KR101811369B1

KR101811369B1 - Composition for improving skin disease

Info

Publication number: KR101811369B1
Application number: KR1020150084887A
Authority: KR
Inventors: 임순호
Original assignee: 동신대학교산학협력단
Priority date: 2015-06-16
Filing date: 2015-06-16
Publication date: 2017-12-21
Also published as: KR20160148206A

Abstract

The present invention relates to a composition for improving skin diseases comprising a compound represented by the following formula (1).
[Chemical Formula 1]

The compound of the present invention is characterized in that it restores skin cell damage caused by UV, has antimicrobial activity against skin bacteria, and inhibits tyrosinase activity to improve skin diseases. Further, the compound of the present invention can be used as a raw material for functional cosmetic for skin improvement.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for improving skin conditions,

The present invention relates to a composition for improving skin diseases comprising a compound represented by a specific formula, more specifically, to a composition for restoring damage to skin cells caused by UV, having antimicrobial activity against skin microorganisms, inhibiting tyrosinase activity The present invention relates to a composition for improving skin diseases comprising a compound consisting of a specific peptide sequence having a characteristic of improving skin diseases.

The skin is an organ that is directly exposed to the external environment and always exposed to ultraviolet rays such as sunlight. If the skin is exposed to excessive UV rays, it will cause inflammation, tissue damage, immunosuppression, loss of DNA and connective tissues, and promoting collagen degradation, wrinkle formation, and aging. Bring it. In addition, recent industrialization has caused frequent exposure of harmful substances to the skin, and there has been an increasing tendency for sensitive skin accompanied by atopy or allergy due to changes in eating habits and life patterns.

On the other hand, various skin diseases may occur even in the skin of the skin. Examples of such skin-bearing bacteria include S. aureus ( Staphylococcus aureus ), P. acnes ( Propionibacterium acnes ) and E. coli ( Escherichia coli ), gram-negative bacteria. In particular, S. aureus is a major cause of skin diseases and causes diseases such as acne, atopy, skin boil, and swelling. As such, the proliferation of skin bacteria and other bacteria may not only cause skin diseases but also cause damage to skin cells or barrier functions. Recently, as the importance of skin infectious bacterium to various skin diseases has been revealed, antimicrobial activity against skin infectious bacteria has been recognized as an important factor for maintaining healthy skin.

In addition, melanin present in the skin is known to protect the skin by blocking UV damage and removing reactive oxygen species (ROS). Tyrosinase, the most important enzyme in melanogenesis, is converted from tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and DOPA quinone by the enzyme. Finally, eumelanin and pheomelanin are synthesized. However, when tyrosinase is activated in the cells and melanin is overproduced, it causes pigmentation and aging of the skin and damage.

The present inventors have made extensive efforts to provide a composition having the function of restoring skin damage caused by UV, having antimicrobial activity against skin bacteria, and inhibiting tyrosinase activity by studying various causes of skin diseases. As a result, Peptide compound exhibited excellent efficacy in improving skin diseases and confirmed that the side effects were small, and the present invention was completed.

It is an object of the present invention to provide a composition for improving skin diseases comprising a compound represented by a specific formula.

It is still another object of the present invention to provide a cosmetic composition comprising a composition for improving skin diseases comprising a compound represented by a specific formula as an active ingredient.

In order to achieve the above object, the present invention provides a composition for improving skin diseases comprising a compound represented by the following formula (1).

[Chemical Formula 1]

The present invention also provides a cosmetic composition comprising, as an active ingredient, a composition for improving skin diseases comprising the compound represented by the above formula (1).

FIG. 1A is a graph of HPLC analysis of the compound of Chemical Formula 1. FIG.
FIG. 1B shows a HPLC analysis graph of a KTTKS (Lysine-Throne-Threonine-Lysine-Serine) peptide.
FIG. 2 is a safety test graph in human skin cells (FIG. 2 (a) is a safety test graph in a human keratinocyte lineage of a compound of Chemical Formula 1, and FIG. 2 (b) is a safety test graph of a compound of Chemical Formula 1 in a human fibroblast cell line ).
3 is a graph showing the cytoprotective effect against UV damage.
4 is a graph showing an antibacterial test against Staphylococcus aureus .
5A is a graph showing a tyrosinase inhibitory activity test.
FIG. 5B is a graph showing the L-DOPA oxidation inhibitory activity test.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

According to an embodiment of the present invention, there is provided a composition for improving skin diseases comprising a compound represented by the following formula (1).

