WO2017116092A1 - Composition for preventing or treating hair loss or promoting hair growth or hair restoration comprising diosmin as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating hair loss or for promoting hair growth or hair restoration; and a quasi-drug, a cosmetic composition, a functional food composition or a food composition for improving hair loss or promoting hair growth or hair restoration, comprising diosmin or a pharmaceutically acceptable salt thereof. According to the present invention, the composition of the present invention not only contains, as an active ingredient, a natural compound having no side effects even when administered for a long period of time for hair loss, which is a chronic disease, but also exhibits excellent and stable effects of hair growth and hair restoration, so that the present invention can be usefully employed as an effective hair loss preventing or treating agent, or as a quasi-drug/cosmetic/functional food/food composition for promoting hair growth or hair restoration.

Description

Hair loss prevention or treatment, or hair growth or hair growth promoting composition comprising diosmin as an active ingredient

This application claims the priority of Korean Patent Application No. 10-2015-0188799, filed December 29, 2015, the entirety of which is a reference of the present application.

The present invention relates to a composition for preventing or treating hair loss or for promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.

According to the National Health Insurance Corporation's Health Insurance Policy Research Institute analyzing the health insurance payment data from 2001 to 2008, the actual number of patients with 'hair loss disease' was 103,000 in 2001 to 142,000 in 2005, and In 2008, it was 165,000, an increase of 60% over the last seven years. By age, 114,000 patients in their 20s and 40s were diagnosed, accounting for 69.5% of patients, and over 22,000 patients were in their teens. As of 2008, there were 84,000 males and 80,000 females, with males slightly more than females. By year, alopecia areata (130,000), alopecia areata (20,000), androgenetic alopecia (9,000), and other non-scarious hair loss (8,000) were followed.

The prevalence of hair loss patients overseas showed that in June 2003, data from the International Hair and Plastic Beauty Research Debate reported that 250 million people were alopecia worldwide, and the incidence of hair loss was 30-65% at 24-50 years of age. As of 2008, there are about 300 million alopecia in China, 30% of male population in their 30s and 50% of male population in their 50s show signs of hair loss, and the number of hair loss patients increases by 10-15% every year. Doing. The incidence of hair loss in Japan was 26.5%, and the estimated population of hair loss patients was estimated at 1,293 million.

Currently, drugs for treating hair loss are largely classified into medicines, quasi-drugs, and cosmetics. Specialty drugs that can be purchased only with a doctor's prescription include Propecia, developed and sold by Merck, USA, and its main ingredient, Finasteride, was approved by the US FDA in December 1997 to treat hair loss. Finasteride is a drug that inhibits the 5-α-reductase enzyme, which converts testosterone into dihydrotestosterone (DHT), and serves to grow soft hair into thick and long hair. It is effective in improving hair loss in the short term, but side effects such as erectile dysfunction, decreased sexual function, and breast enlargement in men have been reported. Minoxidil is a drug that can be purchased without a doctor's prescription because of its safety and effectiveness. In December 1997, it was approved by the US FDA as the first anti-hair loss treatment. This drug has the effect of promoting hair growth by improving blood circulation and opening potassium channels, but it may cause local reactions such as itching and rashes, and tachycardia.

The quasi-drug products that have been approved for hair loss prevention and hair growth by the KFDA include CJ Lion's 'Hair Power Competency', Moracle's 'Hair Tonic', and LG H & H's 'Mo and Moa'. In addition, products are used on the scalp or hair to maintain or promote the health of the hair.

Human hair loss cycle is largely divided into anagen, catagen and telogen. The growth phase is a time when hair growth grows rapidly due to vigorous cell division and cell division. The lifespan of the growing season varies depending on the type of hair, but for hair it is about 3-6 years. Growth hair accounts for 80-90% of the total hair, and hair loss is progressing in people who have a short hair growth period and a long rest period, which reduces the proportion of growth hair in the total hair. The degenerative phase is the end of the growth phase of the hair and the production of the hair gradually slows down, resulting in cell division and growth stopping. The degenerative lifespan is about 1-1.5 months and about 1% of the hair belongs to this stage. Resting phase is the final stage of growth when hair follicles and papillae are completely separated, causing hair follicles to atrophy and hair roots to rise upwards and hair fall out. The rest period lasts 3-4 months and 4-14% of all hairs fall into this stage. After the rest period, the activity of the nipple is active again, new nipples are created, and the hair in the resting period is pushed out of the scalp.

