JP5842640B2 - Phosphodiesterase 3 inhibitor - Google Patents

Description

  The present invention relates to a phosphodiesterase 3 inhibitor containing a processed kuzu flower as an active ingredient.

  Phosphodiesterase (PDE) is an enzyme that hydrolyzes cyclic phosphodiester of cyclic AMP (cAMP) and cyclic GMP (cGMP), which are intracellular signal molecules, and controls their intracellular concentration. In mammals, there are 11 types of isozymes, PDE1 to PDE11, and each subtype exists and is involved in various scenes of intracellular signal transduction. In addition, it is known that specificity is observed in tissue localization by isozymes and respective subtypes. Inhibitors that inhibit the activity of this phosphodiesterase (PDE) can directly control the intracellular concentration of cAMP and cGMP, and have selectivity according to isozymes and their subtypes. As such, it is expected to be applied to medicine and health.

  Among them, phosphodiesterase 3 (PDE3) is mainly present in platelets, heart, and vascular smooth muscle, and as an inhibitor thereof, milrinone, which is a therapeutic agent for acute heart failure, ulcer based on chronic arterial occlusion, pain, coldness, etc. Cilostazol, which is an ameliorating agent for ischemic symptoms, is known.

  Further, Patent Document 1 below discloses that a synergistic amount of (1) sulfonylurea, non-sulfonylurea K + ATP channel blocker, or sulfonylurea and non-non-insulin-dependent diabetes Disclosed is an invention of a method for treating non-insulin-dependent diabetes mellitus comprising the step of administering a sulfonylurea K + ATP channel blocker and (2) a cAMP phosphodiesterase type 3 inhibitor, glyburide and PDE3 inhibitor as sulfonylurea drugs It is described that the secretion of insulin from rat insulin-producing cells can be synergistically enhanced by the combined use with milrinone.

  Patent Document 2 listed below discloses PDE4 or PDE3 / 4 inhibitors and diseases in effective treatment of diseases that can be treated with disease-modifying anti-rheumatic drugs (DMARD) or other anti-rheumatic drugs or anti-arthritic drugs. An invention of the combined use of a modified anti-rheumatic drug (DMARD) or other anti-rheumatic or anti-arthritic drugs is disclosed, by the combined use of the anti-rheumatic drug methotrexate and the PDE3 / 4 inhibitor Pumafentrine It is described that the symptoms of collagen-induced arthritis in mice can be synergistically improved.

  Furthermore, the following Patent Document 3 discloses an invention of a cisplatin effect enhancer comprising a phosphodiesterase III B (PDE3B) inhibitor as an active ingredient, and the combination of a cisplatin that is an anticancer agent and cilostazol that is a PDE3 inhibitor is a cancer-bearing agent. It has been described that the increase in tumor volume of mice can be synergistically suppressed.

  On the other hand, kuzu is a large vine plant of the legume family, and kuzu starch collected from its roots has long been used as a raw material for Japanese sweets. In addition, the roots and flowers are referred to as kudzu and kuzuka, respectively, and are used as a raw material for Chinese medicines for symptoms such as antipyretic drugs, analgesics, antispasmodics, and sweating.

JP 2001-354568 A JP 2005-508983 Gazette JP 2009-242378 A

  In order to further expand the application of phosphodiesterase inhibitors (PDE inhibitors) to medicine and the health and health field, it is desired to develop new active ingredients with little risk of side effects. Accordingly, an object of the present invention is to provide a new active ingredient excellent in the effect of inhibiting phosphodiesterase 3, and to provide a phosphodiesterase 3 inhibitor containing the same.

  As a result of intensive studies to achieve the above object, the present inventors have found a high phosphodiesterase 3 inhibitory activity in the flower portion of kuzu, particularly in its hot water extraction fraction and isoflavone fraction, and have completed the present invention. .

