KR20190064307A - Development of new peptide derivative for cosmetic material with enhanced skin permeability - Google Patents

Abstract

The present invention provides a method for effectively synthesizing a fatty acid-peptide showing skin wrinkle-reducing activity. It was confirmed that the peptide of the present invention inhibits MMP-1 expression in skin cells, promotes collagen production, exhibits skin wrinkle-reducing activity, and promotes proliferation of skin cells. The peptide also has shown an excellent improvement effect on a wrinkle reduction clinical test compared to a control group, and has excellent stability and reduced skin irritation. In addition, the fatty acid-peptide exhibits higher skin permeability compared to conventional peptides. The present invention provides a cosmetic composition for reducing skin wrinkles comprising the fatty acid-peptide.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel peptide derivative for cosmetic materials having improved skin permeability and efficacy. BACKGROUND ART < RTI ID = 0.0 > [0002] <

More particularly, the present invention relates to a peptide derivative having improved skin permeability and improved wrinkle-reducing effect and a cosmetic composition for improving wrinkles containing the same as an active ingredient. .

Skin is the largest tissue in the body and functions to protect the inside of the body from sunlight, physical and chemical stimuli, which is absolutely necessary for the maintenance of human life and is constantly regenerated to maintain homeostasis. The skin is composed of the epidermis, the dermis and the subcutaneous fat in order from the outside. The skin which is the thinnest tissue plays an important function for moisturizing and protecting the skin, and it plays a role to prevent water loss, damage and invasion of bacteria. Skin is a tissue that covers the whole body and has various functions. It has a barrier function between the inside and the outside of the body, so it has an immune system by Langerhans, and it has moisture control function by epidermis. It also synthesizes vitamin D in the proper sunlight and also excretes lipids into the reservoir and sweat glands. The stratum corneum, which is produced by the normal differentiation process in healthy skin, has the function of maintaining the moisturization of the skin and protecting it from the stimulation of the external environment. This function is called skin barrier function and it is the most important role of the epidermis.

One of the greatest areas of interest in cosmetics is wrinkling, pigmentation, loss of elasticity, skin dryness, hair loss, and lack of shine in the hair associated with aging. The skin is subject to various changes through aging. First, the thickness of the epidermis, dermis, and subcutaneous tissues, which are components of the skin, is thinned and the extracellular matrix (ECM) component that gives skin elasticity changes. Among them, collagen which accounts for 70 ~ 80% As the age increases, the production thereof is drastically lowered to have a close relationship with wrinkle formation, and collagen, elastin, proteoglycans, glucosaminoglycan, , Laminin, fibronectin, etc. are oxidized and lose their function, causing the skin to lose its elasticity and wrinkles becoming excessively formed and changing into senile skin.

There are collagen fibers, collagen fibers, reticular fibers, elastic fibers (elastic fibers, elastic fibers) in the connective tissue of the extracellular matrix. Collagen, which accounts for 70% Are mostly formed in fibroblasts of the skin. As you get older, the collagen content in the connective tissue of the skin decreases, which is due to degradation of collagen synthesis and promotion of degradation. Therefore, degradation of collagen biosynthesis and promotion of collagen degradation are the major causes of wrinkles in the skin.

The collagen biosynthesis process is regulated by many factors involved in transcription level and post-translation level, and collagen degradation is triggered by collagenase degrading collagen by ultraviolet rays Promotion of matrix metalloproteases (MMP), such as collagenase, promotes collagen degradation and reduces collagen content. In addition, as the collagen's deformation accelerates due to the external environment, the skin becomes wrinkled and deepened. As a result, skin aging is caused by non-homogenization of cells, loss of elastin, destruction of collagen, reduction and delay of collagen synthesis, and the like. Therefore, the skin aging phenomenon occurs in the epidermis of the skin but can be seen as a phenomenon that occurs in the dermis.

Various cosmetic compositions have been studied for solving the problem of skin aging, and the wrinkle improvement effect of the skin is visible in some parts. For example, there have been various reports on retinoids widely used for the improvement of skin wrinkles, spots, and pigment deposition, and in particular, clinical results of removing skin wrinkles on retinol. Among them, retinol- And skin deflection and elasticity reduction.

