WO2022045635A1 - Composition for preventing hair loss or promoting hair growth containing lagerstroemia indica extract as active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing hair loss or promoting hair growth and, specifically, to a cosmetic composition, a pharmaceutical composition and a health food composition for preventing hair loss or promoting hair growth, containing a Lagerstroemia indica extract as an active ingredient. The Lagerstroemia indica extract can effectively induce or promote the growth of dermal papilla cells by activating WNT signaling and inhibiting TGF-β signaling, thus more effectively preventing hair loss or promoting hair growth. In addition, the extract is a natural substance, and thus has the advantages of fewer side effects and being safe.

Description

A composition for preventing hair loss or promoting hair growth, containing Barerong tree extract as an active ingredient

The present invention relates to a composition for preventing hair loss or promoting hair growth, and more particularly, to a cosmetic composition, a pharmaceutical composition, and a health food composition for preventing hair loss or promoting hair growth, which contain an extract of Barberry tree as an active ingredient.

Alopecia is a phenomenon in which hair does not grow normally, so it falls out easily and becomes thin, and the growth period is shortened and the resting period is lengthened at the same time. Normally, about 50-100 hairs fall out of the skin a day, but if more than 100 hairs fall out continuously every day, you can think of a person with alopecia.

Recently, people's quality of life has improved due to rapid economic growth and improvement of living standards, but modern people are exposed to external stimuli and various diseases due to the occurrence of pollution and environmental pollution. In particular, it has been found that alopecia is caused by local infection, genetic predisposition, endocrine disorders, etc. in relation to hair, but in recent studies, various factors such as stress, side effects due to environmental pollution, and side effects due to chemical products acted. These factors, such as stress, activate immune cells and neutrophils to cause an inflammatory response, destroy hair follicle tissue, and induce cell death of hair cells, leading to hair loss.

The important reason why the medical/pharmaceutical industry is not responding properly to these problems is that the mechanism of hair loss is in hormones based on genetic factors. Of course, there are factors other than hormones that cause hair loss, such as drugs and nutrition, but these are temporary rather than permanent factors. am.

Therefore, the current drugs to treat hair loss are mainly focused on delaying or stopping the progression of hair loss through mechanisms that control hormones that affect hair loss. As a result, existing chemical-based drugs are accompanied by side effects caused by hormone control, affect the wide range of use and development of drugs, and difficulties in the medical/pharmaceutical industry paradoxically affect the cosmetic and procedure markets. The balloon effect was induced. Cosmetics and treatment methods for hair loss include scaling to remove dead skin cells from the scalp or applying an external agent that can supply nutrients to the hair root. In the name of improving skin condition, infrared injections or scalp massage are mainly used as a result of thinning hair or clogging pores due to problems with the skin condition of the scalp.

In order to bridge the gap between the existing medical/pharmaceutical industry and cosmetics and to allow the medical/pharmaceutical and cosmetic industry to regain the leadership of hair loss treatment, it is urgent to develop natural product-based hair products. Testosterone, the male hormone that causes hair loss, controls the rapid progression of hair from the life cycle to the decline phase, and researches target proteins with hair growth/hair growth effects to develop hair products that can control hormones without side effects. If developed, the side effects of existing treatments can be reduced and it will be effective for the shared growth of the medical/pharmaceutical market and the cosmetic market.

It is an object of the present invention to provide a composition for preventing hair loss or promoting hair growth based on a natural product having an excellent effect.

In order to achieve the above object, the present invention Bareong tree ( Lagerstroemia indica ) provides a cosmetic composition for preventing hair loss or promoting hair growth containing the extract as an active ingredient.

The present invention is Bareong tree ( Lagerstroemia indica ) provides a pharmaceutical composition for preventing hair loss or promoting hair growth containing the extract as an active ingredient.

In addition, the present invention is Bareong tree ( Lagerstroemia indica ) provides a health food composition for preventing hair loss or promoting hair growth containing the extract as an active ingredient.

The Barrong tree extract according to the present invention can effectively induce or promote the growth of dermal papilla cells. The extract activates WNT signaling, a hair target protein, and increases the expression of one or more dermal papilla cell growth-related factors selected from the group consisting of β-catenin, TCF/LEF, BCL-2, Gli-1, and VEGF, whereas , by inhibiting the signaling of TGF-β, it can prevent the death of dermal papilla cells and promote the growth of dermal papilla cells.

In addition, the cosmetic composition, the pharmaceutical composition, and the health food composition according to the present invention contain a natural product, Barerong tree extract, as an active ingredient, so they have fewer side effects and are safe, and can more effectively prevent hair loss or promote hair growth through the composition. .

1 is an active ingredient of an extract of Barerong tree according to the present invention, campphenol (kaempferol) and an indicator ingredient, 3,3',4-tri-O-methylellagic acid (3,3',4-Tri-O-methylellagic) acid).

Figure 2 is the index analysis result according to an experimental example of the present invention, Figure 2a shows a chromatogram of the campphenol standard, which is an active ingredient of the Barrong tree extract, Figure 2b is the index component 3,3',4-tri- A chromatogram of O-methyl ellagic acid standard is shown, and FIG. 2c shows a chromatogram of an extract of Prunus oleracea analyzed at 370 nm.

3 is a graph showing the level of ATP luciferase activity according to the concentration of the leaf extract of Barerong tree according to an experimental example of the present invention and campphenol, which is an active ingredient thereof.

4 is a result of real-time monitoring of the effect of the leaf extract of Barong tree leaf extract on the growth of dermal papilla cells according to an experimental example of the present invention.

