WO2013180526A1

WO2013180526A1 - Skin regeneration and hair growth promoter containing edelweiss extract

Info

Publication number: WO2013180526A1
Application number: PCT/KR2013/004833
Authority: WO
Inventors: 김수나; 박필준; 김아름; 서정아; 박원석; 이영란
Original assignee: (주)아모레퍼시픽
Priority date: 2012-05-31
Filing date: 2013-05-31
Publication date: 2013-12-05
Also published as: KR20130135165A; KR102022538B1; KR20130135164A

Abstract

Disclosed is a skin regeneration and hair growth promoter containing an edelweiss extract as an active ingredient. In addition, the present invention relates to a promoter containing an enzyme-treated edelweiss extract, and more specifically, to a promoter containing a cell wall-degrading enzyme-treated edelweiss extract.

Description

Skin regeneration and hair growth promoter including edelweiss extract

The present invention relates to a skin regeneration and hair growth promoter comprising an edelweiss extract.

Hair loss can be classified into resting hair loss and growing hair loss according to the growth cycle, and the resting hair loss includes androgenetic alopecia caused by androgen, postpartum hair loss of women or endocrine disease, and rounded hair growth by circular growth. Hair loss, scar hair loss, or hair loss due to ringworm. In addition, hair loss is also known to occur due to local infection, endocrine disorders, genetic factors and autoimmunity.

Representative anti-hair loss agents currently include Propecia and Minoxidil, which are approved by the US FDA. These drugs have clinically definite effects, but also have side effects such as sexual dysfunction and skin irritation, and women can not use or use only in extremely limited amounts. Therefore, there is a growing interest in natural products formulations that can be safely used without any gender restrictions, but through a new mechanism so as not to have the above disadvantages.

The present invention seeks to provide a skin regeneration accelerator having an excellent skin regeneration promoting effect and a hair growth promoter having an excellent hair growth promoting effect.

It is also an object of the present invention to increase the content of the active substance contained in the edelweiss extract, to provide an accelerator having a further improved skin regeneration and hair growth effect.

One aspect of the present invention provides a skin regeneration accelerator and a hair growth promoter comprising an edelweiss extract as an active ingredient.

Another aspect of the present invention provides a cosmetic composition and a pharmaceutical composition comprising the accelerator.

The present invention also provides a skin regeneration accelerator comprising an extract of Edelweiss treated with cell wall degrading enzyme as an active ingredient.

The formulation according to the present invention may include an edelweiss extract as an active ingredient, thereby promoting Wnt / beta-catenin signal transduction, thereby exhibiting excellent skin regeneration effect, specifically excellent skin wound healing and scar reduction effect. have. In addition, the preparation not only promotes wint / beta-catenin signal transduction, but also activates stem cells of the dermal papilla cells and promotes the proliferation of dermal papilla cells of the hair follicles, thereby exhibiting an excellent hair growth promoting effect, further preventing hair loss and promoting wool. have.

In addition, the accelerator including the extract of the enzyme-treated edelweiss according to the present invention can increase the extraction efficiency of the active ingredient in the plant cell, the content of chlorogenic acid is rapidly increased, and the antioxidant activity is increased with a high content of chlorogenic acid, By further promoting the transmission of Wnt / beta-catenin (Wnt / beta-catenin) signal can exhibit an improved skin regeneration effect, specifically excellent skin wound healing and scar reduction effect, hair growth promoting effect.

1 is a graph showing that Edelweiss extract promotes Wnt / beta-catenin signal transduction of stem cells.

2 is a graph showing that the edelweiss extract maintains and activates the stem cell nature of the dermal papilla cell line.

Figure 3 is a graph showing the hair follicle dermal papilla cell growth promoting effect of the edelweiss extract.

Figure 4 is a graph showing the hair growth promoting effect and the effect of maintaining the hair follicle growth period in the hair follicle of Edelweiss extract.

5 is a graph showing that the enzyme-treated Edelweiss extract promotes Wnt / beta-catenin signal transduction of stem cells.

Figure 6 is a graph showing the hair follicle dermal papilla cell growth promoting effect of the enzyme treatment Edelweiss extract.

Figure 7 is a graph comparing the content of chlorogenic acid as an indicator component contained in the edelweiss extract and the enzyme treatment edelweiss extract.

8 is a graph showing the antioxidant efficacy of chlorogenic acid as an edelweiss index component.

Each tissue in the human body contains adult stem cells that produce the cells that make up that tissue. The cells that make up skin or hair are also derived from stem cells present in the skin or hair follicles.

Stem cells generally refer to pluripotent cells that can develop into any tissue. It has the ability to self-renewal to regenerate itself and to differentiate into cells with specific functions depending on the environment. These stem cells can be roughly divided into embryonic stem cells (adryonic stem cells) and adult stem cells (adult stem cells).

Embryonic stem cells are derived from fertilized eggs produced by fertilization of sperm, a male germ cell and an egg, a female germ cell. It is also called pluripotent stem cell because it has the ability to differentiate into various cell types. Embryonic stem cells are capable of mass proliferation and differentiation into almost all body cells, and have no immune rejection reaction, so that they can be transplanted to other species as well as others. However, it is difficult to control differentiation, which may lead to cancer cells, requiring technical precision, and accompanied by destruction of the fertilized egg, which may be an ethical problem.

