WO2017082690A1 - Multifunctional skin-permeating peptide having whitening, skin elasticity, wrinkle improvement, and wound healing activities - Google Patents

Abstract

The present invention relates to a multifunctional peptide and use of same, the multifunctional peptide having whitening, elasticity, and wrinkle improvement effects based on a skin-permeating function, and more specifically, to a method for utilizing, as a raw ingredient in functional cosmetics and medicine, a multifunctional peptide having the effects of skin permeation, melanin production inhibition, collagen production promotion, and keratinous stem cell proliferation promotion. The multifunctional skin-permeating peptide, according to the present invention, has the characteristic of easily permeating skin and is exceptionally effective in inhibiting melanin synthesis and promoting collagen synthesis and keratinous stem cell proliferation, thus rendering the peptide useful, even independently, as cosmetics or medicine for whitening skin, improving skin elasticity, improving wrinkles, or healing wounds.

Description

 【Specification】

 [Name of invention]

 Multifunctional skin penetration peptide with whitening, skin elasticity, wrinkle improvement and wound healing activity

 Technical Field

 The present invention relates to a multifunctional peptide having a skin-permeating function-based whitening, elasticity and wrinkle improvement effect, and more particularly, a skin permeation function, inhibiting melanin production, promoting collagen production, and function of skin keratin stem cell proliferation. It relates to a method to utilize the multifunctional peptide having a functional cosmetics and pharmaceutical raw materials.

 Background Art

 Skin is the organ with the largest surface area in the human body, and is a route for effective drug delivery when used properly. However, due to the stratum corneum, which is composed of dead cells on the outermost side of the skin, the skin permeability of the foreign material is extremely low, which makes it difficult to deliver substances with high molecular weight and hydrophilicity. In order to overcome the defense mechanism against the foreign substances of the skin barrier in the delivery of the drug to the skin, studies on various skin penetration methods have been attempted, but the delivery of the drug through the properties of the chemical without physically damaging or irritating the skin It is considered to be almost impossible, and the situation is continuously developed due to various advantages that can be expected from the material that overcomes it.

 In response to these demands, a number of studies have been reported on skin permeation delivery materials such as liposomes or nanoparticles and peptide ligands for effective delivery of large molecular weight drugs. Lademarm et al., 2007 (Eur J Pharm Biopharm. 2007 May 66 (2): 159-64.), Chen et al., 2006a (J Pharmacol Exp Ther. 2006 Nov; 319 (2): 765-75.), Chen et al., 2006b (Nat Biotechnol. 2006 Apr ; 24 (4): 455-60.)], Despite the increase in the development cost of the commercialization stage, it is impossible to guarantee the efficiency and stability of the drug to be delivered. This is required.

On the other hand, the skin protects the body from the external environment and plays an important role in maintaining homeostasis. However, in the endless external stimulation and metabolic processes Unnecessary metabolites (metabolites) reduce skin function and accelerate aging.

 Collagen in the skin is a structural protein produced by skin fibroblasts, and it is known that the decrease in production due to aging and the change in its structure are related to wrinkle formation of the skin. Melanin in the skin acts to protect the skin from UV rays and free radicals in the body, but abnormal activation of melanocytes causes hyperpigmentation such as blemishes, freckles and blotch, resulting in aesthetic problems. Skin keratin stem cells present in the basal epidermal layer of the skin are known to play an important role in skin regeneration by controlling the growth and differentiation of keratinocytes as well as the fibroblasts of the dermis. Therefore, in vivo homeostasis related to collagen production, melanin pigment suppression and keratin stem cell growth are intimate factors affecting the health and beauty of the skin. The effectiveness of external treatment materials applied requires a great deal of effort due to the technical difficulties of passing through the epidermis and dermis.

 Under this background, the present inventors have limited application to the problems of reduced efficacy and safety, or increased cost due to the modification of such agonist substances.

As a result of intensive efforts to solve the conventional problems of the peptide, the present invention was completed by discovering a multifunctional peptide material that not only has a skin permeation function, but also inhibits melanin synthesis and promotes collagen synthesis and proliferation of skin keratin stem cells.

[Detailed Description of the Invention]

 Accordingly, one object of the present invention is to provide a skin permeable multifunctional peptide having a function of inhibiting melanin synthesis and collagen synthesis, and promoting the proliferation of skin keratin stem cells and dermal dermal fibroblasts in addition to skin permeation function. .

