JP2015174850A - anti-inflammatory compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anti-inflammatory drug and an anti-inflammatory cytokine inhibitor including a new phospholipid having a high anti-inflammatory action and a specified acidic lipid.SOLUTION: Provided is an anti-inflammatory drug or an inflammatory cytokine production inhibitor including 1-eicosapentaenoyl-2-(3-hydroxy)palmitoylphosphatidyl glycerol and a lipid as fatty acid in which the number of carbons is 20 or higher and the number of double bonds is 4 or more, being a glycero type acidic substance in which two fatty acids are coupled to the 1, 2 positions of a glycerol skeleton.

Description

  The present invention relates to a novel phospholipid, a method for producing an algal extract, and an algal extract obtained by the production method. Furthermore, this invention relates to an anti-inflammatory agent, an inflammatory cytokine production inhibitor, a food composition, cosmetics, and a pharmaceutical composition.

  Inflammatory reaction as a common pathological condition of metabolic syndrome has attracted attention, and it has been pointed out that excessive stress due to chronic inflammation increases the risk of developing lifestyle-related diseases such as arteriosclerosis. In addition, diseases such as neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), autoimmune diseases (such as rheumatism and psoriasis), and cancer are also caused by chronic inflammation.

  Therefore, the development of anti-inflammatory materials (functional foods, functional cosmetics) using special products etc. from the viewpoint of reducing medical expenses, improving quality of life, and developing unique products that make use of local materials. It has been demanded.

  Known algae used as functional foods include chlorella, donariella, spirulina, euglena, and hematococcus. Among these, it is known that hematococcus contains a large amount of astaxanthin used as a supplement.

  As another algae used in the industry, the genus Nannochloropsis sp. Is known. The genus Nannochloropsis is a spherical or oval microalgae having a diameter of 2 to 6 μm (also known as marine chlorella), and has been cultured and sold as a feed for rotifers of cultured fish feed. In addition, the genus Nannochloropsis has a high oil content (20 to 30% per dry weight) and is easy to culture, and thus has attracted attention as a candidate for biofuel that is a renewable energy.

  Moreover, in patent document 1, when a feed containing Nannochloropsis is fed to a chicken, a chicken egg containing EPA and / or DHA at a high concentration can be produced. It has been reported to be excellent. It has also been reported that when this feed is fed, the egg-laying rate does not decrease, so that eggs can be produced efficiently.

  In Patent Document 2, the genus Nannochloropsis or an extract thereof has an antioxidant stress action, an excellent skin tightening effect, and can relieve signs of aging such as sagging and wrinkles. Has been reported to be applicable to skin cosmetics.

  However, until now, there has been no report of utilizing lipids derived from the genus Nannochloropsis as an anti-inflammatory material.

Japanese Patent Laid-Open No. 8-173056 JP 2006-232766

  An object of the present invention is to provide a novel phospholipid having a high anti-inflammatory action, and an anti-inflammatory agent and an inflammatory cytokine inhibitor containing a specific acidic lipid. Another object of the present invention is to provide a method for producing an algal extract containing a high amount of anti-inflammatory lipid, an algal extract obtained by the production method, an anti-inflammatory agent containing the algal extract, and an inflammatory cytokine inhibitor. To do. Furthermore, this invention aims at providing the anti-inflammatory agent and inflammatory cytokine inhibitor containing specific algae or its extract.

  The inventors have identified 1-eicosapentaenoyl-2- (3-hydroxy) palmitoylphosphatidylglycerol (EpPOH-PG), a novel phospholipid, and 1-eicosapentanoyl, a known phospholipid. -2- (3-trans-hexadecenoyl) phosphatidylglycerol (EpHe-PG) has a high anti-inflammatory effect, these phospholipids are contained in algae such as Nannochloropsis, and water content in a specific concentration range It was found that lipids having anti-inflammatory action can be extracted from Nannochloropsis in high yield by using ethanol.