5 is a graph showing the RNA expression level in dermal papilla cells according to an experimental example of the present invention, FIG. 5a is β-catenin and TCF/LEF, FIG. 5b is STAT3 and STAT6, FIG. 5c is BCL-2 and BAX, and 5d shows the expression levels of Gli-1 and VEGF.

6 is a graph showing the measurement result of luciferase activity according to an experimental example of the present invention, where A is the measurement result of TCF/LEF-luciferase activity, and B is the measurement result of SBE-luciferase activity by TGF-β signal transduction indicates.

7 shows the results of observing the effect of the leaf extract of Baerong tree according to an experimental example of the present invention on WNT signaling.

8 shows the results of hair count analysis using a phototrichogram according to an experimental example of the present invention.

Hereinafter, the present invention will be described in detail.

While the present inventors were researching a material with fewer side effects and excellent efficacy in preventing hair loss and promoting hair growth compared to conventional synthetic chemical substances, while the Barong tree extract promotes the growth of dermal papilla cells in human hair follicles and activates WNT signal transduction, , by inhibiting the signal transduction of TGF-β to prevent hair loss or to promote hair growth, thereby completing the present invention.

The present invention is Bareong tree ( Lagerstroemia indica ) provides a cosmetic composition for preventing hair loss or promoting hair growth containing the extract as an active ingredient.

As used herein, " Lagerstroemia indica )" is a deciduous small tree in the family Asteraceae. It is called Zinnia because its flowers bloom for a long time, and it is also called Gancho or Tickle tree because the leaves move when the bark is scratched. Zinnia elegans is an annual plant of the Asteraceae family called Zinnia elegans It is distinct from grass, and its origin is southern China, and there are about 30 species distributed in Korea, Japan, Australia, etc. The flowers have hemostasis and anti-inflammatory effects, and are used in oriental medicine as medicines for hypermenorrhea, enteritis, diarrhea, etc. used, but other effects are unknown.

As used herein, the term "extract" refers to a substance obtained by extracting a component of a natural product regardless of an extraction method, an extraction solvent, an extracted component, or the form of an extract. It may include any material obtainable by processing or processing by the method.

The extract may be extracted according to a method commonly used in the art, for example, hot water extraction method, ultrasonic extraction method, filtration method, reflux extraction method, etc., these are performed alone or in combination of two or more methods. can be

In the present invention, the Barrong tree extract may be extracted with water, C1 to C4 alcohol or a mixed solvent thereof, and for example, methanol, ethanol, propanol, butanol or an aqueous solution thereof, etc. may be included as the solvent. and preferably 40 to 60 wt% ethanol aqueous solution, more preferably 50 wt% ethanol aqueous solution as an extraction solvent, but is not limited thereto.

The extract may be obtained by extracting the roots, stems, leaves, flowers, or a mixture of these parts, and preferably may be extracts from the leaves of the Aeroba tree, but is not limited thereto.

The barberry may be collected or grown in nature, or purchased commercially.

In the present invention, the extract of Barongyi may induce or promote the growth of dermal papilla cells.

As used herein, "Humam dermal papilla (HDP) cells" are cells located in the dermal papilla below the hair follicle, and control the development and growth of hair follicles. Capillaries are distributed in the dermal papilla cells, and through this, nutrients can be supplied to the hair follicles and growth factors and inhibitors are secreted to regulate the growth of hair follicle epithelial cells.

According to an experimental example of the present invention, when treated with the leaf extract of Barongyi, dermal papilla cell growth was induced in a concentration-dependent manner, and when compared with minoxidil or tofacitinib as a positive control, similar or enhanced It was confirmed that the effect of

The extract of Barerong tree can promote and activate the growth of dermal papilla cells by activating WNT signaling, which is a hair target protein, and inhibiting signal transduction of TGF-β, β-catenin, TCF (T At least one selected from the group consisting of cell factor)/lymphoid enhancer factor (LEF), B-cell lymphoma 2 (BCL-2), glioma-associated oncogene homolog 1 (Gli-1), and vascular endothelial growth factor (VEGF). May increase the expression of dermal papilla cell growth-related factors.

As used herein, "WNT protein" is a protein used as a ligand of the Wnt pathway, and controls the stability of β-catenin.

In the present specification, "TGF-β (transforming growth factor-β)" is one of the transforming growth factor-β family and acts as an important ligand for cell activity together with activin, bone morphogenetic factor, etc. . TGF-β signaling is a cytokine that regulates various cellular responses, such as proliferation, differentiation, cell migration, and apoptosis, from metazoan development to mature cell proliferation, and TCF/β-catenin signaling It regulates LEF activity, and when TGF-β signaling is activated, it induces apoptosis of dermal papilla cells, leading to hair loss.

The Barongyi extract can activate Wnt/β-catenin, a signal transduction material that decreases in the process of hair loss, and inhibits TGF-β signal transduction that induces dermal papilla cell death, thereby inhibiting the death of dermal papilla cells and the cells It can induce or promote the growth of, and can be utilized as a cosmetic composition for preventing hair loss or promoting hair growth.

According to an experimental example of the present invention, the activity of TCF/LEF-luciferase, which is a transcription factor involved in promoting the synthesis of β-catenin, increased in a concentration-dependent manner according to the concentration of the Barong tree leaf extract, whereas TGF-β signal transduction It was confirmed that the SBE-luciferase activity was significantly reduced.

