KR101900745B1 - Peptide for improving skin wrinkle and whitening effect, and uses thereof - Google Patents

Abstract

The present invention relates to a peptide for improving skin wrinkles, and relates to a peptide composed of a specific sequence and its use.

Description

FIELD OF THE INVENTION [0001] The present invention relates to peptides having a wrinkle-improving and whitening effect, and their uses. [0002] PEPTIDE FOR IMPROVING SKIN WRINKLE AND WHITENING EFFECT, AND USES THEREOF [

The present invention relates to a novel peptide having an excellent wrinkle-reducing effect.

Skin is the primary barrier of the human body. It protects the internal organs of the body from external stimuli such as changes in temperature and humidity, ultraviolet rays, and pollutants, and plays an important role in the maintenance of body homeostasis such as body temperature control.

Like the other organs of the human body, the skin ages with aging, accompanied by changes in skin elasticity loss, keratinization, wrinkle formation, and skin atrophy.

The cause of skin aging is largely classified into internal factors such as gene mutation and tissue change, and external factors such as ultraviolet rays and humidity.

The phenomenon of aging due to ultraviolet rays is called photoaging. Active oxygen radicals are produced in the cells by ultraviolet rays and reactive oxygen species are activated by a signal transduction system that induces an inflammatory response through proteolytic enzymes such as collagen and elastin (MMP-1, MMP-3, MMP-9 , etc.) (Fisher GJ et al., Nature, Vol. 379, 335-339), thereby reducing the elasticity of the dermis and thereby causing skin wrinkles.

Studies have been made on physiologically active substances having a wrinkle-reducing effect in order to prevent skin aging and maintain elastic skin.

Tretinoin (trans-retinoic acid) was approved by the US FDA for the treatment of aging skin, and wrinkle-improving cosmetics began to be launched in earnest as retinol, which had few side effects, began to be used as cosmetic ingredients in the late 1990s. Afterwards, female hormone-like substances and antioxidants extracted from various plants were introduced into cosmetics as wrinkle-improving raw materials.

However, recently developed cosmetic raw materials have various problems such as insufficient efficacy and skin side effects. Even in the case of excellent efficacy, current cosmetic raw materials have a limited range of action on the skin, and most of them require the customers who want fundamental wrinkle improvement by promoting collagen synthesis, inhibiting collagen decomposition, I could not satisfy it enough.

Therefore, based on the latest theories of skin science, researches on raw materials and techniques that can identify and delay or delay new skin aging mechanisms are actively under way.

Korean Patent No. 10-1093685

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a novel peptide having excellent biostability and anti-aging activity.

According to one aspect of the present invention, there is provided a skin condition improving peptide comprising the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the peptide can promote fibroblast growth and induce collagen biosynthesis of the skin tissue.

In one embodiment, the peptide may inhibit the production of melanin and the activity of tyrosinase.

According to another aspect of the present invention, there is provided a skin condition improving composition comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the composition may be for wrinkle improvement, skin elasticity improvement, skin aging prevention, antioxidant, skin whitening, skin moisturizing, or skin regeneration.

According to another aspect of the present invention, there is provided an external preparation composition comprising the peptide as an active ingredient.

According to the present invention, since the peptides are small in size, they are excellent in skin permeability and wrinkle-improving effect, and can replace conventional anti-wrinkle agents.

The peptide can be used not only for medical purposes for improving wrinkles and anti-aging but also for cosmetic products such as quasi-drug compositions, health functional food compositions, and cosmetic compositions.

It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be deduced from the description of the invention or the composition of the invention set forth in the claims.

As used herein, the terminology used herein is intended to encompass all commonly used generic terms that may be considered while considering the functionality of the present invention, but this may vary depending upon the intent or circumstance of the skilled artisan, the emergence of new technology, and the like. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term, not on the name of a simple term, but on the entire contents of the present invention.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

The numerical range includes numerical values defined in the above range. All numerical limitations of all the maximum numerical values given throughout this specification include all lower numerical limitations as the lower numerical limitations are explicitly stated. All the minimum numerical limitations given throughout this specification include all higher numerical limitations as the higher numerical limitations are explicitly stated. All numerical limitations given throughout this specification will include any better numerical range within a broader numerical range, as narrower numerical limitations are explicitly stated.