That is, this invention provides the phosphodiesterase 3 inhibitor which has the following structures.
[1] A phosphodiesterase 3 inhibitor comprising a processed kuzuhana product as an active ingredient.
[2] The phosphodiesterase 3 inhibitor according to the above [1], wherein the processed kuzuka product comprises an isoflavone-containing composition obtained from kuzuka.
[3] The phosphodiesterase 3 inhibitor according to [1] or [2], which is used for inhibiting phosphodiesterase 3B.
[4] Formula (1) below:

Or the following formula (2):

Or a glycoside thereof, as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the phosphodiesterase 3 inhibitor with few fears of a side effect can be provided by using as an active ingredient the isoflavone contained in a Kuzuka processed material or Kuzuka.

  Hereinafter, preferred embodiments of the phosphodiesterase 3 inhibitor (hereinafter referred to as “PDE3 inhibitor”) of the present invention will be described. First, the processed Kuzuka product, which is an active ingredient of the PDE3 inhibitor of the present invention, will be described.

  Kuzuka, which is the flower part of Kuzu, is rich in flavonoids, especially isoflavones. In the present invention, the kuzu flowers collected at any stage from the bud to the fully opened flower may be used, or the kuzu flowers collected at each stage may be mixed and used. There are no particular restrictions on the type of kuzu, but examples include Pueraria thomsonii, Pueraria lobata, and Pueraria thunbergiana. Use Pueraria thomsonii. Is preferred.

  In the present specification, the “Kuzuhana processed product” is a processed product of the Kuzuka, and includes, for example, those obtained by performing a treatment such as a drying treatment, a pulverization treatment, and an extraction treatment. Moreover, the processed material obtained by giving processes, such as extraction, concentration, fractionation, isolation | separation, a refinement | purification, is included so that content of specific components, such as isoflavones, may be raised.

  Hereinafter, a method for preparing a kuzu flower dried product, a kuzu flower powder (a dry powder and an extract powder), and a kuzu flower extract will be described.

  The dried kuzu flower can be obtained by drying kuzu flowers, preferably kuzu flowers in the cocoon stage, by sun drying, hot air drying or the like. The moisture content is preferably dried to 10% by mass or less.

  The kuzu flower powder (dry powder) can be obtained by pulverizing the dried kuzu flower product. The powderization can be performed by a method commonly used by those skilled in the art, for example, a ball mill, a hammer mill, a roller mill, or the like. Alternatively, the Kuzuka powder (dried powder) is obtained by crushing the collected Kuzuka using a mass colloid, slicer, comitolol, etc. to obtain a Kuzuka crushed product, and drying this Kuzuka crushed product. be able to.

  The kuzu flower extract is prepared by adding a solvent to a kuzu flower dried product such as kuzu flower, kuzu flower crushed product, kuzu flower dried product, or kuzu flower powder (dried powder), preferably kuzu flower dried powder, for example. Accordingly, it can be obtained by heating, performing extraction, and collecting the extract by centrifugation or filtration.

  Examples of the solvent that can be used to obtain the Kuzuhana extract include water, organic solvents, and hydrous organic solvents. Examples of the organic solvent include methanol, ethanol, n-propanol, n-butanol, acetone, hexane, cyclohexane, propylene glycol, ethyl methyl ketone, glycerin, methyl acetate, ethyl acetate, diethyl ether, dichloromethane, edible oils and fats, 1, Examples include 1,1,2-tetrafluoroethane, 1,1,2-trichloroethene and the like. Among these, a polar organic solvent is preferable, ethanol, n-butanol, methanol, acetone, propylene glycol, and ethyl acetate are more preferable, and ethanol is most preferable.

  Examples of the extraction method include a warm extraction method and a supercritical extraction method. In these extraction methods, heating may be performed by applying pressure as necessary. When heating, it is necessary to prevent the solvent added to Kuzuka from volatilizing. When heating, the extraction temperature is preferably 50 ° C. or higher, more preferably 70 ° C. or higher, most preferably 90 ° C. or higher, preferably 130 ° C. or lower, more preferably 100 ° C. or lower.

