WO2021125733A1 - Pharmaceutical composition for preventing or treating hair loss - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating hair loss, which can effectively prevent or treat hair loss by inhibiting NOX expression and reducing the generation of reactive oxygen. The composition according to the present invention comprises two or more compounds among tanshinone I, tanshinone IIA and cryptotanshinone, and has excellent efficacy in preventing or treating hair loss as compared to a case in which a single compound is used.

Description

Pharmaceutical composition for preventing or treating hair loss

The present invention relates to a pharmaceutical composition for preventing or treating hair loss.

Hair is an important indicator of health, and when health deteriorates, hair becomes coarse or falls out. On average, there are 100,000 hairs on the human scalp, and they repeat generation and extinction with periodic changes in the anagen, catagen, telogen, and exogen phases. Hair is composed of 90% keratin composed of 18 amino acids and 10% water, and is mainly composed of carbon, oxygen, nitrogen, hydrogen, and sulfur. About 80 to 100 hairs fall out a day normally, and new hairs are formed in the places where they fall out.

Hair loss refers to a condition in which the number of hairs falling out is greater than the number of new hairs. The causes of hair loss vary from person to person, but the degeneration of scalp cells due to aging including genetic factors, lack of nutrients supplied to the hair roots, and scalp fat Examples include insufficient oxygen supply due to excess, inflammation and pore clogging caused by fat or foreign substances or scalp disease, and stress. In addition, if the blood circulation is good, nutrients and oxygen are smoothly supplied to the hair roots, so that the hair grows healthy. However, if the blood circulation is not smooth due to a high fat diet or smoking, hair loss is promoted. Alopecia occurred only in the middle-aged in the past, but recently, it is also occurring frequently in young people in their 20s and 30s.

Numerous hair loss treatment agents and hair growth promoting agents have been introduced so far, but there is no report that the hair of patients is remarkably promoted and regenerated and long-term hair loss is prevented. In addition, it has been reported that these drugs for hair loss treatment exhibit side effects such as decreased libido and erectile dysfunction in some patients.

Accordingly, there is a need to develop a safe and excellent material for preventing or treating hair loss.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating hair loss.

An object of the present invention is to provide a cosmetic composition for preventing or alleviating hair loss.

An object of the present invention is to provide a food composition for preventing or alleviating hair loss.

1. A pharmaceutical composition for preventing or treating hair loss comprising at least two or more of tanshinone I (Tanshinone I), tanshinone IIA (Tanshinone IIA), and cryptotanshinone.

2. The pharmaceutical composition for preventing or treating hair loss according to the above 1, comprising tansinone I and tansinone ⅡA.

3. The pharmaceutical composition for preventing or treating hair loss according to the above 2, wherein the tansinone I and the tansinone IIA are included in a molar ratio of 3:1 to 1:3.

4. The pharmaceutical composition for preventing or treating hair loss according to the above 1, wherein the hair loss is androgenetic alopecia.

5. The pharmaceutical composition for preventing or treating hair loss according to the above 4, wherein the composition prevents or treats hair loss by lowering the active oxygen level in testosterone.

6. The pharmaceutical composition for preventing or treating hair loss according to the above 1, wherein the composition inhibits the expression of NOX (NADHP Oxidase) in hair follicle cells.

7. The pharmaceutical composition for preventing or treating hair loss according to the above 1, further comprising a 5α-reductase inhibitor.

8. A method for preventing or treating hair loss, comprising administering to a subject a therapeutically effective amount of at least two or more of Tanshinone I, Tanshinone IIA, and cryptotanshinone.

9. The method for preventing or treating hair loss according to 8 above, comprising administering to the subject a therapeutically effective amount of tansinone I and tansinone IA.

10. The method for preventing or treating hair loss according to 9 above, wherein the tansinone I and the tansinone ⅡA are administered in a molar ratio of 3:1 to 1:3.

11. The method for preventing or treating hair loss according to 8 above, wherein the hair loss is androgenetic alopecia.

12. The method of 11 above, wherein the tanshinone Ⅰ (Tanshinone Ⅰ), tanshinone ⅡA (Tanshinone ⅡA) and cryptotanshinone (cryptotanshinone) at least two or more of the therapeutically effective amount of administering to the subject to lower the active oxygen level, hair loss Prevention or treatment methods.

