KR102663334B1 - Peptide for strengthening skin elasticity and barrier, and cosmetic use thereof - Google Patents

Abstract

The present invention relates to peptides for skin elasticity and barrier strengthening effects, and their use in cosmetics. Specifically, the present invention provides a human-derived collagen type V recombinant peptide that has skin elasticity and barrier strengthening effects, and a method using the same. Therefore, when using a cosmetic composition for strengthening skin elasticity and barrier containing the peptide of the present invention, excellent effects can be obtained in strengthening the skin barrier and promoting skin elasticity by i) increasing the expression of collagen type III protein, which contributes to skin elasticity. ii) By increasing the expression of Laminin 5, a component of the basement membrane that firmly fixes the epidermis and dermis and provides stability to the skin, an improvement effect on skin damage caused by exposure to ultraviolet rays, etc. can be expected.

Description

Peptide for strengthening skin elasticity and barrier, and cosmetic use thereof}

The present invention relates to peptides for skin elasticity and barrier strengthening effects, specifically, human-derived collagen type V recombinant peptides with skin elasticity enhancing and barrier strengthening effects, and their use in cosmetics.

Skin is a body organ in the human body that is directly in contact with the external environment. Its main function is to protect the inside of the human body, and it is composed of the epidermis, dermis, and hypodermis in that order from the outside. Among these, the epidermis plays an important role in maintaining moisture within the skin by preventing evaporation of moisture inside the human body, and the dermis contains elastic solid substances (fibers) and viscous liquid substances. These are combined to allow the skin to maintain its inherent elasticity.

In general, it is well known that the increase in skin wrinkles and decrease in elasticity as we age is caused by the deformation and decrease of the extracellular matrix (ECM) present in the dermis, and the fact that the Structural and functional changes in the basement membrane are also known to be involved in skin aging.

Collagen is one of the most abundant proteins in the body and is an essential ingredient that binds tissues such as skin, bones, and joints. Together with elastin, it is a structural protein of the skin that maintains both elasticity and firmness of the skin. In particular, collagen, a fibrous protein, makes up most of the skin and contributes to maintaining the mechanical strength of the tissue.

To date, 28 types of collagen proteins have been discovered, and humans have a large number of types 1 to 5 collagen. Specifically, in the case of type 1 collagen, it accounts for a large portion as the most basic component in forming tissues, and type 5 collagen plays a role in helping form cell surfaces and cross-linking tissues.

In particular, type 5 collagen exists throughout the dermis of the skin and accounts for approximately 2% of collagen in the dermis. In addition, it forms fibrils with type 1 and type 3 collagen to control the size of collagen, and interacts with type 4 collagen to form crosslinks and fibrils in the dermis and epidermis of the skin.

In the existing cosmetics market, research on hydrolysis and low molecular weight collagen using existing fish-derived collagen and research on genetically recombinant collagen peptides are in progress. However, research using recombinant collagen peptides has only been conducted on human collagen types 1 and 3, and research on type 5 collagen is currently not active.

Therefore, with respect to type 5 collagen, which is closely related to the human skin barrier, there is a need to produce type 5 collagen peptides using genetic recombination technology and study how to utilize them.

Republic of Korea Patent Publication No. 10-2003-0074999 (September 22, 2003)

The present inventors made extensive research efforts to develop new recombinant collagen peptides that are effective in strengthening skin elasticity and barrier. As a result, a human-derived collagen type V peptide fragment was produced, which increased skin elasticity by increasing collagen type III expression and induced damage recovery by UVB, thereby strengthening the dermal-epidermal junction (DEJ). By identifying , the present invention was completed.

Therefore, the object of the present invention is to provide a peptide consisting of any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 3.

Another object of the present invention is to provide a polynucleotide encoding the peptide.

Another object of the present invention is to provide a recombinant expression vector containing the above polynucleotide.

Another object of the present invention is to provide a host cell transformed with the above recombinant expression vector.

Another object of the present invention is to provide a method for preparing the peptide of the present invention comprising:

(a) transforming a host cell with a recombinant expression vector expressing the peptide of claim 1;

(b) culturing the transformed host cells; and

(c) Obtaining the peptide of claim 1 from the cultured transformed host cells.

In addition, another object of the present invention is to provide a cosmetic composition containing the above peptide.

The present inventors made extensive research efforts to develop new recombinant collagen peptides that are effective in strengthening skin elasticity and barrier. As a result, 5 human-derived collagen type V peptide fragments were prepared, thereby improving skin elasticity by increasing collagen type III expression and inducing recovery from damage caused by UVB, thereby strengthening the dermal-epidermal junction (DEJ). It was found that it appears.

According to one aspect of the present invention, the present invention provides a peptide consisting of any one amino acid sequence of SEQ ID NOs: 1 to 3.

The present inventors discovered that a specific fragment of human collagen type 5, alpha 1 chain (hCOL5A1) protein exhibits skin elasticity and barrier strengthening effects.

As used herein, the term "collagen type 5 alpha 1 chain (COL5A1) protein" specifically refers to a protein derived from humans, herein referred to as hCOL5A1, and is encoded in the human gene COL5A1. refers to the protein in

The “collagen type 5 alpha 1 chain (hCOL5A1) protein” may consist of the amino acid sequence of SEQ ID NO: 7.