In the present invention, the alopecia is alopecia areata, hereditary androgenetic alopecia, telogen alopecia, traumatic alopecia, hair growth wall, pressure alopecia, genital alopecia, alopecia pityroides, syphilitic alopecia, seborrheic alopecia, symptomatic alopecia, scar alopecia, And it may be one symptom of alopecia selected from the group consisting of congenital alopecia, but is not limited thereto.

As used herein, "cosmetics" may include functional cosmetics having effects such as hair loss improvement and hair growth promotion, and the functional cosmetics are cosmetics emphasizing specific efficacy and effects as defined in the Cosmetics Act. This may include helping with hair loss or alleviating symptoms of hair loss.

In the present invention, the extract of Barrong tree may be included in an amount of 0.01 to 50 parts by weight, preferably 0.1 to 10 parts by weight, more preferably 0.1 to 5 parts by weight, based on 100 parts by weight of the total cosmetic. It is not limited.

The cosmetic composition according to the present invention may be prepared in any formulation conventionally prepared in the art, and may include solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing agents, oils, It may be formulated as a powder, shampoo and spray, but is not limited thereto.

Preferably, the cosmetic composition can be prepared in any form of cosmetics used for hair and scalp, for example, the cosmetic composition is a hair tonic, hair cream, It may be prepared in the form of hair lotion, hair shampoo, hair conditioner, hair spray, hair aerosol, pomade, powder, or gel.

In addition to the extract, the cosmetic composition may further include one or more ingredients acceptable as a hair cosmetic preparation, such as ingredients listed in the list of cosmetic raw materials registered with the Ministry of Food and Drug Safety and the ICID (International Cosmetic Ingredients Collection). For example, alcohols, oils, surfactants, fatty acids, silicone oils, preservatives, humectants, humectants, viscosity modifiers, emulsifiers, stabilizers, sunscreens, coloring agents, fragrances, diluents and the like may be included as carriers. Specific compounds or compositions that can be used as the alcohol, oil, surfactant, fatty acid, silicone oil, preservative, wetting agent, humectant, viscosity modifier, emulsifier, stabilizer, sunscreen, color developer, fragrance, diluent, etc. are already known in the art. Therefore, those skilled in the art can select and use an appropriate compound or composition.

In addition, the cosmetic composition may further include a skin absorption promoter, a scalp or hair protectant, an active agent, etc., or a suitable carrier for application to the hair in order to enhance the effect and minimize side effects, and may be prepared as a scalp or hair protection composition there is. The carrier comprises a solvent and other carriers or vehicle components commonly used in scalp or hair care compositions, and comprises about 0.5% to 99.5% of the total composition, preferably about 5.0% to 99.5%, most Preferably, it may be included in an amount of about 10.0% to 90.0%, but is not limited thereto. The solvent as the carrier may be selected according to the active ingredient used regardless of whether the composition remains on the hair after use such as hairspray, mousse, tonic, etc. or is washed with shampoo, conditioner, etc. For example, water, lower alcohols (ethanol, isopropanol, etc.), hydroalcoholic mixtures, hydrocarbons (isobutane, hexane, decene, etc.), acetone, halogenated hydrocarbons (freon, etc.), hydrocarbon esters (ethyl acetate, dibutyl phthalate, etc.) ), volatile silicone derivatives, siloxanes (phenyl pentamethyl disiloxane, methoxypropyl heptamethyl cyclotetrasiloxane, chloropropyl pentamethyl disiloxane, hydroxypropyl pentamethyldisiloxane, octamethyl cyclotetrasiloxane, decamethyl cyclopentasiloxane, etc. ) and may be selected from the group consisting of mixtures thereof, but is not limited thereto.

The cosmetic composition may be used alone or by being applied in duplicate, or may be used by overlapping with other cosmetic compositions other than the present invention. In addition, the cosmetic composition may be used according to a conventional method of use, and the number of times of use may be varied according to the skin condition or taste of the user.

The present invention is Bareong tree ( Lagerstroemia indica ) provides a pharmaceutical composition for preventing hair loss or promoting hair growth containing the extract as an active ingredient.

The extract may be water, C1 to C4 alcohol, or a mixed solvent thereof, for example, methanol, ethanol, propanol, butanol or an aqueous solution thereof may be included as the solvent, preferably 40 to 60% by weight of an aqueous ethanol solution, more preferably 50% by weight of an aqueous solution of ethanol may be used as an extraction solvent, but is not limited thereto.

The extract may be obtained by extracting the roots, stems, leaves, flowers, or a mixture of these parts, and preferably may be extracts from the leaves of the Aeroba tree, but is not limited thereto.

The Barong tree extract can activate WNT signaling, a hair target protein, and inhibit signal transduction of TGF-β, from the group consisting of β-catenin, TCF/LEF, BCL-2, Gli-1, and VEGF. may increase the expression of one or more selected dermal papilla cell growth-related factors. That is, the extract of Barongyi can induce or promote and activate the growth of dermal papilla cells, and thus can be utilized as a pharmaceutical composition for preventing hair loss or promoting hair growth.

Corresponding features may be substituted for the above-mentioned parts.

As used herein, "pharmaceutical composition" refers to a composition administered to prevent or treat or promote hair growth for the purpose of the present invention.

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier, excipient, etc. in addition to the above-mentioned Barongyi extract as an active ingredient, and parenteral forms of oral dosage forms, external preparations, suppositories and sterile injection solutions according to conventional methods It can be formulated and used as a dosage form, and preferably topical formulations, for example, creams, lotions, ointments (semi-solid external preparations), microemulsions, gels, pastes, transdermal preparations (TTS) (patches, bandages, etc.) ) and the like, but is not limited thereto.

