JP2016065004A - Urokinase inhibitor and angiogenic inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel urokinase inhibitor and angiogenic inhibitor.SOLUTION: The urokinase inhibitor and angiogenic inhibitor comprise extract of an algal body belonging to Nostoc, Nostocales, Cyanophyceae as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a urokinase inhibitor and an angiogenesis inhibitor containing, as an active ingredient, an extract of an algal body belonging to the genus Cyanobacteria Nostock.

  As disclosed in Patent Document 1, as algal bodies belonging to the genus Cyanobacteria Nostock, for example, jellyfish (Nostoc commune), hair vegetables (Nostoc flagelliforme), kuzusen rice (Nostoc sphaericum) are known. . These alga bodies contain abundant proteins, vitamins, polysaccharides and various minerals, and have been used for food in China and Japan since ancient times.

  On the other hand, urokinase is one of serine proteases and is known as an enzyme (urokinase-type plasminogen activator) that converts plasminogen, which is an inactive enzyme precursor, into active plasmin. In addition to its function as a protease, urokinase has an effect of promoting the migration of vascular endothelial cells and an effect of promoting the formation of lumens on Matrigel, and plays an important function in angiogenesis.

JP-A-9-149774

  As a result of diligent research by the present inventors, the present invention shows that an extract of an algal body belonging to the genus Cyanobacter Nostock belongs to the action of inhibiting the activity of urokinase and the action of inhibiting angiogenesis. It was made based on the newly found. An object of the present invention is to provide a novel urokinase inhibitor and angiogenesis inhibitor.

The urokinase inhibitor for achieving the above object is characterized by containing, as an active ingredient, an extract of an algal body belonging to the genus Cyanobacteria Nostock.
In the urokinase inhibitor, it is preferable that the algal body is at least one kind selected from Ishijelly, kana, and katsusen rice.

An angiogenesis inhibitor for achieving the above object is characterized by containing, as an active ingredient, an extract of an algal body belonging to the genus Cyanobacteria Nostock.
In the above-mentioned angiogenesis inhibitor, the algal body is preferably at least one selected from Ishijelly, hair vegetable, and katsusen rice.

  According to the present invention, a novel urokinase inhibitor and angiogenesis inhibitor are provided.

[First embodiment]
Hereinafter, the urokinase inhibitor of this embodiment will be described.
The urokinase inhibitor of this embodiment contains an extract of an algal body belonging to the genus Cyanobacteria Nostock as an active ingredient. As the algae belonging to the genus Cyanobacteria Nostock (hereinafter referred to as the above-mentioned algae), for example, at least one selected from Ishijellya, kana, and kuzusen rice can be used.

  The algal body serving as the raw material of the extract may be a naturally occurring algal body or an artificially cultured algal body. In addition, it is industrially preferable to use an artificially cultured alga body from the viewpoint that stable supply is possible and quality maintenance is easy. Further, the algal bodies may be in any state of being collected, crushed after collection, and crushed and dried after collection.

  As an extraction solvent for extracting an extract from the alga body, for example, an organic solvent and a mixed solvent of an organic solvent and water can be used. Examples of the organic solvent include lower alcohol, ethyl acetate, hexane, dimethyl sulfoxide, acetonitrile, acetone, glycerin, and propylene glycol. Examples of the lower alcohol include methanol, ethanol, propanol, isopropanol, and butanol.

  Moreover, as an organic solvent, single type may be used and the mixed solvent which mixed multiple types may be used. When a mixed solvent of water and an organic solvent is used as the extraction solvent, the content of the organic solvent in the mixed solvent is preferably 50% by volume or more, more preferably 80% by volume or more. In addition, additives such as organic salts, inorganic salts, buffering agents, and emulsifiers may be dissolved in the extraction solvent.

  As an extraction method for extracting the extract from the algal cells, any known extraction method, for example, cooling extraction, room temperature extraction, and heat extraction may be used. The extraction temperature can be appropriately set according to the type of solvent, extraction efficiency, deterioration of components, and the like.

  The extraction operation is performed by immersing the algal bodies as raw materials in the extraction solvent for a predetermined time. At that time, the concentration of the algal bodies in the extraction solvent can be appropriately set according to the type of the extraction solvent, the extraction efficiency, the efficiency of the concentration treatment after extraction, and the like. In such an extraction operation, in order to increase the extraction efficiency, processing such as stirring processing, pressurizing processing, and ultrasonic processing may be further performed as necessary.

  Moreover, extraction operation may be performed only once with respect to the same said algal body, and may be performed repeatedly in multiple times. Then, a solid-liquid separation operation is performed after the extraction operation to separate the extract (extract) from the algal residue. As a method for the solid-liquid separation operation, for example, a known separation method such as filtration or centrifugation can be used. Moreover, you may concentrate or dry the obtained extract (extract) as needed.