Diosmin is a flavonoid glycoside (flavone) -based compound, and the name designated by the International Pure and Applied Chemical Organization (IUPAC) is 5-hydroxy-2- (3-hydroxy-4-methoxyphenyl ) -7-[(2S, 3R, 4S, 5S, 6R) -3,4,5-trihydroxy-6-[[((2R, 3R, 4R, 5R, 6S) -3,4,5-tree Hydroxy-6-methyloxan-2-yl] oxymethyl] oxan-2-yl] oxychromen-4-one [5-Hydroxy-2- (3-hydroxy-4-methoxyphenyl) -7-[(2S , 3R, 4S, 5S, 6R) -3,4,5-trihydroxy6-[[(2R, 3R, 4R, 5R, 6S) -3,4,5-trihydroxy-6-methyloxan-2-yl] oxymethyl] oxan-2-yl] oxychromen-4-one], also called diosmetin. CAS registration number of the diosmin is 520-27-4, the structural formula is C ₂₈ H ₃₂ O ₁₅ , the molecular weight is 608.5 g / mol, the formula is shown in the formula (1).

[Formula 1]

Currently, diosmin is a major component of the drug that is commercially available for the treatment of diseases such as chronic venous insufficiency, hemorrhoids, lymphedema, etc. In addition, dermatitis, wound healing, premenstrual syndrome, viral infections, breast pain ( mastodynia, dermatofibrosclerosis, colitis, and the like (Ramelet AA. Clinical benefits of Daflon 500 mg in the most severe stages of chronic venous insufficiency. Angiology. 2001, 52: S49-S56; Hasanoglu A, et al.Efficacy of micronized flavonoid fraction in healing of clean and infected wounds.International Journal of Angiology.2001, 10: 41-44; Bae EA, et al.In vitro inhibitory effect of some flavonoids on rotavirus infectivity. and Pharmaceutical Bulletin. 2000, 23: 1122-1124; Crespo ME, et al. Antiinflammatory activity of diosmin and hesperidin in rat colitis induced by TNBS.Planta Medica. 1999, 65: 651-653), and various animal models Anticancer and cell proliferation inhibitory effects have been identified in cell line models (Kuntz S, et al. Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines.European Journal of Nutrition. 1999, 38: 133-142; Yang M, et al. Chemopreventive effects of diosmin and hesperidin on N-butyl-N- (4-hydroxybutyl) nitrosamine-induced urinary-bladder carcinogenesis in male ICR mice. I International Journal of Cancer. 1997, 73: 19-24; Tanaka T, et al. Modulation of N-methyl-N-amylnitrosamineinduced rat oesophageal tumourigenesis by dietary feeding of diosmin and hesperidin, both alone and in combination. Carcinogenesis. 1997, 18: 761-769). However, there is no report on the anti-hair loss or hair growth promoting activity of diosmin.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have made diligent research efforts to find naturally occurring compounds that can prevent hair loss or promote hair growth or hair growth without side effects. As a result, the present invention was completed by confirming that diosmin effectively promotes hair growth and hair growth and prevents hair loss.

Therefore, an object of the present invention is a pharmaceutical composition for preventing or treating hair loss, or promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient; And quasi-drugs, cosmetic compositions, functional food compositions or food compositions for improving hair loss or promoting hair growth or hair growth.

In addition, the present invention is to provide a method for preventing or treating hair loss, or promoting hair growth or hair growth, including administering a composition comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.

Furthermore, the present invention is to provide a use of a composition comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient for the prevention or treatment of hair loss, or for the preparation of hair growth or hair growth promoter.

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

According to one aspect of the invention, the present invention provides a pharmaceutical composition for preventing or treating hair loss, or promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.

The present inventors have made diligent research efforts to find naturally occurring compounds that can prevent hair loss or promote hair growth or hair growth without side effects. As a result, it was confirmed that diosmin effectively promotes hair growth and hair growth and prevents hair loss.

As used herein, the term "hair loss" refers to a phenomenon in which the hair completely comes out of the scalp. A person who is undergoing hair loss will have a short hair growth period and a long hair rest period. As demonstrated in the following examples, diosmin converts hair from a resting period to a growing phase (FIG. 1) to prevent, improve or prevent hair loss. It has been found to be effective in treating and promoting wool.

As used herein, the term "hair growth" refers to hair growth in the scalp, and the term "hair growth" as used herein means that the length of the hair is increased (ie, hair growth). As demonstrated in the examples below, diosmin promoted hair growth compared to the negative control (FIG. 1), increased hair length by 267% (FIG. 2), increased the number and diameter of hair follicles (FIGS. 3A to 3). 3c), increasing the expression of growth factors related to hair growth in the skin tissue (Fig. 4a, 4b, 5a and 5b) was confirmed that the hair growth promoting effect.

