KR101900748B1 - Peptide exhibiting efficacies of hair growth, and uses thereof - Google Patents

Abstract

The present invention relates to peptides for improving skin condition such as wrinkles, improving hair loss, or promoting hair growth, and relates to peptides composed of a specific sequence and uses thereof.

Description

PEPTIDE EXHIBITING EFFICACIES OF HAIR GROWTH, AND USES THEREOF FIELD OF THE INVENTION The present invention relates to peptides having hair growth promoting effect,

The present invention relates to a novel peptide having excellent hair growth promoting effect.

Hair is an important part of the process of increasing interest in beauty as the age changes. Hair refers to a thin, exfoliated structure produced on the surface of the skin.

In addition to acting as a cushion against external impacts, hair also protects the body from external stimuli such as direct sunlight, cold, friction, danger, and discharges heavy metals such as arsenic, mercury and zinc harmful to the body. It is emphasized.

However, the number of patients who are alleged to have hair loss due to various causes including dietary change, internal and external stress, and the like are rapidly increasing.

Human hair has about 100,000 to 150,000 hairs, and each hair has a different growth period. The hair growth cycle consists of three stages, specifically anagen stage where hair grows most actively, catagen stage where hair starts to degenerate, and telogen (hair growth) stage.

In general, hair loss refers to the shortening of hair growth rate in the growing period and the increase of hair in the retrograde period or rest period in such a cycle, resulting in abnormal abundance of hair.

DKK-1 (Dickkopf-related protein 1) plays an important role in hair follicle growth inhibition and killing, among the factors expressed in hair follicles by male hormone, because male hormone plays a very important role in hair killing and elimination It is known.

On the other hand, regeneration of hair follicles and activation of stem cells must be performed for periodic hair growth, which is known to be caused by the production and activation of Wnt / β-catenin. In addition, DKK-1 mentioned above is known to be an antagonist of Wnt / [beta] -catenin, so that the above signal system is very important for controlling hair growth.

The herbal remedy for the treatment or prevention of alopecia that has been developed so far includes female hormone-based preparations such as minoxidil, finasteride, tricosaccharide, etc. for promoting blood circulation, enhancing hair follicle function, scalp moisturizing and male hormone suppression, (trichosaccharide), etc., but there are many limitations in use due to adverse effects such as sexual dysfunction, possibility of birth of deformed baby, and general hair growth.

Korean Patent Publication No. 10-2013-0015530

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a novel peptide having excellent biostability and excellent hair and skin condition improving effect.

According to one aspect of the present invention, there is provided a skin condition improving peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In one embodiment, the peptide is capable of promoting the growth of fibroblasts, keratinocytes, or hair follicle cells.

In one embodiment, the peptide is capable of inducing collagen biosynthesis of the dermal tissue.

In one embodiment, the peptide may facilitate the production of laminin or hyaluronic acid.

According to another aspect of the present invention, there is provided a composition for improving hair loss or promoting hair growth comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

According to another aspect of the present invention, there is provided an external preparation for skin comprising as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In one embodiment, the composition may be for improving wrinkles, improving skin elasticity, preventing skin aging, improving skin moisturization, or regenerating skin.

According to one aspect of the present invention, an excellent skin penetration rate can be realized by using a small-sized peptide, thereby improving the hair and skin condition improving effect.

The peptides can be used for beauty products such as medicines, quasi-drugs, health functional foods, and cosmetics due to their activities such as superior hair loss suppression, hair generation and wrinkle improvement.

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 skin condition improving peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In addition, the peptide 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 an effect of improving the skin condition such as the wrinkle improving effect of the peptide, and improving hair loss and promoting hair growth.

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 exhibiting hair loss improvement, hair growth promotion and wrinkle reduction.

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 promotes the growth of fibroblasts or keratinocytes to impart elasticity to the skin, thereby suppressing the formation of wrinkles. Furthermore, the peptide may promote hair follicle growth and induce hair growth.

The keratinocytes and fibroblasts in the skin may be damaged and degenerated 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 can 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 the growth of fibroblast and keratinocyte and promotes collagen synthesis, it can grow the stratum corneum, epithelial layer and dermal layer of the skin and improve wrinkles, skin elasticity, skin aging, skin moisturization, .

In addition, the peptide can promote the production of laminin or hyaluronic acid. Since the laminin and hyaluronic acid function together with the collagen to enhance intercellular binding, the skin elasticity can be enhanced by promoting the production of these.