Hereinafter, embodiments of the present invention will be described in detail, but it should be apparent that the present invention is not limited by the following examples.

One aspect of the present invention provides a skin condition improving peptide comprising the amino acid sequence of SEQ ID NO: 1.

The peptide refers to a polymer composed of one or more amino acids linked by amide bonds (or peptide bonds). For the purpose of the present invention, the peptide may refer to peptides or fragments thereof that exhibit wrinkle-improving and skin elasticity-improving effects.

The general rules for naming the peptides may be based on three letter or single letter amino acid codes, unless specifically indicated otherwise. For example, the center of the amino acid structure is represented by a three-letter code (e.g., Ala, Lys), and a D-stereotype (e.g., D-Ala, D-Lys) It is possible to assume an L-steric form. The amino acid residue constituting the peptide may be a natural or non-natural amino acid residue.

As a result of intensive efforts to discover peptides having a biologically active activity, the present inventors have found an excellent skin improving effect, particularly a skin wrinkle reducing effect, of the peptides.

The peptide may be composed of the amino acid sequence of SEQ ID NO: 1, or may partially include the amino acid sequence.

The keratinocytes and fibroblasts in the skin may be damaged and deteriorated in density due to various causes such as mental stress, external harmful factors, and endogenous aging. In particular, the damage of keratinocytes and fibroblasts due to internal and external stress may lower the density of skin cells and interfere with the synthesis of collagen, which may deepen the formation of skin wrinkles.

The collagen is a major component of the connective tissue and is a component mainly distributed in the bones and skin. It is a light protein that maintains the structure and shape of the skin and provides firm strength and elasticity. When the skin is aged, exposed to ultraviolet rays, heat, etc., collagen synthesis ability decreases and collagen amount can be significantly reduced. When the amount of the collagen decreases, the shape of the muscle can not be maintained, so that a skin wrinkle can be generated.

The peptide is derived from a peptide that constitutes an in vivo growth factor. The peptide has a high molecular weight and a small size, and thus has high skin permeability and is excellent in skin growth promoting effect. Since the peptide promotes fibroblast proliferation and collagen synthesis, the skin stratum corneum, epithelial layer and dermis layer are grown. Thus, it can exhibit effects of skin elasticity improvement, skin aging prevention, skin moisturization improvement, and skin regeneration. Thus, the skin condition can be effectively improved when the peptide is topically applied to the skin.

On the other hand, the peptide can be produced biologically by extracting a protein in vivo and treating it with a protease to give a low molecular weight or by using a gene recombination and protein expression system, or by a chemical synthesis method using a peptide synthesizer or the like .

For example, a gene encoding a fusion partner and a fusion protein composed of the peptide may be prepared through gene manipulation, and the host microorganism may be transformed with the gene and expressed in the form of a fusion protein in the host microorganism.

The fusion protein can be cleaved and separated using proteolytic enzymes or compounds, and the peptide can be obtained. Specifically, a DNA sequence encoding an amino acid residue that can be cleaved by a proteolytic enzyme, such as Factor Xa or an enterokinase, a compound such as CNBr or hydroxylamine, can be inserted between the fusion partner and the gene of the peptide .

The N or C-terminal of the peptide may be bonded with an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group or polyethylene glycol (PEG). The stability of the peptide can be remarkably improved by the modification. The stability is a concept including not only in vivo stability but also storage stability. The protecting group can protect the peptide of the present invention from attack of a protein cleaving enzyme in vivo.