designation amino acid sequence sequence number hCOL5A1 MDVHTRWKARSALRPGAPLLPPLLLLLLLWAPPPSRAAQPADLLKVLDFHNLPDGITKTTGFCATRRSSKGPDVAYRVTKDAQLSAPTKQLYPASAFPEDFSILTTVKAKKGSQAFLVSIYNEQGIQQIGLELGRSPVFLYEDHTGKPGPEDYPLFRGINLSDGKWHRIALSVHKKNVTLILDCKKKKTTKFLDRSDHPMIDINGIIVFG TRILDEEVFEGDIQQLLFVSDHRAAYDYCEHYSPDCDTAVPDTPQSQDPNPDEYYTEGDGEGETYYYEYPYYEDPEDLGKEPTPSKKPVEAAKETTEVPEELTPTPTEAAPMPETSEGAGKEEDVGIGDYDYVPSEDYYTPSPYDDLTYGEGEENPDQPTDPGAGAEIPTSTADTSNSSNPAPPPGEGADDLEGEFTEETIRNLDENYYDPYYDP TSSPSEIGPGMPANQDTIYEGIGGPRGEKGQKGEPAIIEPGMLIEGPPGPEGPAGLPGPPGTMPGPTGQVGDPGERGPPGRPGLPGADGLPGPPGTMLMLPFRFGGGGDA GSKGPMVSAQESQAQAILQQARLALRGPAGPMGLTGRPGPVGPPGSGGLKGEPGDVGPQGPRGVQGPPGPAGKPGRRGRAGSDGARGMPGQTGPKGDRGFDGLAGLPGE KGHRGDPGPSGPPGPPGDDGERGDDGEVGPRGLPGEPGPRGLLGPKGPPGPPGPPGVTGMDGQPGPKGNVGPQGEPGPPGQQGNPGAQGLPGPQGAIGPPGEKGPLGKPGLPGMPGADGPPGHPGKEGPPGEKGGQGPPGPQGPIGYPGPRGVKGADGIRGLKGTKGEKGEDGFPGFKGDMGIKGDRGEIGPPGPRGEDGPEGPKGRGGP NGDPGPLGPPGEKGKLGVPGLPGYPGRQGPKGSIGFPGFPGANGEKGGRGTPGKPGPRGQRGPTGPRGERGPRGITGKPGPKGNSGGDGPAGPPGERGPNGPQGP TGFPGPKGPPGPPGKDGLPGHPGQRGETGFQGKTGPPGPPGVVGPQGPTGETGPMGERGHPGPPGPPGEQGLPGLAGKEGTKGDPGPAGLPGKDGPPGLRGFPG GLPGPVGALGLKGNEGPPGPPGPAGSPGERGPAGAAGPIGIPGRPGPQGPPGPAGEKGAPGEKGPQGPAGRDGLQGPVGLPGPAGPVGPPGEDGDKGEIGEPGQ KGSKGDKGEQGPPGPTGPQGPIGQPGPSGADGEPGPRGQQGLFGQKGDEGPRGFPGPPGPVGLQGLPGPPGEKGETGDVGQMGPPGPPGPRGPSGAPGADGPQGP PGGIGNPGAVGEKGEPGEAGEPGLPGEGGPGPGPKGERGEKGESGPSGAAGPPPGPKGPPGDDGPKGSPGPVGFPGDPGPPGEPGPAGQDGPPGDKGDDGEPGQTGSPGPT GEPGPSGPPGKRGPPGPAGPEGRQGEKGAKGEAGLEGPPGKTGPIGPQGAPGKPGPDGLRGIPGPVGEQGLPGSPGPDGPPGPMGPPGLPGLKGDSGPKGEKGHPGLIGL IGPPGEQGEKGDRGLPGPQGSSGPKGEQGITGPSGPIGPPGPPGLPGPPGPKGAKGSSGPTGPKGEAGHPGPPGPPGPPGEVIQPLPIQASRTRRNIDASQLLDDGNGE NYVDYADGMEEIFGSLNSLKLEIEQMKRPLGTQQNPARTCKDLQLCHPDFPDGEYWVDPNQGCSRDSFKVYCNFTAGGSTCVFPDKKSEGARITSWPKENPGSWFSEFK RGKLLSYVDAEGNPVGVVQMTFLRLLSASAHQNVTYHCYQSVAWQDAATGSYDKALRFLGSNDEEMSYDNNPYIRALVDGCATKKGYQKTVLEIDTPKVEQVPIVDIMFNDF 7

The peptide of the present invention is characterized by containing the amino acid proline in a high proportion compared to other amino acid groups.

Specifically, the present inventors prepared three specific peptide fragments based on sequences rich in the amino acid proline in the human collagen type 5 alpha 1 chain (hCOL5A1) protein. More specifically, the peptide fragments were each S1 (901-1020 aa of hCOL5A1, SEQ ID NO: 1) peptide, S2 (1321-1440 aa of hCOL5A1, SEQ ID NO: 2) peptide, and S3 (1521-1640 aa of hCOL5A1, SEQ ID NO: 3) peptide.

The present inventors treated human dermal fibroblasts (HDF) and keratinocytes with the above peptides, that is, S1 to S3 peptides, and confirmed that the peptide fragments have skin elasticity and barrier strengthening effects. Specifically, the peptide increases skin elasticity by increasing the expression of collagen type III (Col-III) protein and strengthens the adhesion between the epidermis and dermis by increasing the expression of laminin 5 protein, which is reduced due to ultraviolet rays. By doing so, it was confirmed that it has the effect of strengthening the skin barrier. In particular, among the three types of peptides, S1 peptide was confirmed to have the best skin elasticity and barrier strengthening effect.

Therefore, in a specific embodiment of the present invention, the peptide may be a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

According to another aspect of the present invention, the present invention provides a polynucleotide encoding a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3.

In one embodiment of the present invention, the polynucleotide is characterized in that it consists of any one base sequence of SEQ ID NOs: 4 to 6.

As used herein, the term “polynucleotide” refers to a polymer of nucleotides in which nucleotide units are connected in a long chain by covalent bonds, and refers to a DNA or RNA strand of a certain length or more.

The polynucleotide consisting of any one of the base sequences of SEQ ID NOs: 4 to 6 encodes the amino acid sequences of SEQ ID NOs: 1 to 3, specifically, the amino acid sequences constituting the S1 peptide, S2 peptide, and S3 peptide of the hCOL5A1 protein specified by the present inventors. It is done.

The polypeptide encoding the peptide may include, for example, any one of SEQ ID NOs: 4 to 6, or a sequence substantially identical to the base sequence. The above substantial identity can be determined by aligning the base sequence of any one of SEQ ID NOs: 4 to 6 and any other sequence to correspond as much as possible, and analyzing the sequence to determine whether any of the other sequences is the same as any of the bases of SEQ ID NOS: 4 to 6. It means that the peptide encoded therefrom has a sequence homology of at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, and maintains skin elasticity and barrier strengthening efficacy. .

A person skilled in the art may substitute, add or delete one or more bases in the base sequence of the polynucleotide using genetic recombination techniques known in the art to produce the same polynucleotide within the range of the substantial homology. It will be easily understood that a polynucleotide encoding an active peptide can be prepared. This comparison of homologies can be performed by calculating the percentage (%) of homology between two or more sequences using a commercially available computer program.

In addition, according to another aspect of the present invention, the present invention provides a recombinant expression vector containing a polynucleotide encoding a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3.

As used herein, the term "recombinant expression vector" refers to a vector that can express a protein or nucleic acid (DNA or RNA) of interest in a suitable host cell, and operates to express the polynucleotide (gene) insert. It refers to a genetic construct containing essential regulatory elements that can be linked together.

The vector can typically be constructed as an expression vector and is intended to express recombinant peptides or proteins.

The term “recombinant” can be used interchangeably with genetic manipulation, and can be used to create a form that does not exist in nature by using molecular cloning experimental techniques such as modifying, cutting, and linking genes. This means manufacturing genes.

The term “expression” refers to the production of a protein or nucleic acid in a cell.

The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence and a sequence encoding a desired protein or nucleic acid to generally perform a function, by the expression control sequence. It means that genes are connected so that they can be expressed. The “expression control sequence” refers to a DNA sequence that regulates the expression of an operably linked polynucleotide sequence in a specific host cell. These regulatory sequences include a promoter to effect transcription, optional operator sequences to regulate transcription, sequences encoding suitable mRNA ribosome binding sites, sequences controlling termination of transcription and translation, initiation codons, stop codons, and polyadenylation. Includes signals and enhancers.

