JP2015044748A - PDE3 inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical, a quasi drug, a food product, feed and a material which can be blended therewith, having excellent PDE3 inhibitory action and a high level of safety.SOLUTION: This invention comprises (A) spearmint, peppermint and extracts thereof, and (B) one or more selected from the group consisting of lysophospholipid as an active ingredient.

Description

  The present invention relates to a PDE3 (phosphodiesterase 3) inhibitor.

  3 ', 5'-cyclic adenosine monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP) act as second messengers when cells receive hormone and neurotransmitter signals It is known that when the intracellular concentration increases, a signal is transmitted downstream and the cell responds. For example, when prostaglandins and adrenaline act on cell surface receptors, adenylate cyclase is activated, increasing intracellular cAMP concentration and causing various cellular responses such as smooth muscle relaxation.

  Enzymes that hydrolyze cyclic phosphate diesters typified by cAMP and cGMP are called phosphodiesterases (PDEs). When the activity of PDE increases, the concentration of intracellular cAMP and cGMP decreases. Accordingly, since the activity of PDE greatly affects the concentration of intracellular cAMP and cGMP, PDE plays an extremely important role in the activation of the intracellular signal transduction system and the function of cells and tissues.

  There are about 11 types of PDEs. Among them, PDE3 is also called cGMP-inhibited PDE because it has higher affinity for cAMP than cGMP, and it is known to reduce the intracellular concentration of cAMP. It has been. PDE3 is expressed mainly in the heart, vascular smooth muscle, ovary, kidney, adipose tissue, hepatocyte, bronchial smooth muscle and the like.

  Inhibiting the activity of PDE3 present in the lungs, heart and peripheral blood vessels prevents the decrease in cAMP concentration, leading to contraction and relaxation in the lungs, heart and peripheral blood vessels, increasing myocardial strength, and inhibiting platelet aggregation . Therefore, the PDE3 inhibitor is effective in preventing and improving heart failure, chronic arterial occlusion, and thrombosis.

  As such PDE3 inhibitors, for example, amrinone and milrinone are known, and preemptive therapy of these drugs reduces the risk of low cardiac output and low oxygen supply after cardiac surgery. be able to. However, there is a demand for materials derived from natural products that are less active and easy to use.

On the other hand, spearmint is a perennial plant belonging to the family Labiatae, and it is reported that the spearmint or an extract thereof has an anti-obesity effect (Patent Documents 1 to 4).
Peppermint is a perennial plant belonging to the genus Labiatae, and the peppermint or extract thereof has an anti-obesity effect (Patent Documents 1, 3, and 4), and is absorbed from the skin to promote blood circulation based on a mental enhancement action. (Patent Document 5) has been reported to contribute to the decomposition of.

  Moreover, lysophospholipid is widely used as a stable emulsifier having high hydrophilicity, and it has been reported that the lysophospholipid has an anti-obesity effect (Patent Document 6).

  However, the relationship between spearmint, peppermint and lysophospholipid and PDE3 inhibition is not known.

JP 2010-106001A JP 2007-22917 A JP 2006-36788 A JP 2006-16312 A JP 2001-354517 A JP 2008-74794 A

  The present invention relates to providing pharmaceuticals, quasi-drugs, foods, feeds, and materials that can be blended in them, having a PDE3 inhibitory action and high safety.

  As a result of intensive studies to solve the above problems, the present inventors have found that spearmint, peppermint and lysophospholipid have a PDE3 inhibitory action.

That is, the present invention relates to the following.
(1) A PDE3 inhibitor comprising as an active ingredient at least one selected from the group consisting of (A) spearmint, peppermint and their extracts, and (B) lysophospholipid.
(2) A circulatory function improver comprising as an active ingredient at least one selected from the group consisting of (A) spearmint, peppermint and extracts thereof, and (B) lysophospholipid.
(3) A non-therapeutic PDE3 inhibition method in which one or more selected from the group consisting of (A) spearmint, peppermint and their extracts, and (B) lysophospholipid are administered or ingested to humans or animals.
(4) A non-therapeutic method for improving circulatory function, wherein one or more selected from the group consisting of (A) spearmint, peppermint and their extracts, and (B) lysophospholipid are administered or ingested to humans or animals.

