KR20180019966A - Peptide having anti-inflammatory activity and skin condition improvement effect, and uses thereof - Google Patents

Abstract

The present invention relates to peptide having skin condition improving effects and provides peptide for skin condition improvement made of an amino acid sequence of sequence number 1. Therefore, the peptide has excellent biostability and anti-inflammatory activity.

Description

PEPTIDE HAVING ANTI-INFLAMMATORY ACTIVITY AND SKIN CONDITION IMPROVEMENT EFFECT, AND USES THEREOF FIELD OF THE INVENTION [0001]

The present invention relates to peptides for improving skin condition, including inflammatory activity.

Inflammation is a phenomenon that occurs when a cell or tissue is damaged by some cause, to minimize the reaction and to restore the damaged area to the original state. Inflammation can cause nerves and blood vessels, lymphatic vessels, humoral responses, cellular reactions, resulting in pain, swelling, redness, fever, and so on.

The causes of inflammation include physical factors such as trauma, frostbite, burns, radioactivity, chemical factors caused by chemicals such as acid, and immunological factors due to antibody reaction. In addition, due to blood vessel or hormone imbalance .

Cells damaged by external stimuli secrete a variety of biological mediators such as pro-inflammatory cytokines and chemokines, interleukins, and interferons and increase permeability by vasodilation, , Complement, plasma, and phagocytic cells are concentrated at the site of inflammation. This phenomenon can cause erythema.

Anti-inflammatory agents are widely used in cosmetics for the purposes of skin soothing and acne and atopic dermatitis. Nonsteroidal benzindamine, indomethacin, and ibuprofen are used as anti-inflammatory drugs. Steroid-based dexamethasone and hydrocortisone are used.

However, the use of the anti-inflammatory agent is limited due to human safety and side effects, and the effect is insignificant, so that there is a problem that the inflammation relieving effect can not be expected substantially.

Therefore, development of ingredients having skin elasticity, wrinkle improvement, moisturizing and anti-inflammatory effect which are more safe than living body, stable in effectiveness, and more effective than those having existing skin elasticity, wrinkle improvement, moisturizing and anti- Is required.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a novel peptide having excellent biostability and excellent anti-inflammatory activity.

According to one aspect of the present invention, there is provided a skin condition improving peptide comprising the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the peptide can inhibit the expression of inflammatory cytokines.

In one embodiment, the peptide may have an antioxidant activity.

According to another aspect of the present invention, there is provided a skin condition improving composition comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the composition may be for antioxidant, skin wrinkle reduction, skin whitening, anti-inflammation or skin regeneration.

According to another aspect of the present invention, there is provided an external preparation composition comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

According to the present invention, since the peptide is small in size, it is excellent in skin penetration rate and excellent in anti-inflammatory effect and can replace the conventional anti-inflammatory agent.

The peptide can be used as a therapeutic agent for an inflammatory disease, and can be used for various inflammation-relieving products such as a quasi-drug composition, a health functional food composition, and a cosmetic composition.

It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be deduced from the description of the invention or the composition of the invention set forth in the claims.

As used herein, the terminology used herein is intended to encompass all commonly used generic terms that may be considered while considering the functionality of the present invention, but this may vary depending upon the intent or circumstance of the skilled artisan, the emergence of new technology, and the like. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term, not on the name of a simple term, but on the entire contents of the present invention.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

The numerical range includes numerical values defined in the above range. All numerical limitations of all the maximum numerical values given throughout this specification include all lower numerical limitations as the lower numerical limitations are explicitly stated. All the minimum numerical limitations given throughout this specification include all higher numerical limitations as the higher numerical limitations are explicitly stated. All numerical limitations given throughout this specification will include any better numerical range within a broader numerical range, as narrower numerical limitations are explicitly stated.

Hereinafter, embodiments of the present invention will be described in detail, but it should be apparent that the present invention is not limited by the following examples.

One aspect of the present invention provides a skin condition improving peptide comprising the amino acid sequence of SEQ ID NO: 1.