  The extraction time may be a time for sufficiently extracting soluble components from the extraction raw material, and may be set as appropriate according to the extraction temperature and the like. Preferably, it is 30 minutes to 48 hours. For example, when the extraction temperature is less than 50 ° C., it can be 6 hours to 48 hours, and when it is 50 ° C. or more, it can be 30 minutes to 24 hours.

  The obtained extract may be concentrated or dried by a method such as concentration under reduced pressure or freeze-drying, if necessary, to form a liquid, a paste, or a powder (extract powder).

  More specifically, preferable embodiments include 1 to 10000 parts by mass with respect to 100 parts by mass (in terms of dry matter) of Kuzuka, Kuzuka crushed material, Kuzuka dried product, or Kuzuka powder (dried powder). Add hot water, extract the soluble components while maintaining the temperature of hot water at 50-130 ° C. for 0.5-48 hours, filter by centrifugation, then dry by spray drying. Thus, it is preferable to obtain a powdered Kuzuka extract.

  The processed kuzuhana product thus obtained is usually a composition containing 0.01 to 50% by mass, preferably 0.1 to 50% by mass, of isoflavones in terms of dry matter.

  In this invention, the isoflavone containing composition obtained by giving the process which raises content of isoflavones can also be used as a kuzu flower processed material. The treatment may be performed by extraction with a water-containing organic solvent or the like, fractionation by column chromatography or the like, but the Kuzuka extract is methanol or water-containing methanol, preferably containing 0.1 to 99.9% methanol. It is preferable to increase the content of isoflavones by performing a treatment for extraction with hydrous methanol. Thus, typically, an isoflavone-containing composition containing 20 to 100% by mass, preferably 40 to 100% by mass, of isoflavones in terms of dry matter can be obtained.

  It is known that Kuzuka contains a lot of unique isoflavones that are not much contained in other plants. Specific examples include Tectorigenin, Tectorigenin 7-O-xylosylglucoside, 6-hydroxygenistein, 6-hydroxygenistein-6,7-di-O-glucoside, Tectoridin, etc. is there. On the other hand, it is known that genistein, daidzein or glycosides thereof, which are contained in a large amount in plants such as soybeans, are hardly contained in kuzuka. In addition, about the above-mentioned isoflavones, it can confirm quantitatively or qualitatively by the HPLC analysis etc. which used these for the sample.

  Further, 6-hydroxygenistein represented by the following formula (1) or tectorigenin represented by the following formula (2), which is an isoflavone contained in Kuzuhana, is shown in the examples described later. Since it has been confirmed that it has an activity to inhibit phosphodiesterase 3 itself, in the present invention, these isoflavones or glycosides thereof may be used as an active ingredient.

  The PDE3 inhibitor of the present invention contains the above-described processed kuzuka or isoflavone contained in kuzuka as an active ingredient for phosphodiesterase 3 inhibition (PDE3 inhibition).

  In the PDE3 inhibitor of the present invention, in addition to the above active ingredients, there is no particular limitation on blending other materials, and if necessary, a pharmaceutically acceptable base or carrier is added, Depending on the formulation method, for example, it can be used as an oral preparation in the form of tablets, granules, capsules, pills, powders, liquids, powders, jelly-forms, rod-forms and the like. Moreover, it can be used as a skin external preparation in the form of an ointment, cream, gel, lotion or the like. Furthermore, you may make it drink after wrapping a katsuhana processed material in a tea bag shape, and leaching the component in hot water. In addition, the dosage form may be dissolved in water, hot water, milk or the like.