  The PDE3 inhibitor of the present invention has an excellent PDE3 inhibitor action and is highly safe even when ingested for a long period of time. Therefore, a pharmaceutical, a quasi-drug, a food that can exert an effect of improving the circulatory function It is useful as a feed or a material or a preparation for blending them.

In the present invention, “PDE3 inhibition” refers to reducing or eliminating the enzyme activity of PDE3. Specifically, it acts on the active center of PDE3 to prevent reaction initiation and to form a covalent bond with a specific moiety. , Inhibition of reaction catalysis by PDE3, inhibition of reaction progress by binding to PDE3 molecule alone, enzyme-substrate complex or both, and competitive initiation at the same site as substrate to initiate reaction Competitive inhibition that prevents, non-competitive inhibition of the enzyme or enzyme-substrate complex that binds to a different site from the substrate and prevents the reaction from proceeding. It also includes that the enzyme activity of PDE3 is suppressed by acutely or chronically reducing the protein expression level of the PDE3 gene.
Although it is known that there are two types of PDE3, 3A and 3B, both are included in the present invention.

  “Improvement of circulatory function” refers to relaxation and expansion of vascular smooth muscles in the lungs, heart and periphery, suppression of platelet aggregation, increase in myocardial strength, heart failure, failure of vascular function, chronic arterial occlusion, thrombus formation or thrombus This refers to the prevention and improvement of symptoms.

(A) Spearmint, peppermint In the present invention, spearmint refers to Mentha spicata belonging to the genus Labiatae, and peppermint refers to Mentha x piperita belonging to the Labiatae genus.

  Such a plant can be any arbitrary part, for example, whole plant, leaf (leaf blade, petiole, etc.), bark, woody part, branch, fruit, seed, flower (petal, ovary, etc.), root, rhizome, etc. , Or combinations thereof, but spearmint or peppermint is preferably whole plant.

  Such plants can be used by crushing, pulverizing or exploiting them as they are, or they can be used after drying or pulverizing those treated from now on, or using them as extracted extracts. Is preferred.

  The plant may be subjected to an extraction process as it is, or may be subjected to an extraction process after being pulverized, cut or dried to obtain an extract.

  As the above extract, commercially available ones may be used, or various solvent extracts obtained by a conventional method, or diluted solutions thereof, concentrated solutions thereof, dried powders thereof, pastes or activated carbon treatments thereof. There may be. As an example, an extract can be obtained by extraction means, such as extracting the said plant under fixed temperature (low temperature, normal temperature, or warming), or extracting using extraction tools, such as a Soxhlet extractor.

Known extraction methods include, for example, solid-liquid extraction, liquid-liquid extraction, immersion, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, stirring, and the like. In order to shorten the extraction time, solid-liquid extraction with stirring is desirable. Examples of suitable conditions for this solid-liquid extraction include stirring at 10 to 100 ° C. and 100 to 400 rpm / min for 1 to 30 minutes. As a suitable example of immersion, the immersion for 1 hour-14 days is mentioned at 10-50 degreeC. Moreover, when shortening extraction time, solid-liquid extraction with stirring is desirable.
In order to prevent oxidation of the extract, a means for extraction under a so-called non-oxidizing atmosphere while removing dissolved oxygen by bubbling degassing or inert gas such as nitrogen gas may be used in combination.

  As the solvent for extraction, either a polar solvent or a nonpolar solvent can be used. Examples of the extraction solvent include water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate. Linear or cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as squalene, hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as benzene and toluene; dichloromethane And halogenated hydrocarbons such as chloroform, dichloroethane and carbon tetrachloride; pyridines; carbon dioxide, supercritical carbon dioxide; organic solvents such as fats, waxes and other oils; and mixtures thereof. These solvents can be used alone or in combination as a mixture.

