KR102276483B1 - Composition for Preventing or Treating Hair Loss or Stimulating Hair Sprouting or Hair Growth Comprising Complex Extracts of Prunellae spica, Polygonum cuspidata, and Cnidium officinale as an Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating hair loss or stimulating hair sprouting or hair growth, including a combined plant extract of Prunella vulgaris, Polygonum cuspidatum and Cnidium officinale as an active ingredient. The ternary combined extract including Prunella vulgaris, Polygonum cuspidatum and Cnidium officinale according to the present invention stimulates proliferation of human keratinocytes and dermal papilla cells, significantly reduces the expression of DKK1 gene which inhibits the Wnt/β-catenin signaling system, increases the expression of vascular endothelial growth factor (VEGF) which is a hair sprouting-stimulating growth factor inducing extension of anagen of hair growth, directly inhibits production of nitrogen oxide playing an important role in skin inflammation response, increase hair thickness and reduces a water swelling degree of hair, and thus can be used as a composition for sprouting or growing hair, which shows an stable effect of stimulating hair sprouting and hair growth, and can be used effectively for preventing or treating diseases, such as depilation.

Description

A composition for preventing or treating hair loss, or a composition for preventing or treating hair loss or Stimulating Hair Sprouting or Hair Growth Comprising Complex Extracts of Prunellae spica, Polygonum cuspidata, and Cnidium officinale, comprising a plant complex extract as an active ingredient as an Active Ingredient}

The present invention relates to a composition for preventing or treating hair loss, or for promoting hair growth or hair growth, comprising a plant complex extract as an active ingredient. It relates to a composition for preventing or treating hair loss, or for promoting hair growth or hair growth.

In the human body, hair grows from hair follicles in the skin, and hair follicles from which hair grows are complex organs composed of inner root epithelium, outer root epithelial sheath, hair stem, and hair matrix. It is characterized by continuous repetition. In particular, among the cells forming the hair follicle, dermal papilla cells play an important role in hair growth, and during the growth phase of the hair follicle, the cells of the hair follicle including keratinocytes of the matrix surrounding the dermal papilla proliferate. In the degenerative phase of the hair follicle, hair follicle growth is stopped, and apoptosis, condensation of the dermal papilla, and the reorganization of the extracellular matrix occur. Only part remains.

Hair loss refers to an increase in abnormally falling hair due to a higher proportion of hair growing in the follicles in the degenerative or resting phase than the hair growing in the follicles in the growth phase during the hair follicle growth cycle. With the recent trend to emphasize appearance, interest in hair loss is also increasing. As the cause of hair loss, the theory of poor blood circulation, excessive male hormone action, excessive sebum secretion, decreased scalp function due to peroxide and bacteria, genetic factors, aging, stress, etc. have been discussed, but there is still no clear explanation for hair loss. The cause is unknown. In general, the most typical form of hair loss is androgenetic alopecia, which occurs due to excessive androgen activity, and it occurs dependent on androgen, which is a male hormone. Representative androgens include testosterone and dihydrotestosterone (DHT), but in patients with hair loss, DHT is higher. known to work. Testosterone is converted to DHT by 5 alpha-reductase (5-reductase), and this conversion occurs by 5 alpha-reductase of Type 2 among two types of 5 alpha-reductase, Type 1 and Type 2, It has been reported that the gradual reduction of the dermal papilla and hair follicles is progressed by the converted DHT.

Currently, the most widely used in the treatment and prevention of such alopecia is Minoxidil-containing formulation of Upzone, which has been approved by the US FDA, and recently Merck's finasteride (finasteride), which has a 5-alpha-reductase inhibitory effect of Type 2 ) as the main ingredient, Propecia, has been released and used.

In addition, studies based on natural extracts have also been conducted, and extracts such as cheongung (Patent No. 2010-0119761), Sanghwang mushroom (Patent No. 2012-0038426), gold and silver flower (Patent No. 2012-0038426), chemomile, etc. (Patent No. 2012-0015874, Patent No. 2010-0091571), Hasuo (Patent No. 2013-0021464, Patent No. 2011-0097386), Calamus (Patent No. 2012-0115980), Pomegranate (Patent No. 2012-7012465), There are results such as ginkgo leaf extract (Patent No. 2006-7008793). However, when these hair growth agents or hair restorers are actually used in clinical practice, the effects on the scalp are not so excellent, and they are more effective than minoxidil or Propecia, which are reluctant to use due to concerns about side effects, and are made of natural substances with fewer side effects. Research to explore is being actively conducted.

