KR101451340B1 - Composition for hair-growth - Google Patents

Abstract

The present invention relates to a composition for promoting hair growth, in vitro (in In vitro , it was confirmed that hair growth could be positively influenced by increasing the growth of hair cells in the skin. As a result, it was confirmed that the animal hair test showed a substantially effective effect on hair growth.

Description

[Composition for hair-growth]

The present invention relates to a composition for promoting hair growth, and more particularly, to a composition for promoting hair growth using a protein as a main raw material.

Recently, as the interest in the appearance has increased and the desire to buy young has increased, there has been an increasing interest in the hair which has not been directly related to life and has been relatively neglected. People living in the modern age are suffering from a lot of alopecia due to environmental factors such as air pollution, mental stress, excessive use of hair cosmetic products, and the age at which hair loss occurs is gradually decreasing.

The causes of hair loss include genetic factors, progression of aging, medication, radiation therapy, and post-mild stress. From the biochemical and physiological point of view, the main causes of hair loss are scalp blood circulation and nutrients essential for hair metabolism Deficiency and the like.

These factors may cause hair loss alone, but they can be combined with each other to accelerate hair loss and make the symptoms worse. In particular, genetic factors or acquired special conditions can lead to premature baldness when the production, change, recognition, and signal transduction of male hormones are overactive and tend to prematurely age the hair follicles.

The surgical methods of baldness caused by the above factors include scalp flap, reduction of hair loss area, scalp surgery by tissue expanding, and autologous hair transplantation. Surgical methods, however, It has a disadvantage that it is difficult to be widely used because of its great economic and psychological burden.

On the other hand, there are methods of treatment using medicines as medicinal treatment methods. Baldness medicines currently sold on the market have little or no effect on them, and many medicines have not been proven. Rather, there are problems in causing serious side effects to the human body There are a lot. As a typical external drug currently on the market, Minoxidil, which is approved by the US FDA, has a capillary vasodilating function and has an effect of preventing hair loss by applying to the scalp. However, it has to be applied twice daily, and the medicine should stay on the scalp for 3 to 4 hours, which is inconvenient not only to show some effect but also to have a slight effect in terms of its effect, itching and irritation .

Korean Patent Publication No. 10-2011-0006212 (published on Jan. 20, 2011) discloses "copper-peptide-containing low temperature nano extract hair growth accelerating agent and method for producing the same ", and more specifically to copper- Containing nano-extracts, copper-peptide, salicylic acid, bamboo extract, manganese bark extract, waxy extract, waxy extract and waxy extract extracted at a temperature of from 15 to 60 캜 at a nano size of 1000 to 3000 Å using a mixed solvent of an organic solvent and water A nano extract extract containing copper-peptides containing kelp extract and a method for producing the same ". Since the nano-sized hair growth promoting agent is well absorbed by hair follicles and is useful for hair loss prevention, wool, hair growth, hair growth, and copper-peptide, herbal medicine and algae as raw materials, And it is said that it can overcome the shedding phenomenon.

The present invention is to develop and provide a hair growth inhibitor composition prepared by compounding proteins in order to substantially assist hair growth.

The fusion protein PEP-1-EGF formed by fusion of PEP-1 having the amino acid sequence of SEQ ID NO: 2 with EGF having the amino acid sequence of SEQ ID NO: 4, PEP-1 having the amino acid sequence of SEQ ID NO: 2, 6 fusion protein PEP-1-FGF1 formed by fusing FGF1 having the amino acid sequence of SEQ ID NO: 2, PEP-1 having the amino acid sequence of SEQ ID NO: 2 and VEGF having amino acid sequence of SEQ ID NO: VEGF, a fusion protein PEP-1-SOD1 formed by fusion of PEP-1 having the amino acid sequence of SEQ ID NO: 2 with SOD1 having the amino acid sequence of SEQ ID NO: 10, and Glycyl-Histidyl-Lysine A composition for accelerating hair growth, which comprises Cu-peptide in which copper is chelated and bound as an active ingredient. The PEP-1-FGF1 is, for example, an amino acid sequence of SEQ ID NO: 14, and the PEP-1-VEGF is, for example, an amino acid sequence of SEQ ID NO: And PEP-1-SOD1 may be, for example, the amino acid sequence of SEQ ID NO: 18.

Meanwhile, the composition of the present invention preferably contains 5 to 15 μg / mL of each of the elongated PEP-1-EGF, PEP-1-FGF1, PEP-1-VEGF, PEP-1-SOD1 and Cu- .

Meanwhile, the composition of the present invention may be, for example, an external preparation or a cosmetic composition.

Hereinafter, the present invention will be described in more detail below.

