Novel peptide exhibiting skin condition improving activity and cosmetic composition comprising same

Abstract

The present invention relates to a novel peptide exhibiting skin condition improving activity and a cosmetic composition having excellent skin calming, moisturizing and wrinkle improving effects comprising the same. The novel peptide of the present invention has excellent skin-calming, moisturizing, and wrinkle-improving effects, has a short amino acid sequence, is easy to absorb, and has few side effects, and thus can be widely used in cosmetic compositions for improving skin conditions.

Description

Technical Field

[0001] This invention relates to novel peptides exhibiting skin condition-improving activity and cosmetic compositions containing them that have excellent skin-soothing, moisturizing, and wrinkle-improving effects.

[0002] This application claims priority to Korean Patent Application No. 10-2023-0022568, filed on February 21, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0003] As the body's primary defense membrane, the skin protects internal organs from external environmental stimuli such as temperature and humidity changes, ultraviolet radiation, and pollutants, playing a vital role in maintaining biological homeostasis, including body temperature regulation. However, excessive physical and chemical stimuli applied to the skin's cells, as well as stress, nutritional deficiencies, ultraviolet radiation, environmental pollutants, and drastic temperature changes, can damage cells and induce various biochemical changes within them. This can accelerate aging phenomena on the skin's surface, such as dark spots, freckles, blemishes, loss of elasticity, keratinization, and wrinkles.

[0004] Aging causes wrinkles to form on the skin. Typical factors contributing to wrinkle formation include ultraviolet radiation exposure and reduced collagen biosynthesis. Skin aging is broadly divided into intrinsic aging caused by genetic factors and extrinsic aging caused by external environmental factors such as sunlight. Extrinsic aging is known to be prevented, treated, or delayed through methods such as removing reactive oxygen species, promoting fibroblast proliferation, and boosting collagen biosynthesis.

[0005] Against this technological backdrop, extensive research has been conducted on peptides and other substances that can improve skin condition.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Korean Patent Publication No. 10-2022-0156701 (November 28, 2022) Summary of the Invention

[0009] Technical issues

[0010] Thus, as a result of the inventors’ extensive research efforts to develop peptide materials that can be used in cosmetic compositions, they discovered that novel synthetic peptides exhibit excellent skin-soothing, moisturizing, and wrinkle-improving effects, thereby completing this invention.

[0011] Therefore, the object of the present invention is to provide novel peptides and cosmetic compositions comprising them for skin soothing, moisturizing and wrinkle improvement.

[0012] Technical solution

[0013] The present invention provides isolated peptides represented by the amino acid sequences numbered below.

[0014] SEQ ID NO: 1: YGSD

[0015] Furthermore, the present invention provides a polynucleotide encoding the peptide.

[0016] On the other hand, the present invention provides cosmetic compositions comprising the peptide or the polynucleotide.

[0017] The cosmetic composition can be used for skin soothing, moisturizing, or wrinkle improvement purposes.

[0018] The effects of the invention

[0019] The novel peptides of this invention have excellent skin-soothing, moisturizing and wrinkle-improving effects. They have short amino acid sequences that are easily absorbed and have few side effects, so they can be widely used in cosmetic compositions for improving skin condition. Attached Figure Description

[0020] Figure 1 The results show the molecular weight analysis of the peptides prepared in the examples.

[0021] Figure 2 A graph to confirm the intracellular nitric oxide (NO) production of the peptides prepared in the treatment examples.

[0022] Figure 3 A graph to confirm the intracellular hyaluronic acid production of the peptides prepared in the processing examples.

[0023] Figure 4 A graph to confirm the intracellular type 1 procollagen peptide production of the peptides prepared in the treatment examples. Detailed Implementation

[0024] The present invention will now be described in detail. It should be noted that the terms or vocabulary used in this specification and the scope of the invention should not be limited to their commonly understood or dictionary meanings. Rather, they should be interpreted in accordance with the principle that the inventor can appropriately define the concepts of terms to best describe their invention, and should be interpreted as conforming to the technical concept of the present invention. Therefore, the structures described in the embodiments of this specification are merely the most preferred embodiments of the present invention and do not represent all the technical concepts of the present invention. It should be understood that, at the time of filing this application, there are many equivalents and modifications that can replace them.