Under the above background, the present inventors have made intensive research efforts to use it for the prevention or treatment of hair loss symptoms, and as a result, the complex extracts of Hagogi, hojanggeun, and cheongung promote the proliferation of human keratinocytes and dermal papilla cells, and Wnt It significantly reduces the expression of the DKK1 gene, which suppresses the /β-catenin signaling system, increases the expression of vascular endothelial growth factor (VEGF), a hair growth promoting growth factor that induces extension of the hair growth phase, and improves the skin inflammatory response. By directly inhibiting the production of nitric oxide, which plays an important role, increasing the thickness of the hair, and reducing the moisture swelling of the hair, it is confirmed that there is an excellent effect in preventing or treating diseases such as alopecia, and completing the present invention became

Accordingly, an object of the present invention is a pharmaceutical composition for preventing or treating hair loss, or for promoting hair growth or hair growth, comprising a complex extract of hagocho, hojanggeun, and cheongung as an active ingredient; A cosmetic composition for improving hair loss, or promoting hair growth or hair growth; And for improving hair loss, or to provide a functional food composition or food composition for promoting hair growth or hair growth.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for preventing or treating hair loss, or for promoting hair growth or hair growth, comprising a complex extract of hagogi, hojanggeun, and cheongung as an active ingredient.

The present inventors made intensive research efforts to discover natural ingredients that can effectively prevent hair loss or promote hair growth or hair growth. As a result, it was confirmed that the three kinds of complex extracts obtained by the combination of the hagocho, hojanggeun, and cheongung effectively promote hair growth and hair growth and prevent hair loss.

In the present invention, the term "Hagocho (Prunella vulgaris Linn*?*)" means a perennial grass of the dicotyledonous plant order Lamiaceae, and the term "Hojanggeun (Polygonum cuspidata SIEB. et ZUCC.)" is It means perennial herbaceous plants belonging to the family, and "Cnidium officinale" means perennial herbs of the Dicotyledonous plant Dicotyledonous family, Umbilicalaceae, Butteraceae.

As used in the present invention, the term "extract" refers to an extract obtained by the extraction treatment of H. radish root, rhododendron, and chrysanthemum, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, and a prepared or purified product of the extract. , or a mixture thereof, and the like, including extracts of all formulations that can be formed using the extract itself and the extract. The extract of the present invention may be prepared and used in the form of a liquid or dry powder, preferably after extraction.

In the extraction of hagocho, hojanggeun, and cheongung, the method of extracting the extract is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method may include a solvent fractionation method, a centrifugation method, and the like, and these may be performed alone or in combination of two or more methods.

As used herein, the term “hair loss” refers to a phenomenon in which hair is completely pulled out of the scalp. In addition, the term "hair growth" of the present specification means that hair grows from the scalp, and the term "hair growth" of the present specification means that the length of the hair becomes longer and thicker, and "wool" is another term used in the art. used in the same sense as As demonstrated in the examples below, the extract of the present invention Hagocho, H. janggeun, and chrysanthemum complex extract promotes cell proliferation of human hair follicle dermal papilla cells (HFDPC) and keratinocytes (HaCaT) (Fig. 1 and 2), significantly suppresses the expression of the DKK1 gene, which suppresses the Wnt/β-catenin signaling system ( FIG. 3 ), and vascular endothelial growth factor (VEGF), which is a hair growth promoting growth factor that induces extension of the hair growth phase ) increases the expression (FIG. 4), protects the scalp by directly inhibiting the production of nitric oxide, which plays an important role in the skin inflammatory response (FIG. 5), increases the thickness of the hair, and reduces the degree of moisture swelling of the hair By ( FIG. 6 ), it was confirmed that there is an effect of preventing, improving or treating hair loss.

In the pharmaceutical composition for preventing or treating hair loss of the present invention, or for promoting hair growth or hair growth, the extracts of the plantargonaceae, rhizome, and chrysanthemum complex extract are human hair follicle dermal papilla cells (HFDPC) and keratinocytes (HaCaT) promotes cell proliferation.