  Among these solvents, one or more selected from water, alcohols (more preferably having 1 to 5 carbon atoms), ketones, and hydrocarbons are preferable. Among these, water, ethanol, a water-ethanol mixed solution, acetone, and hexane are more preferable. When using a water-ethanol mixed solution, the ethanol concentration (V / V) in the mixed solution is preferably 0.01% by volume, more preferably 20% by volume or more, still more preferably 40% by volume or more, preferably Is less than 100% by volume, more preferably 99.5% by volume or less. Moreover, 0.01 volume% or more and less than 100 volume% are preferable, 20 volume% or more and less than 100 volume% are more preferable, and 40-99.5 volume% is still more preferable.

  As a usage-amount of a solvent, 1-50 mass parts is preferable with respect to 1 mass part of the said plants (dry mass conversion), and 2-30 mass parts is more preferable. As extraction temperature, 0-100 degreeC is preferable, 4-85 degreeC is more preferable, and 4-60 degreeC is still more preferable. The extraction time is preferably 1 minute to 150 days, more preferably 5 minutes to 50 days, and even more preferably 10 minutes to 30 days. In the case of spearmint or peppermint, it is preferably extracted at 10-30 ° C. for 3-10 days.

  Specifically, the spearmint or peppermint extract is used at a temperature of 4 to 85 ° C. (preferably 5 to 15 parts by mass of a solvent of 20 to 80% by volume ethanol-water mixture with respect to 1 part by mass of the plant. 15 to 85 ° C.) for 1 hour to 30 days (preferably 3 hours to 25 days).

  The extract thus obtained may be used as it is, alone or mixed with the extract or fraction, and may be used as a diluted solution diluted with an appropriate solvent, or may be a concentrated extract or a dry powder. A paste prepared may be used. In addition, when lyophilized and used, a solvent usually used for extraction, such as water, ethanol, propylene glycol, butylene glycol, water-ethanol mixed solution, water-propylene glycol mixed solution, water-butylene glycol mixed solution, etc. It can also be used after diluting. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.

  The extract may be a crude product as long as it conforms to food and pharmaceutical acceptable standards and exhibits the effects of the present invention, and the obtained crude product is further purified by a known separation and purification method. These purities may be combined appropriately to remove inactive impurities and increase their purity. Examples of the purification means include organic solvent precipitation, centrifugation, ultrafiltration membrane, high performance liquid chromatograph and column chromatograph, liquid-liquid distribution, gel filtration separation, activated carbon treatment and the like.

(B) Lysophospholipid In the present invention, lysophospholipid is a lyso form in which either one of the α-position or β-position fatty acid residue of glycerophospholipid is removed by hydrolysis. Specifically, lysophosphatidylcholine, lysophosphatidyl Examples include ethanolamine, lysophosphatidylglycerol, lysoplasmanylcholine (lysoPAF), lysophosphatidylinositol, and lysophosphatidic acid.
The lysophospholipid can be obtained by treating the phospholipid with an enzyme such as phospholipase A2. In the present invention, as the phospholipid for obtaining lysophospholipid, for example, soybean, rice, corn, rapeseed, cottonseed, wheat Those extracted from plants such as peanut, sunflower, sunflower, barley, oat, safflower and sesame, and animal tissues such as egg yolk, milk and seafood can be used. Alternatively, phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid may be treated with an enzyme. In this case, the lysophospholipid may be used singly or in combination, or the lyso form and phospholipid may be mixed. Can be used in combination. Moreover, you may use commercial items, such as SLP-white lyso (Tsubaki Oil).

The lysophospholipid can be obtained, for example, by lectin obtained by dispersion or extraction from soybeans, or by purification or enzymatic reaction from squid.
As the solvent used for dispersion or extraction, either a polar solvent or a nonpolar solvent can be used. Examples of the extraction solvent include water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate. Linear or cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as squalene, hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as benzene and toluene; dichloromethane Halogenated hydrocarbons such as chloroform, dichloroethane and carbon tetrachloride; pyridines; carbon dioxide, supercritical carbon dioxide; fats and oils, waxes, and other oils. These solvents can be used alone or in admixture of two or more. Further, an aqueous solution containing an inorganic salt, a buffer solution, or the like may be used.

