KR101679310B1 - Cosmetic composition for skin regeneration and skin soothing comprising novel peptide and growth factor complex - Google Patents

Abstract

The present invention relates to a cosmetic composition for wound regeneration and skin soothing which contains a novel peptide and a growth factor complex, and more particularly to a novel peptide (prospin) having efficacy for wound regeneration and skin soothing, To a cosmetic composition having a wound regeneration and skin soothing effect containing a growth factor complex containing various growth factors.
The complex containing the peptide according to the present invention and further containing EGF, bFGF, SCF and PGF has excellent skin regeneration ability and ability to regenerate skin wound, and is particularly effective for protecting and regenerating skin through application after skin laser treatment .

Description

TECHNICAL FIELD The present invention relates to a cosmetic composition for skin regeneration and skin soothing containing a novel peptide and a growth factor complex,

The present invention relates to a cosmetic composition for wound regeneration and skin soothing which contains a novel peptide and a growth factor complex, and more particularly to a novel peptide (prospin) having efficacy for wound regeneration and skin soothing, To a cosmetic composition having a wound regeneration and skin soothing effect containing a growth factor complex containing various growth factors.

Recently, as people's interest in skin has increased, more and more cases are being treated to treat scars such as acne by laser dermabrasion in dermatology to make clean skin.

Laser peeling is mainly used for the treatment of scars and wrinkles caused by acne or chickenpox. The principle of treatment is to irradiate the skin with exactly one degree of burning by laser light, so that new tissue grows in the dermis layer. Erbium laser and carbon dioxide laser are used for laser peeling. Carbon dioxide gas lasers have a lot of heat damage and cause a lot of damage to the surrounding tissue.

Erbium-YAG lasers have little thermal damage and thus have less side effects such as erythema and pigmentation after treatment than carbonic acid lasers. In contrast, it has been found that heat damage requires some degree of scarring, and recently, it is used in combination with a carbon dioxide gas laser rather than an erbium laser. If you do not get satisfactory results with a single procedure, you will have to try it several times, but it also has side effects such as hypercholesterolemia after the procedure. In addition, since skin burns due to heat damage may occur, it is necessary to use a skin lotion having a wound regeneration activity, which is not irritating in order to calm the skin weakened by heat damage and recover the wound quickly after laser sheathing.

(Korean Patent Laid-open Publication No. 1020130056838), there has been no case in which the novel peptide and the growth factor complex containing the peptide of the present invention are used for wound regeneration and skin relaxation.

Patent Document 1: Korean Patent Publication No. 102013005683

The present inventors have synthesized novel peptides having skin wound regeneration efficacy and sedative ability, and confirmed that the growth factor complex (complex) containing such peptides and growth factors has a skin wound regeneration efficacy and sedative ability, thus completing the present invention.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition containing a peptide having an amino acid sequence of SEQ ID NO: 1 and a skin further containing an epidermal growth factor (EGF), a basic fibroblast growth factor (bFGF), a stem cell factor (SCF) A skin-soothing composition, or a cosmetic composition for applying a laser treatment site.

In order to achieve the above object, the present invention provides a pharmaceutical composition comprising one or more growth factors selected from the group consisting of Epidermal Growth Factor (EGF), Basic Fibroblast Growth Factor (bFGF), Stem Cell Factor (SCF) and Placenta Growth Factor And a peptide having an amino acid sequence of SEQ ID NO: 1.

The present invention also provides a pharmaceutical composition comprising at least one growth factor selected from the group consisting of Epidermal Growth Factor (EGF), Basic Fibroblast Growth Factor (bFGF), Stem Cell Factor (SCF) and Placenta Growth Factor (PGF) The present invention provides a cosmetic composition for skin wound regeneration comprising a peptide having an amino acid sequence of SEQ ID NO:

The present invention also provides a pharmaceutical composition comprising at least one growth factor selected from the group consisting of Epidermal Growth Factor (EGF), Basic Fibroblast Growth Factor (bFGF), Stem Cell Factor (SCF) and Placenta Growth Factor (PGF) The present invention provides a cosmetic composition for application to a skin laser treatment site.

The present invention also provides a skin-soothing cosmetic composition containing the peptide.

The present invention also provides a cosmetic composition for applying a skin laser treatment site containing the peptide.

The present invention also provides a cosmetic composition which further contains not only the peptide but also an epidermal growth factor (EGF), a basic fibroblast growth factor (bFGF), a stem cell factor (SCF) or a placenta growth factor (PGF).

The complex containing the peptide according to the present invention and further containing EGF, bFGF, SCF and PGF has excellent skin regeneration ability and ability to regenerate skin wound, and is particularly effective for protecting and regenerating skin through application after skin laser treatment .

1 is a graph showing the cytotoxicity test results of the composition of the present invention.
FIG. 2 is a microscopic photograph showing the results of the keratin-forming cell line cell proliferative activity test of the composition of the present invention. FIG.
FIG. 3 shows the results of experiments on the ability of the composition of the present invention to proliferate fibroblasts.
Fig. 4 shows the results of the cytoprotection test for UV of the composition of the present invention.

The present invention relates to a novel peptide having wound regeneration efficacy and skin sedative ability and a cosmetic composition containing growth factors of EGF, bFGF, SCF and PGF.

The novel peptide according to the present invention is called a prospin, specifically a hexapeptide having the amino acid sequence of SEQ ID NO: 1, which is a peptide in which the N-terminal of the peptide is bound with nicotinic acid.

