JP7724607B2 - Expression enhancer of intercellular adhesion molecules in lymphatic vessels - Google Patents

Description

The present invention relates to an agent that increases the expression of intercellular adhesion molecules in lymphatic vessels.

Lymphatic vessels are formed from a single layer of endothelial cells to facilitate the absorption of unnecessary waste products, and their structure is maintained by adhesion between endothelial cells. Intercellular adhesion is composed of cell adhesion structures such as tight junctions (TJs), adherens junctions (AJs), and desmosomes (DSs).

Of these, tight junctions exist to function as a barrier that prevents harmful substances from penetrating into endothelial cells, a type of epithelial tissue. The cell membrane proteins claudin and occludin, as well as Zonula occludens protein, which binds to these proteins, have been discovered as factors that make up tight junctions.

It is known that when the factors that make up such tight junctions decrease, the structure of the tight junctions is destroyed, causing various diseases, edema (swelling), and the like.
In recent years, in order to prevent and improve such diseases, etc., components that promote the formation of tight junctions in epithelial tissues have been sought. For example, a tight junction formation promoter containing mandarin orange extract as an active ingredient (Patent Document 1), an epithelial tight junction formation promoter containing processed garlic extract containing a high concentration of sulfur-containing garlic components as an active ingredient (Patent Document 2), and an intercellular adhesion reduction inhibitor containing peach kernel (Prunus persica Batsch) extract as an active ingredient (Patent Document 3) have been disclosed.

Japanese Patent Application Laid-Open No. 2016-222558 JP 2016-113368 A Japanese Patent Application Laid-Open No. 2020-132546

As mentioned above, it has become clear that a decrease in the intercellular adhesion function of lymphatic vessels causes various disorders in the body.
Therefore, an object of the present invention is to provide a novel agent for enhancing the expression of intercellular adhesion factors, which strengthens intercellular adhesion in lymphatic vessels.

The present invention, which solves the above-mentioned problems, provides an agent for enhancing the expression of lymphatic vessel intercellular adhesion factors, which contains star fruit extract as an active ingredient.
The agent for enhancing the expression of an intercellular adhesion factor of the present invention can promote the expression level of an intercellular adhesion factor in lymphatic vessels and strengthen intercellular adhesion. Furthermore, the agent for enhancing the expression of an intercellular adhesion factor in lymphatic vessels of the present invention is useful for preventing or improving a decline in biological function caused by a decline in intercellular adhesion function in lymphatic vessels.

In a preferred embodiment of the present invention, the intercellular adhesion factor is a tight junction-associated factor.

In a preferred embodiment of the present invention, the tight junction-associated factor is tight junction protein-1 (Zonula occludens-1, ZO-1).

In a preferred embodiment of the present invention, the content of the star fruit extract is 0.2 to 4.0% by mass based on the dry mass.
By setting the content as described above, the effect of strengthening adhesion between cells in lymphatic vessels is more pronounced.

The present invention also relates to an external skin preparation for increasing the expression of intercellular adhesion molecules in lymphatic vessels, which contains the above-mentioned agent for increasing the expression of intercellular adhesion molecules.
The topical skin preparation of the present invention can promote the expression of intercellular adhesion factors in lymphatic vessels and strengthen intercellular adhesion in lymphatic vessels.

The agent for enhancing the expression of intercellular adhesion factors in lymphatic vessels of the present invention is highly effective in enhancing the expression of intercellular adhesion factors in lymphatic vessels. Furthermore, the agent for enhancing the expression of intercellular adhesion factors in lymphatic vessels of the present invention strengthens adhesion between cells present in lymphatic vessels, and is effective in preventing or ameliorating lymphatic vessel dysfunction caused by decreased intercellular adhesion, as well as diseases such as lymphedema.

1 is an immunohistochemical staining photograph showing ZO-1 protein expression two days after the addition of star fruit extract to lymphatic endothelial cells in Test Example 1. The scale bar in the photograph is 100 μm. 1 is an immunohistochemical staining photograph showing ZO-1 protein expression 5 days after the addition of star fruit extract to lymphatic endothelial cells in Test Example 1. The scale bar in the photograph is 100 μm. 2 is a graph showing the stained area calculated by extracting the ZO-1 protein stained portion from the immunohistochemical staining image of FIG. 1. 3 is a graph showing the stained area calculated by extracting the ZO-1 protein stained portion from the immunohistochemical staining image of FIG. 2. 1 shows immunohistochemical staining photographs showing ZO-1 protein expression in lymphatic endothelial cells when a lotion containing a star fruit extract or a lotion not containing the extract was added in Test Example 2.