In addition, the plant extract of the present invention is characterized in that it inhibits the expression of DKK-1 (Dickkopf-1) gene, and promotes the production of vascular endothelial growth factor (VEGF).

In the dermal papilla cells of hair loss patients, it was observed that the expression of DKK1 protein was increased by dihydrotestosterone (DHT) formed by 5-alpha-reductase, which has long been considered the main cause of hair loss. In addition, Wnt protein is known as a protein that activates hair follicle stem cells, and thus plays an important role in the formation of hair follicles. Since DKK1 protein is a protein that inhibits Wnt protein signal transduction, it inhibits DKK1 protein expression and thus Wnt protein signal transduction. can increase Therefore, suppression of the expression of DKK1 protein promotes the growth of hair follicles due to the activation of Wnt protein, and thus a hair growth effect can be expected.

On the other hand, vascular endothelial growth factor (VEGF), which plays an important role in various cell functions such as cell survival and proliferation, as well as a major factor in promoting angiogenesis, is a hair growth promoting growth factor that induces extension of the hair growth phase.

In a preferred embodiment of the present invention, the three kinds of complex extracts composed of Hagonia spp. of the present invention, hojanggeun, and cheonggung inhibit DKK-1 (Dickkopf-1) gene expression in a concentration-dependent manner, and VEGF (Vascular endothelial growth factor) production It has an effect of increasing the , and it has been confirmed that there is a synergistic effect by the combination of these three extracts compared to each single extract (see FIGS. 3 and 4).

In addition, the hagocho, hojanggeun, and cheongung complex extract of the present invention has anti-inflammatory activity through inhibition of nitric oxide (NO) production. In a preferred embodiment of the present invention, the three kinds of complex extracts composed of Hagogi, H. rhizome, and cheonggung treat or prevent skin inflammation by directly inhibiting the production of nitric oxide, which plays an important role in the inflammatory response, and, through this, It was confirmed that there is an effect of protecting (see FIG. 5).

In addition, the three kinds of complex extracts composed of hagocho, hojanggeun, and cheongung of the present invention increase the thickness of the hair and reduce the moisture swelling of the hair to protect the hair. In a preferred embodiment of the present invention, it has been confirmed that the three kinds of complex extracts composed of Hagogi, hojanggeun, and cheongung have the effect of protecting the hair by increasing the thickness of the hair and reducing the degree of moisture swelling of the hair (Fig. 6).

Therefore, the composition of the present invention can be applied as a multi-faceted and stable hair loss prevention or treatment, or an effective hair loss prevention or treatment composition, or a hair growth / hair growth agent composition that exhibits hair growth and hair promoting efficacy.

According to the present invention, the composition of the present invention is alopecia areata, androgenetic alopecia, tinea capitis, hypotrichosis, hereditary hypotrichosis simplex, localized alopecia (circumscribed alopecia), alopecia congenitalis, alopecia pubis, alopecia seborrheica, alopecia senilis, alopecia totalis, alopecia universalis, and telogen alopecia (telogen efffluvium) can be used to treat any one of alopecia selected from.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally. According to one embodiment of the present invention, it is administered by a parenteral method, and according to another embodiment of the present invention, it is administered by a transdermal administration method.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity of the patient. can be A preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.0001-100 mg/kg for adults.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

According to another aspect of the present invention, the present invention provides a functional food or food composition for improving hair loss, or for promoting hair growth or hair growth, comprising as an active ingredient three kinds of complex extracts consisting of hagogi, hojanggeun, and cheongung. .

The food composition of the present invention may be included in the form of a fraction or a processed product of three kinds of complex extracts consisting of hagocho, hojanggeun, and cheongung. In addition, the composition may include a food pharmaceutically acceptable food supplement additive in addition to the active ingredient.

In the present invention, "food supplementary additive" means a component that can be added to food as an auxiliary, added to the manufacture of health functional food of each formulation can be appropriately selected and used by those skilled in the art. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.

The food composition of the present invention may include a health functional food. In the present invention, "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful for the human body. Here, "functional" means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body, specifically, obtaining a useful effect for improving hair loss or promoting hair growth or hair growth it means. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art.

