KR102462330B1 - Composition for preventing or ameliorating skin aging or wrinkle - Google Patents

Abstract

The present invention relates to a novel polypeptide fragment and a composition for preventing or improving skin aging or wrinkles comprising the same. The polypeptide of the present invention reduces the expression of MMP-1 that decomposes collagen, and has an excellent effect of increasing the expression of type 1 collagen related to the inhibition of skin wrinkles, skin elasticity, and skin moisture, and prevention of skin aging or wrinkles , can be usefully used as a composition for improvement.

Description

Composition for preventing or improving skin aging or wrinkles {Composition for preventing or ameliorating skin aging or wrinkle}

The present invention relates to a novel polypeptide fragment and a composition for preventing or improving skin aging or wrinkles comprising the same.

The skin is a membrane that covers the outside of the body, and it protects the body from various environmental factors and is responsible for the aesthetic function reflected from the outside. As the skin ages, not only the immune function of the skin decreases, but also external changes such as a decrease in skin elasticity, a change in skin color, and the formation of skin wrinkles are also accompanied. This skin aging process can be classified into intrinsic aging and extrinsic aging. Intrinsic aging is a natural aging process that occurs naturally with aging, and it is difficult to control artificially because it is mainly influenced by genetic factors. It can be said that prevention is possible through Therefore, research on the discovery of functional materials for the prevention and treatment of skin aging, centering on extrinsic aging, is being actively conducted. The dermis of the skin accounts for most of the total skin volume, and reflects skin characteristics such as flexibility, elasticity, and tension.

Republic of Korea Patent Publication No. 10-2016-0011297 (published on Feb. 1, 2016)

The present inventors made intensive research efforts to develop a formulation for wrinkle generation and improvement. As a result, a novel 7mer short polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention was developed, and this polypeptide has excellent inhibitory effect on MMP-1 without showing cytotoxicity, and The present invention was completed by finding out that it promotes the production.

Accordingly, an object of the present invention is to provide a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a cosmetic composition for preventing or improving skin aging or wrinkles, or a composition for external use, comprising the polypeptide as an active ingredient.

Another object of the present invention is to provide a nucleic acid molecule encoding the polypeptide, a recombinant vector comprising the nucleic acid molecule, and a host cell comprising the recombinant vector.

According to one aspect of the present invention, the present invention provides a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the amino acid sequence of SEQ ID NO: 1 of the present invention is "KAIGELD". In one embodiment of the present invention, the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention has no cytotoxicity, inhibits the increase in the expression of collagenase MMP-1 by UVB ultraviolet irradiation, and type 1 collagen increase the expression of Therefore, the polypeptide can be usefully used for preventing or improving skin aging or wrinkles.

In one embodiment of the present invention, the aging includes photoaging. Photoaging reduces the synthesis of collagen I and collagen III, the main components of the dermis, by inhibiting TGF-β and TGF-α by activating AP-1 and by activating AP-1. Activation of -1 and NF-κβ proceeds by activating MMPs, particularly MMP-1, MMP-3, and MMP-9, to promote the breakdown of connective tissue in the dermis. In the case of long-term UV exposure, the thickness of the epidermis increases 2-3 times in the skin tissue, and in the epidermal layer, an increase in pilose cells and polymorphism of keratinocytes are observed. In addition, substances including amorphous elastin are deposited on the epidermis and dermis connecting layer, and the fibrous structure of collagen and elastin is deformed in the dermal layer, and solar elastic fibrosis (elastosis) occurs due to the increase of elastic fibers.

Wrinkles of the skin are caused by activation of MMPs and degradation of collagen. Skin wrinkles are mainly caused by aging and UV rays. Skin exposure due to aging and UV light activates mitogen-activated protein kinase (MAPK). The factor most affected by MAPK is AP-1. AP-1 is a transcription factor composed of Jun and Fos family proteins, and its transcriptional activity is highest when it exists as a heterodimer of c-Jun and c-Fos. When human skin is not exposed to UV light, it exists as a dimer of c-Fos and JunD. However, the expression of c-Jun and c-Fos proteins increases due to UV rays and external environmental stimuli, and c-Jun interacts with JNK and JNK. It is activated by phosphorylation by p38. AP-1 regulates the expression of many genes involved in cell and differentiation and strongly regulates the expression of several MMPs. MMP is an endoprotease that requires zinc ions and is an enzyme that breaks down extracellular matrix proteins. Among MMPs whose expression is regulated by AP-1, MMP-1 is known as collagenase 1, and uses type I and III collagen as substrates. MMP-3, also called stromelysin 1, decomposes type IV collagen in the basement membrane and activates proMMP-1, a zymogen. MMP-9 is gelatinase B, which hydrolyzes the products degraded by collagenase to a smaller extent. By UV light, MMP-1 breaks down into fibillar collagen (types I and III), decomposing collagen, and subsequently increasing MMP-3 and MMP-9.