 Furthermore, another object is to provide a cosmetic composition for skin whitening, skin elasticity enhancement, or wrinkle improvement comprising the skin permeable multifunctional peptide. Another object of the present invention is to provide a skin permeable multifunctional temperide. It provides a pharmaceutical composition for skin whitening, skin elasticity enhancement or wrinkle improvement comprising.

Another object is for wound healing comprising the skin permeable multifunctional peptide It is to provide a pharmaceutical composition.

 In addition, another object of the present invention is to provide a composition for transdermal delivery of a physiologically active substance containing the skin permeable multifunctional peptide or a derivative thereof and the peptide and a bioactive substance.

 Furthermore, another object of the present invention is to provide a method for skin whitening, skin elasticity improvement, wrinkle improvement or wound healing, which comprises using the skin permeable multifunctional tempide or a derivative thereof.

 In addition, another object of the present invention is to provide a method for transdermal delivery of a physiologically active substance, using the skin permeable multifunctional peptide or derivatives thereof.

【Effects of the Invention】

 As one embodiment for solving the above problems, the present invention relates to a skin permeable multifunctional peptide having the amino acid sequence of SEQ ID NO: 1 or derivatives thereof.

 [SEQ ID NO 1] KLTTQMM

 【Effects of the Invention】

 The skin permeable multifunctional peptide according to the present invention has a property of effectively penetrating the skin, and when foreign substances are combined, it can be effectively transferred into the skin, and thus can be used as a tool for mass transfer, and excellent melanin synthesis. Inhibiting ability, collagen synthesis, exfoliation promoting effect of skin keratin stem cells and dermal fibroblasts, the peptide alone can be useful as cosmetics, medicines for skin whitening, skin elasticity improvement, wrinkle improvement or wound healing.

 [Brief Description of Drawings]

 1 is a graph quantitatively confirming the wound healing effect when using a skin permeable multifunctional peptide alone according to the present invention.

 Figure 2 is a graph confirming the skin permeation effect in the case of simple mixing of the skin permeable multifunctional peptide and the permeation target 20mer peptide according to the present invention.

[Best form for implementation of the invention] Hereinafter, the present invention will be described in more detail.

 As one embodiment for solving the above problems, the present invention relates to a skin permeable multifunctional peptide having the amino acid sequence of SEQ ID NO: 1 or derivatives thereof.

 [SEQ ID NO 1] KLTTQMM

 The derivative specifically refers to a peptide having a sequence in which one or more amino acids of the amino acid sequence of SEQ ID NO: 1 are substituted with alanine (Ala).

Preferably means a peptide having an amino acid sequence selected from SEQ ID NOs: 2 to 8.

 Synthesis of the peptides of the invention can be carried out, for example, using a device or using genetic engineering techniques. Automated peptide when synthesized using a device

Fmoc to the desired peptide in a synthesizer (PeptrEX-R48, Peptron, Daejeon, Korea)

It is synthesized by the Fmoc solid-phase method. After isolation of the synthetic peptide from the resin, reverse-phase HPLC using a Shiseido capcell pak Cl 8 analytical RP column (reverse-phase HPLC, Prominence LC-20AB, Shimadzu,

Peptides are purified and analyzed by Japan. Final peptides are identified using a mass spectrometer (HP 1 100 Series LC / MSD, Hewlett-Packard, Roseville, USA). When using genetic engineering techniques, a bacterium lacking protease such as BL21 (^ DE3) and BL21 (DE3) pLys after introducing the corresponding nucleotide sequence of a desired peptide into a protein expression vector (vector). Expression using IPTG

Induce and separate purely.

 In one specific embodiment, the skin permeable multifunctional peptide according to the present invention was identified using phage display technique and selected by verifying the efficacy of the identified peptide. In the step of identifying peptides that penetrate the skin, after applying a random phage display library to the pig skin, the skin epidermis is removed after a certain time and information about peptide sequence and appearance frequency is amplified by remaining phages remaining inside the skin except the epidermis. Can be secured.

Furthermore, in order to verify the efficacy of the identified peptide, 1) the peptide having a skin permeation function 2) melanin synthesis inhibitory effect in mouse melanocytes, 3) human Collagen synthesis in fibroblasts, 4) Proliferation in human dermal keratinocytes 5) Wound healing effect due to the proliferation of human dermal dermal fibroblasts 6) Effectiveness of skin permeation effect of the target substance when mixed with the target substance Verification multifunctional peptide was selected.

