KR102346006B1 - Pentapeptide derived from spider web and composition for anti-aging containing the same - Google Patents

Abstract

The present invention relates to spider silk protein-derived peptides and an anti-aging composition containing the same as an active ingredient. The peptides according to the present invention and a pharmaceutically acceptable salt thereof exhibit an excellent anti-wrinkle effect, a wound healing promoting effect, or a skin regenerating effect, thereby being usefully used in the preparation of the anti-aging cosmetic composition or pharmaceutical composition.

Description

Spider silk-derived pentapeptide and composition for anti-aging containing the same {Pentapeptide derived from spider web and composition for anti-aging containing the same}

The present invention relates to a protein-derived peptide constituting the spider silk and an anti-aging composition containing the same, and more particularly, to a pentapeptide derived from a protein constituting the spider silk and excellent anti-aging, skin regeneration and wound healing by containing it as an active ingredient. It relates to a composition exhibiting a accelerating effect.

The skin is an organ that has a protective function, a barrier function, a temperature control function, an excretory function, a respiratory function, and the like, and consists of the epidermis, dermis, and subcutaneous fat. The epidermis is the thinnest layer and consists of organic bonds between keratinocytes and melanocytes. The dermis occupies about 95% of the skin and is a layer responsible for moisturizing and protecting the skin. Collagen and elastin, which are representative proteins that play an important role in skin elasticity (wrinkle), are distributed like a net, and blood vessels and nerves exist. , mast cells involved in allergic reactions, and natural moisturizing factors such as Na-PCA or hyaluronic acid. Subcutaneous fat plays the role of supplying nutrients to the epidermis and dermis, determining body shape, maintaining body temperature, absorbing external shocks, and protecting cells under the subcutaneous fat.

Such skin undergoes aging in which the skin function rapidly deteriorates due to intrinsic or extrinsic causes as the skin ages. As aging progresses, the epidermis, dermis, and subcutaneous fat, which are components of the skin, become thinner, and collagen and elastin become thinner and loose, resulting in loss of elasticity and wrinkles. In addition, with aging or exposure to ultraviolet rays, liposynthesis in subcutaneous fat cells is suppressed and the skin protection function is lost, indicating rapid skin aging, and the beauty of beauty such as tissue sagging and formation of wrinkles in the face, breast and buttocks also cannot be sought.

Many methods have been researched to solve this deterioration of skin function, and the most important part of them is to find a method for promoting collagen biosynthesis in skin cells. For this purpose, vitamin C and its derivatives, KTKS peptide, copper peptide, and various natural extracts are being selected and used. However, vitamin C and its derivatives have disadvantages of poor stability and low long-term activity, and KTKS peptide and copper peptide have disadvantages in which the collagen biosynthesis enhancing effect is lowered. The difference in activity has a very different disadvantage. Accordingly, there is an urgent need to discover a new concept of collagen biosynthesis enhancing material that can overcome all these shortcomings.

The present inventors have made efforts to develop such a material, and as a result of conducting an experiment to confirm anti-aging activity including wrinkle improvement activity for various types of peptides, in the case of a specific pentapeptide derived from spider silk, strong wound healing (wound) The present invention was completed by confirming that the ability to produce collagen in fibroblasts is excellent along with the healing) promoting effect.

(0001) Republic of Korea Patent Registration No. 10-1565542 (2015.10.28) (0002) Republic of Korea Patent Registration No. 10-1508614 (2015.03.30)

An object of the present invention is to provide a novel peptide excellent in skin wrinkles improvement, skin regeneration and wound healing promoting effects.

In addition, another object of the present invention is to provide a cosmetic composition containing the peptide and exhibiting an excellent skin wrinkle improvement effect, wound improvement or skin regeneration effect.

In addition, it is another object of the present invention to provide a pharmaceutical composition containing the peptide to exhibit an excellent skin wrinkle improvement effect, wound healing or skin regeneration effect.

According to the present invention in order to achieve the above object, there is provided a peptide or a pharmaceutically acceptable salt thereof consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The peptide is derived from Spider silk protein.

According to the present invention to achieve the other object, there is provided a cosmetic composition for improving skin wrinkles containing the peptide or a pharmaceutically acceptable salt thereof. In addition, there is provided a cosmetic composition for wound improvement or skin regeneration containing the peptide or a pharmaceutically acceptable salt thereof.

