KR20180019970A - Peptide having canities improvement effect, and uses thereof - Google Patents

Abstract

The present invention relates to a peptide for improving canities which is composed of a specific sequence, and a use thereof. The peptide is excellent in biostability and a canities improving effect. The peptide for improving canities is composed of an amino acid sequence of a sequence number 1.

Description

PEPTIDE HAVING CANITIES IMPROVEMENT EFFECT, AND USES THEREOF FIELD OF THE INVENTION [0001]

The present invention relates to novel peptides which are excellent in anti-wilt effect.

The canities, also called the gray hair, are caused by the progress of aging. Accordingly, a great deal of research and development has been carried out on dyeing white moths for cosmetics.

However, the dyeing of white moths is only a temporary effect. Therefore, periodic dyeing is required as the white moths grow, and the oxidation type black hair dye widely used at present has a problem of skin damage due to the use of the oxidizing agent.

The main cause of white spot syndrome is the disappearance of melanocyte stem cells and a decrease in the activity of melanocytes. In particular, it is known that white rot caused by senescence is mainly caused by loss of stem cells, and white rot caused by mites is caused by degradation of melanocytes due to environmental and mental stress of modern society.

Recently, it has been confirmed that artificial compounds such as oxidized black hair dye can cause fatal side effects in the human body, and natural materials such as black sesame seeds, walnut seeds, hanuksikcho, honey, This alternative material is attracting attention as a candidate.

However, although the above methods can suppress the occurrence of side effects such as artificial compounds, there is a limit in that the effect of improving blood circulation such as promotion of tyrosinase expression itself can not be satisfied with existing artificial compounds.

Accordingly, there is a need to develop a material for improving the appearance of white ginseng which is capable of exhibiting excellent effects while reducing the occurrence of such side effects.

SUMMARY OF THE INVENTION The present invention has been conceived to solve the above-mentioned problems, and an object of the present invention is to provide a novel peptide which is excellent in biostability and is excellent in white peel improvement.

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

In one embodiment, the peptide can promote melanin production.

In one embodiment, the peptide may facilitate microphthalmia-associated transcription factor (MITF) expression.

In one embodiment, the peptide may facilitate the expression of tyrosinase.

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

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

According to the present invention, since the peptide is small in size, it has excellent penetration of skin and excellent melanin production promoting effect, and thus it can replace the conventional material for improving white ginseng.

The peptide can be used not only for medical purposes for improving white mastication 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.

According to one aspect of the present invention, there is provided a peptide for the amelioration of canities consisting of the amino acid sequence of SEQ ID NO: 1. In addition, the peptide may be composed of the amino acid sequence of SEQ ID NO: 1, or may include the amino acid sequence as a part.

As a result of intensive efforts to discover peptides having a biologically active activity, the present inventors have found that the peptides exhibit a whitening effect and a particularly excellent melanin production promoting effect.

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 peptide may refer to peptides or fragments thereof exhibiting anti-waning 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 anti-waning effect of the peptides.

The term " canities " refers to a physiological aging phenomenon in which whitening or whitening of hair is progressed according to halting of melanin production. Usually, the above-mentioned white mold is found in middle-aged people of 30 to 40 or more, but in some cases, it is also found in 20's with various diseases such as autoimmune disease and hyperthyroidism.

In other words, it can be said that the white moth is caused by the stoppage or disorder of melanin, which is a coloring matter in the body.

The term " melanin " means a blackish brown pigment that functions to protect the skin and maintain body temperature by absorbing a certain amount of ultraviolet light. The melanin is synthesized and secreted in a melanocyte-forming melanocyte distributed in the basal layer of the dermis.

The synthesis of melanin is initiated by the participation of tyrosinase-based proteins such as tyrosinase, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2 / DCT).

The tyrosinase acts as an important catalyst in the oxidation of tyrosine to DOPA (3,4-dihydroxy phenylalanine) and the oxidation of dopa to DOPA-quinone. Further, the dopaquinone is naturally converted into dopachrome.

Then, the TRP2 (DCT) and TRP1 act as a catalyst to convert the dopachrome into indole-5,6-quinone-2-carboxylic acid (5-dihydroxy-indole- , 6-quinone-2-carboxylic acid) to form the final melanin pigment.

Meanwhile, the microphthalmia-associated transcription factor (MITF) is a factor that regulates transcription of tyrosinase, TRP1 and TRP2, and the overall melanin formation pathway can be regulated depending on the expression amount of MITF.

In contrast, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 can promote the production of melanin in the melanocyte by promoting the expression of the tyrosinase. In addition, the peptide may promote melanogenesis more fundamentally by promoting the expression of MITF.

The peptide is derived from a peptide that constitutes an in vivo growth factor and has a high molecular weight and a small size, and thus has a high skin permeability and thus has an excellent melanin production promoting effect.

Therefore, when the peptide is applied locally to the skin, an excellent pigment deficiency disease-improving effect such as improvement in white mastication can be realized.

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 the melanin production promoting 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 composition for improving erythropoietin comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

The above-mentioned peptides related to the meaning, cause of the occurrence, and improvement effect of the white moxibustion are as described above.

As the above-mentioned " comprising as an active ingredient ", for example, a degree of melanin formation in the melanocyte, which is capable of exhibiting a white mite improvement effect, more specifically an activity of promoting the expression of MITF and / or tyrosinase Quot; effective amount ".

Due to the melanin-promoting activity, the peptides can be used in various applications such as pharmaceutical compositions, cosmetic compositions, and external skin preparations. In particular, the pharmaceutical composition may be used for improving white mastication.