 The peptide according to the present invention has a skin permeability and thereby can transport or deliver a variety of active substances such as low molecular weight materials, and at the same time, by itself, multifunctional, particularly excellent skin whitening, skin elasticity increase, wound healing and wrinkle improvement effect. It is characterized by showing, the usefulness regarding a drug or cosmetics is large. Furthermore, in the case of transporting or delivering various active materials, the active materials may be transported or delivered by covalently bonding the active materials and the template according to the present invention, or by simply mixing the active materials with the peptides according to the present invention. Since it is possible to easily deliver the desired active material, in particular, there is an advantage that can effectively avoid problems such as denaturation and the difficulty of synthesis and purification that can occur when covalent bonds, such as peptides. At this time, the method of covalently binding the active substance and the skin permeable multifunctional peptide according to the present invention can be appropriately prepared using known techniques.

 Specifically, the physiologically active substance is a substance that enhances or inhibits the function of a living body in living life, and means any substance including a drug that can exert a desired effect in a living body. The bioactive substance may include, but is not limited to, peptides, polypeptides, polynucleotides, nanoparticles, DNA, RNA, small molecule compounds, and the like. As a specific example, the present inventors confirmed the transdermal delivery by the skin permeable multifunctional peptide according to the present invention using as a physiologically active substance to deliver a random peptide of 20mer.

 According to one embodiment of the present invention, the skin-permeable multifunctional peptide of the present invention not only shows excellent skin permeability compared to the transmittance of the TAT protein known as PTD (Protein transduction domain), but also excellent melanin even when used alone. Biosynthesis inhibitory effect, collagen synthesis effect, keratin stem cell proliferation effect and dermal fibroblast proliferation effect.

Furthermore, as another aspect, the present invention provides a skin permeable multifunctional peptide It relates to a cosmetic composition for skin whitening, skin elasticity enhancement or wrinkle improvement comprising as an active ingredient.

 In the present invention, the term "wrinkle" means a skin line caused by the decay of the skin, can be caused by the cause of the gene, the reduction of collagen and elastin in the dermis of the skin, the external environment. In the present invention, "wrinkle improvement" refers to inhibiting or inhibiting wrinkles from being generated on the skin, or alleviating wrinkles already generated.

 In the present invention, the term "skin elasticity" is represented by the elastic fibers composed of elastin (elastin) present in the dermal layer, '' skin elasticity enhancement 'refers to the elastic fibers composed of elastin and collagen, which are collagen fibers The state of skin elasticity is maintained.

 The cosmetic composition according to the present invention is a solution, an external ointment, a cream, a product, a nourishing cosmetic, a flexible cosmetic, a pack, a flexible water, an emulsion, a makeup base, an essence, a soap, a liquid detergent, a bath, a sunscreen cream, a sun oil suspension, an emulsion Liquids, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, waxes

It may be prepared in a formulation selected from the group consisting of foundations, patches and sprays, but is not limited thereto. In addition, the cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers formulated in general skin cosmetics, and as conventional components, for example, oil, water, surfactants, moisturizers, lower alcohols, Thickening crab, chelating agent, colorant, preservatives, fragrances and the like may be appropriately blended, but is not limited thereto.

As another aspect, the present invention relates to a pharmaceutical composition for skin whitening, wrinkle improvement, skin elasticity enhancement or wound healing, comprising the multifunctional skin penetration peptide. The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The compositions of the present invention can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsulants, suspensions, emulsions, syrups, aerosols, etc., respectively, according to conventional methods. Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbbi, manny, xylly, erythris, malty, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyridone, water, methylhydroxy benzoate,

Propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as commonly used layering agents, extenders, binders, wetting agents, disintegrating agents and surfactants are used. In the case of the composition according to the present invention, the multifunctional skin permeable peptide, which is an active ingredient, is preferably in the form of a transdermal preparation. Preferred dosages of the pharmaceutical compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. Administration may be administered once a day or may be divided several times.

 The cosmetic or pharmaceutical composition according to the present invention may further include at least one substance having a skin whitening, skin wrinkle improvement, skin elasticity enhancement or wound healing function.