The present invention has been completed based on these findings and has been completed and provides the following phospholipids, anti-inflammatory agents, inflammatory cytokine inhibitors, algal extract production methods, algal extracts, and the like. is there.
Item 1.1-Eicosapentaenoyl-2- (3-hydroxy) palmitoylphosphatidylglycerol.
Item 2. A glycero-type acidic lipid in which two fatty acids are bonded to positions 1 and 2 of the glycerol skeleton, and at least one of the constituent fatty acids has 20 or more carbon atoms and 4 or more double bonds An anti-inflammatory agent comprising a lipid.
Item 3. A glycero-type acidic lipid in which two fatty acids are bonded to positions 1 and 2 of the glycerol skeleton, and at least one of the constituent fatty acids has 20 or more carbon atoms and 4 or more double bonds An inflammatory cytokine production inhibitor comprising a lipid.
Item 4.1 A food composition comprising eicosapentaenoyl-2- (3-hydroxy) palmitoylphosphatidylglycerol.
Item 5. A cosmetic comprising 1-eicosapentaenoyl-2- (3-hydroxy) palmitoylphosphatidylglycerol.
Item 6.1. A pharmaceutical composition comprising 1-eicosapentaenoyl-2- (3-hydroxy) palmitoylphosphatidylglycerol.
Item 7. It consists of the genus Nannochloropsis, the genus Mitsodezo, the genus Fusunagi, the genus Ibarakiri, the genus Maca, the genus Usagiri, the genus Abyssa, the genus Duckweed, the genus Eucalyptus, the genus Eucalyptus, the genus Ogonori, the genus Funali and the genus Durus A method for producing an algal extract comprising a step of extracting at least one kind of algae selected from a group using hydrous ethanol having an ethanol concentration of 60 to 90% by volume.
Item 8. Item 8. An algal extract obtained by the production method according to item 7.
Item 9. Item 9. An anti-inflammatory agent comprising the algal extract according to Item 8.
Item 10. Item 9. An inflammatory cytokine production inhibitor comprising the algal extract according to Item 8.
Item 11. Item 10. A food composition comprising the algal extract according to Item 8.
Item 12. Cosmetics containing the algal extract of claim | item 8.
Item 13. Item 9. A pharmaceutical composition comprising the algal extract according to Item 8.
Item 14. It consists of the genus Nannochloropsis, the genus Mitsodezo, the genus Fusunagi, the genus Ibarakiri, the genus Maca, the genus Usagiri, the genus Abyssa, the genus Duckweed, the genus Eucalyptus, the genus Eucalyptus, the genus Ogonori, the genus Funali and the genus Durus An anti-inflammatory agent comprising at least one algae selected from the group or an extract thereof.
Item 15. It consists of the genus Nannochloropsis, the genus Mitsodezo, the genus Fusunagi, the genus Ibarakiri, the genus Maca, the genus Usagiri, the genus Abyssa, the genus Duckweed, the genus Eucalyptus, the genus Eucalyptus, the genus Ogonori, the genus Funali and the genus Durus An inflammatory cytokine production inhibitor comprising at least one algae selected from the group or an extract thereof.

  EpPOH-GP of the present invention is a novel phospholipid and has a high anti-inflammatory action. Moreover, this invention provides the anti-inflammatory agent and inflammatory cytokine production inhibitor containing a specific acidic lipid.

  According to the method for producing an algal extract of the present invention, lipids having an anti-inflammatory action can be extracted from specific algae in a high yield. Therefore, the algal extract obtained by the production method has a high anti-inflammatory effect.

  Furthermore, this invention provides the anti-inflammatory agent and inflammatory cytokine production inhibitor containing specific algae or its extract.

  Since the anti-inflammatory lipid of the present invention can be produced from natural materials such as algae, it is considered to be highly safe.

6 is a photograph showing the results of thin layer chromatography in Test Example 4. 1: 50% aq EtOH extract, 2: 60% aq. EtOH extract, 3: 70% aq. EtOH extract, 4: 80% aq. EtOH extract, 5: 90% aq. EtOH extract, 6 : 95% aq. EtOH extract, 7: 99.9% aq EtOH extract 6 is a graph showing the production inhibition rates of NO and inflammatory cytokines (TNF-α and IL-6) in Test Example 5.

  Hereinafter, the present invention will be described in detail.

Acidic lipid The acidic lipid in the present invention is a glycero-type acidic lipid in which two fatty acids are bonded to positions 1 and 2 of the glycerol skeleton, and at least one of the constituent fatty acids has 20 or more carbon atoms and is double. It is characterized in that the number of bonds is 4 or more fatty acids. The acidic lipid is characterized by having an anti-inflammatory action and / or an inflammatory cytokine suppressing action.

  Examples of the acidic lipid include phosphatidylglycerol, sulfoquinovosyl diacylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylserine, and the like, preferably phosphatidylglycerol, phosphatidylinositol, phosphatidylserine and sulfoquinovosyl diacylglycerol, Preferred is phosphatidylglycerol.

  The fatty acid preferably has 20 to 24 carbon atoms, more preferably 20, and the number of double bonds is preferably 4 to 6, more preferably 4 or 5. Although it does not specifically limit as said fatty acid, For example, eicosapentaenoic acid, arachidonic acid, etc. are mentioned, Preferably it is 5,8,11,14,17-eicosapentaenoic acid.

  Although it does not specifically limit as constituent fatty acids other than the said fatty acid, For example, myristic acid, palmitic acid, 3-hydroxy palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, hexadecenoic acid etc. Preferred are palmitic acid, 3-hydroxypalmitic acid and hexadecenoic acid (particularly 3-trans-hexadecenoic acid).

  Specific examples of the acidic lipid include, for example, 1-eicosapentaenoyl-2- (3-hydroxy) palmitoyl phosphatidylglycerol, 1-eicosapentanoyl-2- (3-trans-hexadecenoyl) phosphatidylglycerol, Examples include 1-palmitoyl-2-arachidonoyl-phosphatidylglycerol (PA-PG), 1-eicosapentaenoyl-2-palmitoyl-sulfoquinovosylglycerol (EpP-SQDG), and the like. Sapentanoenoyl-2- (3-hydroxy) palmitoyl phosphatidylglycerol and 1-eicosapentanoyl-2- (3-trans-hexadecenoyl) phosphatidylglycerol.