According to the present invention in order to achieve the above another object, there is provided a pharmaceutical composition for improving skin wrinkles containing the peptide or a pharmaceutically acceptable salt thereof. In addition, there is provided a pharmaceutical composition for wound healing or skin regeneration containing the peptide or a pharmaceutically acceptable salt thereof.

The spider silk-derived pentapeptide of the present invention has excellent skin wrinkle improvement, wound healing promotion and skin regeneration effects, and can effectively exert its original function without side effects such as skin irritation when applied to the human body, thereby preventing skin aging, wound healing, and skin It can be very usefully used in the manufacture of external pharmaceuticals and cosmetics for regenerative use.

1 shows the results of measuring the cytotoxicity of the peptide according to the present invention.
2 and 3 show the wound healing promoting effect of the peptide according to the present invention.
4 and 5 show the measurement results for the collagen biosynthesis enhancing effect of the peptide according to the present invention.

Hereinafter, the present invention will be specifically described.

According to the present invention, there is provided a peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof.

The peptide is a pentapeptide derived from a protein constituting the spider silk. A spider's web is a structure made by ejecting a spider from an exit projection, and it has good elasticity and a tensile strength that is five times better than iron of the same thickness. Spider silk is made by a complex linkage of spider silk protein. Conventionally, studies on the skin improvement activity of spider silk extract have been actively conducted, but it has been difficult to use due to a difficult extraction process and problems inducing skin allergies.

The present invention is to overcome these difficulties, and the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 according to the present invention and a pharmaceutically acceptable salt thereof have a collagen production promoting effect and wound healing promoting effect. It is characterized by excellent technical characteristics.

In the present invention, the 'pharmaceutically acceptable salt thereof' includes salts derived from pharmaceutically acceptable inorganic acids, organic acids or bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid , benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium and ammonium and the like.

The peptide can be prepared in the following way. Representative synthesis methods are described below, but those skilled in the art can use it with appropriate modifications within the range of techniques known in the art.

According to one embodiment of the present invention, it is synthesized using the Fmoc-chemistry synthesis method according to general solid phase peptide synthesis (SPPS).

The solid-phase peptide synthesis method comprises the steps of 1) loading a protected amino acid into a resin; 2) removing the protecting group of the amino acid; 3) inducing a coupling reaction of amino acids; 4) checking whether the reaction is present (eg, Kaiser test); 5) removing the resin and protecting group; And 6) the synthesis was carried out according to a total of 6 steps of solidifying the peptide.

Hereinafter, the synthesis method will be described in more detail step by step.

Step 1 is a step of sequentially loading amino acids into the resin. Wang resin, chlorotrityl resin, polystyrene resin, etc. may be used as the resin, and an appropriate solvent is added to the resin to swell the resin. For example, methylene chloride (MC) may be used as the solvent, but is not limited thereto. Next, the solvent is removed under reduced pressure, and the protected amino acids dissolved in an appropriate solvent are mixed with a mixed solution of DIC (diisopropylcarbodiimide) and DMAP (4-dimethylaminopyridine) in the order of Glycine, Serine, Glycine, Proline, and Glycine or Glutamine. It is reacted by adding it to a reaction vessel containing the swollen resin. As the solvent, for example, dimethylformamide (DMF) may be used, and Fmoc (9-Fluorenylmethoxycarbonyl) may be used as a protecting group of an amino acid, but is not limited thereto.

Step 2 is the step of removing the protecting group of the amino acid. Removal of the amino acid protecting group may be performed according to a method generally known in the art, and in a specific embodiment of the present invention, the amino acid solution loaded on the resin is removed under reduced pressure and washed, followed by washing with a DMF solution diluted with piperidine. The reaction was carried out to remove the protecting group.

Step 3 is a step inducing a coupling reaction of amino acids. The amino acid coupling reaction may be performed according to a method generally known in the art, for example, N-hydroxybenzotriazole-dicyclohexylcarodiimide (HOBt-DCC) method or N-hydroxybenzotriazole-diisopropylcarbodiimide (HOBt-DIC) method (Wang C. Chan). , Perter D. white, "Fmoc solid phase peptide synthesis", Oxford). In addition, coupling reagents N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), O-benzotriazol-1-yl-N,N, N',N'-tetramethyluronium hexafluorophosphate (HBTU), benzo-triazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (PyBOP), benzo-triazol-1-yl Oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), [O-(7-azabenzotri-azol-1-yl)-N,N,N',N'-tetramethyluroniumhexafluoro -Phosphate] (HATU), lH-hydroxy-benzotriazole (HOBt), lH-hydroxy-7-azabenzotriazole (HOAt), etc. can be used for synthesis, and depending on the coupling reagent, trifluoro An organic base such as roacetic acid (TFA), diisopropylethylamine (DIPEA), N-methylmorpholine (NMM), etc. may be added to proceed the reaction, but is not limited thereto.