[Chemical Formula 1]

The compound according to the present invention is composed of a peptide sequence of Ala-KTTKS (Aminolevulinic acid-Lysine-Thronine-Threonine-Lysine-Serine). The molecular weight of the compound is 676.38.

On the other hand, the compound as having an antimicrobial effect against Staphylococcus aureus (Staphylococcus aureus, S.aureus), there is an effect of improving skin conditions caused by S.aureus (Staphylococcus aureus). Staphylococcus aureus is a major cause of skin diseases, causing diseases such as acne, atopy, skin boils, swelling and urticaria. In particular, it is known that Staphylococcus aureus plays an important role in the deterioration and onset period of atopic dermatitis. When the compound according to one embodiment of the present invention is applied to the skin of a patient suffering from atopic dermatitis, the staphylococcus aureus and superantigen toxin It is effective in improving atopic dermatitis.

In order to confirm the antimicrobial activity against Staphylococcus aureus of the compound according to the present invention, a minimum inhibitory concentration (MIC) test and a paper disc test were carried out. As a result, the growth inhibition was inhibited It was confirmed that the growth of microorganisms was inhibited by about 2 times at 1,000 ppm.

Meanwhile, the compound according to the present invention has activity of restoring skin damage by UV. Ultraviolet rays irradiated to the skin cause photochemical reaction, which causes skin inflammatory lesions. When the skin is exposed to ultraviolet rays for a certain period of time, inflammation such as erythema, edema and pain occurs, and the epidermis and keratin are thickened and the pigmentation reaction is increased. In addition, prolonged exposure leads to severe skin lesions, leading to changes in skin appearance such as wrinkles, skin cell death, and the development of malignant tumors. It is known that active oxygen plays an important role in inducing acute and chronic reactions of these ultraviolet rays. After irradiation with ultraviolet rays, a high concentration of reactive oxygen species is generated in the living body. These active oxygen species cause damage to the cell membrane, causing inflammation of the skin, promoting melanin production, and causing pigmentation.

As a result of measuring the cytoprotective effect of UV damage on the human keratinocytes in the outermost layer of the human skin constituting cells according to the present invention, it was found that the cytoprotective effect by UV was recovered by about 35% at 100 ppm Respectively.

The compound according to the present invention has an activity of restoring skin damage by UV rays, and thus can be used for various diseases caused by skin aging and inflammation, such as acne, atopy, urticaria, psoriasis, inflammation And the like.

In addition, the compound according to the present invention is characterized by having tyrosinase inhibitory activity. Melanin is a widely distributed pigment in skin, hair and eyes. It is synthesized in the human melanocyte called melanocyte in the skin. Tyrosine is a precursor of DOPA (3,4-dihydroxy- phenylalanine) or DOPA quinone by oxidation and polymerization. At this time, excessive production of melanin causes pigmentation, which causes skin aging and damage. The tyrosinase inhibitory activity of the compound according to the present invention was measured to be about 80% when treated at 100 ppm, and was higher than that of arbutin used as a positive control.

As a result of measuring the inhibitory effect of the compound according to the present invention on L-DOPA oxidation, it was confirmed that tyrosinase inhibitory activity was about 70% at all concentrations. That is, the compound effectively inhibits the activity of tyrosinase, prevents skin aging and damage due to pigmentation, and also has a skin whitening effect.

According to another embodiment of the present invention, there is provided a cosmetic composition comprising, as an active ingredient, a composition for improving skin diseases comprising the compound represented by Formula 1 above.

As a result of an experiment on the safety of the compound represented by Chemical Formula 1 according to the present invention in human skin cells, it was confirmed that cytotoxicity was not observed in both human keratinocyte cell line HaCaT cell line and human fibroblast cell line .

The cosmetic composition according to the present invention can be applied to skins, essences, lotions, creams, baby powders, face powders, powder blushers, packs, soaps, baths, Improve skin for various skin improvement effects such as skin improvement, skin irritation reduction, skin whitening effect, skin regeneration, heavy metal adsorption, peeling effect, radioactivity absorption, skin pigment reduction, organic pollutant adsorption, moisturizing and oil absorption, It can be used as a raw material for functional cosmetics.

The cosmetic composition according to the present invention may be prepared by adding various known components conventionally used for the formulation of cosmetics, in addition to the compound of formula (1). These examples may include additives such as skin protectants, moisturizers, chelating agents, skin softening agents, conditioning agents, surfactants, viscosity modifiers, alcohols, neutralizing agents, pH adjusting agents and preservatives, May be added and used without departing from the scope of the present invention.

