KR101725461B1 - Composition for Preventing or Treating Hair Loss or Stimulating Hair Sprouting or Hair Growth Comprising Diosmin as Active Ingredients - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating hair loss or promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof; 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 growth. According to the present invention, the composition of the present invention not only contains a natural compound having no side effects even in chronic treatment for hair loss, which is a chronic disease, but also has an excellent and stable effect on hair growth and hair growth, Cosmetics for the promotion of hair growth or hair growth / cosmetics / functional food / food composition.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing hair loss or hair growth comprising diosmin as an active ingredient, or for promoting hair growth or hair growth.

The present invention relates to a 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.

According to the National Health Insurance Corporation 's Health Insurance Policy Institute' s analysis of health insurance payment data from 2001 to 2008, the number of patients treated with 'hair loss disease' increased from 103,000 in 2001 to 142,000 in 2005, In 2008, it reached 165,000, which has increased by 60% over the past seven years. By age, the number of patients in their 20s and 40s was 114,000, accounting for 69.5% of the patients, and the number of patients under 10 was more than 22,000. As of 2008, the number of patients treated by gender was 84,000 men and 80,000 women, with males slightly more than females. The number of patients treated with 'hair loss' (13,000), scarring alopecia (20,000), androgenic alopecia (9,000), and other non-scarring hair loss (8,000).

The prevalence rate of hair loss in overseas countries is estimated to be 250 million people in the world and 30-65% in the age group of 24-50 years, according to the International Hair and Cosmetic Research Debate on June, 2003. As of 2008, China's hair loss population is about 300 million, 30% of the male population in their 30s, and 50% of the male population in their 50s have hair loss symptoms, and the number of hair loss patients increases by 10-15% . In Japan, the rate of hair loss was 26.5%, and the estimated number of patients with hair loss was estimated to be around 1.293 million.

Currently, treatments for hair loss treatments are classified into pharmaceuticals, quasi-drugs, and cosmetics. The specialty medicine that can be purchased with a doctor's prescription is "Propecia", which is developed and sold by Merck, USA. Its main ingredient, Finasteride, was approved by the US FDA for hair loss treatment in December 1997. Pinasteride is an inhibitor of 5-α-reductase enzymes that convert testosterone to dihydrotestosterone (DHT), which is responsible for the growth of hair into thick, long hair. This is effective in improving hair loss in the short term, but side effects such as erectile dysfunction, sexual dysfunction and male breast enlargement are reported. Minoxidil has been approved as safe and effective and can be purchased without doctor's prescription. In December 1997, it was approved as FDA's first hair loss treatment. This drug has the effect of promoting hair growth by improving blood circulation and opening the potassium channel, but local reactions such as itching, rash, and tachycardia may occur.

Among the quasi-drug products approved by the KFDA for the prevention of hair loss and hair growth, there are CJ Lion's 'Hair Force Competent', Morlacle's 'Hair Tonic' and LG Household & Health Care's 'Mo.Moa'. Cosmetics include shampoo or skin , Products used for scalp or hair to maintain or promote hair health are being sold.

Human hair loss cycle is largely divided into anagen, catagen, and telogen. The growing period is the time when the activity of the breast milk is active and the cell division is vigorous and the hair grows at a high speed. The lifespan of the growing period varies depending on the type of hair, but in the case of hair, it is about 3-6 years. Growing hair accounts for 80-90% of total hair, and the person with hair loss progresses to shortening of growing period and having a long hair cycle, so that the specific gravity of growing hair in total hair is decreased. The regressive period is the period when the growth of the hair ends and the production of the hair is slowed down. As a result, cell division and growth are stopped. The life of the retrograde period is about 1-1.5 months, and about 1% of the total hair belongs to this stage. The resting stage is the final stage of growth, in which the hair follicles and hair follicles are completely separated and the hair follicles are contracted, and the hair follicles are further raised upward and hair is removed. The rest period lasts 3-4 months and 4-14% of total hairs are in this stage. When the dormant period is over and the activity of the dermal papilla becomes active again, the new dermal papilla is made, and the hair at the dormant pillar is pushed out completely out of the scalp.

