JP6857512B2 - A fat-soluble extract that promotes the migration and proliferation of vascular endothelial cells and its extraction method, as well as angiogenesis-promoting agent and wound healing agent. - Google Patents

Description

The present invention comprises a fat-soluble extract that promotes the migration and proliferation of vascular endothelial cells, a method for extracting the fat-soluble extract from Maitake mushrooms, and the fat-soluble extract and the fat-soluble extract as active ingredients. Regarding angiogenesis promoter or wound healing agent.

Endothelial cells play an important role in maintaining vascular homeostasis. When vascular endothelial cells recognize angiogenesis-promoting factors released from ischemic tissue or cells, the vascular endothelial cells migrate and proliferate to induce luminal formation and vascularization. The new blood vessels provide the ischemic tissue or cells with the oxygen and nutrients needed to maintain them, repairing inflammation and healing wounds.

In addition, arteriosclerosis caused by lifestyle habits such as lack of exercise and intake of high-fat diet causes ischemic diseases such as myocardial infarction, angina, cerebral infarction, vascular dementia and peripheral arterial disease. However, an arteriosclerosis promoter is often used as a treatment method.

Patent Documents 1 to 4 and Non-Patent Document 1 disclose documents of angiogenesis-promoting agents related to the present invention. Patent Document 1 discloses a post-ischemic angiogenesis-promoting agent containing a quercetin glycoside as an active ingredient, and Patent Document 2 discloses an angiogenesis-promoting agent containing a cyclobutylpurine derivative as an active ingredient. ing.

Here, quercetin is known to have an inhibitory effect on the drug-metabolizing enzyme CYP2C8, and there are concerns about side effects when used in combination with some anticancer agents. Since it contains bases, there is concern that it may cause hyperuricemia.

Further, Non-Patent Document 1 discloses that the currently performed angiogenesis treatment is limited to injection of an angiogenesis-promoting agent such as a drug or a growth factor into an affected area or gene therapy.

These are considered to have an excellent effect of promoting angiogenesis, but they have a large mental and physical burden on the patient due to side effects, thoracotomy and craniotomy. Therefore, there is a long-awaited angiogenesis-promoting agent that can improve blood flow without imposing a heavy burden on the patient.

On the other hand, it is expected that side effects will be reduced and that it will promote the improvement of ischemic diseases such as myocardial infarction, angina, cerebral infarction, vascular dementia and peripheral arterial disease in the diet instead of injection and gene treatment. Therefore, the search for substances having an angiogenesis-promoting effect from natural substances that can be eaten on a daily basis is also underway.

Patent Document 3 discloses an angiogenesis-promoting agent containing fucosterol extracted from a natural product such as mozuku as a main component, and Patent Document 4 discloses an angiogenesis containing an extract from a natural product winter worm summer grass as an active ingredient. Neonatal promoters are disclosed. All of these are derived from natural products that have been eaten, and it is considered that side effects are reduced.

However, the yield of Cordyceps varies depending on the season, and stable harvesting and supply are difficult. There are various types of Cordyceps sinensis that can be cultivated, but there is no example of artificial cultivation of Cordyceps sinensis according to Patent Document 4. In addition, not all other Cordyceps sinensis have an angiogenesis-promoting effect uniformly, and Cordyceps sinensis parasitizes insects and causes death, so there are concerns about human safety.

Japanese Unexamined Patent Publication No. 2008-266215 Re-table 2010-061931 Japanese Unexamined Patent Publication No. 2011-121917 Japanese Unexamined Patent Publication No. 2008-143868

Therapeutic angiogenesis for revascularization in peripheral artery disease. Gene, 525, 220-228, 2013.

The problem to be solved by the present invention is the migration of vascular endothelial cells from the fresh food Maitake (scientific name: Grifola frontosa), which can stably supply raw materials throughout the year, has abundant eating experience, and ensures safety. To provide a method for obtaining a fat-soluble extract that promotes proliferation, obtain the fat-soluble extract, and further provide an angiogenesis-promoting agent or a wound healing agent in which the fat-soluble extract is used as an active ingredient and side effects are not a concern. There is.

As a result of intensive studies to solve the above problems, the present inventors obtained a primary extract from Maitake with an organic solvent, and partitioned the primary extract with water and ethyl acetate to form an ethyl acetate layer. A secondary extract is obtained from the above, and the secondary extract is further eluted with a gradient solution of hexane and ethyl acetate, and a fraction in which hexane: ethyl acetate is eluted at a ratio of 1: 4 is obtained as a fat-soluble extract. It was.

By this method, the obtained lipophilic extract remarkably promotes the migration and proliferation of vascular endothelial cells, and can be an active ingredient of an angiogenesis promoter or a wound healing agent. The present invention has been completed based on the above findings.

