KR101672883B1 - Microneedle for prevention of depilation, of improvement of hair growth - Google Patents

Abstract

The present invention relates to a percutaneous delivery system for fermentation of peptone for preventing hair loss or promoting hair growth, and more particularly to a percutaneous delivery system for fermenting peptone fermented product or its formulation, preferably by using a micro-needle, / Or a hair tonic, hair promoting or hair health promoting system.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a micro needle for preventing hair loss or promoting hair growth,

The present invention relates to a percutaneous delivery system for fermentation of peptone for preventing hair loss or promoting hair growth, and more particularly to a percutaneous delivery system for fermenting peptone fermented product or its formulation, preferably by using a micro-needle, / Or a system for promoting hair growth, hair growth, or hair health.

The hair of the human body is about 100,000 to 150,000, and it repeats growth and drop through the anagen, catagen, and telogen. About 50-100 of the normal hair fall out naturally, and when hair is dropped more than the number of hair normally falling off due to various causes, it is called hair loss. Recently, the incidence is increasing for men as well as for women due to various complicated causes, and the age group is gradually lowering.

Alopecia is largely classified into male-type alopecia, alopecia areata, and alopecia areata, and it is known that abnormalities of hair cycle due to male hormone action, hair follicle weakening, hair dermal papilla cell and geminal matrix cell Aging, skin diseases, anticancer drugs, mental stress, physical stimulation, malnutrition, pathological complaints (diseases and drugs) and environmental pollution, etc. , Hair loss occurs due to a wide variety of causes, either singly or in combination.

Among them, testosterone (testosterone) is converted to dihydrotestosterone (DHT) by 5 a-reductase, and this hormone acts on the hair follicle, And shrinks the hair follicle to induce hair loss by inducing it to the regenerator phase at the growing stage.

In the case of alopecia areata, it is mainly caused by the decrease of the estrogen level after menopause. Unlike male pattern alopecia, the head of the head does not fall off but only the hair of the middle part is hairless. This phenomenon is due to the low level of male hormone and 5-a-reductase, which is about half of that of males. There is an enzyme called aromatase, mainly in the hair line, and male hormone and 5-alpha-reductase Lt; / RTI >

Alopecia areata is caused by abnormalities of the immune system, mental stress, blood flow disorders, and genetic factors. It is characterized by the appearance of round or oval hair loss, and the appearance of tofu rings or hairy walls. These alopecia areata have different causes than androgenetic alopecia.

A representative treatment for alopecia currently known is the application of minoxidil (6-Amino-1,2-dihydro-1-hydroxy-2-imino-4-phenoxypyrimidine) solution to help blood circulation, 5 alpha-reductase (Finasteride, Propecia (MSD)) medication, and self-hair graft. Minoxidil was first developed as a vasodilator for the treatment of hypertension, but as a side effect, it was developed as a hair growth agent. The mechanism of action of minoxidil on hair growth has not been clarified yet, but it is thought that hair growth is induced by an increase in nutritional supply through vasodilation. In addition, finasteride inhibits the activity of 5-alpha-reductase, an enzyme that acts on the male hormone metabolism, and was originally developed as a treatment for benign prostatic hyperplasia. However, it was also developed as a hair growth inhibitor as a side effect.

However, there is an inconvenience that both of them are effective only when they are used, and they are discontinued and disappear. Therefore, there is a disadvantage that they have to be continuously used. Minoxidil has a side effect such as stickiness feeling and skin irritation. In case of pinasteride, Of the patients were reported to have sexual dysfunction.

In the case of magnetic hair grafting, not only is hair transplantation costly, but also the transplantation period is long and only the hair of the patient can be transplanted, thereby limiting the number of transplanted hair.

Recently, the prospects for hair loss treatment are becoming more and more widespread as many researchers have been actively studying many regulatory factors involved in hair growth and hair loss. Especially, it has been found that the hair growth effect is regulated by various factors related to the hair cycle of growing, regressive and resting periods and by signaling by their receptors.

Hair growth is caused by a complex mechanism caused by various genes, growth factors, receptors, systemic hormones, and the like. Especially, a hair follicular dermal matrix cell and a dermal papilla cell, papilla cell is known to act as a pivotal factor in the formation and growth of hair (see FIG. 1).

The dermal papilla cells are the cells located at the base of the hair follicle. They supply oxygen and nutrients to the cells constituting the hair follicle, and control hair growth and follicle cycle regulation. Therefore, when the growth of the dermal papilla cells is promoted, the hair becomes healthy, hair growth is promoted, and hair loss can be prevented. The growth of hair progresses as the epithelial cells surrounding the dermal papilla divide and form the hair shaft, so the dermal papilla cells play an important role in regulating the division of epithelial cells. In addition, in male alopecia, the region where the male hormone acts on the hair follicle also plays a very important role in hair growth.

The geminal matrix cell, which is the part that touches the papilloma, produces active cell division by using nutrients and oxygen supplied from the papilloma and produces hair. The generated hair is pushed up near the pores, and in the process of passing through the keratinization transition part existing in the lower part of the follicle and the sebaceous gland, the water gradually escapes and changes into an elliptic shape, and cystine crosslinking is formed to stabilize the hair . Through this process, keratinized hair pushed out of the scalp is composed of dead keratin protein, which is composed of dead cells and cell interstitial substances. In particular, in male alopecia, a region where male hormone acts on hair follicle plays a very important role in hair growth, which is hair follicle surrounding hair follicle and papillary hair, and hair follicle forming through active cell division. Therefore, when hair follicle proliferation is promoted, hair becomes healthy, hair growth is promoted, and hair loss can be prevented.

According to a study conducted by Jahoda et al., Induction of hair growth by implantation of cultured dermal papilla cells, Nature, 311, pp. 560-562, 1984), dermal papilla cells Cells were temporarily removed from the dermal papilla cells, and the growth of the hair was continued while the cells were moved from the dermis root to the dermal papilla cells, and the lower part of the hair follicles was reduced to 1/3 or less It is reported that dermal papilla cells are formed from dermis root in the dermis root, mosquito cells are formed from the root of root, and hair growth is continued. However, hair growth stops when removing more than 1/3 of the lower part of the hair follicle have. In addition, after removing the lower third of the hair follicles and reporting that hair growth continued after implantation of the separated dermal papilla cells, the dermal papilla cells and dermal papilla cells forming hair follicles (germinal matrix cell) plays a very important role in hair growth and growth.

In addition to these results, the development of culture methods of various cells constituting hair follicles and hair follicles is used as a useful evaluation model while providing important clues for study on the hair loss mechanism. Currently, hair tissue culture has been performed as a method for measuring the hair growth effect of a test substance in various research fields. In general, human dermal papilla cells and monocyte cells are used.

C57BL / 6 mice are mainly used for evaluation of hair growth induction effect using animal models. C57BL / 6 mouse melanocytes are synthesized melanocyte only in the hair follicle and melanin cells are synthesized only in the hair follicle. Therefore, the skin color of the mouse is black in the growth period, but pink in the regenerating period and resting period. When we look at the hair cycle of C57BL / 6 mice, the mouse's skin enters the dormant state after 6-7 weeks of age. Using these characteristics, it is possible to evaluate the growth-inducing effect of the test substance by observing changes in the skin color of the mouse while treating the test material with water for several weeks after simultaneously removing the mouse hair from the resting state.

1. Korean Patent No. 10-1049776 discloses a cosmetic composition for preventing hair loss and promoting hair growth, a method for producing the same, and a hair care composition containing the same as an active ingredient. 2. Korean Patent No. 10-1103651 (disclosed on Jan. 11, 2012: hair cosmetic composition containing herbal extract having hair loss prevention and hair growth promoting effect)

On the other hand, there are many kinds of hair-related products distributed on the market, but most of them do not satisfy the needs of users because hair loss prevention and hair growth prevention effect are insufficient or temporary, and the data on efficacy and safety are insufficient. Even in the case of minoxidil and propecia, which have been confirmed to be effective, there are many limitations in the use of the drug because it is reported that alopecia are recurred or serious side effects such as sexual dysfunction are reported when the use is stopped. Therefore, it is required to develop natural drugs which have few side effects and are proved to be objective and reproducible.