13. The method of 8 above, wherein a therapeutically effective amount of at least two or more of Tanshinone I, Tanshinone IIA, and Cryptotanshinone is administered to the subject to generate NOX (NADHP Oxidase) in hair follicle cells. ) to inhibit the expression, hair loss prevention or treatment method.

14. The method for preventing or treating hair loss according to 8 above, further comprising administering to the subject a therapeutically effective amount of a 5α-reductase inhibitor.

15. For the prevention or treatment of hair loss, the use of the pharmaceutical composition of 1 to 7 above.

16. A cosmetic composition for preventing or alleviating hair loss comprising at least two of tanshinone Ⅰ, tanshinone ⅡA, and cryptotanshinone.

17. Use of the cosmetic composition of the above 16 for preventing or treating hair loss.

18. Food composition for preventing or alleviating hair loss comprising at least two or more of tanshinone Ⅰ (Tanshinone Ⅰ), tanshinone ⅡA (Tanshinone IA), and cryptotanshinone.

19. Use of the food composition of the above 18 for preventing or treating hair loss.

The present invention relates to a pharmaceutical composition for preventing or treating hair loss, which can effectively prevent or treat hair loss by inhibiting NOX expression and reducing active oxygen production.

In addition, the composition according to the present invention contains two or more compounds of tansinone I, tansinone IA, and cryptotansinone, and has excellent efficacy in preventing or treating hair loss compared to the case of using a single compound.

1 is a view confirming the reduction in active oxygen production according to the inhibition of NOX expression.

Figure 2 is a view confirming the reduction of apoptosis (apoptosis) according to the inhibition of NOX expression.

3 is a view confirming the restoration of hair growth according to the inhibition of NOX expression.

4 is a view confirming the NOX inhibitory efficacy of tansinone I and tansinone ⅡA.

5 is an analysis result of removing the non-target effects of tansinone I and tansinone ⅡA.

6 is a result of glucose oxidase inhibitory activity assay.

7 is a confirmation result of xanthine oxidase inhibitory activity assay.

8 and 9 are diagrams confirming the inhibition of active oxygen production of tansinone I and tansinone ⅡA in vitro.

10 and 11 are diagrams confirming the inhibition of active oxygen production according to the content of tansinone I and tansinone ⅡA in vitro.

12 to 15 are diagrams confirming the reduction in testosterone-induced cell death of tansinone I and tansinone IIA in vitro.

16 and 17 are diagrams confirming hair follicle length growth by tansinone I and tansinone ⅡA.

18 and 19 are photographs confirming hair growth using C57BL/6 mice.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating hair loss comprising at least two or more of tansinone I, tansinone IA, and cryptotansinone.

In the present invention, tansinone I may be represented by the following formula (1).

[Formula 1]

In the present invention, tansinone IA may be represented by the following formula (2).

[Formula 2]

In the present invention, cryptotansinone may be represented by the following formula (3).

[Formula 3]

The composition according to the present invention comprises at least two compounds selected from tansinone I, tansinone IA and cryptotansinone. By using a combination of two or more of the above compounds, the efficacy of preventing or treating hair loss may be enhanced compared to the case of using a single compound.

In one embodiment of the present invention, the composition according to the present invention comprises tansinone I and tansinone IA. The content of tansinone I and tansinone IA included in the composition may be included in, for example, a molar ratio of 5:1 to 1:5, a molar ratio of 3:1 to 1:3, or a molar ratio of 1:1. It is not limited.

The hair loss of the present invention may include androgenetic hair loss, telogen hair loss, anagen phase hair loss, alopecia areata, drug telogen hair loss, and postpartum telogen hair loss. In addition, it may include all kinds of hair loss that forms an abnormal hair loss pattern such as vellus hair growth of terminal hair, short hair growth of group hair, and atrophy of hair follicles due to a series of other factors.

According to an embodiment of the present invention, hair loss may be androgenic alopecia. Androgenetic alopecia is due to hyperandrogen stimulation caused by excessive accumulation of testosterone or androgen hormone in the metabolic system.