(1) Star Fruit Extract The agent for enhancing the expression of an intercellular adhesion factor in lymphatic vessels (hereinafter simply referred to as the agent for enhancing the expression of an intercellular adhesion factor) of the present invention contains a star fruit extract.
The star fruit extract can be obtained by extraction from Averrhoa carambola (Japanese name: gorenshi), a star fruit of the genus Averrhoa in the family Oxalidaceae. The part of the star fruit to be extracted can be appropriately selected depending on the purpose, and examples include leaves, stems, flowers, fruits, and roots. Among these, leaves are particularly preferred.

Star fruit extract can be easily obtained by methods commonly used for plant extraction.
In the extraction, the extracted star fruit part or its dried product is preferably crushed or shredded in advance to improve the extraction efficiency. The extracted part may be dried in the sun or using a commonly used dryer.

Examples of extraction methods include immersing the plant in an extraction solvent at normal, pressurized, or reduced pressure, at room temperature, cooled, or heated, distillation methods such as steam distillation, and squeezing the parts to be extracted to obtain an extract. These methods can be used alone or in combination of two or more.

When extracting by maceration, 1 to 30 parts by weight of solvent is added to 1 part by weight of dried star fruit plant matter, aboveground parts, rhizomes, and/or seeds, and the mixture is macerated for several days at room temperature, or for several hours at a temperature near the boiling point of the solvent. After maceration, the mixture is cooled to room temperature, and after removing any undesired substances as desired, the solvent can be removed by vacuum concentration or other methods. The desired extract can then be obtained by fractionation using column chromatography packed with silica gel or ion exchange resin.

The extraction solvent is preferably a polar solvent, and suitable examples thereof include one or more selected from water, alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butylene glycol, polypropylene glycol, and glycerin, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran, and particularly suitable examples thereof include water, ethanol, and 1,3-butylene glycol.
The extract thus obtained, or an appropriately concentrated version thereof, can be used as the agent for enhancing the expression of an intercellular adhesion-promoting factor of the present invention.

In addition, commercially available star fruit extracts can be used as agents for enhancing the expression of intercellular adhesion-promoting factors of the present invention. For example, "Star Fruit Leaf Extract BG30 (manufactured by Maruzen Pharmaceutical Co., Ltd.)" can be used.

The content of the star fruit extract relative to the total amount of the intercellular adhesion factor expression enhancer of the present invention is preferably 0.2 to 4.0% by mass, more preferably 0.3 to 2.0% by mass, and even more preferably 0.5 to 1.5% by mass, based on the dry mass.

(2) Intercellular Adhesion Factors Intercellular adhesion factors are proteins that form cell-to-cell adhesion. Preferred examples of intercellular adhesion factors in the present invention include factors associated with tight junctions between cells, such as claudins and occludins, which are skeletal proteins of tight junctions, and Zonula occludens protein, which binds to these proteins to form a lining structure.

Tight junction proteins include Zonula occludens protein-1 (ZO-1), Zonula occludens protein-2 (ZO-2), and Zonula occludens protein-3 (ZO-3). The agent for increasing the expression of intercellular adhesion factors of the present invention is particularly effective in increasing the expression of ZO-1.

(3) Dosage Form The agent for enhancing the expression of an intercellular adhesion factor of the present invention may be administered in its undiluted form to the body, or may be diluted to any desired concentration before use. The agent for enhancing the expression of an intercellular adhesion factor of the present invention may be combined with any desired components used in formulation to form an external preparation for skin or an oral preparation.
In the present invention, the agent for enhancing the expression of intercellular adhesion factors is preferably in the form of an external preparation for skin.

Examples of topical skin preparations include cosmetics, quasi-drugs, and topical skin medicines, with cosmetics and quasi-drugs being more preferred because they can be used daily. There are no particular limitations on the dosage form of these preparations.
When used as a cosmetic, it is preferably in the form of a lotion, emulsion, gel, cream, ointment, etc. More specifically, it may be in the form of a lotion, emulsion, cream, gel, pack, hair cream, spray, etc., and it is particularly preferably in the form of a lotion, emulsion, or cream.

When the agent for enhancing the expression of an intercellular adhesion factor of the present invention is used as an oral preparation, it is preferably in the form of a food composition containing the agent for enhancing the expression of an intercellular adhesion factor of the present invention as an active ingredient. More specifically, it is preferably in the form of a supplement in the form of a general food, tablet, granule, drink, or the like.