Although there are many patents on natural hair growth, there are not many natural drugs that have an objective and reproducible examination on the effect. Therefore, there is a lot of room for excavation of new hair growth ingredients or materials from natural products, and the necessity of development is also increased accordingly.

Accordingly, the present inventors have studied and screened for preventing hair loss by various natural substances and improving hair growth. As a result, it has been found that peptone fermented by using peptone-degraded protein degradation activates and promotes proliferation of dermal papilla cells and dermal cells The present invention has been accomplished by confirming the human excitability and the biosafety.

The present invention provides a transdermal delivery system, particularly a scalp delivery system, of fermented peptone.

The peptone fermented product of the present invention has effects of preventing hair loss and / or promoting hair growth, hair growth, or hair health.

The present invention provides a micro-needle transdermal delivery system comprising a composition comprising the peptone fermentation product.

The present invention also provides a hair loss prevention, hair growth promoting, or hair health promoting system for transdermally delivering cosmetic formulations comprising the composition containing the peptone fermented product using a micro-needle.

The present invention also provides a method for preventing hair loss, promoting hair growth, or promoting hair health by transdermally delivering a composition comprising the peptone fermentation product, preferably using micro-needles.

In the present invention, the most important advantage of the puncturing technique using the micro needle is that the penetration and absorption of the active ingredient can be enhanced through the hole made up to the dermal layer in the scalp tissue and the circulatory function of the scalp tissue And the diffusion of the active ingredient is maximized.

In the present invention, the peptone fermented product itself can be applied to the scalp by microneedles in the form of a liposome-loaded form or a formulation such as an emulsion. The microneedles can be applied by using a roller device equipped with a medical or cosmetic rotating body which can be applied to a conventional micro needle procedure.

The peptone fermentation product of the present invention comprises a first step of preparing a culture medium containing a peptone-digested protein degradation product; A second step of inoculating the culture medium prepared in the first step with lactic acid bacteria; A third step of culturing the inoculated lactic acid bacteria in the second step to ferment a peptoned protein to produce a peptone fermented product; A fourth step of removing the cells and insoluble components of the peptone fermentation produced in the third step; And a fifth step of drying the peptone fermentation product from which the cells and insoluble components have been removed, obtained in the fourth step.

In the production process of the present invention, the peptonized protein degradation product of the first step may include at least one selected from the group consisting of soy peptone, wheat peptone, potato peptone, casein peptone, milk peptone, gelatin peptone and fish peptone . In addition, the lactic acid bacteria used in the second step may be lactobacillus strains. Also, in the third step, the fermentation of the peptonized protein degradation product can be carried out at a temperature of 25 to 35 DEG C for 12 to 120 hours.

In addition, the present invention provides a peptone fermented product produced by the above-described method.

The present invention also provides a composition for promoting hair loss prevention, treatment or hair growth comprising the peptone fermented product or a composition for promoting hair health and a transdermal delivery system thereof.

In the present invention, the peptone fermentation product comprises fermenting a peptoned protein, wherein the peptoned protein is selected from the group consisting of soy peptone, wheat peptone, potato peptone, casein peptone, milk peptone, gelatin peptone and fish peptone And may include at least one selected.

In the present invention, the soybean peptone may be at least one selected from the group consisting of Seed Pea, Seomyeutae, Black Pear, Yellow Pear, Kidney bean, Stark beet, Fence bean, Fuda bean, Mung bean, A protein degradation product obtained by subjecting soybean to peptone may be used, and particularly preferably soybean peptone may be used.

In the present invention, the peptone fermentation product can be fermented by lactic acid bacteria, and in particular, it can be fermented by lactic acid bacteria of Lactobacillus sp. Strain. It can also be used after fermentation with lactic acid bacteria and after drying treatment such as freeze-drying.

In the present invention, the term "peptonized protein" or "peptonized protein degradation product" refers to a mixture of peptides having a small molecular weight as a result of decomposition of a protein with an acid, a base or an enzyme. Protein hydrolysis changes protease, peptone, peptide, and finally amino acid sequence. Peptone is lower in protein than protease and is soluble in water, while aqueous solution has almost neutral or weak acidity. It is not solidified by heat and does not precipitate even when ammonium sulfate or tannic acid is added. Peptone is a raw material used as a nitrogen source for the cultivation of organic nutrition bacteria, and it is a material which is not only edible but also can be transdermally applied. Herein, the peptone-degraded protein is a generic term for vegetable peptone and animal peptone, and more specifically, soybean peptone, wheat peptone, potatoes peptone, casein peptone, (milk peptone), gelatin peptone, fish peptone, and the like.

In the present invention, the term " peptone fermentation product "or" fermented peptone "refers to fermentation of a peptized protein degradation product using lactic acid bacteria or the like. It is contemplated that the peptone fermentation product is also included in the peptone fermentation product by further treatment, such as drying, freeze-drying, and the like. In addition, the "peptone fermentation product" of the present invention is intended to include pharmaceutical or pharmacologically acceptable derivatives thereof.

The composition of the present invention can exhibit, for example, prevention of hair loss or promotion of hair growth / hair growth through activation, proliferation or growth of hair cells and / or dermal papilla cells, or hair health promoting effect.

The peptone fermented product of the present invention may be formulated by a known method in the field of cosmetics to be applied to a transdermal delivery system and mixed with an acceptable carrier or an excipient in the field of cosmetics, Transdermal delivery systems, which may be applied to the scalp by perforation techniques in an extracutaneous volume suitable, for example, by a rotating roller with microneedles, once to ten times per day, preferably once to five times per day have.

The present invention can provide a percutaneous delivery system of peptone fermented product having an effect of prevention, prevention, treatment or hair growth / hair growth of excellent hair loss. In addition, the transdermal delivery system of the present invention has an effect of prevention, prevention, treatment or hair growth / hair growth of excellent hair loss.

1 is a view showing the structure of hair.
2 is a diagram showing one embodiment of the method for producing the fermented peptone of the present invention.
FIG. 3 is a graph showing the effect of breeding cells of Examples and Comparative Examples of the present invention.
4 is a graph showing the effect of peptone fermentation on the breeder cells in the example of the present invention.
FIG. 5 is a graph showing the effect of the peptone fermentation product of another embodiment of the present invention on the proliferation of bovine cells.
Figs. 6 and 7 are diagrams showing the effect of growth of the dermal papilla cell according to the embodiment of the present invention.
8 is a view showing the effect of the other peptone fermented product of another embodiment of the present invention on the growth of the papilla cells.
FIG. 9 is a graph showing the results of measurement of organ weights after administration of the composition of the example of the present invention. FIG.
FIG. 10 is a graph showing the result of measuring the hair growth state of a mouse after administration of the composition of the example of the present invention. FIG.
FIG. 11 is a diagram showing the hair growth score per parking lot of a mouse after administration of the composition of the example of the present invention.
12 is a graph showing the average hair weight of a mouse after administration of the composition of the example of the present invention.
FIG. 13 is a graph showing the results of measuring the depth of hair follicles of a mouse after administration of the composition of the example of the present invention. FIG.
Figure 14 is an image of the depth of the hair follicle of a mouse after administration of the composition of the example of the present invention.
FIG. 15 is a graph showing the results of measurement of enzyme activity of skin tissue after administration of the composition of the example of the present invention. FIG.
16 is a diagram showing a skin penetration pattern of a drug through a micro needle.
17 is a view showing a roller device equipped with a micro needle according to one embodiment of the present invention.
18A to 18B are the results of HLPC measurement of the peptone fermented product prepared in Example 1 performed in Test Example 4, in which FIG. 18A shows the results from 0 to 60 minutes, and FIG. 18B shows the results from 0 to 25 minutes .
19A to 19E are the results of MS spectral measurement carried out in Experimental Example 1 and the result of confirming the molecular weight of the portion shown in FIG. 18B.

