JP7411196B2 - Type XVII collagen maintenance and enhancement agent - Google Patents

Description

The present invention relates to an agent for maintaining and enhancing type XVII collagen based on the type XVII collagen production promoting effect and the neutrophil elastase activity inhibiting effect. Furthermore, the present invention also relates to type XVII collagen production promoters and neutrophil elastase activity inhibitors.

The skin is composed of the stratum corneum, epidermis, basement membrane, and dermis. Of these, the epidermis and dermis are composed of epidermal cells, fibroblasts, and extracellular matrices such as collagen, elastin, and hyaluronic acid that are located outside these cells and support the skin structure. On the other hand, the basement membrane is a type of extracellular matrix that exists at the boundary between the epidermis and dermis, and it not only connects the epidermis and dermis, but also plays an important role in maintaining skin function (Non-patent Document (see 1). The main skeleton of the basement membrane has a network structure composed of type IV collagen. A structure called hemidesmosome contributes to the adhesion between the basement membrane and epidermal cells, and cell adhesion molecules such as type XVII collagen, integrin α6β4, and laminin 332 adhere the basement membrane and epidermal cells. In young skin, the interaction between the epidermis and dermis maintains homeostasis through the action of the basement membrane, ensuring moisture retention, flexibility, elasticity, etc., and the skin appears firm, shiny, and fresh. will be maintained.

However, when affected by certain external factors such as ultraviolet irradiation, extremely dry air, excessive skin washing, etc., or when aging progresses, the cell adhesion molecules described above decompose and change in quality, causing the basement membrane structure to deteriorate. is destroyed (see Non-Patent Documents 2 and 3). As a result, the moisturizing function and elasticity of the skin decrease, and the stratum corneum begins to peel off abnormally, causing the skin to lose its firmness and luster, and to exhibit aging symptoms such as roughness and wrinkles. In this way, changes associated with skin aging, such as the formation of wrinkles, loss of texture, and decrease in elasticity, are associated with a decrease in basement membrane components and structural changes in the basement membrane, and type XVII collagen is involved. It is thought that by promoting production, it is possible to prevent and improve skin aging symptoms.

In addition, in recent years, it has been discovered that type XVII collagen expressed in hair follicle stem cells is essential for maintaining hair follicle stem cells, and hair follicle stem cells also maintain pigment stem cells, and type XVII collagen can prevent gray hair and hair loss through maintenance of stem cells. (Refer to Non-Patent Document 4).Furthermore, the expression of neutrophil elastase is induced in hair follicles due to DNA damage response due to aging, etc., and type XVII collagen is degraded by the neutrophil elastase. It has been revealed that hair follicle stem cells lose their stemness and differentiate into epidermal keratinocytes, causing hair follicles to become dwarfed and cause thinning hair loss (see Non-Patent Document 5). The relationship between this and gray hair, thinning hair, etc. is attracting attention. Under these circumstances, hair loss inhibitors, hair depigmentation inhibitors, and the like containing type XVII collagen or its mutants have been proposed (see Patent Document 1).

Furthermore, among the external factors mentioned above, it is known that neutrophils infiltrate into the dermis, particularly due to ultraviolet irradiation. In recent years, it has been reported that the activity of neutrophil elastase increases with the infiltration of neutrophils into the dermis, and that neutrophil elastase is activated by cleaving the precursors of matrix metalloproteinases (MMPs) MMP-1 and MMP-2. It has been reported that this phenomenon occurs (see Non-Patent Document 6). Furthermore, neutrophil elastase has low substrate specificity and is known to degrade extracellular matrix components such as collagen in addition to elastin.
Therefore, if the activity of neutrophil elastase can be inhibited, in addition to inhibiting its own activity, it can also inhibit the activation of MMP-1 and MMP-2. It is thought that it can suppress the decomposition of matrix components and prevent and improve skin aging symptoms, especially wrinkle formation.