[0025] The novel peptide of the present invention may contain the sequence of SEQ ID NO: 1.

[0026] SEQ ID NO: 1: YGSD

[0027] According to the IUPAC-IUB nomenclature, the amino acids mentioned in this specification can be referred to by the following abbreviations.

[0028] Y: Tyrosine (Tyr)

[0029] G: Glycine (Gly)

[0030] S: Serine

[0031] D: Aspartic acid (Asp)

[0032] In this invention, the term "peptide or fragment thereof" refers to a polymer consisting of two or more amino acids linked by an amide bond (or peptide bond).

[0033] To achieve chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broad-spectrum bioactivity), and reduced antigenicity, a protecting group may be bound to the N-terminus or C-terminus of the peptide. In one specific example, the N-terminus of the peptide may be bound to any protecting group selected from the group consisting of acetyl, fluoreonylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl, butoxycarbonyl, allyloxycarbonyl, and polyethylene glycol (PEG), and / or the C-terminus of the peptide may be bound to any protecting group selected from the group consisting of amino (-NH2), tertiary alkyl, and azide (-NHNH2). Furthermore, the peptide may selectively include a targeting sequence, a tag, labeled residues, or an amino acid sequence prepared for a specific purpose, such as increasing half-life or peptide stability.

[0034] The peptides of the present invention can be synthesized by methods well known in the art, such as automated peptide synthesizers, or produced by genetic engineering techniques, but are not limited thereto.

[0035] Furthermore, the present invention provides a polynucleotide encoding the peptide. The peptide can be obtained in large quantities by inserting the polynucleotide encoding the peptide into a vector and expressing it.

[0036] On the other hand, the present invention provides cosmetic compositions comprising the peptide or the polynucleotide.

[0037] The cosmetic composition can be used for skin soothing, moisturizing, or wrinkle improvement purposes.

[0038] The cosmetic compositions of the present invention may contain ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, as well as carriers.

[0039] The cosmetic compositions of the present invention can be prepared in any dosage form commonly prepared in the technical field to which the present invention pertains, for example, as solutions, suspensions, emulsions, pastes, gels, creams, emulsions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, and sprays, but are not limited thereto.

[0040] More specifically, it can be prepared into dosage forms such as softening lotion, nourishing lotion, nourishing cream, massage cream, serum, eye cream, facial foam, facial cleanser, mask, spray or powder.

[0041] When the dosage form of the present invention is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, astragalus gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide can be used as carrier components.

[0042] When the dosage form of the present invention is a powder or spray, lactose, talc, titanium dioxide, aluminum hydroxide, calcium silicate or polyamide powder can be used as carrier components. Especially in the case of spray, it can also contain propellants such as chlorofluorocarbons, propane / butane or dimethyl ether.

[0043] When the dosage form of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier can be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, fatty acid glycerides, polyethylene glycol or fatty acid esters of sorbitol.

[0044] When the dosage form of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene dehydrated sorbitan ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar or astragalus gum, etc., can be used as carrier components.

[0045] In the case where the dosage form of the present invention is a facial cleanser containing surfactants, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurine, sarcosinate, fatty acid amide ether sulfates, alkylamide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters are used as carrier components.

[0046] When the cosmetic composition of the present invention is a soap, a surfactant-containing facial cleanser, or a surfactant-free facial cleanser, it can be wiped off, peeled off, or washed off with water after being applied to the skin. Specifically, the soap can be a liquid soap, powdered soap, solid soap, or oil-based soap; the surfactant-containing facial cleanser can be a facial foam, facial toner, facial wipe, or facial mask; and the surfactant-free facial cleanser can be a facial cream, facial lotion, facial toner, or facial gel, but is not limited thereto.

[0047] Hereinafter, embodiments and experimental examples will be described in detail to specifically illustrate the present invention. However, the embodiments of the present invention can be modified into many other forms, and the scope of the present invention should not be construed as limited to the embodiments detailed below. The embodiments of the present invention are provided merely to provide a more comprehensive explanation of the present invention to those skilled in the art.