In the present invention, collagen is also called collagen fiber and occupies 85-90% of the dermis. The elasticity or moisture of the skin is maintained by the moisture present in the stratum corneum of the epidermis, which is the outermost layer of the skin, and the collagen present in the dermis. Most of the skin collagen is type-1 collagen. The main functions are the mechanical firmness of the skin, the resistance of the connective tissue and the binding force of the tissue, the support of cell adhesion, and the induction of cell division and differentiation. Collagen exists in high concentrations in skin, bone, ligaments, cartilage and teeth, etc., and is not affected by proteolytic enzymes such as trypsin, but is degraded by collagenase.

As used herein, the term “polypeptide” refers to a linear molecule formed by bonding amino acid residues to each other by peptide bonds. Polypeptides of the present invention can be prepared by chemical synthesis methods known in the art, particularly solid-phase synthesis techniques; Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al. , Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid-phase synthesis technology (US Patent No. 5,516,891).

The polypeptide of the present invention may select a portion of the amino acid sequence and induce modifications at the N-terminus or C-terminus to increase its activity. Through this modification, the polypeptide of the present invention can have a high half-life with increased half-life upon in vivo administration.

In another embodiment of the present invention, the C-terminus of the polypeptide of the present invention may be modified with a hydroxyl group (-OH), an amino group (-NH ₂ ), an azide group (-NHNH ₂ ), etc., of the polypeptide A protecting group selected from the group consisting of an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) may be bonded to the N-terminus.

Modification of the above-described amino acids acts to greatly improve the stability of the polypeptide of the present invention. As used herein, the term “stability” refers not only to in vivo stability, but also storage stability (eg, room temperature storage stability). The above-mentioned protecting group functions to protect the polypeptide of the present invention from attack by proteolytic enzymes in vivo.

According to another aspect of the present invention, the present invention provides a cosmetic composition for preventing or improving skin aging or wrinkles comprising the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

According to another aspect of the present invention, the present invention provides an external composition for preventing or improving skin aging or wrinkles, comprising the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

In one embodiment of the present invention, the cosmetic composition, and the composition for topical application are selected in relation to the intended route of administration and standard pharmaceutical or cosmetic application in addition to the polypeptide comprising the amino acid sequence of SEQ ID NO: 1, at least one pharmaceutically and/or cosmetically acceptable excipient, carrier, or diluent. (See, eg, Remington: The Science and Practice of Pharmacy, 19 ^th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA, incorporated herein by reference).

The polypeptides of the invention may be lyophilized for storage and reconstituted with an appropriate carrier prior to use.

As used herein, "pharmaceutically acceptable" means that the preparation is sterile and pyrogen-free. Suitable pharmaceutical carriers are well known in the pharmaceutical art. The carrier(s) should be "acceptable" in the sense of not adversely affecting the efficacy of the active ingredient of the present invention and not harmful to its recipient. Typically, the carrier will be sterile, pyrogen-free water or saline; However, other acceptable carriers may be used. Thus, "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" are merely intended to act as carriers, i.e., not intended to have their own biological activity. compound(s) used to form part of the formulation. Pharmaceutically acceptable carriers or excipients are generally safe and nontoxic. A pharmaceutically acceptable carrier and/or excipient as used herein includes both one or more carriers and/or excipients.

Likewise, the term “cosmetically acceptable” is used to denote an agent suitable for use as a cosmetic. Suitable cosmetic carriers are well known in the art, as are commonly used in shampoos, lotions, creams, sprays and other such products.

The excipient may be one or more carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to the composition, for example, to facilitate lyophilization. Examples of polymers include starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carrageenan, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulfonates, polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degrees of hydrolysis, and polyvinylpyrrolidone of all different molecular weights, which are for example , for viscosity control, to achieve adhesion, or to protect lipids from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, phosphoric acid, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids of all different acyl chain lengths and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are It is added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain advantages such as reduced liquid build-up or advantageous pigment properties.