In addition, the peptide may comprise a functional equivalent of a peptide comprising the amino acid sequence. Said "functional equivalent" means that at least 40%, preferably at least 80%, more preferably at least 90%, more preferably at least 95% sequence identity with said amino acid sequence as a result of addition, substitution or deletion of amino acids Means a protein having homology and having a physiological activity substantially equivalent to the above amino acid sequence.

The "substantially homogenous physiological activity" means a skin condition improving activity such as anti-inflammatory activity due to the structural and functional homology of the peptide. The sequence homology is determined by comparing the two optimally arranged sequences with the comparison region, and some nucleic acid sequences in the comparison region may be added or deleted.

According to another aspect of the present invention, there is provided a skin condition improving composition comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

The composition may be for improving wrinkles, improving skin elasticity, preventing skin aging, antioxidation, skin whitening, skin moisturizing, or skin regeneration. The effect of improving the skin condition includes all the skin-beneficial effects such as prevention or improvement of skin aging and wrinkles, skin protection, skin barrier function improvement, skin irritation mitigation, skin cell proliferation and regeneration ability, antioxidant ability, collagen synthesis promoting ability .

The "wrinkles " refers to the scarring caused by degeneration of the skin, and may be caused by a cause of genes, a reduction of collagen and elastin present in the skin dermis, and an external environment.

The "wrinkle improvement" refers to a phenomenon that inhibits or inhibits the generation of wrinkles on the skin, or alleviates the already generated wrinkles.

The "skin elasticity" means elasticity realized by elastic fibers composed of elastin existing in the dermal layer. Since the elastic fibers have a low elastic modulus like rubber, they are easily deformed by external force, It can be easily restored to its original state.

The elastic fibers are embedded in microfibrils in an amorphous substrate called elastin. The elastin is a protein composed of a unique amino acid found only in elastic fibers such as desmosine and isodesmosine derived from lysine. to be. The desmosine and isodesmosin form cross-links within long peptide chains, and the structure can confer rubber-like properties to elastin.

The "improvement of skin elasticity" means that elastic fibers composed of elastin are present together with collagen, and elastic elasticity is maintained or improved in a state where elastin and collagen are sufficiently present.

The "skin whitening" mentioned above is intended to mitigate the formation of various biomolecules such as melanin, redox hemoglobin, carotene and melanoid affecting the skin color, thereby alleviating skin pigmentation phenomena such as dullness, freckles and stains, It means an effect of alleviating redness and improving skin brightness and uniformity.

As used herein, the term " comprising as an active ingredient " may mean an effective amount that can exhibit a skin improving effect, for example, an amount capable of exhibiting collagen synthesis, elasticity improvement or antioxidative activity associated with wrinkle-

The peptides can be used for various applications such as pharmaceutical compositions, cosmetic compositions, and external skin preparations due to the skin improving activity.

In one embodiment, the pharmaceutical composition may be for use in anti-aging or wrinkle-improving.

Since the composition contains a peptide having excellent skin growth promoting effect, it can provide an anti-aging effect by promoting the production of collagen and alleviating the decrease of collagen caused by skin aging.

The pharmaceutical composition may form pharmaceutically acceptable acid addition salts according to methods conventional in the art. The free acid may be an organic acid or an inorganic acid. For example, the inorganic acid may be hydrochloric acid, bromic acid, sulfuric acid, or phosphoric acid. The organic acid may be citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid But are not limited to, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4- toluenesulfonic acid, benzenesulfonic acid, Nicotinic acid, isonicotinic acid, picolinic acid, glutamic acid, or aspartic acid.

The pharmaceutical composition may further comprise a commonly used carrier, excipient and diluent.

The compositions can be administered in the form of oral delivery , parenteral delivery. The peptide may be administered systemically or topically, and the administration may include oral administration and parenteral administration. When the compound is administered as a composition, it may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide a suitable dosage form.