Among these solvents, one or more selected from water, alcohols (preferably having 1 to 5 carbon atoms), polyhydric alcohols, esters, and hydrocarbons are preferable. Among these, water, ethanol, ethyl acetate, glycerin, acetone, hexane, and chloroform are preferable.
1-50 mass parts is preferable with respect to 1 mass part of raw materials, and, as for the usage-amount of a solvent, 2-30 mass parts is more preferable. 0-100 degreeC is preferable, as for extraction temperature, 4-85 degreeC is more preferable, and 4-60 degreeC is still more preferable. The extraction time is preferably 1 minute to 150 days, more preferably 5 minutes to 50 days, and even more preferably 10 minutes to 30 days.

  As shown in Examples described later, spearmint, peppermint extract, and lysophospholipid have an action of suppressing the activity of PDE3. Therefore, the above (A) spearmint, peppermint or an extract thereof, and (B) lysophospholipid are useful for PDE3 inhibition, and they can be expected to have an effect of improving the circulation function.

  Therefore, the spearmint, peppermint or extract thereof, and lysophospholipid used in the present invention can be used as a PDE3 inhibitor, a circulatory function improving agent (hereinafter referred to as “PDE3 inhibitor etc.”), and It can be used to produce these agents.

  The PDE3 inhibitor or the like itself is a drug for humans or animals, quasi-drugs, foods, or feeds that exerts the effects of PDE3 inhibition and circulatory function improvement when ingested or administered to animals including humans. Alternatively, it may be a material or a preparation used in combination with the pharmaceutical, quasi-drug, food or feed.

The use of the PDE3 inhibitor or the like when administered to humans or animals may be therapeutic use or non-therapeutic use.
Here, “non-therapeutic” is a concept that does not include medical practice, that is, does not include a method of operating, treating, or diagnosing a human by a doctor or a person who has received instructions from a doctor.

  In addition, the foods include foods, functional foods, health foods, sick foods, and foods for specified health that have PDE3 inhibition and circulatory function improvement in middle-aged and elderly people as the concept, and that are indicated as necessary. Is done. Since these foods are foods whose function is permitted, they can be distinguished from general foods.

  (A) Spearmint, peppermint or extract thereof, (B) The dosage form of the above-mentioned pharmaceutical (including quasi-drugs) containing lysophospholipid is tablet, capsule, granule, powder, syrup, ointment , Lotions, creams, poultices, poultices, intravenous injections, intramuscular injections, infusions, infusions, suppositories, inhalants, transdermal absorption agents, eye drops, nasal drops, etc. . The administration form may be either oral administration (internal use) or parenteral administration (transdermal, enteral, transmucosal external use, injection). Of these dosage forms, the preferred form is oral administration. In addition, the said formulation can be manufactured by a conventional method according to the target object which should be mix | blended.

  In order to prepare pharmaceutical preparations of such various dosage forms, the above (A) spearmint, peppermint or an extract thereof, and (B) lysophospholipid alone or a pharmaceutically acceptable carrier are used. And may be used in combination. Such carriers include, for example, excipients, binders, disintegrants, lubricants, diluents, osmotic pressure regulators, pH regulators, emulsifiers, preservatives, stabilizers, antioxidants, colorants, ultraviolet rays. Absorber, moisturizer, thickener, brightener, activity enhancer, anti-inflammatory agent, bactericidal agent, corrigent, flavoring agent, extender, surfactant, dispersant, buffer, preservative, fragrance, coating agent Etc., and other medicinal components can be prepared by appropriately combining them.

  When the above (A) spearmint, peppermint or extract thereof (dried product equivalent), (B) lysophospholipid is used in a preparation for oral administration, its content is usually preferably 0 in the total mass of the preparation. 0.001% by mass or more, more preferably 0.01% by mass or more, further preferably 0.1% by mass or more, preferably 50% by mass or less, more preferably 10% by mass or less, and further preferably 5% by mass or less. . Moreover, Preferably it is 0.001-50 mass%, More preferably, it is 0.01-10 mass%, More preferably, it is 0.1-5 mass%.

