KR101945947B1 - Protocatechuic acid derivative, method for production thereof and external skin composition containing the same - Google Patents

Abstract

The present invention relates to: a compound using protocatechuic acid; a method of manufacturing the same; and a composition for a skin external application composition which contains the same. To be specifically, the present invention relates to a protocatechuic acid derivative represented by chemical formula 1. The protocatechuic acid derivative according to the present invention has high bioaffinity and can maintain a skin wrinkle-reducing effect stably.

Description

Protocatechuic acid derivatives, a method for producing the same, and a composition for external skin application containing the same

The present invention relates to a protocatechuic acid derivative which can be used as a skin aging material or a skin wrinkle improving material, a method for producing the same, and a composition for external application for skin comprising the same.

The skin of a person is constantly changing with age. Skin aging is largely classified into natural aging and external aging. Natural aging is difficult to artificially control because it is affected by genetic factors, while external aging is relatively easy to control because it is affected by environmental factors. A typical external aging factor is ultraviolet rays, and the most prominent external aging phenomenon is wrinkle formation.

The synthesis and degradation of extracellular matrix such as collagen in vivo is appropriately controlled, but its synthesis decreases as aging progresses and the expression of matrix metalloproteinase (MMP), an enzyme that degrades collagen, is promoted, And the wrinkles are formed.

The substrate metalloproteinase is a metalloproteinase family having a zinc at the active site, and is composed of membrane collagen, aggrecan, fibronectin, laminin (laminin) is the enzyme that decomposes and reconstitutes proteins.

Adenosine and retinoic acid are known to be effective in improving skin wrinkles. However, adenosine has little clinical efficacy and retinoic acid can not be used in women who are pregnant, and there is a high possibility of irritation such as erythema.

On the other hand, since peptides are biocompatible and have very high activity, they are conducting research in the cosmetics and pharmaceuticals industry as a new generation of new materials. However, these peptides are difficult to maintain their efficacy because they are very easily degraded to protease, one of the most enzymes in human body. In order to solve this problem, a method of modifying a part of a peptide to slow down the degradation rate is widely used. In general, acetyl group is introduced at the N terminus ( Pharm. Res ., 10 , 1268, (1993) ; Int. J. Cancer , 83 , 326, (1999)).

It is an object of the present invention to provide a method for effectively preventing skin aging and skin wrinkles by applying a peptide acting on an active site of collagenase or elastase, The present invention also provides a protocatechuic acid derivative, a method for producing the protocatechuic acid derivative, and a composition for external application for skin, which can exhibit not only an efficacy but also a skin aging effect and a skin wrinkle improving effect of the peptide.

In order to achieve the above object, an aspect of the present invention relates to a protocatechuic acid derivative represented by the following formula (1), a method for producing the same, and a composition for external application for skin comprising the same.

≪ Formula 1 >

(Wherein X is a peptide consisting of 2 to 6 amino acids, wherein the amino acid is at least one of Pro, Gly, Leu, Arg, Ala and Asp, Y is a C-terminal portion of the peptide, OH or it is NH _2.)

The present invention introduces protocatechuic acid having a high antioxidant ability at the N-terminus of the peptide, thereby improving the stability in vivo and improving the efficacy. The protocatechuic acid derivatives of the present invention can be applied to the human body safely with high biocompatibility while providing a skin anti-aging effect and a skin wrinkle-reducing effect of peptides in vivo in a stable manner, A protocatechuic acid derivative, a method for producing the same, and a composition for external application for skin comprising the same.

The composition of the present invention is effective for collagen production, inhibition of collagenase activity and improvement of skin wrinkles due to inhibition of elastase activity.

The composition of the present invention is less irritating to the skin, is safe to human body including peptide components, and has no side effects even when used for a long time.

The composition of the present invention can be formulated into various formulations.

Figure 1 shows the HPLC analysis result of the Protocatechuic-RA-NH ₂ Prototype category chyusan derivative according to one embodiment of the present invention.
FIG. 2 shows MALDI-TOF analysis results of Protocatechuic-RA-NH _{2 as} a protocatechuic acid derivative according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention provides a protocatechuic acid derivative into which a peptide having a wrinkle-improving mechanism is introduced into a naturally occurring substance.

The protocatechuic acid derivative can introduce the naturally-occurring substance into the N-terminal of the peptide having the skin wrinkle improving mechanism to inhibit the degradation of the peptide in vivo, thereby increasing the stability of the peptide and providing the effect of improving the skin wrinkle. The protocatechuic acid derivative may be represented by the following formula (1).