 As yet another aspect, the present invention is to provide a composition for transdermal delivery of the skin-permeable multifunctional peptide or derivatives thereof and the bioactive material containing the peptide and the bioactive material. Preferably, the skin permeable multifunctional peptide or derivatives thereof and the bioactive material in the transdermal delivery composition may be in a mixed form or in a complex form covalently linked to each other. In still another aspect, the present invention provides a skin whitening, skin elasticity enhancement, wrinkle improvement or wound healing method, using the skin permeable multifunctional peptide and a cosmetic or pharmaceutical composition comprising the same as an active ingredient, and the skin The present invention relates to a method for transdermal delivery of an active substance comprising the use of a permeable multifunctional ramide and a cosmetic or pharmaceutical composition comprising the same as an active ingredient. Depending on the degree of skin condition, the form of the cosmetic or pharmaceutical composition, the administration and route and duration of application, it may be appropriately selected by those skilled in the art.

 [Form for implementation of invention]

Hereinafter, the present invention will be described in detail through examples. These examples are intended to describe the present invention in more detail, the scope of the present invention to these examples It is not limited by.

Example 1 Synthesis of Peptide Derivatives

 In this embodiment, in order to synthesize various peptide derivatives, it proceeded according to the general peptide solid phase ¾ ¾ (Wang C. Chan, Perter D. White, "Fmoc Solid phase peptide synthesis" Oxford). More specifically, using an autosynthesizer (ASP48S, Peptron, Inc.) using a Fmoc-SPPS (9-Fluorenyl methyl oxycarbonyl solid phase peptide synthesis) method was coupled (coupling) one by one from the C- terminal. All monomer raw materials used in the synthesis of peptide derivatives have amino acids protected with Fmoc at the N-terminus and residues protected with trityl (Trt), t-butyloxycarbonyl (Boc) and t-butyl (t-Bu). Used.

Coupling reagent was used as HBTU (2- (lH-Benzotimzloe-1-yl) -1,1,3,3-tetramethyluronium hexafluorophophate I HOBt (Hydroxybenzo triazloe) I NMM (N-methylmorpholine).

 (1) Protected amino acid (8 equivalents) and coupling agent HBTU (8 equivalents) / ΝΜΜ (16 equivalents)

After dissolving in DMF (dimethyl formamide) and adding, the mixture was reacted at room temperature for 2 hours.

 (2) Fmoc removal was performed by adding 20% (v / v) piperidine / DMF and reacting twice at room temperature for 5 minutes.

 After repeating the reaction of (1) and (2) to make the peptide derivative, the fluorescent peptides for confirming skin permeability are 6-amino hexanoic acid as a linker at the N-terminal part of the peptide. ) And fluorescein isothiocyanate (FITC).

Peptide separation from Resin uses TFA (Trifluoroacetic acid) / EDT (l, 2-ethanedithiol) / Thioanisole I TIS (Triisopropylsilane) IH ₂ 0 (mixture ratio (by weight) = 90 1 2.5 1 2.5 1 2.5 1 2.5) Separated.

 The resultant mixed solution contained an excess of diethyl ether solvent stored in storage.

The treatment produces a precipitate. The precipitate obtained was centrifuged to completely precipitate, and excess trifluoroacetic acid, thianizol, ethanedithiol, and the like were firstly removed, and the same procedure was repeated twice to obtain a solid.

The precipitate obtained was subjected to C18 column (250 mm ^χ 22 mm, ΙΟμηι, Vydac Everest, USA). Water-acetonitrile liner containing 0.1 ^o / o (v / v) Trifluoroacetic acid using a high performance liquid chromatography instrument (Shimadzu Prominence HPLC, Japan)

Gradient (Acetonitrile concentration: 10 ~ 75% (v / v)) was separated by the method. The molecular weight of the purified peptide derivative was confirmed using LC / MS (Agilient HP 1 100 series), and pure purified fractions were lyophilized to obtain the following peptide as a TFA (Trifluoroacetic acid) salt in the form of a white powder.

 Specifically, the sequence and molecular weight of the synthesized peptide derivatives are shown below. In the following heptapeptides 2 to 9 are derivatives of heptapeptide 1 (SEQ ID NO: 1) according to the present invention.