  (A) Spearmint, peppermint or extract thereof, (B) Bread, noodles, confectionery, jelly, dairy product, frozen food, instant food, starch processing as the form of the food containing lysophospholipid Products, processed meat products, other processed foods, beverages, soups, seasonings, nutritional supplements, soft drinks, tea-based beverages, coffee beverages, fruit juice beverages, carbonated beverages, juices, wafers, biscuits, sausages, etc. Or any of various foods such as nutritional foods and their raw materials. Furthermore, a nutritional supplement composition in the same form (tablet, pill, capsule, liquid, syrup, powder, granule, etc.) as the above-mentioned oral administration preparation can be mentioned.

  Various types of foods include (A) spearmint, peppermint or extracts thereof, (B) lysophospholipid alone, or other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, fragrances , Stabilizers, colorants, ultraviolet absorbers, antioxidants, humectants, thickeners, active ingredients other than the present invention, and the like can be prepared in appropriate combinations.

The content of the above-mentioned (A) spearmint, peppermint or their extract (in terms of dry matter) and (B) lysophospholipid in the food product varies depending on the form of use, but is usually preferably 0.00 in the form of a beverage. It is 001 mass% or more, More preferably, it is 0.01 mass% or more, More preferably, it is 0.1 mass% or more, Preferably it is 50 mass% or less, More preferably, it is 5 mass% or less, More preferably, it is 1 mass% or less. Moreover, Preferably it is 0.001-50 mass%, More preferably, it is 0.01-5 mass%, More preferably, it is 0.1-1 mass%.
In the form of solid foods such as tablets and processed foods, the content of (A) spearmint, peppermint or their extract (in terms of dry matter) and (B) lysophospholipid is preferably 0.005% by mass or more. Preferably it is 0.05 mass% or more, More preferably, it is 0.1 mass% or more, Preferably it is 50 mass% or less, More preferably, it is 5 mass% or less, More preferably, it is 1 mass% or less. Moreover, Preferably it is 0.005-50 mass%, More preferably, it is 0.05-5 mass%, More preferably, it is 0.1-1 mass%.

  Examples of the feed include feed for livestock used for cattle, pigs, chickens, sheep, horses, pet food used for dogs, cats, and the like, and can be prepared in the same form as the above food.

  When producing feed, in addition to the above (A) spearmint, peppermint or extracts thereof, and (B) lysophospholipid, meat such as cattle, pigs and sheep, protein, grains, bran, rice bran , Sugars, vegetables, vitamins, minerals, and other commonly used feed ingredients, and gelling agents, shape-preserving agents, pH adjusters, seasonings, preservatives, nutritional supplements, etc. They can be used in combination.

  The content of the above (A) spearmint, peppermint or extract thereof (dry matter equivalent), (B) lysophospholipid in the feed varies depending on the form of use, but is usually preferably 0.001% by mass or more. Preferably it is 0.01 mass% or more, More preferably, it is 0.1 mass% or more, Preferably it is 50 mass% or less, More preferably, it is 10 mass% or less, More preferably, it is 5 mass% or less. Moreover, 0.001-50 mass% is preferable, 0.01-10 mass% is more preferable, 0.1-5 mass% is still more preferable.

  When used as an external medicine, quasi-drug or cosmetic including the natural product or extract or purified product thereof according to the present invention, it may be a skin external preparation, a cleaning agent, a bath agent, a makeup cosmetic, etc. Yes, depending on how you use it, serum, lotion, massage agent, lotion, emulsion, gel, cream, ointment, powder, pack, poultice, granule, foundation, lipstick, shampoo, conditioner, hair tonic, It can be provided in various dosage forms such as tablets, capsules, absorbent articles, and sheet products. Such external pharmaceuticals, quasi-drugs or cosmetics in various dosage forms are the above-mentioned natural products of the present invention or extracts, purified products, etc., alone or externally-used pharmaceuticals, quasi drugs or skin cosmetics. Oils or oily substances, moisturizers, powders, pigments, emulsifiers, solubilizers, cleaning agents, UV absorbers, thickeners, medicinal ingredients, fragrances, resins, antibacterial and antifungal agents, It can be prepared by combining other plant extracts (herbal medicine, herbal medicine, herbs), alcohols, polyhydric alcohols, inorganic acids, organic acids, vitamins, water-soluble polymers, surfactants and the like.