Japanese Patent Application Publication No. 2009-161509

Marinkovich MP et al., J. Cell Biol., 1992, vol.199, pp.695-703 Lavker et al., J. Invest. Dermatol., 1979, vol.73, pp.59-66 Langton AK et al., Mech. Aging Dev., 2016, vol.156, pp.14-6 Tanimura S et al., Cell Stem Cell, 2011, vol.8, pp.177-87 Matsumura H et al., Science, 2016, vol.351, aad4395 Takeuchi H et al., J Dermatol Sci. 2010, vol.60, pp.151-8

The present invention has discovered, among ingredients derived from natural products with excellent safety, a substance that has the effect of promoting the production of type XVII collagen and the effect of inhibiting the activity of neutrophil elastase, and uses it as an active ingredient. The purpose of the present invention is to provide an agent for maintaining and enhancing type collagen. Furthermore, it is an object of the present invention to provide a type XVII collagen production promoter and a neutrophil elastase activity inhibitor containing the natural product-derived component as an active ingredient.

In order to solve the above problems, the type XVII collagen maintenance and enhancement agent, the type XVII collagen production promoter, and the neutrophil elastase activity inhibitor according to the present invention are characterized in that they contain an extract from licorice as an active ingredient. .

According to the present invention, a type XVII collagen maintenance and enhancement agent, a type XVII collagen production promoter, and a neutrophil elastase activity inhibitor have excellent effects and are highly safe, by using an extract from licorice as an active ingredient. can be provided.

Embodiments of the present invention will be described below.
The type XVII collagen maintenance and enhancement agent, the type XVII collagen expression promoter, and the neutrophil elastase activity inhibitor of the present embodiment contain an extract from licorice as an active ingredient.

In addition, in this embodiment, "extract" includes an extract obtained from the above plant as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crude product thereof. Both purified products and purified products are included.

The extraction raw material used in this embodiment is licorice (herbal drug name).
Licorice is the root and/or rhizome of licorice, a perennial herb belonging to the genus Glycyrrhiza in the Fabaceae family. It has been used for medicinal purposes and as a raw material for sweeteners since ancient times, and is known to contain glycyrrhizin and other useful ingredients. It is being
Daylilies as plants include Glychyrrhiza glabra, Glychyrrhiza inflata, Glychyrrhiza uralensis, Glychyrrhiza aspera, Glychyrrhiza eurycarpa, Glychyrrhiza paridiflora There are various types, including Glychyrrhiza pallidiflora, Glychyrrhiza yunnanensis, Glychyrrhiza lepidota, Glychyrrhiza echinata, and Glychyrrhiza acanthocarpa. Although any type of licorice may be used as the extraction raw material, it is particularly preferred to use Glychyrrhiza glabra, Glychyrrhiza uralensis, and Glychyrrhiza inflata as the extraction raw material. .

The above-mentioned extract from licorice can be obtained by drying the extraction raw material and then pulverizing it as it is or using a coarse pulverizer and subjecting it to extraction with an extraction solvent. Drying may be performed in the sun or using a commonly used dryer. Alternatively, it may be used as an extraction raw material after being subjected to pretreatment such as degreasing with a nonpolar solvent such as hexane. By performing pretreatment such as degreasing, the extraction treatment using a polar solvent of the above-mentioned extraction raw material can be performed efficiently.

As the extraction solvent, it is preferable to use a polar solvent, such as water, hydrophilic organic solvents, etc., which are used alone or in combination of two or more at room temperature or at a temperature below the boiling point of the solvent. It is preferable.

Examples of water that can be used as an extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as water that has been subjected to various treatments. Examples of treatments applied to water include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, and the like. Therefore, water that can be used as an extraction solvent in this embodiment includes purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

Hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene. Examples include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol, and glycerin.

When a mixture of two or more polar solvents is used as an extraction solvent, the mixing ratio can be adjusted as appropriate. For example, when a mixture of water and lower aliphatic alcohol is used as the extraction solvent, the mixing ratio of water and lower aliphatic alcohol is preferably 9:1 to 1:9 (volume ratio), More preferably, the ratio is 7:3 to 2:8 (capacity ratio). In addition, when using a mixed solution of water and lower aliphatic ketone, the mixing ratio of water and lower aliphatic ketone is preferably 9:1 to 2:8 (volume ratio). When using a mixed solution with a polyhydric alcohol, the mixing ratio of water and polyhydric alcohol is preferably 8:2 to 1:9 (volume ratio).