Hereinafter, the present invention will be described in more detail with reference to a preferred embodiment.

The present invention provides a hair loss prevention, hair growth promoting or hair health promoting system for transdermally delivering a composition containing a peptone fermented product using a micro-needle.

The peptone fermentation product may be one obtained by fermenting a peptoned protein.

The peptone fermented product may include a low molecular weight peptide having a weight average molecular weight of 200 to 500, and the low molecular peptide may include at least one selected from dipeptide, tripeptide and tetrapeptide.

And, the composition may contain 0.25 mg / ml to 12.5 mg / ml, preferably 0.5 mg / ml to 10.0 mg / ml of the peptone fermented product. A composition containing the peptone fermented product in such a concentration range may be more excellent in preventing hair loss, promoting hair growth, or promoting hair health.

The peptonized protein may include at least one selected from the group consisting of soy peptone, wheat peptone, potato peptone, casein peptone, milk peptone, gelatin peptone and fish peptone, and most preferably soy peptone .

The soy peptone may be one derived from one or more soybean selected from the group consisting of Seed Rape, Seomotei, Black Rice, Yellow Rice, Kidney Bean, Fence Soybean, Fuda Bean, Mung Bean, Most preferably from soybeans.

The peptone fermentation product may be fermented by lactic acid bacteria. The lactic acid bacteria may be selected from the group consisting of Lactobacillus rhamnosus or Lactobacillus casei, Lactobacillus casei, Lactobacillus plantarum, etc., and may be selected from at least one of them.

One. Peptoned Proteolytic products  How to ferment lactic acid bacteria using

1. Preparation of fermented peptone

A method for producing a lactic acid bacterium culture product using a peptoned protein degradation product will be described with reference to FIG.

The fermented peptone of the present invention comprises a first step of producing a culture medium containing a peptized protein degradation product; A second step of inoculating the culture medium prepared in the first step with lactic acid bacteria; A third step of culturing the inoculated lactic acid bacteria in the second step to produce a peptone fermentation culture produced by degradation of peptone-degraded protein degradation product; A fourth step of removing cells and insoluble components of the fermented culture of the peptone fermented in the third step; And a fifth step of drying the peptone fermentation product from which the cells obtained in step 4 and the insoluble components have been removed.

In the first step, the medium is characterized in that it comprises a peptoned protein degradation product. The peptonized protein degradation product is a conventional peptonized protein degradation product manufactured and sold for culturing microorganisms, and includes both vegetable and animal products, and desirably is desalted and desalted. In addition, the medium contains not only peptoned protein degradation products but also additional carbon sources and trace elements.

The carbon source is at least one selected from the group consisting of glucose, sucrose, maltose, fructose, lactose, maltose, xylose, galactose, arabinose, and combinations thereof. The amount of the carbon source to be used is preferably 2 to 99.5% by weight, more preferably 2 to 20% by weight based on the total weight of the peptoned protein decomposition product.

The trace elements usable in the medium of the present invention are at least one selected from the group consisting of magnesium sulfate, sodium acetate, potassium phosphate, manganese sulfate, zinc sulfate, cooper sulfate, calcium chloride and combinations thereof. The amount of the trace elements to be used is preferably from 0.5 to 98% by weight, more preferably from 5 to 50% by weight, based on the weight of the pepttonized protein decomposition product.

Preferably, the mixed medium is sterilized and the sterilization temperature is maintained at 90 to 121 캜 for 15 to 30 minutes.

In the second step, the lactic acid bacteria are inoculated on the medium in which the sterilization treatment is completed in the first step.

The lactic acid bacterium used in the present invention is preferably a strain of the genus Lactobacillus, most preferably a strain of Lactobacillus < RTI ID = 0.0 > and such rhamnosus) or Lactobacillus casei (Lactobacillus Casei), Lactobacillus Planta Rum (Lactobacillus Plantarum), may be at least one double-checked. The inoculation amount of the lactic acid bacteria is preferably 10 ⁵ to 10 ⁶ cfu / ml after the inoculation, and the incubation time becomes longer when the number of bacteria is less than 10 ⁵ to 10 ⁶ cfu / ml. .

In the third step, the fermentation is carried out at 25 to 35 ° C. in the medium inoculated with the lactic acid bacteria in the second step. This is because fermentation does not occur easily at lower temperatures, and lactic acid bacteria that are weak to heat are killed at temperatures above that temperature. The fermentation time of the lactic acid bacteria is preferably 12 to 120 hours, more preferably 24 to 72 hours. If the fermentation time is less than 12 hours, the degradation time of the peptone-modified protein degradation product is insufficient and the content of the low molecular weight peptite may be too small. If the fermentation time exceeds 72 hours, the economical efficiency may deteriorate. It is good to proceed.

In a fourth step of removing the cells and the insoluble component from the fermentation culture of the peptone, the cells and / or cells are separated by centrifugation or by a conventional filtration method (for example, a filter press, a membrane filter or the like) Insoluble components can be removed.

The cells and the cultures from which the insoluble matter has been removed are concentrated in vacuo at a temperature of 50 to 70 캜 in order to facilitate drying in the fifth step and then subjected to a conventional drying method (for example, vacuum drying, spray drying spray dry, freeze dry, etc.) to obtain a peptone fermented product.

In addition, the pharmaceutical composition packaged in each formulation may be prepared by mixing the dried peptone fermented product with a sub ingredient. The pharmaceutical composition may take the form of a solution, an emulsion, a viscous mixture or the like.

In addition, the cosmetic composition packaged in each formulation may be prepared by mixing the dried peptone fermented product with a sub ingredient. The cosmetic composition may be in the form of a solution, an emulsion, a viscous mixture or the like.

The cosmetic composition according to the present invention is not particularly limited in its formulation. For example, it may be applied to cosmetic compositions such as emulsions, lotions, creams, lotions, essences, packs, gels and mists, And may have formulations of cosmetics.

In the cosmetic composition of each formulation, the ingredients other than the above-mentioned ingredients may be selected and mixed according to the formulation or use purpose of other cosmetic ingredients.

Examples of the compounding ingredients that may be added include organic solvents such as a preservative component, a moisturizer, an emollient, a surfactant, an organic and inorganic pigment, an organic powder, an ultraviolet absorbent, a preservative, a bactericide, an antioxidant, a plant extract, a pH adjuster, And purified water.

The components to be added in addition to the above components are not limited thereto, and any of the above components may be compounded within a range that does not impair the objects and effects of the present invention.

2. Transdermal delivery system

The present invention also provides a transdermal delivery system in which the peptone fermentation product is maximally absorbed through the micro needle to prevent hair loss and improve hair growth.

The scalp tissue, which is divided into the epidermis, dermis and subcutaneous fat layer from the outer surface, pushes new cells toward the stratum corneum through cell division process and discharges heavy metals, waste materials and toxins in the body. That is, the skin tissue including the scalp does not easily receive the external substance because it has to obey the basic function for protecting human tissue from various external materials and environments. Therefore, the active ingredient applied to the scalp inhibits the absorption of the active ingredient through various routes in order to prevent penetration of the active ingredient into the skin due to the inherent protecting effect of the body tissue immediately after application.

In particular, unhealthy scalp accumulates oxidized lipids and waste products, and has a much thicker aging horny skin than a healthy scalp, resulting in a considerable decrease in absorption of active ingredients. In addition, the microvessel circulation ability is remarkably lowered, and after the transdermal absorption of the active ingredient, the spreading ability is remarkably decreased. Therefore, in order to achieve superior hair loss prevention and hair growth effects, sufficient absorption and diffusion into the scalp tissue must be achieved.

Micro needle is an invasive treatment method using a cylindrical device with fine needle and fine needle than hair. Using a micro needle to physically create a small hole in the skin and then creating a drug penetration pathway, drugs with a high molecular weight or drugs that are difficult to ionize or polarize can effectively penetrate into the scalp, and the concentration of drug / reagent (See FIG. 16).