On the other hand, dihydrogestosterone, a testosterone-converted substance, is known to induce androgenic alopecia.

The dihydrotestosterone binds to the androgen receptor present in the hair follicle cells and enters the dermal papilla cells. In this process, hair follicle cell destruction materials including TGF-beta, DKK-1 and BMP are produced to cause hair loss.

That is, it is possible to reduce the production amount of dihydrotestosterone, block the entry path into hair follicle cells, or achieve hair loss improvement or hair growth effect through regeneration of hair follicle cells.

Among these peptides, the peptide significantly inhibits the production of dihydrootestosterone and improves hair loss or hair growth through the action of growing hair follicle cells.

Therefore, when the peptide is applied locally to the skin, overall effects such as improvement of skin condition such as wrinkle improvement, improvement of hair loss, and promotion of hair growth can be simultaneously 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 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 composition for improving hair loss or promoting hair growth comprising, as an active ingredient, a peptide for improving skin condition comprising an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 / RTI >

The term " hair loss " refers to a state in which hair is normally broken at a site where hair should be present, and generally means that the hair of the scalp is missing. Unlike colorless and thick skin, the Virgin Mary can cause cosmetic problems when it is missing.

Hair loss can be classified into two types, which are clinically scarring and non-scarring. Hair loss (scarring) in which a scar is formed can not be regenerated because hair follicles are destroyed. However, hair loss without scarring Sexuality) is maintained hair follicles, hair is regenerated after the symptoms disappear.

The hair loss to which the composition is applied is selected from the group consisting of alopecia areata, alopecia totalis, alopecia universalis, androgenic alopecia, telogen effluvium, anagen effluvium, ), Or chemotherapy-induced alopecia. ≪ / RTI >

Currently, medicines such as minoxidil, pinastride, and hair transplantation are used for the treatment of hair loss, and in the case of round hair loss, a topical steroid preparation, a whole body steroid preparation, an immunotherapy and the like are used.

In connection with this, the peptide can promote the growth of hair follicle cells, so that it can fundamentally promote hair growth and effectively protect the growing hair. The action of the androgen receptor present in the hair follicle cells and the associated dihydrotestosterone is as described above.

The " improvement of hair loss " or " hair growth " may be used in a similar sense, and may have the same meaning as another term used in the art, promoting wool and promoting hair growth.

According to another aspect of the present invention, there is provided an external preparation for skin comprising as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

Specifically, the composition for external application for skin may be for improving wrinkles, improving skin elasticity, preventing skin aging, improving skin moisturizing, or regenerating skin.

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 the long peptide chain, 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 " means a composition capable of exhibiting an effect of improving hair loss, promoting hair growth, or improving skin such as inhibition of dihydrotestosterone related to hair growth effect, collagen synthesis associated with wrinkle- Quot; effective amount "

The peptides can be used for various purposes such as pharmaceutical composition, cosmetic composition, external preparation for skin due to improvement of hair loss, hair growth or skin condition improving activity.

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.

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 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 1 YSAFSLGYE SEQ ID NO: 1 Example 2 FSPNLWGLE SEQ ID NO: 2

Experimental Example 1: Cell Growth Effect Test

In order to analyze the similar effect and inhibitory effect of the growth factors on the peptides prepared according to Examples 1 and 2, they were measured using SRB (Sulforhodamine B) colorimetric method using HaCaT keratinocyte, NIH3T3 fibroblast, and HHFDPC hair follicle cells [Reference: Rizzino, et al. Cancer Res. 48: 4266 (1988).

The HaCaT keratinocyte, NIH3T3 fibroblast, and HHFDPC hair follicle cells were cultured in DMEM (Dulbecco's modied Eagle's medium) containing 10% fetal bovine serum (FBS) using a 250 ml tissue culture flask .

The cultured cell lines were detached from the bottom of the culture dish with 1% trypsin solution and centrifuged to collect only cell precipitates. The cells were suspended in DMEM culture medium without FBS and then cultured in a 96-well tissue culture plate at 3 × 10 ³ cells / well for 24 hours at 37 ° C. and 5% CO ₂ .

After 24 hours, the medium was replaced with the same culture medium except for serum. For the standard setting, the blank sample and the synthetic peptide were dissolved in 10% DMSO in a sterilized state, and cultured at the concentration of 10 mg / L for 72 hours under the same conditions as above.

