JP7402509B2 - DKK1 expression promoter and whitening agent - Google Patents

Description

The present invention relates to a DKK1 expression promoter and a whitening agent.

The Wnt signal pathway is an intracellular signal pathway that is widely conserved from nematodes to mammals. In the Wnt signal pathway, signals are transmitted when Wnt, a secreted protein, binds to Wnt receptors present on cell membranes. In the epidermis, activation of the Wnt signal pathway is known to promote melanocyte differentiation and proliferation and melanin synthesis.

On the other hand, DKK1 (Dickkopf 1), a secreted protein that inhibits the binding between Wnt and Wnt receptor, is known as a factor that inhibits the Wnt signal pathway. In the skin, DKK1 is secreted by skin fibroblasts in the dermis, and is known to suppress the differentiation and proliferation of melanocytes present in the epidermis, and suppress the synthesis of melanin (see Non-Patent Document 1). It is also known that DKK1 promotes the proliferation of epidermal keratinocytes (epidermal keratinocytes) and suppresses the uptake of melanin from melanocytes (see Non-Patent Document 2).

DKK1 is known to be highly expressed in fibroblasts in the palmoplantar region (palms and soles of the feet), and while skin color generally varies greatly depending on race, the expression of DKK1 in the palmoplantar region differs regardless of race. The reason for the white color is thought to be due in part to the high expression level of DKK1 in the palmoplantar region.

Therefore, if the expression of DKK1 can be increased in skin fibroblasts, it is thought that pigmentation in the epidermis can be suppressed by inhibiting the activation of the Wnt signal pathway.

J. Cell Biol., 2004, vol.165, no.2, pp.275-285 FASEB J., 2008, vol.22, issue 4, pp.1009-1020

The present invention is based on the discovery of a DKK1 expression promoting effect among natural product-derived ingredients with excellent safety, and a DKK1 expression promoter containing this as an active ingredient, and based on the DKK1 expression promoting effect of the natural product-derived ingredient. The purpose is to provide skin whitening agents.

In order to solve the above-mentioned problems, the DKK1 expression promoter of the present invention is extracted from one or more natural products selected from the group consisting of moongour leaf, licorice leaf, seaweed, royal jelly, and loquat. It is characterized by having a substance as an active ingredient. In addition, the skin whitening agent of the present invention is based on the DKK1 expression promoting effect, and is made from one or more natural products selected from the group consisting of mengum leaf, licorice leaf, seaweed, royal jelly, and loquat. It is characterized by having an extract of as an active ingredient.

According to the present invention, by using as an active ingredient an extract from one or more natural products selected from the group consisting of moon peach leaf, licorice leaf, seaweed, royal jelly, and loquat, the effects can be improved. It is possible to provide a DKK1 expression promoter and a whitening agent that have excellent properties and are highly safe.

Embodiments of the present invention will be described below.
The DKK1 expression promoter of the present embodiment has as an active ingredient an extract from one or more natural products selected from the group consisting of moongour leaf, licorice leaf, seaweed, royal jelly, and loquat. It is. Furthermore, the skin whitening agent of the present embodiment is based on the DKK1 expression promoting effect, and contains one or more natural products selected from the group consisting of mengum leaf, licorice leaf, seaweed, royal jelly, and loquat. The active ingredient is an extract from.

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

The extracted raw materials used in the DKK1 expression promoter and whitening agent of this embodiment are Alpinia speciosa (Scientific name: Alpinia speciosa (Wendl.) K. Schum.), Licorice root, Taisou (herbal medicine name), Royal jelly, and Loquat (scientific name: Eriobotrya japonica Lindley).

Alpinia speciosa (Wendl.) K. Schum. is a perennial evergreen herb belonging to the Zingiberaceae family, and is distributed from southern Kyushu to India, and can be easily obtained from these regions. . Moon peach is called sannin in Okinawa and is used in moochie, a traditional sweet that dates back to the Ryukyu Dynasty, and is also used as an herb. The part used as the extraction raw material is the leaf part.

Licorice is a perennial herb belonging to the genus Glycyrrhiza in the Fabaceae family. Licorice plants include Glychyrrhiza glabra, Glychyrrhiza inflata, Glychyrrhiza uralensis, Glychyrrhiza aspera, Glychyrrhiza eurycarpa, and Glychyrrhiza paridiflora. pallidiflora ), Glychyrrhiza yunnanensis, Glychyrrhiza lepidota, Glychyrrhiza echinata, and Glychyrrhiza acanthocarpa. Licorice may be used as the extraction raw material, but it is particularly preferred to use Glychyrrhiza glabra, Glychyrrhiza uralensis, and Glychyrrhiza inflata as the extraction raw material.

In this embodiment, the constituent parts of licorice used as the extraction raw material are leaves. The root of licorice has been used as a raw material for medicinal purposes and sweeteners since ancient times, and it is known to contain glycyrrhizin and other useful ingredients, and the term "licorice" generally refers to the root. However, in the present embodiment, as a result of focusing on the remaining part after using the root part, it was discovered that an extract from the leaf part of licorice has a DKK1 expression promoting effect. Note that glycyrrhizin and other useful components contained in the roots are not contained in the leaves of licorice.