Step 4 is a step to check whether a reaction is present. For example, in a specific embodiment of the present invention, the Kaiser test was used to confirm the coupling reaction of amino acids. The Kaiser test is a qualitative confirmation method that uses ninhydrin to confirm the presence or absence of a functional group of a primary amine by a difference in color change. Specifically, after adding 2-3 drops of Kaiser test solution to a small amount of resin washed after the amino acid coupling reaction, observe the color change of the resin for a certain period of time. If there is no change in the color of the resin, the coupling reaction is considered Then, the next reaction is carried out, and when the color of the resin is blue, it is considered that the unreacted part remains and the amino acid coupling reaction can be performed again.

Step 5 is the step of removing the resin and protecting group. This is a step of removing the synthesized peptide from the resin by repeating steps 3 to 5, and deprotecting the amino acid side chain protecting group. Removal of the resin and the amino acid protecting group may be performed according to a method generally known in the art, and in a specific embodiment of the present invention, trifluoroacetic acid (TFA), triisopropylsilane (TIS), thioanisole, H ₂ O , by adding a cleavage cocktail solution consisting of EDT (ethaneedithiol) to obtain a peptide solution. The composition of the solution may be appropriately modified and used by those skilled in the art according to experimental conditions.

Step 6 is the step of solidifying the peptide. For example, an excess of diethyl ether solvent may be added to form a precipitated solid, but is not limited thereto.

The peptide according to the present invention prepared as described above and a pharmaceutically acceptable salt thereof do not have side effects such as cytotoxicity to skin fibroblasts (FIG. 1), and have excellent wound healing effect in skin fibroblasts was shown (FIG. 2, FIG. 3), and also showed a strong ability to promote collagen biosynthesis (FIG. 4, FIG. 5).

According to the present invention, there is provided a cosmetic composition for skin wrinkle improvement, wound improvement or skin regeneration comprising the peptide or a pharmaceutically acceptable salt thereof as an active ingredient.

The content of the peptide or its salt as an active ingredient can be appropriately adjusted depending on the use, application form, purpose of use, and desired effect of the composition, and in consideration of the effect compared to the content, for example, 0.0001 to 99% by weight based on the total weight of the composition, Preferably, it is 0.001 to 50% by weight, more preferably 0.001 to 1% by weight, and most preferably 0.005 to 0.1% by weight.

The cosmetic composition may be administered orally or parenterally, for example, oral, transdermal, subcutaneous, or intravenous administration is possible, preferably transdermal administration during parenteral administration, more preferably topical administration (topical administration) application) can be applied.

The composition is applied percutaneously to the skin, scalp or hair, and refers to a composition that can be used in the manufacture of all cosmetic products such as basic cosmetics, creams for chest and buttocks, makeup cosmetics, body products, shaving products, and hair products. , warning agents, sprays, suspensions, emulsions, creams, gels, foams, etc. may be formulated in the form, but there is no particular limitation on the form.

Preferably, the cosmetic composition is a softening lotion, astringent lotion, nutrient lotion, nutrition cream, massage cream, eye cream, eye essence, essence, cleansing cream, cleansing lotion, cleansing foam, cleansing water, pack, powder, body lotion, body Creams, body essences, body washes, sunscreen creams, hair dyes, shampoos, rinses, toothpastes, mouthwashes, hair dressings, hair conditioners, lotions, ointments, gels, creams, patches and sprays, including those having a formulation selected from the group consisting of do.

According to the present invention, there is provided a pharmaceutical composition for skin wrinkle improvement, wound healing or skin regeneration comprising the peptide or a pharmaceutically acceptable salt thereof as an active ingredient.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the degree of disease, drug form, and time, but may be appropriately selected by those skilled in the art, and the daily dosage of the composition is preferably 0.001 mg/kg to 50 mg/kg, and may be administered once a day or divided into several times a day as needed.

