim 4,
Characterized in that the protease used in step 2) is selected from the group consisting of protamex, alcalase, flavorzyme and mixtures thereof. &Lt; / RTI &gt; 5. The method of claim 4,
Wherein the protease is used in an amount of 0.1 to 5.0% by weight (w / w) based on the skin of the axal skin in step 2). 5. The method of claim 4,
Wherein the acid used in step 3) is HCl, and the alkali is NaOH. 5. The method of claim 4,
Wherein the alcohol is used in an amount of 1 to 4 times based on the purified solution in step 4).

Images