The compounds according to one embodiment of the present invention are described in more detail below. It will be apparent to those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

[Example]

Preparation of compound of formula (1)

The compound according to the present invention is composed of the sequence of Ala-KTTKS (Aminolevulinic acid-Lysine-Thronine-Threonine-Lysine-Serine) and its chemical structure is as follows.

[Chemical Formula 1]

The molecular weight of the compound was 676.38, which was synthesized using a solid phase synthesis method. Peptide synthesis begins at the C-terminal and proceeds to the N-terminal and is synthesized by forming a peptide bond by the condensation reaction of the amino group and the carboxyl group of the amino acid. The compound represented by the formula (1) synthesizes KTTKS, which is a peptide moiety, and finally binds the amino group of the amino group of Aminolevulinic acid with the carboxyl group of Lysine. As a specific synthesis method, Wang resin having serine attached to the terminal sequence of C-terminal was used. At this time, the solvent used is NMP (N-Methylpyrrolidinone, DAE JUNG). Serine-Wang resin is swelled sufficiently in NMP, and then, to attach the next sequence, K (Lysine), the process of removing Fluorenylmethyloxycarbonyl (Fmoc) protected at the amino group of serine is performed. The solvent used to remove Fmoc is 20% piperidine (Merk) and reacted twice for 20 min. After the reaction is complete, the remaining piperidine is completely removed from the resin by washing three times with MC (Methylene Chloride, DUKSAN) and NMP (N-Methylpyrrolidinone, DAE JUNG). The following sequence, K (Lysine), can activate the peptide bond well. Activation is carried out by incubating the same amount of HOBT (N-hydorxybenzotriazole, beadTech) and DIC (N, N'-Diisoproyl carbodiimide, Alfa Aesar) And reacts sufficiently to activate K (Lysine). Activated K (Lysine) is added to the S-resin from which F-moc is removed and reacted at room temperature for 3-4 hours. In this way, all of the N-terminal K is bound, and finally the aminolevulinic acid is activated and bound to KTTKS-resin. When the coupling is completed, the resin is removed by reacting with 95% TFA (Triflouroacetic acid, DAE JUNG) for 3-4 hours. The solution containing the compound induces the precipitation of the peptide by pouring it into the cold ether in an amount of 10 times or more. The peptides thus obtained are analyzed and purified using HPLC equipment.

Analysis and purification of synthesized peptides

To analyze the synthesized compound of formula (1), HPLC was performed using a Waters 650E Advanced Protein Purification System. The column is Gemini 15u C18 300 A (Phenomenex sk) with dimensions of 250 * 4.60 mm, 5 microns. The solvent used was water with 0.1% TFA and acetonitrile with 0.1% TFA. The wavelength was 215 nm and the flow rate was 1 ml / min.

The slope conditions of the solvent are as follows.

Solvent slope conditions Time (min) Solvent A (%) Solvent B (%) 0 95 5 30 35 65 31 35 65 36 95 5 41 95 5

After the synthesis, two main peaks of the 2 < th > and 18 < rd > peaks were observed (Fig. 1A). To confirm which peak was the peak of the compound of formula 1, KTTKS (Lysine-Thronine- Threonine- Lysine-serine) peptide (Fig. 1B). As can be seen from the graph of FIG. 1B, since KTTKS appears in the second half, the compound of formula (1) can be inferred that the peak of the 18th minute is correct.

[Experimental Example]

Safety test in human skin cells

In order to confirm the safety or cytotoxicity of the synthesized compound of formula (1) in human skin cells, the MTT method for human keratinocyte cell line HaCaT cell line and human fibroblast cell line CCD-986sk was used.

As a specific experimental method, human keratinocyte HaCaT cells and fibroblast CCD-986sk Count the cells in a 24-well plate using 5 × 10 ³ cells / well and 5 × 10 ⁴ cells / well using a heamacytometer. After culturing for 48 hours in DMEM containing 10% FBS and culturing to ~ 50% of the culture vessel surface area, the cells were treated with appropriate concentrations and incubated for a further 24 hours. After incubation, 50 μl of a solution of 2.5 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT, Sigma M5655, USA) was added and cultured for additional 3 hours. Then, 200 μl of dimethyl sulfoxide (DMSO, Sigma D2650, USA) was added to each well and the mixture was treated with 200 μl of each solution. 100 μl of each solution was transferred into 96 wells and subjected to Enzyme-Linked Immunosorbent Assay (ELISA) Absorbance was measured at 570 nm. The extent of promoting toxicity or proliferation to cells was expressed as a percentage based on the absorbance intensity of the control using pure water.