The peptide refers to a polymer composed of one or more amino acids linked by amide bonds (or peptide bonds). For the purposes of the present invention, the peptide may refer to a peptide or fragment thereof that exhibits anti-inflammatory effects.

The general rules for naming the peptides may be based on three letter or single letter amino acid codes, unless specifically indicated otherwise. For example, the center of the amino acid structure is represented by a three-letter code (e.g., Ala, Lys), and a D-stereotype (e.g., D-Ala, D-Lys) It is possible to assume an L-steric form. The amino acid residue constituting the peptide may be a natural or non-natural amino acid residue.

As a result of intensive efforts to discover peptides having a biologically active activity, the present inventors have found an excellent skin improving effect of a peptide composed of the amino acid sequence of SEQ ID NO: 1.

The peptide may be composed of the amino acid sequence of SEQ ID NO: 1 or may partially include the amino acid sequence.

The peptide consisting of the amino acid sequence of SEQ ID NO: 1 may exhibit anti-inflammatory activity through inhibition of the expression of inflammatory cytokines and proliferation of inflammatory cells.

In addition, the peptide may have an antioxidative activity, and the peptide may have various skin improving effects when applied to a human body.

The improvement of the skin condition is a wrinkle improvement, a skin elasticity improvement, a skin aging prevention, a skin moisturization improvement, a wound removal and a skin regeneration effect, more preferably a wrinkle improvement, a skin elasticity improvement, a skin aging prevention, .

Specifically, the peptide promotes the proliferation of fibroblasts and grows the stratum corneum, epithelial layer and dermal layer of the skin, and can exhibit effects of improving skin elasticity, preventing skin aging, improving skin moisturization, and regenerating skin.

Thus, the skin condition can be effectively improved when the peptide is topically applied to the skin.

On the other hand, the peptide can be produced biologically by extracting a protein in vivo and treating it with a protease to give a low molecular weight or by using a gene recombination and protein expression system, or by a chemical synthesis method using a peptide synthesizer or the like .

For example, a gene encoding a fusion partner and a fusion protein composed of the peptide may be prepared through gene manipulation, and the host microorganism may be transformed with the gene and expressed in the form of a fusion protein in the host microorganism. The fusion protein can be cleaved and separated using proteolytic enzymes or compounds, and the peptide can be obtained. Specifically, a DNA sequence encoding an amino acid residue that can be cleaved by a proteolytic enzyme, such as Factor Xa or an enterokinase, a compound such as CNBr or hydroxylamine, can be inserted between the fusion partner and the gene of the peptide .

The N or C-terminal of the peptide may be bonded with an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group or polyethylene glycol (PEG). The stability of the peptide can be remarkably improved by the modification. The stability is a concept including not only in vivo stability but also storage stability. The protecting group can protect the peptide of the present invention from attack of a protein cleaving enzyme in vivo.

In addition, the peptide may comprise a functional equivalent of a peptide comprising the amino acid sequence of SEQ ID NO: 1. The above-mentioned " functional equivalent "means an amino acid sequence having at least 40% or more, preferably 80% or more, more preferably 90% or more, more preferably 95 Or more homologous to the amino acid sequence of SEQ ID NO: 1, and having substantially the same physiological activity as the amino acid sequence of SEQ ID NO: 1.

The "substantially homogenous physiological activity" means a skin condition improving activity such as anti-inflammatory activity due to the structural and functional homology of the peptide. The sequence homology is determined by comparing the two optimally arranged sequences with the comparison region, and some nucleic acid sequences in the comparison region may be added or deleted.

According to another aspect of the present invention, there is provided a skin condition improving composition comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

The skin improvement effect may be improved by skin protection and improvement of skin condition, skin whitening, prevention or improvement of skin aging and wrinkles, skin protection and alleviation of skin inflammation reaction, skin barrier improvement, skin irritation mitigation, , Antioxidant ability, ability to promote collagen synthesis, and so on.

As used herein, the phrase " comprising as an active ingredient " may mean an effective amount of an effective amount capable of exhibiting a skin improving effect, such as an anti-inflammatory activity, collagen synthesis associated with wrinkle- have.