The diosmin of the present invention may be used in the form of a pharmaceutically acceptable salt, and acid salts formed by pharmaceutically acceptable free acid are useful as salts. Inorganic acids and organic acids can be used as the free acid.

Specifically, pharmaceutically acceptable salts of diosmin of the present invention are hydrochloride, bromate, sulfate, phosphate, citrate, acetate, trifluoroacetate, lactate, tartarate, maleate, fumarate, gluconate, Methanesulfonate, glyconate, succinate, 4-toluenesulfonate, gluturonate, embonate, glutamate, or aspartate, but may be selected from the group consisting of, but is not limited thereto All salts formed using various inorganic and organic acids are included. Diosmin of the present invention may also be present in the form of solvates (eg hydrates).

According to one embodiment of the present invention, the composition of the present invention increases β-catenin expression, Wnt10b (Wigless related MMTV integration site 10b), FZD1 (Frizzled receptor 1), LRP5 (Low-density lipoprotein receptor-related protein) 5) Increases expression of genes selected from the group consisting of Insulin-like growth factor 1 (IGF1), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and keratocyte growth facfor (KGF). According to one embodiment of the present invention, the composition of the present invention reduces the expression of glycogen synthase kinase 3β (GSK3β) or Axin. The inventors of the present invention not only increase the expression of growth factors IGF1, VEGF, HGF and KGF that affect hair growth in skin tissue, but also Wnt / β-catenin signaling agents Wnt10b, FZD1, LRP5 and β It also increases catenin expression and activates the Wnt / β-catenin signaling system, which has been shown to play a pivotal role in the development and hair growth of hair follicles by confirming that the expression of GSK3β and Axin, which are inhibited by Wnt10b, is reduced. It was confirmed at the molecular level that the composition of the present invention promotes hair growth and wool (FIGS. 5A, 5B). Therefore, the composition of the present invention can be applied as an effective hair regrowth composition showing multiple and stable hair growth promoting effects.

According to the present invention, the composition of the present invention may be prepared as a pharmaceutical composition for preventing or treating hair loss, or promoting hair growth or hair growth. When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, 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 doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations 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, administered parenterally, and according to another embodiment of the present invention, administered by transdermal administration.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Preferred dosages of the pharmaceutical compositions of the invention are in the range of 0.0001-100 mg / kg on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

According to another aspect of the present invention, the present invention provides a quasi-drug composition for improving hair loss or promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.

When the composition of the present invention is used as an quasi-drug composition, the diosmin or a pharmaceutically acceptable salt thereof may be added as it is, or may be used together with other quasi-drug components, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). According to an embodiment of the present invention, the quasi-drug composition of the present invention may be a disinfectant cleaner, a shower foam, a gagreen, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment, or a filter filler.

According to another aspect of the present invention, the present invention provides a cosmetic composition for improving hair loss, or promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.

The components included in the cosmetic composition of the present invention include components conventionally used in cosmetic compositions in addition to diosmin or a pharmaceutically acceptable salt thereof as the active ingredient, and include, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments and Conventional adjuvants such as perfumes, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

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, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant 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 the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

According to another aspect of the present invention, the present invention provides a functional food composition or food composition for improving hair loss, or promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.

When the composition of the present invention is made of a functional food composition or a food composition, it contains not only diosmin or a pharmaceutically acceptable salt thereof as an active ingredient, but also components commonly added in the manufacture of the functional food or food, for example Examples include proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

When the functional food or food composition of the present invention is prepared with a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, in addition to diosmin or a pharmaceutically acceptable salt thereof, the active ingredient of the present invention, Jujube extract or licorice extract may be further included.

Since the quasi-drugs, cosmetics, functional foods, and food compositions of the present invention share the above-mentioned pharmaceutical composition with active ingredients and uses, common contents in relation to the pharmaceutical composition are to avoid excessive complexity of the present specification. Omit the description.

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

(a) The present invention provides a pharmaceutical composition for preventing or treating hair loss, or promoting hair growth or hair growth; And quasi-drugs, cosmetic compositions, functional food compositions or food compositions for improving hair loss or promoting hair growth or hair growth.

(b) The composition of the present invention not only has a natural compound having no side effects even during long-term administration of hair loss, which is a chronic disease, but also shows excellent and stable effects on hair growth and hair growth, and is effective in preventing or treating hair loss, or hair growth or It can be usefully used as a quasi-drug / cosmetics / functional food / food composition for promoting hair growth.