  The structures of EpPOH-PG and EpHe-PG are shown below.

  Among the above acidic lipids, 1-eicosapentaenoyl-2- (3-hydroxy) palmitoylphosphatidylglycerol is a novel phospholipid.

  The acidic lipid in the present invention can be obtained as a commercially available product or can be produced by a known method, or can be obtained by separation and purification from an organism such as algae containing the acidic lipid.

  For example, EpPOH-PG is included in the genus Yukari, Akabaginanso, Darus, Nannochloropsis, etc., and EpHe-PG is Nannochloropsis, Mitsodezo, Fusutunagi, Ibaranori, Maxa It is included in the genus Usbanori, Amygusa, Yakuzusa, Obakusa, Honda Walla, Yukari, Ogonori, Akabaginanso, Funori, and Darus.

  Among them, the genus Nannochloropsis has a large content of EpPOH-PG and EpHe-PG, and therefore can be particularly suitably used as an algae used for extracting EpPOH-PG and EpHe-PG. There are six known genus Nannochloropsis, N. gaditana, N. granulata, N. limnetica, N. oceanica, N. oculata, and N. salina, and any strain can be suitably used.

  In the present invention, the genus Mizodesozo is the genus Mitsudesozo, the genus Fusutunagi, the Ibaranori genus, the Ibaranori genus, the genus Maxa, the Usubanori genus, the Amygusa genus Amidigusa, the Yahzusa genus As for the genus Amaranthus, as a genus of duckweed, as a genus of duckweed, as for the genus Honda, as for Mametarawa and Honda, as a genus of genus, as a genus of eucalyptus, as a genus of genus, as a genus of genus, as Fukurofunori is preferable as the genus Darus.

  EpPOH-PG and EpHe-PG are, for example, purified by silica gel, purified by synthetic adsorption resin, normal phase partition chromatography, reverse phase partition chromatography, anion exchange chromatography, desalting by electrodialysis, etc. Purification can be carried out using molecular weight fractionation by ultrafiltration membrane or the like, size exclusion chromatography or the like. From the viewpoint of increasing the purification efficiency, a plurality of purification methods may be used in combination.

Method for producing algal extract The method for producing the algal extract of the present invention includes the genus Nannochloropsis, the genus Mitsodesozo, the genus Fusutunagi, the genus Ibaranori, the genus Genus, the genus Usubanori, the genus Abyssa, the genus Ochusa, the genus Honda, the genus Eucalyptus, Characterized in that it comprises a step of extracting at least one algae selected from the group consisting of Ogonori, Akabaginanso, Funori and Dalus using hydrous ethanol having an ethanol concentration of 60 to 90% by volume. .

  According to the production method described above, lipids having an anti-inflammatory action (EpPOH-PG, EpHe-PG) can be extracted with high yield. Therefore, the obtained algal extract has a high anti-inflammatory effect.

  Among the algae mentioned above, extract EpHe-PG when using the genus Mizodezo, Fusutunagi, Ibaranori, Maca, Usubanori, Ajigusa, Yakuzusa, Ochakusa, Honda Walla, Ogonori and Funori However, both EpPOH-PG and EpHe-PG can be extracted when the genus Yukari, Akabaginanso, Dalus and Nannochloropsis are used.

  Among the above algae, the genus Nannochloropsis is preferable because it contains a large amount of EpPOH-PG and EpHe-PG, and can extract a large amount of EpPOH-PG and EpHe-PG.

  The algae used for the extraction may be either a raw state that has not been dried or a dry state. Also, the algae may be in an original state that has not been cut or crushed, or in a sliced or crushed state.

  The ethanol concentration of the water-containing ethanol used for extraction is usually 60 to 90% by volume, preferably 70 to 90% by volume. When the concentration is within this range, the amount of the pigment that is a contaminant is small, and the target lipid can be obtained in a high yield.

  The temperature of the water-containing ethanol at the time of extraction is, for example, 10 to 50 ° C, preferably 20 to 40 ° C. It is desirable to perform extraction at room temperature for convenience. The time for performing the extraction is preferably 5 minutes or more, more preferably 10 minutes or more, and further preferably 10 to 120 minutes. Although the usage-amount of hydrous ethanol is not specifically limited, For example, it is 5-50 weight part with respect to 1 weight part of algae, Preferably it is 5-20 weight part. Extraction may be performed in any state of standing or hydrated ethanol constantly or appropriately stirred.

  The method for recovering the water-containing ethanol after the extraction is completed may be any method that can eliminate the solids contained in the water-containing ethanol, and examples thereof include centrifugation and filtration.

  The water-containing ethanol recovered here may be subjected to solid phase extraction, activated carbon treatment, graphite carbon treatment, activated clay treatment, acid clay treatment, ultrafiltration, normal phase chromatography, reverse phase chromatography, molecular sieve chromatography as necessary. Purification may be carried out by gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, a combination thereof, and the like.