Adult stem cells generally refer to cells that differentiate into cells of specific tissues when needed, and usually only a small amount is present in each tissue of the body. That is, adult stem cells of bone marrow cells are differentiated into hematopoietic cells, skin adult stem cells into skin, and adult stem cells of olfactory neurons into olfactory neurons. These adult stem cells are stable and are unlikely to become cancer cells, and unlike embryonic stem cells, they do not destroy fertilized eggs, so it is not ethically problematic.

Hair follicle stem cells are adult stem cells that exist in the hair follicles and produce the cells that make up the skin and hair, which are derived from hair follicle stem cells and mesenchymal stem cells, which may be derived from epithelial cells, also called bulge stem cells. The hair follicle dermal papilla cells and neural stem cells derived from melanin-forming stem cells can be largely divided into three types.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

One aspect of the present invention provides a skin regeneration accelerator comprising an edelweiss extract as an active ingredient.

Another aspect of the present invention provides a hair growth promoter comprising an edelweiss extract as an active ingredient.

In one aspect of the invention, the edelweiss extract may be an enzyme-treated edelweiss extract.

In the skin regeneration accelerator which is an aspect of the present invention, the promoter may heal a skin wound.

In the skin regeneration accelerator which is an aspect of the present invention, the accelerator may prevent hair loss or promote wool.

In the skin regeneration accelerator, which is an aspect of the present invention, the promoter may activate a Wnt signal.

Wnt / beta-catenin signaling is a signaling system involved in the development of living organisms and is known to act on skin formation and hair follicle development. Furthermore, when a wound occurs on the skin, it is known that wint signal transduction is essential during skin tissue recovery to promote differentiation of epithelial cells. Therefore, if the wint signal is promoted, the differentiation of epithelial cells may be promoted to promote skin regeneration, and further, the healing of skin wounds may be promoted. The enzyme-treated edelweiss extract of the present disclosure promotes the transmission of Wnt / beta-catenin in stem cells. Therefore, the formulation containing the same may promote skin regeneration, and in particular, may promote healing of skin wounds.

Hair repeats growth and fall through the growth-degeneration-rest phase. Among them, hair follicle stem cells differentiate during growth, forming new hair. On the other hand, the treatment of the winte protein in the mouse promotes hair growth, it has been known that hair growth is inhibited in mice knocked down the wint gene. This means that substances that promote wint / beta-catenin signal transduction present in hair follicle stem cells can promote hair growth. Since substances that promote hair growth through this mechanism are not involved in promoting hair growth through the regulation of testosterone and / or blood circulation, severe hair loss and which have not been addressed by methods of inhibiting testosterone or promoting blood circulation and Female hair loss can be improved. In addition, when the stem cells of the dermal papilla cells are activated, the activity of the dermal papilla cells may be promoted to promote hair growth. Edelweiss extract promotes Wnt / beta-catenin signaling in stem cells and activates stem cells in dermal papilla cells. Edelweiss extract also promotes the growth of hair follicle dermal papilla cells. Therefore, the formulation containing the edelweiss extract can promote hair growth, further prevent hair loss and promote wool. In addition, the hair growth accelerator including the edelweiss extract may have a superior hair growth promoting, hair loss prevention and wool promoting effect than minoxidil, which is known as a conventional hair growth promoter, has little side effects such as sexual dysfunction or skin irritation, and has no gender. Can be used regardless.

In one embodiment of the present invention, the edelweiss includes a plant of the genus Leontopoium sp. In one embodiment of the present invention, the edelweiss may be Leontopoium alpinum .

In the present specification, the "Edelweiss" may be a plant of the genus Leontopoium sp. Leontopoium sp. Plants are Leontopoium alpinum , Leontopodium andersonii , Leontopodium artemisiifolium , Leontopoium auran Tiacum ( Leontopodium aurantiacum ), Leontopodium brachyactis , Leontopodium calocephalum , Leontopodium campestre , Leontopoium chuii , Leontopodium chuii , Umm beans Rover lithium (Leontopodium conglobatum), Leon topoyi help Correa num (Leontopodium coreanum), Leon topoyi help Dedeman Ken City (Leontopodium dedekensii), Leon topoyi help to Surabaya yanum (Leontopodium delavayanum), with Leon topoyi help disco (Leontopodium discolor) Leon topoyi help pagin Regensburg (Leontopodium fangingense), Leon topoyi help us this file (Leontopodium fauriei), On topoyi help Forres Tia num (Leontopodium forrestianum), Leon topoyi help program rancheti (Leontopodium franchetii), Leon topoyi help Fuck di (Leontopodium giraldii), Leon topoyi help Haas syeoyi Death (Leontopodium haastioides), Leon topoyi Be Umm Director nenseu (Leontopodium hallaisanense ), Leontopodium haplophylloides , Leontopodium hayachinense , Leontopodium himalayanum , Leontopodium jacotianum and Leontopodium jacotianum and Leontopodium haplophylloides It may include one or more plants selected from the group consisting of Leontopodium japonicum .