1 is a diagram showing the results of observing the hair regrowth as the number of days of administration of each sample in the depilated mouse model.

2 is a graph showing the length of hair grown during the experimental period in the mouse coated with each sample. Each value is the mean ± standard error of 9 mice. ns = p > 0.05, ** = p <0.01, *** = p <0.001 [one-way ANOVA, Tukey's test].

Figure 3 shows the results of histological observation of the skin, such as a mouse coated with each sample for 4 weeks. Figure 3a is a result of confirming the increase in the number of hair follicles, Figure 3b is a result of evaluating the diameter of the hair follicles, Figure 3c is a photograph showing the skin expression of the hair follicles. Each value is the mean ± standard error of 9 mice. ns = p > 0.05, ** = p <0.01 [one-way ANOVA, Tukey's test].

Figure 4 shows the protein expression changes in skin tissue, such as a mouse coated with each sample. 4A is an immunohistochemical result, and FIG. 4B is a Western blot result. Western blot results are mean ± standard error of 3 independent experiments in each group (n = 2 or 3 for each experiment). ns = p > 0.05, ** = p <0.01 [one-way ANOVA, Tukey's test].

Figure 5 is a diagram showing the change in gene expression of skin tissue, such as a mouse coated with each sample. RT-PCR results are the mean ± standard error of two or three independent experiments in each group (n = 2 or 3 for each experiment). ns = p > 0.05, * = p <0.05, ** = p <0.01, *** = p <0.001 [one-way ANOVA, Tukey's test].

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example  One: Diosmin  Hair growth promoting effect

1-1. Breeding of test animals and application of samples

Sample Preparation

Vehicle (Vehichle, ethanol: water: propylene glycol = 5: 3: 2), the control drug minoxidil (MXD), the test material diosmin (DSM) was purchased from Sigma Aldrich Co., Ltd ..

Breeding of Experimental Animals

Twenty-four male C57BL / 6N mice, 6 weeks old, were purchased from Orient Bio Co., Ltd. and adapted to the rearing room environment for two weeks, after which the negative control group (Con group), positive control group (MXD group) and diosmin group (DSM group) The experiment was divided into three groups. The animal cage was maintained at a temperature of 21 ± 2.0 and a relative humidity of 50 ± 5% for 12 hours at day and night. During the experiment, the mice were freely fed with a general solid chow and water.

Skin application and visual observation of the sample

To examine the hair loss prevention, hair growth or wool effect, 8 week old mice with resting hairs with pink dorsal skin color were used. After the hair of the back of the mouse was removed using a mouse clipper, each sample was applied once every four weeks at 4 pm. Diosmin (3 mg / mouse / day), a test substance, and minoxidil (3 mg / mouse / day) used as a control drug were applied by dissolving in a vehicle (ethanol: water: propylene glycol = 5: 3: 2). Only the vehicle was applied to the negative control group.

In order to visually check the state of hair growth, the application was started, and at 1, 2, 3, and 4 weeks, the animals were lightly anesthetized with ether, and the back part was photographed. The hair length was measured using a ruler to evaluate the degree of hair growth.

1-2. Measurement of weight change

The body weight of the test animals was measured every week from just before the sample application to the time when the application was completed. The initial weights of the negative control group (Con group), test group (DSM group), and positive control group (MXD group) were not significantly different between the groups, and the weights measured after 4 weeks of application were not significantly different between the experimental groups.

1-3. Hair growth condition and Hair-length  Change measurement

Visual characteristics of hair growth

While the sample was applied to the back of the mouse for 4 weeks, the pattern of hair growth was confirmed by taking pictures every week. Mice entering Telogen had a pink color on their body surface when epilated. In the case of the negative control group (Con group), almost no hair grew even after 3 weeks, but in the DSM group, the hair began to grow from the 2nd week of application, and the body surface color changed to black, and the hair cycle entered the anagen phase from the resting period to 4 weeks. Later it was observed that the hairs grew evenly and vigorously in more individuals than in the negative control (FIG. 1).

Change in hair length

The length of the hair was measured every week during the experiment (4 weeks). The hair length of the DSM group increased significantly compared to the negative control group at 2 weeks, and after 2 weeks, the hair length of the DSM group was increased by 267% (p <0.001) (FIG. 2). Therefore, it can be seen that diosmin has an excellent effect of promoting hair growth.