Micro-needles are a great tool for effectively penetrating and diffusing natural efficacy into the scalp, but it is also very important to prevent side effects from misuse and understanding. Particularly, scalp has developed hair follicle and capillary blood vessels compared to other skin tissues, and nerve fiber tissue of skin tissue is most developed around isthmus and inferior part of hair follicle, Respond quickly to internal stimuli. In addition, the thickness of the tissue is thinner than the skin tissue of the other region by about 1.5 mm. Especially, the scalp thickness of the region where the hair loss occurs is particularly sensitive because the hair is only 30 to 60% of the scalp tissue in which the hair exists.

Therefore, when developing hair growth components and their formulations to be used with micro needles, it is necessary to fully consider the dynamics of micro needles, formulations and scalp tissues with a thorough understanding of the physical advantages and disadvantages of micro needles.

The inventors of the present invention examined the compatibility and safety of the peptone fermented product with the micro needle as an active ingredient and confirmed that excellent safety can be secured.

The microneedles applicable to the transdermal delivery system of the present invention are as shown in FIG. 17, in which, when microneedles corresponding to 192 or 200 microneedles attached to a rotatable roller are rolled on the scalp, A channel is created and a micro hole is created. Micro needle operation induces penetration of active ingredient by micro hole, maximizing diffusion of active ingredient.

The device for accommodating the micro needle in the transdermal delivery system of the present invention is not particularly limited, and conventional medicinal cosmetic coagulation devices can be used as long as the diffusion of the desired active ingredient into the scalp can be achieved.

For example, a streamlined body; A roller mounted on one end of the body; A detachable portion that is detachably attached to one side of the body and fixes the roller so that the roller is rotatable / rotatable; And a plurality of microneedles rotated together with the rollers and repeatedly arranged on the outer circumferential surface of the roller.

Hereinafter, the present invention will be described in more detail by way of examples, comparative examples and test examples. The following examples and comparative examples are merely illustrative and are not intended to limit the scope of the invention.

Example  One. Of peptone fermentation  Produce

Peptoned protein degradation products were purchased from Fluka (Sigma-Aldrich, St. Quentin Fallavier, France). 500 g of peptone-degraded protein soybean peptone was added to the components and the contents of the following Table 1, and the mixture was sterilized by heating at 121 占 폚 for 15 minutes. After cooling, Lactobacillus rhamnosus was inoculated as lactic acid bacteria so that the initial number of bacteria was 10 ⁶ cfu / ml, and cultured at 30 ° C for 36 hours with stirring at 50 rpm to obtain a fermentation culture of peptone.

N0. Ingredients Content (w / w) One Soybean Peptone 5.000% 2 Glucose 0.500% 3 Potassium phosphate 0.100% 4 Sodium acetate 0.050% 5 MgSO ₄ 0.005% 6 MnSO ₄ 0.005% 7 CuSO ₄ 0.002% 8 Purified water 94.338% Sum 100,000%

The resulting peptone fermentation culture was heated to a final temperature of 95 ° C and centrifuged (3,000 rpm, 15 min) to remove cells and insoluble components. For accurate cell concentration, peptone fermentation broth was lyophilized, and 7.5 g of freeze-dried peptone fermentation product was obtained from 100 g of the fermentation broth of peptone.

Comparative Example  One. Peptoned Protein degradation product

Soybean peptone was used as the peptone-degraded protein degradation product, which was purchased from Fluka (Sigma-Aldrich, St. Quentin Fallavier, France) and dissolved as it was.

Comparative Example  2. Soybean peptone  Excluded lactic acid bacteria culture

In Example 1, a culture solution of lactic acid bacteria was prepared by the same method and the same procedure except for soybean peptone.

Test Example  One. Human origin Moth cells  Proliferation test

[cell culture]

Human-derived mononuclear cells for cell culture were purchased from Innoprot (Spain) and seeded in poly-D-lysine coated 75T flasks and cultured at 37 ° C and 5% CO ₂ . The medium was MSC medium (Mesenchymal stem cell medium-Innoprot) containing 10% FBS, 1% antibiotic and 1% growth supplement, and the medium was changed every 3 days. After 80 ~ 90% of the flask was filled, the cells were stabilized at least once.

[MTS assay]

To each well of a 24-well plate, 200 μl of 4 × 10 ⁴ cells / ml was added to each well and incubated at 37 ° C. in 5% CO ₂ Lt; / RTI > for 24 hours. After 24 hours, the samples were treated at different concentrations and then incubated at 37 ° C in 5% CO ₂ (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit, Cat no. G5421, Promega) was added to each well and incubated at 37 ° C in a 5% -CO ₂ Incubator For 1 hour and then measured at 492 nm. As a control, the same amount of PBS as the sample was treated.

[Create Standard Curve]

The thigh cells 0.5 * 10 ⁵ cells / ㎖ to ⁵ * 10 5 cells / ㎖ in to count the cell number to 0.5 units was dispensed by 200㎕ the 96 well plate, 37 ℃ processes the MTS sample 20㎕, 5% CO ₂ Incubator for 1 hour, and the absorbance was measured with a Microplate Reader at 492 nm.

The absorbance values were obtained for each cell number and the standard curve corresponding to y = ax + b was obtained by using the Excel equation. The OD value obtained in the experiment is an experiment for predicting the actual cell number. All the results are calculated by converting the OD value into the actual cell number obtained by substituting the OD value into the standard curve, and the results are shown in Table 2 below.

Cell number
(* 10 ⁵ cells / ml)
0.5
1.0
1.5
2.0
2.5
3.0
4.0
4.5
5.0 OD value 0.424 + 0.014 0.579 ± 0.008 0.716 ± 0.001 0.868 ± 0.049 1.064 + 0.025 1.147 + 0.040 1.264 + 0.066 1.371 + 0.071 1.597 + - 0.052

[Result statistics processing]

All experimental results are shown by the actual number of cells obtained by substituting the optical density obtained from MTS into the y = ax + b equation obtained from the standard curve experiment. The results were expressed as means ± standard error. The test for each sample was analyzed by Student's t-test. The significance level was expressed as * for p <0.05, ** for p <0.01, p &Lt; 0.001.

Test Example  1-1. Of peptone fermentation Moth cells  Comparison of proliferation effects

The soybean peptone fermented product prepared in Example 1 was dissolved in PBS to a concentration and then treated with 0.2 쨉 m-filtering to confirm the effect of promoting the proliferation of monocyte. Soybean peptone of Comparative Example 1 was also dissolved in PBS in the same concentration and 0.2 .mu.m-filtered and used as a control group after treatment with the same amount of PBS.

24 well plate the cells Momo ⁴ * 10 4 cells / ㎖ after 24 hours incubation under 37 ℃, 5% CO ₂ condition in order to stabilize the frequency division by 200㎕ on, the step-by-step density as the sample with 0.001 ~ 1mg / ㎖ And cultured for 72 hours at 37 ° C and 5% CO ₂ . 20 μl of the MTS sample (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit, Cat no. G5421, Promega) was added and reacted for 1 hour at 37 ° C in a 5% -CO ₂ incubator. Respectively. The OD value was again assigned to the standard curve, converted to the actual cell number, and expressed as a percentage based on the control group, and the cell proliferation promoting effect was compared with the control group. The results are shown in Table 3 and FIG.

Name of sample Treatment concentration
(mg / ml) OD value Cell number
(X 10 &lt; ⁵ &gt; cells / ml) Percentage conversion
(%) Control group
(Untreated group) - 1.579 + 0.043 5.105 ± 0.263 100,000 + - 5.142


Example 1

0.001 1.605 + 0.021 5.326 + 0.155 104.328 ± 3.028 0.010 1.559 + 0.031 5.494 + 0.160 107.609 ± 3.136 0.050 1.666 + 0.022 5.802 0.206 113.657 ± 4.036 0.100 1.779 + 0.027 5.807 ± 0.101 113.752 + 1.957 0.500 2.043 + 0.049 7.256 + 0.407 142.131 + 7.965 1,000 2.070 + 0.026 7.070 + - 0.298 138.485 ± 5.845

Comparative Example 1


0.001 1.551 + 0.010 4.982 + 0.050 97.590 + 0.971 0.010 1.509 + 0.123 5.346 + 0.175 104.723 ± 3.431 0.050 1.565 + 0.049 5.394 + 0.248 105.653 ± 4.867 0.100 1.582 + 0.039 5.233 + 0.223 102.497 + 4.362 0.500 1.693 ± 0.002 5.219 + 0.139 102.225 + - 2.724 1,000 1.731 + 0.030 5.563 + 0.158 108.978 + - 3.105

As can be seen from Table 3 and FIG. 3, soy peptone (Comparative Example 1) had little effect on the hair cell proliferation, but in the case of the peptone fermented product fermented with lactic acid bacteria (Example 1), 0.001 mg / ml It was confirmed that at all concentrations of ~1,000 mg / ml, proliferating mononuclear cells compared to the control group.