In the PDE3 inhibitor of the present invention, in addition to the above active ingredients, food ingredients can also be blended as other ingredients. Examples of food ingredients include royal jelly, propolis, vitamins (A, C, D, E, K, folic acid, pantothenic acid, biotin, derivatives thereof, etc.), minerals (iron, magnesium, calcium, zinc, selenium, etc.) , Α-lipoic acid, lecithin, polyphenols (flavonoids, derivatives thereof, etc.), carotenoids (lycopene, astaxanthin, zeaxanthin, lutein, etc.), xanthine derivatives (eg caffeine), fatty acids, proteins (collagen, elastin, etc.), mucos Polysaccharides (such as hyaluronic acid), amino sugars (such as glucosamine, acetylglucosamine, galactosamine, acetylgalactosamine, neuraminic acid, acetylneuraminic acid, hexosamine, salts thereof), oligosaccharides (isomalto-oligosaccharides, cyclic oligosaccharides, etc.) Contains phospholipids and their derivatives (phosphatidylcholine, sphingomyelin, ceramide, etc.), sulfur-containing compounds (eg, alliin, sepaene, taurine, glutathione, methylsulfonylmethane), sugar alcohols, lignans (eg, sesamin) Animal and plant extracts, root vegetables (turmeric, ginger, etc.) .

  In the PDE3 inhibitor of the present invention, the content of isoflavones contained in the processed kuzuka product or kuzuka is in various forms, taking into account the relationship between the amount used and the effective dose. However, in the case of a solid form, it is usually 0.01 to 100% by mass in terms of the dried product of the Kuzuka process product, and isoflavones. It is 0.000001-100 mass% in quantity conversion. Moreover, in the case of a liquid or jelly-like form, it is 0.001 to 100% by mass in terms of dry matter of the Kuzuka processed product, and 0.0000001 to 100% by mass in terms of isoflavone.

  The administration form of the PDE3 inhibitor of the present invention may be taken orally because it acts from inside the body, or may be applied to the skin. Further, it may be applied to the respiratory system by aspiration, and its administration form is not particularly limited.

  The dose of the PDE3 inhibitor of the present invention is not particularly limited, but typically when orally ingested, it is approximately 0.001 to 100 g per day for adults in terms of dry matter of the Kuzuka treatment product. is there. Moreover, it is about 0.00001-50 g per day in terms of isoflavone.

  Since the PDE3 inhibitor of the present invention has the same action as the PDE3 inhibitor shown in the above-mentioned prior art, for example, an agent for treating acute heart failure, ulcer based on chronic arterial occlusion, pain and cold sensation, etc. It is expected to be used as a drug for improving various ischemic symptoms, a therapeutic agent for non-insulin-dependent diabetes, an anti-rheumatic drug, an anti-arthritic drug, an anti-cancer drug and the like.

  Moreover, since the PDE3 inhibitor of this invention has high inhibitory activity with respect to phosphodiesterase 3B (PDE3B) which is an isozyme of PDE3 as shown in the Example mentioned later, it can be used especially suitably as an inhibitor of phosphodiesterase 3B.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

<Preparation Example 1> (Kuzuhana extract)
The flower part of the kuerzu (Puerariathomsonii) was extracted with hot water to obtain an extract, which was dried to obtain a powdery kuzu flower extract. This extract contains 10% by mass of the isoflavones identified in Test Example 1 below, about 38% by mass of other carbohydrates, about 19% by mass of protein, about 14% by mass of ash, and about 6% by mass of water. And a composition containing about 5% by mass of lipid.

<Preparation Example 2> (isoflavone-containing composition)
The Kuzuhana extract was separated by column chromatography using aqueous methanol and dried to obtain a powdery isoflavone-containing composition. In this composition, the isoflavones content in the composition was increased to about 61% by mass.

<Test Example 1> (Identification / quantification of isoflavone)
According to a conventional method, isoflavones contained in the kuzuhana extract and the isoflavone-containing composition were identified and quantified by an HPLC method using each isoflavone standard as a concentration standard.