EGF used in the present invention is an abbreviation of "epidermal growth factor" and is a growth promoting factor for promoting cell growth. FGF1 used in the present invention is an abbreviation of "fibroblast growth factor 1" and is a fibroblast growth factor. VEGF used in the present invention is an abbreviation of "vascular endothelial growth factor" and is a vascular endothelial growth factor. SOD1 used in the present invention is an abbreviation of "superoxide dismutase 1" and is an antioxidant enzyme.

In the present invention, PEP-1 is fused to EGF, FGF1, VEGF and SOD1. PEP-1 is a type of PTD (protein transduction domain), which is bound together with a protein, It is important to understand that In the present invention, PEP-1 having such a role was used by fusion to the EFG, FGF1, VEGF and SOD1 proteins.

Meanwhile, the present invention includes a Cu-peptide. It is known that it regenerates damaged proteins and changes damaged tissues into normal tissues to exert skin tissue regeneration effect, which is effective for skin regeneration and hair regeneration of hair follicles. The increase in hair follicle size and hair growth rate due to the administration of the copper-peptides provides adequate nutrition to the hair follicles through the change of blood flow, allowing the hair with thick hairy to grow rapidly.

The copper-peptide (Cu-GHK), which is chelated with copper on the Glycyl-Histidyl-Lysine, is a very rare sequence extracted from proteins in the human body and mainly protects tissues and prevents inflammation due to tissue damage. And it plays a role of regenerating damaged protein and replacing damaged tissue with normal tissue, and it is known to promote hair growth.

Meanwhile, the composition for promoting hair growth of the present invention may be an external preparation or a cosmetic composition.

In the case of a composition for external application, there is no particular limitation in the formulation, and for example, it may have a formulation of lotion, ointment, gel, cream, patch or spray. Further, in the external preparation for each formulation, components other than the protein of the present invention can be mixed and selected without difficulty by those skilled in the art depending on the formulation of the external preparation or the purpose of use.

In the case of a cosmetic composition, the formulation is preferably any one selected from hair tonics, hair lotions, hair creams, hair sprays, hair mousses, hair gels, hair conditioners, hair shampoos, hair rinses, hair packs and hair treatments. It may also optionally be applied to the skin in the form of an aerosol, or it may be in solid form, e.g., in the form of a stick. In addition, the cosmetic composition according to the present invention can be used as an adjuvant in cosmetics such as a hydrophilic or lipophilic gelling agent, a hydrophilic or lipophilic active agent, a preservative, an antioxidant, a solvent, a perfume, a filler, ≪ / RTI > The amount of these various adjuvants is an amount commonly used in the art, for example, 0.01 to 20% by weight based on the total weight of the composition. In any case, the adjuvants and their proportions will be chosen so as not to adversely affect the desired properties of the cosmetic composition according to the invention.

Hair growth promoting composition of the present invention in vitro (in In vitro , it was confirmed that hair growth could be positively influenced by increasing the growth of hair cells in the skin. As a result, it was confirmed that the animal hair test showed a substantially effective effect on hair growth.

1 is a schematic diagram of a protein expression vector used in the present invention.
Fig. 2 shows the result of SDS-PAGE analysis of the expressed protein. 1: PEP-1-SOD1 expression, 5: PEP-1-EGF expression, 3: PEP-1-FGF1 expression, VEGF expression.
Figure 3 is a Western blot analysis of the expressed protein.
FIG. 4 is a graph showing the proliferation results of PEP-1-EGF, PEP-1-FGF1 and PEP-1-VEGF.
FIG. 5 shows Western-blot analysis results confirming the expression of phosphorylated-p42 / 44 by protein treatment of each of the present invention. M: size ladder, -: untreated protein (Non-protein treatment), +: protein processing, 1 ^st Ab: phosphorylated -Erk1 / 2, 2 ^nd Ab: anti- and fixes this IgG-HRP.
Figure 6 shows the change in body weight for each sample after application of the test group samples.
Figure 7 shows the visual changes of the wool of each sample after application of the test group samples.
Figure 8 shows quantitative changes in wool for each sample after application of the test group samples.
Figure 9 shows skin color change for each sample after application of the test group samples.
Figure 10 shows the change in hair length for each sample after application of the test group samples.
Figure 11 shows the histological changes of the wool of each sample after application of the test group samples.
Fig. 12 shows the result of visually confirming the effect after applying the composition of the present invention.
Fig. 13 shows the results of calculating the area of the skin area of the dorsal hair after application of the composition of the present invention.
14 is a photograph showing histological observation results after application of the composition of the present invention. H & E dyeing. x100.
Fig. 15 shows the result of measuring the number of cells of hair follicular cells after applying the composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Example  1: Production and in vitro test of proteins which are active ingredients of hair growth promoter ( in vitro test)]

(One) PEP -One- EGF , PEP -One- FGF1 , PEP -One- VEGF  , PEP -One- SOD1  Production of expression vector

EGF, FGF1, VEGF and SOD1 genes were amplified by PCR using primers and templates shown in Table 1 below.

