JP2022023250A - Dkk1 expression promoting agent and whitening agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide DKK1 expression promoting agents containing, as an active ingredient, DKK1 expression promoting action found out of ingredients derived from natural products excellent in safety, and to provide whitening agents based on the DKK1 expression promoting action of the ingredients derived from natural products.

SOLUTION: As an active ingredient of the DKK1 expression promoting agent and the whitening agent based on DKK1 expression action, an extract from one or more natural products selected from the group consisting of Alpinia speciosa leaf, Glycyrrhiza uralensis leaf, jujube, royal jelly, and Eriobotrya japonica is contained.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

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

The Wnt signaling pathway is a widely conserved intracellular signaling pathway from nematodes to mammals. In the Wnt signaling pathway, a secretory protein, Wnt, binds to a Wnt receptor present on the cell membrane to transmit a signal. In the epidermis, it is known that activation of the Wnt signaling pathway promotes melanocyte differentiation / proliferation and melanin synthesis.

On the other hand, as a factor that inhibits the Wnt signaling pathway, DKK1 (Dickkopf 1), which is a secretory protein that inhibits the binding between Wnt and the Wnt receptor, is known. It is known that DKK1 is secreted from the cutaneous fibroblasts of the dermis in the skin, suppresses the differentiation and proliferation of melanocytes present in the epidermis, and suppresses 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).

It is known that DKK1 is highly expressed in fibroblasts in the palms (palms and soles), and in general, the skin color varies greatly depending on the race, whereas the palms are of any race. It is considered that the reason why it is white is partly due to the high expression level of DKK1 in the palm-footed site.

Therefore, if the expression of DKK1 can be enhanced in skin fibroblasts, it is considered that pigmentation in the epidermis can be suppressed by inhibiting the activation of the Wnt signaling 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 has been found to have a DKK1 expression-promoting action among natural product-derived components having excellent safety, and is based on a DKK1 expression-promoting agent containing the same as an active ingredient and a DKK1 expression-promoting action of the natural product-derived component. The purpose is to provide a whitening agent.

In order to solve the above problems, the DKK1 expression promoter of the present invention is extracted from one or more natural products selected from the group consisting of lunar peach leaf part, licorice leaf part, taiso, royal jelly, and loquat. It is characterized by using a substance as an active ingredient. In addition, the whitening agent of the present invention is based on the DKK1 expression promoting action, and is selected from one or more natural products selected from the group consisting of moon peach leaf part, licorice leaf part, taiso, royal jelly, and loquat. It is characterized in that the extract of royal jelly is used as an active ingredient.

According to the present invention, the active ingredient is an extract from one or more natural products selected from the group consisting of moon peach leaf, licorice leaf, taiso, royal jelly, and loquat. It is possible to provide a DKK1 expression-promoting agent and a whitening agent which are excellent in quality and highly safe.

Hereinafter, embodiments of the present invention will be described.
The DKK1 expression promoter of the present embodiment contains an extract from one or more natural products selected from the group consisting of lunar peach leaves, licorice leaves, taiso, royal jelly, and loquat as an active ingredient. Is. In addition, the whitening agent of the present embodiment is based on the DKK1 expression promoting action, and is one or more natural products selected from the group consisting of moon peach leaf part, licorice leaf part, taiso, royal jelly, and loquat. The active ingredient is an extract from loquat.

Here, the "extract" in the present embodiment includes an extract obtained by using the above-mentioned natural product as an extraction raw material, a diluted solution or a concentrated solution of the extract, a dried product obtained by drying the extract, or these. Both crude or refined products of.

The extraction raw materials used in the DKK1 expression-promoting agent and the whitening agent of the present embodiment are shell ginger (scientific name: Alpinia speciosa (Wendl.) K. Schum.), Alpinia, taiso (raw drug name), royal jelly, and biwa (scientific name:: Eriobotrya japonica Lindley).

Shell ginger (Alpinia speciosa (Wendl.) K. Schum.) Is a perennial evergreen herb belonging to the genus Alpinia of the family Zingiberaceae, distributed from southern Kyushu to India, and can be easily obtained from these areas. .. Shell ginger is called Sannin in Okinawa and is used not only for Muchi, a traditional confectionery since the Ryukyu dynasty, but also as an herb. The part used as an extraction raw material is the leaf part.