[0048] Example: Preparation of peptides

[0049] A peptide with the amino acid sequence YGSD (Tyr-Gly-Ser-Asp) of SEQ ID NO: 1 was synthesized using an automated peptide synthesizer (PeptrEX, Peptron Corporation, South Korea). Using C... 18 The purity of the synthesized peptide was confirmed to be 95% by reverse high-performance liquid chromatography (HPLC, SHIMADZU Prominence, Japan). The molecular weight (441) was confirmed by mass spectrometry (Shimadzu LCMS-2020). The results are as follows: Figure 1 As shown.

[0050] The column used for the analysis was a Shiseido Capcell Pak C18 (4.6 × 50 mm, 5 μm, Shiseido, Japan), prepared by dissolving the sample in water at a concentration of 1 mg / mL. The mobile phase consisted of distilled water containing 0.1% trifluoroacetic acid (TFA) and acetonitrile containing 0.1% TFA, at a flow rate of 1.0 mL / min. A UV spectrophotometer (220 nm) was used for detection. Elution conditions were performed in gradient mode by varying the proportion of acetonitrile containing 0.1% TFA for 17 minutes.

[0051] Experimental Example 1: Cell Culture

[0052] Culture macrophages

[0053] RAW264.7 cells, used as macrophages, were obtained from the American Type Cell Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 100 units / mL of penicillin-streptomycin and 10% fetal bovine serum (FBS) at 37°C and 5% CO2, with passages performed at 2-3 day intervals.

[0054] Culture skin keratinocytes

[0055] HaCaT cells, used as keratinocytes, were obtained from Cell Line Service GmbH (Germany). They were cultured in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum in a 37°C, 5% CO2 incubator, and passaged at 2-3 day intervals.

[0056] Culture skin fibroblasts

[0057] Normal human dermal fibroblasts were obtained from Lonza Walkersville, Inc. They were cultured in fibroblast basal medium (FBM) containing 0.1% hFGF-B, insulin, GA-1000, and 2% fetal bovine serum at 37°C and 5% CO2, and passaged at 2-3 day intervals.

[0058] Experimental Example 2: Evaluation of Cytotoxicity

[0059] EZ-cytox analysis (assay)

[0060] The EZ-cytox assay is a typical method for measuring cell viability by utilizing the principle that a water solution of tetrazolium salt (WST) reacts with the dehydrogenase of living cells to generate orange water-soluble formazan.

[0061] Assess cytotoxicity to macrophages

[0062] To confirm cytotoxicity, DMEM medium supplemented with 10% FBS was used at a concentration of 1.5 × 10⁻⁶. 4 RAW264.7 cells were seeded in 96-well plates and cultured for 18 hours at 37°C and 5% CO2. The cultured cells were then treated with the sample to be evaluated. EZ-cytox was then added to each well and reacted at 37°C and 5% CO2 for 30 minutes. The absorbance was measured at 450 nm using a microplate reader. The average absorbance value for each sample group was calculated and compared with the absorbance value of the control group to assess cell viability. No cytotoxicity was observed at the evaluated concentrations.

[0063] Assess cytotoxicity to skin keratinocytes

[0064] To confirm cytotoxicity, DMEM medium supplemented with 10% FBS was used at a concentration of 1.0–1.5 × 10⁻⁶. 4HaCaT cells were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. The cultured cells were then replaced with serum-free DMEM and the sample to be evaluated was processed. EZ-cytox was then added to each well and reacted at 37°C and 5% CO2 for 30 minutes. The absorbance was measured at 450 nm using a microplate reader. The average absorbance value for each sample group was calculated and compared with the absorbance value of the control group to assess cell viability. No cytotoxicity was observed at the evaluated concentrations.