The type of recombinant expression vector of the present invention is not particularly limited as long as it is a vector commonly used in the cloning field, and examples include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. The above plasmids include E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119, pET-22(+)), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (pPICZ, YEp13, YEp24, and YCp50). , the virus may be a retrovirus, an animal virus such as adenovirus or vaccinia virus, an insect virus such as baculovirus, or pcDNA3.1 may be used. Specifically, the pET-28(+) vector can be used as the recombinant expression vector of the present invention.

Accordingly, the recombinant expression vector according to the present invention refers to a genetic construct operably linked so that the polynucleotide encoding the peptide of the present invention can be expressed in an appropriate host cell. Specifically, the recombinant expression vector according to the present invention contains a polynucleotide consisting of any one base sequence selected from the group consisting of base sequences of SEQ ID NOs: 4 to 6.

Additionally, according to another aspect of the present invention, the present invention provides a host cell transformed with the above recombinant expression vector.

The type of host cell that can be transformed with the recombinant expression vector is not particularly limited as long as it is a cell that can be used to express (overexpress) a polynucleotide encoding a specific peptide of the present invention. (Host) cells transformed with the recombinant expression vector according to the present invention can be prokaryotes (e.g. Escherichia coli), eukaryotes (e.g. yeast or other fungi), plant cells (e.g. tobacco or tomato plant cells) ), animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, insect cells, or hybridomas derived therefrom. The host cells may be Host cells with high DNA introduction efficiency and high expression efficiency of the introduced DNA are preferred, and may be prokaryotic cells such as E. coli, which are generally used for transformation to overexpress proteins.

The term “transformation” means introducing DNA into a host cell so that the DNA can be replicated as a chromosomal element or by completing chromosomal integration.

The transformed host cell can be prepared by introducing a recombinant expression vector capable of expressing the peptide according to the present invention into the cell by a general transformation method known in the art. For example, the above transformation methods include transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran- DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and introduction of nucleic acids into or into cells. Any known method for introduction may be included, and is not particularly limited thereto.

The peptide according to the present invention can be overexpressed or mass produced using cells transformed with the recombinant expression vector according to the present invention.

In order to confirm whether the recombinant peptide according to the present invention is expressed in the examples, the present inventors induced overexpression in E. coli and produced the peptide of the present invention by mass producing and separating and purifying the peptide.

Therefore, according to another aspect of the present invention, a method for producing a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3 is provided, comprising the following steps:

(a) transforming a host cell with a recombinant expression vector expressing a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3;

(b) culturing the transformed host cells; and

(c) Obtaining a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3 from the cultured host cells.

Step (a) is a step of transforming a host cell with a recombinant expression vector to which a polynucleotide encoding the peptide is operably linked to produce the peptide according to the present invention.

As described above, the type of the recombinant expression vector is not particularly limited as long as it is a commonly used vector. However, in one embodiment of the present invention, the recombinant expression vector in step (a) is pET-28a(+). It is characterized by

In addition, as described above, the type of host cell is not particularly limited as long as it is a cell that can be used to express the polynucleotide, and a person skilled in the art can select an appropriate host cell and transformation method to perform the steps of the present production method ( a) can be carried out. Specifically, the host cell for carrying out the method of the present invention can be selected without limitation as long as it can overexpress the protein. More specifically, the host cell may be Escherichia coli ( E.coli ) for protein overexpression.

The step (b) is a step of culturing the transformed host cell to overexpress or produce the peptide according to the present invention from the recombinant expression vector introduced into the host cell.

A person skilled in the art can appropriately select the medium composition, culture conditions, culture time, etc. for culturing the selected host cells.

In one embodiment of the present invention, the culture may be carried out until the absorbance (OD ₆₀₀ ) of the culture medium reaches 30 in order to obtain a high concentration of peptide.

The peptide produced in the host cell can be accumulated in the cytoplasm of the cell, secreted outside the cell or into the culture medium by an appropriate signal sequence, or targeted to the periplasm, etc. In addition, the peptide according to the present invention can be refolded using methods known in the art to maintain or improve the properties such as structure and have a functional conformation.

The step (c) is a step of obtaining the peptide produced in the host cell.

A person skilled in the art can appropriately select and adjust the acquisition method by considering the characteristics of the peptide produced in the host cell, the characteristics of the host cell, or the expression method. For example, peptides secreted into the culture medium can be recovered by obtaining a medium (supernatant) in which host cells were cultured and centrifuging to remove impurities. If necessary, in order to release and recover peptides present in a specific organelle or cytoplasm of the cell outside the cell, the cell may be lysed to the extent that it does not affect the functional structure of the peptide. In addition, the obtained peptide may be further subjected to a process of further removing impurities and concentrating through methods such as chromatography, filtration using filters, etc., and dialysis.

In one embodiment of the present invention, the peptide according to the present invention can be obtained by permeating the host cell lysate or lysate through a microfiltration membrane in step (c).

The present inventors confirmed that when treating human dermal fibroblasts (HDF) with the peptide according to the present invention, the expression of Col-III (collagen type III) protein increased compared to the untreated control group.

In this specification, the term 'Collagen type I (Col-III)' is a collagen protein present in skin, ligaments, blood vessels, joints, etc., which creates an elastic network that stores kinetic energy as elastic recoil, and collagen type I It is known to be involved in skin elasticity by working together with .

In addition, the present inventors found that the expression of laminin 5 protein was reduced in keratinocytes by UVB-stimulation, but when treated with the peptide according to the present invention, the decreased expression of laminin 5 protein increased compared to the untreated control group. It was confirmed that this was the case.

As used herein, the term 'Laminin' refers to an extracellular glycoprotein that is an important component of all basement membranes, and is a heterotrimer consisting of α, β, and γ chains (cahin). It is assembled as αβγ, and at least 16 isoforms are known to exist in mammals.

As used herein, the term 'Laminin 5' refers to a heterotrimeric protein consisting of α3, β3, and γ2 chains, and provides skin integrity and resistance to external mechanical forces by attaching the epidermis to the dermis. It is an essential component of the dermal-epidermal junction (DEJ), which is the basement membrane area. Laminin 5 is known to play an important role in adhesion of epithelial tissue to the basement membrane through interaction with two receptors, α6β4 or α3β1 integrin.

In summary, the peptides in the composition of the present invention have an excellent effect in strengthening the skin barrier and improving skin elasticity by increasing the expression of collagen type III protein, which contributes to skin elasticity, and provide stability to the skin by firmly fixing the epidermis and dermis. By increasing the expression of Laminin 5, a component of the basement membrane, it can have the effect of improving skin damage caused by exposure to ultraviolet rays, etc.

Therefore, according to another aspect of the present invention, the present invention provides a cosmetic composition for strengthening the skin barrier and elasticity comprising a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3.

