JP2023102984A - Endo180 production promoter - Google Patents

Abstract

[PROBLEMS] To provide an Endo180 production promoting agent containing the ingredient as an active ingredient by finding one having an excellent Endo180 production promoting action among ingredients derived from natural products.
Kind Code: A1 An extract from one or more natural products selected from the group consisting of wolfberry leaves, salvia, perilla and rosemary is used as an active ingredient of the Endo180 production promoter of the present invention.
[Selection figure] None

Description

TECHNICAL FIELD The present invention relates to an Endo180 production promoter containing a natural product-derived component as an active ingredient.

Quantitative and qualitative changes in dermal collagen fiber structure have been confirmed in photoaging caused by long-term exposure to ultraviolet rays, and are thought to be involved in wrinkle formation. Homeostasis of collagen fiber structure is maintained by metabolic balance due to repeated synthesis and degradation of collagen fibers. Among these, in the degradation process, collagen is fragmented by matrix metalloprotease (MMP), and the fragmented collagen is taken up into fibroblasts and degraded. It is known that collagen receptor "Endo180", which is a membrane protein, is involved in the intracellular uptake of fragmented collagen (Non-Patent Document 1).

Endo180, also known as mannose receptor C2 (MRC2), urokinase plasminogen activator receptor-associated protein (uPARAP) and CD280, is a recycled endocytic receptor that binds and internalizes ligands such as collagen and directs them for degradation in endosomes.

Endo180 has homology with the macrophage mannose receptor family, mannose receptor, phospholipase A2, DEC-205/MR6, and others. Endo180 is unique among the family of mannose receptors in that it is targeted from the plasma membrane to the recycling endosome, rather than to the late endosomal/lysosomal compartment, and functions in cell motility and extracellular matrix remodeling by facilitating cell migration and uptake of collagen for intracellular degradation. As uPARAP, it forms a triple complex with urokinase-type plasmin activator (uPA) and urokinase-type plasmin activator receptor (uPAR), and participates in the generation of plasmin from plasminogen. Endo180 is localized in clathrin-covered pits on the cell surface, endosomes, etc., and is mainly expressed in fibroblasts, endothelial cells, and macrophages. As tissues, it is mainly expressed in lungs, blood vessels, bones and skin (see Patent Document 1).

On the other hand, the dermis of the skin is also a tissue rich in collagen, and the involvement of Endo180 in the remodeling of collagen fibers in the dermis has been reported. So far, it has been known that in photoaged skin, Endo180 expression is decreased in fibroblasts, which accompanies the accumulation of fragmented collagen in the dermal matrix (Non-Patent Document 2). It is also known that IL-1α produced by keratinocytes is involved in the mechanism of UV-induced reduction of Endo180 expression in skin fibroblasts (Non-Patent Document 3). Furthermore, it is known that when the expression of Endo180 is reduced in dermal fibroblasts, fragmented collagen accumulated due to suppressed uptake causes fibroblast dysfunction and induces oxidative stress, resulting in a decrease in collagen production and an increase in MMP-1 production, resulting in inhibition of regeneration of normal collagen fiber structure (Non-Patent Document 4). Based on these findings, it is believed that the decrease in Endo180 is involved in the formation of photoaged skin (eg, formation of wrinkles) caused by long-term exposure to ultraviolet rays.

Therefore, for example, if Endo180 expression can be promoted in fibroblasts, it is thought that regeneration of normal collagen fiber structure can be promoted, and formation of photoaged skin and wrinkles caused by photoaging can be suppressed.

Japanese Patent Publication No. 2012-521217

J. Cell Sci., 2009, vol.122, pp.4042-4048 J. Dermatol. Sci., 2013, vol.70, pp.42-48 Photodermatol. Photoimmunol. Photomed., 2020, vol.36, pp.34-41 Photodermatol. Photoimmunol. Photomed., 2021, DOI:10.1111/phpp.12728

An object of the present invention is to find a highly safe natural product-derived component that has an excellent Endo180 production promoting effect, and to provide an Endo180 production promoting agent containing the same as an active ingredient.

In order to solve the above problems, the Endo180 production promoter of the present invention is characterized by containing, as an active ingredient, an extract from one or more natural products selected from the group consisting of wolfberry leaves, salvia, perilla, and rosemary.

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 wolfberry leaves, salvia, perilla, and rosemary derived from natural products, it is possible to provide an Endo180 production promoter with excellent effects and high safety.

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below.
The Endo180 production promoter of the present embodiment contains, as active ingredients, extracts from one or more natural products selected from the group consisting of wolfberry leaves, salvia, perilla, and rosemary.

Extraction raw materials used in the present embodiment include leaves of wolfberry (scientific name: Lycium chinense Mill, or Lycium barbarum L.), salvia (scientific name: Salvia officinalis L.), perilla (scientific name: Perilla frutescens (L.) Britton var. crispa (Benth.) W. Deane) and rosemary (scientific name: Rosmarinus officinalis L.).