The peptides can be used for various applications such as pharmaceutical compositions, cosmetic compositions, and external skin preparations due to the skin improving activity.

In one embodiment, the pharmaceutical composition may be for the prophylaxis or treatment of inflammatory diseases.

The inflammation refers to a phenomenon that occurs when a cell or tissue is damaged due to any cause, to minimize the reaction and to restore the injured site to the original state. The nerve, the blood vessel, the lymphatic vessel, the humoral response, And as a result, pain, swelling, redness, fever and the like can be referred to as a dysfunction. The disease caused by inflammation may be any of dermatitis, allergy, systemic lupus erythematosis, retinitis, gastritis, hepatitis, enteritis, pancreatitis or nephritis, and the allergy may be anaphylaxis, allergic rhinitis, asthma ), Allergic conjunctivitis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, insect allergies, food allergies and drug allergies.

Said " prevention " means any action which reduces the frequency or degree of occurrence of the pathological phenomenon of an animal. Prevention can be complete or partial. In this case, the disease caused by inflammation in the subject may be reduced as compared with the case where the composition is not used.

The term " treatment " means any clinical intervention to alter the natural course of a subject or cell to be treated, and may be performed during or during the course of a clinical pathology. The desired therapeutic effect may be to prevent the occurrence or recurrence of the disease, to alleviate the symptoms, to reduce all direct or indirect pathological consequences of the disease, to prevent metastasis, to reduce the rate of disease progression, Relieving or temporarily alleviating the condition, or improving the prognosis.

The pharmaceutical composition may form pharmaceutically acceptable acid addition salts according to methods conventional in the art. The free acid may be an organic acid or an inorganic acid. For example, the inorganic acid may be hydrochloric acid, bromic acid, sulfuric acid, or phosphoric acid. The organic acid may be citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid But are not limited to, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4- toluenesulfonic acid, benzenesulfonic acid, Nicotinic acid, isonicotinic acid, picolinic acid, glutamic acid, or aspartic acid.

The pharmaceutical composition may further comprise a commonly used carrier, excipient and diluent.

The compositions can be administered in the form of oral delivery , parenteral delivery. The peptide may be administered systemically or topically, and the administration may include oral administration and parenteral administration. When the compound is administered as a composition, it may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide a suitable dosage form.

In addition, the composition comprising the peptide may further comprise a carrier, an excipient and a diluent which are used in the production of the pharmaceutical composition.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, But are not limited to, cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition, the composition may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, external preparation, suppository and sterilized injection solution.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and the solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Or gelatin. In addition to the above excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, syrups, and the like. In addition to water and liquid paraffin which are simple diluents, various excipients such as wetting agents, sweeteners, have.

The preparation for parenteral administration may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository. Examples of the non-aqueous solvent and suspensions may include injectable esters such as propylene glycol, polyethylene glycol, vegetable oil such as olive oil and ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, and glycerogelatin.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is desirable to administer an amount that allows for all of the above factors to be maximally effected in a minimal amount without side effects, which can be readily determined by one skilled in the art.

On the other hand, the cosmetic composition or external preparation for skin may be formulated by a conventional method. In the formulation of the external preparation for skin, Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can refer to, and in the formulation of the cosmetic composition, International cosmetic ingredient dictionary, 6th ed., The cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995, which is incorporated herein by reference.

Specifically, the cosmetic composition may be prepared in the form of a general emulsified formulation and a solubilized formulation. For example, lotion such as soft lotion or nutrition lotion; Lotion such as facial lotion, body lotion; Nourishing cream, moisture cream, cream such as eye cream; essence; Makeup ointment; spray; Gel; pack; Sunscreen; Makeup base; A foundation such as a liquid type, a solid type or a spray type; powder; Make-up removers such as cleansing creams, cleansing lotions and cleansing oils; Or a cleansing agent such as a cleansing foam, a soap, a body wash, and the like, but is not limited thereto. The external preparation for skin may be formulated into ointments, patches, gels, creams or sprays, but is not limited thereto.