≪ Formula 1 >

Wherein X is a peptide consisting of 2 to 6 amino acids, wherein the amino acid is at least one of Pro, Gly, Leu, Arg, Ala and Asp, Y is a C-terminal portion of the peptide, NH ₂ . NH ₂ means that the C-terminus of the peptide is converted to -CONH ₂ structure.

Is a protocatechuic acid derivative in which the carboxy group of protocatechuic acid is bonded to the N-terminal of the peptide. The protocatechuic acid derivative includes a peptide and is highly biocompatible. The protocatechuic acid derivative is designed to further enhance the efficacy of the peptide material by introducing protocatechuic acid at the N-terminus of the peptide.

Peptides are polymeric molecules of amino acids and polymers of more than 100 amino acids are generally classified as proteins. The peptide bond is a bond that forms a bond between the carboxyl group (-COOH) and the amine group (-NH2), in the form of an amide (-CONH-). That is, the peptide refers to a compound in which two or more amino acids, which are basic units of a protein, are linked by an amide bond. The peptide is a polymer formed by bonding 2 to 50 amino acids, and has a molecular weight of 100,000 or less. The number of peptides with 2, 3, 4, etc. constituent amino acids is referred to as a dipeptide, a tripeptide, a tetrapeptide or the like depending on the number of peptides. About 10 or fewer amino acid polymers are referred to as oligopeptides, and more than one amino acid is linked by an amide bond is referred to as a polypeptide.

Amino acids that constitute the basic unit of peptide are molecules having biological activity with amine group, carboxy group and various residues. The residues of amino acids can be divided into hydrophobic and hydrophilic ones, and amino acids having a hydrocarbon chain or a ring are hydrophobic. Typically, there are 20 types - phenylalanine (F), tyrosine (Y), tryptophan (W), serine (S), threonine (T), aspartic acid (D), asparagine (N), glutamic acid (K), arginine (R), histidine (H), cysteine (C), methionine (M), proline (P), glycine (G), alanine (A) , Leucine (L) and isoleucine (I)), and three-dimensionally, L- and D- structures, respectively. In addition to the typical 20 amino acids, it is also possible to use other amino acids such as beta alanine, sarcosine, norvaline, norleucine, ornisine, alpha-amino adipic acid, N- methyllazine, N- methylphenylalanine, phenylglycine, There are many kinds of amino acids not found in proteins such as beta-hydroxy-leucine, beta-methyl aspartic acid, beta-mercaptolin, lan cyanine, dihydroalanine and dihydrobutyrin. In one embodiment of the present invention, the amino acids constituting X in the formula (1) include proline (P), glycine (G), leucine (L), arginine (R), alanine (A), and aspartic acid .

The protocatechuic acid derivative may shorten, reorder and / or modify the peptide sequence PLGL or YYRADA that binds to the active site of the enzyme collagenase and elastase that affects skin wrinkle formation. Peptides that have been replaced with similar amino acids have been prepared and applied. Since the peptide is the basis for constituting the human body, it can exhibit not only high biocompatibility but also excellent activity. In addition, the peptide acts on the active sites of collagenase and elastase to more effectively inhibit the expression of collagenase or elastase, thereby providing excellent anti-aging and wrinkle-reducing effects .

The peptide is represented by X in which Y is introduced at the C-terminus of the peptide in the above formula (1).

According to one embodiment of the present invention, a compound represented by the following formula (2) may be used.

(2)

In the present invention, the compound of Formula ₂ is represented by Protocatechuic-RA-NH ₂ .

Further, the present invention provides a method for producing protocatechuic acid derivatives including a peptide synthesis step, an organic substance introduction step, and a free step.

The peptide synthesis step (A) produces a peptide by a solid phase synthesis method. The solid phase synthesis method is a method widely used for peptide synthesis, and in the present invention, there is no particular limitation with regard to synthesis conditions. According to one embodiment of the present invention, it can be prepared according to Scheme 1 or 2.

Processes for preparing protocatechuic acid derivatives including the above steps according to Reaction Scheme 1 and Reaction Scheme 2 are respectively as follows.