Recently, human lifespan has been lengthened and the quality of life has been improved, so that the interest of healthy and beautiful people has been greatly increased. Recently, the market for functional cosmetics, functional foods and cosmetics related to skin aging has increased rapidly It is well known that

Skin aging has been divided into extrinsic aging (aging caused by external stimuli such as the sun's rays) and endogenous aging (natural aging with increasing age), and aging skin has increased wrinkles and decreased elasticity. However, understanding, prevention and treatment of the causes of skin aging symptoms have not been developed yet.

As a result, skin aging phenomenon is caused by non-homogenization of cells, disappearance of elastin, destruction of collagen, reduction and delay of collagen synthesis, and thus, skin aging phenomenon occurs in skin epidermis but occurs more in dermis than in skin epidermis.

A variety of cosmetic compositions have been studied to solve the problem of skin aging. Among them, peptides are biomolecules produced through polymerization of amino acids and are known to be harmless to the human body and particularly to control various physiological activities in vivo . In particular, peptides are widely used for pharmaceuticals and pharmacies because they are very stable to heat and pH, and do not easily lose their activity compared to protein, which is a large combination of amino acids. They are also used in cosmetics to improve skin wrinkles and wrinkles . However, various attempts have been made to develop various wrinkle remedies using peptides, but the effect thereof is not so great, and vitamin C-peptide complexes and hydroquinone-peptide complexes for maximizing effects are also difficult to manufacture, It is not easy to apply in the market because of limited use. Particularly, peptides have good solubility in water and they do not dissolve well in oil, so they penetrate into the skin through the skin layer and have a physical property hard to exhibit physiological activity.

The skin is formed in a layered structure composed of various layers such as stratum corneum, epidermal layer and dermis layer. Each layer has its own characteristics and protects the human body from external threats such as ultraviolet rays and microorganisms. It seems to be a function to suppress things.

However, such a structure of the skin prevents penetration of useful ingredients outside the skin into the skin, thereby suppressing the expression of the substance's efficacy. Various methods have been proposed and developed as a solution to the difficulty of penetration of the skin. One of these methods is to improve the permeability of the stratum corneum, which has hydrophobic properties, by reducing the water-solubility characteristics of the substance through chemically bonding the water-soluble substance, which is difficult to penetrate into the skin, with the lipophilic substance. In such a method, the manufacturing method is determined according to the property of the substance to increase the skin permeability.

In order to facilitate peptide synthesis, a protecting group (protecting group) is required. Up to now, a protecting group having various characteristics has been developed and utilized. It has been reported that Fmoc / trt-amino acid derivatives have been successfully used to synthesize some peptides. However, even though Fmoc / trt-amino acid derivatives have an excellent effect on the synthesis of solid phase peptides However, low Fmoc solubility due to the hydrophobicity of Fmoc showed low condensation yield in egg shell peptide synthesis. In contrast, 2- (4-nitrophenyl) sulfonylethoxycarbonyl (Nsc) inhibits the hydrophobic interaction between peptide chains due to increased solubility of amino acids due to the structural characteristics of the nitro group and the sulfonyl group It plays a role. In addition, the stability of Nsc is superior to that of Fmoc under an automation system, which is very advantageous for a long-chain peptide synthesis reaction.

 In order to effectively prevent or improve skin aging and wrinkles, it is necessary to develop a novel wrinkle-improving composition using ingredients that have been proven to be efficacious, and it is important to verify the efficacy with a molecular biological mechanism and to validate it with human clinical trials.

The present inventors have made efforts to search for a peptide derivative exhibiting an excellent wrinkle-reducing effect in a peptide library having a fatty acid-bonded peptide. As a result, they found that some peptides having a fatty acid bond improve skin permeability and improve wrinkles. And a method of producing a peptide having excellent skin permeability, and selecting the peptide.

Accordingly, an object of the present invention is to provide a cosmetic composition for improving skin wrinkles comprising a peptide selected from the group consisting of SEQ ID NOS: 1 to 5 and a fatty acid-peptide complex in which a saturated or unsaturated fatty acid having 10 to 20 carbon atoms is amide bonded, To provide a composition.

It is another object of the present invention to provide a method for producing the above saturated or unsaturated fatty acid having 10 to 20 carbon atoms, wherein the saturated or unsaturated fatty acid is selected from the group consisting of myristic acid, stearic acid, linoleic acid, palmitic acid, Wherein the cosmetic composition is at least one selected from the group consisting of oleic acid and lauric acid.