The cosmetic composition or the external preparation may suitably contain other ingredients in addition to the essential ingredients in the respective formulations within the range not hindering the object of the present invention depending on the type of the formulation or the purpose of use.

The cosmetic composition or the external preparation may contain an acceptable carrier and may be suitably mixed with oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, a chelating agent, a pigment, a preservative, It is not.

The acceptable carrier may vary according to the formulation. For example, when formulated into ointments, pastes, creams or gels, there may be used an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, Mixture can be used

When the cosmetic composition or the external preparation is formulated with a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as a carrier component, Such as propane, butane, or dimethyl ether.

When the cosmetic composition or the external preparation is formulated into a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as the carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, Glycol, and 1,3-butylglycol oil may be used, and fatty acid esters of cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or sorbitan may be used have.

When the cosmetic composition or the external preparation is formulated as a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the cosmetic composition is formulated into soap, it is preferable to use, as a carrier component, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolizate, isethionate, a lanolin derivative, an aliphatic alcohol, a vegetable oil, Can be used.

The cosmetic composition or the external preparation for skin according to the present invention may be formulated into a lipid, an organic solvent, a solubilizer, a thickening agent, a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, a foaming agent ), Perfumes, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants, chelating agents, preservatives, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, Such as any other ingredients used in cosmetics or dermatology.

However, the adjuvant and the mixing ratio thereof can be appropriately selected so as not to affect the preferable properties of the cosmetic composition according to the present invention.

The present invention will be further described with reference to the following examples, but it should be apparent that the present invention is not limited by the following examples.

Preparation Example: Preparation of Peptide (ITLQEIIRC)

Peptides were synthesized using conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group of amino acid. Amino acid residues were extended using N-hydroxybenzotriazole (HOBt) and N, N'-diisopropylcarbodiimide (DIC) as activators (Wang C. Chan , Perter D. White, 'Fmoc-solid phase peptide synthesis', Oxford).

The synthesized peptides were purified by C18 reverse phase high performance liquid chromatography (HPLC, Waters Associates, USA). The column was ACQUITY UPLC BEH300 C18 (2.1 x 100 mm, 1.7 탆, Waters Co, USA).

Experimental Example 1: Antioxidant activity test

Active oxygen decomposition effect

Free radical scavenging activity using DPPH (1, 1-diphenyl-2-picryl hydrazyl).

4 mL of methanol was added to the test tube, and the peptide was added by concentration. 1 mL of 0.15 mM DPPH solution was added, and the reaction was allowed to proceed at room temperature for 30 minutes. Then, the absorbance at 520 nm was measured.

Initial (Ai) and blank (Ab) without substrate and DPPH were measured, and α-tocopherol and BHT were used as positive control. The results are shown in Table 1 below.

division Peptides alpha-tocopherol BHT 0 mg / L 0 0 0 1 mg / L 53.2 45.1 36.9 10 mg / L 68.5 63.7 52.3 50 mg / L 88.3 82.8 66.4

 [DPPH erase rate%]

The peptides were evaluated to have higher free radical scavenging activity than α-tocopherol and BHT, which are well known in the art. Therefore, the degradation of active oxygen can be promoted by the peptide, and the result suggests that the antioxidative activity is effective in preventing skin aging and improving skin condition.

Measurement of reactive oxygen species in cells (DCF-DA assay)

The removal of reactive oxygen species (ROS) was assessed by comparing the amount of reactive oxygen species produced in the cells stimulated with silica after the peptide treatment with the negative control (Cho et al., 1999; Kim et al., 2002).

Raw 264.7 cells were inoculated into 96 well plates at a concentration of 1 × 10 ⁴ cells / well. After replacing the medium with fresh medium, cells were cultured for 24 hours by treatment with a certain concentration of the peptide.

10.0 μL of WST-1 solution (EZ-CYTOX, DOGEN, Korea) was added to each well, and the cells were incubated for 1 hour. The absorbance was measured at 450 nm and 655 nm using an ELISA reader Respectively.