Androgenetic alopecia is known to cause hair loss due to an increase in the level of free radicals caused by testosterone. Specifically, testosterone increases calcium production by binding to the GPRC6A receptor, thereby increasing the activity of NOX to generate reactive oxygen species. Reactive oxygen generated in hair follicle cells can induce hair follicle cell death and cause hair loss (Eunbi Ko et al., Testosterone stimulates Duox1 activity through GPRC6A in skin keratinocytes. Journal of Biol Chem. 2014 oct 17;289(42)) :28835-45.).

NOX or NADPH oxidase is a protein that mediates reactive oxygen production and includes NOX1, NOX2, NOX4, NOX5, DUOX1 and DUOX2.

*69 Reactive oxygen or reactive oxygen species (ROS) are chemically reactive molecules containing oxygen atoms. As a compound of oxygen produced in living organisms, it has strong oxidative power that attacks living tissue and damages cells. When active oxygen increases, cells die, and when antioxidants such as NAC (N-acetylcysteine) or BHA (butylated hydroxyanisole) are treated in the cells with increased active oxygen, apoptosis is inhibited (Sang Won Kang, Role of Reactive) Oxygen Species in Cell Death Pathways, Hanyang Med Rev, 2013;33:77-82).

According to the present inventors, hair loss may be caused by an increase in the level of active oxygen due to overexpression of NOX in hair follicle cells, and when the composition of the present invention is administered, it inhibits the expression of NOX in hair follicle cells to lower the level of active oxygen, and apoptosis The present invention was completed by confirming that it is possible to prevent or treat hair loss by inhibiting.

In one embodiment of the present invention, it was confirmed through TUNEL asssy (Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling assay) that apoptosis induced by testosterone is reduced in cells treated with the composition of the present invention (Figs. 12 to 15). Here, the 'TUNEL assay' is a method for confirming the number of dead cells, and is performed using the property of TdT that labels the blunt end of the double-stranded DNA fragment regardless of the template (Kateryna Kyrylkova et al. ., Detection of apoptosis by TUNEL assay, Methods Mol Biol, 2012;887:41-7), can be visualized according to the introduced label.

In addition, androgenetic alopecia of the present invention, dihydrotestosterone (DHT, androgen hormone) produced from testosterone by 5α-reductase, which is an enzyme that reduces the double bond between the 4th and 5th carbons of testosterone, is the androgen receptor of the hair root. (Shigeki Inui, Satoshi Itami. Molecular basis of androgenetic alopecia: From androgen to paracrine mediators through dermal papilla. Journal of Dermatological Science. 2011 Jan; 61(1):1) -6.).

The composition of the present invention may further comprise a 5α-reductase inhibitor. As a 5α-reductase inhibitor that inhibits the action of this enzyme to inhibit the production of dihydrotestosterone, compounds such as finasteride and dutasteride may be used, but the present invention is not limited thereto.

When the composition of the present invention includes two or more compounds selected from tansinone I, tansinone IA and cryptotansinone and a 5α-reductase inhibitor, it is possible to inhibit each mechanism by NOX and 5α-reductase, It may have excellent efficacy in preventing or treating hair loss.

The pharmaceutical composition for preventing or treating hair loss of the present invention is formulated in the form of oral dosage forms such as powders, granules, tablets, capsule suspensions, emulsions, syrups and aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. can be used for In one embodiment of the present invention, the composition of the present invention is for external use, and it is possible to prepare any formulation that can make the composition come into contact with the skin, such as a liquid or gel type. For example, it may be formulated in the form of ointments, creams, packs and gels such as liniments, patches, or sprays. The liniment agent may be used by applying it to a body part in need of hair growth, and the patch may be used by attaching it to the body part in need of hair growth. In addition, the spray can be used in a manner of spraying a body part in need of hair growth.

Carriers, excipients and diluents that may be contained in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin , calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the compound, for example, starch, calcium carbonate, sucrose or lactose. , gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The usage amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, but may be administered at 0.1 to 100 mg/kg, preferably 1 to 10 mg/kg, once or several times a day. In addition, the dosage may be increased or decreased according to the route of administration, the degree of disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any way.