As used herein, the term 'cosmetic composition' refers to a composition administered by applying or spraying directly to the skin for the purpose of cleaning, beautifying, maintaining health, or promoting health.

As used herein, the term 'improvement of skin elasticity' refers to improving the property of skin deformed by external force to easily return to its original shape when the external force is removed.

As used herein, the term 'skin barrier strengthening' refers to strengthening the barrier function of the stratum corneum, which is located on the outermost layer of the skin and prevents moisture and nutrient loss.

In one embodiment of the present invention, the composition is characterized by increasing the expression of proteins such as Col-III (collagen type III), laminin 5, or a combination thereof.

In addition, according to another aspect of the present invention, the present invention provides a cosmetic composition for promoting collagen production in the skin, comprising a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3.

As used herein, the term 'collagen' refers to a component protein of the extracellular matrix (ECM) that exists in large quantities in vertebrate tissues, and is known to constitute bone, skin, cartilage, connective tissue, etc. depending on the type. In particular, collagen is a large family that includes 29 different types, so numerous collagen synthesis genes are involved in collagen synthesis, and MMPs (matris metalloproteinases) exist that decompose connective tissue components, including collagen. In terms of skin elasticity, collagen synthesis decreases due to aging and the decomposition of collagen increases due to increased activity of MMPs enzymes that decompose collagen, thereby reducing skin elasticity and increasing wrinkles.

As described above, the present inventors have confirmed that the expression of Col-III (collagen type III) protein is increased by the peptide according to the present invention, and thus, an effect of promoting collagen production in cells can be expected.

In addition, according to another aspect of the present invention, the present invention provides a cosmetic composition for skin improvement comprising a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3.

The above ‘improvement’ refers to all actions that maintain or improve the health of the skin.

In one embodiment of the present invention, the skin improvement may include one or more selected from the group consisting of skin moisturizing, skin wrinkle improvement, skin damage improvement from ultraviolet rays, etc.

The term ‘skin moisturizing’ means maintaining skin moisture or preventing moisture loss. In other words, the present invention can exhibit an effective skin moisturizing effect by strengthening the skin barrier to maintain moisture in the skin or prevent its loss.

The 'improvement of skin wrinkles' refers to suppressing or inhibiting the formation of wrinkles by suppressing the expression of factors related to wrinkles from a skin condition caused by loss of skin elasticity, or alleviating wrinkles that have already been formed. That is, the composition of the present invention can exhibit an effective skin wrinkle improvement effect by improving skin elasticity and suppressing or inhibiting the formation of skin wrinkles, or by alleviating already formed wrinkles.

The term 'improving skin damage from ultraviolet rays' means improving or relieving the increase in skin wrinkles and decrease in skin elasticity caused by ultraviolet rays.

In one embodiment of the present invention, the cosmetic composition according to the present invention may include not only the active ingredient, a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3, but also components commonly used in cosmetic compositions, For example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers may be additionally included.

The carrier includes purified water, monohydric alcohols (ethanol or propyl alcohol), polyhydric alcohols (glycerol, 1,3-butylene glycol or propylene glycol), and higher fatty acids ( It may be palmitic acid or linoleic acid), fats and oils (wheat pear oil, camellia oil, jojoba oil, olive oil, squalene, sunflower oil, macadamia peanut oil, avocado oil, or fatty acid glycerides), etc., but must be It is not limited. Additionally, surfactants, disinfectants, antioxidants, ultraviolet absorbers, anti-inflammatory agents, and fresheners may be additionally added as needed.

The surfactant includes polyoxyethylene, hydrogenated castor oil, polyoxyethylene, oleyl ether, polyoxyethylene monooleate, polyoxyethylene, glyceryl monostearate, sorbitan monostearate, polyoxyethylene monooleate, sorbitan, It may be sucrose fatty acid ester, hexaglycerin monolauric acid, polyoxyethylene reduced lanolin, POE, glyceryl pyroglutamic acid, isostearic acid, diester, N-acetylglutamine, and isostearyl ester, but is not limited thereto.

The disinfectant may be hinocthiol, tricrosan, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zinc pyritaone, etc., but is limited thereto. no.

The antioxidant may be used as long as it is at least one selected from the group consisting of butylhydroxyanisole, gallic acid, propyl gallic acid, and erythorbic acid.

The ultraviolet absorbers include benzophenones such as dihydroxybenzophenone, melanin, ethyl para-aminobenzoate, para-dimethylaminobenzoic acid 2-ethylhexyl ester, synoxite, para-methoxycinnamic acid 2-ethylhexyl ester, 2-(2- It may be hydroxy-5-methylphenyl) benzotriazole, urochanic acid, and metal oxide fine particles, but is not limited thereto.

The anti-inflammatory agent may be dipotassium glycyrrhizate, allantoin, etc., and the refreshing agent may be red pepper tincture, 1-menthol, etc., but is not limited thereto.

In one embodiment of the present invention, the cosmetic composition is a solution, external ointment, gel, cream, foam, lotion, skin lotion, skin softener, skin toner, astringent, milk lotion, moisture lotion, nutritional lotion, massage cream, nutrition. Cream, moisture cream, hand cream, body lotion, nourishing lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, nourishing essence, ampoule, hair ampoule, scalp treatment, hair tonic, hair conditioner, hair treatment, Hair lotion, hair shampoo, hair rinse, conditioner shampoo, hair nourishing lotion, hair gel, hair wax, hair spray, hair dye, soap, liquid detergent, cleansing foam, cleansing lotion, cleansing cream, bath salt, body cleanser, sunscreen Can be prepared in a formulation selected from the group consisting of creams, sun oils, suspensions, emulsions, pastes, powders, soaps, surfactant-containing cleansers, oils, foundations, powder foundations, emulsion foundations, wax foundations, patches and sprays. However, it is not limited to this.

The cosmetic composition of the present invention may further include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids, and seaweed extract.

Water-soluble vitamins may be any that can be blended into the cosmetic composition, but preferably include vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H, etc. and their salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbic acid-2-phosphate, magnesium ascorbic acid-2-phosphate, etc.) are water-soluble vitamins that can be used in the present invention. included in Water-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from microbial cultures, enzymatic methods, or chemical synthesis.

Oil-soluble vitamins may be any as long as they can be blended in cosmetic compositions, but preferably include vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol), etc. and their derivatives (ascorbine palmitate, ascorbine stearate, ascorbine dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherol nicotinate, vitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl Ethyl ether, etc.) are also included in the oil-soluble vitamins used in the present invention. Oil-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from microbial culture, enzyme or chemical synthesis.

The polymer peptide may be any peptide as long as it can be blended into a cosmetic composition, but preferably includes collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, and keratin. High molecular weight peptides can be purified and obtained by conventional methods such as purification from microbial culture media, enzyme methods, or chemical synthesis, or they can usually be purified and used from natural products such as dermis of pigs or cows or silk fiber of silkworms. .