Since the angiogenesis-promoting agent according to the present invention contains a fat-soluble extract extracted from Maitake, which is a food material in daily eating habits, as an active ingredient, there is no risk of side effects, and the fat-soluble extract is Since it is a natural substance, there is no need for labor such as synthesis. Furthermore, since Maitake mushrooms can be stably mass-produced throughout the year, materials can be provided in a stable manner.

More specifically, the effect of the invention will be described in more detail. By using the method according to claim 1, a fat-soluble extract that remarkably promotes the migration and proliferation of vascular endothelial cells from Maitake mushrooms that can be stably cultivated throughout the year. Can be easily obtained and a stable supply can be achieved.

The invention according to claim 2 is a fat-soluble extract that remarkably promotes the migration and proliferation of vascular endothelial cells extracted from Maitake mushrooms, and the fat-soluble extract is dried and powdered or liquefied. It can be used as an active ingredient of an angiogenesis promoter or a wound healing agent.

The invention according to claim 3 contains a fat-soluble extract that remarkably promotes the migration and proliferation of vascular endothelial cells extracted from Maitake as an active ingredient, encapsulated in tablets, granules, powders, capsules, and added to an oral solution. By enabling administration in various forms such as, it can be used as an angiogenesis-promoting agent without causing side effects.

The invention according to claim 4 contains a fat-soluble extract that remarkably promotes the migration and proliferation of vascular endothelial cells extracted from Maitake as an active ingredient, encapsulated in tablets, granules, powders, capsules, and added to an oral solution. By enabling administration in various forms such as, it can be used as a wound healing agent without causing side effects.

It is the schematic of the process of the method of extracting the fat-soluble substance which promotes the migration and proliferation of vascular endothelial cells from Maitake mushroom. It is a figure which showed that the fat-soluble extract obtained from Maitake promoted the migration of vascular endothelial cells. It is a figure which showed that the fat-soluble extract obtained from Maitake promoted the proliferation of vascular endothelial cells. It is a figure which showed that the fat-soluble extract obtained from Maitake promoted the luminal-like formation of vascular endothelial cells.

The angiogenesis-promoting agent and the wound-healing agent according to the present invention are composed of a fat-soluble extract that promotes the migration and proliferation of vascular endothelial cells extracted from Maitake mushroom as an active ingredient.

Maitake mushrooms are edible mushrooms of basidiomycetes. According to the statistics of the Ministry of Agriculture, Forestry and Fisheries in 2015, the annual production of Maitake mushrooms is about 48,800 tons, and they are cultivated throughout the year. Regarding the acquisition of Maitake mushroom, which is the material of the present invention, mycelia can be collected by tank culture or the like, but it is easier to obtain commercially available fruiting bodies.

Further, in order to obtain the effect of the present invention, a culture of mycelium can be used, but in order to obtain the effect of the present invention sufficiently, a fat-soluble extract of Maitake mushroom is extracted from the fruiting body of Maitake mushroom. Is preferable.

Next, a method for extracting the fat-soluble extract from Maitake mushroom will be described.

As the fruiting body or mycelium of Maitake mushroom as a material, a dry powdered one can also be used. Considering the extraction yield, it is desirable to dry the Maitake mushroom and then pulverize it, but it is more preferable to use fresh Maitake mushroom because the cost can be reduced by omitting the drying step and the pulverization step.

Further, for drying the fruiting bodies or mycelium of Maitake mushroom, a method such as sun drying, warm air drying, hot air drying or freeze drying can be applied.

Next, the primary extraction will be described.

Fresh Maitake mushrooms or dried Maitake mushroom powder are immersed in a solvent, and then the solvent is suction-filtered to collect the extract. The solvent of the recovered extract can be removed under reduced pressure to obtain a residue as a primary extract.

Here, as the solvent, a polar organic solvent can be used, and more specifically, 1-butanol, 1-propanol, 2-propanol, ethanol, acetone, acetonitrile or ethanol can be used alone or as a mixed solvent of two or more. More preferably, ethanol is used. Further, when extracting from powdered Maitake mushroom without water, a non-polar organic solvent such as ethyl acetate, hexane or toluene can be used.

In addition, the immersion period, the amount of the organic solvent, and the like can be appropriately changed in consideration of the extraction efficiency.

Next, the secondary extraction will be described. The primary extract has an action of promoting the migration and proliferation of vascular endothelial cells, but further fractionation can enhance the action of promoting the migration and proliferation of vascular endothelial cells.

Distilled water and one or more organic solvents selected from low-polarity organic solvents are added to the primary extract, mixed, allowed to stand, and when the mixed solution is separated, the organic solvent layer is removed from the distilled water layer. Leave only. Next, the organic solvent layer is dehydrated with anhydrous sodium sulfate and then concentrated under reduced pressure to obtain a secondary extract.