In addition, the dosage form of the health functional food may also be manufactured without limitation as long as it is a dosage form recognized as a health functional food. The food composition of the present invention can be prepared in various types of dosage forms, and unlike general drugs, natural plant extracts are used as raw materials, so there are no side effects that may occur during long-term administration of the drug, excellent portability, and skin It can be taken as a supplement to increase the moisture content of the skin, reduce the occurrence of inflammation, and enhance the effect of shrinking pores.

There is no limitation on the form that the health functional food of the present invention can take, and it may include any food in a conventional sense, and may be used in combination with terms known in the art, such as functional food. In addition, the health functional food of the present invention can be prepared by mixing known additives with other suitable auxiliary ingredients that may be included in the food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding to juice, tea, jelly, juice, etc. prepared by using the three types of complex extracts composed of Hagogi root, hojanggeun, and cheongung according to the present invention as main components. Also included are foods used as feed for animals.

According to another aspect of the present invention, the present invention provides a cosmetic composition for improving hair loss, or for promoting hair growth or hair growth, comprising as an active ingredient three kinds of complex extracts consisting of hagocho, hojanggeun, and cheongung.

The ingredients included in the cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions in addition to the three kinds of complex extracts composed of Hagonia spp., horjanae root, and chrysanthemum, for example, antioxidants, stabilizers, solubilizers, vitamins, and pigments. and conventional adjuvants such as perfumes, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like can be used.

Since the food composition and the cosmetic composition of the present invention have in common the above-described pharmaceutical composition, active ingredients and uses, common content in relation to the pharmaceutical composition is omitted in order to avoid excessive complexity of the present specification. .

The features and advantages of the present invention are summarized as follows:

(1) The present invention provides a composition for preventing hair loss, or for promoting hair growth or hair growth, comprising a complex extract of sagebrush, hojanggeun, and cheongung as an active ingredient.

(2) The three kinds of complex extracts composed of Hagogoniasis, H. janggeun, and Chunkyung of the present invention promote the proliferation of human keratinocytes and dermal papilla cells, and suppress the Wnt/β-catenin signaling system of DKK1 gene expression. It significantly reduces, increases the expression of vascular endothelial growth factor (VEGF), a hair growth promoting growth factor that induces extension of the hair growth phase, and directly inhibits the production of nitric oxide, which plays an important role in the skin inflammatory response, By increasing the thickness of and reducing the degree of moisture swelling of the hair, it can be usefully used as a hair growth or hair growth composition showing stable hair growth and hair promotion efficacy, and can be effectively used for preventing or treating diseases such as alopecia.

(3) In addition, the composition of the present invention is not only very safe for the human body, but also has excellent stability.

1 is a graph showing the measurement of the cell viability of human keratinocytes (HaCaT) according to the treatment of a single extract or a complex extract thereof of hagocho, hojanggeun, and cheongung.
Figure 2 is a graph showing the measurement of cell viability of human hair follicle dermal papilla cells (HFDPC) according to the treatment of a single extract or a complex extract thereof of Hagogi, hojanggeun, and chrysanthemum.
3 is a graph showing the analysis of the expression level of the DKK-1 gene according to the treatment of a single extract or a complex extract thereof of hagocho, hojanggeun, and cheongung.
4 is a graph showing changes in the expression of vascular endothelial growth factor (VEGF) according to the treatment of a single extract or a complex extract thereof of hagocho, hojanggun, and cheongung.
5 is a graph showing the change in nitric oxide (NO) production according to the treatment of hagocho, hojanggeun, and cheongung single extract or a complex extract thereof.
6 is a graph showing changes in hair thickness and moisture swelling according to treatment with a single extract or a complex extract thereof of hagocho, hojanggeun, and cheongung.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

Example 1. Preparation of hagocho, hojanggeun, and cheongung extract

1-1. Preparation of Hagogi extract

After pulverizing 100 g of dried seaweed, using 3 L of purified water as a solvent, heating and refluxing for 12 hours, followed by cooling, and filtering with filter paper having a permeation size of 1.2 μm to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare a dry powder of hakama.

1-2. Preparation of Hojang root extract

After pulverizing 100 g of dried rhizome, 3 L of purified water was used as a solvent, followed by heating and reflux extraction for 12 hours, followed by cooling, followed by filtration with filter paper having a permeation size of 1.2 μm to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare a dried rhizome.