[0065] Assess cytotoxicity to skin fibroblasts

[0066] To confirm cytotoxicity, DMEM medium supplemented with 10% FBS was used at a concentration of 1.0 × 10⁻⁶. 4 NHDF cells were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. The cultured cells were then replaced with serum-free FBM and the sample to be evaluated was treated. EZ-cytox was then added to each well and reacted at 37°C and 5% CO2 for 30 minutes. The absorbance was measured at 450 nm using a microplate reader. The average absorbance value for each sample group was calculated and compared with the absorbance value of the control group to assess cell viability. No cytotoxicity was observed at the evaluated concentrations.

[0067] Experiment 3: Confirmation of skin soothing effect (nitric oxide (NO) production inhibition effect)

[0068] In 24-well plates, at a concentration of 1.5 × 10⁻⁶ 5 After seeding RAW264.7 cells / wells, they were cultured at 37°C and 5% CO2 for 18 hours. The prepared peptides from the examples were treated with the cultured cells at concentrations of 50-200 μM and cultured for 24 hours. 100 μL of supernatant from each experimental group was recovered from a 96-well plate, and 100 μL of Griess reagent was added. The reaction was carried out at room temperature for 10 minutes. The absorbance was then measured at 540 nm using a microplate reader. The control group was treated with 2-amino-4-methylpyridine (2-AM) at a concentration of 2 μM. The relative amount of NO generated by the treatments in the examples was measured based on the NO generation of the stimulant (lipopolysaccharide (LPS)). The results are shown in Table 1 below. Figure 2 As shown.

[0069] Table 1

[0070] As shown in Table 1 and Figure 2As shown, the peptide concentration in the embodiment confirms that NO generation is inhibited in a concentration-dependent manner.

[0071] Experiment 4: Confirming the effect of increasing the production of the moisturizing factor hyaluronic acid (HA).

[0072] In 24-well culture plates, at 1.0 × 10 5 After HaCaT cells were seeded into cells / wells, they were cultured at 37°C and 5% CO2 for 24 hours. The culture medium was replaced with serum-free DMEM, and the peptides from the examples were treated with 50–200 μM and cultured for 24 hours. Then, the supernatant from each experimental group was used to measure the amount of free hyaluronic acid (HA) produced in the culture medium. The control group was treated with 10 mM N-acetylglucosamine. The hyaluronic acid production was measured using a hyaluronic acid ELISA kit (Cusabio Biotechnology Co., Ltd.) according to the manufacturer's instructions. The hyaluronic acid production results after treatment in the examples are shown in Table 2 below. Figure 3 As shown.

[0073] Table 2

[0074] As shown in Table 2 and Figure 3 As shown, when the peptides of the embodiments are processed, the amount of hyaluronic acid produced increases, confirming that the peptides of the present invention exhibit excellent moisturizing effects.

[0075] Experimental Example 5: Confirmation of the generation of wrinkle-improving factor type I procollagen peptide (PIP)

[0076] In 24-well culture plates, at 2.0 × 10 4After seeding NHDF cells into wells, the cells were cultured at 37°C and 5% CO2 for 24 hours. The culture medium was replaced with serum-free FBM medium, and the peptides from the examples were treated with a concentration of 1–100 μM and cultured for 24 hours. Then, the supernatant from each experimental group was used to measure the amount of free procollagen in the culture medium. The control group was treated with ascorbic acid at a concentration of 0.001 v / v%. The amount of procollagen was measured using a procollagen type I peptide (PIP) EIA kit (Takara Biomedical Co.), according to the method provided by the manufacturer. The quantitative results of the type I procollagen peptide production after treatment in the examples are shown in Table 3 below. Figure 4 As shown.

[0077] Table 3

[0078] As shown in Table 3 and Figure 4 As shown, when the peptides of the embodiments are treated, the amount of procollagen production increases, confirming that the peptides of the present invention exhibit excellent wrinkle improvement and anti-aging effects.

Claims

1. A separated peptide, characterized in that, Represented by the amino acid sequence of SEQ ID NO:

1.

2. A polynucleotide, characterized in that, Encoding the peptide as described in claim 1.

3. A cosmetic composition, characterized in that, It comprises the peptide of claim 1 or the polynucleotide of claim 2.

4. The cosmetic composition according to claim 3, characterized in that, The composition has excellent skin-soothing, moisturizing, and wrinkle-improving effects.

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