In one aspect of the invention, Edelweiss ( Leontopoium alpinum ) is a perennial plant of the dicotyledon plant Campanula Asteraceae is an alpine plant that lives primarily in dry places. The leaves are relatively high in the roots, slightly on the stems, and linear. At the end of the stem, the squirrel gathers, spreads out all over it, with a slight heading in the middle.

As used herein, edelweiss may be included in the form of an extract, or may be included as a ground powder of the herbal medicine, or as a dry ground powder of the herbal medicine, but is not limited thereto. In addition, the edelweiss used in the present specification is not limited to the method of acquiring, can also be grown and used or purchased commercially available, can use some or all of the above-ground parts of the herb, preferably all of the above-ground parts can be used have.

As used herein, the term "extract" includes all materials obtained by extracting the components of natural products, regardless of the extraction method, extraction solvent, extracted components or the form of the extract, and extracts the substances obtained by extracting the components of natural products. It is a broad concept that includes all of the materials that can be obtained by processing or processing in a method.

In the present specification, "edelweiss extract" includes all materials obtained by extracting the components of edelweis, regardless of the extraction method, the extraction solvent, the extracted components or the form of the extract, and heat, acid (acid) in the process of extracting the components. Material obtained through the extraction method including the step of treating with a base, an enzyme, and the like, and a material obtained by extracting an edelweiss component and then processing or treating it by another method. All inclusive is a broad concept.

In one aspect of the invention, the edelweiss extract is a step of washing and drying the edelweiss; Solvent extraction of the dry edelweiss; And it can be obtained through the step of filtering the extracted edelweiss. In the above, the solvent includes at least one selected from the group consisting of water, an organic solvent, and a mixture of water and an organic solvent. In the above, water includes distilled water or purified water, and the organic solvent is selected from the group consisting of alcohol, acetone, ether, ethyl acetate, diethyl ether, ethyl methyl ketone and chloroform, for example, a lower alcohol having ₁ to ₅ carbon atoms. Including but not limited to one or more.

The edelweiss extract may include an edelweiss-C ₁ ~ C ₆ alcohol extract, specifically, the alcohol may be methanol or ethanol.

In the present specification, the edelweiss extract may be a crude extract of water, a C ₁ -C ₆ alcohol, and a solvent selected from the group consisting of a combination thereof. The C ₁ -C ₆ alcohol may be specifically methanol. When extracting the edelweiss with a solvent, it is preferable to extract by adding a solvent corresponding to about 5 to 15 times the edelweiss, specifically, it is preferable to extract by adding about 10 times the solvent, but is not limited thereto. The extraction may be a heat extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction, and the like, there is no limitation if the extraction is obvious to those skilled in the art. The extraction may be performed at room temperature, but for more efficient extraction, the extraction may be performed under heating conditions, preferably at about 40 to 100 ° C., more preferably at about 80 ° C., but is not limited thereto. It is not. The extraction time is preferably about 2 to 4 hours, more preferably about 3 hours, but is not limited thereto, and may vary depending on conditions such as extraction solvent and extraction temperature. The extraction may be extracted one or more times several times in order to obtain a larger amount of the active ingredient, preferably one to five times, more preferably three times the continuous extraction can be used combined extract.

In the present specification, the edelweiss extract may include the crude extract of edelweis as described above, and may include the crude extract as a soluble fraction of the organic solvent obtained by further extracting the organic extract with a low polarity organic solvent. Hexane, methylene chloride, ethyl acetate, n-butanol, and the like may be used as the organic solvent, but is not limited thereto. The extract extracted by the above method or a soluble fraction of the extract may be used as it is, but may be used in the form of an extract by concentrating after filtration, it may be used as a lyophilized form by lyophilization after concentration.

As used herein, the term "skin" refers to a tissue covering the body surface of an animal, and is a broad concept including not only tissues covering the body surface such as the face or body, but also the scalp.

"Promoting skin regeneration" herein may mean promoting the formation of new skin cells and promoting exfoliation of aged skin and includes regeneration of both normal skin cells or wounded skin cells.

As used herein, "hair" may be described as hair as a general term for body hair and hair. The hair includes both human and animal hair.

As used herein, "promoting wool" means not only promoting the production of new hair, but also allowing existing hair to grow thick and healthy.

In one aspect, the present invention relates to a skin regeneration accelerator comprising an extract of an enzyme-treated edelweiss as an active ingredient.

As used herein, the term "enzyme" refers to a substance which is capable of obtaining a high content of an extract when the substance constituting the edelweiss is extracted by physically or chemically modifying the substance.

In one aspect of the present invention, the enzyme is not limited so long as it is an enzyme capable of obtaining an active ingredient of a plant at a high content and high yield rate, and specifically, includes a cell wall degrading enzyme.