In addition, it was confirmed that at a concentration of 0.500 mg / ml to 1.000 mg / ml, the proliferation of the somatic cells was increased by 30% or more at the maximum compared to the control group, and at the concentration of 0.500 mg / ml, .

The peptone fermentation product of the peptone fermentation product was diluted 10 times as much as the product of various formulations such as ampoule, tonic, shampoo, rinse, capsule and liquid, It is inferred that it is advantageous to use less than 10.000 mg / ㎖ when manufacturing various formulations using.

Test Example  1-2. The peptone fermentation product  Have Moth cells  Identifying the cause of the proliferation effect

In order to confirm what the peptone fermentation product (Example 1) had, the pea cell growth effect was compared with the culture solution of lactic acid bacteria fermented except for peptone (Comparative Example 2). All samples were treated with 0.5 mg / ml. The results of the treatment are shown in Table 4 and FIG.

As a result, the culture broth of lactic acid bacteria fermented with the exception of peptone (Comparative Example 2) did not show the effect of proliferation of pea cell, and the proliferation effect of peptone fermented product was not effected by the culture broth of lactic acid bacteria or lactic acid bacteria, It has been found that peptone is an effect of a change (for example, a peptone whose molecular weight has been changed), that is, a peptone fermentation itself.

Name of sample Treatment concentration
(mg / ml) OD value Cell number
(X 10 &lt; ⁵ &gt; cells / ml) Percentage conversion
(%) Control group
(Untreated group) - 1.645 + 0.027 5.314 ± 0.111 100,000 + 2.083 Comparative Example 1 0.500 1.749 + 0.039 5.743 + 0.161 108.066 ± 3.022 Example 1 0.500 2.141 + 0.034 7.364 + 0.142 138.568 ± 2.680 Comparative Example 2
0.100 1.702 + 0.018 5.550 + 0.074 104.430 占 .400 0.500 1.770 + 0.026 5.829 + 0.106 109.697 ± 1.998

Test Example  1-3. Other To peptone fermentation  About Moth cells  Confirmation of proliferation effect

In order to confirm whether peptones other than the soybean peptone fermentation used in Test Example 1 showed the effect of breeding the peel cells after fermentation, similar peptones were purchased and fermented in the same manner as in Example 1 to obtain respective peptone fermentations . For the exact comparison, the peptones used in the experiment were treated with the same concentration of 0.5 mg / ml before fermentation and after fermentation. The follicle proliferation effect upon treatment is shown in Table 5 and FIG.

As a result, it was confirmed that pea cell proliferation effect was exhibited after the same fermentation, and it was confirmed that soy peptone fermentation product of peptone fermented product showed the best proliferation effect of pea cell.

Name of sample OD value Cell number
(X 10 &lt; ⁵ &gt; cells / ml) Percentage conversion
(%) Control group 1.628 ± 0.009 5.170 + 0.036 100,000 + 0.695 Milk peptone 1.846 0.068 6.080 ± 0.282 117.600 + - 5.452 Milk peptone fermentation product 1.926 + 0.068 6.414 ± 0.282 124.052 ± 5.461 Wheat peptone 1.851 + 0.085 6.908 0.354 117.947 ± 6.851 Wheat peptone fermentation product 2.082 + 0.024 7.062 + 0.098 136.581 ± 1.901 Potato peptone 1.694 + 0.064 5.447 ± 0.265 105.354 ± 5.125 Potato peptone fermentation product 1.934 + 0.073 6.447 + 0.304 124.697 + - 5.870

Test Example  2. Human origin Dermal papilla cells  Proliferation test

[cell culture]

Human-derived dermal papilla cells for cell culture were purchased from Innoprot (Spain) and seeded in poly-D-lysine coated 75T flasks and cultured at 37 ° C and 5% CO ₂ . The medium was MSC medium (Mesenchymal stem cell medium-Innoprot) containing 10% FBS, 1% antibiotic and 1% growth supplement, and the medium was changed every 3 days. After 80 ~ 90% of the flask was filled, the cells were stabilized at least once.

[MTS assay]

Dermal papilla cells were seeded in a 24-well plate at a density of 4 × 10 ⁴ cells / ml in 200 μl aliquots and cultured for 24 hours at 37 ° C and 5% CO ₂ for stabilization. After 24 hours, the samples were treated at different concentrations and then cultured for 72 hours (3 days) under the condition of 37 ° C and 5% CO _{2. Then} , MTS samples (CellTiter 96 AQueous Non Radioactive Cell Proliferation Assay kit, Cat No. G5421, Promega) was added thereto, and the mixture was incubated at 37 ° C in 5% -CO ₂ Incubator for 1 hour and then measured at 492 nm. As a control, the same amount of PBS as the sample was treated.

[Create Standard Curve]

The dermal papilla cells 0.5 * 10 ⁵ cells / ㎖ to ⁵ * 10 5 cells / ㎖ in to count the cell number to 0.5 units was dispensed by 200㎕ the 96 well plate, 37 ℃ processes the MTS sample 20㎕, 5% CO ₂ Incubator for 1 hour, and the absorbance was measured with a Microplate Reader at 492 nm.

The absorbance values were obtained for each cell number and the standard curve corresponding to y = ax + b was obtained by using the Excel equation. The OD value obtained in the experiment is an experiment for predicting the actual cell number. All the results are calculated by converting the OD value into the actual cell number obtained by substituting the OD value into the standard curve, and the results are shown in Table 6.

Cell number
(* 10 ⁵ cells / ml)
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0


5.0 OD value 0.473 + 0.014 0.616 ± 0.008 0.775 ± 0.004 0.861 + 0.048 0.993 0.027 1.171 + 0.032 1.241 + - 0.110 1.361 + - 0.051 1.526 + 0.087

[Result statistics processing]

All experimental results are shown by the actual number of cells obtained by substituting the optical density obtained from MTS into the y = ax + b equation obtained from the standard curve experiment. The results were expressed as means ± standard error. The test for each sample was analyzed by Student's t-test. The significance level was expressed as * for p <0.05, ** for p <0.01, p &Lt; 0.001.

Test Example  2-1. Of peptone fermentation Dermal papilla cells  Comparison of proliferation effects

The soybean peptone fermentation product obtained in Example 1 was dissolved in PBS in concentrations and then treated with 0.2 쨉 m-filtering to confirm the promoting effect of the growth of the dermal papilla cells. Soybean peptone of Comparative Example 1 was also dissolved in PBS in the same concentration and 0.2 .mu.m-filtered and used as a control group after treatment with the same amount of PBS.

After dermal papilla cells were seeded in a 24-well plate at a density of 4 × 10 ⁴ cells / ml, the cells were incubated at 37 ° C and 5% CO ₂ for 24 hours for stabilization. The cells were incubated at a concentration of 0.001 to 1 mg / And cultured for 72 hours at 37 ° C and 5% CO ₂ . 20 μl of the MTS sample (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit, Cat no. G5421, Promega) was added and reacted for 1 hour at 37 ° C in a 5% -CO ₂ incubator. Were measured. The OD value was again assigned to the standard curve, converted to the actual cell number, and expressed as a percentage based on the control group, and the cell proliferation promoting effect was compared with the control group. The results are shown in Table 7 and FIG.