[Reaction Scheme 1]

Reaction 1) Reaction using 2-CTC resin (2-chlorotrityl chloride resin) as a support to maintain OH groups at the C-terminal of amino acid

[Reaction Scheme 2]

Scheme 2) to form a -CONH ₂ by introducing the NH ₂ terminus of the amino acid linked to the C- amide linkers (Rink amide linker), the use of a polystyrene series resin that is introduced into the reaction

For example, referring to Scheme 1 and Scheme 2, R represents various side chains of amino acids, and X is a peptide consisting of 2 to 6 amino acids. In the synthesis process, the amine-protected amino acid and the support are reacted in a binding reagent with Fmoc to prevent side reactions. Next, the Fmoc protecting group is removed after washing and filtration. This synthesis process is repeatedly carried out by adding amino acid to form a desired peptide.

One or more of BOP, HOBt and DIEA may be applied to the binding reagent according to the resin and reaction conditions, and 20% peperidine / NMP may be used to remove the Fmoc protecting group.

The step (B) of introducing the organic substance binds the naturally-occurring substance to the N-terminal of the peptide prepared in the binding reagent. The carboxy group of the protocatechuic acid is combined with the N-terminal of the peptide to form a protocatechuic acid derivative. The binding reagent is as mentioned above.

The free phase (C) separates the peptide bound to the resin by a free reaction using reagent K (trifluroacetic acid) after the step of introducing the organic substance.

The protocatechuic acid derivative obtained by the above method can be further washed, purified and dried by a conventionally known method.

Also, according to the present invention, there is provided a composition for external application for skin, which comprises the protocatechuic acid derivative as an active ingredient, for aging skin and improving skin wrinkles.

The skin external aging composition for skin aging and skin wrinkle improvement may contain 0.000001 to 30% by weight, specifically 0.001 to 10% by weight, of the skin aging and skin wrinkle improving compound. When the protocatechuic acid derivative is contained in an amount of less than 0.000001 wt%, the effect of improving skin wrinkles may be insignificant. When the protocatechuic acid derivative is contained in an amount exceeding 30 wt%, the stability of the formulation may deteriorate.

The composition for external application for skin may be a cosmetic composition or a pharmaceutical composition.

The formulation of the cosmetic composition is not particularly limited and may be appropriately selected according to the purpose. For example, it can be manufactured by a formulation such as a soft lotion, a nutritional lotion, a massage cream, a lotion, an essence, a nutrition cream, an eye cream, a foundation, a mask pack, a fact, and the like.

In the cosmetic composition, an acceptable carrier for cosmetic preparations may be included. Herein, " an acceptable carrier for a cosmetic preparation " is a known or used compound or composition that can be contained in a cosmetic preparation, or a compound or composition to be developed in the future, which is toxic, instable or irritating It says nothing.

The carrier may comprise from about 1% to about 99.99% by weight, preferably from about 90% to about 99.99% by weight of the composition, based on the total weight of the composition. However, since the ratio varies depending on the formulations described below in which the composition for external application for skin of the present invention is prepared, and its specific application site (face, neck, etc.) or its preferable application amount and the like, And should not be construed as limiting the scope of the invention.

Examples of the carrier include an alcohol, an oil, a surfactant, a fatty acid, a silicone oil, a wetting agent, a moisturizer, a viscous modifier, an emulsifier, a stabilizer, an ultraviolet scattering agent, an ultraviolet absorber, a coloring agent and a perfume. The compounds / compositions which can be used as the above-mentioned alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscous modifier, emulsifier, stabilizer, ultraviolet scattering agent, ultraviolet absorber, The person skilled in the art can select and use the appropriate substance / composition.

The pharmaceutical composition may be a transdermal dosage form such as, but not limited to, lotions, ointments, gels, creams, patches or sprays. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter, a perfume or the like in the case of oral administration. A solubilizing agent, an isotonic agent, a stabilizer and the like may be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above.

Examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltoditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

In the composition for external application for skin according to each formulation, components other than the above-mentioned composition of the present invention may be appropriately selected and mixed by those skilled in the art according to the formulation or purpose of use of other external preparation for skin.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. This is for the purpose of illustrating the present invention, and thus the scope of the present invention is not limited thereto.

Preparation Example 1 Preparation of Protocatechuic-RA-NH ₂  Produce

1 g of Rink amid resin (substitution ratio: 0.5 mmol / g) was added to the reactor and swelled in a solvent of N-methylpyrrolidone (NMP) for 2 hours. Then, 20% piperidine / N- Add methylpyrrolidone and react for 30 minutes to remove the Fmoc protecting group of the resin. After removing the solution, 20% piperidine / N-methylpyrrolidone is added to the reactor containing the resin and reacted for 1 hour. After removing the solution again, the resin is washed three times each with N-methylpyrrolidone, methanol, dichloromethane (DCM), and finally washed again with N-methylpyrrolidone to remove the excess reaction solution.