As used herein, "pharmaceutically acceptable" means that it is not toxic to cells or humans exposed to the pharmaceutical composition, and the appropriate carrier does not inhibit the activity and properties of the P. , may be selected differently depending on the dosage form and formulation.

The pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (corn starch, potato starch, etc.), cellulose, derivatives thereof (sodium carboxymethyl cellulose, ethylcellulose, etc.), malt, gelatin, talc, solid lubricants. (stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oil (peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyol (propylene glycol, glycerin, etc.), alginic acid, emulsifier (TWEENS), wetting agent (sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, water, saline, phosphate buffered solutions, and the like. One or more suitable carriers may be selected and used according to the formulation of the pharmaceutical composition of the present invention.

The excipient may also be selected and used according to the formulation of the pharmaceutical composition of the present invention, for example, a suspending or dispersing agent such as sodium carboxymethyl cellulose, methyl cellulose, hydropropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, etc. and the like.

The effective dose level of the pharmaceutical composition according to the present invention is determined by the purpose of use, the age, sex, weight and health condition of the patient, the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the administration method, the administration time, the administration route and the discharge. It may be determined differently according to factors including the ratio, duration of treatment, combination or concurrent drugs, and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once to several times a day. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition may be administered to any animal that can cause hair loss, and the animal may include, for example, not only humans and primates, but also livestock such as cattle, pigs, horses, and dogs.

The pharmaceutical composition may be administered by an appropriate administration route according to the form of the formulation, and may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. The administration method is not particularly limited, and for example, oral, rectal or intravenous, intramuscular, subcutaneous, endobronchial inhalation, intrauterine dural or intracerebroventricular injection, etc. may be administered in a conventional manner. , Preferably it can be administered by applying directly to the scalp.

The pharmaceutical composition according to the present invention may be used alone to prevent hair loss or promote hair growth, or may be used in combination with surgery or other drug treatment.

In addition, the present invention is Bareong tree ( Lagerstroemia indica ) provides a health food composition for preventing hair loss or promoting hair growth containing the extract as an active ingredient.

The extract may be water, C1 to C4 alcohol, or a mixed solvent thereof, for example, methanol, ethanol, propanol, butanol or an aqueous solution thereof may be included as the solvent, preferably 40 to 60% by weight of an aqueous ethanol solution, more preferably 50% by weight of an aqueous solution of ethanol may be used as an extraction solvent, but is not limited thereto.

The extract may be obtained by extracting the roots, stems, leaves, flowers, or a mixture of these parts, and preferably may be extracts from the leaves of the Aeroba tree, but is not limited thereto.

The Barong tree extract can activate WNT signaling, a hair target protein, and inhibit signal transduction of TGF-β, from the group consisting of β-catenin, TCF/LEF, BCL-2, Gli-1, and VEGF. may increase the expression of one or more selected dermal papilla cell growth-related factors. That is, the extract of Barongyi can induce or promote and activate the growth of dermal papilla cells, and thus can be utilized as a health food composition for preventing hair loss or promoting hair growth.

Corresponding features may be substituted for the above-mentioned parts.

As used herein, the term "health food" refers to food manufactured and processed using raw materials or ingredients useful in the human body, and is processed to efficiently exhibit bioregulatory functions in addition to nutritional supply. It refers to food with high medical and medical effects. and may be used interchangeably with terms known in the art, such as functional food. For example, it may be prepared as a health supplement, a food for special nutritional supplementation, a functional beverage, or the like, or the composition according to the present invention may be added to natural food, processed food, or the like.

The health food may be prepared in powder, granule, tablet, capsule, syrup or beverage, etc., there is no limitation in the form that the health food can take, and may include any food in a conventional sense. For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc. are possible, and may include all functional foods in a conventional sense. It may also include food used as feed for animals.

The health food composition according to the present invention may be prepared by further including pharmaceutically acceptable food additives and other suitable auxiliary ingredients commonly used in the art. For example, flavoring agents, natural carbohydrates, sweetening agents, vitamins, electrolytes, coloring agents, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc. can In particular, as the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol can be used. , As the sweetener, natural sweeteners such as taumatine and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used. It is preferable that the food additives and other auxiliary ingredients are added and used in a very small amount as long as the use to which they are added can be achieved. When expressed numerically, the trace amount may range from 0.0005% by weight to about 0.5% by weight based on the total weight of the health food composition.

The health food composition according to the present invention has the advantage that there are no side effects that may occur when taking the drug for a long period of time by using food as a raw material, unlike general drugs, and has excellent portability and can be consumed as an auxiliary for preventing hair loss or promoting hair growth. can

Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

< Example 1> Barberry ( Lagerstroemia indica ) extract preparation

1.8 kg of cut Barrong tree leaves (Jeju single-drop herb) was prepared as a raw material, and 50% ethanol of 20 times the raw material of Barerong tree was added and immersed for 72 hours. The extract was filtered with a 2.5 μm filter paper, and the filtered solution was concentrated at 60±5° C. until it became 35-45 brix using a vacuum concentrator. The concentrate was sterilized at 95° C. for 30 seconds, and then lyophilized and pulverized to obtain 278 g of powder of Barongyi extract.