The extraction treatment is not particularly limited as long as the soluble components contained in the extraction raw material can be eluted into the extraction solvent, and can be performed according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent of 5 to 15 times the amount (mass ratio) of the extraction raw material, the soluble components are extracted at room temperature or under reflux heating, and then the extraction residue is removed by filtration. liquid can be obtained. When the solvent is distilled off from the obtained extract, a paste-like concentrate is obtained, and when this concentrate is further dried, a dry product is obtained.

The extract from licorice obtained as described above (hereinafter sometimes simply referred to as "licorice extract") has an excellent effect of promoting type XVII collagen production and inhibiting neutrophil elastase activity. Therefore, it can be used as an active ingredient of type XVII collagen maintenance and enhancement agents, type XVII collagen production promoters, or neutrophil elastase activity inhibitors. The type XVII collagen maintenance and enhancement agent, type XVII collagen production promoter, and neutrophil elastase activity inhibitor according to the present embodiment can be used in a wide range of applications such as pharmaceuticals, quasi-drugs, and cosmetics.

Here, the type XVII collagen maintenance and enhancement agent of the present embodiment is based on both the action of promoting type XVII collagen expression and the action of inhibiting neutrophil elastase activity, and utilizes the fact that licorice extract has both of these actions. are doing.

The agent for maintaining and enhancing type XVII collagen, the type XVII collagen production promoter, and the neutrophil elastase activity inhibitor according to the present embodiment may consist only of licorice extract, or may be a formulation of licorice extract.

The type XVII collagen maintenance and enhancement agent, type XVII collagen production promoter, and neutrophil elastase activity inhibitor according to the present embodiment are prepared using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent. It can be formulated into any desired dosage form such as powder, granules, tablets, and liquid according to conventional methods. In this case, as the auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent, etc. can be used. The type XVII collagen maintenance and enhancement agent, the type XVII collagen production promoter, and the neutrophil elastase activity inhibitor can be used in combination with other compositions (e.g., skin cosmetics, etc.), as well as in tablets and capsules. etc. can be used as

When formulating the type XVII collagen maintenance and enhancement agent, type XVII collagen production promoter, and neutrophil elastase activity inhibitor according to the present embodiment, the content of licorice extract is not particularly limited, and the content of licorice extract is not particularly limited. It can be set as appropriate.

The type XVII collagen maintenance and enhancement agent, the type XVII collagen production promoter, and the neutrophil elastase activity inhibitor according to the present embodiment may have a type XVII collagen production promoting effect or a neutrophil elastase activity inhibiting effect, as necessary. Other natural extracts and the like can be used as active ingredients by blending them with the licorice extract.

Methods for administering the type XVII collagen maintenance and enhancement agent, type XVII collagen production promoter, and neutrophil elastase activity inhibitor to patients according to the present embodiment include oral administration, intravenous administration, etc. Depending on the situation, a method suitable for its prevention or treatment may be selected as appropriate. In addition, the dosage of the type XVII collagen maintenance and enhancement agent, type XVII collagen production promoter, and neutrophil elastase activity inhibitor according to the present embodiment also depends on the type of disease, severity, individual differences between patients, administration method, and administration period. It may be increased or decreased as appropriate.

The type XVII collagen maintenance and enhancement agent according to the present embodiment has the active ingredient licorice extract having both the action of promoting type XVII collagen production and the action of inhibiting neutrophil elastase activity. , can promote the production of type XVII collagen and strengthen the function of type XVII collagen, and can also suppress the degradation of type XVII collagen and maintain the function of type XVII collagen. Due to these effects, the type XVII collagen maintenance and enhancement agent of the present embodiment can maintain hair follicle stem cells and pigment stem cells, and can prevent or improve hair depigmentation (white hair) and hair loss.