The reactor was charged with 297 mg of Fmoc-Ala-OH (2 equivalents of substituent), 442 mg of BOP reagent (equivalent to amino acid reagent), 135 mg of 1-hydroxybenzotriazole (HOBt) And diisopropylethylamine (DIEA) (1.1 equivalents of amino acid reagent) were added to the reaction mixture, and 10 ml of N-methylpyrrolidone was added thereto, followed by reaction for 3 hours. The reaction was terminated using the kaiser's ninhydrin test ( Anal. Biochem ., 1981, 117, 147).

The solution in the reactor was removed and the resin was washed three times with N-methylpyrrolidone, methanol and dichloromethane, respectively, and finally washed again with N-methylpyrrolidone. 20% piperidine / N-methylpyrrolidone was added to the washed resin, and the same reaction as described above was carried out to remove the Fmoc protecting group. Subsequently, Fmoc-Arg-OH was introduced into the resin in the same manner.

180 mg of protocatechuic acid (2 equivalents of substituent), 442 mg of BOP reagent (equivalent to the amount of amino acid reagent), and 1 mg of BOP reagent were added to the resin to which arginine (Arginine, Arg, R) (Equivalent amount of amino acid reagent) and 142 mg of diisopropylethylamine (1.1 equivalents of amino acid reagent) were added to the reaction mixture, and 10 ml of N-methylpyrrolidone was added thereto, followed by reaction for 24 hours. The reaction was terminated using Kaiser's ninhydrin test.

How the protocatechuic-Arg-Ala-NH ₂ prepared on Rink amide resin as described above is known using reagent K (Synthetic Peptides:. A User's Guide (GA Grant, ed), WH Freeman and Company, New York, 1992) . The separated peptide derivatives were precipitated with cold ether, and then separated by centrifugation at 0 ° C. The separated peptide derivatives were centrifuged by adding 20 ml of cold ether under the same conditions and repeated three times to remove impurities. The centrifuged peptide derivative was lyophilized for 24 hours to obtain about 100 mg of a final product having a purity of 95.215%.

The presence of the final product was confirmed by HPLC analysis and MALDI-TOF (Matrix-assisted laser desorption ionization time of flight mass spectrometry) analysis. The results are shown in Fig. 1 to Fig.

<Experimental Example 1> Measurement of intracellular collagen production amount

Human Dermal Fibroblasts (HDFs) cells were used to measure intracellular collagen production. HDFs cells were cultured in a 5% CO ₂ incubator (HERAcell 150i, Thermo, USA) at 37 ° C with DMEM / F12 3: 1 mixed medium containing 1% Antibiotic-Antimycotic and 10% FBS. HDFs cells were inoculated on a 24-well plate at 5 × 10 ⁴ cells / well, cultured for 24 hours, and then cultured for 24 hours in a medium containing 50 μM of the diluted medium. After incubation for 24 hours, the supernatant was taken and the amount of procollagen liberated in the medium was measured at 450 nm using Procollagen Type I C-Peptide (PIP) ELISA Kit. The degree of collagen production was calibrated by total protein amount, and the positive control group was compared using L-ascorbate (151 μM). Protein content was determined by Bradford method using BSA as a standard solution. BSA was diluted stepwise in distilled water at 0, 2, 5, 10, and 20 μg / mL, and 20 μL of each concentration of BSA solution was added to 0.98 mL of Bradford reagent. After reacting for 5 minutes, absorbance was measured at 595 nm and BSA The standard curve was obtained. In the same manner, the absorbance of the cell lysate of HDFs cultured on a 24-well plate was measured and the amount of protein was calculated using a BSA standard curve. As a negative control, a reaction solution to which no sample was added was used. Experimental results of the effect of increasing intracellular collagen production on the negative control group are shown in Table 1.