< production example 1> Contains barberry extract Hair Tonic (BSKLI01) Produce

1. Preparation of undiluted solution containing Barerong tree extract

A undiluted solution containing Barerong tree extract was first prepared by using the Barerong tree extract powder. Based on 100 parts by weight of the total amount, 1.25 parts by weight of banyan tree extract powder, by 50 ethyl alcohol, and by 48.75 in purified water were mixed homogeneously for each measurement, followed by stirring at a rotational speed of 10 to 30 rpm to completely dissolve the extract powder to prepare an undiluted solution containing Barerong tree extract.

2. Addition of undiluted solution containing Barerong tree extract

For the preparation of hair tonic containing Barrong tree extract, 40 parts by weight of the stock solution containing Barerong tree extract prepared in 1 of Preparation Example 1 is added to 60 parts by weight based on the total weight to be homogeneously mixed in purified water and mixed. After giving, the mixture was stirred at a rotational speed of 10 to 30 rpm to prepare a hair tonic containing Barerong tree extract.

Table 1 below shows the ingredients and contents required for preparing the hair tonic of Preparation Example 1.

award	turn	Ingredient name	Content % (w/w)
A	One		Ethyl alcohol	20
	2	water	19.5
	3	Lagerstroemia Indica Extract (powder)	0.5
B	One	water	60

< Experimental example 1> Analysis of indicator components and active ingredients of Barerong tree extract

1 is an active ingredient of an extract of Barerong tree according to the present invention, campphenol (kaempferol) and an indicator ingredient, 3,3',4-tri-O-methylellagic acid (3,3',4-Tri-O-methylellagic) acid).

In order to analyze the indicator components of the Barrong tree extract, first, an appropriate amount of camphenol and 3,3',4-tri-O-methylellagic acid was dissolved in 100% methanol to make 10mg/100mL, and this was used as a standard stock solution. After taking about 0.3 g of the sample, it was placed in a 100 mL volumetric flask and filled up to the mark with 60% methanol solution. After it was sufficiently dissolved in an ultrasonic shaker, it was filtered through a 0.22 μm syringe filter to obtain a test solution.

Index component analysis was quantitatively analyzed using high performance liquid chromatography (HPLC). LC20A (Shimadzu, Japan) was used for HPLC, and sunfire C18 (4.6×250 mm, 5 μm, Waters, USA) was used for the column. The composition of the mobile phase was acetonitrile and 0.1% formic acid in a stepwise concentration gradient, the flow rate was 1.0mL/min, the injection amount was 10μL, the column oven was 35℃, the wavelength was campphenol and 3,3' ,4-tri-O-methylellagic acid was analyzed at 370 nm, respectively.

FIG. 2 is an indicator analysis result according to an experimental example of the present invention. Referring to this, as a result of analyzing the Barong tree extract, the main components campenol and 3,3',4-tri-O-methylellagic acid peaks Retention times were 37 minutes and 40 minutes, respectively, and the contents of camphenol and 3,3',4-tri-O-methylellagic acid in the extracts of Barongyi were found to be 1.4 mg/g and 2.2 mg/g, respectively. .

< Experimental example 2> of human hair follicles mammary gland Human hair dermal follicle papilloma cells, HHDFP ) growth analysis

Human hair dermal follicle papilloma cells (HHDFP) used in an experimental example of the present invention were purchased from abm (Richmond, BC, Canada). Cells were tested and validated cytogenetically prior to storage in nitrogen tanks. HHDFP cells were cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 100 units/mL of penicillin and streptomycin and 10% fetal bovine serum (FBS) in a 37°C, 5% CO ₂ incubator. was maintained

2-1. ATP measurement in cells kit ( CellTiterGlo Cell growth confirmation using luminescent cell viability kit)

The dermal papilla (HDP) cells were inoculated in a 96-well plate (5×10 ³ cell/well) and cultured in DMEM + 10% FBS for 24 hours. After 24 hours, the cells were treated with extracts of banyan tree leaves (3.12-50 mg/mL) by concentration and cultured for an additional 72 hours, and less than 1% dimethyl sulfoxide (DMSO) was used as a negative control.

Cell growth and viability were measured using the celltiter-Glo luminescent cell viability kit according to the manufacturer's instructions. Based on ATP measurement through intracellular luciferase reaction, the product is proportional to the number of viable cells. The cell proliferation inhibition rate was calculated as [(relative luminescence intensity of experimental group/relative luminescence intensity of control group)×100], and the luminescence degree that indirectly reflects cell viability was measured with a Lubi luminometer (bethold TEC GmbH & co, TN, USA). All experiments were repeated three or more times.

3 is a graph showing the degree of ATP luciferase activity according to the concentration of the Barong tree leaf extract and its active ingredient, camphenol, according to an experimental example of the present invention. As a result of observation using the cell ATP measurement kit to confirm the effect, it was found that the B. leaf extract significantly induced the growth of cells in a concentration-dependent manner when compared to the control. In addition, when compared with the positive control minoxidil (minoxidil, Mnx) or tofacitinib (Tofacitinib, Tof), showed a similar or enhanced effect. Through this, it can be confirmed that the leaf extract of Barong tree significantly induces the growth of dermal papilla cells.

2-2. Real-time cell growth measurement device ( xCELLigence Cell growth confirmation using Real Time Cell Analysis Instruments)

The xCELLigence system uses electrical cell sensor array technology to enable label-free, real-time monitoring of cellular processes such as cell proliferation, cytotoxicity, adhesion, survival, invasion and migration. The status of cells is monitored in real time without binding to the label by measuring the electrical impedance passing through the microelectrodes integrated at the bottom of special tissue culture plates (E-Plates, ACEA Biosciences). The impedance of the electrode, expressed as a cell index (CI) value, is used to provide quantitative information about the living state of cells, including cell number, viability, and morphology. Changes in cell status, such as cell morphology, cell adhesion, or cell viability, follow a change in the CI value by quantitative measurement of the number of cells present in a well.