Peptides were diluted with dimethylsulfoxide (DMSO, Sigma-Aldrich, USA) to a final concentration of 20 μM and DMSO was used as a blank test (negative control). The experiment was repeated 3 times to ensure reliability.

The experiment was carried out under the same cell line and the same cell culture conditions as the WST-1 assay, a cell activity assay. The cultured Raw 264.7 cells were washed once with phosphate buffered saline (PBS; sodium chloride 137 mM, phosphate buffer 10 mM, potassium chloride 2.7 mM, all from Biopure, Canada), and Raw 264.7 cells were harvested. The cells were suspended in 15 mL of PBS, and 15 μL of 20 mM 2 ', 7'-Dichlorofluorescein diacetate was added and incubated for 1 hour.

After centrifugation at 1200 rpm for 2 minutes, the cells were washed once with PBS solution, diluted to 1 × 10 ⁵ cells / mL, treated with the control substance and the peptide, and cultured for 10 minutes. 20 mg / mL of silica was treated with 50 μL and incubated for 1 hour. The cell pellet obtained by centrifuging at 6000 rpm for 5 minutes was dispersed in 200 μL PBS solution and inoculated into a 96-well plate.

Fluorescence was measured at 485 nm excitation / 535 nm emission using a fluorescence microplate reader to evaluate the amount of reactive oxygen species.

The peptides treated in the measurement of reactive oxygen species to the concentration of the test solution were used as the concentration of the test solution set through the cell activity measurement.

As a negative control, DMSO was used instead of the sample. As a positive control, ascorbic acid (Sigma-Aldrich) was diluted to 100 mg / mL in DMSO and used at a concentration of 100 μg / mL.

Based on the above results, a test for the measurement of reactive oxygen species at the same concentration was carried out, and the treated peptide was used at a concentration of 50 mg / L. The experiment was repeated 4 times to ensure reliability. The measurement results are shown in Table 2 below.

division Peptides Ascorbic acid Measures 71.4 74.7

 [ROS generation inhibition rate (% of control)]

The peptides were evaluated to have equivalent levels of ascorbic acid and free radical scavenging activity, which are well known in the art.

That is, the above results indicate that the peptides have excellent ability to remove reactive oxygen species, and that they have excellent antioxidative effects due to free radical scavenging activity.

Experimental Example 2: Anti-inflammatory activity test

NF-kB transcriptional repression effect

The NF-kB reporter assay was used to measure the inhibitory effect of NF-kB transcription. INOS, COX-2, IFN-? Or TNF-? Induced by LPS are expressed by the transcription factor NF-kB. The NF-kB is inactivated in complex with IkB in the cytoplasm, and when I-kB-α is phosphorylated by I-kB-α kinase, the bound complex is degraded and activated as NF-kB, )do. Induces the expression of the target gene of NF-kB and causes inflammation.

Also, TRIF, which acts on inflammatory diseases such as multiple sclerosis, is a receptor that responds to the activity of TLRs (toll-like-receptors) in the TIP-domain with receptors that induce interferon-beta, And activates the immune response to the antigen. The receptors recognize a well-preserved pathogenic pattern and activate the signal to stimulate the release of inflammatory cytokines. IRF-3 (interferon-stimulating factor 3) is a transcription factor induced by interferon alpha / beta stimulation, which binds to the interferon-stimulated response sequence (ISRE) in the transcriptional regulatory region of most interferon inducible genes and activates the transcription.

According to the manufacturer's protocol (Jin et al., 2009), 12-well plates were coated with NF-kB according to the presence of receptor molecules such as? -Galactosidase (MyD88 and TRIF) -Luc or TRIF was transfected into HEK293 (1 x ¹⁰⁶ cells / ml) cells and cultured for 24 hours. LPS and the peptide were treated 24 hours after the treatment with luciferase assay Luciferase assays were used to measure the activity of NF-kB and IRF-3 activity. The luciferase assay was performed using a luciferase assay system (Promega Co., USA) reported in the manufacturer's protocol (Jung et al., 2009).

division NF-kB activity IRF-3 activity Control group (Blank group) 1.00 1.00 LPS
100 ng / ml Peptides 0 mg / L 3.80 4.51 1 mg / L 2.63 2.91 10 mg / L 1.91 1.37 50 mg / L 1.62 1.20

 [Fold, Luciferase activity]

Referring to Table 3, the activity of NF-kB and the activity of IRF-3 were significantly decreased when the peptide was treated. These results suggest that the peptide effectively inhibits transcription of NF-kB, thereby inhibiting or alleviating inflammation and preventing and improving inflammatory diseases.