Here, the low-polarity organic solvent includes methylene chloride, ethyl acetate, chloroform, diethyl ether, toluene, benzene, hexane and the like, and preferably any one of ethyl acetate, chloroform and hexane is used alone or in combination of two or more. Used as a solvent. More preferably, ethyl acetate is used.

Next, the fat-soluble extract will be described. The secondary extract has a stronger effect of promoting migration and proliferation of vascular endothelial cells than the primary extract, but can be fractionated to further improve the purity. That is, by adsorbing the secondary extract on silica gel and then eluting the silica gel adsorbent with an organic solvent, it is possible to obtain an action of further strongly promoting the migration and proliferation of vascular endothelial cells.

That is, the secondary extract was adsorbed on the silica gel packed in the open column, and the fractionation operation was performed according to the gradient elution method using a mixed organic solvent of hexane and ethyl acetate, and hexane: ethyl acetate was 1: 4. The fraction eluted at the ratio of can be obtained as a fat-soluble extract. The lipophilic extract has an action of more strongly promoting the migration and proliferation of vascular endothelial cells.

Here, as the organic solvent for eluting the silica gel adsorbent, an organic solvent obtained by mixing two or more organic solvents selected from all of the non-polar organic solvent and the polar organic solvent can be used. Preferably, an organic solvent in which hexane and ethyl acetate are mixed can be used.

Further, as the silica gel to be filled in the open column, various silica gels for filling in normal phase column chromatography can be used, and the particle size of the silica gel is preferably 40 to 200 μm.

By adding the lipophilic extract to the cell culture solution of vascular endothelial cells and _{leaving it in the CO 2} incubator for a predetermined time, it can be observed that the migration and proliferation of vascular endothelial cells were promoted, and further, vascular endothelial cells. It is also possible to confirm the luminal-like formation of.

Lumen-like formation of vascular endothelial cells is a phenomenon that indicates that blood vessels have been regenerated. Therefore, the fat-soluble extract that promotes the migration and proliferation of vascular endothelial cells tertiaryly extracted by the above method is used as an active ingredient in excipients, binders, disintegrants, lubricants, emulsifiers, colorants, fragrances and the like. Can be added as an angiogenesis-promoting agent or a wound healing agent in a food-blended, tableted, granulated or encapsulated state.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the configuration of the present invention is not limited to the contents described in the following examples, and the description of the examples is such that the scope of claims is limited or the scope of claims is reduced. It's not something to understand. The configuration of the present invention can be modified in various ways within the technical scope described in the claims.

The outline of the extraction process of the maitake fat-soluble extract is shown in FIG. 1, and an example of extracting the fat-soluble extract from the fresh maitake fruiting body will be described in detail below.

12.5 kg of fresh Maitake fruiting body was immersed in 21 L of ethanol and left at room temperature for 3 days. Then, the ethanol solution in which the fresh Maitake mushroom was infiltrated was suction-filtered to collect the extract to obtain the first extract. The residue of the filtration was again immersed in 21 L of ethanol, left for 3 days in the same manner, and then suction-filtered to obtain a second extract. The first extract and the second extract were mixed, and the solvent was evaporated from the mixed extract using an evaporator to obtain a residue as a primary extract.

Next, the primary extract was added to a solvent in which ethyl acetate and water were mixed at a ratio of 1: 1 and then stirred and left to stand. After a while, when the ethyl acetate layer and the aqueous layer were separated, the ethyl acetate layer was recovered. Anhydrous sodium sulfate was added to the recovered ethyl acetate layer to carry out a dehydration operation. The solvent was then evaporated from the ethyl acetate layer using an evaporator to give the residue as a secondary extract.

Next, the secondary extract dissolved in ethyl acetate was adsorbed on Celite to be completely dried, and then placed on silica gel packed in an open column to adjust the concentration ratio of ethyl acetate and n-hexane. Was used to elute the concentration gradient. Then, the fraction eluted with a mixing ratio of n-hexane and ethyl acetate of 1: 4 was used as a fat-soluble extract. In this example, 26.9 mg of the fat-soluble extract could be obtained.
It was experimentally confirmed that the primary extract, the secondary extract and the lipophilic extract obtained in this example promote the migration and proliferation of vascular endothelial cells, respectively.

Next, an example of the cell migration test using the fat-soluble extract obtained in Example 1 was described, and the migration rate of the vascular endothelial cells obtained as a result is shown in FIG.