Accordingly, HDP cells were cultured at 20,000 cells/well in E-Plates L8 in 3% FBS medium. Growth curves were automatically recorded in the xCELLigence system in real time, and after 24 hours, the live cells were monitored for 96 hours by adding the P.

4 is a result of real-time monitoring of the effect of the dermal papilla leaf extract in accordance with an experimental example of the present invention on the growth of dermal papilla cells. Thus, it can be confirmed that the growth of dermal papilla cells is significantly increased in a concentration-dependent manner.

< Experimental example 3> RNA isolation and real time PCR

HHDFP cells (5×10 ⁵ cells/well) were inoculated in a 6-well plate, and exposed to WNT3a alone or Barongyi extract at different concentrations for 72 hours. Thereafter, total RNA was isolated using the Aurum total RNA mini kit (Bio-Rad, Hercules, CA, USA). First strand cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase (MMLV-RTase) (Bio-Rad, Hercules, CA, USA) using 1 μg of total RNA.

The synthesized cDNA was synthesized using a GeneAmp PCR 9700 thermocycler (Thermo Fisher Scientific, Waltham, MA, USA) to obtain β-catenin, T-cell factor (TCF)/lymphoid enhancer factor (LEF), signal transducer and STAT3 (signal transducer and activator of transcription 3), STAT6 (signal transducer and activator of transcription 6), BCL-2 (B-cell lymphoma 2), BAX (Bcl-2-associated X protein), Gli-1 (glioma-associated oncogene homolog 1) , VEGF (vascular endothelial growth factor) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was amplified with PCR primers. Quantitative gene expression levels of cytokines were measured using SYBR Green reagents (Bio-Rad, Hercules, CA, USA) on a CFX96 Touch PCR system (Bio-Rad, Hercules, CA, USA). Relative target gene expression was determined after normalization to GAPDH levels. For each experiment, 2 independent experiments were repeated 3 times to calculate the results.

Table 2 below shows primers for RT-PCR.

factor	forward/station	primer sequence	SEQ ID NO:
β-catenin	forward	5’-CCCACTAATGTCCAGCGT TT-3’	One
	reverse	5’-AACCAAGCATTTTCAC CAG G-3’	2
glycogen synthase kinase (GSK)-3β	forward	5’-AACTGCCCGACTAACA ACAC-3’	3
	reverse	5’-ATTGGTCTGTCCACGGT CTC-3’	4
TCF/LEF	forward	5’-AATCATCC CGGCCAGC A-3’	5
	reverse	5’-TGTCGT GGTAGGGCTCCTC-3’	6
BAX	forward	5’-GTTG TCGCCCTTTT CTACT-3’	7
	reverse	5’-GA AGTCCAATGTCCAGCC-3’	8
BCL-2	forward	5’-CACCAGAATCAAGTGTTCC-3’	9
	reverse	5’-GCTATTTTATTGGATGTG CTTTG-3’	10
STAT3	forward	5'-GTTATTGT TGTT GTGTTCTTAGAC-3'	11
	reverse	5’-AATGCCA GGAGTATGTAGC-3’	12
STAT6	forward	5’-CCAGGATGGCT CTCCACAG-3’	13
	reverse	5’-CATGGAGGAATCAGGG GC-3’	14
Gli	forward	5’-TACTCACGCCTCGAAAACCT-3’	15
	reverse	5’-GTCTGCTTTCCTCCCTGATG-3’	16
VEGF	forward	5’-GGAGAGATGAGCTT CCTACAG-3’	17
	reverse	5’-TCACC GCCTTGGCTTGTCACA-3’	18
GAPDH	forward	5’-T GG CAAATTCCATGCAC-3’	19
	reverse	5’-CCATGGTGGTGAAGA CGC-3’	20

5 is a graph showing the amount of RNA expression in dermal papilla cells according to an experimental example of the present invention. Referring to this, the RNA expression levels of β-catenin and TCF/LEF, which are major proteins of WNT signal transduction, are increased compared to the negative control group. And, it can be confirmed that the expression level of STAT6 of JAK3 signal transduction by the cytokine IL-4 is reduced. In addition, it can be seen that the expression of BCL-2 and BAX related to cell survival and death showed an increase in BCL-2, and the expression of Gli-1 and VEGF related to dermal papilla growth was also increased.

< Experimental example 4> luciferase ( luciferase ) activity evaluation

4-1. wint signal transmission ( Wnt TAP, a transcription factor in signaling) of Flash (TOP-Flash) Luciferase activity evaluation

Wnt/β-catenin signaling is important for hair follicle formation, development and growth. Top-flash HDP cells in which T cell factor/lymphoid enhancer factor (TCF/LEF) gene binding site and luciferase reporter gene are fused After inoculation with 1×10 ⁵ cells in a 24-well plate, 24 hours later, TCF-luciferase vector (TCF-luciferase vector) was transfected for 4 hours. Thereafter, the cells were treated with WNT3a alone or with various concentrations of Prunus leaf extract samples for 24 hours.

Several substances are involved in the hair growth process, and it is known that the first signal of hair follicle development comes from the dermis, and β-catenin is estimated to be the most important substance. In addition, it is known that TCF/LEF and WNT signals are also involved in the first signal.