The amino acid sequence of SEQ ID NO: 1 is an inhibitory peptide of PAR2, which is a signal transduction receptor that induces exogenous aging of various environmental pollution and stress, Vitamin B3 (Nicotinic acid, Niacin) which is a vitamin essential for healthy skin condition and young and vital skin , Which is a new concept of advanced anti-wrinkle and anti-aging peptide new materials combined with nicotinic acid.

The amino acid sequence of prospin is FSLLRY-NH2 (phenylalanine-serine-leucine-leucine-arginine-tyrosine-amide) and has been developed to synthesize it based on a solid phase synthesis platform for high efficiency production. Peptides are substances in which two or more amino acids are linked in a chemical bond called a peptide bond. Most of them now use solid phase peptide synthesis (SPPS), which was originally designed by Bruce Merrifield in 1963. In this research development, we have also developed a synthesis platform of Prospin based on this solid phase synthesis method. To this end, amino acids having a protecting group in an insoluble polystyrene resin are sequentially added in sequence to synthesize peptide moieties first, and then chemically synthesized nicotinic acid directly without separating them from the synthetic support, A method for removing nicotinic acid from a reaction body has been developed as a platform.

In one aspect, the present invention relates to cosmetic compositions for skin regeneration, skin regeneration, containing the novel peptide and growth factor complexes.

These growth factors include EGF, bFGF, SCF, and PGF.

EGF (epidermal growth factor) is an epithelial cell growth factor and cell signaling is carried out by EGF receptor. EGF consists of 53 amino acids and is a tertiary structure consisting of three disulfide bonds. The biological mechanism of EGF is known to be involved in cell proliferation, differentiation, and survival.

The cDNA sequence for this EGF is shown in SEQ ID NO: 2, and the protein sequence is shown in SEQ ID NO: 3.

SEQ ID NO: 2: AATAGTGACTCTGAATGTCCCCTGTCCCACGATGGGTACTGCCTCCATGATGGTGTGTGCATGTATATTGAAGCATTGGACAAGTATGCATGCAACTGTGTTGTTGGCTACATCGGGGAGCGATGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGC,

SEQ ID NO: 3: NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

bFGF (basic fibroblast growth factor) is a fibroblast growth factor that not only promotes proliferation and differentiation of fibroblast cells but also acts on proliferation and differentiation of vascular endothelial cells, vascular smooth muscle cells and epidermal cells. Therefore, it is known that bFGF is an essential protein to heal cell damage in the event of cell damage, and is known to exert excellent effects in burn, pressure sores, skin wounds and the like.

The cDNA sequence for this bFGF is shown in SEQ ID NO: 4 and the protein sequence is shown in SEQ ID NO:

SEQ ID NO: 4:

-cDNA: CCCGCCTTGCCCGAGGATGGCGGCAGCGGCGCCTTCCCGCCCGGCCACTTCAAGGACCCCAAGCGGCTGTACTGCAAAAACGGGGGCTTCTTCCTGCGCATCCACCCCGACGGCCGAGTTGACGGGGTCCGGGAGAAGAGCGACCCTCACATCAAGCTACAACTTCAAGCAGAAGAGAGAGGAGTTGTGTCTATCAAAGGAGTGTGTGCTAACCGTTACCTGGCTATGAAGGAAGATGGAAGATTACTGGCTTCTAAATGTGTTACGGATGAGTGTTTCTTTTTTGAACGATTGGAATCTAATAACTACAATACTTACCGGTCAAGGAAATACACCAGTTGGTATGTGGCACTGAAACGAACTGGGCAGTATAAACTTGGATCCAAAACAGGACCTGGGCAGAAAGCTATACTTTTTCTTCCAATGTCTGCTAAGAGCTGA

SEQ ID NO: 5:

- Protein: PALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS)

SCF (stem cell factor) is a major growth factor that binds to c-Kit receptor (CD117) as a growth factor known as kit-ligan, KL or steel factor. SCF is present in both membrane-bound and water-soluble protein forms and is involved in blood formation, spermatogenesis and melanin formation. SCF is an essential element that activates stem cells present in almost all tissues including skin. It is known to prevent aging in skin and promote the formation of new hair follicles in hair.

The cDNA sequence for this SCF is shown in SEQ ID NO: 6, and the protein sequence is shown in SEQ ID NO: 7

SEQ ID NO: 6:

cDNA: ATGAAGAAGACACAAACTTGGATTCTCACTTGCATTTATCTTCAGCTGCTCCTATTTAATCCTCTCGTCAAAACTGAAGGGATCTGCAGGAATCGTGTGACTAATAATGTAAAAGACGTCACTAAATTGGTGGCAAATCTTCCAAAAGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGTTTTGCCAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAGACAGCTTGACTGATCTTCTGGACAAGTTTTCAAATATTTCTGAAGGCTTGAGTAATTATTCCATCATAGACAAACTTGTGAATATAGTGGATGACCTTGTGGAGTGCGTGAAAGAAAACTCATCTAAGGATCTAAAAAAATCATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTTAGAATTTTTAATAGATCCATTGATGCCTTCAAGGACTTTGTAGTGGCATCTGAAACTAGTGATTGTGTGGTTTCTTCAACATTAAGTCCTGAGAAAGGGAAGGCCAAAAATCCCCCTGGAGACTCCAGCCTACACTGGGCAGCCATGGCATTGCCAGCATTGTTTTCTCTTATAATTGGCTTTGCTTTTGGAGCCTTATACTGGAAGAAGAGACAGCCAAGTCTTACAAGGGCAGTTGAAAATATACAAATTAATGAAGAGGATAATGAGATAAGTATGTTGCAAGAGAAAGAGAGAGAGTTTCAAGAAGTGTAA

SEQ ID NO: 7:

protein :

MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKGKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV

PGF (placenta growth factor) is a growth factor belonging to the VEGF family and is a major growth factor that regulates angiogenesis and growth in the embryonic stage. PGF, which is produced most abundantly by placental trophoblast during pregnancy, is a key substance that enables the physiological functions of the commonly referred placenta and placenta extracts.