The part where the composition of the present invention can be included can be applied to any part of the body that requires hair growth as well as the scalp. For example, it can be used to improve the condition of hair or hair damaged by trauma or a wide forehead or M-shaped forehead, eyelashes or eyebrows for the purpose of simple cosmetic effect, and alopecia.

The present invention provides a method for preventing or treating hair loss comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition.

The pharmaceutical composition administered to a subject in the method for preventing or treating hair loss according to the present invention may have the characteristics as described above herein.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field may be determined. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art. The exact therapeutically effective dose of an active ingredient may vary depending on the relative amount of each active ingredient used, the drug used, and the synergistic ratio.

The present invention provides a method for preventing or treating hair loss, comprising administering to a subject a therapeutically effective amount of at least two or more of tanshinone Ⅰ, tanshinone IA, and cryptotanshinone do.

The present invention provides a method for preventing or treating hair loss, comprising administering to a subject a therapeutically effective amount of tansinone I and tansinone ⅡA. In an embodiment of the present invention, the tansinone I and the tansinone IIA may be administered in a molar ratio of 3:1 to 1:3.

The present invention provides a method for preventing or treating androgenetic alopecia.

In one embodiment of the present invention, by administering to the subject a therapeutically effective amount of at least two or more of tanshinone Ⅰ, tanshinone IA, and cryptotanshinone to lower active oxygen levels, hair loss by can be prevented or treated.

In one embodiment of the present invention, by administering to the subject a therapeutically effective amount of at least two or more of the tanshinone I, Tanshinone IIA, and cryptotanshinone, NOX (NADHP) in hair follicle cells Oxidase), thereby preventing or treating hair loss.

In an embodiment of the present invention, a therapeutically effective amount of at least two or more of the tanshinone I, Tanshinone IIA, and cryptotanshinone and a 5α-reductase inhibitor is further administered to the subject. It can be administered to prevent or treat hair loss.

The present invention provides the use of the pharmaceutical composition for preventing or treating hair loss.

The pharmaceutical composition for this use according to the present invention may have the characteristics as described hereinabove.

The present invention provides a cosmetic composition for preventing or alleviating hair loss comprising the composition.

The cosmetic composition is a softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, body It may be formulated as an essence, makeup base, foundation, hair dye, shampoo, conditioner or body cleaner, but is not limited thereto.

When used as a cosmetic composition, a substance may be further added according to the formulation of an external preparation for skin or cosmetics. For example, but not limited thereto, when the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or Zinc oxide and the like may be used, and when the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally propellants such as chlorofluorohydrocarbons, propane/butane or dimethyl ether. In addition, when the formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, preferably water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-Butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan may be used, but is not limited thereto. When the formulation is a suspension, as a carrier component, water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, Aluminum metahydroxide, bentonite, agar or tracanth may be used, and when the formulation is a surfactant containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivatives, methyltaurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester may be, but is not limited thereto.

The present invention provides the use of a cosmetic composition for preventing or treating hair loss.

The composition for this use according to the invention may have the characteristics as described hereinabove.

The present invention provides a food composition for preventing or alleviating hair loss comprising the composition.

The food composition of the present invention includes beverages (including alcoholic beverages), fruits and processed foods thereof (eg, canned fruit, bottled, jam, marmalade, etc.), fish, meat, and processed foods thereof (eg, ham, corned sausage). etc.), breads and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable protein, It may be retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.).

In addition, the food composition of the present invention may be formulated in tablets, pills, powders, granules, powders, capsules, liquid formulations, and the like. They may be formulated to further include one or more of carriers, diluents, excipients and additives.

The food composition may further include an additive. Additives include natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers (cheese, chocolate, etc.), lactic acid and its salts, alginic acid and its salts. At least one component selected from the group consisting of salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonation agents, and pulp may be used.

In addition, the food composition includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain the pulp for the production of natural fruit juice and vegetable beverage. These components may be used independently or in combination.

The present invention provides the use of a food composition for preventing or treating hair loss.

The composition for this use according to the invention may have the characteristics as described hereinabove.