The cosmetic composition and pharmaceutical composition of the present invention are, in addition to the active ingredients, all kinds of ingredients that can be used for conventional commercialization or formulation, such as fragrances, pigments, bactericides, antioxidants, preservatives, humectants, thickeners, excipients, diluents, inorganic Salts and synthetic polymers may be additionally included, and the type and content may be appropriately adjusted according to the purpose and purpose of the final product.

In addition, the composition of the present invention may include a solvent commonly included in the form to be applied, for example, ethanol, glycerin, butylene glycol, propylene glycol, polyethylene glycol, 1,2,4-butanetriol, sorbitol Ester, 1,2,6-hexanetriol, benzyl alcohol, isopropanol, butanediol, diethylene glycol monoethyl ether, dimethyl isosorbide, N-methyl-2-pyrrolidone, propylene carbonate, glycerose-26, methyl One selected from glucose-20, isocetyl myristate, isocetyl octanoate, octyldodecyl myristate, octyldodecanol, isostearyl isostearate, cetyl octanoate and neopentyl glycol dicaprate, etc. may include more than one. In the case of preparing the composition of the present invention using such a solvent, the solubility of the compound in the solvent is slightly different depending on the type of compound or the mixing ratio of the solvent. It can be applied by appropriately selecting the type and amount of use.

In addition, the composition of the present invention may contain various substances for enhancing transdermal penetration upon transdermal administration. For example, laurocepram derivatives and ester derivatives of oleic acid, monooleate derivatives, adapalene, tritinoin, retinaldehyde, tazarotene, salicylic acid, azilaic acid, glycolic acid, ethoxydiglycol, tween 80, lecithin organogel, and the like. In addition, the composition of the present invention is a co-surfactant, a surfactant, an anti-dandruff agent, an emollient, a blood circulation promoter, and a cell activator within a range that does not impair the effect of the composition that imparts a moisturizing effect of the present invention in order to impart an additional function. , a refreshing agent, a moisturizer, an antioxidant, a pH adjuster, purified water, etc. may be added, and may contain additives such as an appropriate flavoring agent, colorant, preservative, and excipient depending on the form to be applied.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1: Preparation of GPGSG peptide

In the present invention, a peptide derivative was obtained using a solid-phase synthesis method using Fmoc (9-luorenylmethoxycarbonyl) as a protecting group of Nα-amino acid as a general peptide synthesis method, and specific examples thereof are as follows.

First, the amount of Wang resin (1.1 mmol/g, Novabiochem Corporation) with respect to 1 mmol was weighed and placed in a reaction vessel, and then 30 ml of MC was added to swell the resin for 10 minutes. The solvent was removed under reduced pressure, and the amino acids dissolved in the DMF solvent were added in the order of Fmoc-Glycine, Fmoc-Serine, Fmoc-Glycine, Fmoc-Proline, and Fmoc-Glycine (each 4 eq.), DIC (4 eq.; Diisopropylcarbodiimide) & HOBt (4 eq.; N-hydroxybenzotriazole) coupling solution was added, and the reaction was performed at room temperature for 4 hours to induce an amino acid coupling reaction. In each reaction step, the amino acid solution that was not bound to the resin was removed under reduced pressure, the resin was washed with DMF and MC for 30 ml X 5 times each, and the amino acid-coupled resin was treated with 20% (v/v%) piperidine. After deprotection by reacting for 10 minutes with DMF solution diluted in After the amino acid coupling reaction, Kaiser Test Solutions A, B, C [Reagent A: Ninhydrin (5 g) in ethanol (100 ml), Reagent B: Phenol (80 in ethanol (20 ml)) g), Reagent C: 0.1M KCN (2 ml) in pyridine (98 ml)] was added 2-3 drops, and the color change of the resin was observed while maintaining 100° C. for 10 minutes. If there was no change in the color of the resin, the coupling reaction was considered to have progressed and the next reaction was carried out.

In order to remove the peptide synthesized in this way from the resin and deprotect the amino acid side chain protecting group, it is composed of TFA (trifluoroacetic acid), TIS (triisopropylsilane), thioanisole, H ₂ O, and EDT (ethaneedithiol). The mixture was added in a ratio of 90:2.5:2.5:2.5:2.5 to produce a precipitated solid, and the precipitate thus obtained was centrifuged _{to remove TFA, TIS, thioanisole, H 2} O, EDT, etc. of the filtrate and , was repeated three or more times to obtain a GPGSG peptide (SEQ ID NO: 1, spider pentapeptide-1).