Increase in collagen production (%) Example 1 Protocatechuic-PLGL-OH 2 Example 2 Protocatechuic-RA-NH ₂ 26.5 Example 3 Protocatechuic-PG-OH 11 Example 4 Protocatechuic-RADA-NH ₂ 15.6 Example 5 Protocatechuic-ADA-OH 9.8 Example 6 Protocatechuic-RAD-NH ₂ 19 Comparative Example 1 PLGL -24.45 Comparative Example 2 YYRADA -8.37 Comparative Example 3 Protocatechuic-Y-NH ₂ 1.8 Comparative Example 4 Protocatechuic-P-NH ₂ -12.5 Comparative Example 5 Protocatechuic-YW-NH ₂ -3.9 Comparative Example 6 Protocatechuic-YP-NH ₂ 0.5 Comparative Example 7 L-ascorbate (151 μM) 147

As shown in Table 1, the collagen production effect of the protocatechuic acid derivatives of the present invention was superior to that of the peptides alone. In the case of two to six of the preferred amino acids, proline (P), glycine (G), leucine (L), arginine (R), alanine (A) and aspartic acid (D) (%) Of the collagen production. Based on this, it was expected that the composition for external application for skin of the present invention effectively promotes collagen, thereby improving wrinkles of the skin.

Experimental Example 2 Inhibition of intracellular collagenase (MMP-1) activity

Human Dermal Fibroblasts (HDFs) were inoculated on a 24-well plate at 5 × 10 ⁴ cells / well for 24 h. After incubation, the cells were exchanged with HBSS to induce the activity of collagenase, UVA (6.3 J / cm ² ) was irradiated, and the medium was replaced with medium containing 50 μM of the diluted material and cultured for 24 hours. After 24 hours incubation, the activity of collagenase was measured at 450 nm using MMP-1 Human ELISA Kit. MMP-1 activity was calibrated by total protein content and compared with Epigallocatechin gallate (EGCG) (5 μM) as a positive control. Protein content was determined by Bradford method using BSA as a standard solution. BSA was diluted stepwise in distilled water at 0, 2, 5, 10, and 20 μg / mL, and 20 μL of each concentration of BSA solution was added to 0.98 mL of Bradford reagent. After reacting for 5 minutes, absorbance was measured at 595 nm and BSA The standard curve was obtained. In the same manner, the absorbance of the cell lysate of HDFs cultured on a 24-well plate was measured and the amount of protein was calculated using a BSA standard curve. As a negative control, a reaction solution to which no sample was added was used. Experimental results of inhibition of intracellular collagenase (MMP-1) activity on the negative control group are shown in Table 2.

Collagenase (MMP-1) activity inhibition rate (%) Example 1 Protocatechuic-PLGL-OH 2 Example 2 Protocatechuic-RA-NH ₂ 19.3 Example 3 Protocatechuic-DA-NH ₂ 14.8 Example 4 Protocatechuic-RADA-NH ₂ 13.3 Example 5 Protocatechuic-ADA-NH ₂ 13.9 Example 6 Protocatechuic-PG-OH 15 Example 7 Protocatechuic-RAD-NH ₂ 12 Comparative Example 1 PLGL -16.76 Comparative Example 2 YYRADA -22.31 Comparative Example 3 Protocatechuic-Y-NH ₂ -8.3 Comparative Example 4 Protocatechuic-P-NH ₂ -2.6 Comparative Example 5 Protocatechuic-YW-NH ₂ 0.7 Comparative Example 6 Protocatechuic-YP-NH ₂ 1.1 Comparative Example 7 Epigallocatechin gallate (EGCG) (5 [mu] M) 78

From the above results, it was confirmed that the effect of the protocatechuic acid derivative of the present invention on collagenase (MMP-1) activity inhibition was superior to that of the peptide alone, and the preferred amino acids proline (P), glycine (%) Of collagenase (MMP-1) activity is higher than that when mixed with other amino acids, when it is composed of 2 to 6 of amino acids (L), arginine (R), alanine (A) and aspartic acid I could see what was visible. Based on this, the composition for external application for skin of the present invention can effectively inhibit collagenase (MMP-1) activity, thereby improving skin wrinkles.

<Experimental Example 3> Elastase activity inhibition experiment

Human Dermal Fibroblasts (HDFs) cells were inoculated into 100 mm culture dish with 5 × 10 ⁴ cells / dish. The cultured cells were dissolved in 0.1% Triton X-100 * 0.2 M Tris-HCl (pH 8.0) The cell lysate was centrifuged at 10,000 rpm for 30 minutes at 4 ° C, and the supernatant was used as an enzyme solution containing elastase. The elasthase enzyme solution was quantified, and 100 μg of the solution was added to a 96-well plate, and the volume was adjusted to 80 μL by adding 0.2 M Tris-HCl (pH 8.0) buffer solution. L-alanyl-L-alanyl-L-alanyl p-nitroanilide (STANA) (10 μL) was added to each well and incubated at 37 ° C (Oven period, JSOF-150, JSR, Korea) for 90 minutes, and absorbance was measured at 405 nm to evaluate elastase activity. Phosphoramidon (10 μM) was used as a positive control. The experimental results of the inhibitory effect of elastase activity on the negative control group are shown in Table 3. &lt; tb &gt;&lt; TABLE &gt;