As described above, the composition according to the present invention can be usefully used as a raw material for the development of a pharmaceutical (external) product capable of preventing or treating hair loss or promoting hair growth.

Hereinafter, examples will be given to describe the present invention in detail.

Experimental methods and results

1. Confirmation of reduction in reactive oxygen production when NOX expression is inhibited

Primary mouse keratinocytes were isolated from wild type mice and Duox1 knockout mice, respectively, and cultured in a 35 mm culture dish, followed by serum starvation for 16-18 hours. After testosterone stimulation, cells were incubated for 10 minutes in Hanks' balanced salt solution (HBSS) containing 10 μM 2',7'-dichlorofluorescin diacetate (DCF-DA). The fluorescence level of these cells was measured at a wavelength of 488 nm in a laser-scanning confocal microscope (LSM 510, Carl Zeiss).

The results are shown in FIG. 1 . Referring to FIG. 1 , it was confirmed that active oxygen production was reduced in Duox1 knockout mice.

2. Confirmation of reduction in apoptosis upon inhibition of NOX expression

Primary keratinocytes of wild type mice and Duox1 knockout mice were treated with 70% ethanol or testosterone in serum free medium and incubated for 24 hours. After that, it was fixed with 3.5% paraformaldehyde at room temperature for one hour, and permeabilized in 0.5% triton X-100 for 10 minutes. Apoptotic cells were stained for 1 hour using an in situ cell death detection kit (Roche). Then, it was stained with DAPI for 10 minutes. Wash with 1X PBS between each step. The stained cells were analyzed by measuring the fluorescence through a confocal microscope, and the number of apoptotic cells compared to DAPI was analyzed using the Image J program.

The results are shown in FIG. 2 . As shown in FIG. 2 , it was confirmed that apoptosis was reduced as the TUNEL-positive cells of the Duox1 knockout mice were decreased.

3. Confirmation of hair growth upon inhibition of NOX expression

In 8-week-old mice of wild type and Duox1 knockout, the back skin was depilated using wax to synchronize the mouse hair cycle. After depilation, 200 μg/100 μl of testosterone was applied to the back skin of mice for 7 days from the 8th day. After that, mice were sacrificed to obtain skin tissues, and H&E (hematoxylin & eosin) staining was performed to analyze the number of hairs per unit length.

The results are shown in FIG. 3 . It was found that the inhibition of hair growth by testosterone in Duox1 knockout mice was restored ( FIG. 3A ). In addition, it was confirmed that Duox1 knockout mice had a higher number of hair follicles per unit length ( FIG. 3B ). This suggests that inhibition of NOX expression is effective in treating hair loss.

4. Confirmation of NOX Inhibition by Tansinone I and Tansinone IIA

For Drosophila whole body high expression, membranes for each Nox lineage were obtained from a transgenic fly expressing human Nox isozyme in the daughterless (Da)-GAL4 promoter, respectively. Tansinone I and Tansinone IIA were dispensed on this membrane and reacted for 10 minutes. A mixture of 500μM NADPH (β-Nicotinamide adenine dinucleotide phosphate hydrate) and 400μM lucigenin (N,N'-Dimethyl-9,9'-biacridinium dinitrate) in 1X HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer was made and placed in the membrane. The degree of lucigenin chemiluminescence was measured at 1 minute intervals for 10 minutes using a Spectramax i3x instrument. Ki value analysis was performed using the Graph-Pad Prism 5 program.

The NOX inhibitory efficacy of tansinone I and tansinone IA was confirmed by measuring _{IC 50 .} The results are shown in FIG. 4 and Table 1.