As used herein, "cell wall degradation enzyme" may refer to an enzyme that is involved in degradation of the plant cell wall. The cell wall is generally composed of cellulose, hemicellulose, pectin substance, lignin, protein, magnesium, calcium and the like. Cellulolytic enzymes include exo-β, 4 glucanase (fibrinase C1), endo-β, 4 glucanase (fibrinase Cx); Hemicellulose degrading enzymes include xylanase, 1,3-xylanase, α-L-arbinofuranosidase, and the like; Pectinase is a group of enzymes that cleave α-1,4 bonds of the main chains of pectin and pectinic acid (endo-polymethylgalacturonase (endo-PMG), endo-polygalacturonase (endo-). PG), endo-pectintranslylimase (endo-PTE), endo-polygalacturonic acid trans-iriminase (endo-PATE), exo-polymethylgalacturonase (exo-PMG), exo- Pectin liberating the methyl group of polygalacturonase (exo-PG), exo-Pectintranslylimase (exo-PTE), exo-polygalacturonic acid trans-Iliaminase (exo-PATE) and pectin Methyl ester hydrolase, but is not limited thereto.

Plant cells, unlike animal cells, are wrapped in a rigid cell wall. The cell wall is composed of pectin and cellulose at the beginning, but is then cottoned to contain many lignin and hemicellulose in addition to cellulose. Cellulase is an enzyme that breaks down cellulose, and processing it increases the efficiency of extracting active ingredients from plants as the hard cell wall is partially broken down. Therefore, when the cellulase is reacted with the cell wall degrading enzyme before extraction of edelweiss, the efficiency of extracting the active ingredient in the plant cell may be increased as compared with the case where the enzyme is not treated, thereby increasing the efficacy of the raw material.

In the present specification, "enzyme-treated edelweiss extract" may be obtained by treating cell edase on edelweis and extracting it.

In the present specification, the enzyme-treated edelweiss extract, the edelweiss extract, washing and drying the edelweiss; Treating the enzyme with edelweiss; Solvent extraction of edelweiss; And it can be obtained through the step of filtering the extracted edelweiss. In the above, the solvent includes at least one selected from the group consisting of water, an organic solvent, and a mixture of water and an organic solvent. In the above, water includes distilled water or purified water, and the organic solvent is selected from the group consisting of alcohol, acetone, ether, ethyl acetate, diethyl ether, ethyl methyl ketone and chloroform, for example, a lower alcohol having ₁ to ₅ carbon atoms. Including but not limited to one or more.

In one embodiment of the present invention, the enzyme may be selected from the group consisting of cellulase, xylanase, and arabinofuranosidase.

In one aspect of the present invention, the extract may be obtained by adding 0.01 to 1% by weight of the cell wall degrading enzyme relative to the total weight of the edelweiss.

The cell wall degrading enzyme may be contained in 0.001 to 10% by weight relative to the total weight of the edelweiss. If the cell wall degrading enzyme is contained in less than 0.001% by weight, the increase in the content of chlorogenic acid, an active ingredient included in the edelweiss extract is insignificant, and if it contains more than 10% by weight, the content of other components is limited by the content of the cell wall degrading enzyme Done. In view of the above, the edelweiss extract is 0.0011 to 9% by weight, 0.0012 to 8% by weight, 0.0013 to 7% by weight, 0.0014 to 6% by weight, 0.0015 to 5% by weight, 0.0016 to 4% by weight based on the total weight of the composition %, 0.0017 to 3% by weight, 0.0018 to 2% by weight or 0.0019 to 1% by weight.

In one aspect of the present invention, the enzyme-treated edelweiss extract may contain 6 ppm or more of chlorogenic acid. In the case of containing such a range of chlorogen, it can have a better skin regeneration, hair growth promoting effect than the edelweiss extract without enzyme treatment. In view of this, the extract obtained from the edelweiss treated with the cell wall degrading enzyme of the present specification is at least 6.5ppm, at least 7ppm, at least 7.5ppm, at least 8ppm, at least 8.5ppm, at least 9ppm, based on the total extract weight of chlorogenic acid as an active ingredient. It may include more than 9.5ppm, more than 10ppm, more than 10.5ppm, more than 11ppm or more than 11.5ppm, may contain less than 100,000ppm or less than 10,000ppm.

Accelerators according to one aspect of the invention may comprise from 0.01% to 10% by weight, specifically from 0.1% to 5% by weight, more specifically from 0.5% to 3% by weight, based on the total weight of the accelerator. have. When included in the above range is not only suitable for showing the intended effect of the present invention, it can satisfy both the stability and safety of the composition, it may be appropriate to include in the above range in terms of cost-effectiveness. Specifically, when the edelweiss extract is less than 0.01% by weight, sufficient skin regeneration and hair growth promoting effect may not be obtained, and when it exceeds 10% by weight, safety and formulation stability may be lowered.

In another aspect, the present invention relates to a cosmetic composition comprising the edelweiss extract. The cosmetic composition has a skin regeneration effect, specifically it can heal wounds and reduce scars. In addition, the cosmetic composition may promote hair growth, further prevent hair loss and promote wool.

Cosmetic compositions according to the invention may be provided in all formulations suitable for topical application. For example, it may be provided in the form of a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, a solid, a gel, a powder, a paste, a microneedle, a foam, or an aerosol composition. have. Compositions of such formulations may be prepared according to conventional methods in the art.