Here, the "extract" in the present embodiment includes an extract obtained by using the natural product as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or any of these crudely purified products or purified products.

Wolfberry is a deciduous shrub belonging to the Solanaceae family. The wolfberry is distributed in Japan, Korea, China, Taiwan, etc., and is easily available from these regions. Since ancient times, the fruits, root bark, and leaves of wolfberry have been used as foods, herbal medicines, or herbal medicines. Thus, wolfberry is a highly safe plant.

The species of wolfberry is not particularly limited and can be appropriately selected depending on the intended purpose. , Lycium chinense Mill. var. potinii A. M. Lu, Lycium Barbarum L.; var. auraticarpum K. F. Ching, Lycium ruthenicum, Lycium truncatum Y.; C. Wang, Lycium dasystemum Pojark, Lycium dasystemum Pojark var. rubricaulium A. M. Lu, Lycium eylindricum Kuang et A.; M. Lu and the like. These may be used individually by 1 type, and may use 2 or more types together. Among these, Lycium chinense Mill (hereinafter sometimes referred to as "Maruhakuko"), Lycium barbarum L. (hereinafter, sometimes referred to as "Nagabakuko") is preferred.
As the wolfberry leaf extract, one prepared from wolfberry leaves may be used, or a commercially available product may be used.

The method for preparing the wolfberry leaves is not particularly limited, and can be appropriately selected according to the intended purpose. The drying may be performed in the sun, or may be performed using a commonly used dryer.

The wolfberry leaf extract can be easily obtained by a method commonly used for plant extraction. The form of the wolfberry leaf extract is not particularly limited and can be appropriately selected according to the intended purpose.

The method for extracting the wolfberry leaves is not particularly limited and can be appropriately selected according to the intended purpose. Examples thereof include a method in which the wolfberry leaves, which are raw materials for extraction, are put into a treatment tank filled with an extraction solvent, and if necessary, soluble components are eluted while stirring appropriately, and then the extraction residue is removed by filtration to obtain an extract.
In addition, 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, the extraction treatment with a polar solvent can be efficiently performed.

The conditions for extracting the wolfberry leaves (extraction time and temperature), the extraction solvent, and the amount used thereof are not particularly limited, and can be appropriately selected according to the purpose.

The solvent for extracting the wolfberry leaves is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include water, hydrophilic solvents, and mixed solvents thereof.

The water is not particularly limited and can be appropriately selected according to the purpose. Examples include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, purified water, hot water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered physiological saline, and the like. These may be used individually by 1 type, and may use 2 or more types together.

The hydrophilic solvent is not particularly limited and can be appropriately selected depending on the purpose. Examples include lower 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; polyhydric alcohols having 2 to 5 carbon atoms such as 1,3-butylene glycol, propylene glycol and glycerin. These may be used individually by 1 type, and may use 2 or more types together.

The amount of the hydrophilic solvent used relative to the water in the mixed solvent is not particularly limited, and can be appropriately selected according to the purpose. When using the lower alcohol, it is preferable to add 1 part to 90 parts by mass with respect to 10 parts by mass of water.

The temperature of the solvent for extracting the wolfberry leaves is not particularly limited and can be appropriately selected depending on the intended purpose.

Among these, hot water and a mixed solvent of water and lower alcohol are preferable as the solvent for extracting the wolfberry leaves.

The obtained wolfberry leaf extract may be subjected to dilution, concentration, drying, purification, etc. according to a conventional method in order to obtain a dilution, concentrate, dried, crudely purified, purified, etc. of the wolfberry leaf extract.

The method for purifying the wolfberry leaf extract is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include purification methods such as liquid-liquid partition extraction, various chromatography, and membrane separation.

The salvia is a plant belonging to the Labiatae family distributed in North America, South America, Asia, etc., and can be easily obtained from these regions. Constituent parts of salvia that can be used as raw materials for extraction include, for example, leaves, stems, roots, flowers, whole plants, and mixtures thereof, with leaves being preferred.

The perilla is a perennial herb belonging to the family Labiatae and the genus Perilla, which is cultivated throughout Japan, and can be easily obtained from these regions. Constituent parts of perilla that can be used as raw materials for extraction include, for example, leaves, branches, stems, flowers, buds, seeds, above ground parts, whole plants, or mixtures thereof, and preferably leaves.

The rosemary is an evergreen shrub belonging to the genus Ganoderma of the Labiatae family distributed in Europe, Japan, etc., and can be easily obtained from these regions. Constituent parts that can be used as raw materials for extraction include, for example, above-ground parts such as leaves, branches, bark, stems, stems, fruits, seeds, and flowers, roots, and mixtures of these parts, etc., but leaf parts are preferred.