Heptapeptide 1 (SEQ ID NO: 1): KLTTQMM: Rt = 6.317 min (0.01% (v / v) from 5% (v / v) to 100% (v / v) acetonitrile / water containing TFA Various concentration gradients over 30 minutes); MS (ESI) 851 m / e, [M + H] ⁺ = 851

Heptapeptide 2 (SEQ ID NO: 2): ALTTQMM: Rt = 6.575 min (0% to 1% (v / v) from 5% (v / v) to 100% (v / v) acetonitrile / water Various concentration gradients over 30 minutes); MS (ESI) 794.98 m / e, [M + H] ⁺ = 795

Heptapeptide 3 (SEQ ID NO: 3): KATTQMM: Rt = 5.352 min (0.01% (v / v) from 5% (v / v) to 100% (v / v) acetonitrile / water containing TFA Various concentration gradients over 30 minutes); MS (ESI) 809.99 m / e, [M + H] ⁺ = 810

Heptapeptide 4 (SEQ ID NO: 4): KLATQMM: Rt = 6.210 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Varying concentration gradients); MS (ESI) 822.05 m / e, [M + H] ⁺ = 823

Heptapeptide 5 (SEQ ID NO: 5): KLTAQMM: Rt = 6.070 min (() .01% (v / v) 5% (v / v) to 100% (v / v) acetonitrile / water containing TFA Concentration gradient over 30 minutes); MS (ESI) 822.05 m / e, [M + H] ⁺ = 823

Heptapeptide 6 (SEQ ID NO: 6): KLTTAMM: Rt = 6.252 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA) Varying concentration gradients); MS (ESI) 795.03 m / e , [M + H] + = 796

Heptapeptide 7 (SEQ ID NO: 7): KLTTQAM: Rt = 5.720 min (() .01% (v / v) 5% (v / v) to 100% (v / v) acetonitrile / water containing TFA Concentration over 30 minutes from gradient); MS (ESI) 791.96 m / e, [M + H] ⁺ = 792

Heptapeptide 8 (SEQ ID NO: 8): KLTTQMA: Rt = 5.694 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Varying concentration gradients); MS (ESI) 791.86 m / e, [M + H] ⁺ = 792

Heptapeptide 9 (SEQ ID NO: 9): GLTTQMM: Rt = 6.283 min (from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Various concentration gradients over 30 minutes); MS (ESI) 780.35 m / e, [M + H] ⁺ = 781 fluorescent heptapeptide 1: FITC-linker-KXTTQMM: Rt = 7.250 min (0.01% (v / v) 5% containing TFA (v / v) ) Varying concentration gradients over 30 minutes from acetonitrile / water of from 100% (v / v)); MS (ESI) 1353.97 m / e, [M + H] ⁺ -1354

Fluorescent heptapeptide 2: FITC-linker-ALTTQMM: Rt = 7.542 min (from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Various concentration gradients over 30 minutes); MS (ESI) 1296.92 m / e, [M + H] ⁺ = 1296

Fluorescent heptapeptide 3: FITC-linker-KATTQMM: Rt = 6.258 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA) Varying concentration gradients); MS (ESI) 131 1.93 m / e, [M + H] ⁺ = 1312

Fluorescent heptapeptide 4: FITC-Iinker-KLATQMM: Rt = 7.042 min (from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01 ^ο / _ο (ν / ν) TFA Various concentration gradients over 30 minutes); MS (ESI) 1323.96 m / e, [M + H] ⁺ = 1324

Fluorescent heptapeptide 5: FITC-Unker-KLTAQMM: Rt = 7.008 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Varying concentration gradients); MS (ESI) 1323.96 m / e, [M + H] ⁺ = 1324

Fluorescent heptapeptide 6: FITC-linker-KLTTAMM: Rt = 7.158 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water with 0.01% (v / v) TFA) Varying concentration gradients); MS (ESI) 1296.95 m / e, [M + H] ⁺ = 1297

Fluorescent heptapeptide 7: FITC-linker-KLTTQAM: Rt = 6.533 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Varying concentration gradients); MS (ESI) 1293.97 m / e, [M + H] ⁺ = 1294 Fluorescent heptapeptide 8: FITC-linker-KLTTQMA: Rt = 6.533 min (30 min from 5% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) TFA Varying concentration gradients); MS (ESI) 1293.97 m / e, [M + H] ⁺ = 1294

Fluorescent heptapeptide 9: FITC-linker-GLTTQMM: Rt = 7.175 min (() .01% (v / v) 5% (v / v) to 100% (v / v) acetonitrile / water containing TFA Concentration gradient over 30 minutes); MS (ESI) 1282.90 m / e, [M + H] ⁺ = 1282