The cosmetic composition according to the present invention may include other ingredients in addition to the above-mentioned substances within the range not impairing the main effect, preferably giving a synergistic effect to the main effect. In addition, the cosmetic composition according to the present invention may further include a moisturizer, an emulsifier, a surfactant, a ultraviolet absorber, a preservative, a bactericide, an antioxidant, a pH adjuster, an organic and an inorganic pigment, a perfume, a cooling agent, or a restricting agent. The blending amount of the above components can be easily selected by those skilled in the art within the range that does not impair the object and effect of the present invention, the blending amount may be 0.01 to 5% by weight, specifically 0.01 to 3% by weight based on the total weight of the composition have.

In another aspect, the present invention relates to a pharmaceutical composition comprising the edelweiss extract. The pharmaceutical composition has a skin regenerating effect, and may specifically heal wounds and reduce scars. In addition, the pharmaceutical composition may promote hair growth, further prevent hair loss and promote wool.

The pharmaceutical composition according to one aspect of the present invention may be administered orally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously, and the like.

Formulations for oral administration may be, but are not limited to, tablets, pills, soft and hard capsules, granules, powders, granules, solutions, emulsions or pellets. These formulations may contain, in addition to the active ingredient, diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose or glycine), glidants (e.g. silica, talc, stearic acid or polyethylene glycol), or binders (e.g. magnesium aluminum Silicates, starch pastes, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose or polyvinylpyrrolidine). It may optionally contain pharmaceutical additives such as disintegrants, absorbents, colorants, flavors, or sweeteners. The tablets can be prepared by conventional mixing, granulating or coating methods.

Formulations for parenteral administration may be, but are not limited to, injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays.

The active ingredient of the pharmaceutical composition according to one aspect of the present invention will vary depending on the age, sex, weight, pathology and severity of the subject to be administered, the route of administration or the judgment of the prescriber. Dosage determination based on these factors is within the level of one skilled in the art and its daily dosage may be, for example, from 0.1 mg / kg / day to 100 mg / kg / day, more specifically from 5 mg / kg / day to 50 mg / kg. May be, but is not limited to.

In another aspect, the present invention relates to a health food composition comprising an edelweiss extract. The food composition has a skin regenerating effect, and may specifically heal wounds and reduce scars. In addition, the food composition may promote hair growth, further prevent hair loss and promote wool.

The formulation of the food composition is not particularly limited, but may be, for example, formulated into a liquid such as tablets, granules, powders, drinks, caramels, gels, bars, and the like. In addition to the active ingredient, the food composition of each formulation may be suitably selected by a person skilled in the art according to the formulation or purpose of use in addition to the active ingredient, and may be synergistic when applied simultaneously with other raw materials.

Determination of the dosage of the active ingredient is within the level of those skilled in the art, the daily dosage of which is for example from 0.1 mg / kg / day to 5000 mg / kg / day, more specifically from 50 mg / kg / day to 500 mg / kg / day, but is not limited thereto, and may vary depending on various factors such as age, health condition, and complications of the subject to be administered.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to Examples and Test Examples. However, these examples and test examples are provided only for the purpose of illustration in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

Example 1 Preparation of Edelweiss Extract

Edelweiss ( Leontopoium alpinum ) was washed with ultrapure water and filtered over a 250 mesh filter and dried at 50 ° C for 24 hours. 20 L of distilled water is added to 1 kg of dry edelweiss and extracted for 48 hours under a temperature condition of 4 ° C. After that, the precipitate is filtered through a 250 mesh filter, and then again filtered through a 0.5 μm filter. After decolorization and deodorization with activated carbon, the edelweiss extract is obtained by filtration through a 0.5 μm filter.

Example 2 Preparation of Extract of Edelweiss Treated with Enzyme

Edelweiss ( Leontopoium alpinum ) was washed with ultrapure water and filtered over a 250 mesh filter and dried at 50 ° C for 24 hours. To 1 kg of dry Edelweiss, 2 L of distilled water is added and 100 g of cellulase is added to adjust the pH to 4.8. The reaction is carried out at 40 ° C. for 72 hours. Then 18 L of distilled water is added and extracted for 48 hours under the temperature condition of 4 ℃. After that, it is filtered through a 250 mesh filter and once again filtered through a 3μm filter. After decolorization and deodorization with activated carbon, the edelweiss extract is obtained by filtration through a 0.5 μm filter.

Test Example 1 Evaluation of the Wint / Betacatenin Signal Transduction Promoting Effect of Stem Cells

When Wnt signals are given to stem cells, protein complexes (GSK3, APC and Axin) that inhibit intracellular beta-catenin are inhibited and beta-catenin moves into the cell nucleus and is involved in development and hair growth. Various genes are expressed.

Beta-catenin is a transcriptional regulator attached to a specific nucleotide sequence of the chromosome, and a plasmid vector obtained by artificially combining the beta-catenin attachment nucleotide sequence followed by a Gaussian luciferase gene sequence is prepared for HEK293 (human embryos). Renal cell line). The transformed cell line thus prepared is resistant to G418 antibiotics, and when cultured in a medium containing G418, only the transformed cell line can be selectively cultured. The transformed HEK293 cell line having the Wint / Beta catenin signal transduction system thus obtained was treated with the Edelweiss extract of Example, and then Gaussian luciferase assay kit (BioLabs) increased the expression of Gaussian luciferase. Confirmed.