The extract from the natural product can be obtained by drying the raw material for extraction, either as it is or pulverizing it using a coarse crusher, and subjecting it to extraction with an extraction solvent. Drying may be performed in the sun or using a commonly used dryer. In addition, 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, the extraction treatment of the raw material for extraction 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, 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.

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, osmotic adjustment, buffering, and the like. Therefore, water that can be used as an extraction solvent in the present embodiment includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered physiological saline, and the like.

Hydrophilic organic solvents that can be used as an 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 glycol, propylene glycol, and polyhydric alcohols having 2 to 5 carbon atoms such as glycerin.

When using a mixture of two or more polar solvents as the extraction solvent, the mixture ratio can be adjusted as appropriate. For example, when a mixture 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), more preferably 7:3 to 2:8 (volume ratio). Further, when a mixed solution of water and lower aliphatic ketone is used, the mixing ratio of water and lower aliphatic ketone is preferably 9:1 to 2:8 (volume ratio), and when using a mixed solution of water and 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 raw material for extraction can be eluted into the extraction solvent, and can be carried out 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 filtered to remove the extraction residue. An extract can be obtained. A paste-like concentrate is obtained by distilling off the solvent from the obtained extract, and a dried product is obtained by further drying the concentrate.

The extracts from wolfberry leaves, salvia, perilla, and rosemary obtained as described above have an excellent Endo180 production-promoting effect, and therefore can be used as an active ingredient of an Endo180 production-promoting agent. The Endo180 production promoter of the present embodiment can be used in a wide range of food and drink applications such as pharmaceuticals, quasi-drugs, and cosmetics.

The Endo180 production promoter of the present embodiment may consist solely of extracts from wolfberry leaves, salvia, perilla, rosemary or a mixture thereof, or may be formulated from extracts or mixtures thereof from wolfberry leaves, salvia, perilla, rosemary.

The Endo180 production promoter of the present embodiment can be formulated into any dosage form such as powder, granule, tablet, liquid, etc. according to a conventional method, using a pharmaceutically acceptable carrier such as dextrin, cyclodextrin, or any other adjuvant. At this time, as auxiliary agents, for example, excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents and the like can be used. The Endo180 production promoter can be used by blending it with other compositions (eg, skin cosmetics, food and drink, etc.), and can also be used as ointments, external liquids, patches, and the like.

When the Endo180 production promoter of the present embodiment is formulated, the content of extracts from wolfberry leaves, salvia, perilla, rosemary, or a mixture thereof is not particularly limited, and can be appropriately set according to the purpose.

In addition, the Endo180 production promoter of the present embodiment can be used as an active ingredient by blending other natural extracts having Endo180 production promoting action with extracts from wolfberry leaves, salvia, perilla, rosemary, or mixtures thereof, if necessary.

Methods for administering the Endo180 production promoter of the present embodiment to patients include transdermal administration, oral administration, and the like, and suitable methods for prevention and treatment may be appropriately selected depending on the type of disease. In addition, the dosage of the Endo180 production promoter of the present embodiment may also be appropriately increased or decreased depending on the type and severity of disease, individual patient differences, administration method, administration period, and the like.

The Endo180 production promoter of the present embodiment promotes the production of Endo180 in fibroblasts through the Endo180 production-promoting action of the extracts of wolfberry leaf, salvia, perilla, and rosemary, which are active ingredients, thereby promoting the uptake of fragmented collagen by endocytosis, promoting regeneration of normal collagen fiber structure, and preventing, treating, or improving skin photoaging symptoms such as wrinkle formation. In addition, the Endo180 production promoter of the present embodiment promotes regeneration of normal collagen fiber structure, and can be used for purposes such as prevention, treatment, or improvement of arthritis such as rheumatoid arthritis, osteoarthritis, septic arthritis, gouty arthritis, traumatic arthritis, and osteoarthritis. However, the Endo180 production-enhancing agent of the present embodiment can be used for all uses other than these uses that are significant in exhibiting the above effects.

In addition, since the Endo180 production promoting agent of the present embodiment has an excellent Endo180 production promoting action, it is suitable for blending in, for example, external preparations for skin and foods and drinks. In this case, an extract from wolfberry leaf, salvia, perilla, rosemary or a mixture thereof may be blended as it is, or an Endo180 production promoter formulated from an extract from wolfberry leaf, salvia, perilla or rosemary or a mixture thereof.

Here, the term "external preparation for skin" is not limited to any category, and includes a wide range of percutaneously used skin cosmetics, quasi-drugs, pharmaceuticals, etc. Specific examples include ointments, creams, milky lotions, lotions, serums, lotions, gels, beauty oils, packs, foundations, lip balms, bath salts, hair tonics, hair lotions, shampoos, rinses, soaps, body shampoos, and the like.