Licorice is a perennial herb belonging to the genus Glycyrrhiza of the family Leguminosae. For licorice, Glychyrrhiza glabra, Glychyrrhiza inflata, Glychyrrhiza uralensis, Glychyrrhiza uralensis, Glychyrrhiza aspera, Glychyrrhiza aspera, Glychyrrhiza aspera, Glychyrrhiza aspera ), Glychyrrhiza yunnanensis, Glychyrrhiza lepidota, Glychyrrhiza echinata, Glychyrrhiza acanthocarpa, etc. Licorice may be used as an extraction raw material, but it is particularly preferable to use Glychyrrhiza glabra, Glychyrrhiza uralensis, and Glychyrrhiza inflata as extraction raw materials.

In the present embodiment, the constituent part of licorice used as an extraction raw material is a leaf part. Here, it is known that the root of licorice has been used as a raw material for medicinal or sweeteners since ancient times and contains 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 portion after utilizing the root portion, it was found that the extract from the leaf portion of licorice has a DKK1 expression promoting action. In addition, glycyrrhizin and other useful components contained in the root part are not contained in the leaf part of licorice.

Jujube (scientific name: Zizyphus jujuba Miller var.inermis Rehder) is a mature fruit part of the buckthorn family Jujube (scientific name: Zizyphus jujuba Miller var.inermis Rehder). Jujube is a deciduous small tree that grows naturally in East Asia such as Southern Europe and China, and Taiso can be easily obtained from these regions. Taisou is known to have effects such as relief, tonicity, and diuresis, and is used for anorexia, diarrhea, palpitation, and the like.

Royal jelly is the pharynx of a young worker bee among the European honey bee (scientific name: Apis melifera L., also known as Western honey bee) or the western honey bee (scientific name: Apis indeca Radoszkowski), which belongs to the honey bee genus of the honey bee family, which is distributed in Europe and Africa. It is a secretion from the glands and is fed to honeybees that become honeybees and honeybees that become adults.

Loquat (Eriobotrya japonica Lindley) is an evergreen tree belonging to the genus Eriobotrya of the Rosaceae family, native to southwestern China, cultivated in Nagasaki, Chiba, Kagoshima, etc., and can be easily obtained from these areas. Examples of the part that can be used as the extraction raw material include a leaf part, a stem part, a branch part, a flower part, a fruit part, a seed part, a root part, or a mixture of these parts, and the leaf part is preferable.

The above-mentioned extract from a natural product can be obtained by drying the extraction raw material, pulverizing it as it is or using a coarse crusher, and subjecting it to extraction with an extraction solvent. Drying may be carried out in the sun or by using a commonly used dryer. Further, it may be used as an extraction raw material after being subjected to pretreatment such as degreasing with a non-polar solvent such as hexane. By performing pretreatment such as degreasing, extraction treatment with a polar solvent can be efficiently performed.

As the extraction solvent, it is preferable to use a polar solvent, for example, water, a hydrophilic organic solvent and the like, and these are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. Is preferable.

Water that can be used as an extraction solvent includes pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmoregulation, buffering and the like. Therefore, the water that can be used as the extraction solvent in the present embodiment includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.

Hydrophilic organic solvents that can be used as the extraction solvent 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 thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.

When a mixed solution of two or more polar solvents is used as an extraction solvent, the mixing ratio can be appropriately adjusted. For example, when a mixed solution of water and a lower aliphatic alcohol is used as an extraction solvent, the mixing ratio of water and the lower aliphatic alcohol is preferably 9: 1 to 1: 9 (volume ratio). It is more preferably 8: 2 to 2: 8 (volume ratio). When a mixed solution of water and a lower aliphatic ketone is used, the mixing ratio of water and the lower aliphatic ketone is preferably 9: 1 to 2: 8 (volume ratio), and water and a large amount are used. When a mixed solution with a valent alcohol is used, the mixing ratio of water and the polyhydric alcohol is preferably 5: 5 to 1: 9 (volume ratio).

The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be carried out according to a conventional method. For example, the extraction material is immersed in an extraction solvent having an amount (mass ratio) of 5 to 15 times that of the extraction material, the soluble component is extracted at room temperature or under reflux heating, and then the extract is filtered to remove the extraction residue. You can get the liquid. Distilling off the solvent from the obtained extract gives a paste-like concentrate, and further drying the concentrate gives a dried product.