Specifically, the transgenic HEK293 cells, 10% bovine serum serum (Fetal bovine serum) (FBS) and G418 500μg / Dulbecco's modified Eagle's medium with ml containing; a (DMEM Gibco BRL, Gaithersburg, MD , USA) 5% CO 2 Incubated for 24 hours in an incubator at 37 ℃. Cells cultured in 96-well plates for 24 hours were treated with 2 ppm and 5 ppm of edelweiss extract of Example 1 and 2ppm and 5ppm of enzyme-treated edelweiss extract of Example 2, respectively, and then cultured in an incubator for 48 hours. . As a positive control, 16.6 ppm, 50 ppm and 83 ppm of sodium valproic acid (VPA), which is widely used as a glycogen synthase kinase 3-beta (GSK-3β) inhibitor, were used. Luciferase expression levels of the cultured cells were measured using a Gaussian luciferase assay kit to evaluate the effect of Edelweiss extract on wint / beta catenin signal transduction. The results are shown in FIGS. 1 and 5.

As can be seen in Figure 1, the edelweiss extract promotes the wint / beta catenin signal transduction of stem cells. It can be seen that the edelweiss extract has the effect of promoting skin regeneration and hair growth.

In addition, as shown in Figure 5, the edelweiss extract and the enzyme-treated edelweiss extract promotes the wint / beta catenin signal transmission of stem cells, it can be seen that the enzyme-treated edelweiss extract is better than the general edelweiss extract. The results show that the treatment of cellulase enzyme can increase the skin regeneration and hair growth promoting effect of the edelweiss extract.

Test Example 2 Evaluation of Alkaline Phosphatase Activity

In order to evaluate whether Edelweiss extract can maintain the stem cellularity of human dermal papilla cell lines, we applied an in vitro system to determine the alkaline phosphatase enzymes involved in maintaining the stem cell lines after application of Edelweiss extract. Activity was evaluated.

Human immortalized dermal papilla, iDPc, is an immortalized dermal papilla cell prepared by transducing human dermal papilla cells with a vector containing Simian virus 40 large T (SV40T) and c-myc. cell) was used. The cell lines were incubated for 24 hours in an incubator at 5% CO ₂ , 37 ° C. in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Gaithersburg, MD, USA) containing 10% bovine serum serum (FBS), respectively. 10 ppm and 20 ppm edelweiss extracts of 1 were treated. DMSO was used as a negative control, and 83 ppm of sodium valproic acid (VPA), which is widely used as a glycogen synthase kinase 3 beta (GSK-3β) inhibitor, was used as a positive control. 166 ppm was used. After 48 hours of treatment with the test material, the cells were washed twice with PBS Phosphate buffered saline on ice, and then 300 μl ALP assay buffer (ALP assay kit, BioVision, Catalog # K412-500) was placed on the cells and scraped. The scrapes were collected in eppendorf tubes. The collected cells were awakened on a probe sonicator on ice, centrifuged at 4 ° C., 12,000 rpm for 5 minutes, and the supernatant was taken. Using the Bio-rad DC protein kit (Biorad, cat.no. 500-0111) based on Lowry's method, included in the supernatant according to the manufacturer's recommended method The concentration of cellular protein was measured. The alkaline phosphatase activity of the test substance was evaluated in terms of the measured concentration values.

Specifically, nitrophenyl phosphate (pNPP), which is a substrate of alkaline phosphatase enzyme, is dissolved in pNPP tab (ALP assay kit, BioVision, Catalog # K412-500) assay buffer, prepared at a concentration of 5 mM, and reacted with cellular proteins. Put it. The pNPP dissolved in 1mM concentration was added to 4 ~ 20 농도 by concentration, and the standard solution was prepared by adding an ALP enzyme to cause the enzymatic degradation reaction of pNPP. In order to cause the above enzymatic reaction, a 96-well plate containing test material and standard solution was placed in a 25 ° C. incubator for 1 hour, and then, a stop solution was added, and the absorbance was measured at 405 nm. The activity value of the enzyme was determined by the amount of pNP produced by degradation. The results are shown in FIG.

As can be seen in Figure 2, the edelweiss extract increased the alkaline phosphatase activity in the dermal papilla cells. In other words, it can be seen that the edelweiss extract can promote hair production by maintaining and activating the stem cell nature of the dermal papilla cell line.

Test Example 3 Evaluation of hair follicle papilla cell proliferation effect

The keratin proteins that make up hair are produced in hair root keratinocytes, which are differentiated from dermal papilla cells. Therefore, if the edelweiss extract promotes the activity of the dermal papilla cells, it may promote the activity of keratinocytes differentiated therefrom and further promote hair production.

In order to evaluate the effect of promoting the dermal papilla cell activity of the edelweiss extract, iDPc (Human immortalized dermal papilla cell) cell line was used. The cell line is an immortalized papillary cell prepared by transducing human dermal papilla cells with a vector containing Simian virus 40 large T (SV40T) and c-myc. The cell lines were incubated for 24 hours in an incubator maintained at 5% CO ₂ , 37 ° C. in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Gaithersburg, MD, USA) containing 10% bovine serum serum (FBS). The 2ppm and 5ppm edelweiss extracts of Example 1 and the 2ppm and 5ppm enzymatically treated edelweiss extracts of Example 2 were treated. DMSO was used as a negative control. 24 hours after the test material treatment, the cell proliferation (%) was measured using the WST-1 kit (Roche). The results are shown in FIGS. 3 and 6.