  The above-mentioned natural product of the present invention or an extract or purified product thereof in the total amount of the external medicine, quasi-drug, and cosmetic is usually 0.0001 to 30% by mass in terms of dry matter, 0.001 10 mass% is preferable, 0.01-5 mass% is more preferable, 0.1-3 mass% is further more preferable.

  Moreover, in the total amount of the bath agent, the natural product of the present invention or an extract or purified product thereof is usually 0.0001 to 30% by mass, preferably 0.001 to 10% by mass in terms of dry matter, 0.01-5 mass% is more preferable, and 0.1-3 mass% is further more preferable.

  The intake of a PDE3 inhibitor, etc. of the present invention may vary according to the subject's species, weight, sex, age, condition or other factors. The dose, route, interval of administration, and the amount and interval of ingestion can be appropriately determined by those skilled in the art, but per day for an adult (60 kg), (A) spearmint, peppermint or an extract thereof (dried) Product conversion), (B) The lysophospholipid is usually preferably 0.01 mg or more, more preferably 0.1 mg or more, still more preferably 1 mg or more, preferably 5000 mg or less, more preferably 1000 mg or less, still more preferably 500 mg or less. is there. Moreover, Preferably it is 0.01-5000 mg, More preferably, it is 0.1-1000 mg, More preferably, it is 1-500 mg. Moreover, although the said formulation can be administered according to arbitrary administration schedules, it is preferable to administer once to several times per day.

  The subject of administration or ingestion is not particularly limited as long as it is a person who needs or desires it regardless of whether it is sick or healthy, but middle-aged and elderly are preferred. In addition, the PDE3 inhibitor of the present invention is caused not only by patients with circulatory function diseases such as heart failure, chronic arterial occlusion and thrombosis, but also by subjects who do not suffer from circulatory function diseases, for example, due to decreased circulatory function. It can be applied to subjects who suffer from coldness, swelling, and stiff shoulders and want to improve them.

With respect to the above-described embodiment, the following aspects are disclosed in the present invention.
<1> A PDE3 inhibitor comprising as an active ingredient at least one selected from the group consisting of (A) spearmint, peppermint and extracts thereof, and (B) lysophospholipid.
<2> A circulatory function improver comprising, as an active ingredient, one or more selected from the group consisting of (A) spearmint, peppermint and extracts thereof, and (B) lysophospholipid.
<3> One or more uses selected from the group consisting of (A) spearmint, peppermint and their extracts, and (B) lysophospholipid for producing a PDE3 inhibitor.
<4> One or more uses selected from the group consisting of (A) spearmint, peppermint and their extracts, and (B) lysophospholipid for producing a circulatory function improving agent.
<5> One or more selected from the group consisting of (A) spearmint, peppermint and their extracts, and (B) lysophospholipid for use in inhibiting PDE3.
<6> One or more selected from the group consisting of (A) spearmint, peppermint and their extracts, and (B) lysophospholipid for use in improving the circulation function.
<7> One or more selected from the group consisting of (A) spearmint, peppermint and an extract thereof, and (B) lysophospholipid according to <5> or <6> for non-therapeutic use.
<8> A method for inhibiting PDE3, wherein one or more selected from the group consisting of (A) spearmint, peppermint and extracts thereof, and (B) lysophospholipid are administered or ingested to humans or animals.
<9> A method for improving circulatory function, wherein one or more selected from the group consisting of (A) spearmint, peppermint and their extracts, and (B) lysophospholipid are administered or ingested to a human or an animal.
<10> The method according to <8> or <9>, which is a non-therapeutic method.
<11><2>,<4>,<6>,<7>,<9>,<10> Preferably, the circulatory function improvement is preferably heart failure improvement, chronic arterial occlusion improvement and thrombosis It is 1 or more types chosen from improvement.
<12> The lysophospholipid according to any one of <1> to <11> is preferably lysophosphatidylcholine, lysophosphatidylglycerol, or lysoplasmanylcholine.
<13> In any one of <1> to <12>, the extract is preferably an aqueous ethanol extract.