Elastase activity inhibition rate (%) Example 1 Protocatechuic-PLGL-OH 5 Example 2 Protocatechuic-RA-NH ₂ 20.4 Example 3 Protocatechuic-DA-NH ₂ 14.6 Example 4 Protocatechuic-RADA-NH ₂ 17.8 Example 5 Protocatechuic-ADA-NH ₂ 13.4 Example 6 Protocatechuic-PG-OH 14 Example 7 Protocatechuic-RAD-NH ₂ 14 Comparative Example 1 PLGL 4.3 Comparative Example 2 YYRADA 3.8 Comparative Example 3 Protocatechuic-Y-NH ₂ 2.2 Comparative Example 4 Protocatechuic-P-NH ₂ 3.1 Comparative Example 5 Protocatechuic-YW-NH ₂ 2 Comparative Example 6 Protocatechuic-YP-NH ₂ 2.4 Comparative Example 7 Phosphoramidon (10 μM) 81

From the above results, it was confirmed that the effect of the protocatechuic acid derivative of the present invention on elastase activity was superior to that of the peptides alone, and the preferable amino acids proline (P), glycine (G), leucine (%) Of elastase activity when compared with the case where it is mixed with other amino acids, when it is composed of 2 to 6 of arginine (R), alanine (A) and aspartic acid (D) I could. Based on this, the composition for external application for skin of the present invention effectively inhibits the activity of elastase, thereby improving the skin wrinkles.

<Experimental Example 4> Cytotoxicity test

Cell viability was analyzed using the principle of MTT. Human Dermal Fibroblasts (HDFs) were inoculated into a 24-well plate at a concentration of 5 × 10 ⁴ cells / well, cultured for 24 hours, and then cultured for 24 hours. After incubation for 24 hours, 100 μL of 0.5% MTT solution was added to each well and cultured for 4 hours. After removing the culture medium, 500 μL of DMSO was added, and the mixture was shaken for 10 minutes. Then, the absorbance was measured at 570 nm (BioTek, USA). As a negative control, a reaction solution to which no sample was added was used. The cell viability by each sample solution was calculated according to the following equation (1), and the measurement results are shown in Table 4 below.

(1)

Cell viability (%) = A / B X 100

A: Absorbance of the sample

B: absorbance of negative control group

Cell survival rate (%) Example 2 _{Protocatechuic-RA-NH 2 (10} μM) 98.18 _{Protocatechuic-RA-NH 2 (25} μM) 98.89 _{Protocatechuic-RA-NH 2 (50} μM) 103 Example 3 Protocatechuic-DA-NH ₂ (10 μM) 97.9 _{Protocatechuic-DA-NH 2 (25} μM) 101.5 _{Protocatechuic-DA-NH 2 (50} μM) 106.6

From the results shown in Table 4, it was confirmed that the cytotoxicity of the protocatechuic acid derivative of the present invention was very low, and based on this, it is judged that the composition for external application for skin of the present invention is safe for skin.

Experimental Example 5: Skin stimulation experiment

A human patch test was performed to evaluate the degree of irritation to the skin. The skin patch test was carried out on 20 normal subjects aged 20 years or older. The sample was made into 0.01% 0.1%, 1%, 10% aqueous solution and then attached to the inside of the upper abdomen using a Finn chamber for 48 hours The first readings were taken after the removal, and the second readings were taken 72 hours later to determine skin reaction results. Calculation of evaluation criteria and stimulation index and the results are shown in Tables 5 and 6.

reaction weight Evaluation standard - 0.0 No reaction ± 0.5 Suspicious Positive Response + 1.0 Weakly positive reaction ++ 2.0 Strong positive reaction +++ 3.0 Polar positive reaction

(2)

Skin irritation index = Σ (number of weighted X responses) / (maximum weighted X total number of subjects) × 100

Skin irritation index Degree of stimulation Example 2 Protocatechuic-RA-NH ₂ 0.01% 0.0 No stimulation Protocatechuic-RA-NH ₂ 0.1% 0.0 No stimulation Protocatechuic-RA-NH ₂ 1% 0.0 No stimulation Protocatechuic-RA-NH ₂ 10% 0.4 No stimulation

As a result, the protocatechuic acid derivatives of the present invention showed no irritation to the skin, and it was judged that there was no irritation to the skin when used as a raw material for external application for skin.