As can be seen in Figure 3, the edelweiss extract significantly increased the proliferation of dermal papilla cells. It can be seen that the edelweiss extract may promote the growth of dermal papilla cells, and further promote the activity and hair production of keratinocytes.

As can be seen in Figure 6, the enzyme treated edelweiss extract increased the growth of dermal papilla cells compared to before the enzyme treatment. It can be seen that the enzyme-treated edelweiss extract can promote the growth of dermal papilla cells and further promote the activity and hair production of keratinocytes.

Test Example 4 Evaluation of Hair Growth Promoting Effect Using Human Hair Follicle Organs

Hair follicles were isolated from the scalp and scalp tissue of a 55 year old male with William's E medium (L-glutamine (2 mM), insulin (10 µg / ml), hydrocortisone (40 ng / ml), antibiotics (1%), fungizon (1%). )). After 3 days of culture, the hair follicles were grown, cut to 3 mm, and the selected hair follicle tissues were treated with no drug in the control group, and the experimental group was treated with minoxidil 10 μM and the Edelweiss extract of Example 1 50 ppm and 100 ppm. After 3 days, 7 days, 11 days, and 14 days after drug treatment, length measurement and photographing were performed, respectively. The results are shown in FIG.

As can be seen in Figure 4, when treated with 50ppm edelweiss extract in the hair length increase in hair follicle organs showed a hair growth promoting effect similar to the minoxidil positive control. Moreover, when 100ppm of edelweiss extract was treated, the effect was superior to minoxidil. All of these are statistically significant. Therefore, it can be seen that the edelweiss extract has an effect of promoting the length of hair growth in the hair follicles.

In addition, the length of the growth occurs when the hair is in the anagen phase of the hair cycle, and the hair begins to fall out when it is transferred to the catagen. In the Edelweiss treatment group, even after 14 days of treatment, the ratio of hair follicles in the growing stage was higher than that of minoxidil at both concentrations, indicating an effect of reducing hair follicles that metastasize to hair growth.

Test Example 5 Analysis of Indicator Material Content in Edelweiss Extract

Chlorogenic acids, luteolin, apigenin, leontopodic acid and the like are known as compounds present in the edelweiss extract. The indicators can be qualitatively and quantitatively analyzed by high performance liquid chromatography (HPLC) analysis. Chlorogenic acid is a kind of polyphenolic compound contained in coffee in large quantities and is known to have an antioxidant effect. Since the content of the chlorogenic acid was the highest when the content of the indicator substance in the edelweiss extract was analyzed, the content of chlorogenic acid was used in the present invention to analyze the extraction efficiency of the enzyme treatment extraction method.

Detailed analysis method is as follows. The edelweiss extract of Example 1 and the enzyme treatment of the edelweiss extract of Example 2 were analyzed the stock solution, and chlorogenic acid used as a standard was dissolved in 50% methanol at a concentration of 5 g / ml. The HPLC instrument used in the analysis was a Water Alliance 2695E model HPLC, the detector was a PDA detector 2998 model from Water, and the column was Mightysil-RP18 (4.6 x 250, 5 um). Standard was prepared at 2,5,10 μg / mL to draw a 3-point calibration curve to quantify the chlorogenic acid in the edelweiss extract.

10 μl of the extract was injected and the analytical solvent was flowed A (20 mM KH ₂ PO ₄ (pH 2.5)) / B (ACN) into the following gradient. The flow rate is 1 mL / min.

Table 1

time (min)	A	B
0	96	4
One	96	4
60	55	45
65	40	60
70	40	60
75	96	4
82	96	4

The retention time of chlorogenic acid is 17 minutes and the detection wavelength is 326 nm.

As shown in Figure 7, after the cellulase enzyme treatment it can be seen that the amount of chlorogenic acid in the edelweiss extract increased significantly compared to before treatment. In other words, by treating the cellulase enzyme, as the Edelweiss cell wall was decomposed, it was found that the extraction efficiency of the indicator component in the plant was increased. Therefore, it can be seen that the enzyme-treated edelweiss extract obtained by the extraction method including the present enzyme treatment may contain a large amount of the active ingredient compared to the extract without the enzyme treatment.

Test Example 6 Evaluation of Antioxidant Effect of Edelweiss Index Component

The DPPH antioxidant assay was used to evaluate the antioxidant effect of chlorogenic acid, an edelweiss index component. Oxidation reactions occurring in the body are mainly caused by free radicals, and this action is known to lead to the aging of cells and the development of cancer. Therefore, inhibition of free radical production in the body may eventually be associated with antioxidant and anti-aging in the body, and since this antioxidant effect is difficult to experiment with free radicals in the body, DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals may be It is used universally. DPPH radicals have a deep purple color. When they react with antioxidant compounds, they turn to pale yellow color and measure their absorbance at 517 nm. Higher antioxidants have a lighter yellow color and lower absorbance.