  As a result, Kuthana has tectigenin (Tectorigenin), tectorigenin 7-O-xylosylglucoside (Tectorigenin 7-O-xylosylglucoside), 6-hydroxygenistein, 6-hydroxygenistein-6,7-di-O-glucoside as isoflavones, It was confirmed that tectoridin and the like were included. In addition, the composition ratio of isoflavones was hardly different between the kuzu flower extract extracted with hot water and the isoflavone-containing composition separated by column chromatography using water-containing methanol.

<Test Example 2> (PDE3B inhibition experiment)
About the said Kuzuka extract and isoflavone containing composition, PDE3B inhibition experiment using them was conducted. Moreover, the PDE3B inhibition experiment using these was also performed about the compound shown below.
Cilostazol (PDE3 selective inhibitor)
・ 6-hydroxygenistein (Kuzuhana isoflavone)
・ IBMX (3-isobutyl-1-methylxanthine) (PDE inhibitor)
・ Quercetin (plant flavonoid; PDE inhibitor)
・ Tectorigenin (Kuzuhana isoflavone)
・ Genistein (soy isoflavone)
・ Daidzein (soy isoflavone)
·caffeine

  The PDE3B inhibition experiment was performed using a kit “PDE3B Assay Kit” manufactured by PBS Bioscience. First, a diluted sample was prepared by serially diluting the 10 mM stock sample (2 mM stock sample in the case of Kuzuka extract) of each sample using the buffer included in the kit. Specifically, in the case of Kuzuka extract, isoflavone-containing composition, cilostazol, and 6-hydroxygenistein, a dilution series of 0.5, 1, 5, 10, 25, 50, 100, 250 μM was prepared, and IBMX, quercetin In the case of 1, 10, 25, 50, 100, 200, 300, 600μM dilution series is prepared, and in the case of tectorigenin and genistein, 10, 100, 250, 500, 1000, 2000, 3000, 6000μM In the case of daidzein, the dilution series of 500, 1000, 2000, 3000, 4000, 5000, 6000 μM was created, and in the case of caffeine, 10, 33, 66, 100, 333, Dilution series of 666, 1000, 3333, 6666 μM were prepared.

  Next, 20 μL of 4 pg / μL phosphodiesterase 3B (PDE3B) as the enzyme solution, 25 μL of 200 nM FAM-Cyclic-3 ′, 5′-AMP (fluorescently labeled cAMP) as the substrate solution, 5 μL of each of the above dilution series, Were mixed in each well of a 96-well microplate and incubated at room temperature for 1 hour. Thereafter, 100 μL of a 100-fold diluted binding reagent (“Binding Agent”) attached to the kit was added and incubated for 1 hour with shaking.

  Using a fluorescence polarization degree measuring device ("Flex Station 3" manufactured by Molecular Devices), the sample in each well after the above reaction was polarized with an excitation wavelength of 485 nm, and the resulting fluorescence with a fluorescence wavelength of 538 nm was measured. The degree of polarization P shown in the following formula (1) was determined.

  Here, the enzymatic degradation product of FAM-Cyclic-3 ', 5'-AMP (fluorescently labeled cAMP) that has been enzymatically degraded by PDE3B binds to the binding reagent, and the degree of polarization of fluorescence increases. On the other hand, if the activity of PDE3B is lowered by the inhibitor, the amount of the enzymatic degradation product is also reduced. For this reason, the amount of enzymatic degradation products bound to the binding reagent is also reduced, and as a result, the degree of polarization of fluorescence is reduced as compared with the case where no inhibitor is added. Therefore, in this kit, the degree of polarization can be used as an indicator of reaction progress, and the decrease can be used as an indicator of reaction inhibition. Thus, the degree of polarization was plotted on the vertical axis, the inhibitor concentration was plotted on the horizontal axis, and the results of the dilution series for each test substance were plotted, and the IC50 value was determined according to a conventional method. The results are summarized in Table 1.