1-4. Histological analysis of the back skin

Four weeks after the application of each sample, the animals were sacrificed, and the skin tissue samples were removed using scissors and forceps around the site where the test substance was applied, and then fixed with formalin. After dehydration with alcohol and xylene step by step, embedded in paraffin, 5 μm sections were made using a microtome and paraffin was removed again with alcohol and xylene. Hematoxylin-Eosin (H & E) staining was performed, and histological changes of hair follicle tissues were observed by light microscopy. Pathologists examined the hair growth cycle, and the number and diameter of hair follicles were measured on an optical microscope.

 The result of measuring the number of hair follicles in each test group under an optical microscope at 40 magnification is shown in FIG. 3. The number of hair follicles was significantly increased in the DSM group and the MXD group compared to the negative control group (FIG. 3A). As a result of evaluating the hair follicle diameter of each test group through the image analysis, it was significantly increased in the DSM group and the MXD group compared to the negative control group (Fig. 3b). In addition, in the DSM group and the MXD group, hair follicles were prolonged and exposed to the skin, thereby confirming the hair growth promoting effect of diosmin and minoxidil (FIG. 3C).

Example  2: Diosmin  Regulation of protein and gene expression in mouse skin tissue

2-1. Expression of protein related to hair growth promotion in skin tissue

Immunohistochemical Analysis

Recently, it has been found that intracellular signaling activators such as Wnt / β-catenin and the like play a role in hair growth and dropping, and thus, these factors have attracted attention as new targets of drugs for inhibiting hair loss and promoting hair growth. Therefore, the immunohistochemical study was conducted to determine whether the Wnt / β-catenin signaling system involved in preventing hair loss and promoting hair growth (or hair growth) in the back skin tissue was applied by 4 weeks of sample application.

In order to observe the expression level of IGF1 and β-catenin related to hair growth in skin tissues, primary antibodies against IGF1 and β-catenin were diluted 1:50 and added to tissue sections and reacted at room temperature for 12 hours. . Dilution of the primary antibody was used by mixing 1% normal goth serum (Vector Laboratories Inc.) and 0.3% Triton X-100 (Sigma) in 0.1 M phosphate buffer (PB). Tissue sections were washed twice with 0.1 M PB for 15 minutes at room temperature and again reacted with a secondary antibody [Biotinylated anti-rabbit IgG (Vector Laboratories Inc.) diluted 1: 200 at room temperature for 1 hour. After washing twice with 0.1M PB for 15 minutes, it was immersed in peroxidase-labeled ABC (avidin-biotin complexex) solution and reacted at room temperature for 1 hour. Afterwards, washed twice with 0.1M PB for 15 minutes, 30 mg of 3-3'diaminobenzidine was dissolved in 150 ml of 0.1M PB for 5 minutes, and then hydrogen peroxide was added at a concentration of 0.005%. Brown color reaction was performed for about 5 minutes. After completion of the reaction, the tissues were washed several times with 0.1 M PB and counterstained with hematoxylin for 20 seconds. After dehydration and clarification according to a conventional method, the tissues were encapsulated and observed with an optical microscope.

As a result, more β-catenin and IGF1 were detected in the DSM group than in the negative control group. The increase effect of β-catenin and IGF1 expression in the back skin tissue by such diosmin was observed to be more excellent than minoxidil (Fig. 4a).

Weston Blot  Method

In a mortar, a certain amount of skin tissue was homogenized with liquid nitrogen and lysis buffer, and then centrifuged at 13,000 × g, 4 for 20 minutes, the middle layer was taken, and protein was quantified by Bradford method. 50 μg of protein was electrophoresed on SDS polyacrylamide gel, followed by electroblotting onto PVDF hyperfilm and the corresponding antibody, β-catenin and β-actin (all from Cell-signaling Technology, USA) And reacted with each. The signal of each protein was visualized with a chemiluminescent detection system (Amersham) and the thickness of the band was quantified using Quantity One Analysis Software (Bo-Rad Laboratories).

As a result, it was observed that β-catenin protein expression was significantly increased in the DSM group compared to the negative control group (FIG. 4B).

