JP7630764B2 - Hair Anti-Aging Agent - Google Patents

Description

The present invention relates to an agent for preventing or improving gray hair or senile alopecia, an agent for suppressing or improving DNA damage in hair follicle constituent cells, and an agent for suppressing the decrease in melanocyte stem cells.

The pigment (melanin) in hair is produced by pigment cells present in the hair bulb, and epithelial cells that are supplied with this pigment form hair, resulting in the development of colored hair. When hair falls out and is replaced, the hair bulb disappears, and with it the pigment cells in the hair bulb. Hair follicle stem cells and melanocyte stem cells reside in a part of the hair follicle called the bulge region, and when a new hair cycle begins and new hair is produced, the hair follicle stem cells supply epithelial cells to the hair bulb, and the melanocyte stem cells supply melanocytes to the hair bulb, and the cycle above is repeated once again.

There are several causes of gray hair, including aging, stress, and vitiligo, and it occurs when an abnormality occurs in one of the above-mentioned processes. In the past, it was believed that gray hair could be prevented or improved by maintaining or improving the pigment production ability of the pigment cells in the hair bulb, and many substances were discovered that activate enzymes such as tyrosinase, which is involved in pigment synthesis in pigment cells (Patent Documents 1 to 5).

In recent years, the mechanism of hair graying due to aging has been elucidated. Melanocyte stem cells that have accumulated DNA damage to an extent that cannot be repaired differentiate while still in the bulge region (this differentiation is called ectopic differentiation), lose their self-maintenance function, and disappear (Non-Patent Document 1). As a result, pigment cells cannot be supplied to the hair bulb, and epithelial cells that do not have pigment produce hair, resulting in gray hair. In other words, in the majority of gray hair caused by aging, there are no pigment cells in the hair bulb to begin with, and activation of enzymes involved in pigment synthesis cannot improve the condition. Gray hair with pigment cells remaining in the hair bulb is thought to be able to be improved with traditional approaches, but survey results show that only about 20% of gray hair (not necessarily caused by aging) has pigment cells in the hair bulb (Non-Patent Document 2).

It has also been elucidated that melanocyte stem cells are maintained by the function of hair follicle stem cells (Non-Patent Document 3). Hair follicle stem cells produce factors including TGF-β and WNT ligands, creating an environment (melanocyte stem cell niche) in which melanocyte stem cells can exist while maintaining their stemness, and controlling the maintenance and activation of melanocyte stem cells (Non-Patent Documents 3, 4). In other words, in order to prevent or improve gray hair, it is important to maintain hair follicle stem cells and further maintain the melanocyte stem cell niche function to prevent the disappearance of melanocyte stem cells.

It is known that by inflicting DNA damage in black mice using X-rays, it is possible to induce gray hair that reflects the above-mentioned age-related mechanisms, and this is widely used as an age-related mouse gray hair model (Patent Document 6). In this model, X-rays damage both hair follicle stem cells and melanocyte stem cells, but it has been elucidated that the primary target is hair follicle stem cells (impairment of melanocyte stem cell niche function) (Non-Patent Document 5).

JP 2001-288052 A JP 2001-131032 A JP 2007-8888 A JP 2011-157317 A JP 2002-47130 A JP 2015-193550 A

Inomata K, Aoto T, Binh NT, Okamoto N, Tanimura S, Wakayama T, Iseki S, Hara E, Masunaga T, Shimizu H, Nishimura EK, Genotoxic stress abrogates renewal of melanocyte stem cells by triggering their differentiation, Cell.2009;137(6):1088-99. "Anti-aging Series 1: The reality of gray hair, hair loss, and hair growth," by Tatsuro Ideta et al., NTS, July 4, 2005, p.49-61 Tanimura S, Tadokoro Y, Inomata K, Binh NT, Nishie W, Yamazaki S, Nakauchi H, Tanaka Y, McMillan JR, Sawamura D, Yancey K, Shimizu H, Nishimura EK, Hair follicle stem cells provide a functional niche for melanocyte stem cells, Cell Stem Cell. 2011;8(2):177-87. Rabbani P, Takeo M Chou W, Myung P, Bosenberg M, Chin L, Taketo MM, Ito M, Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration, Cell. 2011 Jun 10;145(6):941-955. Aoki H, Hara A, Motohashi T, Kunisada T, Keratinocyte stem cells but not melanocyte stem cells are the primary target for radiation-induced hair graying, J Invest Dermatol. 2013;133(9):2143-51. Matsumura H, Mohri Y, Binh NT, Morinaga H, Fukuda M, Ito M, Kurata S, Hoeijmakers J, Nishimura EK, Hair follicle aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis, Science. 2016;351(6273):aad4395.