Name of sample Treatment concentration
(mg / ml) OD value Cell number
(X 10 &lt; ⁵ &gt; cells / ml) Percentage conversion
(%) Control group
(Untreated group) - 1.612 + 0.063 5.105 ± 0.263 100,000 + - 5.142


Example 1

0.001 1.665 + 0.037 5.326 + 0.155 104.328 ± 3.028 0.010 1.705 + 0.038 5.494 + 0.160 107.609 ± 3.136 0.050 1.780 + 0.049 5.802 0.206 113.657 ± 4.036 0.100 1.781 + 0.024 5.807 ± 0.101 113.752 + 1.957 0.500 2.128 + 0.098 7.256 + 0.407 142.131 + 7.965 1,000 2.084 + 0.072 7.070 + - 0.298 138.485 ± 5.845

Comparative Example 1


0.001 1.583 + 0.012 4.982 + 0.050 97.590 + 0.971 0.010 1.670 + 0.042 5.346 + 0.175 104.723 ± 3.431 0.050 1.682 + 0.060 5.394 + 0.248 105.653 ± 4.867 0.100 1.643 + - 0.053 5.233 + 0.223 102.497 + 4.362 0.500 1.640 + 0.033 5.219 + 0.139 102.225 + - 2.724 1,000 1.722 + 0.038 5.563 + 0.158 108.978 + - 3.105

As shown in Table 7 and FIG. 6, soybean peptone (Comparative Example 1) had little effect on the growth of the papilla cells, but in the case of the peptone fermented product fermented with the lactic acid bacterium (Example 1), 0.001 mg / It was confirmed that at all concentrations of mg / ml proliferation of dermal papilla cells compared to control.

Furthermore, it was confirmed that at a concentration of 0.500 mg / ml to 1.000 mg / ml, the proliferation of the dermal papilla cells by 30% or more was greater than that of the control group, and at 0.500 mg / ml, .

The peptone fermentation product of peptone fermentation product was prepared by diluting peptone fermentation product at a concentration 10 times as diluted as that of various products such as actual ampule, tonic, shampoo, rinse, capsule, and liquid product. It is inferred that it is advantageous to use less than 10.000 mg / ㎖ when manufacturing various formulations using.

Test Example  2-2. The peptone fermentation product  Have Dermal papilla cells  Identifying the cause of the proliferation effect

In order to confirm what the effect of the peptone fermentation product (Example 1) on the growth of the dermal papilla cell was due to, a comparative experiment was conducted with the culture solution of lactic acid bacteria fermented except for peptone (Comparative Example 2). All samples were treated with 0.5 mg / ml. The results of the treatment are shown in Table 8 and FIG.

Name of sample Treatment concentration
(mg / ml) OD value Cell number
(X 10 &lt; ⁵ &gt; cells / ml) Percentage conversion
(%) Control group (untreated group) - 1.538 + 0.037 4.797 + 0.127 100,000 ± 2.664 Example 1 0.500 2.023 + 0.018 6.817 + 0.061 142.099 + - 1.271 Comparative Example 1 0.500 1.529 + 0.045 4.756 + 0.152 99.146 ± 3.172 Comparative Example 2
0.100 1.538 + 0.033 4.798 + 0.113 100.006 + - 2.359 0.500 1.597 + 0.039 5.043 + 0.132 105.122 ± 2.751

As a result, the culture solution of lactic acid bacteria fermented with the exception of peptone (Comparative Example 2) did not show the effect of proliferation of the papilla cells, and the effect of the peptone fermentation on the development of the papilla cells was not effected by the culture solution of the lactic acid bacteria or lactic acid bacteria, It has been found that peptone is an effect of a change (for example, a peptone whose molecular weight has been changed), that is, a peptone fermentation itself.

Test Example  2-3. Other To peptone fermentation  About Dermal papilla cells  Confirmation of proliferation effect

To confirm whether peptones other than soybean peptone fermentation used in Test Example 1 exhibited the effect of proliferating papilla cells after fermentation, similar peptones were purchased and fermented in the same manner as in Example to obtain each of the peptone fermented products. For the exact comparison, the peptones used in the experiment were treated with the same concentration of 0.5 mg / ml before fermentation and after fermentation. The effect of treatment on the dermal papilla cell growth is shown in Table 9 and FIG.

As a result, it was confirmed that the growth of the dermal papilla cell was observed after fermentation, and that the fermented soybean peptone in the peptone fermented product showed the most excellent dermal papilla cell proliferation effect.

Name of sample OD value Cell number
(X 10 &lt; ⁵ &gt; cells / ml) Percentage conversion
(%) Control group 1.616 + 0.045 5.120 + 0.185 100,000 + 3,623 Milk peptone 1.654 + 0.072 5.279 + 0.301 103.117 ± 5.872 Milk peptone fermentation product 1.730 0.068 5.594 + 0.283 109.265 ± 5.524 Wheat peptone 1.720 0.010 5.554 + 0.043 108.481 ± 0.845 Wheat peptone fermentation product 1.840 + 0.033 6.053 + 0.136 118.239 + - 2.660 Potato peptone 1.665 ± 0.079 5.282 + 0.327 103.177 ± 6.396 Potato peptone fermentation product 1.807 + 0.069 5.916 + 0.288 115.551 ± 5.633

Test Example  3. Mouse C57BL / 6 Hair force  And Biotoxicity  exam

Samples used in the test were dissolved in sterilized water to a concentration of 5% by weight of the sample prepared according to Example 1, and a 5% by weight minoxidil solution, which is a widely used drug for hair growth, was used as a positive control. Animals used in this study were evaluated for their hair growth by transdermal administration of test substance once a day for 4 weeks using C57BL / 6 mice. The groups were divided into three groups: male control (untreated), positive control (5 wt% minoxidil) and test group (5 wt% - peptone fermented product).

Weight, feed, and water intake were measured once a week for 4 weeks, and the degree of hair growth was photographed and evaluated. After 4 weeks of transdermal administration, the autopsy was carried out the next day. The degree of hair growth was photographed and the weight of hair was measured at the time of autopsy. Some of the skin tissues were frozen and then biochemically assayed for ALP and γ-GT enzymes and the skin tissue, thymus, spleen, genitalia (male-testis and epididymis, female-ovary) Respectively.

This will be described in detail below.

6 weeks old mouse C57BL / 6 female and male were selected from the Orient by selecting individuals with similar weights within 3 days from the date of birth, and they were divided into two groups, each having an adaptation period of 1 week at 25 ° C and 50% humidity, Week. The mouse was placed in a desiccator containing ether and anesthetized for 1 minute, and then the hair was removed by epilating the back part of the mouse with an electric animal clipper (2 cm x 4 cm). After 1 day, mice not injured at the back region were selected and only those mice whose hair follicles were adjusted to the resting period were re-selected. Three groups of 3 rats were divided into three groups according to the nodule method, and the samples were transdermally administered once a day. The dose was 100 쨉 l per dose. The animals were housed in a standard breeding environment with a 12-hour light / dark cycle controlled at night and day, temperature and humidity maintained appropriately, and the feed and water quantities were supplied without restriction throughout the experiment. All of the experiments were conducted under the guidelines of the Asan Institute for Life Science 's animal experiment ethics committee (approval number: 2013-04-059). The animals were sacrificed for 4 weeks and then autopsied the next day. They were fasted for 12 hours and euthanized by ether anesthesia. Spleen and thymus were weighed and fixed in 10% neutral buffered formalin solution. Male genitalia (testis, epididymis) and female reproductive organs (ovary) were additionally extracted and fixed in 10% neutral buffered formalin solution. Skin tissues were excised and fixed in 10% neutral buffered formalin solution for histological changes, and some were frozen in nitrogen and stored frozen at -70 ° C for use in enzyme activity and gene expression assays.

Test Example  3-1. Long-term weighing

After 4 weeks of transdermal administration, autopsy was performed the following day. At the time of autopsy, the spleen and thymus were removed, washed with physiological saline, and water was removed with a filter paper, and the weight was measured. The skin, spleen and thymus, genitalia (male-testis, epididymis, female-ovary) were observed under a microscope and the weight of the spleen and thymus was measured and compared to the body weight. As a result, There was no significant difference between the groups and it was confirmed that there was no biotoxicity in the test group.

