KR20180019972A - Peptide with high whitening activity, and uses thereof - Google Patents

Abstract

The present invention relates to a peptide for skin whitening and skin condition improvement, and more particularly, to a peptide consisting of a specific sequence, and the user of the peptide. The peptide consists of an amino acid sequence of SEQ ID NO: 1.

Description

FIELD OF THE INVENTION [0001] The present invention relates to peptides having excellent whitening activity,

The present invention relates to a novel peptide having excellent skin whitening 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.

As the industry develops, environmental pollution becomes serious, and there is a growing interest in skin aging prevention and whitening. In particular, human skin constantly undergoes changes, the most representative of which is degradation of the skin caused by aging and a reduction in visual beauty. The final appearance of skin appearance as a result of skin aging is wrinkle formation, skin elasticity decrease, senile spot formation, and wrinkle formation is considered to be the most typical phenomenon.

Lipids, proteins, polysaccharides and nucleic acids, which are major components of the skin, can be oxidized to destroy skin cells and tissues, and the skin may eventually age. Particularly, oxidation of proteins can cause collagen, hyaluronic acid, elastin, proteoglycan, fibronectin, etc., which are connective tissues of the skin, to be cleaved and hyperinflammation reaction. In the case of further deepening, mutation due to DNA mutation, Cancer induction can be promoted.

Therefore, it is necessary to protect the cell membrane by eradicating reactive oxygen species which are mediated by reactive oxygen species, ultraviolet ray irradiation, and inflammation reaction occurring during the metabolism of the body, regenerate damaged cells through active metabolism, and restore skin health desirable.

On the other hand, melanin is converted from tyrosine to dopa and dopaquinone by the action of tyrosinase existing in the pigment cells, and is produced through dopachrome and the like. Melanin protects the body from harmful factors such as ultraviolet rays and regulates hormone secretion in the body.

However, since melanin is overproduced, it can promote the formation of spots, freckles, and aging of the skin, and it can participate in the induction of skin cancer, so research and development for inhibiting the excessive production of melanin is actively performed.

(Japanese Patent Laid-open No. 4-9320), hydroquinone (Japanese Patent Laid-open No. 6-192062), kojic acid (Japanese Patent Laid-open No. 56-7710), arbutin Rosinase inhibitory activity and was used as a whitening cosmetic. However, these ingredients are poor in stability among cosmetic formulations, may be decomposed to cause coloration or odor, and their safety or efficacy in vivo is unclear and their use is limited.

Meanwhile, the cosmetics industry is actively developing products using natural materials to reduce skin irritation caused by chemical substances. However, cosmetic materials derived from natural materials are not easily realized in cosmetic compositions and are not fully functional in spite of environmentally friendly characteristics, or research on natural materials is saturated, and new materials can not be easily found.

Therefore, new skin improvement mechanisms have been identified based on the latest skin science theory, and cosmetic materials using the same have actively been developed.

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 excellent skin whitening activity.

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

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

In one embodiment, the peptide is capable of promoting the growth of fibroblasts and keratinocytes.

In one embodiment, the peptide can promote the production of laminin, collagen or hyaluronic acid.

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 skin whitening, antioxidant, wrinkle improvement, skin elasticity improvement, skin aging prevention, or skin regeneration.

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

According to the present invention, since the peptides are small in size, they are excellent in skin permeability and are excellent in skin whitening effect, thereby replacing existing skin whitening materials.

Due to its superior skin whitening activity, the peptides can be used for skin care products such as medicines, quasi-drugs, health functional foods and cosmetics.

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.

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

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

The peptide refers to a polymer composed of one or more amino acids linked by amide bonds (or peptide bonds). For the purposes of the present invention, the peptides may refer to peptides or fragments thereof that exhibit antioxidant and skin whitening 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.

The peptide can inhibit the production of melanin and the activity of tyrosinase. The melanin is a dark brown pigment present in eyes, skin and hair, and protects the body by protecting ultraviolet rays from penetration by absorbing a certain amount or more of ultraviolet rays from the body, and maintains body temperature. However, excessive exposure to ultraviolet light can result in excessive secretion of melanin and discoloration of the skin color. Melanin can be produced by the combined action of melanogenesis enzymes and hormones, including tyrosinase.

The peptide effectively inhibits the activity of tyrosinase and inhibits the production of melanin, thereby achieving skin whitening effect.

In addition, since the peptide promotes the growth of fibroblasts and keratinocytes, it can impart elasticity to the skin and suppress the formation of wrinkles.

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 excellent skin growth promoting effect.