Another object of the present invention is to provide a cosmetic composition characterized in that the fatty acid-peptide complex is a complex in which a peptide represented by SEQ ID NO: 4 and a myristic acid or palmitic acid are amide bonded to each other will be.

Another object of the present invention is to provide a cosmetic composition, wherein the fatty acid-peptide complex is prepared using a compound represented by the following formula (1).

[Chemical Formula 1]

In Formula 1,

R 1 and R 2 are each independently hydrogen or a linear or branched hydrocarbon having 1 to 20 carbon atoms.

Another object of the present invention is to provide a cosmetic composition characterized in that the fatty acid-peptide complex inhibits MMP-1 expression.

Another object of the present invention is to provide a cosmetic composition characterized in that the fatty acid-peptide complex activates collagen biosynthesis in skin cells.

Another object of the present invention is to provide a cosmetic composition characterized in that the fatty acid-peptide complex has a skin permeability (based on 24 hours as an experiment) of at least 0.01 mg / ml using a Franz diffusion cell.

In order to accomplish the above object, the present invention provides a peptide-peptide conjugate comprising one peptide selected from the group consisting of SEQ ID NOS: 1 to 5 and a saturated or unsaturated fatty acid having 10 to 20 carbon atoms, The present invention provides a cosmetic composition for improving skin wrinkles.

As used herein, the term " peptide " refers to a linear molecule in which amino acid residues are joined together by peptide bonds, and a fatty acid-peptide refers to a form in which a fatty acid is bonded to the N-terminus of the peptide.

As a result of efforts to search for a peptide showing a wrinkle-improving effect in a peptide library having a fatty acid-bound peptide, the inventors found that some peptides to which fatty acid-linked peptides inhibit the expression of MMP-1 protein, promote collagen production, Which is effective for improving skin wrinkles.

The present inventors have completed the present invention by preparing various peptide libraries containing various fatty acids at the N-terminus, preparing candidate peptides, and screening fatty acid-peptide complexes excellent in skin permeability and wrinkle-reducing activity among candidate peptides.

According to a preferred embodiment of the present invention, the peptide of the present invention can induce deformation by binding a fatty acid to the N-terminus. Through such modification, the peptide of the present invention may have increased half-life due to increased stability in vivo.

According to a preferred embodiment of the present invention, an amino group (-NH2) may be bonded to the C-terminal of the peptide to induce a modification.

In the present invention, the fatty acid-peptide complex may be prepared by using a compound represented by the following formula (1) as a protecting group.

[Chemical Formula 1]

In Formula 1,

R 1 and R 2 are each independently hydrogen or a linear or branched hydrocarbon having 1 to 20 carbon atoms.

According to an embodiment of the present invention, when the compound of Formula 1 is used as a protecting group and a fatty acid-peptide complex is used, the yield of the complex is significantly higher than that of Fomc (9-fluorenylmethyloxycarbonyl group) It was confirmed that various kinds of fatty acid-peptide complexes could be prepared.

Preferably, the compound of Formula 1 may be 2- (4-nitrophenyl) sulfonylethoxycarbonyl.

The above-mentioned modification of the amino acid serves to greatly improve the stability of the peptide of the present invention. As used herein, the term " stability " refers not only to in vivo stability, but also to storage stability (e.g., room temperature storage stability).

In the present invention, the fatty acid is not particularly limited as long as it is a saturated or unsaturated fatty acid having 10 to 20 carbon atoms. Preferably, the saturated or unsaturated fatty acid having 10 to 20 carbon atoms is selected from the group consisting of myristic acid, stearic acid ), Linoleic acid, palmitic acid, oleic acid, and lauric acid. [0027] The term " acid anhydride "

According to one embodiment of the present invention, it has been found that the peptides of SEQ ID NOS: 1 to 5 significantly improve the wrinkle of skin when a complex is formed through the amide bond with the fatty acid.

The peptides represented by SEQ ID NOS: 1 to 5 were found to exhibit a particularly improved wrinkle-reducing effect when they were combined with a fatty acid of a certain kind among fatty acids having 10 to 20 carbon atoms to form a complex.