The term "diluent" is intended to mean an aqueous or non-aqueous solution with the purpose of diluting a peptide in pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or an oil (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The diluent may also function as a buffer. The term “buffer” is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilizing the pH. Examples of buffers include Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate , AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolamic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES to be.

The cosmetic composition or composition for topical application of the present invention may also be in the form of a polymer gel or microneedle made of a polymer, wherein the polymer is starch, cellulose ether, cellulose carboxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginate, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonate, and the like.

The polypeptide of the present invention may be formulated at various concentrations depending on the efficacy or toxicity of the active ingredient used. Preferably, the composition comprises 1 nM to 1 M, for example 0.1 μM (micromole) to 1 mM, 1 μM to 100 μM, 5 μM to 50 μM, 10 μM to 50 μM, 20 μM to 40 μM and optionally and the polypeptide at a concentration of about 30 μM. For ex vivo and in vitro applications, the composition may comprise a lower concentration of the polypeptide, for example, between 0.0025 μM and 1 μM.

It will be appreciated by those skilled in the art that the compositions of the present invention may be administered by a variety of routes, for example, topical, subcutaneous, parenteral or oral administration.

Preferably, the composition of the present invention is suitable for topical or intracutaneous administration. Accordingly, the compositions of the present invention may be administered topically to the skin (eg, the scalp). For example, the composition may be provided in the form of an ointment containing the active polypeptide suspended or dissolved in a mixture having, for example, one or more of: mineral oil, liquid petrolatum, white petrolatum, propylene glycols, polyoxyethylene polyoxypropylene compounds, emulsifying waxes and water. Alternatively, the polypeptide may be formulated as a suitable lotion or cream, spray, gel, emulsion, paste, soap, powder, or combination thereof, etc., for example, suspended or dissolved in a mixture of one or more of the following: Mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Optionally, compositions for topical administration may include a penetration enhancer (eg, Osborne & Henke, 1997, Pharmaceutical Technology, November: 58-82 and the disclosure of which is incorporated herein by reference). As described in Patan & Setty, 2009, Tropical Journal of Pharmaceutical Research 8(2): 173-179).

Alternatively, the compositions of the present invention may be administered parenterally, for example intradermally. Such compositions are best used in the form of a sterile aqueous solution which may contain sufficient salt or glucose to make the solution isotonic with other substances, for example, blood. The aqueous solution should be suitably buffered (preferably to pH 3-9), if necessary. The preparation of suitable parenteral preparations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

A composition comprising the polypeptide of the present invention is administered to a subject in an effective amount. As used herein, 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective' refers to an effect of preventing or improving (reducing) skin aging and wrinkles. amount to provide This is a predetermined amount of an active substance calculated to produce the desired prophylactic or ameliorating (therapeutic) effect. As will be appreciated by those skilled in the art, the amount of an active ingredient may vary depending on its specific activity. Appropriate dosages may contain a predetermined amount of active substance calculated to produce the desired prophylactic or ameliorating (therapeutic) effect in association with the required diluent. In the methods and uses for the preparation of the compositions of the present invention, a prophylactically or ameliorating (therapeutically) effective amount of an active ingredient is provided. A prophylactically or ameliorated (therapeutically) effective amount can be determined by a practitioner of ordinary skill in the medical or veterinary field based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art. .

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 described above.

As used herein, the term "nucleic acid molecule" has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules. Analogs are also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

It is apparent to those skilled in the art that the nucleotide sequence encoding the polypeptide of the present invention is not limited to a specific nucleotide sequence, as long as it is a nucleotide sequence encoding an amino acid sequence constituting the polypeptide. This is because, even if a nucleotide sequence mutation occurs, the protein sequence does not change when the mutated nucleotide sequence is expressed as a protein in some cases. This is called codon degeneracy. Thus, the nucleotide sequence is a functionally equivalent codon or codon encoding the same amino acid (e.g., due to codon degeneracy, there are six codons for arginine or serine), or a codon encoding a biologically equivalent amino acid It contains a nucleotide sequence comprising a.