Zizyphus jujuba (herbal medicine name) is the mature fruit part of the jujube (scientific name: Zizyphus jujuba Miller var. inermis Rehder) of the Rhamnaceae family. Jujube is a small deciduous tree that grows naturally in southern Europe, China, and other East Asia, and jujube can be easily obtained from these regions. It is known to have relaxing, tonic, diuretic, and other effects, and is used to treat anorexia, diarrhea, palpitations, and the like.

Royal jelly is produced in the pharynx of young worker bees of the European honey bee (scientific name: Apis mellifera L., also known as the Western honey bee) or the eastern honey bee (scientific name: Apis indeca Radoszkowski), which belong to the Apis family Apis family and are distributed in Europe and Africa. It is a secretion from glands that is fed to larvae that become queen bees and to adult queen bees.

Loquat (Eriobotrya japonica Lindley) is an evergreen tall tree belonging to the genus Loquat in the Rosaceae family, and is native to southwestern China, cultivated in Nagasaki, Chiba, Kagoshima, etc., and can be easily obtained from these regions. Parts that can be used as extraction raw materials include, for example, leaves, trunks, branches, flowers, fruits, seeds, roots, and mixtures of these parts, with leaves being preferred.

The above-mentioned extract from the natural product 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, extraction treatment using a polar solvent 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 8:2 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 5:5 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 extracts obtained as described above from moongour leaves, licorice leaves, Japanese radish, royal jelly, or loquats have excellent DKK1 expression promoting and whitening effects, and therefore can be used as DKK1 expression promoters and whitening agents. It can be used as an active ingredient in drugs. In the present embodiment, the skin whitening effect of the extracts from moongour leaves, licorice leaves, Japanese radish, royal jelly, and loquats is exerted based on the DKK1 expression promoting effect.

The DKK1 expression promoter and whitening agent of this embodiment can be used in a wide range of applications such as pharmaceuticals, quasi-drugs, and cosmetics.

In addition, in this embodiment, any one of the extracts from moontooth leaves, licorice leaves, seaweed, royal jelly, or loquat may be used as the above-mentioned active ingredient, or two or more of these may be used. They may be mixed and used as the above active ingredient. When extracts from moontooth leaf, licorice leaf, seaweed, royal jelly, or loquat are mixed together and used as the above-mentioned active ingredient, the blending ratio should be: It may be adjusted as appropriate depending on the degree of the DKK1 expression promoting effect of the extract.

The DKK1 expression promoter and whitening agent of the present embodiment may consist only of extracts from moontooth leaf part, licorice leaf part, sea bream, royal jelly, or loquat, or a mixture thereof; It may also be a formulation of extracts from , Japanese turmeric, royal jelly, or loquat, or a mixture thereof.

The DKK1 expression promoter and whitening agent of this embodiment can be prepared in the form of powder, granules, tablets, liquid, etc. using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent according to a conventional method. It can be formulated into any dosage form. 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 whitening agent can be used in combination with other compositions (for example, skin cosmetics, etc.), and can also be used as an ointment, an external liquid, a patch, etc.

When the DKK1 expression promoter and whitening agent of the present embodiment are formulated, there are no particular limitations on the content of extracts from moontooth leaves, licorice leaves, seaweed, royal jelly, or loquat, or mixtures thereof. It can be set as appropriate depending on the purpose.

In addition, the DKK1 expression promoter and whitening agent of the present embodiment may be prepared by adding other natural extracts having a DKK1 expression promoting effect, etc., as necessary, from moongweed leaf, licorice leaf, Japanese radish, royal jelly, or loquat. It can be used as an active ingredient in combination with extracts or mixtures thereof.

Methods for administering the DKK1 expression promoter and whitening agent of the present embodiment to patients include transdermal administration, oral administration, etc., but methods suitable for prevention and treatment may be selected as appropriate depending on the type of disease. do it. Further, the dosage of the whitening agent of the present embodiment may be appropriately increased or decreased depending on the type of disease, severity, individual differences among patients, administration method, administration period, etc.

The DKK1 expression promoter of the present embodiment promotes the expression of DKK1 in skin fibroblasts through the DKK1 expression-promoting effect of the active ingredients, which are extracts from stingray leaf, licorice leaf, seaweed, royal jelly, or loquat. However, due to the Wnt signal pathway inhibiting effect of DKK1, it is possible to suppress differentiation and proliferation of melanocytes in the epidermis, suppress melanin production, and suppress melanin uptake from melanocytes in keratinocytes (epidermal keratinocytes). be able to. Therefore, according to the DKK1 expression promoting effect of the present embodiment, pigmentation in the epidermis can be prevented, treated, or improved by promoting the expression of DKK1 in dermal fibroblasts. Furthermore, the above-mentioned abnormal activation of the Wnt signal pathway is considered to be a factor in carcinogenesis in colon cancer, and it is known that DKK1 is inactivated in colon cancer cells. Therefore, the DKK1 expression promoter of the present embodiment can also be used to treat or improve colorectal cancer through inactivation of the Wnt signal pathway by promoting the expression of DKK1. However, the DKK1 expression promoter of the present embodiment can be used for all other uses in which it is meaningful to exhibit a DKK1 expression promoting effect in addition to these uses.