Example 2 Measurement of Skin Permeability

The excised pig skin (1.77 cm ²⁾ Franz (Franz) cell proliferation (FCDV-15, Labfine,

And the isophosphate buffer (pH 7.4) equally 13 ^ in the receiver chamber. The temperature of the receiver chamber is maintained ^at 37 ± 0.5 ^° C, and the magnetic bar is stirred at a speed of 600 rpm and the conditions are maintained until the end of the experiment. Hepta peptides labeled with fluorescence (FITC) and derivatives thereof and TAT labeled with positive control fluorescence were applied to the pig skin surface in contact with the donor chamber and peptides and derivatives thereof were transferred to the receiver chamber after 20 hours. Peptide quantification was _{spectrophotometer;:} was used (Nanodrop2000 Thermoscientific USA). Table 1 shows the results of analyzing the permeability of heptapeptide, and Table 2 shows the results of performing alanine scanning as a method for verifying the effective sequence of heptapeptide.

 Table 1

【표 2]

[Table 2]

표 1에서 알 수 있는 바와 같이, 형광이 표지된 헵타펩타이드

As can be seen in Table 1, fluorescently labeled heptapeptides

It was confirmed that the skin permeability of 2.09 g / cm ² / hr of l (KLTTQMM), as can be seen in Table 2, the skin transmittance (%) between the derivatives of heptapeptide l (KLTTQMM) is fluorescent heptapeptide 1 Compared with (FITC-linker-KLTTQMM), fluorescent heptapeptide 2 was 3.06 times, fluorescent heptapeptide 3 was 1.69 times, fluorescent heptapeptide 4 was 0.95 times, fluorescent heptapeptide 5 was 1.98 times, and fluorescent heptapeptide 6 was 1.96 times, respectively. , The fluorescent heptapeptide 7 was 0.92 fold, the fluorescent heptapeptide 8 was 1.31 fold change.

Example 3 Measurement of Melanin Biosynthesis Inhibitory Effect

 In addition to tyrosinase inhibitory substances, which are cosmetic materials for whitening, substances that induce melanin synthesis inhibition in skin cells also have a great potential for skin whitening. Thus, whitening can be expected as an inhibitory effect on melanin biosynthesis of melanoma cells.

 B16F10 melanoma cells (ATCC) containing 10% FBS

6-well plates containing DMEM (Dulbecco's modified eagle medium, Gibco) medium were inoculated (IX 10 ⁵ cells / well) and incubated at 37 ^° C for 24 hours in a C0 ₂ incubator at 5% concentration. After 24 hours, the medium was removed from each well, and the medium containing Arbutin (Sigma Aldrich) as a positive control group, the medium containing 20 ppm heptapeptide as the experimental group,

Normal medium was treated as a negative control and incubated for 48 hours. After 48 hours, The cells from which the medium was removed were washed with PBS (phosphate buffer saline) and treated with Trypsin-EDTA to recover the cells. The recovered cells were centrifuged at 13,000 rpm for 3 minutes, and then the supernatant was removed to obtain a precipitate. The obtained precipitate was washed once with PBS and the supernatant was removed. Then, 200 ul of lN NaOH containing 10% DMSO was added and allowed to stand at 60 ^° C for 1 hour to dissolve intracellular melanin. The solution was transferred to a microplate, and the absorbance was measured at 405 nm to determine the amount of melanin. The experimental results are shown in Table 4.

 As a result, as shown in Table 3, heptapeptide was shown to inhibit melanin biosynthesis in melanoma cells not only as a negative control group but also as arbutin, a positive control group known to have an effect on whitening.

 Table 3

Example 4 Measurement of Collagen Synthesis of Human Dermal Fibroblasts The amount of collagen synthesis of fibroblasts is an indicator of wrinkle formation in the skin. Wrinkles are known to occur due to decreased collagen synthesis and degradation of existing collagen. As a result, promoting collagen synthesis of fibroblasts can have an effect of inhibiting wrinkle formation and improving existing wrinkles.

Fibroblasts (human dermal fibroblast (ATCC)) are seeded in 96-well plates containing fibroblast basal medium (PCS-201-020; ATCC) medium (3 x 10 3 cells / well) and incubated in a C0 ₂ incubator at 5% concentration. Incubated at 37 ^° C for 24 hours. After 24 hours, the medium was removed from each well, followed by a medium containing vitamin C as a positive control group and 20 ppm as an experimental group.