Diosmin is a flavonoid glycoside (flavone) based compound, designated by the International Pure and Applied Chemistry Agency (IUPAC) as 5-hydroxy-2- (3-hydroxy-4-methoxyphenyl ) - 7 - [(2S, 3R, 4S, 5S, 6R) -3,4,5-trihydroxy- Hydroxy-2- (3-hydroxy-4-methoxyphenyl) -7 - [(2S , 3S, 5R, 6S) -3,4,5-trihydroxy-6-methyloxan-2-yl] oxymethyl] oxan-2-yl] oxychromen-4-one], also called diosmetin. DIOS seeds CAS registry number 520-27-4, the structural formula is C ₂₈ H ₃₂ O _15, molecular weight is 608.5 g / mol, the formula is shown in formula (1).

Formula 1

Currently, diosmin is a major component of drugs that are marketed and marketed for the treatment of diseases such as chronic venous insufficiency, hemorrhoids, and lymphedema. In addition, diosmin is a major component of dermatitis, wound healing, premenstrual syndrome, mastodynia, dermatofibrosclerosis, colitis, etc. (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) (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. IInternational Journal of Cancer. 1997, 73: 19-24; Tanaka T, et al. Modulation of N-methyl-N-amylnitrosamineinduced rat oesophageal tumourigenesis by dietary mining of diosmin and hesperidin, both alone and in combination. Carcinogenesis. 1997, 18: 761-769). However, there has been no report on the activity of preventing Diosmin hair loss or promoting hair growth.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made intensive researches in order to discover natural-derived compounds that can prevent hair loss or promote hair growth or hair growth without side effects. As a result, the present inventors have completed the present invention by confirming that Diosmin effectively promotes hair growth and hair growth and prevents hair loss.

Accordingly, an object of the present invention is to provide 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 a quasi-drug, a cosmetic composition, a functional food composition or a food composition for improving hair loss or 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, there is provided a pharmaceutical composition for the prevention or treatment of hair loss or for promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.

The present inventors have made intensive researches in order to discover natural-derived 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 promoted hair growth and hair growth and prevented hair loss.

As used herein, the term " hair loss " refers to a phenomenon in which the hair completely comes out of the scalp. People who are undergoing hair loss have shorter hairy periods and longer hairy periods. As demonstrated in the examples below, diosmin converts hair from resting to growth phase (FIG. 1) to prevent, improve, or prevent hair loss It was confirmed that there was an effect of treating and promoting wool.

As used herein, the term " hair growth " means that the hair grows in the scalp, and the term " hair growth " As demonstrated in the following example, diosmin promotes hair growth (Fig. 1), increased hair length by 267% (Fig. 2), increased number and diameter of hair follicles 3c), and the expression of growth factors associated with hair growth in skin tissue was increased (Figs. 4a, 4b, 5a and 5b).

The diosmin of the present invention can be used in the form of a pharmaceutically acceptable salt, and the acid addition salt formed by a pharmaceutically acceptable free acid is useful as a salt. As the free acid, inorganic acid and organic acid can be used.

Specifically, the pharmaceutically acceptable salts of the diosmin of the present invention include, but are not limited to, hydrochloride, bromate, sulfate, phosphate, citrate, acetate, trifluoroacetate, lactate, tartrate, maleate, fumarate, But are not limited to, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, gluturonate, ebonate, glutamate, or aspartate salts, And salts formed using various inorganic acids and organic acids. The diosmin of the present invention may also exist in the form of a solvate (for example, a hydrate).