(Unit: μM)

NOX isozyme	NOX1	NOX2	NOX4	NOX5	DUOX1	DOUX2
Tanshinone I	12.9	14.1	4.9	3.9	6.5	2.6
Tanshinone ⅡA	2.2	3.4	7.2	1.9	2.8	3.6

*132

5. Confirmation of non-target effects of tansinone Ⅰ and tanshinone ⅡA

For scavenging assay, ROS scavenging ability of Nox inhibitor was confirmed by adding tansinone I and tansinone ⅡA to 1X PBS at concentrations of 1 and 10 μM _{and reacting with H 2} O _{2 500 mM and lucigenin 400 μM.} Chemiluminescence was measured with a Promega GloMax 20/20 Luminometer at 10-second intervals for 1 minute. The average values were compared with NAC (N-Acetylcysteine) 500 μM and 1 mM as positive controls. In all experiments, the measured values obtained by reacting only the Nox inhibitor with lucigenin without adding _{H 2} O _{2 were subtracted.}

Amplex red glucose/glucose oxidase assay kit and Amplex red xanthine/xanthine oxidase assay kit (Thermo Fisher) were used for glucose oxidase activity and xanthine oxidase activity. When tanshinone I and tanshinone ⅡA were treated at concentrations of 1 and 10 μM, fluorescence values were measured at absorbance at a wavelength of 560 nm. This was compared with the case of NAC 500 μM, 1 mM treatment.

The results are shown in FIGS. 5 to 7 . As a result of FIG. 5, it was confirmed that tansinone I and tansinone IA are not substances that directly reduce active oxygen. In addition, since tansinone I and tansinone IA have no inhibitory activity of glucose oxidase (Fig. 6), and no or very low inhibitory activity of xanthine oxidase (Fig. 7), it is known that they are compounds having specific inhibitory activity on NOX. could

6. Confirmation of reduction of active oxygen production of tansinone Ⅰ and tanshinone ⅡA

^{1.5x10 5} Ker-CT cells (ATCC) and ^{2x10 6} primary mouse keratinocytes were cultured in a confocal 35mm culture dish, followed by serum starvation for 16-18 hours. Tansinone Ⅰ and tanshinone ⅡA, or the same amount of DMSO was added and incubated for 30 minutes, then the existing medium was removed and the cells were washed with 1X PBS. Then, the cells were stained with 5 μM PO-1 (Peroxy-Orange 1; TOCRIS) diluted in 1X PBS for 15 minutes. After 5 minutes of PO-1 treatment, testosterone was added and stimulation was given for a total of 10 minutes. After washing with 1X PBS, the fluorescence intensity was measured under conditions of excitation 580 nm and emission 545-750 nm using a confocal microscope. A total of 5 zones per dish were measured and the average value was analyzed.

The results are shown in FIGS. 8 and 9 . Reactive oxygen production was reduced in both tansinone I and tansinone IIA.

7. Confirmation of reduction in active oxygen production according to the content of tansinone Ⅰ and tanshinone ⅡA

The level of reactive oxygen production according to the concentration of tansinone I and tanshinone IIA was measured in Ker-CT cells in the same manner as in Experiment 6. Tansinone I and Tanshinone ⅡA were treated with 10 nM, 100 nM, 1 μM, and 10 μM, respectively.

The results are shown in FIGS. 10 and 11, and both tansinone I and tansinone IA decreased the production of active oxygen in a concentration-dependent manner.

8. Testosterone-induced cell death reduction efficacy of tansinone Ⅰ and tanshinone ⅡA confirmed

Wild type primary mouse keratinocytes were cultured and treated with 100 and 500 nM of tansinone I and tansinone ⅡA in serum free medium, respectively. Then, it was incubated for 24 hours after treatment with 70% ethanol or testosterone. After that, it was fixed with 3.5% paraformaldehyde at room temperature for one hour, and permeabilized in 0.5% triton X-100 for 10 minutes. Apoptotic cells were stained for 1 hour using an in situ cell death detection kit (Roche). Then, it was stained with DAPI for 10 minutes. Wash with 1X PBS between each step. The stained cells were analyzed by measuring the fluorescence through a confocal microscope, and the number of apoptotic cells compared to DAPI was analyzed using the Image J program.

The results are shown in FIGS. 12 to 15 . Since TUNEL-positive cells were decreased in both tansinone I and tansinone IIA, it was found that cell death was reduced.