Algae or extract thereof As described above, the genus Nannochloropsis, the genus Mitsodezo, the genus Fusutunagi, the genus Ibaranori, the genus Usa, the genus Usagiri, the genera genus, the duckweed genus, the genus Honda, the genus Eucalyptus, the genus Akabaginan At least one kind of algae selected from the group consisting of Sola, Funori and Dulsu or extracts thereof are useful as active ingredients of anti-inflammatory agents and inflammatory cytokine inhibitors because they contain the above-mentioned acidic lipids It is.

  The algae may be in a raw state that has not been dried or in a dried state. Also, the algae may be in an original state that has not been cut or crushed, or in a sliced or crushed state.

  Examples of the extraction solvent used to produce the algae extract include water, lower alcohols (methanol, ethanol, n-propanol, isopropanol, etc.), ketones (acetone, methyl ethyl ketone, methyl butyl ketone, etc.), squalane, Examples include glycols (ethylene glycol, propylene glycol), acetonitrile, glycerin, tetrahydrofuran, acetamide, acetic acid, propionic acid, dimethylformamide, dimethyl sulfoxide, and the like. These extraction solvents can be used singly or in combination of two or more.

  Extraction is carried out by adding a part or all of the above algae to the solvent, extracting at 0 ° C to the temperature at which the solvent boils, usually at a temperature of 100 ° C or less, standing for 5 minutes to 3 days, soaking or shaking, and then insoluble. This can be done by removing the. The solvent extract thus obtained may be further subjected to a separation and purification step.

Anti-inflammatory agent The anti-inflammatory agent of the present invention is characterized by containing the acidic lipid or the algal extract.

  Further, the anti-inflammatory agent of the present invention includes a genus Nannochloropsis, a genus Mitsodezo, a genus Fusutunagi, a genus Ibaranori, a maca, a genus Usubanori, a genus Amygusa, a genus Dendrobium, a genus Ossa, a genus Honda, a genus Eucalyptus, a genus Akabaginan. It contains at least one kind of algae selected from the group consisting of Sou, Funori, and Dalus, or an extract thereof.

  Since the acidic lipid exhibits a strong anti-inflammatory action, it is useful as an active ingredient of an anti-inflammatory agent. The algal extract containing the acidic lipid is also useful as an active ingredient of an anti-inflammatory agent.

  The anti-inflammatory agent of the present invention can be suitably used in the fields of food compositions, pharmaceutical compositions, cosmetics and the like.

  The content of the acidic lipid in the anti-inflammatory agent of the present invention is not particularly limited because the final content of the acidic lipid when contained in a food composition, a pharmaceutical composition, cosmetics, etc. is important. 1 to 100% by weight, preferably 5 to 100% by weight. The content of the acidic lipid in the anti-inflammatory agent added to food compositions, pharmaceutical compositions, cosmetics and the like will be described later. The content of the acidic lipid in the anti-inflammatory agent may be 100% by weight.

  In this document, the content of algal extract, algae or algae extract in anti-inflammatory agents, inflammatory cytokine inhibitors, food compositions, pharmaceutical compositions, cosmetics, etc., unless otherwise specified, Expressed as the total amount (% by weight) of the acidic lipid in inhibitors, food compositions, pharmaceutical compositions, cosmetics and the like.

Inflammatory Cytokine Inhibitor The inflammatory cytokine inhibitor of the present invention comprises the acidic lipid or the algal extract.

  Furthermore, the inflammatory cytokine inhibitor of the present invention is a genus Nannochloropsis, genus Mitsodezo, genus Fusutunagi, genus Ibaranori, genus Usbano, genus Usagiri, genus Azagusa, genus genus, Honda genus, Eucalyptus, Ogonori, It contains at least one kind of algae selected from the group consisting of the genus Akabaginanso, Funori and Durus, or an extract thereof.

  The acidic lipid is useful as an active ingredient of an inflammatory cytokine inhibitor because it exhibits a strong inflammatory cytokine inhibitory action. Algae containing the above acidic lipids and extracts thereof are also useful as active ingredients of inflammatory cytokine inhibitors.

  The inflammatory cytokine inhibitor of the present invention can be suitably used in the fields of food compositions, pharmaceutical compositions, cosmetics and the like.

  The content of the acidic lipid in the inflammatory cytokine inhibitor of the present invention is not particularly limited because the final content of the acidic lipid when contained in a food composition, a pharmaceutical composition, a cosmetic or the like is important. Usually, it is about 1 to 100% by weight, preferably about 5 to 100% by weight. The content of the acidic lipid in the inflammatory cytokine inhibitor added to food compositions, pharmaceutical compositions, cosmetics and the like will be described later. The content of the acidic lipid in the inflammatory cytokine inhibitor may be 100% by weight.

Food Composition The food composition of the present invention is characterized by containing EpPOH-PG or the above algal extract. The food composition of the present invention includes any food composition that can be ingested by animals (including humans).