Specifically, the peptide of SEQ ID NO: 1 binds to myristic acid or stearic acid, and when the peptide of SEQ ID NO: 2 binds oleic acid, linoleic acid or palmitic acid, the peptide of SEQ ID NO: The peptide of SEQ ID NO: 4 when bound to stearic acid, myristic acid or palmitic acid, and the peptide of SEQ ID NO: 5 is bound to lauric acid or myristic acid, when bound to uric acid, oleic acid or stearic acid, The synergistic effect of the wrinkle-improving effect was found to be particularly remarkable.

According to one embodiment of the present invention, the fatty acid-peptide complex of the present invention does not show toxicity to human-derived cells, and thus is highly utilized as a cosmetic composition for improving skin wrinkles. According to the present invention, when the fatty acid peptides were treated with HaCat cells and HDF cells at a concentration of 100 ng / ml to 1000 μg / ml, no significant cytotoxicity was measured, and even when the morphology of the cells was confirmed visually, (Fig. 6).

According to the present invention, it was confirmed that the fatty acid-peptide complex exhibited excellent thermal stability even at a temperature of 50 캜 and was stable even in daylight (Figs. 4 and 5). Thus, the fatty acid-peptide complexes of the present invention can be advantageously applied to products requiring long-term storage such as cosmetics.

According to one embodiment of the present invention, the fatty acid-peptide complex of the present invention has the ability to inhibit MMP-1 expression. In accordance with the present invention, the inhibitory effect of MMP-1 on the expression of MMP-1 was examined by irradiating UV light to skin dermal cells (Fibroblast) to promote MMP-1 expression and then treating and culturing the fatty acid-peptide complex of the present invention. Peptide complex inhibited MMP-1 expression compared to the control group.

According to another embodiment of the present invention, the wrinkle-improving effect of the wrinkle-improving cosmetic composition comprising the fatty acid-peptide complex of the present invention was evaluated clinically, and as a result, (Table 5 and Fig. 7).

In addition, in the case of a composition containing the peptide of the present invention, various formulations can be used to effectively use it for improving skin wrinkles.

The fatty acid-peptide complex used as an active ingredient in the composition of the present invention exhibits a low HLB (hydrophilic lipophilic balance) as compared with a peptide not bound to a fatty acid, and thus has excellent skin permeability. Therefore, when the composition of the present invention is locally applied to the skin, the effect of improving the wrinkles of the skin can be maximized due to the high penetration of the skin.

Specifically, the fatty acid-peptide complex of the present invention may have a skin permeability (based on 24 hours of the experiment) using Franz diffusion cells of not less than 0.01 mg / ml, preferably not less than 0.05 mg / ml .

The cosmetic composition containing the fatty acid-peptide complex according to the present invention as an active ingredient can be applied to cosmetic compositions such as a gel type, a skin type, a cream type, an ointment type and the like, but is not limited thereto. The above composition can be suitably prepared by known methods, adding appropriate conventional softening agents, emulsifying agents, thickening agents or other materials known in the art depending on the type thereof.

The gel-type composition may be prepared by adding a softening agent such as trimethylolpropane, polyethylene glycol or glycerin, a solvent such as propylene glycol, ethanol, isocetyl alcohol, or the like.

The skin-type composition may contain at least one of fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol and isocetyl alcohol, butylene glycol, 2-sodium, xanthan gum, dimethicone, polyethylene glycol-60 hydrogeneate castor oil, polysorbate 60 and purified water.

The cream-type composition may be formulated by dissolving or dispersing the above-mentioned cream type composition in an aqueous medium such as alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol and the like, lecithin, phosphatidylcholine, phosphatidylethanolamine, Emulsifiers such as glyceryl stearate, sorbitan palmitate, and sorbitan stearate; natural fats or oils such as avocado oil, apricot oil, barba water oil, glass fat oil and camellia oil; Propylene glycol and the like, purified water, and the like.

The ointment type composition may be prepared by adding a softening agent, an emulsifying agent, and a wax such as microcrystalline wax, paraffin, ceresin, wax, spermaceti, vaseline or the like.

The present invention may also be provided as a formulation comprising the fatty acid-peptide complex as the active ingredient and at least one cosmetically acceptable carrier or excipient.