Example 2: Preparation of GPGSQ peptide

In the present invention, a peptide derivative was obtained using a solid-phase synthesis method using Fmoc (9-luorenylmethoxycarbonyl) as a protecting group for Nα-amino acid as a general peptide synthesis method, and specific examples thereof are as follows.

First, the amount of Wang resin (1.1 mmol/g, Novabiochem Corporation) with respect to 1 mmol was weighed and placed in a reaction vessel, and then 30 ml of MC was added to swell the resin for 10 minutes. The solvent was removed under reduced pressure, and the amino acids dissolved in the DMF solvent were mixed with DIC (4eq.; Diisopropylcarbodiimide) & HOBt (4eq.; N-hydroxybenzotriazole) coupling solution was added, and the reaction was performed at room temperature for 4 hours to induce an amino acid coupling reaction. In each reaction step, the amino acid solution that was not bound to the resin was removed under reduced pressure, the resin was washed with DMF and MC for 30 ml X 5 times each, and the amino acid-coupled resin was treated with 20% (v/v%) piperidine. After deprotection by reacting for 10 minutes with DMF solution diluted in After the amino acid coupling reaction, Kaiser Test Solutions A, B, C [Reagent A: Ninhydrin (5 g) in ethanol (100 ml), Reagent B: Phenol (80 in ethanol (20 ml)) g), Reagent C: 0.1M KCN (2 ml) in pyridine (98 ml)] was added 2-3 drops, and the color change of the resin was observed while maintaining 100° C. for 10 minutes. If there was no change in the color of the resin, the coupling reaction was considered to have progressed and the next reaction was carried out.

In order to remove the peptide synthesized in this way from the resin and deprotect the amino acid side chain protecting group, it is composed of TFA (trifluoroacetic acid), TIS (triisopropylsilane), thioanisole, H ₂ O, and EDT (ethaneedithiol). The mixture was added in a ratio of 90:2.5:2.5:2.5:2.5 to produce a precipitated solid, and the precipitate thus obtained was centrifuged _{to remove TFA, TIS, thioanisole, H 2} O, EDT, etc. of the filtrate and , was repeated three or more times to obtain a GPGSQ peptide (SEQ ID NO: 2, spider pentapeptide-2).

Test Example 1: Measurement of cytotoxicity of spider pentapeptides

In order to examine the cytotoxicity of the peptides synthesized in Examples 1 and 2, an experiment was conducted as follows. Cytotoxicity was measured using MTT assay, and relative cytotoxicity was calculated as 100% of the sample not added as a control. The detailed experimental method is as follows.

Normal human fibroblasts (Human Dermal Fibroblast neonatal) were uniformly aliquoted in a 96-well plate for cell culture at the number of 3,000 cells, and in DMEM (Dulbecco's Modified Eagle Media, Gibco BRL) containing 10% Fetal bovine serum (FBS). Incubated in an incubator at 37° C., 5% CO _{2 conditions for 24 hours.} Dissolve each test substance in water at a concentration of 10mM to make a concentrate, and dilute it with DMEM containing 0.5% FBS to a concentration of 100uM and 10uM. After each treatment, cultured for 48 hours, cytotoxicity was measured.

In the case of MTT assay for cytotoxicity measurement, 20ul each of 3-(4,5-dimethylthiazo1-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dissolved in PBS at 5mg/ml in each well was added, and reacted for 3 hours at 37° C., 5% CO _{2 conditions.} Thereafter, the supernatant was carefully removed, and the intracellularly precipitated MTT formazan was dissolved in 100ul DMSO, and then the absorbance at OD 570 was measured using an ELISA reader.

The results obtained above are shown in FIG. 1 . As can be seen from Figure 1, the peptides synthesized in Examples 1 and 2 of the present invention had no toxicity to cells.

Test Example 2: Measurement of wound healing promoting ability of spider pentapeptide

In order to examine the wound healing promoting ability of the peptides synthesized in Examples 1 and 2, an experiment was conducted as follows. To measure the degree of wound healing, after forming an artificial wound on the cells, the degree of recovery was calculated by analyzing the difference in volume from the initial wound after image analysis by photographing. The detailed experimental method is as follows.