The nucleic acid molecule of the present invention encoding the polypeptide is construed to include a nucleotide sequence exhibiting substantial identity to the above-described nucleotide sequence. The substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. , more preferably at least 90% homology, most preferably at least 95%, 97%, 98%, or 99% homology.

In consideration of the above-described variation having biological equivalent activity, it is construed that the nucleic acid molecule encoding the polypeptide of the present invention includes a sequence exhibiting substantial identity to the sequence listed in the sequence listing. The substantial identity is at least 61% when the sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. means a sequence that exhibits homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981) ; Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10(1990)) is accessible from the National Center for Biological Information (NBCI), etc. It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. BLAST can be accessed through the BLAST page of the ncbi website. A method for comparing sequence homology using this program can be found on the BLAST help page of the ncbi website.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the above-described polypeptide.

According to one embodiment of the present invention, the expression vector is a vector into which a nucleic acid molecule encoding the polypeptide is inserted, is operatively linked to the nucleotide sequence of the nucleic acid molecule, and is an RNA molecule in a host cell. It is a recombinant vector for host cell expression comprising a promoter that forms it and a poly A signal sequence that acts in the host cell to cause polyadenylation of the 3'-end of the RNA molecule.

As used herein, the term "operatively linked" refers to a functional association between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, By this, the regulatory sequence controls the transcription and/or translation of the other nucleic acid sequence.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). , this document is incorporated herein by reference.

Vectors of the invention can typically be constructed as vectors for cloning or as vectors for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of propagating transcription (eg, pL ^λ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) , a ribosome binding site for initiation of translation and a transcription/translation termination sequence. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024 (1984)) and the left-handed promoter of phage λ (pL) The ^λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445 (1980)) can be used as a regulatory region.

On the other hand, vectors that can be used in the present invention include plasmids (eg, pSK349, pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series and pUC19, etc.), phage (eg, λgt) which are often used in the art. λ4B, λ-Charon, λΔz1, and M13) or viruses (eg, SV40, etc.) can be manufactured.

The vector of the present invention may be fused with other sequences to facilitate purification of the polypeptide expressed therefrom. The sequence to be fused includes, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA). Because of the additional sequences for purification, the protein expressed in the host is rapidly and easily purified via affinity chromatography.

The vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and a resistance gene to tetracycline.

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is a host, a promoter derived from the genome of a mammalian cell (eg, a metallotionine promoter) or a promoter derived from a mammalian virus (eg, adeno viral late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

Optionally, the vector may additionally carry genes encoding reporter molecules (eg, luciferase and -glucuronidase).

According to another aspect of the present invention, the present invention provides a host cell transformed with the recombinant vector.

As a host cell capable of stably and continuously cloning and expressing the vector of the present invention, any host cell known in the art may be used, for example, E. coli Origami2, E. coli JM109, E. coli BL21 (DE3). ), E. coli strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus genus strains such as Bacillus subtilis, Bacillus thuringiensis, and Enterobacteriaceae and strains such as Salmonella typhimurium, Serratia marcesens and various Pseudomonas species.

In addition, when the vector of the present invention is transformed into eukaryotic cells, as host cells, yeast (Saccharomyce cerevisiae), insect cells and animal cells (eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) , HepG2, 3T3, RIN and MDCK cell lines) can be used.

The method of delivering the vector of the present invention into a host cell is, when the host cell is a prokaryotic cell, the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)) ), Hanahan method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166:557-580). (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145 (1988)). In addition, when the host cell is a eukaryotic cell, the microinjection method (Capecchi, MR, Cell, 22:479 (1980)), the calcium phosphate precipitation method (Graham, FL et al., Virology, 52:456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bambadment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)) ), and the like, to inject the vector into the host cell.

In the present invention, the recombinant vector injected into the host cell can express the above-mentioned polypeptide recombined in the host cell, and in this case, a large amount of the polypeptide is obtained. For example, when the expression vector includes the lac promoter, the host cell may be treated with IPTG to induce gene expression.

The transformed host cell can be cultured by a known host cell culture method or a modified method thereof. For example, if the host cell is E. coli , a natural medium or a synthetic medium can be used as the medium for culturing the transformed host cell if it contains a carbon source, nitrogen source, inorganic salt, etc. that can be efficiently used by E. coli. have. Carbon sources that can be used include carbohydrates such as glucose, fructose, sucrose; starch, a hydrolyzate of starch; organic acids such as acetic acid and propionic acid; alcohols such as ethanol, propanol, glycerol, and the like. The nitrogen source is ammonia; ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate; peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean extract, soybean hydrolyzate; various fermented cells and their lysates; and the like. Inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, manganese sulfate, copper sulfate, calcium carbonate, and the like.