  The food composition of the present invention includes, as necessary, minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants. Agents, colorants, fragrances, stabilizers, preservatives, sustained release regulators, surfactants, solubilizers, wetting agents and the like can be blended. For these additives, the “Designated Items Food Additives Handbook 1999 Edition” edited by Shinnosuke Kishi, published on December 10, 1999, or “New Revised Version = Well-Knowning Life” Among these, the additives described in “Japan Food Additives Association, Published December 20, 1996” can be used.

  Examples of food compositions include processed meat products such as ham and sausage, processed marine products such as octopus and salmon, processed milk products such as butter, cheese and yogurt, udon, soba, Chinese soba, spaghetti and instant ramen Noodles, tofu, soy milk, soy flour, soy sauce, bean confectionery, processed soybean products such as soy protein isolate, processed agricultural products, processed fruit products such as jam, prune extract, puree, seasonings, sparkling wine, wine, beer, etc. Preferable examples include alcoholic beverages, soft drinks / carbonated drinks / coffee / tea drinks, pickles, bread, confectionery, frozen desserts, prepared dishes, retorts, rice products, and the like.

  The content of EpPOH-PG and / or EpHe-PG in the food composition of the present invention is the type, form, fat content, processing state, consumption or expiration date set, desired effect, desired effect of the food composition. Although it is not limited because it varies depending on the storage form, coexisting components, and the like, for example, 0.00001 to 80% by weight, preferably 0.0001 to 50% by weight, and more preferably 0.0001 to 30% by weight.

  In another preferred embodiment of the present invention, the food composition of the present invention comprises a disease or symptom caused by an inflammatory reaction (e.g., metabolic syndrome, arteriosclerotic disease, cancer / neoplastic disease, neurodegeneration). It is a functional food composition that can improve and / or prevent diseases (such as Alzheimer's disease and Parkinson's disease) and autoimmune diseases (such as rheumatism and psoriasis). As the functional food composition, for example, a food composition such as powder, tablet, granule, tablet, chewable tablet, capsule, soft capsule, troche, candy, candy, gum, jelly, gummy, biscuit, cookie, beverage, etc. is suitable. Illustrated.

  As a functional food composition, the food composition of the present invention can improve and / or prevent the above-mentioned diseases or symptoms caused by an inflammatory reaction.

Cosmetics The cosmetics (including hair care cosmetics) of the present invention are characterized by containing EpPOH-PG or the above algal extract. The cosmetics of the present invention include all cosmetics applied to the skin, mucous membranes, body hair, head hair, scalp, nails, teeth, face skin, lips, etc. of animals (including humans).

  In addition to EpPOH-PG, which is an essential component, or the above algal extract, the cosmetics of the present invention include powders, pigments, fats and oils, moisturizers, surfactants, antioxidants, thickeners, washings as necessary. Ingredients used in normal cosmetics such as agents, excipients, emulsifiers, plasticizers, preservatives, antifungal agents, pH adjusters, UV absorbers, amino acids, and fragrances are suitable as long as the effects of the present invention are not impaired. Can be blended. For these additives, reference can be made to JP-A-9-87133 and JP-A-3-264510, the entirety of which is incorporated herein.

  The dosage form of the cosmetic of the present invention is arbitrary, and is a dosage form such as a solution system, a solubilization system, an emulsification system, a powder dispersion system, a water-oil two-layer system, a water-oil-powder three-layer system, and the like. obtain. The pH is preferably adjusted to about 4 to 8 from the viewpoint of safety to the skin.

  The use of the cosmetics of the present invention is also optional, such as facial cosmetics such as lotion, milky lotion, cream, cosmetic liquid, pack, shampoo, hair rinse, hair tonic, hair cream, hair mousse, hair spray etc. Makeup cosmetics such as foundation, lipstick, gloss, eye shadow, eyebrow, teak, mascara, body cosmetics such as body lotion, body cream, cleansing foam, facial cleanser, hand soap, body soap etc., perfume, It can be used for aromatic makeup such as deodorant, nail polish, base coat, top coat, nail cosmetics such as a light removal liquid, bathing agent, and the like.

  The content of EpPOH-PG and / or EpHe-PG in the cosmetics of the present invention is particularly different because it varies depending on the purpose of use of the cosmetics, dosage form, composition, set expiration date, set storage state, coexisting ingredients, etc. Although not limited, it is 0.000001-50 weight%, for example, Preferably it is 0.00001-50 weight%, More preferably, it is 0.0001-30 weight%.

  The cosmetic of the present invention can treat and / or prevent the aforementioned diseases or symptoms caused by inflammatory reactions.

Pharmaceutical Composition The pharmaceutical composition of the present invention comprises EpPOH-PG or the above algal extract. The pharmaceutical composition of the present invention is administered to mammals including humans.

  The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier, excipient, extender, binder, wetting agent, disintegrant, surfactant, lubricant, dispersant, buffer as necessary. Ingredients used in normal pharmaceutical compositions such as preservatives, preservatives, solubilizers, preservatives, flavoring agents, flavoring agents, stabilizers, etc. can be appropriately blended within a range that does not impair the effects of the present invention. As these additives, for example, the additives described in “Pharmaceutical Additives Encyclopedia 2000” edited by the Japan Pharmaceutical Additives Association, published on April 28, 2000, which is incorporated herein in its entirety, can be used.