  Hereinafter, examples and test examples will be given to specifically describe the present invention, but the present invention is not limited to these examples.

Example 1
To 100 g of dried spearmint whole plant (Tochimoto Tenkaido), add 10 liters of 1 volume of 50 vol% ethanol-water mixed solution, and extract with a flask while stirring with a magnetic stirrer at about 20 ° C. for 7 days. After filtering the insoluble matter of the obtained extract, a spearmint extract having a solid concentration of 2.1% was obtained.

Example 2
After freeze-drying 6.25 g of Falcolex peppermint B (Ichimaru Falcos), a whole plant water-containing ethanol extract of dried peppermint, add 3.75 mL of 50 vol% ethanol-water mixed solution so that the solid content concentration becomes 1%. In addition, a peppermint extract was obtained.

Example 3
To 30 g of soybean-derived L-α-Lecitin (CALBIOCHEM), ion exchange water 120 mL, 200 mM TRIS hydrochloric acid buffer (pH = 8.5) 100 mL, 100 mM calcium chloride aqueous solution 30 mL were added and dispersed with a magnetic stirrer. Thereto, 1 mL (10000 units) of phospholipase A2 (Lecitase 10L, manufactured by novo) was added and incubated at 40 to 50 ° C. for 3 days. During this time, the pH was adjusted to 8.5 with 28% aqueous ammonia. After the reaction for 3 days, chloroform (700 mL) and methanol (350 mL) were added, liquid-liquid distribution was performed, and the lower layer was collected and concentrated. To the resulting concentrate, 400 mL of hexane, 200 mL of ethanol, 150 mL of ion-exchanged water, and 50 mL of 1N hydrochloric acid were added, and the liquid was partitioned and the upper layer was removed. The obtained upper layer was further subjected to liquid-liquid partitioning operation four times using 400 mL of hexane. The remaining lower layer was neutralized with 40 mL of saturated aqueous sodium bicarbonate, concentrated, and extracted again with chloroform-methanol. The extract was concentrated, dispersed in ion-exchanged water, and lyophilized to obtain 17.76 g of lysophosphatidylcholine (pale yellow powder, yield 89%). The obtained lysophosphatidylcholine was prepared by dissolving in 99.5% ethanol (concentration 10 mM).

Example 4
To 30 g of soybean-derived L-α-Lecitin, 1300 mL of ethyl acetate, 525 mL of 200 mM sodium acetate buffer (pH = 5.6) and 4.4 g of calcium chloride dihydrate were added and dispersed. Thereto, 225 mL of glycerin and phospholipase D220unit were added and incubated at 40 ° C. for 3 days. Three days later, 250 mL of 1N hydrochloric acid was added, and the liquid was partitioned and the aqueous layer was removed. Further, it was washed successively with 300 mL of 1N hydrochloric acid and 300 mL of brine. The mixture was neutralized with saturated aqueous sodium hydrogen carbonate, washed twice with 300 mL of brine, and concentrated to obtain 29.94 g of phosphatidylglycerol sodium salt.
The obtained phosphatidylglycerol sodium salt was converted to lysophosphatidylglycerol sodium salt by the same operation as the preparation method of lysophosphatidylcholine of Example 3. Purification was performed by silica gel column chromatography (chloroform / methanol). The yield was 11.8 g (58% yield). The obtained lysophosphatidylglycerol sodium salt was prepared by dissolving in 99.5% ethanol (concentration 10 mM).