After putting two culture-inserts in a 12-well plate for cell culture of human dermal fibroblast neonatal cells, the cells were uniformly dispensed at the number of 300,000 cells per well, and 10% Fetal bovine serum (FBS) was added. DMEM (Dulbecco's Modified Eagle Media, Gibco BRL) for 24 hours at 37 ℃, 5% CO ₂ Conditions were cultured in an incubator. After removing the culture-insert, the test substance was treated at a concentration of 100 uM and incubated for 48 hours, and the degree of cell migration was measured by taking a picture of each well, which was analyzed using image-j software.

The results obtained above are shown in FIGS. 2 and 3 . As can be seen from FIGS. 2 and 3, the peptides synthesized in Examples 1 and 2 showed a strong wound healing promoting effect, indicating that the wound healing or skin regeneration effect was excellent.

Test Example 3: Measurement of the ability of spider pentapeptide to promote collagen biosynthesis

In order to examine the ability of the peptides synthesized in Examples 1 and 2 to promote collagen biosynthesis, an experiment was conducted as follows. The biosynthesized collagen was measured using the Masson's Trichrome staining method, and the relative ability to generate collagen bundles was calculated with 100% of the sample not added as a control. The detailed experimental method is as follows.

Human normal skin tissue (native skin, Genoskin) fixed with agarose medium was transferred to a 12 well plate containing 1 ml of maintenance medium (Genoskin), and then cultured in an incubator at _{37 ° C., 5% CO 2 conditions for 12 hours.} After transferring the cultured tissue to a new 12-well plate containing 1 ml each of the maintenance medium, carefully process 20 μl of each sample dissolved in the maintenance medium. While culturing this at 37 ° C., 5% CO ₂ conditions, the maintenance medium and each sample were replaced in the same way once a day, and cultured for 48 hours. After completion of the culture, the tissue was washed with PBS (Gibco BRL), placed in 4% neutral buffered formalin (Sigma Aldrich), fixed for 16 hours, and washed with running water for 16 hours. Thereafter, dehydration was carried out through an alcohol hydrolysis process, the solvent was substituted using xylene (Sigma Aldrich), and paraffin (Sigma Aldrich) was permeated.

For deparaffination, the probes were probed in the order of concentration of 100%, 80%, 70%, and 50% of xylene, fixed at 60°C with Bouin's solution (Sigma Aldrich), stained with weigert's iron hematoxylin solution (Sigma Aldrich) for 10 minutes, and then washed in running water. Washing, staining with Biebrich Scarle-Acid Fuchsin solution (Sigma Aldrich) for 10 minutes, washing with running water, reacting with Phosphomolybdic-Phosphotungstic acid solution (Sigma Aldrich) for 10 minutes, washing with running water, 5 with Aniline Blue solution (Sigma Aldrich) Minute-dyeing, washing in running water, reaction with 1% acetic acid solution (Sigma Aldrich) for 2 minutes, washing in running water, and transparent process using xylene, confocal microscopy (Zeiss), and analysis did

The results obtained above are shown in FIGS. 4 and 5 . As can be seen from FIGS. 4 and 5 , the peptides synthesized in Examples 1 and 2 exhibited excellent collagen biosynthesis-increasing efficacy, indicating that the skin wrinkle improvement effect was excellent.

Example 3: Preparation of cream containing spider pentapeptide

A cream containing the peptide prepared in Example 1 was prepared in a conventional manner according to the composition of Table 1 below.

Raw material content (wt%) Peptide (Example 1) 2.0 shea butter 2.0 Polysorbate 60 1.5 Sorbitan Sesquiolate 0.8 mineral oil 10.0 dimethicone 3.0 Sorbitan Stearate 0.5 lipophilic monostearate glycerin 1.0 cetearyl alcohol 2.0 Glyceryl Stearate/PEG-400 Stearate 1.0 propylene glycol 3.0 carboxy polymer 0.25 triethanolamine 0.25 Phenoxyethanol appropriate amount Purified water To 100

<110> PIONTECH <120> Pentapeptide derived from spider web and composition for anti-aging containing the same <130> PP-21001 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial peptide spider pentapeptide-1 derived from spider silk protein <400> 1 Gly Pro Gly Ser Gly 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial peptide spider pentapeptide-2 derived from spider silk protein <400> 2 Gly Pro Gly Ser Gln 1 5

Claims (6)

delete delete A cosmetic composition for improving skin wrinkles containing a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. delete A pharmaceutical composition for improving skin wrinkles containing a pentapeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. delete

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