The polymer polysaccharide may be any one that can be blended into a cosmetic composition, but preferably includes hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or its salts (sodium salt, etc.). For example, chondroitin sulfate or its salt can usually be purified and used from mammals or fish.

The sphingolipid may be any one that can be incorporated into a cosmetic composition, but preferably includes ceramide, phytosphingosine, and sphingolipid. The sphingolipids can be purified by conventional methods from mammals, fish, shellfish, yeast, or plants, or obtained by chemical synthesis.

Any seaweed extract may be used as long as it can be blended in a cosmetic composition, but preferred examples include brown algae extract, red algae extract, and green algae extract, as well as calageenan, arginic acid, and sodium arginate purified from these seaweed extracts. Lime, potassium arginate, etc. are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained by purifying seaweed by a conventional method.

The cosmetic composition of the present invention may also contain other ingredients that are blended in ordinary cosmetic compositions.

Other ingredients that may be added include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohol, colorants, fragrances, Examples include blood circulation promoters, cooling agents, antiperspirants, and purified water.

Examples of the fat component include ester fat, hydrocarbon fat, silicone fat, fluorine fat, animal fat, and vegetable oil.

Examples of ester-based oils include glyceryl tri2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, and isostearate. Til, butyl stearate, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, disebacic acid. Ethyl, diisopropyl adipate, isoalkyl neopentanoate, tri(caprylic, capric acid)glyceryl, trimethylolpropane tri2-ethylhexanoate, trimethylolpropane triisostearate, tetra2-ethylhexanoic acid. Pentaelislitol, cetyl caprylate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinooleate, Isostearyl uric acid, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, 2-ethyl Cetostearyl hexanoate, 2-ethylhexanoate stearyl, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicapric acid, di(capryl, capric acid)propylene glycol, dicaprylic acid. Propylene glycol, neopentyl glycol dicapric acid, neopentyl glycol dioctanate, glyceryl tricaprylate, glyceryl triundecylate, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, octane Isostearyl acid, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglycerol oleic acid ester, polyglycerol iso. Stearic acid ester, triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyltriethyl citrate, acetyltributyl citrate, tri citrate Octyl, diisostearyl malate, 2-ethylhexyl hydroxystearate, di2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesteryl stearate, isostearic acid. Cholesteryl, cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, phytsteryl isostearate, phytsteryl oleate, 12-stealoyl hydroxystearate isocetyl, 12- It may be an ester type such as stearyl stealoyl hydroxystearate or isostearyl 12-stealoyl hydroxystearate.

Examples of hydrocarbon-based fats and oils include squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybudene, microcrystalline wax, and petroleum jelly.

Silicone oils include, for example, polymethyl silicone, methylphenyl silicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane/methylcetyloxysiloxane copolymer, dimethylsiloxane, and methylstealoxysiloxane. It may be a copolymer, alkyl-modified silicone oil, amino-modified silicone oil, etc.

Examples of fluorine-based fats and oils include perfluoropolyether.

Examples of animal or plant oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, birdflower oil, soybean oil, corn oil, rapeseed oil, apricot oil, palm kernel oil, palm oil, castor oil, and sunflower. Oil, grape seed oil, cottonseed oil, palm oil, kukuinut oil, wheat germ oil, rice germ oil, shea butter, moonshine colostrum, marker damia nut oil, meadow oil, egg yolk oil, beef tallow, horse oil, mink oil, orange raspberry. It may be animal or plant oil such as oil, jojoba oil, candelier wax, carnaba wax, liquid lanolin, and hydrogenated castor oil.

The humectant may be, for example, a water-soluble low-molecular moisturizer, an oil-soluble molecular moisturizer, a water-soluble polymer, or an oil-soluble polymer.

Water-soluble low-molecular-weight moisturizers include, for example, serine, glutamine, sorbitol, mannitol, sodium pyrrolidone-carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, and polyethylene glycol B (degree of polymerization n = 2 or more) ), polypropylene glycol (degree of polymerization n = 2 or more), polyglycerin B (degree of polymerization n = 2 or more), lactic acid, lactate, etc.

Examples of fat-soluble low-molecular-weight moisturizers include cholesterol, cholesterol ester, and the like.

Examples of water-soluble polymers include carboxyvinyl polymer, polyaspartate, tragacanth, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble chitin, chitosan, It may be dextrin, etc.

Examples of oil-soluble polymers include polyvinylpyrrolidone/eicosene copolymer, polyvinylpyrrolidone/hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, polymer silicone, etc.

Examples of the emollient agent include long-chain acyl glutamic acid cholesteryl ester, hydroxystearic acid cholesteryl ester, 12-hydroxystearic acid, stearic acid, rosin acid, and lanolin fatty acid cholesteryl ester.

The surfactant may be, for example, a nonionic surfactant, anionic surfactant, cationic surfactant, or amphoteric surfactant.

Nonionic surfactants include, for example, self-emulsifying glycerin monostearate, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, and POE sorbitol. Fatty acid ester, POE glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hydrogenated castor oil, POE castor oil, POEㆍPOP (polyoxyethyleneㆍpolyoxypropylene) copolymer, POEㆍPOP alkyl ether, polyether modified silicone , lauric acid alkanolamide, alkylamine oxide, hydrogenated soybean phospholipid, etc.

Anionic surfactants include, for example, fatty acid soap, alpha-acylsulfonate, alkylsulfonate, alkylallylsulfonate, alkylnaphthalenesulfonate, alkyl sulfate, POE alkyl ether sulfate, alkyl amide sulfate, alkyl phosphate, POE alkyl ginseng. Salt, alkylamide phosphate, alkyloyl alkyl taurine salt, N-acylamino acid salt, POE alkyl ether carboxylate, alkyl sulfosuccinate, sodium alkyl sulfoacetate, acylated hydrolyzed collagen peptide salt, perfluoroalkyl phosphate ester. It may be, etc.

Cationic surfactants include, for example, alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium bromide, cetostearyltrimethylammonium chloride, distearyldimethylammonium chloride, stearyldimethylbenzylammonium chloride, and behenyl bromide. It may be trimethylammonium, benzalkonium chloride, diethylaminoethylamide stearate, dimethylaminopropylamide stearate, quaternary ammonium salt of lanolin derivative, etc.

Examples of amphoteric surfactants include carboxy beta type, amide beta type, sulfo beta type, hydroxy sulfo beta type, amide sulfo beta type, phosphobeta type, aminocarboxylate type, imidazoline derivative type, and amide type. It may be an amphoteric surfactant such as an amine type.

Organic and inorganic pigments include, for example, silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium-coated mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, and oxide. Inorganic pigments such as titanium, aluminum oxide, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine blue, chromium oxide, chromium hydroxide, calamine and complexes thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene/styrene copolymer, It may be organic pigments such as silk powder, cellulose, CI pigment yellow, and CI pigment orange, and complex pigments of these inorganic pigments and organic pigments.