In addition, the composition comprising the peptide may further comprise a carrier, an excipient and a diluent which are used in the production of the pharmaceutical composition.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, But are not limited to, cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition, the composition may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, external preparation, suppository and sterilized injection solution.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and the solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Or gelatin. In addition to the above excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, syrups, and the like. In addition to water and liquid paraffin, simple diluents, various excipients such as wetting agents, sweeteners, have.

The preparation for parenteral administration may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository. Examples of the non-aqueous solvent and suspensions may include injectable esters such as propylene glycol, polyethylene glycol, vegetable oil such as olive oil and ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, and glycerogelatin.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is desirable to administer an amount that allows for all of the above factors to be maximally effected in a minimal amount without side effects, which can be readily determined by one skilled in the art.

On the other hand, the cosmetic composition or external preparation for skin may be formulated by a conventional method. In the formulation of the external preparation for skin, Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can refer to, and in the formulation of the cosmetic composition, International cosmetic ingredient dictionary, 6th ed., The cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995, which is incorporated herein by reference.

Specifically, the cosmetic composition may be prepared in the form of a general emulsified formulation and a solubilized formulation. For example, lotion such as soft lotion or nutrition lotion; Lotion such as facial lotion, body lotion; Nourishing cream, moisture cream, cream such as eye cream; essence; Makeup ointment; spray; Gel; pack; Sunscreen; Makeup base; A foundation such as a liquid type, a solid type or a spray type; powder; Make-up removers such as cleansing creams, cleansing lotions and cleansing oils; Or a cleansing agent such as a cleansing foam, a soap, a body wash, and the like, but is not limited thereto. The external preparation for skin may be formulated into ointments, patches, gels, creams or sprays, but is not limited thereto.

The cosmetic composition or the external preparation may suitably contain other ingredients in addition to the essential ingredients in the respective formulations within the range not hindering the object of the present invention depending on the type of the formulation or the purpose of use.

The cosmetic composition or the external preparation may contain an acceptable carrier and may be suitably mixed with oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, a chelating agent, a pigment, a preservative, It is not.

The acceptable carrier may vary according to the formulation. For example, when formulated into ointments, pastes, creams or gels, there may be used an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, Mixture can be used

When the cosmetic composition or the external preparation is formulated with a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as a carrier component, Such as propane, butane, or dimethyl ether.

When the cosmetic composition or the external preparation is formulated into a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as the carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, Glycol, and 1,3-butylglycol oil may be used, and fatty acid esters of cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or sorbitan may be used have.

When the cosmetic composition or the external preparation is formulated as a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the cosmetic composition is formulated into soap, it is preferable to use, as a carrier component, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolizate, isethionate, a lanolin derivative, an aliphatic alcohol, a vegetable oil, Can be used.

The cosmetic composition or the external preparation for skin according to the present invention may be formulated into a lipid, an organic solvent, a solubilizer, a thickening agent, a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, a foaming agent ), Perfumes, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants, chelating agents, preservatives, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, Such as any other ingredients used in cosmetics or dermatology.

However, the adjuvant and the mixing ratio thereof can be appropriately selected so as not to affect the preferable properties of the cosmetic composition according to the present invention.

The present invention will be further described with reference to the following examples, but it should be apparent that the present invention is not limited by the following examples.

Preparation Example: Preparation of peptide

Peptides were synthesized using conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group of amino acid. Amino acid residues were extended using N-hydroxybenzotriazole (HOBt) and N, N'-diisopropylcarbodiimide (DIC) as an activator (Reference: Wang C. Chan, Perter D. White, 'Fmoc-solid phase peptide synthesis', Oxford].

The synthesized peptides were purified by C18 reverse phase high performance liquid chromatography (HPLC, Waters Associates, USA). The columns were ACQUITY UPLC BEH300 C18 (2.1 x 100 mm, 1.7 μm, Waters Co, USA).

The peptides synthesized according to the above method are shown in Table 1 below

division order Remarks Example YCAEKWHSLCN SEQ ID NO: 1

Experimental Example 1: Test for promoting growth of human dermal fibroblast

In order to examine the effect of improving the skin beauty, it was confirmed that the fibroblast growth which synthesizes collagen as a component of the dermis is promoted by the peptide.