The cDNA sequence for this PGF is shown in SEQ ID NO: 8, and the protein sequence is shown in SEQ ID NO:

PGF

SEQ ID NO: 8:

 - cDNA:

ATGCCGGTCATGAGGCTGTTCCCTTGCTTCCTGCAGCTCCTGGCCGGGCTGGCGCTGCCTGCTGTGCCCCCCCAGCAGTGGGCCTTGTCTGCTGGGAACGGCTCGTCAGAGGTGGAAGTGGTACCCTTCCAGGAAGTGTGGGGCCGCAGCTACTGCCGGGCGCTGGAGAGGCTGGTGGACGTCGTGTCCGAGTACCCCAGCGAGGTGGAGCACATGTTCAGCCCATCCTGTGTCTCCCTGCTGCGCTGCACCGGCTGCTGCGGCGATGAGAATCTGCACTGTGTGCCGGTGGAGACGGCCAATGTCACCATGCAGCTCCTAAAGATCCGTTCTGGGGACCGGCCCTCCTACGTGGAGCTGACGTTCTCTCAGCACGTTCGCTGCGAATGCCGGCCTCTGCGGGAGAAGATGAAGCCGGAAAGGTGCGGCGATGCTGTTCCCCGGAGGTAA

SEQ ID NO: 9:

 - protein sequence:

MPVMRLFPCFLQLLAGLALPAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLVDVVSEYPSEVEHMFSPSCVSLLRCTGCCGDENLHCVPVETANVTMQLLKIRSGDRPSYVELTFSQHVRCECRPLREKMKPERCGDAVPRR

The growth factors are produced by using a microorganism fermentation system using the human gene as an origin. The gene sequence of each growth factor was synthesized, inserted into an expression vector, and injected into E. coli. When the microorganism had an OD value of about 0.5 to 0.8, IPTG (Isopropyl-D-1-thiogalactopyranoside) Induction. The resulting pellet was gently suspended in 20 ml of lysis buffer per g, sonicated for 2 hours with a sonicator, and the membrane of the microorganism was rinsed with sonicator for 2 hours. Thereafter, the supernatant is centrifuged at 12,000 rpm, and the supernatant is purified by chromatography (purification purity of 95% or more).

In the present invention, the content ratio of the peptide and the growth factors in the composition is not particularly limited. For example, the ratio of the EGF: bFGF: SCF: PGF: peptide is 2: 5: 1 to 5: 1 to 5: ≪ / RTI > In other words, EGF: bFGF: SCF: PGF is 2 to 5, 2 to 5, 2 to 5, and 2 to 5 parts by weight based on 100 parts by weight of the peptide.

  The cosmetic composition of the present invention may contain an acceptable carrier in cosmetic preparations. Herein, "an acceptable carrier for a cosmetic preparation" is a known or used compound or composition that can be contained in a cosmetic preparation, or a compound or composition to be developed in the future, which is toxic, instable or irritating It says nothing.

The carrier may be included in the cosmetic composition of the present invention in an amount of about 1% by weight to about 99.99% by weight, preferably about 90% by weight to about 99.99% by weight of the composition, based on the total weight thereof. However, since the ratio varies depending on the formulations described below in which the composition for external application for skin of the present invention is prepared, and its specific application site (face, neck, etc.) or its preferable application amount and the like, And should not be construed as limiting the scope of the invention.

Examples of the carrier include an alcohol, an oil, a surfactant, a fatty acid, a silicone oil, a wetting agent, a moisturizer, a viscous modifier, an emulsifier, a stabilizer, an ultraviolet scattering agent, an ultraviolet absorber, a coloring agent and a perfume. The compounds / compositions which can be used as the above-mentioned alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscous modifier, emulsifier, stabilizer, ultraviolet scattering agent, ultraviolet absorber, The person skilled in the art can select and use the appropriate substance / composition.

In one embodiment of the present invention, the composition according to the present invention may contain, in addition to the growth factor complex, glycerin, butylene glycol, propylene glycol, polyoxyethylene hardened castor oil, ethanol, triethanolamine, , Coloring agent, purified water and the like can be contained as needed.

The cosmetic composition according to the present invention can be prepared in various forms such as lotion, essence, gel, emulsion, lotion, cream (water-in-oil type, water-in-water type, multiphase), solution, suspension (Soft capsules, hard capsules) with a coating such as anhydrous products (oil and glycol), gel, mask, pack, powder, or gelatin.

In the present invention, the cosmetic composition of the present invention is not only a face but also a composition for external application of the skin including scalp and whole body. It is a composition that can be applied to such a scalp and includes a shampoo, a rinse, a treatment, a hair removal agent, And the like can be manufactured in various forms.