Examples of organic powder include metal soap such as calcium stearate; Alkyl phosphate metal salts such as sodium zinc cetilate, zinc laurylate, and calcium laurylate; Acyl amino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc, and N-lauroyl glycine calcium; Amidesulfonic acid polyvalent metal salts such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; N such as N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoylizine, N-alpha-paritoylolnithine, N-alpha-lauroylarginine, N-alpha-hydrogenated beef tallow fatty acid acylarginine, etc. -Acyl basic amino acid; N-acyl polypeptides such as N-lauroylglycylglycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; It may be polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene/styrene copolymer, ethylene tetrafluoride, etc.

Examples of ultraviolet absorbers include para-aminobenzoic acid, ethyl para-aminobenzoate, amyl para-aminobenzoate, octyl para-aminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, and homosalicinate. Menthyl, benzyl cinnamic acid, 2-ethoxyethyl paramethoxycinnamic acid, octyl paramethoxycinnamic acid, mono-2-ethylhexane glyceryl diparamethoxycinnamic acid, isopropyl paramethoxycinnamic acid, diisopropyl, diisopropyl series. Pinic acid ester mixture, urocanic acid, ethyl urocanic acid, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and its salts, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone disulfonate sodium, Dihydroxybenzophenone, tetrahydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino-p-(carbo-2'-ethylhexyl-1' -Oxy)-1,3,5-triazine, 2-(2-hydroxy-5-methylphenyl)benzotriazole, etc.

Disinfectants include, for example, hinokitiol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zincphyllithione, chloride. It may be benzalkonium, photosensitizer No. 301, sodium mononitroguaiacol, undecirenic acid, etc.

Examples of antioxidants include butylhydroxyanisole, propyl gallate, and elisorbic acid.

Examples of the pH adjuster include citric acid, sodium citrate, malic acid, sodium malate, fumalic acid, sodium fumalate, succinic acid, sodium succinate, sodium hydroxide, sodium monohydrogen phosphate, etc.

The alcohol may be a higher alcohol such as cetyl alcohol.

In addition, the ingredients that can be added are not limited to this, and any of the above ingredients can be mixed within a range that does not impair the purpose and effect of the present invention, but is preferably 0.01 to 5% by weight relative to the total weight, more preferably 0.01 to 5% by weight. It can be mixed at 0.01 to 3% by weight.

The cosmetic composition of the present invention may take the form of a solution, emulsion, viscous mixture, etc.

Ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetics in addition to the extract as an active ingredient, for example, conventional auxiliaries such as stabilizers, solubilizers, vitamins, pigments and fragrances, and Contains a carrier.

When the cosmetic composition formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier ingredients. It can be used.

When the cosmetic composition formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the cosmetic composition formulation of the present invention is a spray, chlorofluorohydride may be additionally used. May contain propellants such as carbon, propane/butane or dimethyl ether.

When the cosmetic composition formulation of the present invention is a solution or emulsion, a solvent, solvating agent, or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, and propylene glycol. , 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan.

When the cosmetic composition formulation of the present invention is a suspension, the carrier ingredients include 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, and miso. Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the cosmetic composition formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, Fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linoline derivative, or ethoxylated glycerol fatty acid ester can be used.

Since the cosmetic composition of the present invention includes the peptide, which is another aspect of the present invention described above, to avoid excessive complexity of the description in this specification, redundant content is used and the description is omitted.

The present invention provides a human-derived collagen V type recombinant peptide having skin elasticity and barrier strengthening effects, and a cosmetic composition using the same. When using a cosmetic composition for strengthening skin elasticity and barrier containing the peptide of the present invention, i) excellent effects can be obtained in strengthening the skin barrier and improving skin elasticity by increasing the expression of collagen type III protein, which contributes to skin elasticity, ii) By increasing the expression of Laminin 5, a component of the basement membrane that firmly fixes the epidermis and dermis and provides stability to the skin, an improvement effect on skin damage caused by exposure to ultraviolet rays, etc. can be expected.

Figure 1 shows a schematic diagram of the hCOL5A1 chain protein and the location of candidate peptides for plasmid construction.
Figure 2 shows the results of confirming the expression of S1 peptide, S2 peptide, and S3 peptide, which are candidate peptide fragments identified from hCOL5A1 protein.
Figure 3 shows the results of confirming the expression level of fragment protein according to OD value in the peptide fragment of the present invention.
Figure 4 shows the results of confirming the effect of enhancing skin elasticity according to hCOL5A1 peptide fragments (S1, S2, and S3 peptides) by confirming the expression level of COL-III in fibroblasts (human dermal fibroblasts).
Figure 5 shows the effect of strengthening the skin barrier according to hCOL5A1 peptide fragments (S1, S2, and S3 peptides) by confirming the recovery level of Laminin 5 expression after irradiating UVB to the keratinocyte A431 HEK cell line. Shows the results.

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 means (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.

Example 1. Selection of hCOL5A1 peptide fragment candidates, and preparation of each peptide fragment

Example 1.1. Candidate peptide fragment selection

To select peptide candidates with the effect of strengthening the skin barrier within the human hCOL5A1 protein domain, three types of peptides were identified based on the proline-rich part.

Specifically, amino acid sequence information for the selected peptide candidates is shown in Table 2 below, and a schematic diagram of the hCOL5A1 chain protein and the positions of the candidate peptides specified therefrom are shown in Figure 1. Each peptide fragment is 120 aa long and has a size of approximately 17.7 kDa.

Amino acid sequence information of candidate peptides designation amino acid sequence sequence number S1 peptide
(901-1020aa) GTPGKPGPRGQRGPTGPRGERGPRGITGKPGPKGNSGGDGPAGPPGERGPNGPQGPTGFPGPKGPPGPPGKDGLPGHPGQRGETGFQGKTGPPGPPGVVGPQGPTGETGPMGERGHPGPP One S2 peptide
(1321-1440aa) GPPGPKGPPGDDGPKGSPGPVGFPGDPGPPGEPGPAGQDGPPGDKGDDGEPGQTGSPPGPTGEPGPSGPPGKRGPPGPAGPEGRQGEKGAKGEAGLEGPPGKTGPIGPQGAPGKPGPDGLR 2 S3 peptide
(1521-1640aa) TGPSGPIGPPGPPGLPGPPGPKGAKGSSGPTGPKGEAGHPGPPGPPGPPGEVIQPLPIQASRTRRNIDASQLLDDGNGENYVDYADGMEEIFGSLNSLKLEIEQMKRPLGTQQNPARTCK 3

Example 1.2. Preparation of recombinant expression strains of candidate peptide fragments

Each recombinant expression vector to express the candidate peptide selected in Example 1.1 was prepared.