Heptapeptide-containing medium, serum-free medium as negative control, and incubated for 48 hours. After 48 hours, cell culture medium was collected to prepare a sample. The amount of collagen in the sample was determined using a collagen measurement kit (Procollagen Type I C-peptide EIA kit; MK101; Takara, Kyoto, Japan). The amount of PICP) was measured. The experiment was performed according to the method described in the manual. The experimental results are shown in Table 5.

 As can be seen from the results of Table 4, heptapeptide 1 increased the collagen synthesis of fibroblasts to the same degree as that of the positive control group.

 Table 4

<Example> 5> Determination of the growth promoting activity of epidermal keratinocyte progenitor cells

Sustained division of keratin stem cells in the basal layer of the epidermis decreases with age, which leads to aging effects such as decreased elasticity due to degeneration of skin regeneration. Continuous skin elasticity may be improved by promoting the growth of keratin stem cells associated with skin regeneration and elasticity with aging. Human skin keratinocytes were purchased from Cell & Tech (CdlnTec; USA) and cultured according to Cell & Tech guidelines. The cell medium was used by adding the growth factor cnt-57 to 500 cnt-BM medium according to Cell &Tech's method. Human skin keratinocytes were inoculated in 96-well poolate (5 X 10 ³ cells / well). Incubated for 24 hours at 37 ^° C in 5% C <incubator. After 24 hours, the medium was removed from each well, followed by the routine containing Rutin (Sigma Aldrich) as a positive control, the 20 ppm heptapeptide containing medium as the experimental group,

Treated with normal medium as negative control and incubated again for 72 hours. The degree of cell proliferation was confirmed using a CCK-8 kit (CK04; Dojindo), and the experimental method was performed according to the kit instructions. Table 5 shows the results of the experiment.

From the results of Table 5, it can be confirmed the keratin stem cell proliferation effect of heptapeptide at a concentration of 20 ppm. Table 5

Example 6 Measurement of Cell Viability

Human skin keratinocytes were purchased from CellnTec (USA), and the growth factor Cnt-57 was added to 500 m of Cnt-BM medium according to the instructions, and used for culture. Human skin keratinocytes were seeded in 96-well folate (5 × 10 ³ cells / well) and incubated at 37 ^° C. for 24 hours in a CO ₂ incubator at 5% concentration. After 24 hours, the cells were treated at 100, 200, 400 ppm concentrations to confirm the cytotoxicity of the heptapeptide, and after 48 hours, the degree of cytotoxicity was confirmed by CCK-8 kit (CK04; Dojindo). As a positive control, 20 ppm of doxorubicin hydrochloride (sigma) was treated under the same time condition. From the results of Table 6, heptapeptide was found to be cytotoxic at 100-200 ppm.

 Table 6

<실시예 7> 상처치유능력 측정

Example 7 Wound Healing Ability Measurement

In order to confirm the wound healing effect of hepta ¾ tide according to the present invention, the heptapeptide according to the present invention was treated to human dermal fibroblasts at a concentration of 20 ppm, and the extent of wound healing was confirmed by quantifying the movement distance of cells after 48 hours. It was. Human dermal fibroblasts were purchased from ATCC, and cultured media for fibroblasts were incubated at 37 ^° C for 24 hours in a C0 ₂ incubator at 5% concentration. Human dermal fibroblasts were seeded in 6-well plates and incubated for 24 hours before heptapeptide treatment.

 Vitamin C as a positive control was treated at the same concentration of 20ppm, the same time conditions, the results are shown in Table 7 and FIG.

 [Table 7]

상기 표 7에서 확인할 수 있는 바와 같이, 본 발명에 따른

As can be seen in Table 7, according to the present invention

Heptapeptide 1 (KLTTQMM) has its own effects on wound healing _: it is associated with vitamin C, a positive control group known to have a wound healing effect.

 It was confirmed that it is much superior to comparison.

Example 8 Measurement of Skin Permeability Improvement Effect

 Heptapeptide 1 (KLTTQMM: SEQ ID NO: 1) and derivatives thereof, including the drug according to the present invention, heptapeptide 9 (GLTTQMM: SEQ ID NO: 9)

In order to confirm the effect of improving skin permeability, experiments were carried out using 20mer peptides. Specifically, as a material for skin permeation, a random peptide of 20mer was prepared by the same method as Example 1 and fluorescently labeled with FITC.