Since the composition contains a peptide having an excellent melanin production promoting effect, it can provide an excellent white peel improvement effect caused by a deficiency of a melanin pigment.

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, formic acid, wherein the organic acid is selected from the group consisting of acetic acid, acetic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4- toluenesulfonic acid, benzenesulfonic acid, salicylic acid, nicotinic acid, isonicotinic acid, Or aspartic acid, but are not limited thereto.

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.

Example: Preparation of peptides

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 activating agents [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 LYSHAVSGNG SEQ ID NO: 1

Experimental Example 1: Melanin production promoting effect test

The melanin production promoting effect of the peptides prepared according to the above examples was measured.

(Rosewell Park Menorial Institute) supplemented with 5% fetal bovine serum (FBS), 100 IU penicillin G and 0.2 μM TPA (12-O-tetradecanoyl phorbol-13-acetate) Melanocytes (melan-a) were plated on a 24-well plate at a concentration of 5x10 ⁴ cells / well.

After 24 hours, the cells were treated with different concentrations of the peptides of the above examples, treated with 0.1% DMSO (dimethyl sulfoxide) as a negative control and 100 μM IBMX (3-isobutyl-1-methylxanthine) as a positive control And then cultured at 37 ° C for 3 days.

Each of the cultured samples was washed with PBS (phosphate buffered saline) and 100 1 of 1N NaOH was added to dissolve the melanin in the cells. The absorbance of dissolved melanin was measured at 405 nm using a microplate reader.

The results are shown as relative values based on the negative control values, and the results are shown in Table 2 below.

division density Melanin production (%) Example
(Peptide) 1 mg / L 99 10 mg / L 129 50 mg / L 156 The negative control (DMSO) - 100 Positive control (IBMX) 100 μM 149

Referring to Table 2 above, the peptides of the above examples promoted melanin production in a concentration-dependent manner. In particular, when the concentration was 100 mg / L, the melanin production promoting effect was higher than that of the positive control group IBMX.

The results suggest that the peptide of SEQ ID NO: 1 can enhance melanin production by promoting melanin production in melanocytes.

Experimental Example 2: Tyrosinase activity promoting test

Stimulating activity of the tyrosinase activity by the peptides of the examples in the melanin-producing cells was measured. Cells were plated on a 60 mm plate at a concentration of 1.5x10 ⁵ cells / well, treated with 50 μg / ml of the peptide of the above example in each well, treated with 0.1% DMSO as a negative control and 100 μM IBMX as a positive control Respectively.

After incubation at 37 ° C for 48 hours, the cells were dissolved in 450 μl of dissolution buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) at 4 ° C for 20 minutes. 320 μl of supernatant and 80 μl of 10 mM L-DOPA were reacted at 37 ° C. for 30 minutes, and the absorbance of the resulting DOPA chromium was measured at 450 nm.

The activity increase rate of tyrosinase is shown in Table 3 as a relative value based on the results of the negative control group.

division density Tyrosinase activity (%) Example
(Peptide) 1 mg / L 158 10 mg / L 176 50 mg / L 189 The negative control (DMSO) - 100 Positive control (IBMX) 100 μM 174

Referring to Table 3, the peptide of the above example promotes the oxidation of L-DOPA to DOPA chromium in the melanin-producing cells, and the promoting effect is further improved as the concentration thereof is increased.

This result suggests that the peptide of SEQ ID NO: 1 not only promotes the expression of tyrosinase but also promotes its activity, thereby more effectively promoting the production of melanin.

Experimental Example 3: Test for promoting microphthalmia-associated transcription factor (MITF) and tyrosinase expression

The expression-promoting effect of the peptide of the above example on MITF and tyrosinase in melanocytes was measured.

The 501mel cell line was divided into 6-well plates at a concentration of ⁵ x 10 cells / well. The peptides of the above example were treated at a concentration of 50 mg / L in each well, and 0.1% DMSO as a negative control and 100 μM IBMX as a positive control After incubation, the protein was obtained after culturing at 37 DEG C for 24 hours and 48 hours.

For each of the above proteins, western blotting was performed using MITF and tyrosinase antibodies. At this time, protein extraction and Western blotting were routinely performed by standard methods used by those skilled in the art.

The results of Western blotting are shown in Table 4 as relative values based on the results of negative control.

time
(hr) MITF expression level (%) Tyrosinase expression level (%) Negative control group
(DMSO) Positive control group
(IBMX) Example
(Peptide) Negative control group
(DMSO) Positive control group
(IBMX) Example
(Peptide) 24 100 177 182 100 238 232 48 100 121 125 100 168 161

Referring to Table 4, the peptide of the above example promotes the expression of MITF and tyrosinase in melanocytes, and the promoting effect is further improved as the concentration thereof is increased.

In particular, it can be seen that the peptides can express both proteins at similar levels compared to the positive control, IBMX.

The results suggest that the peptides can promote melanin synthesis in melanocytes and can effectively improve white mastication.

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 HAVING CANITIES IMPROVEMENT EFFECT, AND USES THEREOF <130> 16PP11172 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> SEQ ID NO 1 <400> 1 Leu Tyr Ser His Ala Val Ser Gly Asn Gly   1 5 10

Claims (6)

A peptide for improving canities comprising the amino acid sequence of SEQ ID NO: 1. The method according to claim 1,
Peptides that promote melanin production. 3. The method of claim 2,
A peptide that promotes microphthalmia-associated transcription factor (MITF) expression. 3. The method of claim 2,
A peptide that promotes the expression of tyrosinase. A composition for improving white mastication comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. A composition for external application for skin comprising the peptide of any one of claims 1 to 4 as an active ingredient.