The extract from lunar peach leaf part, licorice leaf part, taiso, royal jelly, or loquat obtained as described above has an excellent DKK1 expression promoting action and whitening action, and thus has an excellent DKK1 expression promoting action and whitening action. It can be used as an active ingredient of an agent. In the present embodiment, the whitening effect of the extract from the moon peach leaf part, licorice leaf part, taiso, royal jelly, and loquat is exhibited based on the DKK1 expression promoting action.

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

In this embodiment, any one of the lunar peach leaf part, the licorice leaf part, the taiso, the royal jelly, and the extract from the loquat may be used as the above active ingredient, and two or more of these may be used. It may be mixed and used as the above-mentioned active ingredient. When an extract from lunar peach leaf, licorice leaf, taiso, royal jelly, or loquat is mixed and used as the above active ingredient, the blending ratio is from lunar peach leaf, licorice leaf, taiso, royal jelly, and loquat. It may be appropriately adjusted depending on the degree of DKK1 expression promoting action of the extract.

The DKK1 expression-promoting agent and whitening agent of the present embodiment may consist only of an extract from loquat, licorice leaf, taiso, royal jelly, or loquat, or a mixture thereof, or the moon peach leaf portion and licorice leaf portion. , Licorice, royal jelly, or an extract from loquat or a mixture thereof may be formulated.

The DKK1 expression-promoting agent and the whitening agent of the present embodiment use a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent, and follow a conventional method in powder form, granule form, tablet form, liquid form and the like. It can be formulated into any dosage form of. At this time, as the auxiliary agent, for example, excipients, binders, disintegrants, lubricants, stabilizers, flavoring / odorizing agents and the like can be used. The whitening agent can be blended with other compositions (for example, skin cosmetics and the like) and used, and can also be used as an ointment, an external liquid, a patch and the like.

When the DKK1 expression-promoting agent and the whitening agent of the present embodiment are formulated, the content of the extract from the lunar peach leaf part, licorice leaf part, taiso, royal jelly, or loquat or a mixture thereof is not particularly limited. However, it can be set as appropriate according to the purpose.

In the DKK1 expression-promoting agent and the whitening agent of the present embodiment, if necessary, other natural extracts having the DKK1 expression-promoting action can be obtained from the moon peach leaf part, licorice leaf part, taiso, royal jelly, or loquat. It can be used as an active ingredient in combination with an extract or a mixture thereof.

Examples of the administration method of the DKK1 expression-promoting agent and the whitening agent of the present embodiment to patients include transdermal administration and oral administration, and a method suitable for prevention / treatment thereof is appropriately selected according to the type of disease. do it. In addition, the dose of the whitening agent of the present embodiment may be appropriately increased or decreased depending on the type of disease, severity, individual difference of patients, administration method, administration period and the like.

The DKK1 expression-promoting agent of the present embodiment promotes the expression of DKK1 in skin fibroblasts through the DKK1 expression-promoting action of an extract from the active ingredients such as moon peach leaf, licorice leaf, taiso, royal jelly, or biwa. However, due to the Wnt signal pathway inhibitory action of DKK1, it is possible to suppress the differentiation and proliferation of melanocytes in the epidermis, suppress the production of melanin, and suppress the uptake of melanin from melanocytes in keratinocytes (epidermal keratinized cells). be able to. Therefore, according to the DKK1 expression promoting action of the present embodiment, it is possible to prevent, treat or improve pigmentation in the epidermis by promoting the expression of DKK1 in the fibroblasts of the dermis. Further, the above-mentioned abnormal activation of the Wnt signaling pathway is considered to be one of the causes of carcinogenesis in colorectal cancer, and it is known that DKK1 is inactivated in colorectal cancer cells. Therefore, the DKK1 expression-promoting agent of the present embodiment can also be used for the treatment or amelioration of colorectal cancer through the inactivation of the Wnt signaling pathway by promoting the expression of DKK1. However, the DKK1 expression-promoting agent of the present embodiment can be used for all uses other than these uses, which are significant in exerting the DKK1 expression-promoting action.

The whitening agent of the present embodiment promotes the expression of DKK1 in the fibroblasts of the dermis through the DKK1 expression promoting action possessed by the extract from the active ingredients such as moon peach leaf part, licorice leaf part, taiso, royal jelly, or biwa. Thereby, pigmentation in the epidermis can be prevented, treated or ameliorated.