Gene name Forward primer Reverse anti-primer Template EGF aatagtgactctgaatgt ttagcgcagttcccacca cDNA Library, Human Liver (Takara # 9505) FGF1 atgtttaatctgcctcct atcagaagagactggcag HS68 cells RNA VEGF atgctcgaggcaccca tcaccgcctcggcttgtc cDNA Library, Human Liver (Takara # 9505) SDO1 atggcgacgaaggccgtg ttattgggcgatcccaat cDNA Library, Human Liver (Takara # 9505)

The gene sequences of the amplification products were SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, respectively. The amino acid sequence of the protein encoded by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9 was SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO:

The amplified PCR product was then cloned into the promega pGEM-T easy vector. Then, the EGF, FGF1, VEGF and SOD1 genes were ligated to an oligomer (SEQ ID NO: 1) capable of synthesizing a PEP-1 peptide (SEQ ID NO: 2) and amplified again to obtain a PCR product. The gene sequences of the amplification products PEP-1-EGF, PEP-1-FGF1, PEP-1-VEGF and PEP-1-SOD1 were SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: The amino acid sequences of the proteins encoded by SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17 were SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18, respectively.

After obtaining the PCR product, the amplified PCR product and the expression vector were cut using a restriction enzyme, and the two were cloned. A schematic diagram of each protein expression gene vector is shown in FIG. Gene cloned into the gene expression vector was transformed to determine the gene by DNA sequencing in the final E. col i BL21 (DE3) and Rosetta gami2 (DE3) Com peteon bit cells (competent cell) to obtain an end-clone.

 (2) Production of recombinant protein

The transformed bacterial cells were inoculated into the 5 ml LB + Ampicillin medium overnight. The following morning, 100 ml of fresh LB + ampicillin medium was inoculated at 1% and cultured at 37 DEG C with shaking at 200 rpm. After 3 hours, it was measured every 20 minutes at OD ₆₀₀ , and when it reached 0.6, 1 mM IPTG was added to overexpress. At this time, the temperature of the shaking incubator was lowered to 25 째 C and further cultured, and cultured for 24 hours. The overexpressed cells were collected and disrupted using ultrasonic wave intensity, and then confirmed by SDS-PAGE to examine the degree of protein expression (Figure 2)

As shown in Fig. 2, in the case of the compent cell in which the expression vector was not inserted, the over expressed protein did not appear, and in the case of the compontent cell into which the expression vector was inserted, the over expressed protein appeared in the respective protein sizes. As a result, it was found that the inserted protein was well expressed.

On the other hand, a Western-blot test method was used to identify over-expressed proteins. Western-blot confirmed protein expression by identifying the 6x histidine protein inserted in the vector. The same sample of the protein analyzed by SDS-PAGE was subjected to electrophoresis, the protein was transferred to a nitrocellulose membrane, blocked with 5% skim milk, Mouse IgG antibody was used as a primary antibody and mouse anti-His tag mAb IgG was used as a primary antibody and a goat anti-mouse IgG-HRG antibody was used as a secondary antibody Western blot analysis of protein expression was performed.

As a result of the experiment, it was confirmed that the target proteins expressed on SDS-PAGE were expressed with histidine binding as shown in the result of FIG. 3, and it was confirmed that the target proteins were well generated.

 (3) PEP -One- EGF , PEP -One- FGF1 , PEP -One- VEGF , PEP -One- SOD1  Identification of protein activity and cell proliferation ability

The activity of the PEP-1-SOD1 protein was measured using a SOD1 activity assay kit from Abcam (USA). The test was carried out according to the test method described in the kit. The activity units of each protein were expressed in units / mg units.

The activity of the proteins of PEP-1-EGF, PEP-1-FGF1 and PEP-1-VEGF was measured using Takara's WST assay kit. The test was carried out according to the test method described in the kit. The activity of PEP-1-EGF, PEP-1-FGF1 and PEP-1-VEGF was measured by using cell proliferation ability. The ED ₅₀ value of the growth rate was converted into 1 unit and converted into units per mg.

PEP-1-EGF, PEP-1-FGF1, mouse fibroblast cell, NIH3T3 cell line, PEP-1-VEGF, human vascular endothelial cell line HUVEC cell line, and 1% ). ≪ / RTI > Cells were treated with each concentration, and after 24 hours, the amount of cells was graphed using a Takara WST assay kit and the ED ₅₀ Respectively. The activity of each of the measured proteins is shown in Tables 2 and 3.