According to an embodiment of the present invention, the composition of the present invention increases β-catenin expression or increases the expression of Wnt10b (Wigess related MMTV integration site 10b), FZD1 (Frizzled receptor 1), LRP5 (Low-density lipoprotein receptor- 5), insulin-like growth factor 1 (IGF1), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and keratocyte growth facor (KGF). According to one embodiment of the present invention, the composition of the present invention reduces the expression of GSK3? (Glycogen synthase kinase 3?) Or Axin. The present inventors not only increase the expression of IGF1, VEGF, HGF and KGF, growth factors affecting hair growth in skin tissue, but also found that Wnt / β-catenin signaling substances Wnt10b, FZD1, LRP5 and β -Catenin expression and to decrease the expression of GSK3β and Axin, which are inhibited by Wnt10b, thereby activating the Wnt / β-catenin signaling system, which has been found to play a central role in hair follicle development and hair growth To confirm that the composition of the present invention promotes hair growth and wool at the molecular level (Fig. 5a, 5b). Accordingly, the composition of the present invention can be applied as an efficient hair growth composition exhibiting a multifaceted and stable hair growth promoting effect.

According to the present invention, the composition of the present invention can be manufactured from a pharmaceutical composition for prevention or treatment of hair loss, or for promoting hair growth or hair growth. When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. 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, and may be administered parenterally according to one embodiment of the present invention, and transdermally administered according to another embodiment of the present invention.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . A preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.0001-100 mg / kg on an adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, there is provided 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 a quasi-drug composition, the diosmin or a pharmaceutically acceptable salt thereof may be directly added or used together with other quasi-drugs, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). According to one embodiment of the present invention, the quasi-drug composition of the present invention may be a disinfectant cleaner, a shower foam, a gagrin, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment agent or a filter filler.

According to another aspect of the present invention, there is provided 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 contained in the cosmetic composition of the present invention include components commonly used in cosmetic compositions in addition to diosmin or its pharmaceutically acceptable salt as an active ingredient, and examples thereof include antioxidants, stabilizers, solubilizers, vitamins, pigments and Customary adjuvants such as perfumes, and carriers.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto.

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

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. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters.

According to still another aspect of the present invention, there is provided a functional food composition or food composition for improving hair loss, 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 manufactured from a functional food composition or a food composition, it includes not only diosmin or its pharmaceutically acceptable salt as an active ingredient, but also components which are conventionally added in the production of a functional food or a food, For example, it includes proteins, carbohydrates, fats, nutrients, seasoning and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings.

When the functional food or the food composition of the present invention is prepared as a drink, the diosmin or its pharmaceutically acceptable salt, which is an active ingredient of the present invention, may be used in addition to citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, 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 the active ingredients and uses thereof, the common content in relation to the pharmaceutical composition is not limited to the above- The description is omitted.

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

(a) a pharmaceutical composition for preventing or treating hair loss, or for promoting hair growth or hair growth; And a quasi-drug, a cosmetic composition, a functional food composition or a food composition for improving hair loss, or for promoting hair growth or hair growth.

(b) The composition of the present invention not only contains a natural compound having no side effects even in chronic treatment for alopecia, but also exhibits an excellent and stable effect on hair growth and hair growth, thus showing an effective hair loss preventing or treating agent, Cosmetics / functional foods / food compositions for accelerating hair growth.

FIG. 1 is a diagram showing a result of observing regrowth of hair with the elapsed days of administration of each sample in a hair model mouse model. FIG.
FIG. 2 is a graph showing hair lengths grown during the experimental period in a mouse to which each sample is applied. Each value is the mean ± SE of 9 mice. ns = p > 0.05, ** = p <0.01, *** = p <0.001 [one-way ANOVA, Tukey's test].
Fig. 3 shows the result of histological observation of skin such as a mouse to which each sample was applied for 4 weeks. FIG. 3 (a) is a result of confirming the increase in the number of hair follicles, FIG. 3 (b) is a result of evaluating the hair follicle diameter, and FIG. Each value is the mean ± SE of 9 mice. ns = p > 0.05, ** = p <0.01 [one-way ANOVA, Tukey's test].
Fig. 4 shows protein expression changes in skin tissues such as mice coated with each sample. 4A is an immunohistological result, and FIG. 4B is a Western blot result. Western blot results are the mean ± SE of three 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].
FIG. 5 is a graph showing changes in gene expression of skin tissues such as mice to which each sample is applied. The RT-PCR results are the mean ± SE of 2 or 3 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. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1 Effect of Diosmin on Hair Growth Promotion

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

Preparation of sample

(Vehichle, ethanol: water: propylene glycol = 5: 3: 2), the reference drug minoxidil (MXD) and the test substance diosmin (DSM) were purchased from Sigma Aldrich.