3T3-WNT cells in which the TCF/LEF-luciferase gene was stably expressed were used to evaluate whether the B. leaf extract induces the activity of the transcription factor TCF by increasing β-catenin through WNT signaling activity. . The 3T3-WNT cell line was maintained in a DMEM medium containing puromycin (3 μg/mL), streptomycin (100 units/mL) and 10% FBS at 37° C. and 5% CO ₂ incubator.

6A is a graph showing the measurement results of TCF/LEF-luciferase activity according to an experimental example of the present invention. Referring to this, it can be seen that the Barerong tree leaf extract acts on the TCF/LEF-luciferase activity in a concentration-dependent manner. can In particular, in the sample 40 μg/mL, it was found that the TCF/LEF-luciferase activity was increased by about 1.5 times compared to the control. Through this, it can be seen that the leaf extract of Barerong tree inhibits the degradation of β-catenin by phosphorylation and is involved in promoting dermal papilla cell growth by activating β-catenin.

4-2. TGF -β signaling ( TGF -b signaling), a transcription factor Smad's SBE ( Smad binding elements)- luciferase activity evaluation

Inhibition of transforming growth factor beta (TGF-β) signaling pathway signaling promotes hair follicle development and hair growth. After inoculating HEK 293 cells in which the SBE-luciferase vector is stably present in a 24-well plate at 1×10 ⁵ cells, 24 hours later, the cytokine TGF-β (10 μg/ml) alone or at various concentrations It was exposed to the tree leaf extract sample for 24 hours.

6B is a graph showing the measurement result of SBE-luciferase activity by TGF-β signaling according to an experimental example of the present invention. Referring to this, Smad-SBE signal transduction by the cytokine TGF-β is appeared to be regulated. Apoptosis is induced by TGF-β signaling, and it can be confirmed that the leaf extract of Prunus oleifera affects the survival of dermal papilla cells by inhibiting TGF-β signaling.

< Experimental example 5> Immune ( Immunoblot ) blot analyze

After appropriate treatment, cells were lysed with cell lysis buffer [20 mM Tris pH 7.5, 150 mM NaCl, 1 mM Na ₂ EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM sodium vanadate, 1 mg/mL leupeptin and 1 mM phenylmethylsulfonyl fluoride (PMSF)] for 30 min on ice.

After centrifugation at 20,817 g for 15 minutes, a supernatant was obtained and the protein concentration was measured. Whole cell protein extracts were separated by SDS-PAGE and polyvinylidene fluoride (PVDF) in 20 mM Tris-HCl (pH 8.0) containing 150 mM glycine and 20% (v/v) methanol. transferred to the membrane. The membrane was blocked with 5% non-fat dry milk dissolved in 1x TBS (TBS-T buffer) containing 0.05% Tween 20, and reacted with appropriate antibodies. The blots were washed 3 times with 1x TBS-T buffer and then reacted with the appropriate HRP-linked IgG. Hybridized proteins were visualized using an enhanced chemiluminescence (ECL) detection system.

Figure 7 is the result of observing the effect of the leaf extract of Baerong tree according to an experimental example of the present invention on WNT signal transduction. Referring to this, the expression of phosphorylated β-catenin (phospho-beta-catenin, p-β-catenin) The amount was shown to decrease and the expression level of β-catenin was shown to increase, thereby confirming that the activity in TCF/LEF was increased after entering the nucleus.

< Experimental example 6> Hair loss Simplified human application test for symptom relief

This experimental example is a simple human application test for alleviating hair loss symptoms, and to evaluate the effect of relieving hair loss symptoms by using the hair tonic (BSKLI01) containing the extract of B. It proceeded [control number: CRA20-CT1001].

1. Test product: Hair tonic ( BSKLI01 )

- Ingredient and properties: Contains 0.5% of Barberry leaf extract, liquid type [Product Control Number: SJM20-1001]

- Storage method: Store at room temperature, avoiding high temperature and direct sunlight.

- Part to be used: scalp

- How to use: Use twice a day (morning, afternoon). Shake the hair tonic thoroughly before use, then spray 4 to 5 times (approximately 1 mL) on the scalp including the hair loss area, and use your fingertips for 5 minutes for absorption. Dry naturally after massage.

2. Test method

In order to evaluate the effect of the test product BSKLI01 on alleviating hair loss symptoms, 12 adults aged 18 to 54 years diagnosed with androgenetic alopecia were allowed to use the test product for 12 weeks. Subjects were selected under the judgment of a dermatologist according to the guidelines of the Ministry of Food and Drug Safety (Guideline-0760-02) and internal regulations of CRA Korea.

At the initial visit, basic information of all subjects was collected, and the test site was marked by tattooing the hair loss area in the form of a dot with a diameter of about 1 mm. Subjects visited the test center at the time of evaluation and participated in the test under constant temperature and humidity conditions (22±2℃, 50±10% RH).

The evaluation was conducted before product use (week 0), and at 6 weeks and 12 weeks after product use, the total number of hairs using a phototricogram, photo shoot, and expert visual evaluation by photo shoot were conducted. After using the product, questionnaire evaluation was conducted at all evaluation points, and adverse reactions were evaluated through the interview of the person in charge of the test. The test results were compared and evaluated before and after depending on the period of use of the product.

This test passed the review of the Institutional Bioethics Review Board (IRB) (IRB Approval No. 2020021201-202004-HR-002-01), and was conducted in compliance with bioethics with the subject's voluntary consent.