  The algal extract contained in the urokinase inhibitor of this embodiment has urokinase inhibitory activity. Urokinase is an enzyme involved in various physiological processes such as angiogenesis, bone remodeling, embryo implantation in the uterus, infiltration of immune cells into inflamed sites, ovulation, and spermatogenesis. Therefore, the urokinase inhibitor of the present embodiment is used for obtaining an inhibitory effect on urokinase, for example, a drug, a quasi-drug, and a food and drink for the purpose of treating and preventing a disease caused by an abnormality of the physiological process described above. It can be applied as an additive for use in experiments or as a research reagent for the purpose of studying the above physiological processes.

  In addition, when using the urokinase inhibitor of the present embodiment by applying it to pharmaceuticals or quasi drugs, in addition to administration by ingestion (oral intake), intravascular administration, transdermal administration, mucosal administration, etc. Any administration method, and a method of applying to the skin or article can be employed. Although it does not specifically limit as a dosage form, For example, a powder, a powder agent, a granule, a tablet, a capsule, a pill, a suppository, a liquid agent, an injection, an application agent etc. are mentioned.

  Moreover, the urokinase inhibitor of this embodiment may contain components other than the said algal extract in the range which does not impair the urokinase inhibitory action. Examples of components other than the algal extract include excipients, bases, emulsifiers, solvents, stabilizers, fragrances, and sweeteners.

Next, the effect of this embodiment will be described below.
(1) The urokinase inhibitor of this embodiment contains an extract of an algal body belonging to the genus Cyanobacteria Nostock as an active ingredient. According to the said structure, the activity of urokinase can be inhibited.

(2) Since the active ingredient of the urokinase inhibitor of this embodiment is a natural ingredient contained in a specific algal body, it can be more safely applied to a living body.
[Second Embodiment]
Hereinafter, the angiogenesis inhibitor of this embodiment will be described.

  The angiogenesis inhibitor of this embodiment contains an extract of an algal body belonging to the genus Cyanobacteria Nostock as an active ingredient. Since the extract of the alga belonging to the genus Cyanobacteria Nostock is contained in the angiogenesis inhibitor of the present embodiment, it is the same as that contained in the urokinase inhibitor of the first embodiment. Specific description is omitted.

  The extract of an algal body belonging to the genus Cyanobacteria Nostock, contained in the angiogenesis inhibitor of the present embodiment, has an inhibitory activity on angiogenesis. Therefore, the angiogenesis inhibitor of the present embodiment is an additive for pharmaceuticals, quasi-drugs, and foods and drinks for the purpose of obtaining an anti-angiogenic effect, and for experiments aimed at research on angiogenesis. -It can be applied as a research reagent.

  In addition, when the angiogenesis inhibitor of this embodiment is applied to pharmaceuticals and quasi-drugs, in addition to administration by taking (oral intake), intravascular administration, transdermal administration, mucosal administration, etc. It is possible to employ any of the above-mentioned administration methods and methods of applying to the skin or articles. Although it does not specifically limit as a dosage form, For example, a powder, a powder agent, a granule, a tablet, a capsule, a pill, a suppository, a liquid agent, an injection, an application agent etc. are mentioned.

  Moreover, the angiogenesis inhibitor of this embodiment may contain components other than the extract of the algal body which belongs to the genus Cyanobacteria Nostock in the range which does not impair the angiogenesis inhibitory action. Examples of components other than extracts of algal bodies belonging to the genus Cyanobacteria Nostock include excipients, bases, emulsifiers, solvents, stabilizers, fragrances, sweeteners and the like.

Next, the effect of this embodiment will be described below.
(3) The angiogenesis inhibitor of the present embodiment contains an extract of an algal body belonging to the genus Cyanobacteria Nostock as an active ingredient. According to the above configuration, angiogenesis can be inhibited.

(4) Since the active ingredient of the angiogenesis inhibitor of this embodiment is a natural ingredient contained in a specific alga body, it can be applied more safely to the living body.
Each of the above embodiments can be modified and embodied as follows.

  -You may use the urokinase inhibitor of 1st Embodiment, and the angiogenesis inhibitor of 2nd Embodiment for animals other than humans, such as a horse, a cow, and a bird, for example.

Next, the above embodiment will be described more specifically with reference to test examples.
<Preparation of algal extract>
N-Hexane (20 ml) was added to dried sea jellyfish (1 g) and subjected to solvent extraction at room temperature for 1 hour while applying ultrasonic waves, and then separated into an extract and a residue using filter paper. . The same extraction process was performed again using the residue, and this was repeated twice (a total of three extraction processes were performed). The extract obtained was dried under reduced pressure to obtain a hexane extract of Ishijelly.

  Ethyl acetate (20 ml) is added to the algal body residue after the above hexane extraction, and after 1 hour of solvent extraction while applying ultrasonic waves at room temperature, it is separated into the extract and the residue using filter paper. did. The same extraction process was performed again using the residue, and this was repeated twice (a total of three extraction processes were performed). The extract obtained was dried under reduced pressure to obtain an ethyl acetate extract of Ishijelly.