The skin whitening agent of the present embodiment promotes the expression of DKK1 in fibroblasts of the dermis through the DKK1 expression-promoting effect of the active ingredients, which are extracts from stingray leaf, licorice leaf, Japanese radish, royal jelly, or loquat. By doing so, pigmentation in the epidermis can be prevented, treated or improved.

Here, as conventional skin whitening agents, in addition to those based on the effect of suppressing melanin production in melanocytes and the effect of suppressing differentiation and proliferation of melanocytes, there are also those based on the effect of promoting proliferation of epidermal keratinocytes and suppressing melanin uptake. . These all act on the epidermis and suppress pigmentation such as melanin caused by exposure to ultraviolet rays. In contrast, the DKK1 expression promoter and whitening agent of the present embodiment act on fibroblasts in the dermis, and also increase melanin regardless of the presence or absence of exposure to ultraviolet rays, such as in the palmoplantar region. It is different from conventional skin whitening agents in that it can prevent skin lightening.

The DKK1 expression promoter and whitening agent of the present embodiment have an excellent DKK1 expression promoting effect and are therefore suitable for being incorporated into external skin preparations or food and drink products, for example. In this case, extracts or mixtures thereof may be blended as they are from moontooth leaf, licorice leaf, seaweed, royal jelly, or loquat; A DKK1 expression promoter or whitening agent prepared from an extract of or a mixture thereof may be added.

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, serums, lotions, packs, foundations, lip balms, bath salts, hair tonics, hair lotions, soaps, and body shampoos.

In addition, food and beverages are not limited to administrative categories such as foods, pharmaceuticals, quasi-drugs, etc., but are general foods that are ingested orally, health foods, functional foods, and foods with health claims ( It includes a wide range of foods such as foods for specified health uses, foods with functional claims, foods with nutritional function claims), quasi-drugs, and pharmaceuticals.

Furthermore, since the DKK1 expression promoter and whitening agent of the present embodiment have an excellent DKK1 expression promoting effect, they can also be suitably used as reagents for research on the mechanism of action of these agents.

Although the DKK1 expression promoter and whitening agent of the present embodiment are preferably applied to humans, they may also be applied to animals other than humans (e.g., mice, rats, It can also be applied to hamsters, dogs, cats, cows, pigs, monkeys, etc.).

Hereinafter, the present invention will be specifically explained using test examples, but the present invention is not limited to the following examples. In the following test examples, freeze-dried products (samples 1 to 5) of the products shown in Table 1 were used as test samples.

[Test Example 1] DKK1 mRNA expression promoting effect test The above natural product extracts (samples 1 to 5) were tested for DKK1 mRNA expression promoting effect as follows.

Human normal skin fibroblasts (NB1RGB) were cultured using Dulbecco's MEM (DMEM) medium containing 10% FBS, and then the cells were collected by trypsin treatment. The collected cells were diluted with DMEM medium containing 10% FBS to a cell density of 1.0 × 10 ⁵ cells/mL, and then seeded in 5 mL portions in 60 mm petri dishes (manufactured by FALCON) (5 × 10 ⁵ cells/mL). ) and cultured overnight.

After culturing, remove the medium, add 5 mL of 10% FBS-containing DMEM medium in which test samples (samples 1 to 5, see Table 2 below for sample concentrations) have been dissolved to each petri dish, and incubate at 37°C with 5% _CO2. The cells were cultured for 24 hours. As a control, DMEM medium containing 10% FBS to which no sample was added was used and cultured in the same manner. After culturing, remove the medium, extract total RNA using ISOGEN (manufactured by Nippon Gene Co., Ltd., Cat. No. 311-02501), measure the amount of each RNA using a spectrophotometer, and adjust the amount to 200 ng/μL. Total RNA was prepared.

Using this total RNA as a template, the expression levels of mRNA were measured for DKK1 and GAPDH as an internal standard. Detection was performed using a real-time PCR device Smart Cycler (manufactured by Cepheid) and a real-time 2-Step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (manufactured by Takara Bio, code No. RR063A). The expression level of DKK1 mRNA was determined by correcting the value of GAPDH based on total RNA preparations prepared from cells cultured with "test sample addition" and "no sample addition", respectively. From the obtained values, the DKK1 mRNA expression promotion rate (%) was calculated using the following formula.

DKK1 mRNA expression promotion rate (%) = A/B x 100
Each term in the formula represents the following.
A: Correction value with addition of test sample B: Correction value without addition of sample The results are shown in Table 2.

As shown in Table 2. ) was confirmed to have an excellent DKK1 mRNA expression promoting effect.

The DKK1 expression promoter and whitening agent of the present invention, unlike conventional whitening agents, act on the dermis and can greatly contribute to the prevention or improvement of pigmentation such as skin darkening, age spots, and freckles.

Claims (1)

A DKK1 expression promoter comprising an extract from licorice leaves and/or loquat as an active ingredient.