“Food and drink” refers to those that have little risk of harming human health and are taken orally or through the gastrointestinal tract in normal social life, and are not limited to administrative divisions such as foods, pharmaceuticals, and quasi-drugs. Therefore, the "food and drink" in the present embodiment includes a wide range of orally ingested general foods, health foods (functional foods and drinks), foods with health claims (foods for specified health uses, foods with nutrient function claims), quasi-drugs, pharmaceuticals, and the like.

In addition, since the Endo180 production promoting agent of the present embodiment has an excellent Endo180 production promoting action, it can be suitably used as a reagent for research on these mechanisms of action.

Although the Endo180 production promoter of the present embodiment is preferably applied to humans, it can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as the respective effects are exhibited.

Production examples and test examples of the present invention will be described below, but the present invention is not limited to the following examples.

Test samples used in the test examples described later were produced or prepared as follows.

(Production Example 1: Lycium chinense leaf hot water extract)
To 20 g of dried Lycium chinense leaves, 10 times the amount (w/v) of water as a solvent was added to the dried Lycium chinense leaves, and reflux extraction was performed at 80° C. for 2 hours. Then, after filtration under reduced pressure, the solvent was removed to obtain a hot water extract from the leaf of Chinese husk. Yield was 27.8%.

(Production Example 2: Lycium chinense leaf 50% by volume ethanol extract)
To 20 g of dried Lycium chinense leaves, 10 times the amount (w/v) of 50% by volume ethanol as a solvent was added to the dried Lycium chinense leaves, and reflux extraction was performed at 80° C. for 2 hours. Then, after filtration under reduced pressure, the solvent was removed to obtain a 50% by volume ethanol extract from the leaf of Chinese husk. Yield was 29.4%.

(Production Example 3: 80% by volume ethanol extract of Lycium chinense leaves) To 20 g of dried Lycium chinense leaves, 80% by volume ethanol of 10 times the amount (w/v) of the dried Lycium chinense leaves was added as a solvent, and reflux extraction was performed at 80°C for 2 hours. Then, after filtration under reduced pressure, the solvent was removed to obtain an 80% by volume ethanol extract from the leaf of Chinese husk. Yield was 19.8%.

(Production Example 4: Production of 50% by volume ethanol extract from Lycium barbarum leaves)
A 50% by volume ethanol extract of Lycium barbarum leaves was prepared in the same manner as in Preparation Example 2, except that Lycium chinense leaves were changed to Lycium barbarum leaves. Yield was 29.1%.

(Production Example 5: Production of 80% by volume ethanol extract from Lycium barbarum leaves)
An 80% by volume ethanol extract of Lycium barbarum leaves was prepared in the same manner as in Preparation Example 3, except that Lycium chinense leaves were changed to Lycium barbarum leaves. Yield was 19.2%.

(Production Examples 6-8)
As the following test samples, commercial freeze-dried products were prepared.
Production Example 6: Salvia extract BG-J (manufactured by Maruzen Pharmaceutical Co., Ltd.)
Production Example 7: Perilla extract BG-J (manufactured by Maruzen Pharmaceutical Co., Ltd.)
Production Example 8: Rosemary extract BG-J (manufactured by Maruzen Pharmaceutical Co., Ltd.)

[Experimental Example 1] Endo180 production promoting effect test The Endo180 production promoting effect was tested as follows using the product produced or prepared in the above production example as a test sample.

Human normal skin fibroblasts (NB1RGB) were cultured using Dulbecco's MEM containing 10% FBS, and then the cells were collected by trypsinization. After diluting the collected cells in the above medium to a cell density of 1.0×10 ⁵ cells/mL, 100 μL of each well was seeded in a 96-well microplate and cultured for two nights.

After the culture was completed, the medium was removed, and 100 μL of a test sample dissolved in 0.25% FBS-containing Dulbecco's MEM (see Tables 1 and 2 below for sample concentrations) was added to each well and cultured for 24 hours. As a control, Dulbecco's MEM containing 0.25% FBS to which no sample was added was used and cultured in the same manner. After culturing, the amount of Endo180 present on the cell surface of each well was measured by ELISA. From the measurement results, the Endo180 production promotion rate (%) was calculated by the following formula.

Endo180 production promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Endo180 production amount with addition of test sample B: Endo180 production amount with no sample addition Tables 1 and 2 show the results.

As shown in Tables 1 and 2, it was confirmed that the wolfberry leaf extract, salvia, perilla, and rosemary all have an excellent Endo180 production promoting effect.

The Endo180 production promoter of the present invention can greatly contribute to the prevention, treatment or improvement of skin photoaging symptoms such as wrinkle formation; the prevention, treatment or improvement of various types of arthritis; and the like.

Claims (1)

An Endo180 production promoter comprising, as an active ingredient, an extract from one or more natural products selected from the group consisting of wolfberry leaves, salvia, perilla and rosemary.