9. Confirmation of hair follicle length growth by tansinone Ⅰ and tanshinone ⅡA

The back hairs of 8-week-old C57BL/6J mice were shaved, and the hairs were completely removed by applying an appropriate amount of depilatory cream. Mice without a back wound were selected, divided into groups, and 200 μg/100 μl of testosterone was applied to the back for 3 days from the day after hair removal. After 3 days, the final concentrations of tansinone I and tansinone ⅡA were diluted in 70% ethanol to 50 and 100 μM and applied together with testosterone for 12 days. In the control group, DMSO in the same amount as tansinone was diluted in 70% ethanol and applied. On the 12th day after drug application, mice were sacrificed to obtain dorsal skin tissue, which was frozen section and H&E stained. The length of hair follicles in each group was analyzed using Image J program.

The results are shown in FIGS. 16 and 17 . Both tansinone I and tansinone ⅡA increased in hair follicle length in a concentration-dependent manner.

10. Confirmation of hair growth using C57BL/6 mice

Examples and Comparative Examples are shown in Table 2 below.

	Tanshinone I	Tanshinone ⅡA	Cryptotansinone
Example 1	10 μM	10 μM	-
Example 2	10 μM	-	10 μM
Example 3	-	10 μM	10 μM
Comparative Example 1	20 μM	-	-
Comparative Example 2	-	20 μM	-
Comparative Example 3	-	-	20 μM

The back hairs of 8-week-old C57BL/6J mice were shaved, and the hairs were completely removed by applying an appropriate amount of depilatory cream. Mice without a back wound were selected, divided into groups, and 200 μg/100 μl of testosterone was applied to the back for 3 days from the day after hair removal. Examples and Comparative Examples, as shown in Table 2, tansinone I and tansinone IIA (Example 1), tanshinone I and cryptotansinone (Example 2), tansinone IIA and cryptotansinone (Example 3) , tansinone I (Comparative Example 1), tanshinone ⅡA (Comparative Example 2), and cryptotansinone (Comparative Example 3) were prepared by diluting in 70% ethanol so that the final concentration of 20 μM. Each Example and Comparative Example was applied to the back from the 4th day (day 0 of drug treatment) after hair removal, and the control group was applied by diluting DMSO in the same amount as tancinone in 70% ethanol. Mice were sacrificed 12 days after drug treatment. The hair condition of the mice was confirmed by photographing once every 2 days from the 0th day of the drug treatment to the 12th day. The ratio of the hair growth area to the total hair removal area was measured using the Image-Pro Analyzer 7.0 program. The results are shown in FIGS. 18 and 19 . When tansinone I and tansinone ⅡA were mixed, the synergistic effect of hair growth was excellent compared to when each compound was used alone.

However, when tansinone I and cryptotansinone were mixed, hair growth efficacy was similar to that when using each single compound, and when tansinone ⅡA and cryptotansinone were mixed, hair growth was higher than when each single compound was used. efficacy was reduced.

Claims (9)

1. A pharmaceutical composition for preventing or treating hair loss comprising at least two or more of tanshinone I (Tanshinone I), tanshinone IIA (Tanshinone IIA), and cryptotanshinone.
2. The pharmaceutical composition for preventing or treating hair loss according to claim 1, comprising tansinone I and tansinone ⅡA.
3. The pharmaceutical composition for preventing or treating hair loss according to claim 2, wherein the tansinone I and the tansinone IA are included in a molar ratio of 3:1 to 1:3.
4. The pharmaceutical composition for preventing or treating hair loss according to claim 1, wherein the hair loss is androgenetic alopecia.
5. The pharmaceutical composition for preventing or treating hair loss according to claim 4, wherein the composition prevents or treats hair loss by lowering the level of active oxygen.
6. The pharmaceutical composition for preventing or treating hair loss according to claim 1, wherein the composition inhibits the expression of NOX (NADHP Oxidase) in hair follicle cells.
7. The pharmaceutical composition for preventing or treating hair loss according to claim 1, further comprising a 5α-reductase inhibitor.
8. A cosmetic composition for preventing or alleviating hair loss comprising at least two or more of tanshinone I (Tanshinone I), tanshinone IIA (Tanshinone IIA), and cryptotanshinone.
9. A food composition for preventing or alleviating hair loss comprising at least two or more of tanshinone I (Tanshinone I), tanshinone IIA (Tanshinone IIA), and cryptotanshinone.

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