Human Dermal Fibroblasts (nHDF) were obtained from Lonza (Switzerland).

Cell culture media were supplemented with 10% fetal bovine serum (FBS), 10% penicillin (100 units / mL) and streptomycin (100 g / mL) in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, USA). The culture conditions were incubated at 37 ° C, 95% humidity, 5% CO ₂ incubator, and cells between 3 and 10 generations were used for the experiment.

In order to examine the degree of cytotoxicity of peptides treated with human dermal fibroblast nHDF cells, peptides were cultured for 24 hours after treatment with 1, 10, and 50 mg / L of peptides, and MTT And the viability was measured. The measurement results are shown in Table 2 below.

division 24 hours elapsed 48 hours elapsed 72 hours elapsed Example 1 mg / L 101.2 104.8 107.1 10 mg / L 102.8 111.5 120.6 50 mg / L 105.2 126.1 139.2

 [nHDF cell viability (% of control)]

According to the peptide treatment of the above example, cell growth was accelerated depending on the peptide concentration. In particular, there was no significant difference in cell viability after about 24 hours, but cell proliferation was markedly accelerated as time progressed.

That is, the above results indicate that the peptide is excellent in stability against skin or human body, and effectively promotes the growth of fibroblasts that synthesize collagen, and thus can be utilized as a skin beauty, particularly a skin wrinkle improvement.

Experimental Example 2: Assay for inducing collagen biosynthesis

It was confirmed that collagen synthesis was promoted by the growth of fibroblasts.

Human dermal fibroblasts at a concentration of 1.0 × 10 ⁴ cells / mL were dispensed into 96-well plates in an amount of 100 μL each, and cultured at 37 ° C., 5% CO ₂ , and humidified conditions for 3 days.

Medium 100 S (manufactured by Kurashiki Boshiki) was used a medium containing 10% by weight of LSGS (Low Serum Growth Supplement, Kurashiki Boshiki Co., Ltd.) per well.

The medium was replaced with DMEM (Dulbecco's Modified Eagle's Medium, Sigma), and the peptide of the above example was treated at a concentration of 50 mg / L and then cultured. The control group was prepared by adding purified water without treating the above peptides.

After the cells were cultured for 7 days, the culture solution was collected and the concentration of secreted type I collagen in the culture solution was quantified by enzyme-linked immunosorbent assay (Procollagen type I c-peptide EIA Kit, Takara Bio).

The amount of collagen in each test sample culture liquid was calculated based on the amount of type I collagen in the control group (100%). The measurement results are shown in Table 3 below.

division Collagen production (%) Example 198.2

 [% of control]

Experimental Example 3: Measurement of expression of COL1A1 and MMP1

The expression of COL1A1, one of typical collagen expressed by human dermal fibroblasts, ELN, one of elastic proteins, and MMP1, a collagenase, were measured.

In order to confirm the extent of expression of collagen expression by the peptides, human pea fibroblasts were treated with 50 mg / L of peptide and cultured for 24 hours.

Changes in gene expression were measured by qRT-PCR (quantitative real-time PCR), which binds the fluorescent substance to the DNA product amplified by polymerase chain reaction (PCR) and continuously detects the fluorescent substance.

To quantify the changes of COL1A1 and MMP1 gene expression, fluorescence values of PCR products were measured by SYBR green I (Invitrogen). A reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris / HCl pH 8.4, 0.8 mM dNTP, 0.5 U Extaq DNA polymerase, 3 mM MgCl ₂ and 1 × SYBR green in a PCR tube.

Denaturation, annealing, and polymerization were performed at 94 ° C for 30 seconds, at 58 ° C for 30 minutes, and then for 3 minutes at 94 ° C using Linegene K (BioER, China) Sec., 72 ° C for 30 sec., And fluorescence intensity was measured after completion of each cycle.