Since the peptide promotes the growth of fibroblast and keratinocyte and promotes collagen synthesis, it grows the stratum corneum, epithelial layer and dermis layer of skin, and improves skin elasticity, skin aging, skin moisturization, skin regeneration and antioxidant .

Thus, the skin condition can be dramatically improved with skin whitening effect when the peptide is topically applied to the skin.

The peptide may be composed of the amino acid sequence of Formula 1, or may include the amino acid sequence as a part.

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.

Specifically, the composition may be for skin whitening, antioxidation, wrinkle improvement, skin elasticity improvement, skin aging prevention, or skin regeneration.

The effect of improving the skin condition may be a skin whitening effect, an antioxidant effect, prevention or improvement of skin aging and wrinkle, skin protection, improvement of skin barrier function, skin irritation mitigation, skin cell proliferation and regeneration ability, All can be covered.

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.

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 matrix called elastin and the elastin is a protein composed of a unique amino acid found only in elastic fibers such as desmosine and isodesmosine derived from lysine . 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 above-mentioned " comprising as an active ingredient " indicates collagen synthesis, elasticity improvement or antioxidative activity related to the wrinkle-reducing effect, for example, a degree capable of exhibiting whitening efficacy due to reduced skin melanin synthesis, Quot; effective < / RTI > amount. &Quot;

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 skin whitening.

Since the composition contains a peptide having an excellent inhibitory effect on the production of melanin and tyrosinase activity, it can effectively alleviate the skin deposition or color change caused by the excessively produced melanin.

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 which are 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). Columns were ACQUITY UPLC BEH300 C18 (2.1 x 100 mm, 1.7 μM, Waters Co, USA).

Peptides synthesized according to the above method are shown in Table 1 below.

division order Remarks Example YCRFFQASCY SEQ ID NO: 1

Experimental Example 1: 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 a wavelength of 450 nm using a microplate reader, and 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 2 below.

[Mathematical Expression]

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

division Melanin production inhibition rate (%) Example 0 mg / L 0 1 mg / L 25.1 10 mg / L 36.7 50 mg / L 48.0 Positive control group 29.2

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

Experimental Example 2: Inhibition activity of tyrosinase

The tyrosinase inhibitory activity was measured in vitro to evaluate the whitening effect of the peptide.

The sample was dissolved in an appropriate solvent, and 220 μL of 0.1 M phosphate buffer (pH 6.5), 20 μL of the sample solution and 20 μL of tyrosinase (1500 U / mL to 2000 U / mL) were sequentially added. 40 μl of a 1.5 mM tyrosinase solution was added thereto and reacted at 37 ° C for about 15 minutes.

Absorbance was measured at 490 nm using an ELISA reader. A 0.1 M phosphate buffer (pH 6.5) was used instead of the sample solution.

As a control, Kojic acid at a concentration of 50 uM was used and tyrosinase inhibitory activity was calculated according to the following equation. The evaluation results are shown in Table 3 below.

[Mathematical Expression]

Tyrosinase inhibitory activity (%) = 100 x (1 - (absorbance of each test substance / absorbance of negative control))

division Tyrosinase inhibitory activity (%) Example 0 mg / L 0 1 mg / L 21.4 10 mg / L 37.1 50 mg / L 51.1 Positive control group 29.3

The peptide of the above example inhibited the activity of tyrosinase in a concentration-dependent manner, and the tyrosinase inhibitory activity was evaluated to be higher than that of the positive control (kojic acid).

Experimental Example 3: Measurement of Expression of Melanin Synthesis Related Gene

The inhibition of the expression of the related gene inducing the synthesis of melanin was examined.

In order to confirm the expression level of the melanin synthesis gene, melanin synthesized cells treated with α-MSH were treated with different concentrations of the peptides 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.

The expression of tyrosinase and MITF gene by peptide was examined by measuring the fluorescence value of the PCR product using 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 the 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'-CGACAGGATGCAGAAGGAG-3 ' 5'-ACATCTGCTGGAAGGTGGA-3 ' MITF 5'-GGAACAGCAACGAGCTAAGG-3 ' 5'-TGATGATCCGATTCACCAGA-3 ' Tyrosinase 5'-GGCAGATTGTCTGTAGCCGA-3 ' 5'-CCTTGGGGTTCTGGATTTGT-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 MITF gene and tyrosinase mRNA expression associated with tyrosinase expression was decreased in a dose dependent manner.