  The administration form and formulation form of the pharmaceutical composition are not particularly limited. Examples of the dosage form include oral administration, intramuscular administration, intravenous administration, enteral administration, rectal administration, and subcutaneous administration. Formulation forms include, for example, tablets, pills, granules, capsules, soft capsules, troches, suppositories, powders, powders and other solid or semisolid agents, liquids, suspensions, emulsions, syrups, lotions And liquids such as sachets, aerosols, dipping agents, and decoction.

  The content of EpPOH-PG and / or EpHe-PG in the pharmaceutical composition is not limited because it varies depending on the purpose of use, formulation form, composition, set expiration date, coexisting components, etc., for example, 0.00001-100 wt%, Preferably it is 0.00001 to 90 weight%, More preferably, it is 0.0001 to 90 weight%.

  The dosage of the pharmaceutical composition of the present invention can be appropriately determined according to various conditions such as the body weight, age, sex, and symptoms of the patient.

  The pharmaceutical composition of the present invention is effective for the treatment and / or prevention of the above diseases or symptoms caused by inflammatory reactions.

  Examples are given below to illustrate the present invention in more detail. However, the present invention is not limited to these examples.

  In the test examples, cytokine production inhibition rate, NO production inhibition rate, and cytotoxicity were measured according to the following methods.

[Method for measuring cytokine production inhibition rate and NO production inhibition rate]
Cell tests were performed using the mouse-derived macrophage-like cell line RAW264.7 (provided by RIKEN BioResource). The cells were subcultured at 37 ° C. in the presence of 5% CO _{2 in} DMEM medium containing 1% penicillin / streptomycin and 10% FBS. LPS-induced inflammatory cytokine (TNF-α / IL-6) and NO production tests were performed under the following conditions. The cells were diluted with a medium to a cell density of 2 × 10 ⁵ cells / mL, dispensed 200 μL each into a 96-well plate for cell culture, pre-cultured for 16 hours, and the cells were allowed to adhere to the plate.

  Remove the culture supernatant from the cells, wash with fresh medium, add 180 μL of medium in which the sample is dispersed at the specified concentration, and then add 20 μL of LPS at a concentration of 100 ng / mL 15 minutes later, for a total volume of 200 μL (N = 3). Culture supernatants after 6 hours and 19 hours of culture were collected, and cytokine (TNF-α and IL-6) concentrations (6 hours after culture) and NO concentrations (19 hours after culture) were measured. The sample was once dissolved in DMSO at a concentration of 15 mmol / L after the solvent was dried, and then dispersed in the medium to a predetermined concentration. Considering the cytotoxicity of DMSO, the final concentration of DMSO in the medium was not allowed to exceed 0.2%.

  The cytokine concentration was determined by ELISA method. The level of cytokine production inhibitory activity (inhibition) was obtained by adding a sample-free medium adjusted to the same DMSO concentration instead of the sample-added medium as a control, and adding a LPS-free medium instead of an LPS-containing medium as a blank. Rate) was calculated by the following formula.

Cytokine production inhibition rate (%) = {1- (Sa-Sb) / (Sc-Sb)} × 100
here,
Sa: Cytokine concentration in sample-added medium
Sb: Blank cytokine concentration
Sc: Control cytokine concentration

The amount of NO production was calculated by measuring the concentration of nitrite ion (NO ₂ ⁻ ) accumulated in the culture supernatant by the Griess method. That is, 125 μL of the culture supernatant was collected on a 96-well microplate, 125 μL of Griess reagent was added and rapidly stirred, left at room temperature for 10 minutes, and then at a wavelength of 500 nm and 650 nm using a microplate reader (M5 manufactured by Molecular devices). Absorbance difference (ΔA) was measured.

A calibration curve was prepared using NO ₂ ⁻ dissolved in the medium as a standard solution, and the NO ₂ ⁻ concentration in the medium was determined. The NO production inhibition rate was calculated by the following formula in the same manner as the cytokine production inhibition rate.

NO production inhibition rate (%) = {1- (ΔAa-ΔAb) / (ΔAc-ΔAb)} × 100
here,
ΔAa: Absorbance difference of sample-added medium ΔAb: Absorbance difference of blank ΔAc: Absorbance difference of control

[Method for measuring cytotoxicity]
In order to confirm the validity of NO / TNF-α production suppression by the cell test, the cytotoxicity of the sample was measured using a cell activity measurement kit (Cell Counting Kit-8). Cultivate the cells for 20 hours in the presence of the sample and LPS under the same culture conditions as above.After removing the medium, culture for 6 hours in a medium containing Cell Counting Kit-8 (2 μL reagent / 98 μL medium). The difference in absorbance (ΔA) at wavelengths of 450 nm and 650 nm was measured. The degree of cytotoxicity was expressed as the ratio of ΔA in the sample-added medium group to ΔA in the control group.