The method for preparing the composition according to the present invention is not limited to the above-mentioned preparation method. Any person skilled in the art will be able to prepare the composition according to the present invention can do.

In particular, the compositions may be prepared in the form of conventional emulsification and solubilization formulations, in addition to the preparation methods specifically disclosed in the present invention, using a conventionally known preparation method.

When the cosmetic composition is manufactured from a cosmetic composition, the cosmetic composition of the emulsified formulation includes nutritional lotion, cream, essence and the like. It can also be prepared in the form of adjuvants which can be applied topically or systemically, which are conventionally used in the field of dermatology by containing a dermatologically acceptable medium or base.

In addition, suitable cosmetic formulations include, for example, solutions, gels, solid or kneaded anhydrous products, emulsions obtained by dispersing an oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) May be provided in the form of a follicular dispersing agent of the type, cream, skin, lotion, powder, ointment, spray or conical stick. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

The composition of the present invention can further comprise at least one of a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a perfume, Any other ingredients commonly used in cosmetics, such as nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, Such as < RTI ID = 0.0 > cosmetics < / RTI > or dermatology. And, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

Such a composition according to the present invention includes forms of functional cosmetics such as skin soothing, skin wound healing, and anti-aging.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

1.1 Novel peptide synthesis

Prospin is a new substance combined with a peptide consisting of 6 amino acids and nicotinic acid. The peptide sequence is a sequence known to inhibit the PAR-2 signal by FSLLRY. As a synthesis method, it was synthesized by solid phase synthesis, and the order of amino acid binding was synthesized in the N-terminal direction at the C-terminal. Specifically, 2-chlorotritylchloride resin (1.4 mmole / g, bead tech) is weighed into a 710 mg (1 mM) balance and reacted sufficiently with DMF (N, N'-DiMethyl Formamide, DAE JUNG) to swell. After 30 minutes of activation with HOBT (N-hydorxybenzotriazole, beadTech) and DIC (N, N'-Diisoproyl carbodiimide, Alfa Aesar), tyrosine (Y) protected with F-moc was calculated as 5 equivalents And then react with resin already swelled for 3 to 4 hours. After the reaction, 20% Piperidine (Merk) is applied to remove Fmoc and allowed to react for about 15 minutes. It then binds the next sequence, Argine (R), to the last sequence, Nicotinic acid, by repeating the above method with Tyrosine. The synthesized peptide is purified by reverse phase (RP) -HPLC with Bondapack C18 column to a purity of 95% or more.

1.2 Growth factor preparation

Growth factors of EGF, bFGF, SCF, and PGF were produced by using microorganism fermentation system using each human gene as an origin to obtain cosmetic composition for wound restoration and skin soothing using growth factor complex used in this study. Each origin is synthesized (bioneer), and in detail, to obtain EGF (Epidermal Growth Factor), human genetic information

cDNA (SEQ ID NO: 2: AATAGTGACTCTGAATGTCCCCTGTCCCACGATGGGTACTGCCTCCATGATGGTGTGTGCATGTATATTGAAGCATTGGACAAGTATGCATGCAACTGTGTTGTTGGCTACATCGGGGAGCGATGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGC, Protein sequence: SEQ ID NO: 3: NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR) were cloned into the expression by the vector into the PET41b + bacteria (BL21). The restriction enzymes used were cloned into PET41b + (Novagen) vector using NdeI (NEB, Cat #: R0111S) and XhoI (NEB, Cat #: R0146S). The vector with the human gene inserted was reacted at 42 ° C for 30 seconds in order to transform BL21 (NEB, Cat #: C25271). Bacteria with the protein gene inserted were cultured in a 500 ml culture medium and then treated with 0.5 mM IPTG (Isopropyl bD-1-thiogalactopyranoside, Sigma, Cat #: 16758-1G) at OD 0.6 to induce protein expression Respectively. After induction with IPTG, the cells were further cultured at 37 ° C. for 3 hours. After completion of the culture, microbial pellets were obtained by centrifugation at 6,000 rpm for 20 minutes using a high-speed viscous centrifuge (VS-24SMTi). Thereafter, the cells were suspended in 20 ml of lysis buffer (5 mM sodiumphosphate buffer) per 1 g, followed by sonication to solubilize the proteins. Subsequently, the protein was purified through chromatography to a purity of 95% or more. The remaining growth factors were also purified using the same experimental method, and the respective genetic and protein information were as follows.

bFGF

-cDNA: SEQ ID NO: 4: CCCGCCTTGCCCGAGGATGGCGGCAGCGGCGCCTTCCCGCCCGGCCACTTCAAGGACCCCAAGCGGCTGTACTGCAAAAACGGGGGCTTCTTCCTGCGCATCCACCCCGACGGCCGAGTTGACGGGGTCCGGGAGAAGAGCGACCCTCACATCAAGCTACAACTTCAAGCAGAAGAGAGAGGAGTTGTGTCTATCAAAGGAGTGTGTGCTAACCGTTACCTGGCTATGAAGGAAGATGGAAGATTACTGGCTTCTAAATGTGTTACGGATGAGTGTTTCTTTTTTGAACGATTGGAATCTAATAACTACAATACTTACCGGTCAAGGAAATACACCAGTTGGTATGTGGCACTGAAACGAACTGGGCAGTATAAACTTGGATCCAAAACAGGACCTGGGCAGAAAGCTATACTTTTTCTTCCAATGTCTGCTAAGAGCTGA