1-3. Preparation of cheongung extract

After grinding 100 g of dried chrysanthemum roots, using 3 L of purified water as a solvent, heating and refluxing extraction for 12 hours, cooling, and filtering with filter paper having a 1.2 μm permeation size to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare a dried cheongung powder.

1-4. Manufacture of complex extracts of hago grass, hojanggeun, and cheongung

After grinding 100 g of a mixed material mixed with the same amount of dried seaweed, rhododendron and chrysanthemum, 3L of 70% ethanol was used as a solvent, heated and refluxed for 12 hours, and cooled, followed by cooling, and filter paper having a 1.2 μm permeation size. Filtration gave a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare dry powder of three kinds of plant materials.

Example 2. Human Keratinocyte Cell Viability Test

The effects of single or complex extracts of Haggis, hojanggeun and chrysanthemum on the growth of human immortalized keratinocytes (HaCaT) were confirmed by the following method.

^{First, human keratinocytes were inoculated in a 24-well cell culture dish at a concentration of 1×10 5} in DMEM (Dulbeccos modified Eagles medium) medium, which is a dedicated medium containing 10% FBS (fetal bovine serum, Cambrex), for 24 hours. , 37° C., 5% CO ₂ Incubated under humid conditions. Thereafter, the medium was removed and the single or complex extracts of H. rhododendrons prepared in Examples 1-1 to 1-4 diluted with serum-free DMEM medium were treated by concentration and cultured for 24 hours. 24 hours later, 1 mg/mL of MTT was treated, and 2 hours later, formazan generated by MTT treatment in cells was dissolved in DMSO and absorbance was measured at 570 nm.

As a result, it was confirmed that the cell viability of human keratinocytes was further increased as the concentration of treatment increased when single or complex extracts of Hagonia spp., hojanggeun and cheongung were treated on human keratinocytes, which are cells constituting hair. Human keratinocytes showed the highest survival rate in the group treated with the complex extracts of , H. spp., hojanggeun, and cheongung.

Through the above results, it could be confirmed that single or complex extracts of Hagogi, hojanggeun and cheongung increase cell survival without being toxic to the human body (see FIG. 1).

Example 3. Evaluation of hair growth efficacy through human dermal papilla cell (HFDPC) proliferation activity assay

In order to confirm the hair growth promoting effect of the three kinds of complex extracts of Hagogi, hojanggeun and cheongung, prepared in Example 1, cells of human hair follicle dermal papilla cells (HFDPC) of hair follicles involved in hair generation By analyzing the proliferative activity, the hair growth efficacy of the complex extract of hagocho, hojanggeun and cheongung was evaluated.

Human dermal papilla cells (HFDPC) were seeded in 96-well plates at a density of ^{2×10 3 cells/well and cultured overnight.} After changing to a serum-free medium, the single or complex extracts prepared in Example 1 were treated at concentrations of 1, 10, and 100ug/mL, cultured for 3 days, and 4mg/ml MTT solution was added 100μl each. The wells were treated and reacted for 4 hours. After dissolving the generated formazan through DMSO treatment, absorbance at 560 nm was measured using a microplate reader.

Referring to Figure 2, it was confirmed that the proliferation of dermal papilla cells was increased in a concentration-dependent manner in the group to which the three types of complex extracts of hagocho, hojanggeun and cheongung of the present invention were added, and at a concentration of 100ug/mL, in the sample untreated group. It was confirmed that the maximum increase of 131% compared On the other hand, in the group treated with the single extracts of Hagogia, Hojanggeun and Chunkyung, the proliferative activity of dermal papilla cells was found to be somewhat insignificant compared to these complex extracts, and based on the concentration of 100ug/mL, the complex extracts of Hagoca, Hojanggeun and Chunkyung were found to be insignificant. According to the treatment, it was confirmed that the proliferative activity of dermal papilla cells increased by 112%, 113%, and 114%, respectively, compared to their single extract.

In summary, it can be confirmed that the three kinds of complex extracts composed of Haggocho, hojanggeun and cheongung of the present invention increase the growth of human dermal papilla cells in a concentration-dependent manner, and through this, it can be effectively used for preventing or treating diseases such as alopecia. was possible (see FIG. 2).