The cell treatment concentration of the compound of formula (1) was 100 ppm, 50 ppm, 25 ppm, 12.5 ppm, and 6.25 ppm, starting from a maximum of 100 ppm and diluting twice. As a result, it was confirmed that no cytotoxicity was observed even at 100 ppm compared to the control group (FIG. 2).

Cell protection test by UV damage

For the compound of formula (1), the cytoprotective ability of human keratinocytes in the outermost layer of human skin cells due to UV damage was examined. To this end, human keratinocyte cell line HaCaT cells Count the cells using a heamacytometer in a 24-well plate at 5 × 10 ⁴ cells / well. When cultured in DMEM containing 10% FBS for ~ 48 hours and cultured to ~ 80% of the culture vessel surface area, irradiation with UV at 50 mJ caused damage to the cells. After that, each alapeptide was diluted twice with 100 ppm as the highest concentration and further cultured for 24 hours.

After incubation, 50 μl of a solution of 2.5 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT, Sigma M5655, USA) was added and cultured for additional 3 hours. Then, 200 μl of dimethyl sulfoxide (DMSO, Sigma D2650, USA) was added to each well and the mixture was treated with 200 μl of each solution. 100 μl of each solution was transferred into 96 wells and subjected to Enzyme-Linked Immunosorbent Assay (ELISA) Absorbance was measured at 570 nm. The degree of cell recovery by UV damage was expressed as a percentage based on the absorbance of the control using pure water.

As a result, it was confirmed that the cell damage by UV was recovered by about 35% at 100 ppm (FIG. 3).

Staphylococcus aureus ( Staphylococcus aureus ) Antibacterial test

The minimum inhibitory concentration (MIC) test and the paper disc test were performed to confirm the antimicrobial activity against Staphylococcus aureus , an indigenous bacterium of chronic infection in atopic dermatitis.

S. aureus was cultured in LB (Luria-Broth, Conda) medium and cultured to an OD ₆₀₀ of 0.2-0.3. After that, the microbial culture was mixed with the sample at a ratio of 1: 1, followed by further incubation for 24 hours to quantify the degree of microbial growth. At this time, a sample-free culture medium was used as a control.

As a result, it was confirmed that the compound of formula (I) inhibited the growth of S. aureus strain at a concentration-dependent manner and showed a growth inhibition effect of about 2 times as much as that of the control at 1000 ppm (FIG. 4).

Tyrosinase inhibitory activity test

Tyrosinase inhibitory activity was measured by L-tyrosin and L-DOPA. L-tyrosine and L-DOPA were dissolved in 0.175 M phosphate buffer (pH 6.8) and the mushroom tyrosinase was used at 2000 (U / ml). ALA (5-Aminolevulinic acid), ALA-pro (compound of formula (1) of the present invention) were added to each well of a 96-well plate by adding 80 μl each of 1.5 mM L-DOPA and 1.5 mM L- , ALA-skin (Aminolevulinic acid-Arginine-Glycine-Aspartic acid) were treated at 6.25, 12.5, 25, 50, and 100 ppm and albutin, a positive control, was added to 100 uM of tyrosinase 20 ㎕ was added and reacted at 37 캜 for 10 minutes, and the absorbance at 450 nm was measured. Tyrosinase inhibitory activity was calculated as follows.

Inhibition rate (%) = [1- (absorbance of sample-added group - absorbance of non-additive) / absorbance of non-additive] x 100

The tyrosinase inhibitory activity of the compound of formula (1) was measured to be about 80% inhibition activity at the treatment of 100 ppm, and it was confirmed that it exhibited a very high inhibitory activity when compared with the arbutin used as the positive control (FIG. 5A).

In addition, as a result of measuring the inhibitory effect of the compound of Chemical Formula 1 on L-DOPA oxidation, it showed inhibitory activity of about 70% at all concentrations and higher inhibitory activity than arbutin, which is a positive control group showing about 40% (Fig. 5B).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

A staphylococcus aureus, a staphylococcus aureus, a staphylococcus aureus, a staphylococcus aureus, and a staphylococcus aureus, which have antibacterial activity against Staphylococcus aureus, And skin diseases caused by tyrosinase.
[Chemical Formula 1]

The composition of claim 1, wherein the compound is a peptide sequence of Ala-KTTKS (Aminolevulinic acid-Lysine-Threonine-Threonine-Lysine-Serine).

The composition according to claim 1, wherein the skin disease is at least one disease selected from the group consisting of acne, atopy, skin boar, swelling and urticaria.

A cosmetic composition comprising the composition of any one of claims 1 to 3 as an active ingredient.

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