Specifically, each encoded DNA sequence (base sequence information of the candidate peptide is shown in Table 3 below) to prepare a candidate peptide fragment was synthesized, and in order to apply the encoded DNA sequence to a vector, the C-terminus of the DNA was added. Synthesis was performed by introducing restriction enzyme BamH1 and Xho1 at the N terminus. The synthesized DNA was inserted into an amplification vector (pUC_GW_Amp) and introduced into XB cells, and then amplification was performed by culturing the XB cells in LB broth containing ampicillin. Next, plasmid DNA was extracted from cultured XB cells and treated with BamH1 and Xho1 to obtain a large amount of encoded DNA. Afterwards, the encoded DNA sequence was inserted into the expression vector (pET-28a(+)) and transformed into the expression strain E.coli Clearcoli BL21 (LPS free) to obtain the expression strain.

Base sequence information of candidate peptides designation base sequence sequence number S1 peptide GGATGGATCCATGGGCACCCCGGGCAAGCCGGGCCCGCGTGGTCAGCGGGGTCCAACCGGGCCAAGGGGCGAAAGAGGACCGCGCGGCATTACCGGCAAGCCTGGACCGAAAGGGAATAGCGGGGGAGACGGACCGGCTGGTCCGCCCGGCGAACGAGGACCGAACGGACCGCAAGGTCCAACCGGCTTTCCGGGCCCGAAGGGCCCTCCGGGCCCGCCCGGGAAAGATGGGTTGCCGGGCCATCCGGGTCA GCGCGGTGAAACCGGCTTTCAAGGCAAAACCGGACCACCGGGCCCCCCGGCGTAGTAGGCCCGCAAGGCCCAACCGGCGAAACCGGGCCTATGGGCGAGCGCGGTCATCCGGGCCCGCCGCTCGAG 4 S2 peptide GGATCCATGGGGGCCCCCGGGCCCAAAGGGACCTCCGGGGGACGACGGACCAAAAGGGTCCCCTGGTCCGGTGGGCTTTCCGGGCGACCCGGGCCCCCCCGGTGAGCCGGGCCCGGCTGGGCAAGACGGACCGCCCGGCGACAAGGGCGACGATGGAGAGCCGGGCCAAACTGGATCTCCGGGACCTACGGGGTGAACCGGGTCCAAGTGGGCCCCCTGGCAAAAGAGGACCTCCGGGCCCGGCGGGCCCGGAG GGTCGCCAAGGCGAAAAAGGCGCGAAAGGGGAGGCGGGCCTGGAAGGGCCTCCGGGCAAAACCGGCCCAATTGGCCCACAAGGCGCGCCGGGTAAACCTGGCCCGGATGGCCTGCGCCTCGAG 5 S3 peptide GGATCCATGGGGGCCCCCGGGCCCAAAGGGACCTCCGGGGGACGACGGACCAAAAGGGTCCCCTGGTCCGGTGGGCTTTCCGGGCGACCCGGGCCCCCCCGGTGAGCCGGGCCCGGCTGGGCAAGACGGACCGCCCGGCGACAAGGGCGACGATGGAGAGCCGGGCCAAACTGGATCTCCGGGACCTACGGGGTGAACCGGGTCCAAGTGGGCCCCCTGGCAAAAGAGGACCTCCGGGCCCGGCGGGCCCGGAG GGTCGCCAAGGCGAAAAAGGCGCGAAAGGGGAGGCGGGCCTGGAAGGGCCTCCGGGCAAAACCGGCCCAATTGGCCCACAAGGCGCGCCGGGTAAACCTGGCCCGGATGGCCTGCGCCTCGAG 6

Example 1.3. Preparation of candidate peptide fragments

The strain into which pET-28a(+)-hCOL5A1 prepared in Example 1.2 was introduced was cultured to express each candidate peptide fragment in large quantities and purify to isolate pure protein fragments.

Specifically, each of the above-prepared expression strains was cultured in a medium containing 50 μg/mL kanamycin at 37°C and 400 rpm until the OD value reached 1.0. Afterwards, IPTG was added to a final concentration of 1 mM to induce expression of the fragment peptide, and then cultured for an additional 10 hours at 37°C and 400 rpm. The culture medium was centrifuged (10,000 rpm, 10 minutes, 4°C) to obtain bacterial cells, suspended in 50 mM Tris-buffer (pH 8.0), and then disrupted using a high-pressure dispersion device. The lysate was centrifuged (12,000 rpm, 30 minutes, 4°C), the pellet was discarded, and only the supernatant was taken. Next, the supernatant was purified using a His-Binding column, and residual ions were removed using ultrafiltration to obtain pure peptide protein.

The obtained peptide protein was quantified by BCA assay, and its molecular weight and purity were confirmed through SDS-PAGE.

The results are shown in Figure 2 below, confirming that the peptide fragment of recombinant hCOL5A1 with a size of approximately 17-20 kDa was effectively overexpressed in the E. coli Clearcoli BL21 (LPS free) strain and was obtained as a pure recombinant protein through a purification step. I was able to.

Example 2. High-concentration cultivation and protein expression confirmation of hCOL5A1 peptide fragment

Example 2.1. High-concentration cultivation of hCOL5A1 peptide fragments

<Seed cultivation>

The strain into which pET-28a(+)-hCOL5A1 was introduced was cultured for 6 hours at 37°C and 100 rpm in a medium containing 50 μg/mL kanamycin, and the culture medium was used as a seed culture for large-scale main culture.

<Main culture>

The seed culture was inoculated at a level of 1/50 into a culture medium that did not contain kanamycin and contained a high concentration of nutrients, and was then cultured at 37°C and 100 rpm until the OD value reached 8. Afterwards, IPTG was added to a final concentration of 1 mM to induce expression of the fragment peptide, and further culture was performed at 37°C and 400 rpm. During additional culturing, a 50% glucose solution was periodically added to provide additional nutrients, and the culture was performed until the OD value reached 30.

<Confirmation of expression level of fragment peptide protein>

To confirm the protein expression level of the mass-cultured peptide fragment, the culture medium was centrifuged (10,000 rpm, 10 minutes, 4°C) to obtain bacterial cells, which were then suspended in 50 mM Tris-buffer. Then, the suspension was crushed using a high-pressure dispersion device, and the crushed liquid was centrifuged (12,000 rpm, 30 minutes, 4°C) to obtain the supernatant. The supernatant was purified using a His-Binding column, and residual ions were removed using ultrafiltration to obtain pure protein fragments.

The obtained protein was quantified using BCA assay, and the expression level of the fragment protein was calculated according to the OD value.

The results are shown in Figure 3 below.

As shown in Figure 3, it was confirmed that the expression level of the protein increased as the OD value increased, and it was confirmed that the highest expression level was maintained constant in the OD value range of 25-30.

Example 3. Confirmation of the skin barrier and elasticity strengthening effect of hCOL5A1 peptide fragment

In order to confirm that the hCOL5A1 peptide fragment of the present invention has the effect of strengthening the skin barrier and elasticity, the activity of factors related to the skin barrier and elasticity was evaluated using this fragment.