  As shown in Table 1, the Kuzuka extract obtained by hot water extraction from the flower portion of Kuzu showed higher PDE3B inhibitory activity than cilostazol, which is a selective inhibitor of PDE3. Moreover, the isoflavone containing composition which extracted the isoflavone fraction from the Kuzuhana extract also showed PDE3B inhibitory activity comparable to cilostazol. The inhibitory activity was higher than IBMX (3-isobutyl-1-methylxanthine) and quercetin, which are known as general PDE inhibitors. Furthermore, it was revealed that 6-hydroxygenistein and Tectorigenin, isoflavones contained in Kuzuhana, exhibit PDE3B inhibitory activity by themselves. In particular, the PDE3B inhibitory activity of 6-hydroxygenistein was remarkably high.

  On the other hand, Genistein and Daidzein, which are soybean isoflavones, could not detect PDE3B inhibitory activity. Therefore, it was suggested that the isoflavone peculiar to Kuzuka contributed to the PDE3B inhibitory activity by the said Kuzuka extract or an isoflavone containing composition.

<Production Example 1>
A powdered beverage containing the Kuzuka extract obtained in Preparation Example 1 was produced with the formulation shown in Table 2 below.

<Production Example 2>
A powdered beverage containing the isoflavone-containing composition obtained in Preparation Example 2 was produced with the formulation shown in Table 3 below.

<Production Example 3>
Soft capsules containing the Kuzuka extract obtained in Preparation Example 1 were produced with the formulation shown in Table 4 below.

<Production Example 4>
Soft capsules containing the isoflavone-containing composition obtained in Preparation Example 2 were produced with the formulation shown in Table 5 below.

<Production Example 5>
Tablets containing the Kuzuka extract obtained in Preparation Example 1 were produced with the formulation shown in Table 6 below.

<Production Example 6>
Tablets containing the isoflavone-containing composition obtained in Preparation Example 2 were produced with the formulation shown in Table 7 below.

<Production Example 7>
A drink containing the kuzu flower extract obtained in Preparation Example 1 was produced with the formulation shown in Table 8 below.

<Production Example 8>
A drink containing the isoflavone-containing composition obtained in Preparation Example 2 was produced with the formulation shown in Table 9 below.

<Production Example 9>
A powdered beverage containing 6-hydroxygenistein (Katsuhana isoflavone; aglycon) was prepared according to the formulation shown in Table 10 below. In addition, as 6-hydroxygenistein, what was obtained by separating and purifying from Kuzuka extract using column chromatography was used.

<Production Example 10>
Soft capsules containing Tectorigenin (Kuzuka Isoflavone; Aglycon) were prepared with the formulation shown in Table 11 below. In addition, as tectorigenin, what was obtained by separating and purifying from Kuzuka extract using column chromatography was used.

<Production Example 11>
Tablets containing 6-hydroxygenistein-6,7-di-O-glucoside (Kuzuka isoflavone; glycoside) were produced with the formulation shown in Table 12 below. As 6-hydroxygenistein-6,7-di-O-glucoside, a product obtained by separating and purifying from Kuzuka extract using column chromatography was used.

<Production Example 12>
A drink containing Tectorigenin 7-O-xylosylglucoside (Katsuka Isoflavone; glycoside) was prepared with the formulation shown in Table 13 below. In addition, as tectorigenin 7-O-xylosyl glucoside, what was obtained by separating and purifying from Kuzuka extract using column chromatography was used.

Claims (4)

Containing Kuzuhana treated as an active ingredient, phosphodiesterase 3 inhibitors, characterized in that for the treatment or prevention of acute cardiac insufficiency.   The phosphodiesterase 3 inhibitor according to claim 1, wherein the processed kuzuhana product comprises an isoflavone-containing composition obtained from kuzuka.   The phosphodiesterase 3 inhibitor according to claim 1 or 2, which is used for inhibiting phosphodiesterase 3B. The processed kuzuhana product has the following formula (1):

And / or the following formula (2):

The phosphodiesterase 3 inhibitor as described in any one of Claims 1-3 which contains the compound represented by these, or its glycoside.