In this example, human umbilical vein endothelial cells (hereinafter referred to as HUVEC) were used as vascular endothelial cells. HUVEC subcultured in advance was suspended in HuMedia-EG2, which is a culture medium, and seeded in RadiusTM 96-well Cell Migration Assay (manufactured by Cell Biolabs) so that ^{HUVEC was 2 × 10 4 cells / well.} .. After culturing for 24 hours by a conventional method, the fat-soluble extract was dissolved in dimethyl sulfoxide and added to the culture medium after the culturing. The concentration of the fat-soluble extract at this time was adjusted to 0, 10 μg / mL. In addition, as a positive control, a test group was set up in which vascular endothelial growth factor (hereinafter referred to as VEGF) was added at 20 ng / mL. 16 hours after each addition, RadiusTM Gel was removed, and 8 hours later, cell staining was performed. The cell staining was photographed and the cell migration rate (%) was calculated from the image using the image processing software ImageJ.

As a result of this example, the cell migration rate of HUVEC was as shown in FIG. 2 , and the lipophilic extract activated the migration of HUVEC.

Next, an example of the cell growth test using the fat-soluble extract obtained in Example 1 was described, and the cell growth rate obtained as a result is shown in FIG.

HUVEC subcultured in advance was suspended in HuMedia-EG2, which is a culture medium, and seeded on a ^{collagen-coated 96-well plate so that HUVEC was 1 × 10 4 cells / well.} Then, after culturing HUVEC for 24 hours by a conventional method, a fat-soluble extract dissolved in dimethyl sulfoxide was added to the culture medium. The concentrations of the fat-soluble extract at this time were adjusted to 0, 2.4, 12, and 60 μg / mL, respectively. After 24 hours had passed since the lipophilic extract was added, a cell growth measuring reagent was added to each of them, and after 3 hours had passed, the absorbance of each was measured, and the cell growth rate (%) was calculated from the measured values.

As a result of this example, the cell proliferation rate of HUVEC was as shown in FIG. The cell growth rate of HUVEC increased depending on the concentration of the fat-soluble extract, and the result was obtained that the fat-soluble extract promoted the growth of HUVEC.

Next, an example of the cavity-like formation test of HUVEC using the fat-soluble extract obtained in Example 1 was described, and the total length of the cavity-like obtained as a result is shown in FIG.

HUVEC subcultured in advance is suspended in HuMedia-EG2 (manufactured by Kurabo Industries Ltd.), which is a culture medium, and seeded on a collagen-coated 96-well plate so that HUVEC is 1.5 × 10 ⁴ cells / well. did. After culturing for 2 hours by a conventional method, the culture medium was removed, and 50 μL of a collagen solution (manufactured by Koken Co., Ltd.) was added to each well for gelation. Then, the fat-soluble extract was dissolved in dimethyl sulfoxide, suspended in the culture medium, and added to each gelled well. The concentration of the fat-soluble extract at this time was adjusted to 0, 10 μg / mL, respectively. In addition, a test group was also set up in which VEGF was added to 20 ng / mL as a positive control. After 48 hours of the addition, the results of the formation of the lumen-like shape were photographed in each test group, and the total length (mm) of the lumen-like shape was measured using ImageJ, an image processing software.

As a result of this example, the total length of the HUVEC tube-like was as shown in FIG. It was shown that the lipophilic extract elongates the lumen of HUVEC and promotes angiogenesis.

According to the present invention, the fat-soluble extract obtained from Maitake mushroom promoted the migration and proliferation of HUVEC, and luminal-like formation was remarkably observed. From this, it was clarified that the lipophilic extract according to the present invention promotes luminal-like formation of vascular endothelial cells and causes angiogenesis.

The method for extracting a fat-soluble extract according to the present invention can obtain a fat-soluble extract that promotes the migration and proliferation of vascular endothelial cells from Maitake mushrooms, and the fat-soluble extract is an angiogenesis-promoting agent or a wound healing agent. In addition, since the fat-soluble extract is an ingredient extracted from Maitake, a fresh food with abundant eating experience, it can be used in the fields of pharmaceuticals and health foods with no side effects and safety. It is possible to do.

Claims (2)

A primary extract is obtained from Maitake with an organic solvent, and the primary extract is partitioned between water and ethyl acetate to obtain a secondary extract from the ethyl acetate layer. A method for extracting an angiogenesis-promoting component , which comprises obtaining a fat-soluble extract fraction eluted with a gradient solution of hexane: ethyl acetate at a ratio of 1: 4 as a component for promoting angiogenesis. A primary extract is obtained from Maitake using an organic solvent, and the primary extract is partitioned between water and ethyl acetate to obtain a secondary extract from the ethyl acetate layer. A lipophilic extract fraction eluted with a gradient solution of hexane: ethyl acetate at a ratio of 1: 4 is obtained as an angiogenesis-promoting component, and an angiogenesis-promoting agent containing the angiogenesis-promoting component as a main component .

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