Test Example  3-2. Gross observation

In order to see the hair growth of the epilated area, we took photographs of the first week, the second week, the second week, the third week, and the fourth week of the experiment. The hair growth effect of each group was visually observed in each animal, and it was 0-19% (0), 20-39% (1), 40-59% (2), 60-79% (3) , And 80-100% (4), respectively, and the mean values were calculated for each group. The results are shown in the following Tables 11, 10 and 11.

(5% by weight - minoxidil) were the highest in males and females, followed by the test group (5% by weight - peptone fermented product) and the negative control group. In the test group (5 wt% - peptone fermented product), the hair growth effect was superior to the negative control on the day of the autopsy, and the hair growth effect was close to 90% as compared with the positive control.

Test Example  3-3. Hair weighing

After ether anesthesia on the day of the autopsy, hair was weighed with a fine scale by pushing the hair of the site (2 cm x 4 cm) with an electric clipper. As shown in Table 12 and Fig. 12, the hair weights were observed to be the highest in males and females (5% by weight - minoxidil) in all the females, followed by the test group (5% . In the test group (5 wt% - peptone fermented product), the weight of the hair of the control group was less than that of the positive control but the hair weight was 5 times that of the negative control group.

Test Example  3-4. Depth measurement of hair follicles

At autopsy, the skin tissue was cut into 2 cm x 3 cm, fixed on cardboard of the same size to prevent wrinkles on the skin, and fixed in 10% neutral formalin solution for 24 hours. Thereafter, the cells were immersed in paraffin after being washed, dehydrated, transparent, and permeated according to a conventional method. The embryonic tissue was stained with hematoxylin and eosin (H & E) stained with a 3-ml-thick section, and examined under an optical microscope. Changes in the depth of the hair follicles were measured randomly at 10 times at a magnification of 40x per sample using an image analysis program . The measurement results are shown in Tables 13, 14, 13, and 14 below.

In the case of pore depth, there was a difference between the populations, but both male and female showed the deepest depth of pores in the test group, and it was confirmed that the test group contributes to deeply deposit pores and form healthy hair .

Test Example  3-5. Measurement of enzymatic activity of skin tissue - Alkaline  phosphatase ( ALP )

At the time of autopsy, the skin tissue was cut, immediately cooled in liquid nitrogen, and stored at -70 ° C. The skin tissue was then divided into 3 groups per group. Tissue lyser was used to make a homogeneous solution by adding 4 times phosphate buffer solution (PBS). This homogenate was centrifuged at 12,000 rpm for 20 minutes using a centrifuge at 4 ° C, and the supernatant was collected and analyzed for the amount of ALP enzyme using an automatic biochemical analyzer.

Biochemical tests showed that the levels of ALP were lowered on average compared to the negative control and no hepatotoxicity occurred.

Test Example  3-6. Enzyme activity measurement of skin tissue - γ- Glutamyl  transpeptidase (γ- GT )

At the time of autopsy, the skin tissue was cut, immediately cooled in liquid nitrogen, and stored at -70 ° C. The skin tissue was then divided into 3 groups per group. Tissue lyser was used to make a homogeneous solution by adding 4 times phosphate buffer solution (PBS). The homogenate was centrifuged at 12,000 rpm for 20 minutes using a centrifuge at 4 ° C. The supernatant was collected and analyzed for the amount of γ-GT enzyme using an automatic biochemical analyzer.

As can be seen in the following Tables 15, 16 and 15, biochemical tests showed that the γ-GT level was lowered on average compared to the negative control and no hepatotoxicity occurred.

Test Example  3-7. Immunohistochemical observation of skin tissue

The embryos were cut into 3 μm thick slices and the tissues were immunostained using an automatic immune stainer (BenchMark XT, Ventana) according to the manual of the instrument. Ventana Ultraview DAB Kit was used for the measurement. Antibodies used for immunohistochemical staining were IGF-1, VEGF, TGFβ1 and Caspase-3 from Abcam.

As a result of the test, it was confirmed that all the values did not cause any significant difference between the groups and did not participate in inflammation.

Test Example  4. High performance liquid chromatography ( HPLC ) And MS ( Mass spectrometry ) Molecular weight confirmation test

In order to confirm the molecular weight of the peptone fermentation product of Example 1, high performance liquid chromatography and MS spectrum were measured.

Accela UHPLC (manufacturer: Thermo Scientfic) was used for high performance liquid chromatography measurement. The lyophilized peptone fermented product was dissolved in 0.1% formic acid, and the temperature was determined as TR (Room Temperature). The column used was Water BEH C18 (2.1 × 100 ml, 1.7 μm) and the flow rate was maintained at 300 μl / min. The sample analysis time was up to 60 minutes. The measurement results from 0 to 60 are shown in FIG. 18A, and the measurement results from 0 to 25 minutes are shown in FIG. 18B.

MS spectra were also measured using an LTQ Orbitrap XL mass spectrometer (manufactured by Thermo Scientific). Five peaks each having a large area among the single components separated by liquid chromatography were arbitrarily selected, and the molecular weight was confirmed by MS spectrum. The results are shown in Figs. 19A to 19E, respectively.

19A to 19E are data obtained by measuring the molecular weights of the peaks of the display portions 1 to 5 in Fig. 18B, and the results of (1) to (5) in Fig. 19 show that most of the peaks have a weight average molecular weight of 100 to 500 or less Can be estimated and / or confirmed to be a peptide composed of 2 to 4 amino acids as a peptide having a weight average molecular weight of 200 to 300.

Example  2. The peptone fermentation product  A variety of uses Formulation Example

The compositions of various types of hair and scalp application compositions containing the peptone fermented product obtained according to Example 1 of the present invention are as follows.

Example  2-1. ample

10 parts by weight of the peptone fermentation product of Example 1 was mixed with 100 parts by weight of the mixture of components shown in Table 17 below to prepare ampoules for hair and scalp application.

Example  2-2. tonic

100 parts by weight of the mixture of the ingredients of Table 18 below were mixed with 5 parts by weight of the peptone fermented product of Example 1 to prepare a tonic for hair and scalp application.

Example  2-3. shampoo

100 parts by weight of the mixture of the ingredients shown in the following Table 19 were mixed with 3 parts by weight of the peptone fermented product of Example 1 to prepare a shampoo for applying hair and scalp.

Example  2-4. Rinse

100 parts by weight of the mixture of the ingredients shown in Table 20 were mixed with 3 parts by weight of the peptone fermentation product of Example 1 to prepare a hair and scalp application rinse.

Example  2-5. Capsule

200 mg of the peptone fermentation product of Example 1

Lactose 100mg

Starch 93mg

Talc 2mg

Magnesium stearate qs

The above components were mixed and filled in gelatin capsules according to the conventional preparation method of capsules to prepare capsules.

Example  2-6. Capsule

100 mg of the peptone fermentation product of Example 1

Lactose 100mg

Starch 93mg

Talc 2mg

Magnesium stearate qs

The above components were mixed and filled in gelatin capsules according to the conventional preparation method of capsules to prepare capsules.

Example  2-7. Liquid

200 mg of the peptone fermentation product of Example 1

Sugar 20g

20g per isomer

Lemon incense quantity

Purified water was added to the entire 100 ml

The above components were mixed according to a conventional method for preparing a liquid preparation, filled in a 100 ml brown bottle, and sterilized to prepare a liquid preparation.

Test Example  5. Bad wool  Or clinical trial for patients with hair loss

To investigate the effects of hair loss prevention and hair growth, clinical trials were conducted in patients with papillion or alopecia. Subjects who were 20 years old or older and who had significant hair loss or alopecia at the time of visual examination were first screened and then examined by scalp diagnostic (ARAMO-SG, Aram Huvis) for scalp analysis (scalp condition, hair density, (More than 120 pieces / 1 cm 2) of the hair density (the number of hairs per 1 cm 2 area) is less than 70% or the hair density is normal Eight patients were re-screened for hair loss (more than 0.5 mm) that was significantly thinner than normal (0.075 mm or more) hair thickness, even though the hair thickness was close.