Test Example 1: Comparison of anti-inflammatory action in phospholipids Various phospholipids and glycolipids (EpPOH-PG, EpHe-PG, EpP-SQDG, PA-PG, PO-PG, diP-PG, PO-PC, diEp- The anti-inflammatory effects of PC, POPS, PO-PE, PO-PA and P-LPC) were compared. Various lipids were dissolved in the medium to 300 nmol / mL and added to the cells at a final concentration of 30, 10, 3 nmol / mL. The anti-inflammatory effect was expressed as NO production inhibition rate, TNF-α production inhibition rate, and IL-6 production inhibition rate of macrophage-like cells RAW264.7 stimulated by LPS. The results are shown in Tables 1 and 2.

  As shown in Tables 1 and 2, lipids having stronger anti-inflammatory activity than the previously reported anti-inflammatory phospholipid PO-PG are EpPOH-PG, EpHe-PG, EpP-SQDG and PA- There were 4 types of PG. In addition, EpPOH-PG had the strongest anti-inflammatory effect.

  The WST activity indicating the activity of the cells was not changed as compared with the case where no lipid was added, and cytotoxicity due to the addition of lipid was not observed.

Test Example 2: Detection of anti-inflammatory lipids in various seaweed-derived lipids The ethanol extract was extracted from various algae and the anti-inflammatory action was strongest using high performance liquid chromatography-mass spectrometer (LC-ESIITMS). The existence of EpPOH-PG and EpHe-PG was investigated. The algae shown in Table 2 were dried and powdered. 500 mg of this powder was put in a 25 ml screw-cap test tube, 10 mL of ethanol was added, and the mixture was stirred for 1 hr at room temperature, and a supernatant was obtained by centrifugation. To the residue, 10 mL of ethanol was added again, the mixture was stirred for 30 min, and a supernatant was obtained by centrifugation. These were combined and concentrated by centrifugation to obtain an ethanol extract.

  The obtained extract was dissolved in ethanol to a concentration of 0.1 mg / mL to remove fine particles, and then subjected to LC-ESIITMS. LC conditions are as follows: LC, Agilent LC1100 series; column, Cadanza CW-C18 (Imtakt Co., φ2 × 250 mm); column temp., 40 ℃; mobile phase-A, water / acetic acid / 1mmol / L ammonium acetate = 100 / 0.07 / 3; mobile phase-B, methanol / acetic acid / 1mmol / L ammonium acetate = 100 / 0.07 / 3; gradient program, 0 min (B80%) → 30 min (B100% with linear gradient ) → 50 min (B100%); injection volume, 3 μL. The conditions for ITMS detection are as follows: ion source, electrospray ionization; polarity, negative; scan range, 50-1200 m / z.

  When LC-MS was performed under these conditions, EpPOH-PG was detected at a retention time of 29.5 min and EpHe-PG was detected at a retention time of 31.5 min at m / z 783 and m / z 765, respectively. The area of this peak was taken as the relative content, and the case of the genus Nannochloropsis was expressed as 100. The results are shown in Table 3.

  In the ethanol extract, EpPOH-PG was contained in the genus Yukari, Akabaginanso, Durus, and Nannochloropsis. In addition, EpHe-PG is included in the genus Mitsudezozo, Funori, Fusutunagi, Ibaranori, Maca, Usubanori, Ajigusa, Yakuzusa, Ochakusa, Honda Walla, Yukari, Ogonori, Akabaginanso Genus, Darus and Nannochloropsis. The relative content per dry weight of algal bodies was highest for Nannochloropsis. This shows that the genus Nannochloropsis is most suitable as an extraction source of the anti-inflammatory material containing EpPOH-PG and EpHe-PG.

Test Example 3: Anti-inflammatory action of algae The anti-inflammatory action of ethanol extracts obtained from various algae was evaluated in an in vitro test. The ethanol extract was dissolved in the medium and added at a final concentration of 100 μg / mL. The results are shown in Table 3.

  All algae in which EpPOH-PG was detected (genus Eucalyptus, Akabaginanso, Darus, Nannochloropsis) had a high anti-inflammatory effect.

Test Example 4: Extraction test of anti-inflammatory lipid A method of extracting lipids having anti-inflammatory activity from the genus Nannochloropsis, which was most suitable as an extraction source, was examined. 1 g of dried Nannochloropsis powder was placed in a 25 mL screw-cap test tube, and 20 mL of various ethanols having different moisture contents were added and stirred for 30 min. The extract was obtained by centrifugation, ethanol was added again to the residue and stirred for 20 min, and the extract was obtained by centrifugation. These were combined and dried by centrifugal concentration to obtain an extract.

  The evaluation of the extraction method is as follows: (1) recovery rate of lipid in the extract, (2) content of pigments (chlorophyll and carotenoid) as impurities, and (3) content of EpPOH-PG and EpHe-PG as target products The evaluation was conducted focusing on these three points. The recovery rate of lipid in the extract was calculated by measuring the weight of the fraction distributed to the lower layer in the chloroform / methanol / water system. The contents of the pigment and the target lipid were confirmed by TLC. The results are shown in Table 4 and FIG.