After the culture was completed, the supernatant was removed, cells were fixed with ethanol, and then washed three times with PBS (phosphate buffer saline). After removing the washing solution, the cells were treated with a colorimetric SRB solution. After washing with 1% acetic acid, the cells were observed under a microscope to observe the viable cell state. At this time, the absorbance at a wavelength of 590 nm was measured and the viability of the cells was measured.

That is, the cell proliferation rate of each of the cells treated with the peptides of Examples 1 and 2 and the cells without any treatment (negative control) was compared, and the relative values thereof were shown in Table 2 below.

division HaCaT keratinocytes NIH3T3 fibroblast HHFDPC hair follicle cells Cell proliferation rate
(%) Example 1 Example 2 Control group Example 1 Example 2 Control group Example 1 Example 2 Control group 134 141 100 143 139 100 151 155 100

Referring to Table 2, the peptides composed of the amino acid sequences of Examples 1 and 2 significantly improved the growth of keratinocytes, fibroblasts, and hair follicle cells, and more than 1.5 times in the case of hair follicular cells.

The above results suggest that the peptide composed of the amino acid sequence of SEQ ID NO: 1 or 2 can improve the skin condition improving effect such as wrinkle improvement and can achieve superior hair loss improving or hair producing effect.

Experimental Example 2: 5 alpha-reductase inhibitory activity test

The inhibitory activity of the peptide of the above example on 5 alpha-reductase (5 alpha-reductase), which is known to be the main cause of hair loss, was evaluated.

Rat dermal papilla cells (DPCs) were divided into 24 well plates at a concentration of ⁵ x ¹⁰ cells / well and cultured for 24 hours. Then, the peptides of Examples 1 and 2 were treated with 10 mg / L and 50 mg / L Each well was treated and stimulated with testosterone at a concentration of 50 nM after 1 hour. The supernatant was separated from the cultured solution to obtain DPCs pellets.

In the following procedure, the concentration of dihydrotestosterone (DHT) was measured using an enzyme-linked immunosorbent assay (ELISA).

Rabbit anti-DHT antibody-coated DHT ELISA 100 쨉 l of the DPCs pellet was dispensed into microtiter strips, and 25 쨉 l of DHT antiserum was dispensed into each well and reacted at 2-8 째 C for 15-20 hours, followed by washing three times Respectively.

100 μl of DHT-HRP enzyme conjugate was added to each well, and reacted at room temperature for 30 minutes on shaker, followed by washing three times. 100 μl of the substrate was dispensed, reacted at room temperature for 20 to 30 minutes, treated with 100 μl of Stop solution, and the absorbance at 450 nm was measured to show the amount of DHT produced. The amount of DHT produced relative to the control without peptide treatment was calculated as inhibition (%), and the results are shown in Table 3 below.

division Sample concentration (mg / L) 5 Alpha-Reductase Inhibition (%) Example 1 0 0 One 11 10 44 50 71 Example 2 0 0 One 14 10 41 50 74

Referring to Table 3, it can be seen that the medium treated with the peptides of Examples 1 and 2 showed an increase in 5-alpha-reductase inhibition rate in a concentration-dependent manner, as compared with the control group in which no peptide was treated.

That is, the above results suggest that the peptides of Examples 1 and 2 inhibit the production of dihydrotestosterone and inhibit the activity of 5-alpha-reductase, thereby achieving excellent hair loss improvement or hair growth effect.

Experimental Example 3: Estimation test of hair growth function

In order to evaluate the hair growth function of the peptides prepared according to Examples 1 and 2, the following clinical trials were conducted on adult males and females having hair loss symptoms.

A total of 20 adult men and women aged 30 to 60 years (15 males and 5 females) were divided into two groups and a 5% minoxidil product and a liquid tonic prepared with the composition of Experimental Example 1 were used once per day 5 ml) was applied to hair loss site for 3 months, and the result was observed.