Growth factor activity (PEP1-EGF, PEP1-FGF, PEP1-VEGF activity) Growth Factor Experimental I.U. ED ₅₀ density PEP1-EGF 1 x 10 < ⁶ > U / mg 1 ng / ml PEP1-FGF1 2.7 x 10 < ⁶ > U / mg 0.37 ng / ml PEP1-VEGF 1 x 10 < ⁵ > U / mg 10 ng / ml IU Definition: Concentration indicating ED ₅₀ in cell proliferation experiments

PEP1-SOD1 activity result S.U. Protein concentration PEP1-SOD1 1421 U / mg 0.44 mg / ml

The results of the experiments (Table 2 and Table 3) confirmed that PEP-1-SOD1 exhibited 1421 U / mg of activity. On the other hand, PEP-1-EGF and PEP-1-FGF1 showed ED ₅₀ values of less than 1 ng / ml, whereas PEP-1-VEGF exhibited ED ₅₀ (Fig. 4).

(4) PEP -One- EGF , PEP -One- FGF1 , PEP -One- VEGF , Cu - The peptide  Effect on cell proliferation signaling

In order to evaluate the effect of PEP-1-EGF, PEP-1-FGF1 and PEP-1-VEGF on signal transduction of cell proliferation factors, p42 / 44 of mitogen-activated protein kinase (MAPK) Western blot tests were performed on the phospho-form of the Erk protein.

In the case of the p42 / 44 protein, it is known as a protein that promotes cell growth. For the experiment, each cell was cultured for 24 hours in a 1% serum. After 10 minutes of treatment with each protein, all cells were sampled by SDS-PAGE sample buffer and subjected to SDS-PAGE and Western blotting . Mouse anti-phospho-p42 / p44 IgG antibody was used as the primary antibody and Goat anti-mouse IgG HRP antibody was used as the secondary antibody.

As shown in FIG. 5, it was confirmed that Erk signal was increased by PEP-1-EGF, PEP-1-FGF1 and PEP-1-VEGF treatment. At this time, in the case of VEGF, the Erk signal of the NIH3T3 cell, an epidermal fibroblast cell, was not affected.

This result suggests that PEP-1-EGF, PEP-1-FGF1, and PEP-1-VEGF increase the growth of hair cells in the skin by Erk signaling that affects cell growth. This means that it can have a positive effect on the hair growth.

[ Example  2: search for the optimal blending ratio of the candidate hair growth agent]

In this example, a growth factor candidate GFM (a mixture of EP-1-EGF, PEP-1-FGF1 and PEP-1-VEGF in a uniform ratio), PEP-1-SOD1 And Cu-peptide.

 (1) Test method

7 week old male C57BL / 6 mice whose skin color was white or pink were used. When the test animals were introduced, they were used for the experiment after a purifying period of about one week, and maintained four cages per cage. Each group was labeled with an ear puncture and a stationary pen. Animal chambers were in the condition of temperature 20 ~ 26 ℃, humidity 40 ~ 70%, light condition 12 hours light, 12 hours dark. Feed and water were free, Animal experiments were conducted in compliance with the IACUC regulation of Hallym University and animal experiment regulations.

Test groups were negative control (drug dilution solvent), positive control (5% minoxidil, MXD), and each combination of drug groups (see Table 4 below).

Test group samples group Marie Drug group Administration volume Route of administration Number of doses (except weekends) G1 12 Vehicle
20 mM Tris-10%
glycerol (pH 8.0)
buffer 150 μl Skin application Once a day G2 12 The positive control (5% MXD) 150 μl Skin application Once a day G3 12 Growth Factor Mix (GFM) 150 μl Skin application Once a day G4 12 PEP-1-SOD1 150 μl Skin application Once a day G5 12 Cu-peptide 150 μl Skin application Once a day G6 12 GFM + PEP-1-SOD1 150 μl Skin application Once a day G7 12 GFM + Cu-peptide 150 μl Skin application Once a day G8 12 PEP-1-SOD1 + Cu-peptide 150 μl Skin application Once a day

Each test group consisted of 12 mice for photographing and follicular observation. After shaving, the hairs of mice and the like were completely removed using cyanoacrylate, and the test material was applied to the depilated area once a day for 3 weeks. During the test period, if possible, the mouse was epilated as far back as possible to prevent licking of the test material on the back. The drug was treated with a total volume of 150 μl once a day. The drug was administered 15 times for 3 weeks, excluding Saturdays and Sundays.

 (2) Observations

The effects of drugs on skin irritation after epilation were compared.

In addition, visual observation of the hair growth and photographing (6 rats) were carried out twice a week.

In addition, tissue extracts were performed once a week for histologic observation (0 day, 7 day, 14 day, 21 day difference) of hair follicles, and compared with normal hair follicles by treatment group.