Example 3.1. Evaluation of collagen III production-promoting activity of hCOL5A1 peptide fragment

First, the activity of promoting the production of Collagen type III (Col-III), a skin elasticity-related factor, by the recombinant hCOL5A1 peptide fragment isolated and purified in Example 1 was evaluated.

Specifically, human dermal fibroblasts (HDF) were cultured in IMDM (Iscove's modified Dulbecco's medium) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotic-antimycotic. Cultured human skin fibroblasts were seeded on a 60 mm plate at a density of 1X10 ⁶ cells and cultured in an incubator at 37°C and 5% CO ₂ for 24 hours for cell attachment and environmental adaptation. Then, the medium was replaced with a serum-free medium, and the cells were treated with different types of recombinant hCOL5A1 peptide fragments (experimental groups) and cultured for 26 hours. Here, the untreated group was used as a negative control, and the vitamin C treated group was used as a positive control. After washing the cultured cells with 1 ) was carried out. The separated proteins were added to Bolt™ Bis-Tris 8% gel and subjected to SDS-PAGE, and then the proteins were transferred using the iBlot2 dry blotting system (Invitrogen). Then, the cells were blocked in 5% skim milk for 2 hours, treated with collagen type III (Santa Cruz) antibody diluted 1:500, and reacted at 4°C for one day. Next, it was washed three times with 1XTBST, treated with anti-mouse-HRP (Bio-rad) at a ratio of 1:1,000, and reacted at room temperature for 1 hour. Finally, after washing three times with 1XTBST, the cells were treated with ECL solution (west save gold, Abforntier), and protein detection was performed using Atto II Chemi Doc (ATTO, Japan).

The results are shown in band images and graphs in Figure 4 below, and the quantified results are shown in Table 4 below.

Collagen type III (Col-III) protein expression Sample Cont. (㎍/㎖) Protein expression rate (%) p-value Average Stdev. Control (untreated group) 100.0 0.00 1.0000 COLV_S1 10 260.3 5.52 0.0000 COLV_S2 10 219.0 8.13 0.0000 COLV_S3 10 179.5 7.80 0.0001 Vitamin C 100 140.0 3.37 0.0000

As shown in Figure 4 and Table 4, based on the Col-III protein expression ratio in the negative control group of 100%, it was confirmed that the expression of S1 peptide increased by 260.3%, S2 peptide increased by 219.0%, and S3 peptide increased by 179.5%. did. In particular, it was confirmed that among the three candidate peptides, the expression rate of S1 peptide was the best.

Therefore, it is believed that the S1 peptide of the present invention exerts an effect on skin elasticity by increasing the components of the fibrous tissue of the dermis within the skin.

Example 3.2. Evaluation of dermal-epidermal junction (DEJ) strengthening efficacy of hCOL5A1 peptide fragment

In addition, to evaluate the strengthening effect of the dermal-epidermal junction (DEJ) by the recombinant hCOL5A1 fragment protein isolated and purified in Example 1, the degree of recovery of the expression of Laminin 5, the expression of which was reduced by UVB irradiation, was evaluated. was evaluated.

Specifically, A431 (human epidermoid carcinoma cell), a keratinocyte cell line, was cultured in MEM (minimum essential medium) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotic-antimycotic. . Cultured A431 (human epidermoid carcinoma cells) were seeded on a 60 mm _plate at a density of ² did. Then, the cells were washed with ¹ Cultivation was performed during. Here, the UVB non-irradiated group was used as a comparison group, the untreated group was used as a negative control, and the vitamin C treated group was used as a positive control. After washing the cultured cells with 1 ) was carried out. The separated proteins were added to NUPAGE™ Tris-acetate 7% gel and subjected to SDS-PAGE, and then the proteins were transferred using the iBlot2 dry blotting system (Invitrogen). Then, 5% skim milk was added for blocking for 2 hours, and laminin 5 (Santa Cruz) antibody diluted 1:1,000 was added and reacted at 4°C for one day. Afterwards, it was washed three times with 1XTBST, treated with anti-mouse-HRP (Bio-rad) at a ratio of 1:1,000, and reacted at room temperature for 1 hour. Finally, after washing three times with 1XTBST, the cells were treated with ECL solution (west save gold, Abforntier), and protein detection was performed using Atto II Chemi Doc (ATTO, Japan).

The results are shown in band images and graphs in Figure 5 below, and the quantified results are shown in Table 5 below.

Laminin 5 protein expression Sample UVB 50 mJ/ ^cm2 Protein expression rate (%) p-value Average Stdev. Control (untreated group) - 324.9 25.50 0.0008 + 100.0 11.38 1.0000 COLV_S1 + 235.3 10.87 0.0001 COLV_S2 + 195.3 10.20 0.0025 COLV_S3 + 133.3 5.77 0.0345 Vitamin C (75㎍/㎖) + 269.4 6.28 0.0003

As shown in Figure 5 and Table 5, based on the expression recovery level of Laminin 5 in the negative control group as 100%, the S1 peptide showed an expression recovery level of 235.3%, the S2 peptide showed an expression recovery level of 195.3%, and the S3 peptide showed an expression recovery level of 133.3%. Confirmed. In particular, among the three candidate peptides, it was confirmed that the S1 peptide treatment group showed the best level of restoration of Laminin 5 expression.

Therefore, it is believed that the S1 peptide of the present invention can help skin recovery by strengthening the interaction between the dermis and epidermis in the skin and effectively act on skin elasticity.

Sequence list electronic file attached

Claims (13)

A peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 3.
A polynucleotide encoding the peptide of claim 1.
The polynucleotide according to claim 2, wherein the polynucleotide consists of any one base sequence of SEQ ID NOs: 4 to 6.
A recombinant expression vector containing the polynucleotide of claim 2.
A host cell transformed with the recombinant expression vector of claim 4.
A method of producing the peptide of claim 1, comprising the following steps:
(a) transforming a host cell with a recombinant expression vector expressing the peptide of claim 1;
(b) culturing the transformed host cells; and
(c) Obtaining the peptide of claim 1 from the cultured host cells.
The method according to claim 6, wherein the recombinant expression vector in step (a) is pET-28a(+).
The method of claim 6, wherein in step (c), the peptide is obtained by permeating host cell lysate or lysate through a microfiltration membrane.
A cosmetic composition for strengthening the skin barrier and elasticity comprising the peptide of claim 1.
The cosmetic composition for strengthening the skin barrier and elasticity according to claim 9, wherein the composition increases the expression of proteins such as Col-III (collagen type III), laminin 5, or a combination thereof.
A cosmetic composition for promoting collagen production in the skin, comprising the peptide of claim 1.
A cosmetic composition for improving skin wrinkles comprising the peptide of claim 1.
A cosmetic composition for improving skin damage from ultraviolet rays, comprising the peptide of claim 1.