Quantification of inflammation-related protein mRNA

TNF-a, iNOS and COX-2 expression levels were assayed to evaluate the anti-inflammatory activity of the peptides.

RNA was prepared from RAW264.7 cells treated with LPS. The method was prepared using Trizol Reagent (Gibco BRL) as described in Lee et al., 2008. All RNAs were stored at -70 ° C before use. Semi-quantitative RT reactions were performed using MuLV reverse transcriptase.

All RNA (1 μg) was incubated with oligo-dT15 at 70 ° C for 5 minutes and mixed with 5 × first strand buffer, 10 mM dNTPs and 0.1 M DTT. The reaction mixture was further incubated at 37 ° C for 5 minutes and then incubated for 60 minutes after addition of MuLV reverse transcriptase (2 U). The reaction was terminated by incubating at 70 DEG C for 10 minutes. (Conditions: 2 μL cDNA, 4 μM 5 'and 3' primer, 10 × buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl and 0.1 % Triton X-100), 250 μM dNTPs, MgCl 2 25 mM , and Taq DNA polymerase 1unit (Promega, USA). PCR reactions after annealing and denaturation at 94 ℃ 30 seconds, 55 to from 60 ℃ 30 seconds, 72 Deg.] C for 42 seconds, and finally polymerized at 72 [deg.] C for 5 minutes.

The primers of TNF-a, iNOS, and COX-2 are shown in Table 4 below.

division Forward primer Reverse primer iNOS 5-FGGAGCCTTTAGACCTCAACAGA-3 5-RTGAACGAGGAGGGTGGTG-3 TNF-a 5-FTGCCTATGTCTCAGCCTCTTC-3 5-RGAGGCCATTTGGGAACTTCT-3 COX-2 5-FCACTACATCCTGACCCACTT-3 5-RATGCTCCTGCTTGAGTATGT-3

The results of measurement of mRNA expression levels by the above peptide treatment are shown in Table 5 below. Analysis of RAW264.7 cell mRNA revealed that the expression of TNF-α, iNOS, and COX-2 was increased by the addition of LPS. On the other hand, the expression of TNF-a, iNOS, and COX-2 induced by the LPS decreased in a concentration-dependent manner when the peptide was treated.

division iNOS
Expression level TNF-a
Expression level COX-2 expression level Control group (Blank group) 1.00 1.00 1.00 LPS
100 ng / ml Peptides 0 mg / L 3.55 2.90 3.30 1 mg / L 2.55 2.25 2.40 10 mg / L 2.05 1.75 2.15 50 mg / L 1.60 1.40 1.95

 [Fold, GAPDH normalized]

That is, the above results suggest that the peptides can be utilized as a skin improving agent due to their excellent anti-inflammatory and antioxidant activity.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

<110> AHN, INSOOK <120> PEPTIDE HAVING ANTI-INFLAMMATORY ACTIVITY AND SKIN CONDITION          IMPROVEMENT EFFECT, AND USES THEREOF <130> 16PP11168 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> SEQ ID NO 1 <400> 1 Ile Thr Leu Gln Glu Ile Ile Arg Cys   1 5

Claims (6)

A peptide for improving skin condition comprising the amino acid sequence of SEQ ID NO: 1. The method according to claim 1,
A peptide that inhibits the expression of inflammatory cytokines. The method according to claim 1,
Peptides with antioxidant activity. 1. A composition for improving skin condition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. 5. The method of claim 4,
Antioxidant, skin wrinkle improvement, skin whitening, anti-inflammation or skin regeneration. A composition for external application for external use comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.