The results were as follows: 1) Average daily amount of hair loss, visual acuity, and subjective symptom improvement were evaluated by 5 steps. At this time, each rating scale was set as 5 points and each grade was set as 1 point unit. The specific evaluation criteria are shown in Table 4 below, and the evaluation results are shown in Table 5 below.

score Visual judgment Subjective symptoms Amount of hair loss One worse worse increase 2 maintain maintain maintain 3 Slight improvement Slight improvement Small amount reduction 4 Normal improvement Normal improvement Medium reduction 5 Significantly improved Significantly improved Mass reduction

division Visual judgment Subjective symptoms Amount of hair loss Overall improvement Example 1 4.2 3.8 4.2 4.06 Example 2 4.1 3.7 4.4 4.06 Minoxidil 5% product 4.2 3.9 4.2 4.10

Referring to Table 5, the compositions containing the peptides according to Examples 1 and 2 were equivalent to those of the control group, minoxidil, in terms of visual determination, subjective symptoms, and hair loss on curing.

These results suggest that the peptides according to Examples 1 and 2 can exert an excellent effect on the hair growth promoting function as compared with the products currently available on the market and improve the overall satisfaction of the user.

Experimental Example 4: Test for inhibiting prostaglandin E2 (PGE2)

Since the inhibition of the activity of prostaglandin E2 (PGE2) can prevent hair loss due to inflammation, the inhibitory effect of the peptides prepared according to Examples 1 and 2 on the production of prostaglandin PGE2 was confirmed. Prostaglandin E2 EIA Kit Were used.

RAW 264.7 cells were treated with 0.1%, 0.1, 1, 10 and 50 mg / L of the peptides of Examples 1 and 2 for 24 hours, respectively. Cell culture supernatants were treated with goat anti-mouse 96 50 占 퐇 of the first antibody solution and 50 占 퐇 of PGE2 conjugate were added thereto, followed by reaction at 4 占 폚 for 18 hours. After washing with washing solution 5 times, Ellman's reagent was treated with 200 쨉 l of each solution, reacted for 60 minutes with stirring, and absorbance was measured at 405-420 nm. The results are shown in Table 6 below. At this time, the value was expressed as a relative value (%) relative to the amount of PGE2 produced in the negative control to which no peptide was added.

division Peptide concentration (mg / L) PGE2 production rate (%) Example 1 0 100 One 95 10 72 50 45 Example 2 0 100 One 93 10 68 50 39

Referring to Table 6, it can be seen that the amount of PGE2 produced by LPS stimulation is inhibited by the peptides of Examples 1 and 2, and the inhibitory effect is enhanced in a concentration-dependent manner, And 45% and 39%, respectively, in Examples 1 and 2, respectively.

These results suggest that the peptides of Examples 1 and 2 can effectively inhibit the inflammation caused by prostaglandin and thus exhibit excellent hair loss improving effect.

Experimental Example 5: 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 quantified 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 group (100%). The measurement results are shown in Table 7 below.

division Collagen production (%) Example 1 187.6 Example 2 192.9

 [% of control]

The above results suggest that the peptide composed of the amino acid sequence of the above Example is excellent in not only the hair growth promoting effect but also the skin elasticity improvement and skin wrinkle improving effect.

Experimental Example 6: Test for promoting the production of laminin and hyaluronic acid

HaCaT cells cultured for 48 hours were treated with the peptides of Examples 1 and 2, 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 Examples 1 and 2 and HaCaT cells (negative control) without any treatment were compared, and the relative values thereof are shown in Table 8 below.

division Laminin Hyaluronic acid density
(%) Example 1 Example 2 Control group Example 1 Example 2 Control group 165 167 100 151 149 100

Referring to Table 8, it can be seen that both the laminin and hyaluronic acid concentrations of the peptides composed of the amino acid sequence of SEQ ID NO: 1 or 2 (Examples 1 and 2) were significantly increased compared with the negative control.

The above results suggest that the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2 exhibits excellent wrinkle-reducing effect and thus can improve the overall skin condition.

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 EXHIBITING EFFICACIES OF HAIR GROWTH, AND USES THEREOF <130> 16PP11170 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> SEQ ID NO 1 <400> 1 Tyr Ser Ala Phe Ser Leu Gly Tyr Glu   1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> SEQ ID NO 2 <400> 2 Phe Ser Pro Asn Leu Trp Gly Leu Glu   1 5

Claims (7)

A peptide for improving hair loss and promoting hair growth comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The method according to claim 1,
A peptide that promotes the growth of fibroblasts, keratinocytes, or hair follicle cells. 3. The method of claim 2,
Peptides that induce collagen biosynthesis of skin tissue. The method according to claim 1,
Peptides that promote the production of laminin or hyaluronic acid. A composition for improving hair loss and promoting hair growth comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. A composition for external application for improving skin wrinkles comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. delete