- protein seqence: SEQ ID NO: 5: PALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS)

SCF

cDNA: SEQ ID NO: 6: ATGAAGAAGACACAAACTTGGATTCTCACTTGCATTTATCTTCAGCTGCTCCTATTTAATCCTCTCGTCAAAACTGAAGGGATCTGCAGGAATCGTGTGACTAATAATGTAAAAGACGTCACTAAATTGGTGGCAAATCTTCCAAAAGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGTTTTGCCAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAGACAGCTTGACTGATCTTCTGGACAAGTTTTCAAATATTTCTGAAGGCTTGAGTAATTATTCCATCATAGACAAACTTGTGAATATAGTGGATGACCTTGTGGAGTGCGTGAAAGAAAACTCATCTAAGGATCTAAAAAAATCATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTTAGAATTTTTAATAGATCCATTGATGCCTTCAAGGACTTTGTAGTGGCATCTGAAACTAGTGATTGTGTGGTTTCTTCAACATTAAGTCCTGAGAAAGGGAAGGCCAAAAATCCCCCTGGAGACTCCAGCCTACACTGGGCAGCCATGGCATTGCCAGCATTGTTTTCTCTTATAATTGGCTTTGCTTTTGGAGCCTTATACTGGAAGAAGAGACAGCCAAGTCTTACAAGGGCAGTTGAAAATATACAAATTAATGAAGAGGATAATGAGATAAGTATGTTGCAAGAGAAAGAGAGAGAGTTTCAAGAAGTGTAA

protein sequence: SEQ ID NO: 7:

MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKGKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV

PGF

 - cDNA: SEQ ID NO: 8:

ATGCCGGTCATGAGGCTGTTCCCTTGCTTCCTGCAGCTCCTGGCCGGGCTGGCGCTGCCTGCTGTGCCCCCCCAGCAGTGGGCCTTGTCTGCTGGGAACGGCTCGTCAGAGGTGGAAGTGGTACCCTTCCAGGAAGTGTGGGGCCGCAGCTACTGCCGGGCGCTGGAGAGGCTGGTGGACGTCGTGTCCGAGTACCCCAGCGAGGTGGAGCACATGTTCAGCCCATCCTGTGTCTCCCTGCTGCGCTGCACCGGCTGCTGCGGCGATGAGAATCTGCACTGTGTGCCGGTGGAGACGGCCAATGTCACCATGCAGCTCCTAAAGATCCGTTCTGGGGACCGGCCCTCCTACGTGGAGCTGACGTTCTCTCAGCACGTTCGCTGCGAATGCCGGCCTCTGCGGGAGAAGATGAAGCCGGAAAGGTGCGGCGATGCTGTTCCCCGGAGGTAA

 - Protein sequence: SEQ ID NO: 9:

MPVMRLFPCFLQLLAGLALPAVPPQQWALSAGNGSSEVEVVPFQEVWGRSYCRALERLVDVVSEYPSEVEHMFSPSCVSLLRCTGCCGDENLHCVPVETANVTMQLLKIRSGDRPSYVELTFSQHVRCECRPLREKMKPERCGDAVPRR

1.3 Composition Preparation

The peptide obtained in 1.1 and the growth factor obtained in 1.2 were mixed in purified water at room temperature at the content ratio shown in Table 1 below to prepare a composition.

Raw material INCI name content
(%) EGF (epidermal growth factor) sh-oligopeptide-1 0.0002 Basic fibroblast growth factor (bFGF) sh-polypeptide-1 0.0001 SCF (Stem cell factor) sh-polypeptide-4 0.0001 PGF (placenta growth factor) sh-polypeptide-16 0.0001 Prospin nicotinoyl hexapeptide-44 0.005

Experimental Example 1: Cytotoxicity test

1.1 Human keratinocyte culture

Human keratinocyte HaCaT cell line was cultured for 37 days in a 5% CO ₂ incubator. Cells were detached by trypsin treatment, and then counted at 5 × 10 ³ cells / cm ² . Cells were cultured in Delbecco's Modified Eagle Medium (DMEM, GIBCO, Cat) supplemented with 10% Fetal Bovine Serum (FBS, GIBCO, Cat. No. 26140-079, USA) and 100 U / ml penicillin and 100 μg / ml streptomycin No. 11995-065, USA). A 75-T flask (NUNC, Cat. No. 156499, Danmark) was used for subculture and 24 well plates (NUNC, Cat. No. 142475, Danmark) were used for cytotoxicity.

1.2 Cytotoxicity test

In order to confirm whether the composition according to the present invention can exhibit an improvement effect without skin irritation even during long-term use, the cytotoxicity of human keratinocytes was examined. Human keratinocytes were counted in a 24-well plate at 5 × 10 ³ cells / well using a heamacytometer. After culturing for 48 hours in DMEM containing 10% FBS and culturing for 25-30% of the surface area of the culture dish, the cells were further replaced with FBS-free DMEM containing the present composition and cultured for another 24 hours. After incubation, 50 μl of a solution of 2.5 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT, Sigma M5655, USA) was added and cultured for additional 3 hours. 100 μl aliquots were transferred into 96 wells and eluted with Enzyme-Linked Immunosorbent Assay (ELISA) at 570 cells / well. The cell lysates were discarded and 200 μl of dimethyl sulfoxide (DMSO, Sigma D2650, USA) nm absorbance was measured. The degree of cytotoxicity was expressed as a percentage based on the absorbance intensity of the control using pure water.

