PDE4 inhibitors

Abstract

To provide a PDE4 inhibitor including propolis.SOLUTION: A PDE4 inhibitor includes propolis produced in the State of Bahia, Brazil.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to PDE (phosphodiesterase) 4 inhibitors, and more particularly to PDE4 inhibitors containing propolis. [Background technology]

[0002] Phosphodiesterase (PDE) is a general term for enzymes that hydrolyze phosphate diesters to phosphate monoesters, and there are 11 types of PDEs (PDE1 to PDE11).

[0003] PDEs degrade and inactivate substances (second messengers) that play a role in intracellular signal transmission. Preventing the degradation of these second messengers is thought to be effective in treating various diseases, and PDE inhibitors (drugs) have been developed to suppress PDE. Among these, PDE4 degrades cAMP (cyclic adenosine monophosphate) to produce AMP. PDE4 is present in many inflammatory cells, immune cells, and the central nervous system, and is involved in the production of substances that cause inflammation.

[0004] High levels of PDE4 can lead to immune imbalances and inflammation, so PDE4 inhibitors have been shown to restore balance and suppress inflammation by suppressing PDE4 activity. Therefore, PDE4 inhibitors are being studied for the treatment of chronic obstructive pulmonary disease (COPD) and rheumatoid arthritis. PDE4 inhibitors are currently being used to treat psoriasis and atopic dermatitis.

[0005] Furthermore, PDE4 inhibitors are known to have cognitive enhancing and neuroprotective effects, and are being studied for the treatment of central nervous system diseases such as depression, memory impairment, schizophrenia, Parkinson's disease, and Alzheimer's disease.

[0006] Compounds having PDE4 inhibitory activity have been reported in, for example, Non-Patent Document 1. Non-Patent Document 1 reports that 3-O-methylquercetin selectively and competitively inhibits PDE4, and that the inhibitory effect is not strong.

[0007] Propolis is a glue-like substance made by mixing tree sap, plant buds, and exudates collected by honeybees with beeswax, and is known to have antibacterial and antioxidant properties. Propolis, which contains natural antibacterial substances, is used in health foods and other products.

[0008] The components of propolis are influenced by the type and composition of the plant from which it is made, and it is known that the types and amounts of active ingredients contained in propolis, as well as their physiological activities, vary greatly depending on the country or region where it is collected.

[0009] In recent years, research into the components of propolis from different origins has been progressing. For example, the main components of Brazilian propolis, which originates from Baccharis doracunclifolia, are p-coumaric acid, artepillin C, drupanin, baccharin, and other cinnamic acid derivatives. On the other hand, the main components of propolis from China, Europe, and Australia, which originates from poplar, are flavonoids such as chrysin and galangin. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Planta Med. 2003 Apr;69(4):310-5. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention aims to provide a PDE4 inhibitor containing propolis. [Means for solving the problem]

[0012] Since propolis is made from plants harvested by honeybees, its components vary depending on the place of origin, and therefore its effects are expected to vary depending on the place of origin. Therefore, the present inventors conducted extensive research to achieve the above-mentioned object, and as a result, they discovered that propolis from the State of Bahia, Federative Republic of Brazil (hereinafter sometimes abbreviated as Bahia propolis) contains three components: 3-O-methylquercetin, 3,6-dimethoxyapigenin, and 6-methoxyapigenin, and that a synergistic effect in PDE4 inhibitory activity can be achieved by combining the two components, 3-O-methylquercetin and 3,6-dimethoxyapigenin, or the three components, 3-O-methylquercetin, 3,6-dimethoxyapigenin, and 6-methoxyapigenin.

[0013] The present invention was completed based on these findings and through further investigation, and provides the following PDE4 inhibitors.