In addition, the length of hair was measured on the 14th day and the 21st day, and the length of the hair per drug was measured using a microscope by 2 mice (20 per mouse) per each experimental group.

 (3) Analysis of results

The ratio of the area where the hair grows in the total area (total area of the abrasion area to the black area) was calculated.

After one week of epilation, changes in the skin color of the experimental animals were measured. The score for skin color is 1-10. 0: light pink skin, 1-5: hazy gray skin increase, 6-9: black skin increase, but not perfect black skin, 10: black skin all over.

In addition, skin histology was performed once a week, four times (0, 7, 14, 21 days) including the start date of the test, and the condition of the hair follicles was evaluated.

 (4) Results of the test

1) Measurement of change in body weight

The result of the test was as shown in FIG. 6. The body weight of C57BL / 6 mice gradually increased with the test period, and there was no significant difference between the test substance administration group and the control group.

2) Visual change of wool

The visual change of the wool was shown in Fig. After 7 days of hair loss, the skin color was changed to darker color than the 5% MXD positive control group in GFM treatment group. After 10 days, it was confirmed that hair growth started, and hair growth in GFM treated group was dominant. In addition, after 14 days, it was confirmed that the hair growth was faster in GFM, GFM + PEP-1-SOD1 and PEP-1-SOD1 + Cu-peptide treated groups. After 17 days, it was confirmed that hair growth was much progressed except PEP1-SOD1, Cu-peptide and GFM + Cu-peptide treatment group.

  3) Quantitative change of wool

For the quantitative measurement of wool area, the density was measured using image J software. As shown in FIG. 8, the GFM treatment group showed the best effect and GFM + PEP-1-SOD1, The PEP1-SOD1 + Cu-peptide-treated group had a dominant effect over the PEP-1-SOD1, Cu-peptide, and GFM + Cu-peptide groups.

4) Change in skin color

As shown in FIG. 9, the C57BL / 6 mouse had a skin color after pinking, gray, and black after hair loss, and then the skin color . GFM, GFM + PEP-1-SOD1, and PEP-1-SOD1 + Cu-peptide treatment groups.

5) Measurement of length of hair

On the 14th day and 21st day after hair loss, 10 hair pieces per mouse were randomly selected from the mice, and the total length of the hair was measured using a microscope (Olympus) length measuring program (cellsens, Olympus) We could not find a big difference in the length.

6) Histological changes in wool

As shown in FIG. 11, GFM, GFM + PEP-1, and GFM + PEP-1 were observed after the test material was applied and examined by autopsy to investigate the effect of the test substance on the growth of hair follicle and hairy root of the skin. 1-SOD1, and PEP-1-SOD1 + Cu-peptide treated group, and the location of the hair follicle was also rapidly shifted to the dermis from the fat cell layer,

[ Example  3: Preparation and efficacy of the hair growth inhibitor composition of the present invention]

In order to examine the hair growth effect of the composition of the present invention, an animal test was conducted as follows. The test materials were prepared as follows based on the results of Example 2 above. PEP-1-FGF1 of SEQ ID NO: 14, PEP-1-VEGF of SEQ ID NO: 16, PEP-1-SOD1 of SEQ ID NO: 18, and PEP- The Cu-peptide (Cu-GHK) was mixed with 20 mM Tris-10% glycerol at a concentration of 10 μg / ml to prepare a test substance. At this time, only 20 mM Tris-10% glycerol buffer was administered to the control. 6-week-old C57BL / 6 mice were purchased from Coatec Co., Ltd., and the test was started at 7 weeks of age through a weekly purging period.

 (1) Visual observation

The test animals were lightly anesthetized using Solarell and rumpun, and then the dorsal hair was removed using a commercially available hair removal agent, the above test material was applied twice a day, and the hair was massaged for 20 and 30 seconds. Photographs were taken at 1, 5, 7, 10, and 15 days after the start of the test to confirm the gross hair growth state, and the test results are shown in Fig.

As shown in the results of FIG. 12, black individuals were observed in the test group on the dorsal side from the 5th day after the application of the test material, and hair cells were found to grow faster than the control group on the 7th day from the control group. On the 10th day and 15th day, it was obvious that the hair growth was different by eyes.

In this experiment, it was confirmed that the growth of hair follicle cells occurs rapidly by applying the test substance. From these results, it was confirmed from the result of animal test that the test substance helps hair growth.

On the other hand, we can see that the dorsal skin changes to black color as follicular cells grow from the inside of the skin. To determine the area of black color on the dorsal side of the 7th and 10th day of tea, After cutting 3 cm in length and width, the optical microscope program Axiovision Rel. The area of the black area of the skin was calculated using a 4.6 (Zeizz, Greman) automated image measurement program and expressed in pixels.