Thus, the preparation may contain a cosmetically acceptable carrier, diluent, excipient, or a combination thereof if necessary. These agents facilitate administration of the active ingredient into the organism.

The cosmetic composition comprising the fatty acid-peptide complex of the present invention as an active ingredient is excellent in skin permeability, inhibits MMP-1 expression, promotes collagen production in skin cells, promotes skin cell proliferation, Effect.

FIG. 1 is a graph showing the results of a high performance liquid chromatography analysis of the synthesized fatty acid peptides (FIG. 1A: Palmitoyl-RKDVY complex, FIG. 1b: Palmitoyl-GPQGPQ complex, FIG. 1C: Myristoyl-GPQGPQ complex, Complex, Figure 1 e: Myristoyl-AFPG).
FIG. 2 is a graph showing the results of experiments of inhibition of intracellular MMP-1 expression of five synthesized fatty acid-peptide complexes.
FIG. 3 is a graph showing the results of an experiment for promoting intracellular collagen production of four synthesized fatty acid-peptide complexes.
4 is a graph showing the results of measuring the light stability of a fatty acid-peptide complex.
5 is a graph showing the results of measuring the thermal stability of a fatty acid-peptide complex.
FIG. 6 is a graph showing MTT assay results for confirming skin cell toxicity after treatment of two skin cells (HDF, HACAT) with a fatty acid-peptide complex.
FIG. 7 is a graph showing the results of a comparison of skin permeability of a fatty acid-peptide complex and a peptide.
FIG. 8 is a graph comparing clinical efficacy of wrinkle improvement of fatty acid-peptide and fatty acid through clinical trials (left: control group, right: test group, A: test group, B: control group).

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 describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1

fatty acid- Peptides  Synthesis of libraries

(Nsc-Ala, nsc-Arg (pbf), nsc-Asp (OtBu), and nsc-Asp) using 2- (4-nitrophenyl) sulfonylethoxycarbonyl as a protecting group to synthesize a fatty acid- Nsc-Asn (trt), nsc-Gly, nsc-Glu (OtBu), nsc-Gln (trt), nsc-His (trt) (Boc), nsc-Trp (Boc), nsc-Leu, nsc-Ile, nsc-Val, nsc-Phe, nsc-Met and nsc-Lys Amino acid (Fmoc-Ala, Fmoc-Arg (pbf), Fmoc-Asp (pbp)) using Fmoc- (OtBu), Fmoc-Asn (trt), Fmoc-Gly, Fmoc-Glu (OtBu), Fmoc-Gln (trt), Fmoc- (Boc), Fmoc-Tyr (tBu), Fmoc-Trp (Boc), Fmoc-Leu, Fmoc-Ile, Fmoc-Val, Fmoc-Phe, Fmoc-Met, Fmoc- Chloro trityl chloride resin (CTL resin, Novabiochem Cat No. 01-64-0021), Lau Into a 96-well Teflon reactor, 50 mg of each of 19 lines was added, and 1 ml of methylene chloride (MC) was added thereto, followed by stirring for 3 minutes . The solution was removed, and 1 ml of dimethylformamide (DMF) was added. After stirring for 3 minutes, the solvent was removed again. The prepared fatty acid-peptidyl resin was washed three times with DMF, MC and methanol, dried slowly by flowing nitrogen air, and then completely dried under reduced pressure in a vacuum under P2O5. The prepared resin was immersed in a solution of maltitol solution (Trifluroacetic acid (TFA) 81.5%, distilled water 5%, thioanisole 5%, phenol 5%, EDT (1,2-Ethanedithiol) 2.5% (Containing 1% of triisopropylsilane)] was added, and the reaction was kept for 1 hour in an ice water bath with occasional shaking at room temperature. The resin was filtered, washed with a small amount of TFA solution, and then combined with the mother liquor. Thereafter, a fatty acid-peptide complex was obtained.

As a result of synthesis and purification, fatty acid-peptide complex species having peptides with different sequences and fatty acids were obtained (Table 1).