[0014] Item 1. PDE4 inhibitor containing propolis from the state of Bahia, Federative Republic of Brazil. Item 2. The PDE4 inhibitor according to Item 1, wherein the propolis contains at least one member selected from the group consisting of 3-O-methylquercetin or a salt thereof, and 3,6-dimethoxyapigenin or a salt thereof, and 6-methoxyapigenin or a salt thereof. Item 3. The PDE4 inhibitor according to Item 1 or 2, which is used for improving or preventing at least one condition selected from the group consisting of dementia, Parkinson's disease, inflammatory diseases, psychiatric disorders, and tumor diseases. Item 4. The PDE4 inhibitor according to Item 3, wherein the inflammatory disease is at least one selected from the group consisting of respiratory inflammatory diseases, skin inflammatory diseases, interstitial pneumonia, and rheumatism. Item 5. The PDE4 inhibitor according to Item 3, wherein the psychiatric disorder is at least one selected from the group consisting of depression, memory impairment, and schizophrenia. Item 6. The PDE4 inhibitor according to Item 3, wherein the tumor disease is colorectal cancer and / or acute lymphocytic leukemia. Item 7. The PDE4 inhibitor according to Item 4, wherein the respiratory inflammatory disease is asthma and / or chronic obstructive pulmonary disease (COPD). Item 8. The PDE4 inhibitor according to Item 4, wherein the inflammatory skin disease is psoriasis and / or atopic dermatitis. Item 9. The PDE4 inhibitor according to any one of Items 1 to 8, which is a cosmetic, a food or drink, a pharmaceutical product, or a quasi-drug. [Effects of the Invention]

[0015] Propolis from the state of Bahia has excellent PDE4 inhibitory activity and is therefore useful as an active ingredient in PDE4 inhibitors.

[0016] Furthermore, propolis is a naturally derived ingredient and is therefore highly safe. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a graph showing the PDE4 inhibition rate (%) at an equivalent of 50 μg / mL of propolis from Bahia. DETAILED DESCRIPTION OF THE INVENTION

[0018] Hereinafter, embodiments of the present invention will be described in detail.

[0019] In this specification, the term "comprise" encompasses the meanings of "essentially consist of" and "consist of only."

[0020] The PDE4 inhibitor of the present invention is characterized by containing propolis from the state of Bahia.

[0021] Propolis is a resinous or waxy substance that forms the walls of beehives, and is made by mixing plant shoots or resins collected by bees with their saliva. In the present invention, propolis produced in the state of Bahia is used. In addition to propolis produced in the state of Bahia, propolis from other producing regions, such as China, Europe, Oceania, and America, may also be used in combination.

[0022] The propolis produced in Bahia is not particularly limited as long as it is collected in Bahia. Among them, propolis produced in Bahia containing at least one selected from the group consisting of 3-O-methylquercetin or a salt thereof, 3,6-dimethoxyapigenin or a salt thereof, and 6-methoxyapigenin or a salt thereof is preferred, and propolis containing 3-O-methylquercetin or a salt thereof, 3,6-dimethoxyapigenin or a salt thereof, and 6-methoxyapigenin or a salt thereof is more preferred. 3-O-methylquercetin, 3,6-dimethoxyapigenin, and 6-methoxyapigenin are compounds represented by the following structural formulas, respectively.

[0023] [ka]

[0024] Salts of 3-O-methylquercetin, 3,6-dimethoxyapigenin, and 6-methoxyapigenin are not particularly limited and include, for example, inorganic salts with sodium, potassium, magnesium, calcium, aluminum, etc.; salts with organic bases such as methylamine, ethylamine, ethanolamine, etc.; salts with basic amino acids such as lysine, ornithine, arginine, etc.; and ammonium salts. The salts may be acid addition salts, and specific examples of such salts include acid addition salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, and ethanesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid. Furthermore, 3-O-methylquercetin, 3,6-dimethoxyapigenin, and 6-methoxyapigenin and salts thereof in the present invention also include hydrates, solvates, crystalline polymorphs, and the like.

[0025] The propolis of the present invention includes, for example, raw propolis, its pulverized product, supercritical extract, and extract with water and / or hydrophilic organic solvent. Among these, extract with water and / or hydrophilic organic solvent and supercritical extract are preferred, with extract with water and / or hydrophilic organic solvent being more preferred. Extract with water and / or hydrophilic organic solvent is preferred because it extracts propolis components efficiently and in a balanced manner in a short time and does not require large-scale equipment for scale-up. The propolis extract preferably contains at least one selected from the group consisting of 3-O-methylquercetin or a salt thereof, 3,6-dimethoxyapigenin or a salt thereof, and 6-methoxyapigenin or a salt thereof, and more preferably contains 3-O-methylquercetin or a salt thereof, 3,6-dimethoxyapigenin or a salt thereof, and 6-methoxyapigenin or a salt thereof.