TLC development conditions Solvent: Chloroform: Methanol: Water: Triethylamine = 30/35/7/35 (v / v / v / v)
Thin plate: Merck silica gel 60
Development tank: A filter paper was passed around the inner dimensions of 12 x 12 x 4 cm and saturated overnight.
Sample coating amount: 50 μL of the extract was centrifuged and dissolved with 10 μL of CM2: 1. A line was drawn 1.5 cm from the lower end and the upper end, and the sample was applied in a band at 1 cm intervals.
Development time: About 22 minutes, at room temperature, developed to 1.5 cm from the top and dried immediately.
Coloration: 50% sulfuric acid Heating was performed in an oven (160 ° C., 5-10 min).

  As shown in Table 4, the yield of lipid fraction in the extract was highest when the ethanol concentration of the extraction solvent was in the range of 70 to 90% by volume. On the other hand, as shown in FIG. 1, the amount of the dye increased as the ethanol concentration increased and reached a certain amount at 90% by volume or more. In addition, the amounts of EpPOH-PG and EpHe-PG increased as the ethanol concentration increased, and reached a certain level at 70% by volume or more. From these results, the most suitable method for extracting lipids having an anti-inflammatory effect from the genus Nannochloropsis has a low content of pigments as impurities and an ethanol concentration of 70 to 90 with a high yield of the target product. It was found to be an extraction method in the volume% range.

Test Example 5: Comparison with free eicosapentaenoic acid
The anti-inflammatory effect of EpPOH-PG was compared with free eicosapentaenoic acid (EPA), which is a constituent fatty acid of EpPOH-PG. EpPOH-PG or EPA (Sigma-Aldrich) was dissolved in the medium and added to the cells at a concentration of 10 μmol / L. LPS is added to a final concentration of 10 ^-4 to 10 ^-1 μg / mL to stimulate macrophages, and the inhibition rate of production of NO and inflammatory cytokines (TNF-α and IL-6), which are indicators of inflammatory response, Compared. As a result, as shown in Fig. 2, EpPOH-PG showed significant NO production suppression in the LPS concentration range of 10 ^-2 μg / mL or less, but EPA almost showed NO suppression effect. I couldn't. Similarly, in TNF-α, EpPOH-PG produced a production inhibitory effect in all measured LPS concentration ranges, but EPA showed little inhibitory effect. As for IL-6, EpPOH-PG showed a remarkable IL-6 production inhibitory effect when LPS concentration was 10 ^-2 μg / mL or less, but EPA showed little inhibitory effect. .

Claims (15)

  1-Eicosapentaenoyl-2- (3-hydroxy) palmitoyl phosphatidylglycerol.   A glycero-type acidic lipid in which two fatty acids are bonded to positions 1 and 2 of the glycerol skeleton, and at least one of the constituent fatty acids has 20 or more carbon atoms and 4 or more double bonds An anti-inflammatory agent comprising a lipid.   A glycero-type acidic lipid in which two fatty acids are bonded to positions 1 and 2 of the glycerol skeleton, and at least one of the constituent fatty acids has 20 or more carbon atoms and 4 or more double bonds An inflammatory cytokine production inhibitor comprising a lipid.   A food composition comprising 1-eicosapentaenoyl-2- (3-hydroxy) palmitoylphosphatidylglycerol.   A cosmetic comprising 1-eicosapentaenoyl-2- (3-hydroxy) palmitoylphosphatidylglycerol.   A pharmaceutical composition comprising 1-eicosapentaenoyl-2- (3-hydroxy) palmitoylphosphatidylglycerol.   It consists of the genus Nannochloropsis, the genus Mitsodezo, the genus Fusunagi, the genus Ibarakiri, the genus Maca, the genus Usagiri, the genus Abyssa, the genus Duckweed, the genus Eucalyptus, the genus Eucalyptus, the genus Ogonori, the genus Funali and the genus Durus A method for producing an algal extract comprising a step of extracting at least one kind of algae selected from a group using hydrous ethanol having an ethanol concentration of 60 to 90% by volume.   An algal extract obtained by the production method according to claim 7.   An anti-inflammatory agent comprising the algal extract according to claim 8.   The inflammatory cytokine production inhibitor containing the algal extract of Claim 8.   A food composition comprising the algal extract according to claim 8.   A cosmetic comprising the algal extract according to claim 8.   A pharmaceutical composition comprising the algal extract according to claim 8.   It consists of the genus Nannochloropsis, the genus Mitsodezo, the genus Fusunagi, the genus Ibarakiri, the genus Maca, the genus Usagiri, the genus Abyssa, the genus Duckweed, the genus Eucalyptus, the genus Eucalyptus, the genus Ogonori, the genus Funali and the genus Durus An anti-inflammatory agent comprising at least one algae selected from the group or an extract thereof.   It consists of the genus Nannochloropsis, the genus Mitsodezo, the genus Fusunagi, the genus Ibarakiri, the genus Maca, the genus Usagiri, the genus Abyssa, the genus Duckweed, the genus Eucalyptus, the genus Eucalyptus, the genus Ogonori, the genus Funali and the genus Durus An inflammatory cytokine production inhibitor comprising at least one algae selected from the group or an extract thereof.

Images