The type XVII collagen production promoter according to the present embodiment can promote the production of type XVII collagen through the type XVII collagen production promoting action of the active ingredient licorice extract. This can induce reconstruction of the basement membrane structure and prevent or improve skin aging symptoms such as wrinkle formation, loss of texture, and decrease in elasticity, as well as treat or improve skin damage. Can be done. In addition, the type XVII collagen production promoter according to the present embodiment can maintain hair follicle stem cells and pigment stem cells through the type XVII collagen production promoting effect of licorice extract, thereby causing hair depigmentation (white hair). and hair loss can be prevented or improved. However, in addition to these uses, the type XVII collagen production promoter according to the present embodiment can be used for any purpose in which it is meaningful to exhibit the type XVII collagen production promoting effect.

The neutrophil elastase activity inhibitor according to the present embodiment can suppress the degradation of type XVII collagen through the neutrophil elastase activity inhibiting effect of licorice extract, which is an active ingredient. It can maintain pigment stem cells and prevent or improve hair depigmentation (white hair) and hair loss. In addition, the neutrophil elastase activity inhibitor according to the present embodiment suppresses the decomposition of extracellular matrix components such as elastin and collagen through the neutrophil elastase activity inhibitory effect of licorice extract, thereby inhibiting skin aging. Symptoms, especially wrinkle formation, can be prevented or ameliorated. However, in addition to these uses, the neutrophil elastase activity inhibitor according to the present embodiment can be used for any purpose in which it is meaningful to exert the effect of inhibiting neutrophil elastase activity.

The agent for maintaining and enhancing type XVII collagen, the type XVII collagen production promoter, and the neutrophil elastase activity inhibitor of the present embodiment have an excellent effect of promoting type XVII collagen production or inhibiting neutrophil elastase activity, and therefore can be used in skin It is suitable for blending into external preparations and food and drink products. In this case, the licorice extract may be blended as is, or a type XVII collagen maintenance and enhancement agent, a type XVII collagen production promoter, or a neutrophil elastase activity inhibitor formulated from the licorice extract may be blended. .

Here, there are no restrictions on the category of external skin preparations, and they include a wide range of skin cosmetics, quasi-drugs, pharmaceuticals, etc. that are used transdermally, and specifically include, for example, ointments, Examples include creams, milky lotions, lotions, serums, lotions, gels, beauty oils, packs, foundations, lip balms, bath salts, hair tonics, hair lotions, soaps, body shampoos, etc.

Food and beverages refer to foods that have little risk of harming human health and are ingested orally or through gastrointestinal administration in normal social life, and are categorized as foods, drugs, quasi-drugs, etc. by administrative classification. It is not limited to. Therefore, "food and drink" in this embodiment refers to general food that is orally ingested, health food (functional food and drink), food with health claims (food for specified health uses, food with nutritional function), quasi-drugs, and pharmaceuticals. It includes a wide range of things such as The food and drink according to this embodiment is preferably a food and drink that can display the favorable effects of licorice extract on the food and drink or its packaging, and is preferably a food with health claims (food for specified health uses, food with functional claims). Foods, functional nutritional foods), quasi-drugs, and pharmaceuticals are particularly preferred.

In addition, the type XVII collagen maintenance and enhancement agent, type XVII collagen production promoter, and neutrophil elastase activity inhibitor according to the present embodiment have excellent type XVII collagen production promoting effects and neutrophil elastase activity inhibiting effects, so It can also be suitably used as a reagent for research on these mechanisms of action.

The type XVII collagen maintenance and enhancement agent, the type XVII collagen production promoter, and the neutrophil elastase activity inhibitor according to this embodiment are preferably applied to humans, but their respective effects may not be effective. It can also be applied to animals other than humans (for example, mice, rats, dogs, cats, cows, pigs, monkeys, etc.) as long as it is suitable.

Hereinafter, the present invention will be specifically explained using test examples, but the present invention is not limited to the following examples. In this test example, licorice extract powder (manufactured by Maruzen Pharmaceutical Co., Ltd., sample 1) was used as the licorice extract.