Here, as conventional whitening agents, those based on the melanin production inhibitory action in melanocytes and the differentiation / proliferation inhibitory action of melanocytes, as well as those based on the proliferation promoting action of epidermal keratinocytes and the melanin uptake inhibitory action have been proposed. .. All of these act on the epidermis and suppress pigmentation of melanin and the like caused by exposure to ultraviolet rays. On the other hand, the DKK1 expression-promoting agent and the whitening agent of the present embodiment act on the fibroblasts of the dermis, and increase melanin regardless of the presence or absence of exposure to ultraviolet rays, as in the palm-plantar region. It differs from conventional whitening agents in that it can be prevented.

Since the DKK1 expression-promoting agent and the whitening agent of the present embodiment have an excellent DKK1 expression-promoting action, they are suitable for blending with, for example, external skin preparations or foods and drinks. In this case, the extract from the moon peach leaf part, the licorice leaf part, the taiso, the royal jelly, or the loquat or a mixture thereof may be blended as it is, or from the moon peach leaf part, the licorice leaf part, the taiso, the royal jelly, or the loquat. A DKK1 expression-promoting agent or a whitening agent formulated from the extract of loquat or a mixture thereof may be blended.

Here, the external skin preparation is not limited in its classification, and includes a wide range of skin lotions, non-pharmaceutical products, pharmaceuticals, etc. used percutaneously, and specifically, for example, an ointment. Examples include creams, milky lotions, beauty liquids, lotions, packs, foundations, lip creams, bathing agents, hair tonics, hair lotions, soaps, body shampoos and the like.

In addition, food and drink is not limited to foods, pharmaceuticals, non-pharmaceutical products, etc. under the administrative classification, but is not limited to foods, general foods, health foods, functional foods, and health functional foods that are taken orally ( It includes a wide range of foods for specified health use, foods with functional claims, foods with nutritional function), non-pharmaceutical products, pharmaceuticals, etc.

Further, since the DKK1 expression-promoting agent and the whitening agent of the present embodiment have an excellent DKK1 expression-promoting action, they can be suitably used as reagents for research on their mechanism of action.

Although the DKK1 expression-promoting agent and the whitening agent of the present embodiment are suitably applied to humans, animals other than humans (for example, mice, rats, etc.) as long as their respective actions and effects are exhibited. It can also be applied to hamsters, dogs, cats, cows, pigs, monkeys, etc.).

Hereinafter, the present invention will be specifically described with reference to 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) shown in Table 1 were used as test samples.

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

Human normal skin fibroblasts (NB1RGB) were cultured in Dulbecco MEM (DMEM) medium containing 10% FBS, and then the cells were recovered by trypsin treatment. The collected cells are diluted with DMEM medium containing 10% FBS so as to have a cell density of 1.0 × 10 ⁵ cells / mL, and then 5 mL each is seeded in a 60 mm petri dish (manufactured by FALCON) (5 × 10 ⁵ cells / petri dish). ), Incubated overnight.

After culturing, the medium was removed, and 5 mL of DMEM medium containing 10% FBS in which the test samples (samples 1 to 5 and the sample concentrations were shown in Table 2 below) were dissolved was added to each petri dish at 37 ° C. and 5% CO ₂ . Was cultured for 24 hours. As a control, DMEM medium containing 10% FBS without sample was used for the same culture. After culturing, the medium is removed, total RNA is extracted with ISOGEN (Nippon Gene Co., Ltd., Cat. No. 311-02501), and the amount of each RNA is measured with a spectrophotometer so that the total RNA is 200 ng / μL. Total RNA was prepared.

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

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

As shown in Table 2, moon peach leaf extract (sample 1), licorice leaf extract (sample 2), taiso extract (sample 3), royal jelly extract (sample 4), and biwa extract (sample 5). ) Was confirmed to have an excellent DKK1 mRNA expression promoting action.

Unlike conventional whitening agents, the DKK1 expression-promoting agent and whitening agent of the present invention act on the dermis and can greatly contribute to the prevention or improvement of skin darkening, age spots, freckles and other pigmentation.

Claims (2)

A DKK1 expression promoter characterized by containing an extract from royal jelly as an active ingredient. A whitening agent based on a DKK1 expression promoting action, which comprises an extract from royal jelly as an active ingredient.