As shown in Fig. 13, the area of the black part of the dorsal skin of the 7th and 10th day tea was increased with increasing date, and the group to which the test substance was applied was significantly increased .

 (2) histological observation

For histological observation of the hair growth state, the following tests were performed. The skin of the animal was sampled on the 1st, 5th, 7th, 10th, 12th, and 15th days of the test, and 4% para After fixing with paraformaldehyde, it was paraffin treated with an automatic tissue processor (Leica, German) and paraffin block samples were made with a paraffin waxer (Leica, German). The paraffin block was sampled with a microtissue slicer (Leica, German) at 7 μm. Paraffin slice samples were stained with H & E (Hematoxylin & Eosin). After removing paraffin from xylin, it was treated with EtOH series (100%, 95%, 90%, 80%, 70%, 50%) and stained with hematoxylin and eosin After dehydration with EtOH series, cover slides were mounted and observed with an optical microscope (Zeizz, German).

As a result, hair follicle cells were observed to grow in the epidermis at the 7th day as shown in FIG. 14, and the number of cells and mature hair follicle cells I could.

From day 10, it was observed that the number of hair follicle cells was about twice that of the hair follicles, and that the cells formed in the epidermal part fell down to the dermis and the hair was grown. On days 12 and 15, the number of cells and the degree of mature cells were significantly different.

As shown in FIG. 15, the number of hair follicle cells was counted. As a result, it was found that the number of cells was significantly increased in the test group from 7 days to 10 days, there was.

These results indicate that PEP-1-EGF, PEP-1-FGF1, PEP-1-VEGF, PEP-1-SOD1 and Cu-peptide rapidly restore growth and maturation of hair cells, And it is anatomically confirmed that the test substance of the present invention helps hair growth.