[0026] Examples of hydrophilic organic solvents include lower alcohols such as ethanol, methanol, and propanol, ketones (acetone, methyl ethyl ketone, methyl butyl ketone, etc.), esters (methyl acetate, ethyl acetate, etc.), glycols (ethylene glycol, propylene glycol, etc.), glycerin, tetrahydrofuran, acetonitrile, acetic acid, propionic acid, acetamide, dimethylformamide, and dimethyl sulfoxide, with lower alcohols being preferred and ethanol being particularly preferred.

[0027] These extraction solvents can be used alone or in any combination of two or more to form a mixed solution. Water, lower alcohol (preferably ethanol), or a mixture thereof (aqueous alcohol, preferably aqueous ethanol) is preferred. When aqueous alcohol (preferably aqueous ethanol) is used as the extraction solvent, the alcohol concentration is not limited, but is preferably 50 to 100% by volume, and more preferably 70 to 100% by volume.

[0028] For extraction, raw propolis, raw propolis washed with alcohol, etc. can be used, and the extraction efficiency can be improved by using raw propolis that has been cut or crushed to an appropriate size. The volume of the extraction solvent used is, for example, 1 to 20 times, preferably 2 to 10 times the mass of the raw propolis.

[0029] Extraction can be carried out by adding propolis to a solvent and heating it at, for example, 0°C to the boiling point of the solvent (usually a temperature of 100°C or less), preferably 2 to 40°C. Extraction can be carried out at the above temperature for a predetermined time (for example, 1 hour or more) with stirring or by leaving the mixture to stand. After extraction, an extract can be obtained by performing a process such as filtration to remove insoluble matter.

[0030] The solvent extract thus obtained can be used as is, or, if necessary, can be further purified to increase the content of active ingredients by ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, or a combination thereof.

[0031] The solvent extract can also be concentrated and dried by freeze-drying, spray-drying, etc. Concentration, freeze-drying, and spray-drying can be carried out according to conventional methods. The propolis extract of the present invention also includes powders obtained by powdering such extracts and granules obtained by granulating the powders.

[0032] The PDE4 inhibitor of the present invention may contain other known ingredients as appropriate, as long as they do not impair the effects of the present invention.

[0033] The content of propolis in the PDE4 inhibitor of the present invention is not particularly limited as long as it is within a range in which the effects of the present invention can be obtained, and can be appropriately adjusted depending on the final form, etc., and can be, for example, 0.00001% by mass or more, 0.0001% by mass or more, 0.001% by mass or more, 0.01% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, It may be 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, or 90% by mass or more, and 100% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or more. Below, it may be 55% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, or 0.5% by mass or less.

[0034] Furthermore, propolis is preferably applied periodically to inhibit PDE4, particularly preferably once a day or in divided doses. Although not particularly limited, propolis can be used at a daily dose of 1 μg to 1,000 mg, preferably 9 μg to 300 mg, and more preferably 18 μg to 100 mg, of solid content for a 60 kg adult. The dose can be appropriately set within the above range depending on factors such as the recipient's health condition, the administration method, and the combination with other agents.

[0035] The PDE4 inhibitor of the present invention can be used as cosmetics, foods and beverages (particularly foods and beverages intended for health, health maintenance, or promotion, etc. (e.g., health foods, functional foods, nutritional compositions, dietary supplements, supplements, health foods, foods for specified health uses, foods with nutrient functions, or foods with functional claims)), quasi-drugs, pharmaceuticals, etc. The PDE4 inhibitor of the present invention also encompasses the meaning of additives that impart PDE4 inhibitory activity.

[0036] In addition to the propolis, the above cosmetics may contain ingredients commonly used in cosmetics, such as disinfectants, preservatives, surfactants, alcohols, aqueous components, water, colorants, pH adjusters, solubilizers, abrasives, foaming agents, enzymes, flavorings, chelating agents, excipients, thickeners, bases, emulsifiers, solvents, stabilizers, oils, cleaning agents (lactic acid bacteria), etc., as needed.