The culture is usually carried out under aerobic conditions, such as by shaking culture or rotation by a rotary machine. The culture temperature is preferably in the range of 10 to 40°C, and the culture time is generally 5 hours to 7 days. The pH of the medium is preferably maintained in the range of 3.0 to 9.0 during culture. The pH of the medium can be adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like. During culture, if necessary, antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin and tetracycline may be added for maintenance and expression of the recombinant vector. When culturing a host cell transformed with a recombinant expression vector having an inducible promoter, if necessary, a suitable inducer may be added to the medium. For example, if the expression vector contains a lac promoter, IPTG (isopropyl-beta-D-thiogalactopyranoside) may be added, and if the expression vector contains a trp promoter, indoleacrylic acid may be added to the medium.

The present invention provides a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, and a cosmetic composition for preventing or improving skin aging or wrinkles comprising the same, and an external composition. The polypeptide of the present invention inhibits the expression of the MMP-1 enzyme that decomposes collagen and has an excellent effect of promoting the expression of type 1 collagen, and thus can be usefully used as a composition for the above-mentioned use.

1 is a diagram showing the cytotoxicity test results of the test substance (AMPEP-AA-1) of the present invention confirmed in Example 1.
2 is a diagram showing the effect of the test substance (AMPEP-AA-1) of the present invention confirmed in Example 2 on the expression of the collagen-degrading enzyme MMP-1.
3 is a diagram showing the effect of the test substance (AMPEP-AA-1) of the present invention confirmed in Example 3 on the expression of type 1 collagen.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (vol/vol) %.

Experimental methods and materials

The test substance of the present invention is a polypeptide consisting of the amino acid sequence of KAIGELD (SEQ ID NO: 1), and was named "AMPEP-AA-1" by the present inventors. The test substance (AMPEP-AA-1) of the present invention was dissolved in 1 ml of purified water and used as sample A in the following test example.

Example 1. Cytotoxicity test

In order to confirm the presence or absence of cytotoxicity of the test substance of the present invention, the toxicity of the samples at 5 concentrations (0.1, 1, 5, 10, and 50 μg/ml) to CCD-986SK cells was evaluated through the following experiment. Confirmed.

First, CCD-986SK cells were seeded at 1x10 ⁴ /well (100 μl) in a 96-well cell culture plate. After culturing for 24 hours in a CO ₂ incubator, it was confirmed whether the cells were evenly distributed on the bottom of the 96-well plate using a microscope and grown (confluency). Remove the medium from each well, and dispense a medium containing the test substance of the present invention at a concentration of 0, 0.1, 1, 5, 10, and 50 μg/ml each by 100 μl/well, and in a CO ₂ incubator 24 incubated for hours. After removing the medium from each well, 50 μl/well of MTT reagent (1 mg/ml) was dispensed, and the plate was incubated at 37° C. for 2 hours after blocking light. After 2 hours, it was confirmed whether a purple precipitate was formed in the cells. After removing the MTT reagent from each well, 100% isopropanol was added at a time of 100 μl/well to dissolve the precipitate. Finally, the absorbance of the plate was measured at the detection wavelength (570 nm) and the reference wavelength (650 nm) using a spectrophotometer. When the number of living cells decreases, the amount of MTT reagent converted to formazan by mitochondrial dehydrogenase decreases. Accordingly, the survival rate was calculated as follows.

The results are shown in FIG. 1 . In the results of Figure 1, since the decrease in the number of living cells leads to a decrease in metabolic activity, the lower the viability (%), the higher the potential cytotoxicity of the sample, and the viability is less than 70% (<70%) of the blank test solution. If reduced, it is considered cytotoxic.

As shown in FIG. 1 , the test substance of the present invention had a survival rate of 70% or more at all concentrations (0.1, 1, 5, 10, and 50 μg/ml), confirming that there was no toxicity.

Example 2 and Example 3. CCD-986SK intracellular MMP-1 and Collagen I quantitative test

In order to confirm the effect of the test substance of the present invention on wrinkle improvement, CCD-986SK cells were treated with the test substance of the present invention by concentration with UVB ultraviolet rays, and the expression levels of MMP-1 and Collagen I were quantitatively measured.