* SEQ ID NO: 1: AFPG

* SEQ ID NO: 2: RKDVY

* SEQ ID NO: 3: EEMQRR

* SEQ ID NO: 4: GPQGPQ

* SEQ ID NO: 5: FVAPDP

The yield of the synthesized fatty acid-peptide complexes showed a large difference also in the yield due to the property difference depending on the sequence of the peptide (the yield of 4% or less was not indicated separately). In some cases, the synthesis itself was not possible depending on the kinds of Fmoc and NSC protecting groups and fatty acids. As can be seen from the following Table 1, it was confirmed that the yield of the fatty acid-peptide complex was excellent when it was prepared by using 2- (4-nitrophenyl) sulfonylethoxycarbonyl (NSC) as a protecting group.

The yield of fatty acid and peptide complexes was predicted to be determined by the length and polarity of the peptide, the length of the fatty acid and the degree of nonpolarity, but not by the sterical hindrance due to the three dimensional structure. The elution time for the separation of the column was also different, but the peak was confirmed at 25 to 35 minutes (FIG. 1).

Example 2

fatty acid- Of peptide  Confirmation of skin wrinkle improvement effect

In order to confirm the effect of improving the skin wrinkles of the fatty acid peptides, the in vitro collagenase activity inhibition experiments of the fatty acid-peptide complexes prepared in Table 1 were carried out as follows. The results are shown in Table 2 Respectively.

 Collagenase activity inhibition test method is prepared by dissolving collagen in phosphate buffer (100 ppm). At this time, adenosine is prepared at the same concentration as a positive control group. The reaction is terminated by adding 25mM citric acid to 400μl / tube. Then add 1.5 ml / tube of ethyl acetate and vortex. Transfer supernatant to sodium sulfate 150 mg / ep-tube, vortex and centrifuge at 10,000 rpm for 3 minutes. Take supernatant 300 μl / ep-tube and transfer to quartz 96-well plate and measure absorbance at 320 nm.

The results showed that the fatty acid - peptide complex showed better collagenase inhibition than fatty acid - free peptide and adenosine.

As shown in Table 2, it was confirmed that some of the complexes having the peptides of SEQ ID NOS: 1 to 5 and the fatty acid-bound complexes showed better collagenase inhibitory ability than the peptides not bound to fatty acids.

Namely, it has been confirmed that each of the peptides of SEQ ID NOS: 1 to 5 exhibits a remarkable improvement in the wrinkle-reducing effect only when a complex is formed with a specific fatty acid. When a complex is formed with other fatty acids other than these peptides, It was confirmed that the collagenase inhibitory effect was lowered.

Example 3:

MMP -1 (Matrix Metalloproteinase  - 1) active  Inhibition experiment

MMP-1 activity inhibition test was performed on the combination of five selected compounds by collagenase inhibition experiment.

Human dermal fibroblast (HDF) cells were cultured in DMEM (Dulbecco's modified Eagle's media, Sigma) supplemented with 10% fetal bovine serum (Sigma) at 37 ° C and 5% CO2. Cells were cultured in a 24-well plate at a concentration of 2 × 10 ⁵ cells / well. After confirming cell attachment, only the solvent was added to the control group without any treatment. In the dish, 5 kinds of test groups were selected, and retinol And treated at a concentration of 10 ug / ml. The test material was added to each dish and cultured for 2 days. After 2 days, the medium was harvested and the inhibitory activity of MMP-1 was measured by Matrix Metalloproteinase-1 (MMP-1), Human, biotrak and ELISA kit (GE healthcare RPN2610).

MMP-1 inhibitory effect was measured after 5 treatment groups. The amount of melanin was found to be reduced in most experimental groups compared to the control group, retinol (Fig. 2). From these results, it can be concluded that fatty acid-peptide complexes can easily penetrate into the cell and inhibit MMP-1 production, thereby suppressing the wrinkles of the skin when UV as a factor inducing MMP-1 production in skin is applied .

Example 4

Intracellular collagen Generation  exam

The intracellular collagen production was tested for the three fatty acid-peptide complexes selected in the MMP-1 inhibition test. Human dermal fibroblast (HDF) cells were cultured in Medium 106 (cascade biologics, M-106-500) supplemented with 1X LSGS (Low Serum Growth Supplement, cascade biologics S-003-10) C, 5% CO2. Cells were cultured in a 24-well plate at a concentration of 1 × 10 ⁵ cells / well. After the cell adhesion was confirmed, only the solvent was added to the control group without any treatment. In the dish, 3 kinds of test groups and 10 μg / ml of TGF . ≪ / RTI > The test material was added to each dish and cultured for 2 days. After 2 days, the medium was collected and collagen production was measured by Procollagen Type I C-peptide (PIP) EIA kit (takara MK101).