2-1. Investigator selection

Among male and female subjects aged 18 to 54 years diagnosed with androgenetic alopecia, subjects satisfying the selection and exclusion criteria through pre-screening were selected and tested. Twelve people were recruited and tested so that more than 10 people could complete the test, and the final 12 people completed the test normally.

2-2. data analysis

The statistical significance of the measurement results was tested using SPSS Subscription (IBM SPSS Statistics, USA). Normality was tested through the Shapiro-Wilk test. If normality was satisfied, a parametric test was performed, and if normality was not satisfied, a non-parametric test was performed. Statistical analysis results were confirmed to be significant at the 95% confidence interval.

For the before-and-after comparison, the paired t-test was used as a parametric test, and the Wilcoxon Signed-rank test was used as a non-parametric test.

2-3. Test methods and results

2-3-1. Phototrichogram. total number of hairs used

After uniformly shaving the hair within a 1cm ² circle centered on the point tattoo on the hair loss area to be evaluated, the image was taken at 14 times magnification with a magnifying device, Folliscope® 5.0 (LeadM, Korea). The total number of hairs before (0 weeks), 6 weeks, and 12 weeks after using the product was analyzed using the phototricogram analysis system.

8 is a result of hair count analysis using a phototrichogram according to an experimental example of the present invention. As a result of the final 12 subjects, compared with before (0 weeks) using the product, 6 after using the product. It can be seen that the total number of hairs increased statistically significantly at week 12 and week 12 (p<0.05).

2-3-2. Subject's questionnaire evaluation

Efficacy questionnaire evaluation was conducted at 6 and 12 weeks after using the product, and usability questionnaire evaluation was conducted at 12 weeks after using the product. Efficacy questionnaire evaluation was conducted on a 7-point scale (1: very bad, 2: bad, 3: slightly worse, 4: no change, 5: slightly improved, 6: improved, 7: very good). The questionnaire evaluation was conducted on a 5-point scale (1: not good at all, 2: not good, 3: average, 4: good, 5: very good).

Tables 3 and 4 below are the results of the subject's questionnaire evaluation regarding efficacy and the positive response rate.

Tables 5 and 6 below are the questionnaire evaluation results and positive response rates of subjects regarding usability.

2-3-3. safety assessment

At each evaluation point, subjective skin irritation and objective skin irritation were evaluated by the questionnaire and the examiner's observation.

Tables 7 and 8 below are the results for the adverse reaction according to an experimental example of the present invention, and no special adverse reaction was observed during the test period.

3. Conclusion

As a result of the “Simple Human Application Test for Relief of Hair Loss Symptoms of BSKLI01” at CRA Korea Co., Ltd. at the request of the applicant, BK Bio, the total number of hairs was analyzed compared to before (0 weeks) before using the product. The total number of hairs increased statistically significantly at 6 weeks and 12 weeks after use.

As a result of the questionnaire evaluation on efficacy, the positive response rate for all 6 questions related to hair loss symptom relief at 6 weeks and 12 weeks after using the product was 50% or more. In the 12th week after using the product, the positive response rate to the question that the hair loss area improved and the crown became abundant was 91.67%. As a result of the usability questionnaire evaluation, 83.33% of the subjects responded positively to the usability of the product. From the results of the above 12 test subjects, it is judged that BSKLI01 tends to help alleviate the symptoms of hair loss.

The test product commissioned by BK Bio Co., Ltd. did not show any special skin adverse reactions during the test period.

As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

1. Lagerstroemia ( Lagerstroemia ) indica ) extract as an active ingredient,

   The extract is a cosmetic composition for preventing hair loss or promoting hair growth, characterized in that the leaves are extracted with an aqueous ethanol solution.
2. The method of claim 1,

   The Barerong tree extract,

   A cosmetic composition for preventing hair loss or promoting hair growth, characterized in that it induces or promotes the growth of dermal papilla cells.
3. The method of claim 1,

   The Barerong tree extract,

   A cosmetic composition for preventing hair loss or promoting hair growth, characterized in that it activates WNT signal transduction, which is a hair target protein, and inhibits TGF-β signal transduction.
4. The method of claim 1,

   The Barerong tree extract,

   β-catenin, T cell factor (TCF)/lymphoid enhancer factor (LEF), B-cell lymphoma 2 (BCL-2), glioma-associated oncogene homolog 1 (Gli-1), and vascular endothelial growth factor), a cosmetic composition for preventing hair loss or promoting hair growth, characterized in that it increases the expression of one or more dermal papilla cell growth-related factors selected from the group consisting of.
5. The method of claim 1,

   The Barerong tree extract,

   A cosmetic composition for preventing hair loss or promoting hair growth, characterized in that it is included in an amount of 0.01 to 50 parts by weight based on 100 parts by weight of the total cosmetic.
6. The method of claim 1,

   The composition is

   Hair tonic, hair cream, hair lotion, hair shampoo, hair rinse, hair spray, hair aerosol, pomade, a cosmetic composition for preventing hair loss or promoting hair growth, characterized in that it has a powder or gel formulation.
7. Contains Lagerstroemia indica extract as an active ingredient,

   The Barerong tree extract is a pharmaceutical composition for preventing hair loss or promoting hair growth, characterized in that the Barerong tree leaves are extracted with an aqueous ethanol solution.
8. Contains Lagerstroemia indica extract as an active ingredient,

   The Barerong tree extract is a health food composition for preventing hair loss or promoting hair growth, characterized in that the Barerong tree leaves are extracted with an aqueous ethanol solution.

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