&Lt; Experimental Example 6 > Measurement of skin wrinkle improvement effect

The cream compositions of Examples 1 to 3 and Comparative Examples 1 to 4 were prepared with the composition shown in Table 7 below (unit: wt%).

Ingredients Content (% by weight) Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Protocatechuic-RA-NH ₂ 0.01 - - - - - - Protocatechuic-ADA-OH - 0.01 - - - - - Protocatechuic-RADA-NH ₂ - - 0.01 - - - - Protocatechuic-Y-NH ₂ - - - 0.01 - - - Protocatechuic-P-NH ₂ - - - - 0.01 - - Protocatechuic-YW-NH ₂ - - - - - 0.01 - Protocatechuic-YP-NH ₂ - - - - - - 0.01 glycerin 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Stearic acid 8.0 8.0 8.0 8.0 8.0 8.0 8.0 Squalane 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Self emulsifying glycerin monostearate 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Polyoxyethylene sorbitan monostearate 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Propylene glycol 4.0 4.0 4.0 4.0 4.0 4.0 4.0 Stearyl glycyrrhetinate 0.2 0.2 0.2 0.2 0.2 0.2 0.2 vaseline 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Antioxidant Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Spices Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount antiseptic Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Purified water Balance Balance Balance Balance Balance Balance Balance

To examine the effect of the protocatechuic acid derivatives of the present invention on the skin wrinkle of the human body, two human women were subjected to human body application tests.

The creams prepared in Examples 1 to 3 and Comparative Examples 1 to 4 were used for 10 weeks per group and then used for 4 weeks after morning cleansing and overnight cleansing twice a day.

The degree of improvement of skin wrinkles before and after use was measured using a facial stage DM-3 (Moritex, Japan) and the change in wrinkle area after 4 weeks was analyzed using an analysis program. The results are shown in Table 8.

(3)

Wrinkle area change amount = Wrinkle area before use - Wrinkle area after 4 weeks use

Wrinkle area variation (mm ² ) Example 1 13.2 Example 2 13 Example 3 12.9 Comparative Example _{1 (Protocatechuic-Y-NH 2} ) 2.3 Comparative Example 2 (Protocatechuic-P-NH ₂ ) 5 Comparative Example 3 (Protocatechuic-YW-NH ₂ ) 1.8 Comparative Example _{4 (Protocatechuic-YP-NH 2} ) -2.2

Based on the results of the above Experimental Example, various formulation examples using Protocatechuic-RA-NH ₂ are shown below as Tables 9 to 18. However, it should be apparent to those skilled in the art that these formulation examples are for illustrating the present invention and that the formulation of the present invention is not limited to these formulation examples.

&Lt; Formulation Example 1 > Soft skin length (skin toner)

Ingredients Content (% by weight) Protocatechuic-RA-NH ₂ 0.01 glycerin 5.0 1,3-butylene glycol 3.0 ethanol 5.0 Polyoxyethylene nonylphenyl ether 0.5 Spices Suitable amount antiseptic Suitable amount Purified water Balance

&Lt; Formulation Example 2 >

Ingredients Content (% by weight) Protocatechuic-RA-NH ₂ 0.01 glycerin 3.0 Citric acid 0.1 ethanol 10.0 Polyoxyethylene oleyl ether 1.0 Sorbitol 2.0 Spices Suitable amount antiseptic Suitable amount Purified water Balance

Formulation Example 3 Lotion (emulsion)

Ingredients Content (% by weight) Protocatechuic-RA-NH ₂ 0.01 glycerin 5.0 1,3-butylene glycol 8.0 Squalane 10.0 Polyoxyethylene sorbitan monooleate 2.0 Triethanolamine 1.5 Glyceryl stearate 0.5 Stearyl glycyrrhetinate 0.2 Carboxyvinyl polymer 0.1 Arginine 0.1 Spices Suitable amount antiseptic Suitable amount Purified water Balance