[Test Example 1] Type XVII collagen production promoting effect test The licorice extract (sample 1) was tested for type XVII collagen production promoting effect as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte growth medium (KGM), and then the cells were collected by trypsin treatment. The collected cells were diluted with KGM to a cell density of 2×10 ⁵ cells/mL, then seeded at 100 μL per well in a 96-well plate, and cultured for 6 hours. After culturing, remove the medium and use normal human epidermal keratinocyte proliferation basal medium (Humedia-KB2, hereinafter referred to as "KBM", the above KGM without the addition of growth factors (hEGF, BPE, insulin)). 100 μL of was added to each well and cultured overnight.

After the culture was completed, 100 μL of the test sample (Sample 1, see Table 1 below for sample concentration) dissolved in KBM was added to each well and cultured for 48 hours. As a control, KBM to which no sample was added was used and cultured in the same manner. After culturing, remove the medium, fix the cells on the plate, and measure the amount of type XVII collagen expressed on the cell surface by ELISA using an anti-human type XVII collagen α1 chain polyclonal antibody (manufactured by FLAREBIO BIOTECH). did. From the measurement results, type XVII collagen production promotion rate (%) was calculated using the following formula.

Type XVII collagen production promotion rate (%) = A/B x 100
Each term in the formula represents the following.
A: Amount of type XVII collagen with addition of test sample B: Amount of type XVII collagen without addition of sample The results are shown in Table 1.

As shown in Table 1, the licorice extract (sample 1) was confirmed to have an excellent effect of promoting type XVII collagen production.

[Test Example 2] Human neutrophil elastase activity inhibitory effect test The licorice extract (sample 1) was tested for human neutrophil elastase activity inhibitory effect as follows.

In a 96-well plate, 50 μL of the test sample (Sample 1, see Table 2 below for sample concentration) prepared with 0.1 mol/L HEPES buffer (pH 7.5) and 6 μg/mL human neutrophil elastase ( ELASTIN PRODUCTS) solution (25 μL) was mixed. Then, 25 μL of a solution of substrate (manufactured by SIGMA, N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide) prepared at 2 mmol/L with the above buffer was added, and the mixture was heated at 25°C for 15 minutes. Made it react. After the reaction was completed, the absorbance at a wavelength of 415 nm was measured. In addition, a blank test without enzyme addition was performed in the same manner and correction was made. From the obtained results, the human neutrophil elastase activity inhibition rate (%) was calculated using the following formula.

Human neutrophil elastase activity inhibition rate (%) = {1-(CD)/(AB)}×100
Each term in the formula represents the following.
A: Absorbance at a wavelength of 415 nm with no sample added and enzyme added B: Absorbance at a wavelength of 415 nm with no sample added and enzyme added C: Absorbance at a wavelength of 415 nm with test sample added and enzyme added D: Test sample added and enzyme added Absorbance results at a wavelength of 415 nm without additives are shown in Table 2.

As shown in Table 2, the licorice extract (sample 1) was found to have an excellent inhibitory effect on human neutrophil elastase activity. It was also confirmed that the degree of inhibition of human neutrophil elastase activity can be adjusted by the concentration of licorice extract.

From the results of Test Examples 1 and 2 above, licorice extract (sample 1) can promote the production of type XVII collagen and suppress its degradation, and the function of type XVII collagen can be improved from two approaches. It was confirmed that it can be maintained and strengthened.
As mentioned above, it is known that the decomposition of type XVII collagen by neutrophil elastase greatly contributes to the dwarfing of hair follicles (non-patent documents 4 and 5 mentioned above), and licorice extract is effective against gray hair and It is recognized that it is particularly suitable for use in preventing hair loss.

The type XVII collagen production promoter and neutrophil elastase activity inhibitor according to the present invention can prevent gray hair; prevent hair loss; reconstruct basement membrane structure; prevent or improve skin aging symptoms including wrinkle improvement; It can greatly contribute to the treatment or improvement of injuries.

Claims (2)

A type XVII collagen maintenance-enhancing agent that contains an extract from licorice as an active ingredient and has a type XVII collagen production promoting effect and a neutrophil elastase activity inhibiting effect. A type XVII collagen production promoter characterized by containing an extract from licorice as an active ingredient.