These results suggest that the peptides can reduce the expression of tyrosinase as well as the activity, thereby effectively inhibiting the production of melanin.

division MITF expression level Tyrosinase expression level Example 0 mg / L 1.00 1.00 1 mg / L 0.97 0.94 10 mg / L 0.81 0.69 50 mg / L 0.47 0.34

 [Fold, β-actin normalized]

Experimental Example 4: Test for promoting growth of fibroblasts and keratinocytes

In order to investigate the effect of improving skin cosmetics, it was confirmed that fibroblasts and keratinocyte cell growth, which are components of dermis, collagen are promoted by peptides.

Human keratinocyte cells (HaCaT) were cultured in an ATCC (American Type Culture Collection), and human dermal fibroblasts (nHDF) were obtained from Lonza (Switzerland).

Each cell was inoculated into a 24 well culture plate at a concentration of 1 x 10 ⁴ cells / mL. 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).

When the cells were cultured in DMEM containing 10% FBS for 48 hours and cultured for 25 to 30% or more of the surface of the culture vessel, the cells were further exchanged with FBS-free DMEM containing the peptide for 24 hours.

50 μL of a solution (2.5 mg / mL) of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Sigma M5655, USA) The supernatant was removed.

200 μL of DMSO (dimethylsulfoxide, Sigma D2650, USA) solution was added to each well and stirred for 20 minutes to dissolve formazan crystals. Then, 100 μL of the solution was taken in 96 wells and subjected to enzyme-linked immunosorbent assay (ELISA) ).

The degree of proliferation activity of skin cells was expressed by the following percentages based on the absorbance of the control group using purified water. The measurement results are shown in Table 6 below.

division Peptide concentration 1 mg / L 10 mg / L 50 mg / L Fiber
cell 24 hours elapsed 101.2 102.7 104.2 48 hours elapsed 102.9 113.4 121.8 72 hours elapsed 105.4 122.3 145.6 corneous
formation
cell 24 hours elapsed 101.4 104.0 110.2 48 hours elapsed 105.1 116.7 129.2 72 hours elapsed 108.3 129.3 144.7

 [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 suggest that the peptide is excellent in stability against skin or human body, effectively promotes the growth of fibroblasts and keratinocytes that synthesize collagen, and can be used as a skin cosmetic, particularly for improving skin wrinkles do.

Experimental Example 5: Test for promoting the production of laminin and hyaluronic acid

HaCaT cells cultured for 48 hours were treated with the peptide of the above example, and after 72 hours, the concentrations of laminin and hyaluronic acid, which are markers of skin wrinkle improvement, were measured. At this time, the concentration was measured using a Laminin ELISA kit and a Hyaluronic acid ELISA kit.

That is, the concentrations of laminin and hyaluronic acid in HaCaT cells treated with the peptides of the examples and HaCaT cells (negative control) without any treatment were compared, and the relative values thereof are shown in Table 7 below.

division Laminin Hyaluronic acid density
(%) Example Control group Example Control group 158 100 146 100

Referring to Table 7, it can be seen that the concentration of laminin and hyaluronic acid in the peptide (example) composed of the amino acid sequence of SEQ ID NO: 1 was significantly increased compared with the negative control.

The above results suggest that the peptide composed of the amino acid sequence of the above-mentioned example exhibits not only a skin whitening effect but also a wrinkle-reducing effect, thereby improving the overall skin condition.

Experimental Example 6: 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 x 10 ⁴ cells / mL were dispensed in a volume of 100 μL into a 96-well plate and cultured for 3 days at 37 ° C, 5% CO ₂ , and humidified conditions.

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 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 quantitated by enzyme-linked immunosorbent assay (Procollagen type I c-peptide EIA Kit, Takara Bio).

The amount of collagen in each test sample culture fluid was calculated based on the amount of type I collagen in the control (100%). The measurement results are shown in Table 8 below.

division Collagen production (%) Example 179.5

 [% of control]

These results suggest that the peptide has excellent skin whitening effect as well as skin elasticity improving effect.

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 WITH HIGH WHITENING ACTIVITY, AND USES THEREOF <130> 16PP11174 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> SEQ ID NO 1 <400> 1 Tyr Cys Arg Phe Phe Gln Ala Ser Cys Tyr   1 5 10

Claims (7)

A peptide consisting of the amino acid sequence of SEQ ID NO: 1. The method according to claim 1,
Peptides that inhibit the production of melanin and the activity of tyrosinase. The method according to claim 1,
Fibroblasts and peptides that promote the growth of keratinocytes. The method according to claim 1,
Peptides that promote the production of laminin, collagen or hyaluronic acid. 1. A composition for improving skin condition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. 6. The method of claim 5,
Skin whitening, antioxidation, wrinkle improvement, skin elasticity improvement, skin aging prevention, or skin regeneration. 1. An external preparation for skin or cosmetic composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.