Example 5
1 kg of dried squid meat was taken, 3 L of hexane-ethanol (1: 1) mixture was added, and the mixture was extracted by stirring at room temperature for 3 hours. After filtration, 3 L of a hexane-ethanol (1: 1) mixed solution was again added to the filtration residue, followed by stirring and extraction at room temperature for 3 hours. The two extracts were combined and concentrated under reduced pressure, and then 500 mL of hexane and 50 g of Radiolite Deluxe W-50 (Showa Chemical Industry) were added and dissolved, followed by filtration and concentration under reduced pressure. As a result, 97 g of squid meal extract was obtained.
40 g of the obtained squid meal extract was taken, 800 mL of acetone was added, and the mixture was dispersed with a homomixer under ice cooling. Thereafter, the acetone-soluble fraction was removed by centrifugation. After the same operation was repeated twice more, the precipitate was dissolved in hexane and collected. This was concentrated and dried to obtain acetone insoluble fraction 1 (10 g).
Furthermore, 200 mL of a 0.4 mol / L KOH-MeOH solution was added to the obtained acetone-insoluble fraction 1, and the mixture was stirred at 37 ° C. for 4 hours to perform a hydrolysis reaction. After completion of the reaction, 400 mL of chloroform and 0.9% saline were added to perform extraction operation. The obtained chloroform layer was concentrated under reduced pressure to obtain 7.6 g of an alkali stable fraction.
To the obtained alkali-stable fraction, 100 mL of acetone was added again and dispersed with a homomixer under ice cooling. Thereafter, the acetone-soluble fraction was removed by centrifugation. After the same operation was repeated twice more, the precipitate was dissolved in hexane and collected. This was concentrated and dried to obtain acetone insoluble fraction 2 (2.0 g).
Finally, acetone-insoluble fraction 2 was dissolved in chloroform and then subjected to silica gel column chromatography (Silica-gel 60 (Merck) 100 g). Elution with chloroform-methanol (30/70) gave 0.1 g of Lyso-PAF purified product. The obtained Lyso-PAF was prepared by dissolving in 99.5% ethanol (concentration 10 mM).

Test example
Using PDE-Glo Phosphodiesterase Assay (Promega), 10 μL of the solution prepared with the PDE-Glo reaction solution so that human recombinant phosphodiesterase 3B was 0.8 μg / mL, and 2.5 μL of the solutions of the above examples were mixed. Thereafter, an enzyme reaction was carried out by adding a cAMP solution prepared to a concentration of 2 μM. The obtained reaction solution was reacted at 37 ° C. for 10 minutes, and then 12.5 μL of PDE-Glo Termination Buffer was added to stop the reaction. Thereto, 12.5 μL of PDE-Glo detection solution was added, and after 10 minutes, 50 μL of Kinase-Glo solution was added, and the amount of chemiluminescence was measured. The PDE3 activity was determined from the measured luminescence amount by the following formula, and the results are shown in Table 1.
PDE3 activity (%) = (light emission amount of any of Examples 1 to 5 and Comparative Example 2 / light emission amount of Comparative Example 1) × 100
In Comparative Example 1, the PDE-Glo reaction solution was not added with the solution of the above example, and in Comparative Example 2, caffeine was added to the prepared solution instead of the above example. .

As shown in Table 1, in Examples 1 to 5 and Comparative Example 2 using caffeine, PDE3 activity was suppressed as compared to Comparative Example 1. It is clear that spearmint, peppermint and lysophospholipid have excellent PDE3 inhibitory action.
Caffeine is a known material having a PDE3 inhibitory action.

Claims (7)

  (A) A PDE3 inhibitor comprising one or more selected from the group consisting of spearmint, peppermint and extracts thereof, and (B) lysophospholipid as an active ingredient.   The PDE3 inhibitor according to claim 1, wherein the lysophospholipid is lysophosphatidylcholine, lysophosphatidylglycerol, or lysoplasmanylcholine.   (A) A circulatory function improving agent comprising one or more selected from the group consisting of spearmint, peppermint and extracts thereof, and (B) lysophospholipid as an active ingredient.   The circulatory function improving agent according to claim 3, which is one or more selected from a heart failure improving agent, a chronic arterial occlusion improving agent and a thrombosis improving agent.   The circulatory function improving agent according to claim 3 or 4, wherein the lysophospholipid is lysophosphatidylcholine, lysophosphatidylglycerol, or lysoplasmanylcholine.   (A) Non-therapeutic PDE3 inhibition method which administers or ingests one or more selected from the group consisting of spearmint, peppermint and extracts thereof, and (B) lysophospholipid to humans or animals.   (A) A non-therapeutic method for improving circulatory function, wherein one or more selected from the group consisting of spearmint, peppermint and extracts thereof, and (B) lysophospholipid are administered or ingested to humans or animals.