The object of the present invention is to provide a means for preventing or improving gray hair. It is also to provide a means for preventing or improving hair loss caused by aging. It is also to provide an agent for suppressing or improving DNA damage in hair follicle constituent cells. It is also to provide an agent for suppressing the decrease in melanocyte stem cells.

As a result of intensive research, the inventors have discovered that berberrubine and its derivatives, as well as salts thereof, have excellent preventive or ameliorative effects on gray hair or age-related alopecia, and that these effects are attributable to their suppression or amelioration of DNA damage in hair follicle constituent cells and their suppression of the decline in melanocyte stem cells, thus completing the present invention.

That is, the present invention provides
(1) An agent for preventing or improving gray hair, comprising at least one active ingredient selected from the group consisting of berberrubine, its derivatives (excluding berberine), and salts thereof;
(2) An agent for preventing or improving senile alopecia, comprising as an active ingredient at least one selected from the group consisting of berberrubine, its derivatives, and salts thereof;
(3) An agent for suppressing or improving DNA damage in hair follicle constituent cells, comprising at least one active ingredient selected from the group consisting of berberrubine, its derivatives, and salts thereof;
(4) The agent for suppressing or ameliorating DNA damage according to (3), wherein the hair follicle-constituting cells are hair follicle stem cells or melanocyte stem cells.
(5) An agent for suppressing a decrease in pigment stem cells, comprising at least one active ingredient selected from the group consisting of berberrubine, its derivatives, and salts thereof.
It is.

The agent for preventing or improving gray hair and hair loss of the present invention can prevent or improve the occurrence of gray hair and age-related hair loss by suppressing or improving DNA damage in hair follicle constituent cells or by suppressing the decrease in melanocyte stem cells.

FIG. 1 is a graph showing the lightness of mouse back hair when berberrubine was evaluated in Test Example 1. FIG. 2 is a graph showing the lightness of mouse back hair when berberine was evaluated in Test Example 1. FIG. 3 is a graph showing the hair growth score of mice 15 days after hair removal when evaluating berberine in Test Example 2. FIG. 4 is a graph showing the hair growth score of mice 17 days after hair removal when evaluating berberine in Test Example 2.

Berberrubin in the present invention is a compound represented by 5,6-dihydro-9-hydroxy-10-methoxybenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium.
The derivative in the present invention refers to a compound obtained by changing a small part in the molecule of the original compound. An example of a derivative is, but is not limited to, berberine (2,3-methylenedioxy-9,10-dimethoxy-5,6-dihydrodibenzo[a,g]quinolizinium), in which one of the hydroxyl groups of berberrubine is changed to a methoxy group.
The salt in the present invention includes, but is not limited to, for example, chlorides, salts with hydrochloric acid, sulfuric acid, and tannic acid.

Berberbine, its derivatives, and their salts used in the present invention can be produced by known synthesis methods, or commercially available products may be used. Commercially available products include berberbine chloride (manufactured by Nagara Science Co., Ltd. and Kishida Chemical Co., Ltd.), berberine chloride (manufactured by Sigma-Aldrich), berberine hydrochloride (manufactured by Nacalai Tesque), berberine chloride hydrate (manufactured by Alps Pharmaceutical Co., Ltd.), and berberine tannate (manufactured by Alps Pharmaceutical Co., Ltd.).

In the present invention, hair follicle-constituting cells refer to any cell that constitutes hair follicle tissue. Examples include epithelial cells such as hair follicle stem cells, outer root sheath cells, inner root sheath cells, and hair matrix cells, pigment cells such as melanocyte stem cells, chromatoblasts, and pigment cells, and mesenchymal cells such as hair papilla cells and dermal root sheath cells.

In the present invention, hair follicle stem cells refer to epithelial cell stem cells that exist in the bulge region of the hair follicle and in the secondary hair germ.

In the present invention, melanocyte stem cells refer to stem cells of pigment cells that exist in the bulge region of the hair follicle and in the secondary hair germ.

The preventive or ameliorative effects of the present invention on gray hair and age-related alopecia are believed to be due to the effect of maintaining or improving the niche function of epithelial cells on pigment cells, the effect of inhibiting ectopic differentiation of melanocyte stem cells, and the effect of inhibiting the decline of hair follicle stem cells and melanocyte stem cells. Ectopic differentiation refers to the phenomenon in which melanocyte stem cells differentiate into melanocytes while remaining in the hair follicle bulge region where they should normally exist as stem cells. Niche function refers to the function of certain cells to create and control the environment in which other cells exist.

The agent for preventing or improving gray hair of the present invention can be provided as a quasi-drug, medicine, food or drink, etc. It can also be used as a reagent (positive control, etc.). The form of administration is not particularly limited, but examples include external application and internal administration, and is preferably external application to the skin including the scalp.