Three different groups were treated by concentration and collagen production rate was measured. The amount of collagen was found to increase in most experimental groups compared to the control (Fig. 3). These results suggest that fatty acid - peptide complex can significantly inhibit wrinkles of skin by increasing collagen production.

Example 5

Experiment to confirm stability of wrinkle-improving composition

In order to confirm the heat and light stability of the fatty acid-peptide complex of the present invention, the composition was dissolved in a 50 mM Tris-HCl (pH 8.0) buffer solution so that the wrinkle-reducing composition was 10 mg / The loss of peptide components in the composition by heating was measured and the stability was measured in daylight conditions by the same method. The loss of peptide by heat was measured and compared with daylight conditions (Fig. 4 and 5). As a result, it was confirmed that the peptide showed less than 10% peptide loss up to 60 days and had high stability in heat and storage period (Figs. 4 and 5).

Example 6

Cytotoxicity, Playability  Experiment

In order to confirm whether the fatty acid-peptide complex of the present invention shows toxicity to keratinocytes, cytotoxicity and regeneration ability were evaluated by MTT assay. HACAT cells and HDF cells were seeded on a 24-well plate at 4 × 10 ⁴ cells / 24 well per well and incubated at 37 ° C and 5% CO 2 for 24 h. After washing twice with PBS, the cultured synthetic fatty acid-peptide complex was treated with concentration (1 mg ~ 1 ug / mL) and incubated for 24 h. After incubation, MTT 0.5% in DPBS was mixed with the culture medium at a ratio of 1: 9 (v / v). Then, formazan was dissolved in DMSO for 2 h and incubated in a CO2 incubator for 2 h at 570 nm using ELISA. As shown in FIG. 6, there was no decrease in cell number due to the composition administered at a low concentration to a high concentration on HaCat cells, a kind of keratinocyte, and HDF cells, a kind of fibroblasts, No significant changes were observed in the microscopic observation of the cells

Thus, it can be seen that the fatty acid peptides of the present invention have no toxicity to the skin-related cells and promote skin cell proliferation. Thus, it can be predicted that the present wrinkle-improving composition will not have a great influence even if it is treated on the skin .

Example 7

fatty acid- Of peptide  Confirm skin permeability improvement

In order to confirm the enhancement of the skin permeability of the fatty acid-peptide, a skin permeability comparative experiment using a Franz diffusion cell was conducted on the epidermis of the human body as described below. The results are shown in FIG. 8 .

[Experimental method of skin permeability]

1. Fill the receptor chamber of the Franz diffusion cell with 6.5 ml of phosphate buffer solution and place the horny layer face up on the skin of the human body between the donor chamber and the receptor chamber. (cadaver). The epidermis skin of Hans Biomed was used as the cadaver used.

2. Donor chambers were prepared with an aqueous solution of 1 mg / ml of a general peptide as a control group and a 1 mg / ml solution of caffeine as a positive control group.

3. During the experiment, the temperature was maintained at 35 ± 1 ℃ and the magnetic stirring was continued.

4. After 0.5, 1, 3, 5, 8, 12 and 24 hours, each sample in the receptor chamber was sampled at a sampling port.

5. The amount of peptide and caffeine in the sample by time was checked by HPLC analysis, and the skin permeation rate and skin permeability were determined, and the comparison group and the control group were compared.

7, a skin permeability comparative experiment using Franz diffusion cells showed that the general peptides not bound to fatty acids had a large molecular weight and hardly penetrated into the skin. However, fatty acid-peptide complexes showed a tendency to increase in proportion to time Was found in the receptor chamber through the cadaver between the receptor chamber of the Franz diffusion cell and the donor chamber. In this example, it was confirmed that the fatty acid peptides smoothly permeate the skin horny layer of the peptide and significantly promote the skin absorption of the peptide.

Example 8

Clinical wrinkle improvement test

In order to confirm the clinical effect of improving the wrinkles of the cosmetic composition comprising the fatty acid-peptide complex according to the present invention, the wrinkle-improving effect of the cosmetic formulation prescribed with the composition was confirmed by an authorized certification body.