When the agent for enhancing the expression of an intercellular adhesion factor of the present invention is used as a topical skin preparation, the agent for enhancing the expression of an intercellular adhesion factor of the present invention can be contained in an amount of preferably 0.0001% to 10% by mass, more preferably 0.001% to 5% by mass, and even more preferably 0.01% to 3% by mass, based on the total amount of the topical skin preparation. When a solvent-removed product (dried product) of a plant extract is used, the agent can be contained in an amount of preferably 0.0000001% to 10% by mass, more preferably 0.0001% to 5% by mass, and even more preferably 0.001% to 3% by mass, based on the total amount of the topical skin preparation. It is believed that if the amount is equal to or greater than the above-mentioned lower limit, the effect of the topical skin preparation of the present invention will be exerted, and if the amount is equal to or less than the above-mentioned upper limit, the plateau of the effect can be avoided.

(4) Other Components When applied to external skin preparations such as cosmetics, the following describes components that can be contained in the external skin preparation. For example, oily components such as hydrocarbons, esters, triglycerides, fatty acids, and higher alcohols, surfactants such as anionic surfactants, amphoteric surfactants, cationic surfactants, and nonionic surfactants, polyhydric alcohols, thickeners/gelling agents, antioxidants, ultraviolet absorbers, colorants, preservatives, powders, and the like can be optionally blended.
Furthermore, as long as the effect of the agent for enhancing the expression of an intercellular adhesion factor of the present invention is not impaired, the agent may contain other active ingredients in addition to the agent for enhancing the expression of an intercellular adhesion factor of the present invention. The active ingredients include, but are not limited to, whitening ingredients, anti-wrinkle ingredients, anti-inflammatory ingredients, and extracts derived from animals and plants.

The topical skin preparation of the present invention can be prepared by treating the intercellular adhesion factor expression promoter of the present invention and the optional ingredients described above using standard methods.

The agent for increasing the expression of intercellular adhesion factors of the present invention can be used to prevent or improve diseases caused by intercellular adhesion of lymphocytes, and is particularly suitable for diseases caused by a decrease in the barrier function of endothelial cells due to weakening of tight junctions between cells.
It can also be used to prevent or improve various skin diseases caused by leakage of lymph fluid due to instability in the structure of lymphatic vessels caused by decreased intercellular adhesion, such as edema (swelling).

The present invention will be explained in detail below using examples, but these are merely illustrative of the present invention and the scope of the present invention is not limited to these examples.

Test Example 1: Immunohistochemical staining of tight junction-associated factors in lymphatic endothelial cells using star fruit extract (1) Cell preparation Frozen lymphatic endothelial cells (HLEC) were seeded in a flask containing Endothelial Cell Medium (Cosmo Bio Co., Ltd.) and cultured. The medium was changed the next day, and five days later, the cells were seeded in a 4-well slide chamber and continued to be cultured.
Star fruit extract (star fruit leaf extract BG30 (Maruzen Pharmaceuticals Co., Ltd.)) was added to new endothelial cell culture media to concentrations of 0.5%, 0.25%, and 0.125% (0.0045, 0.00225, and 0.00125%, respectively, on a dry mass basis). Two days after seeding on the slide chamber, the lymphatic endothelial cells were transferred to the medium containing the extract and further cultured, and two and five days after transfer to the extract-containing medium, the lymphatic endothelial cells were recovered. As a control, cells were cultured in the same manner using a culture medium without star fruit extract.

(2) Cell fixation and immunohistochemical staining The collected lymphatic endothelial cells were washed with PBS and fixed with 4% paraformaldehyde. PBS containing 0.5% Triton X-100 was added and the cells were cultured for 10 minutes for permeabilization, followed by blocking with 10% Block Ace for 30 minutes.

Subsequently, immunohistochemical staining was performed using the following antibodies. Specifically, lymphatic endothelial cells were washed with PBS, then primary antibodies were added and cultured overnight at 4°C. After washing again with PBS, secondary antibodies were added and cultured for 30 minutes. Nuclear staining and slide mounting were performed using Dapi-Fluoromount-G (registered trademark) (Southern Biotechnology Associates, Inc.). The resulting lymphatic endothelial cells were observed using a fluorescence microscope, and images were captured for analysis. The results two days after the addition of starfruit extract are shown in Figure 1, and the results five days after addition are shown in Figure 2.
(antibody)
Primary antibody: Rabbit anti ZO-1 (diluted 200 times with 10% Block Ace)
Secondary antibody: Alexa 488 (diluted 250 times with 10% Block Ace)

(3) Analysis of Expression of Tight Junction-Associated Factors ZO-1 protein-stained areas were extracted from images taken using a fluorescence microscope using Image J, and the percentage of the area of ZO-1 protein-stained areas in the images was calculated as the stained area (%) of intercellular adhesion. The results are shown in Figures 3 and 4.