The cytotoxicity of the skin regeneration and skin soothing cosmetic composition as compared with the control group was as shown in Fig. 1 when 5 x 10 ³ cells of the initial culture per well were cultured by 25 to 30% of the surface area of the culture container. As a result of treatment up to 10%, the growth was 150.58 ± 6.98% compared to the untreated group. In the treatment of 5% and 1%, 125.4 ± 2.74%, 116.28 ± 3.01% and 105.11 ± 8.05%, respectively, were slightly proliferative compared to the untreated group. The results showed that even when the composition was used for a long time, it did not induce skin irritation due to cytotoxicity and induces proliferation of human keratinocytes.

Experimental Example 2: Wound regeneration test

Human keratinocytes were counted in a 6-well plate at a density of 2 × 10 ⁵ cells / well using a hemocytometer. When cultured for 48 hours in DMEM containing 10% FBS and cultured to 90-100% of the surface area of the culture dish, cross-shaped wounds were induced in each cell using a sharp tool, and then 10% of the composition of the present invention was contained in FBS-free DMEM And cultured for another 48 hours. Thereafter, the amount of regeneration after wounding was compared with that of the control (untreated group) through a microscope. As a result, it was confirmed by microscopy that the wound regeneration ability was significantly increased in the human keratinocyte cell line compared with the control group without any treatment (Fig. 2)

Experimental Example 3: Fibroblast proliferative capacity test

Proliferation of fibroblasts must be essential for wound regeneration. Fibroblasts are cells that produce skin collagen such as hyaluronic acid, collagen, and elastin. For the improvement of wrinkles and elasticity, fibroblasts must be proliferated and differentiated. For this experiment, fibroblast cells were counted in a 6-well plate at a density of 2 × 10 ⁴ cells / well using a hemocytometer and then dispensed. After culturing for 24 hours in DMEM containing 10% FBS and culturing for 40-50% of the surface area of the culture dish, the culture medium containing 10% of the composition of the present invention was replaced with FBS-free DMEM for 24 hours. As a result of comparing the increase amount of fibroblasts with the control (untreated group) through the microscope after the cell staining, it was found that the composition containing 10% significantly increased the fibroblast proliferating ability (Fig. 3B) , Which promoted proliferation of fibroblasts by about 230% (FIG. 3A).

Experimental Example 4: Skin irritation experiment

In order to measure the degree of skin irritation of the skin regeneration and skin soothing composition used in the present study, a patch test was conducted on 10 subjects. A patch containing 100ul of the present composition and a control group was attached to a clean area inside the arm of the subject, and after 48 hours, it was rinsed with flowing water and the degree of stimulation after 30 minutes was judged. The negative control was pure water and the positive control was 5% SLS (Sodium Laureth Sulfate).

The evaluation of the skin reaction was made by dividing the stimulation value into five steps. As a result, it was confirmed that the skin irritation of the composition was 0 in the negative control, 40 in the positive control, 2.5 in the present composition and almost no skin irritancy.

division Number of stimulation Skin irritation
Average (%) Irritability judgment 4 3 2 One 0 Negative control group 0 0 0 0 10 0 One Positive control group 0 One 5 3 One 40 4 Composition 0 0 0 One 9 2.5 One

Experimental Example 5: Test method for protecting UV cell damage

The cytotoxicity induced by UV-B was reduced when HaCaT, a human keratinocyte cell line, was treated with cosmetic composition for wound regeneration and skin soothing after inducing cell damage with UV-B, And the effect on sedation was tested. As a specific test method, HaCaT cells, a human keratinocyte cell line, a kind of human skin cells, were cultured in Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS), 1% Antibiotic-antimycotic (GIBCO, Cat No. 15240-062 ) In a 75 Cm ² flask at 37 ° C and 5% CO ₂ . When the cells were confluence of more than 80%, the cells were subcultured and cultured at 6 × 10 ⁴ at a density of 1 × 10 ⁴ cells. Cell culture was continued until confluence reached 80% or more. After the medium was discarded, the UV was irradiated with UV-B at 50 mJ. The composition prepared in Example 1 was then further cultured for 48 hours after treatment. After that, MTT solution (6.6 mg / ml, 3- (4,5-dimethylthiazol-2yl) -2,5 diphenyl-2H-tetrazolium bromide solution) was added to each well. After removing the culture medium, add 200 μl of dimethylsulfoxide (DMSO, Amresco, 0231-500ML) and shake for 10 minutes. Take 100 μl of the solution in 96-wells and measure absorbance at 540 nm with an ELISA reader (Enzyme-Linked Immunosorbent Assay). The degree of cell recovery and soothing was expressed as a percentage based on the absorbance intensity of the control using pure water.

Cell recovery and authenticity (%) = (absorbance of test group / absorbance of control group) x100

As a result, as shown in Fig. 4, when the cells were treated with UV-B, the cells were damaged by about 40% as compared with the control group not treated. The treatment with 1%, 5%, and 10% of the composition of Example 1 restored the UV damage caused by most of the concentrations. Particularly, in the 10% treated group, the cell damage was completely recovered. Cell proliferative activity was about 20% higher than that when it was not.

Preparation of cosmetic composition

Cosmetics such as emulsified formulations such as nutritional lotion, cream, essence, etc. and cosmetics of solubilized form such as softened longevity were prepared with the cosmetic composition containing the peptide and the growth factor complex of the present invention as an active ingredient.