PCR results were verified by melting curve for each result. The threshold cycle (Ct) value of each gene was normalized to the Ct value of β-actin, and the amount of change in the Ct value was compared and analyzed.

The Ct value is the number of cycles when the amount of fluorescence generated by the PCR product (the number of amplified PCR products) reaches a certain reference value above the basal value, and the amount of gene expression can be confirmed through the Ct value. The primer sequences used in the experiments are shown in Table 4 below.

Gene Forward primer Reverse primer β-actin 5'-GGATTCCTATGTGGGCGACGA-3 ' 5'-CGCTCGGTGAGGATCTTCATG-3 ' COL1A1 5'-AGGGCCAAGACGAAGACATC-3 ' 5'-AGATCACGTCATCGCACAACA-3 ' MMP1 5'-TCTGACGTTGATCCCAGAGAGAGAG-3 ' 5'-CAGGGTGACACCAGTGACTGCAC-3 '

The results of the measurement of the expression level according to the treatment of the peptides are shown in Table 5 below. That is, the amount of COL1A1 mRNA expression was increased in a concentration-dependent manner, while the amount of MMP1 mRNA expression was decreased in a concentration-dependent manner.

The above results suggest that the peptide can promote collagen production and improve skin elasticity and wrinkles, thereby being useful as a skin cosmetic application.

division COL1A1 expression level MMP1 expression level Example 0 mg / L 1.00 1.00 1 mg / L 1.21 0.96 10 mg / L 1.89 0.77 50 mg / L 2.77 0.35

[Fold, β-actin normalized]

Experimental Example 4: Melanin contents assay

Melanin contents assay was used to evaluate the inhibition of melanogenesis by the peptides (Eisinger M et al, 1982; Siegrist W and Eberle AN, 1986).

B16F10 cells were inoculated in a 60 mm culture dish at a concentration of ^1.5 × 10 ⁵ cells / well and cultured for 24 hours. Α-MSH (Sigma-Aldrich, St. Louis, Mo., USA), a hormone that promotes melanin formation, and the peptide were added to the medium by concentration (Virador VM et al, 1999).

After 48 hours, the cells were harvested and washed once with PBS. Then, 120 μL of 1 N NaOH lysis buffer was added and the cells were lysed by heating at 95 ° C. for 10 minutes. Absorbance was measured at 450 nm wavelength using a microplate reader. Arbutin (100 μg / mL) was used as a control.

The inhibition rate of melanin production was calculated according to the following equation, and the evaluation results are shown in Table 6 below.

[Mathematical Expression]

Melanin production inhibition rate (%) = 100 x (1 - (absorbance of each test substance / absorbance of control))

division Melanin production inhibition rate (%) Example 1 0 mg / L 0 1 mg / L 23.9 10 mg / L 37.9 50 mg / L 48.2 Positive control group 32.8

The peptides of the examples effectively inhibited the production of melanin and inhibited melanogenesis to a higher level as compared to albutin, the whitening effect being well known.

Experimental Example 5: Effect of decomposition of active oxygen

Free radical scavenging activity using DPPH (1, 1-diphenyl-2-picryl hydrazyl).

4 mL of methanol was added to the test tube, and the peptide was added by concentration. 1 mL of 0.15 mM DPPH solution was added, and the reaction was allowed to proceed at room temperature for 30 minutes. Then, the absorbance at 520 nm was measured.

Initial (Ai) and blank (Ab) without substrate and DPPH were measured, and α-tocopherol and BHT were used as positive control. The results are shown in Table 7 below.

division 0 mg / L 1 mg / L 10 mg / L 50 mg / L Example 0 48.2 69.6 88.4 alpha-tocopherol 0 45.0 63.9 79.6 BHT 0 38.3 53.4 66.9

 [DPPH erase rate%]

The peptides of the above examples were evaluated to have higher free radical scavenging activity than α-tocopherol and BHT, which are well known in the art. These results suggest that the peptide can effectively promote the decomposition of active oxygen, and that it is excellent in preventing skin aging and improving skin condition due to antioxidant activity.