&Lt; 110 > ADbiotech Co., Ltd. <120> Composition for hair-growth <130> AP-2012-0114 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 63 <212> DNA <213> homo sapiens sapiens <400> 1 aaagaaacct ggtgggaaac ctggtggacc gaatggtctc agccgaaaaa aaaacgtaaa 60 gtg 63 <210> 2 <211> 21 <212> PRT <213> homo sapiens sapiens <400> 2 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val              20 <210> 3 <211> 159 <212> DNA <213> homo sapiens sapiens <400> 3 aatagtgact ctgaatgtcc cctgtcccac gatgggtact gcctccatga tggtgtgtgc 60 atgtatattg aagcattgga caagtatgca tgcaactgtg ttgttggcta catcggggag 120 cgatgtcagt accgagacct gaagtggtgg gaactgcgc 159 <210> 4 <211> 53 <212> PRT <213> homo sapiens sapiens <400> 4 Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His   1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn              20 25 30 Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys          35 40 45 Trp Trp Glu Leu Arg      50 <210> 5 <211> 423 <212> DNA <213> homo sapiens sapiens <400> 5 atgtttaatc tgcctccagg gaattacaag aagcccaaac tcctctactg tagcaacggg 60 ggccacttcc tgaggatcct tccggatggc acagtggatg ggacaaggga caggagcgac 120 cagcacattc agctgcagct cagtgcggaa agcgtggggg aggtgtatat aaagagtacc 180 gagactggcc agtacttggc catggacacc gacgggcttt tatacggctc acagacacca 240 aatgaggaat gtttgttcct ggaaaggctg gaggagaacc attacaacac ctatatatcc 300 aagaagcatg cagagaagaa ttggtttgtt ggcctcaaga agaatgggag ctgcaaacgc 360 ggtcctcgga ctcactatgg ccagaaagca atcttgtttc tccccctgcc agtctcttct 420 gat 423 <210> 6 <211> 141 <212> PRT <213> homo sapiens sapiens <400> 6 Met Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr   1 5 10 15 Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val              20 25 30 Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser          35 40 45 Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln      50 55 60 Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro  65 70 75 80 Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn                  85 90 95 Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu             100 105 110 Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln         115 120 125 Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp     130 135 140 <210> 7 <211> 504 <212> DNA <213> homo sapiens sapiens <400> 7 atgctcgagg cacccatggc agaaggagga gggcagaatc atcacgaagt ggtgaagttc 60 atggatgtct atcagcgcag ctactgccat ccaatcgaga ccctggtgga catcttccag 120 gagtaccctg atgagatcga gtacatcttc aagccatcct gtgtgcccct gatgcgatgc 180 gggggctgct gcaatgacga gggcctggag tgtgtgccca ctgaggagtc caacatcacc 240 atgcagatta tgcggatcaa acctcaccaa ggccagcaca taggagagat gagcttccta 300 cagcacaaca aatgtgaatg cagaccaaag aaagatagag caagacaaga aaatccctgt 360 gt; tcctgcaaaa acacagactc gcgttgcaag gcgaggcagc ttgagttaaa cgaacgtact 480 tgcagatgtg acaagccgag gcgg 504 <210> 8 <211> 168 <212> PRT <213> homo sapiens sapiens <400> 8 Met Leu Glu Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu   1 5 10 15 Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile              20 25 30 Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr          35 40 45 Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys      50 55 60 Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr  65 70 75 80 Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu                  85 90 95 Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp             100 105 110 Arg Ala Arg Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys         115 120 125 His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn     130 135 140 Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr 145 150 155 160 Cys Arg Cys Asp Lys Pro Arg Arg                 165 <210> 9 <211> 462 <212> DNA <213> homo sapiens sapiens <400> 9 atggcgacga aggccgtgtg cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat 60 ttcgagcaga aggaaagtaa tggaccagtg aaggtgtggg gaagcattaa aggactgact 120 gaaggcctgc atggattcca tgttcatgag tttggagata atacagcagg ctgtaccagt 180 gcaggtcctc actttaatcc tctatccaga aaacacggtg ggccaaagga tgaagagagg 240 catgttggag acttgggcaa tgtgactgct gacaaagatg gtgtggccga tgtgtctatt 300 gaagattctg tgatctcact ctcaggagac cattgcatca ttggccgcac actggtggtc 360 catgaaaaag cagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gacaggaaac 420 gctggaagtc gtttggcttg tggtgtaatt gggatcgccc aa 462 <210> 10 <211> 154 <212> PRT <213> homo sapiens sapiens <400> 10 Met Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln   1 5 10 15 Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val              20 25 30 Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val          35 40 45 His Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His      50 55 60 Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg  65 70 75 80 His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala                  85 90 95 Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys             100 105 110 Ile Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly         115 120 125 Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg     130 135 140 Leu Ala Cys Gly Val Ile Gly Ile Ala Gln 145 150 <210> 11 <211> 231 <212> DNA <213> Artificial Sequence <220> <223> fusion protein <400> 11 atgaaagaaa cctggtggga aacctggtgg accgaatggt ctcagccgaa aaaaaaacgt 60 aaagtgctcg agaatagtga ctctgaatgt cccctgtccc acgatgggta ctgcctccat 120 gatggtgtgt gcatgtatat tgaagcattg gacaagtatg catgcaactg tgttgttggc 180 tacatcgggg agcgatgtca gtaccgagac ctgaagtggt gggaactgcg c 231 <210> 12 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> fusion protein <400> 12 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro   1 5 10 15 Lys Lys Lys Arg Lys Val Leu Glu Asn Ser Asp Ser Glu Cys Pro Leu              20 25 30 Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu          35 40 45 Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu      50 55 60 Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg  65 70 75 <210> 13 <211> 516 <212> DNA <213> Artificial Sequence <220> <223> fusion protein coding <400> 13 atgtttaatc tgcctccagg gaattacaag aagcccaaac tcctctactg tagcaacggg 60 ggccacttcc tgaggatcct tccggatggc acagtggatg ggacaaggga caggagcgac 120 cagcacattc agctgcagct cagtgcggaa agcgtggggg aggtgtatat aaagagtacc 180 gagactggcc agtacttggc catggacacc gacgggcttt tatacggctc acagacacca 240 aatgaggaat gtttgttcct ggaaaggctg gaggagaacc attacaacac ctatatatcc 300 aagaagcatg cagagaagaa ttggtttgtt ggcctcaaga agaatgggag ctgcaaacgc 360 ggtcctcgga ctcactatgg ccagaaagca atcttgtttc tccccctgcc agtctcttct 420 gatggatcca aagaaacctg gtgggaaacc tggtggaccg aatggagcca gccgaaaaaa 480 aaacgcaaag tgctcgagca ccaccaccac caccac 516 <210> 14 <211> 172 <212> PRT <213> Artificial Sequence <220> <223> fusion protein coding <400> 14 Met Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr   1 5 10 15 Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val              20 25 30 Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser          35 40 45 Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln      50 55 60 Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro  65 70 75 80 Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn                  85 90 95 Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu             100 105 110 Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln         115 120 125 Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp Gly Ser Lys     130 135 140 Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys Lys 145 150 155 160 Lys Arg Lys Val Leu Glu His His His His His                 165 170 <210> 15 <211> 633 <212> DNA <213> Artificial Sequence <220> <223> fusion protein coding <400> 15 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 ataaaagaaa cctggtggga aacctggtgg accgaatggt ctcagccgaa aaaaaaacgt 120 aaagtgtata tgctcgaggc acccatggca gaaggaggag ggcagaatca tcacgaagtg 180 gtgaagttca tggatgtcta tcagcgcagc tactgccatc caatcgagac cctggtggac 240 atcttccagg agtaccctga tgagatcgag tacatcttca agccatcctg tgtgcccctg 300 atgcgatgcg ggggctgctg caatgacgag ggcctggagt gtgtgcccac tgaggagtcc 360 aacatcacca tgcagattat gcggatcaaa cctcaccaag gccagcacat aggagagatg 420 agcttcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc aagacaagaa 480 aatccctgtg ggccttgctc agagcggaga aagcatttgt ttgtacaaga tccgcagacg 540 tgtaaatgtt cctgcaaaaa cacagactcg cgttgcaagg cgaggcagct tgagttaaac 600 gaacgtactt gcagatgtga caagccgagg cgg 633 <210> 16 <211> 211 <212> PRT <213> Artificial Sequence <220> <223> fusion protein coding <400> 16 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Ile Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu              20 25 30 Trp Ser Gln Pro Lys Lys Lys Arg Lys Val Tyr Met Leu Glu Ala Pro          35 40 45 Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met      50 55 60 Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp  65 70 75 80 Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser                  85 90 95 Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu             100 105 110 Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg         115 120 125 Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln     130 135 140 His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu 145 150 155 160 Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln                 165 170 175 Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys             180 185 190 Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys         195 200 205 Pro Arg Arg     210 <210> 17 <211> 534 <212> DNA <213> Artificial Sequence <220> <223> fusion protein coding <400> 17 atgaaagaaa cctggtggga aacctggtgg accgaatggt ctcagccgaa aaaaaaacgt 60 aaagtgctcg agatggcgac gaaggccgtg tgcgtgctga agggcgacgg cccagtgcag 120 ggcatcatca atttcgagca gaaggaaagt aatggaccag tgaaggtgtg gggaagcatt 180 aaaggactga ctgaaggcct gcatggattc catgttcatg agtttggaga taatacagca 240 gt; gatgaagaga ggcatgttgg agacttgggc aatgtgactg ctgacaaaga tggtgtggcc 360 gatgtgtcta ttgaagattc tgtgatctca ctctcaggag accattgcat cattggccgc 420 acactggtgg tccatgaaaa agcagatgac ttgggcaaag gtggaaatga agaaagtaca 480 aagacaggaa acgctggaag tcgtttggct tgtggtgtaa ttgggatcgc ccaa 534 <210> 18 <211> 178 <212> PRT <213> Artificial Sequence <220> <223> fusion protein coding <400> 18 Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro   1 5 10 15 Lys Lys Lys Arg Lys Val Leu Glu Met Ala Thr Lys Ala Val Cys Val              20 25 30 Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe Glu Gln Lys          35 40 45 Glu Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys Gly Leu Thr      50 55 60 Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr Ala  65 70 75 80 Gly Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser Arg Lys His                  85 90 95 Gly Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu Gly Asn Val             100 105 110 Thr Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser Val         115 120 125 Ile Ser Leu Ser Gly Asp His Cys Ile Ile Gly Arg Thr Leu Val Val     130 135 140 His Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr 145 150 155 160 Lys Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val Ile Gly Ile                 165 170 175 Ala Gln        