[0037] Cosmetics include any cosmetic product that is applied to the skin, mucous membranes, body hair, head hair, scalp, nails, teeth, facial skin, lips, etc. of animals (including humans).

[0038] Cosmetics can take a wide range of formulations, including aqueous solutions, solubilized solutions, emulsions, powders, oils, gels, ointments, aerosols, two-layer water-oil systems, and three-layer water-oil-powder systems.

[0039] The use of the cosmetics is also arbitrary, and examples thereof include basic cosmetics such as face wash, lotion, emulsion, cream, gel, essence, beauty serum, pack, and mask; makeup cosmetics such as foundation, lipstick, blush, eye shadow, eyeliner, and mascara; nail cosmetics such as nail polish, base coat, top coat, and nail polish remover; and other examples include face wash, (paste or liquid) dentifrice, mouthwash, massage agent, cleansing agent, aftershave lotion, preshave lotion, shaving cream, body soap, soap, shampoo, rinse, hair treatment, hair styling product, hair tonic, hair growth agent, antiperspirant, and bath additives.

[0040] The propolis can be used as is in the above-mentioned foods and beverages, or, if necessary, can be blended with minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavorings, stabilizers, gelling agents, preservatives, sustained-release regulators, surfactants, solubilizers, humectants, etc.

[0041] Food and beverage products include any food and beverage product that can be consumed by animals (including humans). The types of food and beverage products are not particularly limited, and examples include dairy products; fermented foods (yogurt, etc.); beverages (soft drinks such as coffee, juice, and tea drinks, dairy drinks, lactic acid bacteria drinks, drinks containing lactic acid bacteria, yogurt drinks, carbonated drinks, sake, Western liquor, and alcoholic beverages such as fruit wine); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western confectionery products (chocolate, donuts, pies, cream puffs, gum, gummy candies, jellies, candies, cookies, cakes, puddings, etc.); Japanese confectionery products (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, popsicles, sorbets, etc.); foods (curry, beef bowls, rice porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); and seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

[0042] The method for producing the food or drink is not particularly limited, and any known method can be used as appropriate.

[0043] The dosage unit form when used as a supplement is not particularly limited and can be selected as appropriate, and examples include chewable tablets, troches, capsules, granules, liquids, powders, syrups, pastes, drinks, gummies, etc.

[0044] The propolis may be used alone in the above-mentioned medicines, or may be mixed with other medicinal ingredients such as vitamins and herbal medicines listed in the Japanese Pharmacopoeia.

[0045] When the PDE4 inhibitor of the present invention is prepared as a pharmaceutical, the propolis can be prepared together with pharmaceutical acceptable ingredients into the form of tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (dispersed medicines), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, injections (including the case where the propolis is mixed with distilled water or infusions such as amino acid infusions or electrolyte infusions at the time of use to prepare a liquid), etc., to form a pharmaceutical preparation.

[0046] Pharmaceuticals may be administered locally or systemically. There are no particular limitations on the administration method, and they may be administered orally or parenterally. Parenteral administration routes include subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous or intraarterial administration, and transdermal administration.

[0047] In addition to propolis, the pharmaceutical of the present invention may contain, as needed, pharmaceutically acceptable ingredients such as excipients, binders, disintegrants, lubricants, colorants, suspending agents, thickeners, antioxidants, absorption enhancers, pH adjusters, preservatives, antiseptics, stabilizers, surfactants, sweeteners, flavoring agents, and fragrances.

[0048] The cosmetics and pharmaceuticals of the present invention also include quasi-drugs.

[0049] The PDE4 inhibitor of the present invention as explained above is applicable to mammals including humans (preferably humans).

[0050] The propolis produced in Bahia, which is contained as an active ingredient in the PDE4 inhibitor of the present invention, has PDE4 inhibitory activity as shown in the examples described below, and is therefore useful as an active ingredient in PDE4 inhibitors.