  Methanol (20 ml) was added to the above-described relative residue after extraction with ethyl acetate, and solvent extraction was performed for 1 hour while applying ultrasonic waves at room temperature, and then separated into an extract and a residue using filter paper. . The same extraction process was performed again using the residue, and this was repeated twice (a total of three extraction processes were performed). The extract obtained was dried under reduced pressure to obtain a methanol extract of Ishijelly.

  Moreover, the hexane extract, ethyl acetate extract, and methanol extract of hair vegetables were obtained by performing the same extraction process with respect to the dried alga body of hair vegetables. Moreover, the hexane extract, the ethyl acetate extract, and the methanol extract of Kaksen rice were obtained by performing the same extraction process with respect to the dry alga body of Kaksen rice.

<Urokinase inhibition test>
A urokinase inhibition test was performed on each of the extracts of Ishijelly, kana and katsusen rice.
Extracts of ishi jellyfish, kana and katsusen rice were dissolved in dimethyl sulfoxide to prepare 1 mg / ml test solutions, which were used as sample solutions. As a buffer solution, trishydroxymethylaminomethane (1.5 g) was dissolved in ion-exchanged water (220 ml), and polyoxyethylene sorbitan monooleate (Tween 80) and polyethylene glycol 8000 each had a final concentration of 0.01 mass. %, And adjusted to pH 8.8 with hydrochloric acid or aqueous sodium hydroxide solution.

  A urokinase solution prepared by diluting 178407 U / ml urokinase with a buffer solution to 3750 U / ml was prepared. A substrate solution prepared by diluting a 5 mM Spectrozyme UK solution with a buffer solution to 1 mM was prepared.

  Buffer solution (20 μl), sample solution (10 μl), and urokinase solution (20 μl) were dispensed into a 96-well microplate, stirred for 30 seconds on a plate shaker, and reacted for 10 minutes in a 37 ° C. incubator. The substrate solution (50 μl) was added at the timing when 10 minutes had passed after removing the microplate from the incubator, and the mixture was stirred for 30 seconds on a plate shaker, and then reacted for 10 minutes in a 37 ° C. incubator. The absorbance at 405 nm was measured using a multi-label reader at the timing when 15 minutes had passed after removing the microplate from the incubator. Then, the urokinase inhibitory activity (%) of each test solution was determined according to the following formula. The results are shown in Table 1.

(Calculation formula)
Urokinase inhibitory activity (%) = [1− (AC) / B] × 100
A: Absorbance of sample solution + urokinase solution + substrate solution B: Absorbance of urokinase solution + substrate solution C: Absorbance of sample solution

As shown in Table 1, urokinase inhibitory activity could be confirmed in both hexane extract, ethyl acetate extract, and methanol extract for Ishijelly and Katsusen rice. For hair vegetables, urokinase inhibitory activity could be confirmed in the ethyl acetate extract and methanol extract.

<Angiogenesis inhibition test>
An angiogenesis inhibition test was conducted on the ethyl acetate extract and methanol extract of Ishijelly. The angiogenesis inhibition test was performed using an angiogenesis kit (Kurashiki Boseki Co., Ltd., KZ-2000) according to the instruction manual attached to the kit. The jellyfish ethyl acetate extract and methanol extract were added to the medium so that the final concentrations were 0.10 mg / ml and 1.00 mg / ml, respectively.

  As a result, in the test added at a final concentration of 0.10 mg / ml, the ethyl acetate extract showed no inhibitory activity against angiogenesis, but the methanol extract was confirmed to show inhibitory activity against angiogenesis. We were able to. Further, in the test added at a final concentration of 1.00 mg / ml, it was confirmed that both the ethyl acetate extract and the methanol extract clearly showed an inhibitory activity on angiogenesis.

  In addition, the same angiogenesis inhibition test was conducted using an enzyme-treated product obtained by digestion enzyme treatment (30 minutes pepsin treatment and 2 hour pancreatin treatment) on ethyl jellyfish extract and methanol extract. Went. Each enzyme-treated product was added to the medium so that the final concentration was 1.00 mg / ml.

  As a result, an angiogenesis inhibitory activity could not be confirmed for the enzyme-treated product of the ethyl acetate extract. On the other hand, the angiogenesis inhibitory activity could be confirmed for the enzyme-treated product of methanol extract.

  From these results, among the extracts of algal bodies belonging to the genus Cyanobacteria Nostock, such as medusa, the extract with lower alcohols such as methanol has high angiogenesis inhibitory activity and resistance to the enzyme-treated product. It is thought that it is particularly excellent in terms of

Claims (4)

  A urokinase inhibitor comprising, as an active ingredient, an extract of an algal body belonging to the genus Cyanobacteria Nostock.   The urokinase inhibitor according to claim 1, wherein the algal body is at least one selected from medusa jellyfish, hair vegetables, and katsusen rice.   An angiogenesis inhibitor comprising, as an active ingredient, an extract of an algal body belonging to the genus Cyanobacteria Nostock.   The angiogenesis inhibitor according to claim 3, wherein the algal body is at least one selected from medusa jellyfish, hair vegetables, and katsusen rice.