Claims (5)

A fusion protein PEP-1-EGF formed by fusion of PEP-1 having the amino acid sequence of SEQ ID NO: 2 and EGF having the amino acid sequence of SEQ ID NO: 4,
The fusion protein PEP-1-FGF1 formed by fusion of PEP-1 having the amino acid sequence of SEQ ID NO: 2 and FGF1 having the amino acid sequence of SEQ ID NO: 6,
A fusion protein PEP-1-VEGF in which PEP-1 having the amino acid sequence of SEQ ID NO: 2 and VEGF having the amino acid sequence of SEQ ID NO: 8 are fused,
A fusion protein PEP-1-SOD1 formed by fusion of PEP-1 having the amino acid sequence of SEQ ID NO: 2 and SOD1 having the amino acid sequence of SEQ ID NO: 10,
A composition for promoting hair growth, which comprises, as an active ingredient, a Cu-peptide in which copper is chelated to Glycyl-Histidyl-Lysine.
The method according to claim 1,
The PEP-1-EGF is an amino acid sequence of SEQ ID NO: 12,
Wherein said PEP-1-FGF1 is the amino acid sequence of SEQ ID NO: 14,
The PEP-1-VEGF is the amino acid sequence of SEQ ID NO: 16,
Wherein the PEP-1-SOD1 is the amino acid sequence of SEQ ID NO: 18.
The method according to claim 1,
The composition may comprise,
PEP-1-EGF, PEP-1-FGF1, PEP-1-VEGF, PEP-1-SOD1 and Cu-peptide in amounts of 5 to 15 μg / mL, respectively.
The method according to claim 1,
The composition may comprise,
A composition for promoting hair growth, which is a composition for external application.
The method according to claim 1,
The composition may comprise,
A composition for promoting hair growth, which is a cosmetic composition.

Images