Breeding of experimental animals

Twenty-four male C57BL / 6N mice were purchased from Orient Biotech Co., Ltd. and were adapted to the breeding environment for 2 weeks. Thereafter, a negative control (Con group), a positive control group (MXD group) and a diosmin group Three groups were used for the experiment. The animals were kept at a temperature of 21 ± 2.0 ° C and a relative humidity of 50 ± 5% for 12 hours. During the experimental period, the mice were fed free of general solid feed (chow) and water.

Skin application and gross observation method of sample

In order to investigate hair loss prevention, hair growth or wool effect, 8-week-old mice with resting hairs whose pink color was seen on the dorsal skin were used. After removing the hairs of the mouse back plate using a clipper for a mouse, each sample was applied once a day at 4:00 PM for 4 weeks. Minoxidil (3 mg / mouse / day) used as a test substance and diosmin (3 mg / mouse / day) as an experimental substance were dissolved and applied in a vehicle (ethanol: water: propylene glycol = 5: 3: 2) Only the vehicle was applied to the negative control group.

At the end of 1, 2, 3, and 4 weeks of application, the animals were lightly anesthetized with ether and photographed. Hair length was measured using a ruler to evaluate the extent of hair growth.

1-2. Measurement of weight change

The weight of the experimental animals was measured weekly from immediately before application of the sample to the end of application. There was no significant difference in the initial body weights of the negative control group (Con), the test group (DSM group), and the positive control group (MXD group).

1-3. Measurement of changes in hair growth and hair length

Gross features of hair growth

The sample was applied to the back part of the mouse for 4 weeks, and the appearance of the hair growing each week was photographed and confirmed. The mice that entered the telogen were pink when the hair was removed. In the negative control group (Con group), hair did not grow even after 3 weeks. 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 from the resting period. Thereafter, it was observed that the hair grows evenly and vigorously in a larger number of individuals than in the negative control group (Fig. 1).

Change in hair length

The length of the hair was measured every week for the duration of the experiment (4 weeks). The hair length of the DSM group was significantly increased from the 2nd week to the negative control group, and increased by 267% after 4 weeks compared to the negative control group (p <0.001) (Fig. 2). Therefore, it can be seen that diosmin is excellent in promoting hair growth.

1-4. Histological analysis of skin

Four weeks after the application of each sample, the test animal was sacrificed and the dermal tissue sample was extracted with scissors and forceps around the area to which the test substance was applied, and fixed with formalin. Stepwise dehydration treatment with alcohol and xylene, embedding with paraffin, and 5 μm slice using a microtome were repeated to remove paraffin with alcohol and xylene. Hematoxylin-eosin (H & E) staining was performed and histological changes of the hair follicles were observed under an optical microscope. Histopathologists were examined to evaluate the hair growth cycle, and hair follicle volume and diameter were measured on an optical microscope.

 The results of measurement of the number of hair follicles in each test group under an optical microscope of 40 magnification are shown in FIG. In the DSM and MXD groups, the number of hair follicles was significantly increased compared to the negative control group (Fig. 3a). Image analysis showed that the hair follicle diameter of each test group was significantly increased in the DSM and MXD groups as compared to the negative control group (FIG. 3B). In addition, in the DSM group and the MXD group, hair follicles were prolonged and the skin was expressed, thereby confirming the hair follicle stimulating effect of diosmin and minoxidil (FIG. 3C).

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

2-1. Changes in Protein Expression Associated with Hair Growth Promotion in Skin Tissue

Immunohistochemical analysis

Recently, it has been shown that the intracellular signal transduction activating factors such as Wnt /? -Catenin plays a role in hair growth and elimination, and the factor is attracting attention as a new target of drugs for suppressing hair loss and promoting hair growth. Therefore, we investigated the activation of Wnt / β-catenin signal transduction system, which is involved in the prevention of hair loss and promotion of hair growth (or hair growth), in the dorsal skin tissue by 4 weeks of application of the sample by immunohistochemical method.