In the clinical trial, the ampoule of Example 2-1 was used. After shampooing, the hair was applied to the hair loss area once a day. The amount of application was varied depending on the amount of hair loss or hair loss area, 3 to 5 ml was applied. The total duration of the study was from 3 months to 4 months. The effect was evaluated by assessing the degree of improvement of the subjective symptom, and the overall hair growth and hair loss prevention effect was confirmed.

The soybean fermented product was also fermented in the same manner as in Example 1 except that the soybean itself was replaced with lactobacillus lactobacillus lambosus. The soybean fermented product was used as an active ingredient of the ampule of Example 2-1, Ampoules were prepared and comparative tests were carried out.

The evaluation results of the subjective symptoms improvement of the ampule of Example 2-1 are shown in the following Table 21, and the evaluation results of the subjective symptom improvement of Example 2-1 and Comparative Example 2-1 are shown in Table 22 (only the average score is shown) . As can be seen from the results, the hair growth and hair loss prevention effect according to Example 2-1 was found to be very excellent. In addition, no adverse effects were found in all subjects during the period of use in clinical trials, and it was found to be safe for human body.

Test Example  6. On the micro needle  Conformity check by

In the same manner as in Test Example 5, 10 subjects who were alopecia are selected. In this test, the suitability of the micro needle of the ampule of Example 2-1 was confirmed. To confirm the micro needle compatibility of the ampule of Example 2-1, the ampoule of Example 2-1 was applied twice to the skin inside the subject's arm and then infiltrated using a micro needle roller and observed. The microneedle roller was a microneedle therapy system (MTS) with 192 gold-plated microspheres using a Jenny Tree skin-maker micro-needle roller.

In order to confirm the suitability of the micro needle, it was confirmed that erythema, irritation, itching and inflammation were induced. The degree of pain or irritation felt during or after the micro needle operation is 0 to 4 (0: no pain or irritation 1: pain or irritation but insufficient 2: pain or irritation but tolerable 3: (0: no erythema, 1: erythema), and the degree of redness after micro needle operation was visually observed through visual observation. 4: erythema was very severe), and the degree of itching or persistence after the micro needle procedure was 0 to 4 (0: itching) 3, the itching was severe, 4, the itching was very severe), and the degree of inflammation after the micro needle operation was visually observed 0 to 4 Was evaluated as (inflammation is very severe 0: No inflammation; 1: incomplete, but the inflammation; 2, but also good overall inflammation; ::; 34 severe inflammation) system. The measurement results are shown in Table 23 (only the average value is shown).

Example 2-1 Ampoule Pain or irritation 0.1 Erythema 0.5 Itching 0.2 Inflammation 0.1

As shown in Table 23, it was confirmed that the peptone fermented product of the present invention is suitable for micro needle.

As can be seen from the above results, the peptone fermented product according to the present invention has an effect of prevention, prevention, treatment and hair growth of excellent hair loss. In addition, the peptone fermented product of the present invention exhibits excellent hair health promoting effect. In addition, the peptone fermented product of the present invention is well suited for microneedle transdermal delivery systems.

Claims (12)

The composition containing the peptone fermented product is filled and transdermally delivered,
Wherein the peptone fermented product has a concentration of 0.5 to 1.0 mg / ml, a proliferation rate of a bovine cell treated with 0.5 mg / ml of the peptone fermented product measured by the following method 1 is 134% or more,
A hair growth promoting or hair health promoting micro-needle characterized in that a composition having a growth rate of dermal papilla cells treated with 0.5 mg / ml of peptone fermentation product measured by the following Measuring Method 2 is 134% or more;
[Measurement method 1]
24-well plate for one cell MOMO 4 * 10 ⁴ after 24 hours incubation under 37 ℃, 5% CO ₂ conditions the cells / ㎖ for stabilization and frequency division by the 200㎕, peptone fermentation process the water to 0.5 mg / ml, and then And cultured again for 72 hours at 37 ° C and 5% CO ₂ . The OD value was measured at 492 nm, and the OD value was substituted into the standard curve to be converted into the actual cell number. Thereafter, the OD value was measured at 492 nm, As a percentage, to measure the proliferation rate of mononuclear cells compared to the control group,
[Measurement method 2]
For 24 hours under 24 the dermal papilla cells on a well plate ⁴ * 10 4 cells / ㎖ by the frequency division by 200㎕ 37 ℃ for stabilization, 5% CO ₂ conditions After the culture, the fermentation process the peptone water to 0.5 mg / ml, and then And cultured again for 72 hours at 37 ° C and 5% CO ₂ . The OD value was measured at 492 nm, and the OD value was substituted into the standard curve to be converted into the actual cell number. Thereafter, the OD value was measured at 492 nm, To measure the proliferation rate of the dermal papilla cells relative to the control. The micro needle according to claim 1, wherein the peptone fermentation product is fermented with a peptoned protein, to prevent hair loss, promote hair growth or promote hair health. delete The micro needle according to claim 1, wherein the peptone fermented product comprises a low molecular weight peptide having a weight average molecular weight of 200 to 500, for promoting hair loss, promoting hair growth or promoting hair health. The method of claim 1, wherein the peptoned protein comprises at least one selected from the group consisting of soy peptone, wheat peptone, potato peptone, casein peptone, milk peptone, gelatin peptone, and fish peptone, Needle micro needles. 6. The micro-needle according to claim 5, wherein the peptoned protein comprises soy peptone, hair loss promoting or hair health promoting micro needles. The micro needle according to claim 1, wherein the peptone fermentation product is fermented by lactic acid bacteria, to prevent hair loss, promote hair growth or promote hair health. A cosmetic formulation comprising a composition comprising a peptone fermentation product is filled and delivered transdermally,
Wherein the peptone fermented product has a concentration of 0.5 to 1.0 mg / ml, a proliferation rate of a bovine cell treated with 0.5 mg / ml of the soybean peptone fermented product measured by the following method 1 is 134%
A hair growth promoting or hair health promoting micro-needle characterized in that the composition having the growth rate of the dermal papilla cell treated with 0.5 mg / ml of the soybean peptone fermentation product measured by the following Measuring Method 2 is 134% or more.
[Measurement method 1]
24-well plate for one cell MOMO 4 * 10 ⁴ after 24 hours incubation under 37 ℃, 5% CO ₂ conditions the cells / ㎖ for stabilization and frequency division by the 200㎕, peptone fermentation process the water to 0.5 mg / ml, and then And cultured again for 72 hours at 37 ° C and 5% CO ₂ . The OD value was measured at 492 nm, and the OD value was substituted into the standard curve to be converted into the actual cell number. Thereafter, the OD value was measured at 492 nm, As a percentage, to measure the proliferation rate of mononuclear cells compared to the control group,
[Measurement method 2]
For 24 hours under 24 the dermal papilla cells on a well plate ⁴ * 10 4 cells / ㎖ by the frequency division by 200㎕ 37 ℃ for stabilization, 5% CO ₂ conditions After the culture, the fermentation process the peptone water to 0.5 mg / ml, and then And cultured again for 72 hours at 37 ° C and 5% CO ₂ . The OD value was measured at 492 nm, and the OD value was substituted into the standard curve to be converted into the actual cell number. Thereafter, the OD value was measured at 492 nm, To measure the proliferation rate of the dermal papilla cells relative to the control. The micro needle according to claim 8, wherein the cosmetic formulation is an emulsion, a lotion, a cream, an essence, a pack, a gel, a mist, a shampoo, a rinse or a soap. 6. The method of claim 1, wherein the transdermal delivery comprises: a body; A roller detachably attached to one end of the body; And a plurality of microneedles rotated together with the rollers and repeatedly disposed on an outer circumferential surface of the roller, wherein the microneedles are used for hair loss prevention, hair growth promotion or hair health promotion. delete delete

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