Example 4. DKK-1 inhibitory activity induced by DHT

Activation of Wnt/β-catenin promotes hair growth, whereas male hormone or dihydrotestosterone (WAKO, Japan) promotes the expression of Dickkopf related protein 1 (DKK-1) gene, thereby reducing Wnt/β-catenin. Acts as an antagonist.

Therefore, in order to confirm that the three kinds of complex extracts composed of hagocho, hojanggeun, and cheongung of the present invention can suppress the DKK-1 gene, which is an antagonist of Wnt/β-catenin, and promote hair growth, , after treatment with three types of complex extracts of , hojanggeun and cheongung and 100 nM of dihydrotestosterone for 24 hours, using the primers (Bioneer, Korea) of the following [Table 1], the expression change of DKK-1 was RT-PCR method was confirmed. The primers used at this time are shown in [Table 1] below.

[Table 1]

Result analysis was Comparative CT(ΔΔCT) / Experiment[(Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods, 25, 402 408 (2001)] method, and GAPDH was used as an internal control, and the analysis results are shown in FIG. 3 .

As a result of the experiment, the expression of the DKK-1 gene was 9%, 17%, and 28, respectively, compared to the dihydrotestosterone-treated group at 1, 10, and 100 ug/mL concentrations according to the treatment with the complex extracts of Hagogi, Hojanggeun and Chunkyung. % decreased.

In summary, it was confirmed that the treatment of the three types of complex extracts of Hagogi, hojanggeun and cheongung remarkably reduced the DKK-1 gene expression increased by dihydrotestosterone treatment, thereby promoting hair growth. .

Example 5. Measurement of expression change of hair growth promoting growth factor

VEGF (vascular endothelial growth factor), which is not only a major factor in promoting angiogenesis, but also important for various cell functions such as cell survival and proliferation, is a hair growth promoting growth factor that induces extension of the hair growth phase.

Growth hormone expression changes in human keratinocytes (HaCaT, Human immortalized keratinocytes) according to the treatment of single or complex extracts of Hagogia, H.

^{First, human keratinocytes were inoculated in a 24-well cell culture dish at a concentration of 1×10 5} in DMEM (Dulbeccos modified Eagles medium) medium, which is a dedicated medium containing 10% FBS (fetal bovine serum, Cambrex), for 24 hours. , 37° C., 5% CO ₂ Incubated under humid conditions. Thereafter, the medium was removed and the single or complex extracts of H. rhododendrons prepared in Examples 1-1 to 1-4 diluted with serum-free DMEM medium were treated by concentration and cultured for 24 hours. The normal control group was treated with only the medium. After the culture was completed, the supernatant was taken and the amount of vascular endothelial growth factor (VEGF) production was analyzed using an enzyme-linked immunosorbent assay (ELISA, Enzyme-Linked Immunosorbent Assay) kit.

Referring to FIG. 4, in the group to which three kinds of complex extracts of hagocho, hojanggeun and cheongung of the present invention were added, it was confirmed that the production amount of VEGF, a hair growth factor, increased in a concentration-dependent manner by the treatment of the complex extract, 100ug At the /mL concentration, it was confirmed that the maximum increase was 192% compared to the sample untreated group. On the other hand, in the group treated with single extracts of Hagogia, Hojanggeun and Chungung, the activity of increasing VEGF production was found to be somewhat insignificant compared to these complex extracts, and at 100ug/mL concentration, the complex extracts of H. showed 142%, 152%, and 164% increased VEGF expression activity, respectively, compared to the single extract.

Through the above results, it was confirmed that the three kinds of complex extracts composed of hagocho, hojanggeun, and cheongung of the present invention exhibited a synergistic effect by showing a significantly improved VEGF expression increase effect compared to the single extract treatment group. .

Example 6. Anti-inflammatory activity

In order to confirm the anti-inflammatory effect of the complex extract of Hagogi, hojanggeun and cheonggung of the present invention, the inhibition rate of the production of nitric oxide (NO), a representative cytotoxic substance involved in inducing inflammation, was measured.