&Lt; Formulation Example 4 >

Ingredients Content (% by weight) Protocatechuic-RA-NH ₂ 0.01 Sorbitol 8.0 Polyoxyethylene glycol 1500 6.0 ethanol 5.0 glycerin 3.0 1,3-butylene glycol 3.0 Polyoxyethylene oleyl alcohol ether 1.0 olive oil 0.3 Hyaluronic acid 0.2 Antioxidant Suitable amount Spices Suitable amount antiseptic Suitable amount Purified water Balance

&Lt; Formulation Example 5 >

Ingredients Content (% by weight) Protocatechuic-RA-NH ₂ 0.01 ethanol 10.0 glycerin 4.0 Propylene glycol 4.0 Polyoxyethylene hardened castor oil 0.1 Carboxy polymer 0.3 Triethanolamine 0.3 Antioxidant Suitable amount Spices Suitable amount antiseptic Suitable amount Purified water Balance

&Lt; Formulation Example 6 > ointment &

Ingredients Content (% by weight) Protocatechuic-RA-NH ₂ 0.01 Cetostearyl alcohol 2.0 Self emulsifying monostearic acid 2.0 Stearic acid 1.0 Wax 4.0 Squalane 7.0 Monostearin glycerin 3.0 Sorbitan monostearate 1.0 Polysorbate 80 3.0 glycerin 5.0 Propylene glycol 4.0 Spices Suitable amount Vaseline Balance

&Lt; Formulation Example 7 >

Ingredients Content (% by weight) Protocatechuic-RA-NH ₂ 0.01 glycerin 5.0 Stearic acid 8.0 Squalane 5.0 Self emulsifying glycerin monostearate 2.5 Polyoxyethylene sorbitan monostearate 1.5 Propylene glycol 4.0 Stearyl glycyrrhetinate 0.2 Vaseline 2.0 Antioxidant Suitable amount Spices Suitable amount antiseptic Suitable amount Purified water Balance

&Lt; Formulation Example 8 >

Ingredients Content (% by weight) Protocatechuic-RA-NH ₂ 0.01 Palm oil fatty acid 1.0 Titanium dioxide 0.3 Disodium ethylenediamine tetraacetate 0.1 Spices Suitable amount Pigment Suitable amount Soap chip base Balance

&Lt; Formulation Example 9 >

Ingredients Content (% by weight) Protocatechuic-RA-NH ₂ 0.01 Allantoin 0.2 ethanol 5.0 Nonyl phenyl ether 0.3 Sodium carboxymethylcellulose 0.2 Polyvinyl alcohol 13.0 Pigment Suitable amount Spices Suitable amount Purified water Balance

&Lt; Formulation Example 10 > Body cleanser

Ingredients Content (% by weight) Protocatechuic-RA-NH ₂ 0.01 Sodium laureth sulfate 10 Cocoamidopropyl betaine 2 Coconut diethanolamide 2 Polyquaternium-7 0.3 Spices dose antiseptic dose Purified water Balance

In the present invention, the protocatechuic acid derivatives having various formulations using Protocatechuic-RA-NH ₂ as shown in Tables 9 to 18 above were obtained by introducing a naturally occurring substance into the N-terminus of a peptide having a wrinkle-improving mechanism, It is possible to suppress decomposition to increase the stability of the peptide, to provide a continuous wrinkle-improving activity, and to exhibit excellent skin wrinkle-reducing effect.

Claims (9)

A protocatechuic acid derivative represented by the following formula (1).

&Lt; Formula 1 >
Protocatechuic-XY

(Ala-Asp-Ala), RAD (Arg-Ala), PG (Pro-Gly) and one type of the peptide selected from the group consisting of (Arg-Ala-Asp), Y is a C- terminal portion of the peptide, is OH or NH ₂₎
delete delete A composition for external application for skin for skin aging and skin wrinkle improvement, which comprises the protocatechuic acid derivative of claim 1 as an active ingredient.
5. The method of claim 4,
Wherein the protocatechuic acid derivative is contained in an amount of 0.000001% by weight to 30% by weight based on 100% by weight of the composition.
A pharmaceutical composition for improving skin wrinkles comprising the composition according to claim 4.
The method according to claim 6,
Wherein the pharmaceutical composition is a formulation of any one of lotion, ointment, gel, cream, patch or spray.
A cosmetic composition for skin aging and skin wrinkle improvement, which comprises the composition according to claim 4.
9. The method of claim 8,
Wherein the cosmetic composition is a formulation of any one of a soft lotion, a convergent lotion, a nutritional lotion, a massage cream, a lotion, an essence, a nutritional cream, an eye cream, a foundation, a mask pack and a lotion. A cosmetic composition.

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