When the present invention is applied externally, examples of the dosage form include lotions, liquids, creams, ointments, gels, sprays, shampoos, conditioners, soaps, etc., and when the present invention is applied internally, examples of the dosage form include tablets, powders, granules, liquids, capsules, dry syrups, jellies, liquid foods, semi-solid foods, solid foods, etc.
These can be produced by known methods. During production, various additives that can be contained in cosmetics, quasi-drugs, medicines, foods and beverages, or reagents can be blended within the range that does not impair the effects of the present invention.

Furthermore, the agent for preventing or improving gray hair of the present invention can also be used in combination with hair growth substances such as Swertia japonica extract, carrot extract, capsicum tincture, ginger tincture, loquat leaf extract, glycyrrhetinic acid, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, pantothenic acid, panthenol, vitamin E and its derivatives, hinokitiol, salicylic acid, piroctone olamine, minoxidil, adenosine, t-flavanone, cytopurine, pentadecanoic acid glyceride, allantoin, and nicotinamide. By using the agent in combination, hair production is accelerated, and the effect of preventing or improving gray hair can be obtained at an early stage.

When the berberrubine derivatives and their salts of the present invention are provided as cosmetics, quasi-drugs, pharmaceuticals, food and beverages, or reagents, the blending amount of each is 0.000001 to 20% by mass, preferably 0.0001 to 10% by mass, and more preferably 0.01 to 1% by mass, based on the total composition.

In addition, the agent for preventing or improving gray hair, the agent for preventing or improving senile alopecia, the agent for inhibiting or improving DNA damage to hair follicle constituent cells, and the agent for inhibiting the decrease in melanocyte stem cells of the present invention can be labeled on a product (medicine, quasi-drug, food, beverage, reagent, etc.) containing the agent or its instructions to the effect of preventing or improving gray hair, preventing or improving senile alopecia, inhibiting or improving DNA damage to hair follicle constituent cells, inhibiting or improving DNA damage to hair follicle stem cells, inhibiting or improving DNA damage to melanocyte stem cells, or inhibiting the decrease in melanocyte stem cells. Here, "labeled on a product or its instructions" means that a label has been attached to the main body, container, packaging, etc. of the product, or that a label has been attached to instructions, package inserts, promotional materials, application documents, other printed matter, or advertisements disclosing information about the product. In addition, these labels can include information on the use for preventing or improving gray hair, preventing or improving senile alopecia, or information on the use for preventing or treating diseases or symptoms caused by DNA damage to hair follicle constituent cells, DNA damage to hair follicle stem cells, DNA damage to melanocyte stem cells, or inhibiting the decrease in melanocyte stem cells.

The present invention will be explained in more detail below with reference to examples and test examples, but the present invention is not limited to these examples.

(Test Example 1) Evaluation of anti-hair graying effect using X-ray mouse gray hair model <Test method>
Seven-week-old C57BL/6 mice were anesthetized and the hair on their backs was shaved off with clippers, leaving a few millimeters. After shaving, mice with good back condition were selected and assigned to groups through stratified random allocation (using EXSUS Professional Version 7.7.1 (CAC Exicare)) so that the mean and variance of body weight in each group were approximately equal.
Berberubine chloride or berberine hydrochloride was dissolved in ethanol to a concentration of 0.1% (w/v), and the vehicle ethanol was applied to the mice once a day at 200 μL for one week, starting the day after hair shaving. Six days after hair shaving, hair removal was performed on the back under anesthesia. Hair removal was performed by applying a hair remover (Epilat (Kracie)) to the shaved area. The day after hair removal, the mice were irradiated with a total dose of 5 Gy of X-rays using an X-ray irradiator (RX-650 (Faxitron X-ray)).
After raising the mice until all the hair had grown in, the probe of a color difference meter (CR-400 (Konica Minolta)) was placed on the hair to measure the lightness (L value) and used as an index of the white hair rate.

<Test Results>
Figures 1 and 2 show the results of evaluating berberrubin and berberine, respectively. When DNA damage was caused to hair follicle stem cells and melanocyte stem cells by X-ray irradiation, the number of melanocyte stem cells decreased, leading to an increase in hair brightness and induction of hair whitening (Non-Patent Document 1). However, application of berberrubin or berberine inhibited the increase in hair brightness and inhibited hair whitening. In other words, berberrubin and berberine were confirmed to have the effect of preventing and improving gray hair.
It is presumed that both compounds suppress and improve DNA damage in hair follicle stem cells and melanocyte stem cells, thereby maintaining and improving the melanocyte stem cell niche function of hair follicle stem cells, and as a result, ectopic differentiation of melanocyte stem cells is suppressed and melanocyte stem cells are maintained (Non-Patent Document 1).
In other words, both compounds are presumed to have the effects of inhibiting and improving DNA damage, maintaining and improving the niche function of epithelial cells for pigmentary cells, and inhibiting and maintaining ectopic differentiation of melanocyte stem cells.
It has been confirmed that berberrubine and berberine do not directly absorb or interfere with X-rays.