Production Example 2-1: Lotion

According to the following formulation, it was prepared according to the conventional lotion preparation method.

Raw material name Weight% (w / w) glycerin 5.0 Dipropylene glycol 3.0 Hyaluronic acid 0.5 Polyoxyethylene hardened castor oil 0.1 Polyethylene oleyl ethyl 0.1 ethanol 5.0 antiseptic 0.15 Beauty Suitable amount flash Suitable amount Peptide and Growth Factor Complex 2.0 Purified water to 100

Production Example 2-2: Essence

According to the following prescription, it was prepared according to a conventional method for producing essence.

Raw material name Weight% (w / w) Cetostearyl alcohol 1.0 Self emulsifying monostearic acid 1.0 Wax 0.5 Squalane 5.0 Isocetyl octanoate 3.0 Dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 8.0 glycerin 4.0 Propylene glycol 0.2 Carboxy polymer 0.22 Triethanolamine 0.25 antiseptic Suitable amount Spices Suitable amount flash Suitable amount Peptide and Growth Factor Complex 7.0 Purified water to 100

Production Example 2-3: Lotion

According to the following formulation, it was prepared according to a conventional lotion preparation method.

Raw material name Weight% (w / w) Cetostearyl alcohol 0.8 Self emulsifying monostearic acid 1.0 Wax 0.5 Stearic acid 0.5 Liquid paraffin 7.0 Squalane 5.0 Macadamia oil 3.0 Isocetyl octanoate 2.0 Dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 1.2 glycerin 4.0 Propylene glycol 4.0 Betaine 4.0 Carboxy polymer 0.12 Triethanolamine 0.15 antiseptic 0.25 Spices Suitable amount flash Suitable amount Peptide and Growth Factor Complex 5.0 Purified water to 100

Production Example 2-4: Cream

According to the following formulation, it was prepared according to a conventional cream production method.

Raw material name Weight% (w / w) Cetostearyl alcohol 3.0 Self emulsifying monostearic acid 1.5 Chin type monostearate 1.5 Wax 0.5 Liquid paraffin 8.0 Squalane 7.0 Isocetyl octanoate 4.0 Refined jojoba oil 4.0 Dimethylsiloxane 0.3 Sorbitan monostearate 1.0 Polyethylene glycol monostearate 1.2 glycerin 6.0 Propylene glycol 4.0 Betaine 4.0 Xanthan gum 0.06 Triethanolamine 0.10 antiseptic 0.25 Spices Suitable amount flash Suitable amount Peptide and Growth Factor Complex 7.0 Purified water to 100

Production Example 2-5: Gel

According to the following prescription, it was prepared according to a conventional gel preparation method.

Raw material name Weight% (w / w) glycerin 4.0 Propylene glycol 4.0 ethanol 10 Polyoxyethylene hardened castor oil 0.1 Carboxy polymer 0.30 Triethanolamine 0.30 antiseptic Suitable amount Spices Suitable amount flash Suitable amount Peptide and Growth Factor Complex 1.0 Purified water to 100