To observe the expression of IGF1 and β-catenin associated with hair growth in skin tissue, the primary antibody against IGF1 and β-catenin was diluted 1:50, respectively, and then added to tissue sections and allowed to react at room temperature for 12 hours . Dilution of the primary antibody was performed by mixing 1% normal goat serum (Vector Laboratories Inc.) and 0.3% Triton X-100 (Sigma) in 0.1 M phosphate buffer (PB) The tissue sections were washed with 0.1 M PB twice for 15 min at room temperature and then reacted with secondary antibody (biotinylated anti-rabbit IgG (Vector Laboratories Inc.)) diluted 1: 200 for 1 h at room temperature. After washing twice with 0.1 M PB for 15 minutes, the cells were immersed in peroxidase-labeled ABC (avidin-biotin complexex) solution and reacted at room temperature for 1 hour. After washing with 0.1 M PB for 15 minutes twice, 30 mg of 3-3 'diaminobenzidine was dissolved in 150 ml of 0.1 M PB for 5 minutes. 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, tissues were washed again with 0.1 M PB several times, counterstained with hematoxylin for 20 seconds, dehydrated and clarified according to a conventional method, sealed, and observed with an optical microscope.

As a result, beta -catenin and IGF1 were detected more in the DSM group than in the negative control group. It was observed that the increased effect of diosmin on β-catenin and IGF1 expression in dorsal skin tissue was more excellent than that of minoxidil (FIG. 4A).

Western blot analysis

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

As a result, the expression of the β-catenin protein was significantly increased in the DSM group as compared with the negative control group (FIG. 4B).

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

RNA isolation using the Trizol method

To quantify the total RNA in skin tissue of experimental animals, 1 mL of the trizol solution was added to 50-100 mg of skin tissue, homogenized, and centrifuged at 12,000 × g for 10 minutes at 4 ° C. The supernatant was transferred to a new tube, and then 200 μL of chloroform was added and vortexed. This process was repeated twice, then the supernatant was transferred to a new tube and isopropanol and supernatant were added at a ratio of 1: 1. After 10 minutes of shaking, the mixture was allowed to stand at room temperature for 10 minutes, and centrifuged at 12,000 × g for 10 minutes at 4 ° C. After removing the supernatant, 1 ml of 70% ethanol was added to the remaining precipitate to obtain 7,500 × g, Lt; / RTI &gt; for 5 minutes. 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 RNA samples extracted at 260 nm and 280 nm wavelengths was measured using a UV / VIS spectrophotometer (Beckman coulter, DU730) and agarose gel electrophoresis was performed to confirm the integrity of the RNA samples.

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

RNA was isolated and reverse transcribed using oligo dT primer and Superscript reverse transcriptase (GIBCOBRL, Gaithersburg, USA) to synthesize cDNA. PCR was carried out using the cDNA obtained through reverse transcription as a template and the 5 'and 3' flanking sequences of the cDNA of the gene to be amplified as primers. The necessary primers were used for the synthesis of biona Co., Ltd. These primer sequences are shown in Table 1 below. Add 1 μL each of the 10X reaction buffer [100 mM KCL, 20 mM Tris-HCl (pH 8.0), 2.5 mM MgCl ₂ ], 4 μL of 10 mM dNTP, 0.2 μM sense and antisense primer, And 2.5 units of Taq polymerase (Takara, Japan) were added to each well, and the volume was adjusted to 50 μL with distilled water. PCR was performed using PCR instrument. PCR conditions were 94 ° C (4 min), 94 ° C (30 sec), 30 cycles [52 ° C (30 sec), 72 ° C (45 sec)] and 72 ° C (10 min). 1 μL of the amplified product was electrophoresed on 1% agarose gel to confirm the DNA band to evaluate gene expression amount.