Experimental Example 6: Measurement of active oxygen species in cells (DCF-DA assay)

The removal of reactive oxygen species (ROS) was assessed by comparing the amount of reactive oxygen species produced in the cells stimulated with silica after the peptide treatment with the negative control (Cho et al., 1999; Kim et al., 2002).

Raw 264.7 cells were inoculated into 96 well plates at a concentration of 1 × 10 ⁴ cells / well. After replacing the medium with fresh medium, cells were cultured for 24 hours by treatment with a certain concentration of the peptide.

10.0 μL of WST-1 solution (EZ-CYTOX, DOGEN, Korea) was added to each well and incubated for 1 hour. Absorbance was measured with a measurement wavelength of 450 nm and a reference wavelength of 655 nm using an ELISA reader Respectively.

Peptides were diluted with dimethylsulfoxide (DMSO, Sigma-Aldrich, USA) to a final concentration of 20 μM and DMSO was used as a blank test (negative control). The experiment was repeated 3 times to ensure reliability.

The experiment was carried out under the same cell line and the same cell culture conditions as the WST-1 assay, a cell activity assay. The cultured Raw 264.7 cells were washed once with phosphate buffered saline (PBS; sodium chloride 137 mM, phosphate buffer 10 mM, potassium chloride 2.7 mM, all from Biopure, Canada), and Raw 264.7 cells were harvested. The cells were suspended in 15 mL of PBS, and 15 μL of 20 mM 2 ', 7'-Dichlorofluorescein diacetate was added and incubated for 1 hour.

After centrifugation at 1200 rpm for 2 minutes, the cells were washed once with PBS solution, diluted to 1 × 10 ⁵ cells / mL, treated with the control substance and the peptide, and cultured for 10 minutes. 20 mg / mL of silica was treated with 50 μL and incubated for 1 hour. The cell pellet obtained by centrifuging at 6000 rpm for 5 minutes was dispersed in 200 μL PBS solution and inoculated into a 96-well plate.

Fluorescence was measured at 485 nm excitation / 535 nm emission using a fluorescence microplate reader to evaluate the amount of reactive oxygen species.

The peptides treated in the measurement of reactive oxygen species to the concentration of the test solution were used as the concentration of the test solution set through the cell activity measurement.

As a negative control, DMSO was used instead of the sample. As a positive control, ascorbic acid (Sigma-Aldrich) was diluted to 100 mg / mL in DMSO and used at a concentration of 100 μg / mL.

Based on the above results, a test for the measurement of reactive oxygen species at the same concentration was carried out, and the treated peptide was used at a concentration of 50 mg / L. The experiment was repeated 4 times to ensure reliability. The measurement results are shown in Table 8 below.

division Measures Example 82.7 Ascorbic acid 75.2

 [ROS generation inhibition rate (% of control)]

The peptides of the above examples were evaluated at an equivalent level of free radical scavenging activity than ascorbic acid, which is well known in the art.

That is, the above results suggest that the peptide is excellent in the ability to remove reactive oxygen species and has excellent antioxidative effect due to free radical scavenging activity.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

<110> AHN, INSOOK <120> PEPTIDE FOR IMPROVING SKIN WRINKLE AND WHITENING EFFECT, AND USES          THEREOF <130> 16PP11165 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> SEQ ID NO 1 <400> 1 Tyr Cys Ala Glu Lys Trp His Ser Leu Cys Asn   1 5 10

Claims (6)

A peptide for improving wrinkles and skin whitening comprising the amino acid sequence of SEQ ID NO: 1. The method according to claim 1,
A peptide that promotes the growth of fibroblasts and induces collagen biosynthesis of the skin tissue. The method according to claim 1,
Peptides that inhibit the production of melanin. A composition for external application for improving wrinkles comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. 1. A cosmetic composition for skin whitening comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. delete