[0051] The PDE4 inhibitor of the present invention can be used for the treatment, improvement, and prevention of various abnormalities, disorders, diseases, etc. caused by the presence of large amounts of PDE4. Examples of such abnormalities, disorders, diseases, etc. include dementia (Alzheimer's disease, etc.), Parkinson's disease, respiratory inflammatory diseases (asthma, chronic obstructive pulmonary disease (COPD), etc.), inflammatory skin diseases (psoriasis, atopic dermatitis, etc.), interstitial pneumonia, inflammatory diseases such as rheumatism, psychiatric diseases (depression, memory impairment, schizophrenia, etc.), and tumor diseases (colon cancer, acute lymphocytic leukemia, etc.).

[0052] Furthermore, the PDE4 inhibitor of the present invention is highly safe because it contains naturally occurring components as active ingredients. [Example]

[0053] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0054] Test Example <Identifying sample components> Propolis from Bahia, provided by Yamada Bee Farm, was extracted with aqueous ethanol and analyzed for its components using HPLC and LC-MS. The aqueous ethanol extract was also used to measure the PDE4 inhibitory activity described below.

[0055] As a result, the following compounds were identified as components of propolis produced in Bahia. Standard specimens of the identified components were purchased and the following activity tests were carried out. 3-O-methylquercetin: C 16 H 12 O7 3,6-Dimethoxyapigenin: C 17 H14 O7 · 6-Methoxyapigenin: C 16 H 12 O6 Also, the quantitative values of each component in the propolis produced in the state of Bavaria were as follows. · 3-O-Methylquercetin: 1.6% by mass · 3,6-Dimethoxyapigenin: 3.2% by mass · 6-Methoxyapigenin: 5.8% by mass

[0056] <Measurement of PDE4 inhibitory activity> The PDE4 inhibitory activity of various materials was evaluated by fluorescence polarization using a PDE4B Assay Kit (BPS Bioscience). The procedure was carried out according to the instruction manual attached to the kit, but the volume was customized for a 384-well plate for evaluation. Rolipram was used as a positive control (IC 50 2 μg / mL).

[0057] The PDE4 inhibitory activity of the propolis produced in the state of Bavaria (BPP) and the PDE4 inhibitory activity of each component alone or in combination are shown in Table 1 and Figure 1 (comparison at 50 μg / mL equivalent of BPP). From the results in Table 1, the main active component was 3-O-methylquercetin.

[0058]

Table 1

[0059] · Propolis produced in the state of Bavaria: BPP · 3-O-Methylquercetin: 3O · 3,6-Dimethoxyapigenin: 3,6DI · 6-Methoxyapigenin: 6M

Claims

1. A PDE4 inhibitor comprising propolis produced in the state of Bahia, Federative Republic of Brazil, which contains 3-O-methylquercetin or a salt thereof, and 3,6-dimethoxyapigenin or a salt thereof.

2. The PDE4 inhibitor according to claim 1, wherein the propolis further contains 6-methoxyapigenin or a salt thereof.

3. The PDE4 inhibitor according to claim 1 or 2, which is used for improving or preventing at least one disease selected from the group consisting of dementia, Parkinson's disease, inflammatory diseases, psychiatric diseases, and tumor diseases.

4. The PDE4 inhibitor according to claim 3, wherein the inflammatory disease is at least one selected from the group consisting of respiratory inflammatory diseases, skin inflammatory diseases, interstitial pneumonia, and rheumatism.

5. The PDE4 inhibitor according to claim 3, wherein the psychiatric disorder is at least one selected from the group consisting of depression, memory impairment, and schizophrenia.

6. The PDE4 inhibitor according to claim 3, wherein the tumor disease is colon cancer and / or acute lymphocytic leukemia.

7. The PDE4 inhibitor according to claim 4, wherein the respiratory inflammatory disease is asthma and / or chronic obstructive pulmonary disease (COPD).

8. The PDE4 inhibitor according to claim 4, wherein the inflammatory skin disease is psoriasis and / or atopic dermatitis.

9. The PDE4 inhibitor according to claim 1 or 2, which is a cosmetic, a food or drink, a pharmaceutical or a quasi-drug.

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