(Test Example 2) Evaluation of anti-alopecia effect using X-ray mouse senile alopecia model It has been elucidated that X-ray can induce alopecia by the same mechanism as age-related alopecia (senile alopecia) in addition to age-related gray hair (Non-Patent Document 6).Senior alopecia is different from male pattern alopecia caused by male hormones, and is caused by the accumulation of DNA damage in hair follicle stem cells with aging, which gradually causes hair follicle stem cells to be lost, resulting in miniaturization of hair follicles.X-ray irradiated mice can also be used as this senile alopecia model.
<Test Method>
Seven-week-old C57BL/6 mice were anesthetized and the hair on their backs was shaved off with clippers, leaving a few millimeters. After shaving, mice with good back condition were selected and assigned to groups through stratified random allocation (using EXSUS Professional Version 7.7.1 (CAC Exicare)) so that the mean and variance of body weight in each group were approximately equal.
The administration solution, prepared by dissolving berberine hydrochloride in ethanol to a concentration of 0.1% (w/v), or the solvent ethanol was applied to the mice once a day at 200 μL for one week from the day after hair shaving. Six days after hair shaving, hair removal was performed on the back under anesthesia. Hair removal was performed by applying a hair remover (Epilat (Kracie)) to the shaved area. The day after hair removal, the mice were irradiated with a total dose of 5 Gy of X-rays using an X-ray irradiator (RX-650 (Faxitron X-ray)).
On the 15th and 17th days after hair removal, when differences in the degree of hair growth were observed, a hair growth score was given according to the following scoring criteria to evaluate the degree of hair growth.

Score 1: Pink skin Score 2: Gray skin (less than 30% of shaved area)
Score 3: Gray discoloration of the skin (30% to less than 60% of the shaved area) or hair growth (less than 30% of the shaved area)
Score 4: Gray discoloration of the skin (more than 60% of the shaved area) or hair growth (30% to less than 60% of the shaved area)
Score 5: Hair growth is observed (more than 60% of the shaved area)

<Test Results>
Figures 3 and 4 show the results of evaluating berberine. When DNA damage was caused to hair follicle stem cells by X-ray irradiation, the generation of new hair was delayed. However, the application of berberine eliminated the delay in the generation of new hair. In other words, berberine was confirmed to have the effect of preventing and improving hair loss associated with aging, including senile alopecia.
This is because berberine suppresses and improves DNA damage in hair follicle stem cells, thereby maintaining hair follicle stem cells. Based on the results of Test Example 1, berberrubin, which has the effect of suppressing and improving DNA damage in hair follicle stem cells, also has these actions.

The agent for preventing or improving gray hair and hair loss of the present invention can be used in the fields of quasi-drugs, medicines, or food and beverage products for preventing or improving gray hair and hair loss caused by aging or DNA damage. In addition, the agent for inhibiting or improving DNA damage in hair follicle constituent cells and the agent for inhibiting the decrease in melanocyte stem cells can also be used in the fields of quasi-drugs, medicines, or food and beverage products that require these effects.

Claims (5)

An agent for preventing or improving gray hair, comprising at least one active ingredient selected from the group consisting of berberrubine and its salts (however, excluding agents for preventing or improving gray hair containing a Coptis japonica extract and agents for preventing or improving gray hair containing Phellodendron Bark or Phellodendron Bark extract ). An agent for preventing or improving senile alopecia, comprising at least one active ingredient selected from the group consisting of berberrubine, berberine, and salts thereof (excluding, however, an agent for preventing or improving senile alopecia containing a Coptis japonica extract , and an agent for preventing or improving senile alopecia containing Phellodendron Bark or an extract of Phellodendron Bark ). An agent for suppressing or improving DNA damage in hair follicle constituent cells, comprising at least one active ingredient selected from the group consisting of berberrubine, berberine, and salts thereof (excluding agents for suppressing or improving DNA damage in hair follicle constituent cells containing Coptis japonica extract , and agents for suppressing or improving DNA damage in hair follicle constituent cells containing Phellodendron Bark or Phellodendron Bark extract ). The agent for suppressing or ameliorating DNA damage according to claim 3 , wherein the hair follicle constituent cells are hair follicle stem cells or melanocyte stem cells. An inhibitor of the decrease in melanocyte stem cells, comprising at least one active ingredient selected from the group consisting of berberrubine, berberine, and salts thereof (excluding inhibitors of the decrease in melanocyte stem cells containing a coptis japonica extract and inhibitors of the decrease in melanocyte stem cells containing Phellodendron Bark or an extract of Phellodendron Bark ).