2-2. Analysis of hair related gene expression in skin tissue

Trizol Triz method  RNA isolation using

In order to analyze and quantify total RNA in skin tissue of experimental animals, 1 mL of trizol solution was added to 50-100 mg of skin tissue, homogenized, and centrifuged at 4 ° C. and 12,000 × g for 10 minutes. The supernatant was transferred to a new tube and 200 μL of chloroform was added and vortexed. After repeating this process twice, the supernatant was transferred to a new tube, and isopropanol and supernatant were added at a 1: 1 ratio. After shaking vigorously 10 times and left for 10 minutes at room temperature, centrifuged at 12,000 × g and 4 ° C for 10 minutes, the supernatant was removed, and 1 ml of 70% ethanol was added to the remaining precipitate to obtain 7,500 × g, 4 ° C. Centrifuge for 5 minutes at. After removing the ethanol, the tube containing the RNA precipitate was dried at room temperature for 5 minutes and the RNA precipitate was dissolved using nuclease free water. The concentration of the RNA sample extracted at 260 nm and 280 nm was measured using a UV / VIS spectrophotometer (Beckman coulter, DU730), and agarose gel electrophoresis was performed to confirm the integrity of the RNA sample.

RT- PCR (Reverse Transcription-Polymerase Chain Reaction) Method

After the RNA was isolated, cDNA was synthesized by performing reverse transcription using oligo dT primer and Superscript reverse transcriptase (GIBCOBRL, Gaithersburg, USA). PCR was performed using cDNA obtained through reverse transcription as a template and using 5 'and 3' flanking sequences of cDNA of the gene to be amplified as a primer. At this time, the necessary primers were used for the synthesis of Bioneer Co., Ltd., and these primer sequences are shown in Table 1 below. 2 μL of reaction to a mixture of 1 μL of 10 × reaction buffer [100 mM KCL, 20 mM Tris-HCL (pH 8.0), 2.5 mM MgCl ₂ ], 5 μL of 10 mM dNTP, 0.2 μM sense and antisense primer, respectively After adding the cDNA reaction mixture and 2.5 units of Taq polymerase (Takara, Japan), the volume was adjusted to 50 μL with distilled water, and PCR was performed using a PCR device. PCR conditions were set to 94 (4 minutes), 94 (30 seconds), 30 cycles of [52 (30 seconds), 72 (45 seconds)], and 72 (10 minutes). Gene expression was evaluated by electrophoresing 1 μL of the amplified product on 1% agarose gel to identify DNA bands.

Expression Control Results of Hair Growth Related Genes

Changes in the expression of Wnt / β-catenin signaling agents known as hair loss prevention, hair growth (or hair growth) promoting mechanisms in the back skin tissues of mice coated with experimental materials for 4 weeks were analyzed by RT-PCR analysis. The expression of the frizzled receptor 1 (FZD1) and the low-density lipoprotein receptor-related protein 5 (LRP5) were significantly increased in the DSM group compared to the negative control group. As a result, GSK3β (glycogen synthase kinase 3β) and Axin, which are inhibited by Wnt10b, were significantly decreased in the DSM group compared to the negative control group. The expression of β-catenin was significantly increased in the DSM group compared to the negative control group by activation of the Wnt / β-catenin signaling system (FIG. 5A).

In addition, the expression changes of the endocrine factors affecting hair growth were evaluated to investigate the mechanism of hair loss prevention, hair growth or hair growth promoting action of diosmin. Gene expression levels of IGF1 (insulin-like growth factor 1), VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), and KGF (keratocyte growth facfor) As a result of the evaluation, the expression of all four genes in the DSM group was significantly increased compared to the negative control group (FIG. 5B).

Thus, diosmin accelerates hair follicle length growth, thereby activating hair follicles back to the growth phase, and also promotes hair regrowth by increasing the expression of IGF1, HGF, VEGF and KGF in skin tissues, preventing hair loss, hair growth and hair growth. It could be seen that it promotes.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (7)

1. A pharmaceutical composition for preventing or treating hair loss, or promoting hair growth or hair growth, comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.
2. The method of claim 1,

   The composition increases β-catenin expression, Wnt10b (Wigless related MMTV integration site 10b), FZD1 (Frizzled receptor 1), LRP5 (Low-density lipoprotein receptor-related protein 5), IGF1 (Insulin-like growth factor 1) ), β-catenin, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and KGF (keratocyte growth facfor)

   A pharmaceutical composition for preventing or treating hair loss or promoting hair growth or hair growth.
3. The method of claim 1,

   The composition is characterized in that to reduce the expression of GSK3β (glycogen synthase kinase 3β) or Axin

   A pharmaceutical composition for preventing or treating hair loss or promoting hair growth or hair growth.
4. A quasi-drug for improving hair loss or promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.
5. A cosmetic composition for improving hair loss or promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.
6. Functional food composition for improving hair loss, or promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.
7. Food composition for improving hair loss, or promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.

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