^{First, human keratinocytes were inoculated in a 24-well cell culture dish at a concentration of 1×10 5} in DMEM (Dulbeccos modified Eagles medium) medium, which is a dedicated medium containing 10% FBS (fetal bovine serum, Cambrex), for 24 hours. , 37° C., 5% CO ₂ Incubated under humid conditions. Thereafter, the medium was removed and the single or complex extracts of Hagogia, H. japonica, and chrysanthemums prepared in Examples 1-1 to 1-4 diluted with serum-free DMEM medium were added to concentrations of 1, 10, and 100 μg/ml, respectively. The diluted sample was added, and a fresh medium containing LPS (1 μg/ml), known as an endotoxin, was treated and cultured for 24 hours. After that, 100 μl of the cell culture supernatant and 100 μl of Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphtylethylenediamine in 2.5% (v/v) phosphoric acid] were mixed in a 96-well plate for 10 After reacting for a minute, the amount of nitric oxide produced was measured by measuring the absorbance at 540 nm using an ELISA reader.

The concentration of the produced nitrite (nitrite) was calculated using a standard curve in which sodium nitrite (sodium nitrite) was dissolved in DMEM medium. The nitric oxide production inhibitory activity of each sample was confirmed based on the difference in the amount of nitrite produced in the LPS-treated control group and the LPS-untreated control group.

As a result of the experiment, it was confirmed that the three types of complex extracts had a better nitric oxide production inhibitory effect compared to the single extracts of hagogi, hojanggeun and cheongung.

Specifically, the complex extracts of hagocho, hojanggeun and cheongung were 5% (1㎍ / ㎖), 22% (10㎍ / ㎖) and 52% (100㎍ / ㎖) reduced levels, respectively, compared to the LPS-treated control group It was observed that by inhibiting the production of nitric oxide to show a superior synergistic effect in inhibiting the production of nitric oxide compared to their single extract (see FIG. 5).

Through the above results, it was confirmed that the complex extract of Hagogi, hojanggeun and cheonggung of the present invention can effectively act in the treatment and prevention of inflammation-related diseases by directly inhibiting the production of nitric oxide, which plays an important role in the inflammatory response. could check

Example 7. Measurement of hair change

Maintaining the health of the hair and preventing the hair from falling out is called wool, and the condition of the wool is evaluated as a good condition as the hair is shiny, the thickness is appropriate, and the condition of thinning is abundant. In addition, the improvement of the hydrophilicity of the proteins constituting the hair, that is, the swelling of the hair, may cause protein hydrolysis or loss of lipids in the hair, so it is important to suppress the swelling of the hair for the protection of the hair.

In order to evaluate the health of the hair, the three types of complex extracts of the present invention were treated on the collected hair, and the thickness of the hair and the degree of water swelling were measured.

About 300 to 400 hairs were cut at a distance of 10 mm from the back of the head in the same direction as the hair root and stored in a zipper pack prepared in advance. To measure the physical change of the hair, the hair thickness and water swelling degree were checked. .

To the collected hair, 15 g each of the single or complex extracts of Hagogi, hojanggeun, and cheongung were applied at a concentration of 3% (v/v) and 7% (v/v), and left for 15 minutes. Thereafter, hair thickness was measured by sampling 10 strands of the hair of the prepared sample and measuring the diameter with a hair diagnostic device.

On the other hand, the moisture swelling degree of the hair was measured by putting 0.1 g of a hair sample into a 50 ml tube, adding 15 ml of distilled water, immersing the hair sample, and leaving it for 30 minutes, then inserting the hair into 4 pieces of filter paper and compressing it to absorb moisture and weighing the hair. was measured, and the result was obtained according to the swelling degree calculation method.

As a result of the experiment, compared to the untreated negative control group (Control, 57㎛), the experimental group treated with the three types of complex extracts of the present invention, consisting of H. rhododendron, H. japonica, and Chunkyung, had a hair thickness of 3% (v/v) and 7%. (v/v) was measured to be 68 μm and 71 μm, respectively, and it was confirmed that the thickness was 119% and 125% thicker. In addition, the complex extracts of hagocho, hojanggeun, and cheongung were 117%, 113% and 111%, respectively, at 3% (v/v) concentrations, and at 7% (v/v) concentrations compared to their single extracts. 111%, 113% and 109% thicker hair thicknesses were measured, respectively (see FIG. 6 ).