 20mer random peptide (SEQ ID NO: 10): FITC-linker-DLDLEALAPYIPADDDFQLR: Rt = 5.650 min (5% (v / v) to 100% (v / v) containing TFA)

Various concentration gradients over 30 minutes from acetonitrile / water); 1 \ (£ 81) 2793.08 1 ^ 6, [+ ¾ ⁺ = 1397 (M + l) / 2

This is heptapeptide 1 (SEQ ID NO: 1) or heptapeptide according to the present invention Simple mixing with 9 (SEQ ID NO: 9), wherein the mixing ratio was heptapeptide 1 or heptapeptide 9: random peptide = 1: 5, and 45% EtOH / PBS (v / v) buffer was used.

The removed dorsal dorsal skin (1.77 cm ² ) of the Balb / c mouse was fixed between the donor chamber and the receiver chamber of a Franz diffusion cell (FCDV-15, Labfme, Korea) and isotonic phosphate in the receiver chamber. 13 mL of layer solution (H 7.4) were provided in the same manner. And the temperature of the receiver chamber was maintained ^at 37 ± 0.5 ^° C, the magnetic bar was stirred at a speed of 600 rpm and the conditions were maintained until the end of the experiment. Heptapeptide 1 or heptapeptide 9 and fluorescence (FITC) labeled 20mer peptides and their negative controls, each random peptide alone, were applied to the epidermal skin surface of Balb / c mouse in contact with the donor chamber and 20 hours later Fluorescently labeled random peptides transferred to the chamber were measured. Peptide quantitative analysis was performed using spectrophotometer (Nanodrop2000;

Thermoscientific, USA). The results are shown in FIG.

 As can be seen in Figure 2, by the heptapeptides according to the present invention it was confirmed that the permeability of the desired 20mer peptide significantly increased by 190%, 300%, respectively.

Claims

[Range of request]  [Claim 1]  A skin permeable multifunctional peptide having the amino acid sequence of SEQ ID NO: 1 or a derivative thereof.  [Claim 2]  The method of claim 1, wherein the derivative is one or more of the amino acid sequence of the skin permeable multifunctional peptide is substituted with alanine, a skin permeable multifunctional peptide or derivatives thereof.  [Claim 3]  The skin permeable multifunctional peptide or derivative thereof according to claim 2, wherein the derivative has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-9.  [Claim 4]  A transdermal multifunctional peptide or derivative thereof according to any one of claims 1 to 3, the composition for transdermal delivery of a bioactive substance.  [Claim 5]  According to claim 4, wherein the skin permeable multifunctional peptide or derivatives thereof, and the bioactive material is in the form of covalently bonded to each other, the composition for transdermal delivery of a bioactive material. [Claim 6]  The composition for transdermal delivery of a bioactive substance according to claim 4, wherein the skin permeable multifunctional peptide or a derivative thereof, and the bioactive substance are in a mixed form.  [Claim 7]  Claims 1 to 3, wherein the multifunctional peptide or derivative thereof, comprising a cosmetic composition for skin whitening, skin elasticity or wrinkle improvement.  [Claim 8]  The cosmetic composition of claim 7, further comprising a cosmetically acceptable carrier.  [Claim 9] According to claim 7, wherein the composition is a solution, external ointment, cream, foam, nutrient cosmetics, Softener, Pack, Softener, Latex, Makeup Base, Essence, Soap, Liquid Cleanser, Bath, Sunscreen Cream, Sun Oil, Suspension, Emulsion, Paste, Gel, Lotion, Powder, Soap, Surfactant-containing Cleansing ， A cosmetic composition having a formulation selected from the group consisting of oils, powder foundations, emulsion foundations, wax foundations, patches and sprays.  [Claim 10]  Claims 1 to 3, wherein the multifunctional peptide or derivative thereof, comprising a pharmaceutical composition for skin whitening, skin elasticity, wrinkle improvement or wound healing. [Claim 1 11]  A method for skin whitening, skin elasticity, wrinkle improvement or wound healing, comprising using the multifunctional peptide of any one of claims 1 to 3 or a derivative thereof.  [Claim 12]  A method for transdermal delivery of an active substance, characterized by using the multifunctional peptide of any one of claims U to 3 or a derivative thereof.