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> BIO FDNC          MEDIANS <120> Cosmetic composition for skin regeneration and skin soothing          a novel peptide and growth factor complex <130> P-20141028 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Prospin peptide <400> 1 Phe Ser Leu Leu Arg Tyr   1 5 <210> 2 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> cDNA of EGF <400> 2 aatagtgact ctgaatgtcc cctgtcccac gatgggtact gcctccatga tggtgtgtgc 60 atgtatattg aagcattgga caagtatgca tgcaactgtg ttgttggcta catcggggag 120 cgatgtcagt accgagacct gaagtggtgg gaactgcgc 159 <210> 3 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Protein of EGF <400> 3 Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His   1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn              20 25 30 Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys          35 40 45 Trp Trp Glu Leu Arg      50 <210> 4 <211> 441 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > cDNA of bFGF <400> 4 cccgccttgc ccgaggatgg cggcagcggc gccttcccgc ccggccactt caaggacccc 60 aagcggctgt actgcaaaaa cgggggcttc ttcctgcgca tccaccga cggccgagtt 120 gacggggtcc gggagaagag cgaccctcac atcaagctac aacttcaagc agaagagaga 180 ggagttgtgt ctatcaaagg agtgtgtgct aaccgttacc tggctatgaa ggaagatgga 240 agattactgg cttctaaatg tgttacggat gagtgtttct tttttgaacg attggaatct 300 aataactaca atacttaccg gtcaaggaaa tacaccagtt ggtatgtggc actgaaacga 360 actgggcagt ataaacttgg atccaaaaca ggacctgggc agaaagctat actttttctt 420 ccaatgtctg ctaagagctg a 441 <210> 5 <211> 146 <212> PRT <213> Artificial Sequence <220> <223> Protein of bFGF <400> 5 Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His   1 5 10 15 Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu              20 25 30 Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp          35 40 45 Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser      50 55 60 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly  65 70 75 80 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu                  85 90 95 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Ser Lys Tyr Thr             100 105 110 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser         115 120 125 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala     130 135 140 Lys Ser 145 <210> 6 <211> 738 <212> DNA <213> Artificial Sequence <220> <223> cDNA of SCF <400> 6 atgaagaaga cacaaacttg gattctcact tgcatttatc ttcagctgct cctatttaat 60 cctctcgtca aaactgaagg gatctgcagg aatcgtgtga ctaataatgt aaaagacgtc 120 actaaattgg tggcaaatct tccaaaagac tacatgataa ccctcaaata tgtccccggg 180 atggatgttt tgccaagtca ttgttggata agcgagatgg tagtacaatt gtcagacagc 240 ttgactgatc ttctggacaa gttttcaaat atttctgaag gcttgagtaa ttattccatc 300 atagacaaac ttgtgaatat agtggatgac cttgtggagt gcgtgaaaga aaactcatct 360 aaggatctaa aaaaatcatt caagagccca gaacccaggc tctttactcc tgaagaattc 420 tttagaattt ttaatagatc cattgatgcc ttcaaggact ttgtagtggc atctgaaact 480 agtgattgtg tggtttcttc aacattaagt cctgagaaag ggaaggccaa aaatccccct 540 ggagactcca gcctacactg ggcagccatg gcattgccag cattgttttc tcttataatt 600 ggctttgctt ttggagcctt atactggaag aagagacagc caagtcttac aagggcagtt 660 gaaaatatac aaattaatga agaggataat gagataagta tgttgcaaga gaaagagaga 720 gagtttcaag aagtgtaa 738 <210> 7 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Protein of SCF <400> 7 Met Lys Lys Thr Gln Thr Trp Ile Leu Thr Cys Ile Tyr Leu Gln Leu   1 5 10 15 Leu Leu Phe Asn Pro Leu Val Lys Thr Glu Gly Ile Cys Arg Asn Arg              20 25 30 Val Thr Asn As Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro          35 40 45 Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu      50 55 60 Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser  65 70 75 80 Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser                  85 90 95 Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val             100 105 110 Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys         115 120 125 Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe     130 135 140 Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr 145 150 155 160 Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Gly Lys Ala                 165 170 175 Lys Asn Pro Pro Gly Asp Ser Ser Leu His Trp Ala Ala Met Ala Leu             180 185 190 Pro Ala Leu Phe Ser Leu Ile Ile Gly Phe Ala Phe Gly Ala Leu Tyr         195 200 205 Trp Lys Lys Arg Gln Pro Ser Leu Thr Arg Ala Val Glu Asn Ile Gln     210 215 220 Ile Asn Glu Glu Asp Asn Glu Ile Ser Met Leu Gln Glu Lys Glu Arg 225 230 235 240 Glu Phe Gln Glu Val                 245 <210> 8 <211> 450 <212> DNA <213> Artificial Sequence <220> <223> cDNA of PGF <400> 8 atgccggtca tgaggctgtt cccttgcttc ctgcagctcc tggccgggct ggcgctgcct 60 gctgtgcccc cccagcagtg ggccttgtct gctgggaacg gctcgtcaga ggtggaagtg 120 gtacccttcc aggaagtgtg gggccgcagc tactgccggg cgctggagag gctggtggac 180 gtcgtgtccg agtaccccag cgaggtggag cacatgttca gcccatcctg tgtctccctg 240 ctgcgctgca ccggctgctg cggcgatgag aatctgcact gtgtgccggt ggagacggcc 300 aatgtcacca tgcagctcct aaagatccgt tctggggacc ggccctccta cgtggagctg 360 acgttctctc agcacgttcg ctgcgaatgc cggcctctgc gggagaagat gaagccggaa 420 aggtgcggcg atgctgttcc ccggaggtaa 450 <210> 9 <211> 149 <212> PRT <213> Artificial Sequence <220> <223> Protein of PGF <400> 9 Met Pro Val Met Arg Leu Phe Pro Cys Phe Leu Gln Leu Leu Ala Gly   1 5 10 15 Leu Ala Leu Pro Ala Val Pro Pro Gln Gln Trp Ala Leu Ser Ala Gly              20 25 30 Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Gln Glu Val Trp Gly          35 40 45 Arg Ser Tyr Cys Arg Ala Leu Glu Arg Leu Val Asp Val Val Ser Glu      50 55 60 Tyr Pro Ser Glu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu  65 70 75 80 Leu Arg Cys Thr Gly Cys Cys Gly Asp Glu Asn Leu His Cys Val Pro                  85 90 95 Val Glu Thr Ala Asn Val Thr Met Gln Leu Leu Lys Ile Arg Ser Gly             100 105 110 Asp Arg Pro Ser Tyr Val Glu Leu Thr Phe Ser Gln His Val Arg Cys         115 120 125 Glu Cys Arg Pro Leu Arg Glu Lys Met Lys Pro Glu Arg Cys Gly Asp     130 135 140 Ala Val Pro Arg Arg 145

Claims (4)

(EGF), Basic Fibroblast Growth Factor (bFGF), Stem Cell Factor (SCF), Placenta Growth Factor (PGF), and a peptide having the amino acid sequence of SEQ ID NO: 1,
Wherein said peptide is an N-terminal nicotinic acid-
A cosmetic composition for skin soothing.
(EGF), Basic Fibroblast Growth Factor (bFGF), Stem Cell Factor (SCF), Placenta Growth Factor (PGF), and a peptide having the amino acid sequence of SEQ ID NO: 1,
Wherein said peptide is an N-terminal nicotinic acid-
Is a cosmetic composition for regenerating skin wounds.
(EGF), Basic Fibroblast Growth Factor (bFGF), Stem Cell Factor (SCF), Placenta Growth Factor (PGF), and a peptide having the amino acid sequence of SEQ ID NO: 1,
Wherein said peptide is an N-terminal nicotinic acid-
A cosmetic composition for application to a skin laser treatment site.
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