The primer sequences used for RT-PCR gene primer The sequence (5 '- &gt; 3') Annealing temperature (캜) PCR product
(bp) Wigless related MMTV integration site 10b (Wnt10b) F TTTTGGCCACTCCTCTTCCT 61 183 R TCCTTTTCCAACCGAAAACC Frizzled receptor 1 (FZD1) F TTCTATGAACAGGCCTTTCGTTCT 55 484 R CCTCGTGTAGAACTTCCTCC Low-density lipoprotein receptor-related protein 5 (LRP5) F AAGGGTCCACAAGGTCAAGG 55 520 R AGAAGCACAGATGGCTGCAC Glycogen synthase kinase 3? (GSK3?) F TGTGTGAGGAACAGAACTCA 48 244 R CCTACAGCCCTAGTCATCAG Axin F TGCAGAGTCCCAAAATGAATG 55 108 R GAGCCTGTCCTTGTGTAC β-Catenin F ATGGCTACTCAAGCTGAC 55 298 R CAGCACTTTCAGCACTCTGC Insulin-like growth factor (IGF1) F TCAACAAGCCCACAGGGTAT 60 280 R ACTCGTGCAGAGCAAAGGAT Hepatocyte growth factor (HGF) F CGAGGCCATGGTGCTATACT 54 290 R ACACCAGGGTGATTGAGACC Vascular endothelial growth factor (VEGF) F TCTTCAAGCCATCCTGTGTG 60 165 R GCGAGTCTGTGTTTTTGCAG Keratocyte growth facfor (KGF) F GACATGGATCCTGCCAACTT 54 686 R AATTCCAACTGCCACTGTCC Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) F AGAACATCATCCCTGCATCC 60 321 R TCCACCACCCTGTTGCTGTA

Regulation of Expression of Hairy Related Gene

Analysis of the expression of Wnt / β-catenin signaling agent, which is known as a mechanism of hair loss prevention, hair growth (or hair growth), in the dorsal skin of mice coated with the experimental material for 4 weeks was analyzed by RT-PCR analysis. And Wnt10b receptors, FZD1 (frizzled receptor 1), and LRP5 (low-density lipoprotein receptor-related protein 5) were significantly increased in the DSM group compared to the negative control group. Thus, GSK3β (glycogen synthase kinase 3β) and Axin, which were inhibited by Wnt10b, were significantly decreased in the DSM group compared to the negative control group. The expression of [beta] -catenin was significantly increased in the DSM group as compared with the negative control according to the activation of the Wnt / [beta] -catenin signal transduction system (Fig. 5A).

In addition, the expression of endocrine factors affecting hair growth was evaluated to examine hair loss prevention, hair growth or hair growth promoting action of diosmin. (VEGF), HGF (hepatocyte growth factor), and KGF (keratocyte growth fac- tor), which are growth factors affecting hair growth and wool in skin tissue As a result, expression of the four genes was significantly increased in the DSM group as compared with the negative control group (Fig. 5B).

Therefore, diosmin accelerates the growth of hair follicles, thereby activating hair follicles to return to their growth phase. In addition, by increasing the expression of IGF1, HGF, VEGF and KGF in skin tissue, hair regrowth is promoted to prevent hair loss, .

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

A pharmaceutical composition for promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.
The composition of claim 1, wherein the composition increases β-catenin expression or inhibits Wnt10b (Frizzled receptor 1), LRP5 (low-density lipoprotein receptor-related protein 5), IGF1 Wherein the expression of the gene selected from the group consisting of insulin-like growth factor 1, beta -catenin, vascular endothelial growth factor (HGF), hepatocyte growth factor (HGF) and keratocyte growth facor (KGF) .
2. The composition of claim 1, wherein the composition reduces the expression of GSK3? (Glycogen synthase kinase 3?) Or Axin.
A quasi-drug for promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.
A cosmetic composition for promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.
A functional food composition for promoting hair growth or hair growth comprising diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.
Diosmin or a pharmaceutically acceptable salt thereof as an active ingredient.

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