In this experiment, 0.1mM DPPH solution was added to a 96-well plate, and the absorbance was measured at 517 nm by reacting the positive control vitamin C and chlorogenic acid, respectively. At this time, water was used as a solvent, vitamin C was treated in the amount of 25, 50, 100μg / ml, chlorogenic acid was treated in the amount of 1.25, 2.5, 5, 10ppm. The reaction was carried out for 30 minutes in a 37 ℃ incubator. The results are shown in FIG.

As can be seen in Figure 8, when treated with chlorogenic acid was confirmed to show an antioxidant effect in a concentration-dependent. Through this, it can be seen that in the case of the enzyme treatment Edelweiss extract with an increased content of chlorogen, the antioxidant effect can be increased compared to before the treatment.

Examples of the formulation of the composition according to one aspect of the present invention will be described below, but can be applied to various other formulations, which are intended to explain in detail only, not intended to limit the invention.

Formulation Example 1 Preparation of Ointment

Ointments are prepared according to conventional methods with the compositions described in the table below.

TABLE 2

ingredient	Content (% by weight)
Purified water	Remaining amount
glycerin	8.0
Butylene glycol	4.0
Liquid paraffin	15.0
Beta Glucan	7.0
Carbomer	0.1
Edelweiss extract	5.0
Caprylic Capric Triglycerides	3.0
Squalene	1.0
Cetearyl Glucoside	1.5
Sorbitan stearate	0.4
Stearyl alcohol	1.0
antiseptic	Quantity
incense	Quantity
Pigment	Quantity
beeswax	4.1

Formulation Example 2 Preparation of Hair Lotion

A hair lotion is prepared according to a conventional method with the composition described in the table below.

TABLE 3

ingredient	Content (% by weight)
Purified water	Remaining amount
glycerin	3.0
Butylene glycol	3.0
Liquid paraffin	7.0
Beta Glucan	7.0
Carbomer	0.1
Edelweiss extract	2.0
Caprylic Capric Triglycerides	3.0
Squalene	5.0
Cetearyl Glucoside	1.5
Sorbitan stearate	0.4
Polysorbate	1.2
antiseptic	Quantity
incense	Quantity
Pigment	Quantity
Triethanol amine	0.1

Formulation Example 3 Preparation of Cream

A cream is prepared according to a conventional method with the composition described in the table below.

Table 4

ingredient	Content (% by weight)
Purified water	Remaining amount
glycerin	8.0
Butylene glycol	4.0
Liquid paraffin	45.0
Beta Glucan	7.0
Carbomer	0.1
Edelweiss extract	1.5
Caprylic Capric Triglycerides	3.0
beeswax	4.0
Cetearyl Glucoside	1.5
Sesqui oleic acid sorbitan	0.9
Vaseline	3.0
antiseptic	Quantity
incense	Quantity
Pigment	Quantity
Triethanol amine	0.1

Formulation Example 4 Tablets

10 mg of edelweiss extract, 50 mg of soybean extract, 100 mg of glucose, 50 mg of red ginseng extract, 96 mg of starch and 4 mg of magnesium stearate are mixed and granules are formed by adding ethanol, dried and tableted.

Formulation Example 5 Healthy Drink

Edelweiss extract ............ 100 mg

Citric Acid ................................... 1000 mg

Oligosaccharide ......................... 100 g

Taurine .................................... 1 g

Purified water ..................

The above ingredients are mixed according to a conventional method for preparing a health beverage, then stirred and heated at 85 ° C. for about 1 hour, and then the resulting solution is filtered and sterilized.

Claims

Skin regeneration accelerator comprising edelweiss extract as an active ingredient.
Hair growth promoter comprising an edelweiss extract as an active ingredient.
The promoter of claim 1, wherein the edelweiss extract is an enzyme treated edelweiss extract.
The promoter of claim 2, wherein the edelweiss extract is an enzyme treated edelweiss extract.
The promoter according to claim 3 or 4, wherein the enzyme is a cell wall degrading enzyme.
The promoter according to claim 3 or 4, wherein the enzyme is selected from the group consisting of cellulase, xylanase and arabinofuranosidase.
The promoter according to claim 3 or 4, wherein the extract is obtained by adding an enzyme in an amount of 0.001 to 1% by weight based on the total weight of edelweiss.
The promoter according to any one of claims 1 to 4, wherein the promoter comprises an edelweiss extract or an extract of the enzyme treated edelweiss in an amount of 0.01 to 10% by weight based on the total weight of the promoter.
The skin regeneration accelerator according to claim 1 or 3, wherein the accelerator heals a skin wound.
The hair growth promoter according to claim 2 or 4, wherein the accelerator prevents hair loss or promotes wool.
The promoter according to claim 3 or 4, wherein the promoter has an antioxidant capacity.
The accelerator according to claim 3 or 4, wherein the edelweiss extract contains chlorogenic acid, based on the total extract weight, of 6 ppm or more.
5. The promoter according to claim 1, wherein the edelweiss is Leontopoium sp.
5. The promoter of claim 1, wherein the edelweiss is Leontopoium alpinum . 6.
The method according to any one of claims 1 to 4,

A promoter is a promoter that activates a Wnt signal in a cell.
Cosmetic composition containing an accelerator according to any one of claims 1 to 4.
A pharmaceutical composition comprising an accelerator according to any one of claims 1 to 4.