First, CCD-986SK cells were treated with a test substance and RNA was extracted as follows. CCD-986SK cells were seeded in a 6-well plate at 5Х10 ⁴ cells/well and cultured for 24 hours. After UVB 20 mJ/cm ² irradiation, the test substance AMPEP-AA-1 (Sample A) of the present invention was treated with each concentration (0, 1, 10, 50 μg/mL). After removing the medium supernatant, 500 μl of trizol lysis buffer was dispensed into each well and the cells were homogenized. The homogenized solution was left at room temperature (15-25 ° C. for 5 minutes. 200 μL of chloroform was dispensed into each well, shaken every 15-20 seconds, and left for 3 minutes. Then, centrifuged at 12,000 x g for 15 minutes. The separated clear layer, the aqueous phase, was transferred to a new tube.500 μl of 100% isopropanol was added to the transferred tube and then left at room temperature for 15 minutes Centrifuged at 12,000 xg for 10 minutes Removed all supernatant except for the cell pellet After adding 1 mL of 70% ethanol made of DEPC.DW to the tube containing the pellet, centrifugation at 7,500 x g for 5 minutes, the process of removing all the supernatant except the pellet was repeated twice. The tube was dried at room temperature for 5-10 minutes.In the tube remaining only the pellet, 20-30 μl of DEPC.DW was added and mixed. 1-2 μl of RNA was taken and quantified with a nano-drop.

Next, in order to measure the mRNA expression levels of MMP-1 and Collagen I in CCD-986SK cells treated with the test substance of the present invention, mRNA was quantified using the primer sequences shown in Table 1 below.

RT PCR (reverse transcription PCR) was performed as follows. Green GoTaq Flexi buffer, MgCl ₂ , PCR nucleotide mix (10 mM), primer, GoTaq DNA polymerase, and nuclease free water were added to the synthesized cDNA in a PCR tube. The expression level was measured by performing PCR on the samples of each experimental group under the conditions of 95 °C 30 sec, 56 °C, 60 sec, 72 °C 1 min (40 cycles) for each gene (MMP-1, Collagen I, GAPDH). Expression levels of MMP-1 and Collagen I were quantitatively analyzed by correcting with GAPDH.

The results of Example 2 (expression level of MMP-1) are shown in FIG. 2 . As shown in Figure 2, compared with the group irradiated with UVB 20mJ/cm ² MMP-1 of the test group treated with the test substance of the present invention at a significant level at all concentrations (1, 5, 10 μg/mL) expression decreased.

The results of Example 3 (expression level of Collagen I) are shown in FIG. 3 . As shown in Figure 3, compared with the group irradiated with UVB 20mJ/cm ² Collagen I expression in the test group treated with the test substance of the present invention at a significant level at all concentrations (1, 5, 10 μg/mL) amount increased.

From the above results, the test substance of the present invention reduces the expression of MMP-1 that decomposes collagen, and has an excellent effect of suppressing skin wrinkles and increasing the expression of type 1 collagen related to skin elasticity and skin moisture. Or it was found that it can be usefully used as a composition for preventing and improving wrinkles.

<110> AMMEDICS Co., Ltd. <120> Composition for preventing or ameliorating skin aging or wrinkle <130> AM2-102020063 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of AMPEP-AA-1 <400> 1 Lys Ala Ile Gly Glu Leu Asp 1 5 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 2 ggtgaaggtc ggtgtgaacg 20 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 3 ctcgctggaa gatggtg 17 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Collagen I forward primer <400> 4 cctggaaaga atggagatga tg 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Collagen I reverse primer <400> 5 atccaaacca ctgaaacctc tg 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MMP-1 forward primer <400> 6 tgaaaagcgg agaaatagtg g 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MMP-1 reverse primer <400> 7 gaggacaaac tgagccacat c 21

Claims (5)

A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.
A cosmetic composition for preventing or improving skin aging or wrinkles comprising the polypeptide of claim 1 as an active ingredient.
An external composition for preventing or improving skin aging or wrinkles comprising the polypeptide of claim 1 as an active ingredient.
A nucleic acid molecule encoding the polypeptide of claim 1 .
A recombinant vector comprising the nucleic acid molecule of claim 4 .

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