One kind of fatty acid-peptide complex which had a high skin wrinkle-reducing effect in vitro was selected and a cream containing the above-mentioned fatty acid-peptide complex was prepared in the cream composition shown in Table 3 below. . The test product was applied to the facial area according to the method of use, and the effect on the improvement of the eye wrinkles was evaluated. Evaluation was carried out in accordance with the SOP of the evaluation institution, and matters not specified in the Korea Food and Drug Administration's safety guidelines were referred to the reference documents.

The wrinkles were measured using the PRIMOS (Phaseshift Rapid In-vivo Measurement of Skin) and the overlay function was used to ensure that the existing measurement area and the new measurement area were identical for accurate reproducibility of the test area .

PRIMOS is a device for measuring fine wrinkles. A fringe fraction made from a DMD (Digital Micromirror Device) is projected onto the skin. The projected image is represented by three-dimensional profile information and analyzed by PRIMOS program (GFM, Germany) through a filter process. Ra and R3z were selected as PRIMOS analysis variables.

The arithmetic roughness average is an arithmetic average value of the roughness cross-sectional peaks of the wrinkles measured by PRIMOS. The lower the Ra value, the lower the wrinkle depth of the wrinkles, and the wrinkles are improved.

Base roughness depth (R3z) is expressed as an arithmetic average of five single roughness depths from R3z1 to R3z5. The single roughness depth means the vertical distance between the third highest peak and the third deepest curve at a single evaluation distance (Ir) of the roughness section. The smaller the value of R3z, the lower the wrinkle depth of the skin, which means that the wrinkles are improved.

Analysis of the changes in the eye wrinkles between the two groups showed that there was a statistically significant improvement in the wrinkles of the eye at 8 weeks after using the product in Ra and R3z compared to the control group (Fig. 8).

The preferred embodiments of the present invention have been described in detail above. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing detailed description, and all changes or modifications derived from the meaning, range, and equivalence of the claims are included in the scope of the present invention Should be interpreted.

<110> A and PEP INC.          Hannam University Institute for Industry-Academia Cooperation <120> DEVELOPMENT OF NEW PEPTIDE DERIVATIVE FOR COSMETIC MATERIAL WITH          ENHANCED SKIN PERMEABILITY <130> NP17-11167 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> PEPTIDE-1 <400> 1 Ala Phe Pro Gly   One <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> PEPTIDE-2 <400> 2 Arg Lys Asp Val Tyr   1 5 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> PEPTIDE-3 <400> 3 Glu Glu Met Gln Arg Arg   1 5 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> PEPTIDE-4 <400> 4 Gly Pro Gln Gly Pro Gln   1 5 <210> 5 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> PEPTIDE-5 <400> 5 Phe Val Ala Pro Asp Pro   1 5

Claims (7)

A cosmetic composition for improving skin wrinkles comprising a peptide-peptide complex comprising a peptide selected from the group consisting of SEQ ID NOS: 1 to 5 and a saturated or unsaturated fatty acid having 10 to 20 carbon atoms as an active ingredient. The method according to claim 1,
The saturated or unsaturated fatty acid having 10 to 20 carbon atoms is preferably selected from the group consisting of myristic acid, stearic acid, linoleic acid, palmitic acid, oleic acid, Wherein the cosmetic composition is at least one selected from the group consisting of lauric acid. The method according to claim 1,
Wherein the fatty acid-peptide complex is an amide-bonded complex of a peptide represented by SEQ ID NO: 4 with stearic acid, myristic acid or palmitic acid. The method according to claim 1,
Wherein the fatty acid-peptide complex is prepared by using a compound represented by the following formula (1) as a protecting group.
[Chemical Formula 1]

In Formula 1,
R 1 and R 2 are each independently hydrogen or a linear or branched hydrocarbon having 1 to 20 carbon atoms. The method according to claim 1,
Wherein the fatty acid-peptide complex inhibits MMP-1 expression. The method according to claim 1,
Wherein the fatty acid-peptide complex activates collagen biosynthesis in skin cells. The method according to claim 1,
Wherein the fatty acid-peptide complex has a skin permeability (based on 24 hours of the experiment) of at least 0.01 mg / ml using a Franz diffusion cell.

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