In addition, compared to the untreated negative control group (Control, 20㎛), the experimental group treated with the three types of complex extracts composed of Hagonia spp., hojanggeun, and chrysanthemum of the present invention had a water swelling degree of 3% (v/v) and 7% (v). /v) was found to be reduced by 24% and 32%, respectively. In addition, the complex extracts of hagocho, hojanggeun, and cheongung were 21%, 20%, and 14%, respectively, at 3% (v/v) concentrations, and at 7% (v/v) concentrations compared to their single extracts. 27%, 25% and 29% reduction in water swelling was measured, respectively (see FIG. 6 ).

According to the above results, the three kinds of complex extracts composed of Hagogi, hojanggeun, and chrysanthemum of the present invention promote the proliferation of human keratinocytes and dermal papilla cells, and the DKK1 gene that suppresses the Wnt/β-catenin signaling system. increases the expression of vascular endothelial growth factor (VEGF), a growth factor promoting hair growth that directly inhibits the production of nitric oxide, which plays an important role in the skin inflammatory response, and induces extension of the hair growth phase. By increasing the thickness of the hair and reducing the degree of moisture swelling of the hair, it can be usefully used as a hair growth or hair growth composition that shows stable hair growth and hair promoting effect, and it can be confirmed that it can be effectively used for preventing or treating diseases such as alopecia. could

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

Containing the complex extracts of hagocho, hojanggeun, and cheongung as active ingredients,
The hagocho, hojanggeun, and cheongung are characterized in that they are blended in a weight ratio of 1:0.5-2:0.5-2,
The composition is characterized in that it exhibits anti-inflammatory activity through inhibition of nitric oxide (NO) production,
The complex extract of hagocho, hojanggeun, and cheongung is characterized in that it is an extract obtained by heating and refluxing using ethanol as a solvent,
A pharmaceutical composition for preventing or treating hair loss, for promoting hair growth or hair growth, and for anti-inflammatory. delete According to claim 1,
The composition is characterized in that it promotes cell proliferation of human hair follicle dermal papilla cells (HFDPC) and keratinocytes (HaCaT),
A pharmaceutical composition for preventing or treating hair loss, for promoting hair growth or hair growth, and for anti-inflammatory. According to claim 1,
The composition inhibits DKK-1 (Dickkopf-1) gene expression, characterized in that it promotes VEGF (Vascular endothelial growth factor) production,
A pharmaceutical composition for preventing or treating hair loss, for promoting hair growth or hair growth, and for anti-inflammatory. delete According to claim 1,
The composition increases the thickness of the hair, characterized in that it protects the hair by reducing the degree of moisture swelling of the hair,
A pharmaceutical composition for preventing or treating hair loss, for promoting hair growth or hair growth, and for anti-inflammatory. According to claim 1,
The composition is alopecia areata, androgenetic alopecia, tinea capitis, hypotrichosis, hereditary hypotrichosis simplex, circumscribed alopecia, congenital alopecia (alopecia congenitalis), alopecia pubis, seborrheic alopecia (alopecia seborrheica), alopecia senilis, alopecia totalis, alopecia universalis, and telogen effluvium Characterized in treating one alopecia,
A pharmaceutical composition for preventing or treating hair loss, for promoting hair growth or hair growth, and for anti-inflammatory. Containing the complex extract of hagocho, hojanggeun, and cheongung as an active ingredient,
The hagocho, hojanggeun, and cheongung are characterized in that they are blended in a weight ratio of 1:0.5-2:0.5-2,
The composition is characterized in that it exhibits anti-inflammatory activity through inhibition of nitric oxide (NO) production,
The complex extract of hagocho, hojanggeun, and cheongung is characterized in that it is an extract obtained by heating and refluxing using ethanol as a solvent,
A functional food composition or food composition for preventing hair loss, promoting hair growth or hair growth, and for anti-inflammatory. Containing the complex extract of hagocho, hojanggeun, and cheongung as an active ingredient,
The hagocho, hojanggeun, and cheongung are characterized in that they are blended in a weight ratio of 1:0.5-2:0.5-2,
The composition is characterized in that it exhibits anti-inflammatory activity through inhibition of nitric oxide (NO) production,
The complex extract of hagocho, hojanggeun, and cheongung is characterized in that it is an extract obtained by heating and refluxing using ethanol as a solvent,
A cosmetic composition for preventing hair loss, promoting hair growth or hair growth, and for anti-inflammatory.

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