1 to 4 show that when cells were cultured in a medium containing 0.5% starfruit extract, ZO-1 protein expression increased on both days 2 and 5 after the addition of the extract, compared to when the extract was not added. Furthermore, even when cells were cultured in a medium containing 0.125% starfruit extract, increased ZO-1 protein expression was confirmed on day 5 after the addition of the extract. When cells were cultured in a medium containing 0.25% starfruit extract, increased ZO-1 protein expression was observed only on day 2 after the addition of the extract.
Therefore, it was revealed that star fruit extract has the effect of strengthening intercellular adhesion of lymphatic vessel cells due to increased expression of tight junction-related factors.

Test Example 2: Evaluation of tight junction-related factors in lymphatic endothelial cells using a lotion containing starfruit extract as an active ingredient (1) Preparation of lotion A lotion for external use on the skin was prepared according to the following formulation. Specifically, the ingredients were heated to 80°C, stirred and solubilized, and then stirred and cooled to obtain the lotion of Example 1. Comparative Example 1 (no starfruit extract added) was also prepared in which the starfruit extract described below was replaced with an equal amount of water.

(2) Evaluation of Tight Junction-Associated Factors (2-1) Cell Preparation Frozen lymphatic endothelial cells (HLECs) were seeded in a flask containing Endothelial Cell Medium (Cosmo Bio Co., Ltd.) and cultured. The medium was replaced the next day, and 5 days after seeding, the cells were seeded in a 4-well slide chamber and continued to be cultured.
To a new endothelial cell medium, Example 1 or Comparative Example 1 was added so as to be diluted 32-fold to prepare a lotion-containing medium. Two days after seeding on the slide chamber, the lymphatic endothelial cells were transferred to each lotion-containing medium and further cultured, and five days after transfer to each lotion-containing medium, the lymphatic endothelial cells were collected.

(2-2) Cell Fixation and Immunohistological Staining The collected lymphatic endothelial cells were washed with PBS and fixed with 4% paraformaldehyde. PBS containing 0.5% Triton X-100 was added and the cells were cultured for 10 minutes for permeabilization, followed by blocking with 10% Block Ace for 30 minutes.

Subsequently, immunohistochemical staining was performed using the same antibodies as in Test Example 1 (2). Specifically, the lymphatic endothelial cells were washed with PBS, then the primary antibody was added and incubated overnight at 4°C. After washing again with PBS, the secondary antibody was added and incubated for 30 minutes. Nuclear staining and slide mounting were performed using Dapi-Fluoromount-G (registered trademark) (Southern Biotechnology Associates, Inc.). The resulting lymphatic endothelial cells were observed using a fluorescence microscope, and images were taken for analysis (Figure 5).

(3) Results
As can be seen from Figure 5, when Example 1 containing star fruit extract was added (Figure 5(b)), the expression of ZO-1 protein increased compared to when Comparative Example 1 not containing the extract was added (Figure 5(a)).
Therefore, it was shown that lotion containing star fruit extract has the effect of strengthening intercellular adhesion of lymphatic vessel cells due to increased expression of tight junction-related factors.

This invention can be applied to pharmaceuticals, cosmetics, etc.

Claims (5)

An agent that increases the expression of intercellular adhesion factors in lymphatic vessels, with star fruit leaf extract as the active ingredient. The agent for increasing the expression of lymphatic intercellular adhesion factors according to claim 1, wherein the intercellular adhesion factor is a tight junction-associated factor. The agent for increasing the expression of lymphatic intercellular adhesion factors according to claim 2, wherein the tight junction-associated factor is tight junction protein-1 (Zonula occludens-1, ZO-1). The agent for enhancing the expression of lymphatic intercellular adhesion factors according to any one of claims 1 to 3, characterized in that the content of the star fruit leaf extract is 0.2 to 4.0% by mass, based on the dry mass of the solvent-removed product. The agent for enhancing the expression of lymphatic intercellular adhesion factors according to any one of claims 1 to 4 , which is an external preparation for skin .