JP2006347985A - Medicinal composition containing crepidiastrum lanceolatum nakai and/or fermentation product of crepidiastrum lanceolatum nakai - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicinal composition and a functional food which each effectively uses Crepidiastrum lanceolatum Nakai having physiological activities and/or a fermentation product of the Crepidiastrum lanceolatum Nakai, is safe for living bodies, little produces side effects, and has an active oxygen production-inhibiting activity, a leucotriene compound production-inhibiting activity, a histamine liberation-inhibiting activity, an IL-1β liberation-inhibiting activity, and an IL-6 liberation-inhibiting activity. <P>SOLUTION: The medicinal composition or the functional food contains the extract of Crepidiastrum lanceolatum Nakai and/or a fermentation product of the Crepidiastrum lanceolatum Nakai. The fermentation product is preferably a fermentation product fermented with lactobacillus, lactobacillus and yeast, lactobacillus and Bacillus subtilis, or lactobacillus, yeast and Bacillus subtilis. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a pharmaceutical composition or a functional property having an active oxygen production inhibitory activity, a leukotrienes production inhibitory activity, a histamine release inhibitory activity, an IL-1β release inhibitory activity, an IL-6 release inhibitory activity derived from a Japanese plant growing in Okinawa. Regarding food.

  In recent years, inflammatory or allergic diseases such as hay fever, atopic dermatitis, bronchial asthma, allergic rhinitis, food allergies and urticaria have become serious social problems. In these diseases, symptoms accompanied by itching appear, leading to physical or mental exhaustion of the patient, and depending on the symptoms, there are cases that become serious enough to interfere with daily life.

  An allergic reaction is an immune reaction that is detrimental to the living body with an overexpression of a biological defense reaction against the invasion of bacteria, viruses, or foreign substances, and is classified into four types (types I to IV) according to the onset mechanism. . A type I allergic reaction is a reaction mainly involving immunoglobulin E (IgE) antibodies. The type I allergic reaction is an immediate type reaction that reacts with the IgE antibody bound to the Fc receptor on the surface of mast cells or basophils, and then reacts with the IgE antibody on the cell surface to cross-link between the antibodies. , Which triggers degranulation of mast cells and basophils, releasing chemical mediators such as histamine, serotonin, SRS-A, ECF-A, serotonin, and prostaglandin contained in the granules To cause tissue damage. Diseases that develop due to type I allergic reactions include anaphylactic shock, bronchial asthma, allergic rhinitis, urticaria, and atopic dermatitis.

  In the type II allergic reaction, IgG or IgM antibody binds to the cell membrane itself or an antigen attached to the cell membrane, and a hole is made in the cell by the action of complement to dissolve the cell. Diseases that develop due to type II allergic reactions include hemolytic anemia, idiopathic thrombocytopenia, myasthenia gravis, Gut Pascher syndrome, and organ transplant rejection. In type III allergic reaction, immune complexes that bind antigen and IgG antibody cannot be processed by phagocytic cells and are deposited in tissues. Complements, macrophages, neutrophils, etc. gather there, causing inflammation and tissue damage Is. Diseases that develop due to type III allergic reactions include acute glomerulonephritis, rheumatoid arthritis, viral hepatitis, systemic lupus erythematosus, cryoglobulinemia and the like. In the type IV allergic reaction, T cells sensitized to an antigen by a delayed reaction release rehocaine or the like, causing tissue damage by lymphocytes, macrophages and the like. Diseases that develop due to type IV allergic reactions include contact dermatitis, tuberculosis, malignant tumors, and rejection after organ transplantation. Among these allergic reactions, type I allergy involving immunoglobulin E (IgE) antibody is the most frequently occurring allergy.

  On the other hand, it is known that various physiologically active components are contained in plants and / or extracts thereof, and they are applied to pharmaceuticals because of their high safety to living bodies. It is also used for the prevention or treatment of diseases caused by allergic reactions. Specifically, one or more selected from apigenin, chrysoeriol, luteolin and rosmarinic acid in alcohol extract of perilla seeds are active ingredients A histamine release inhibitor (for example, refer to Patent Document 1), an antiallergic composition (for example, refer to Patent Document 2) characterized by containing an extract of petals of impatiens textori Miq. Catechins and / or glycosides thereof, flavones and / or glycosides thereof, flavonols and / or glycosides thereof, flavanones and / or glycosides thereof, isoflavones and / or glycosides thereof, Coumarins and / or glycosides thereof, mushrooms belonging to the genus Agaricus, Vitis genus, Plantago genus, Linam genus, Synapis genus, Casamas genus Of plants belonging to the genus Perilla, Gossium, Ricinas, Oenocera, Rosemary, Camellia, Banzakuro, Eucalyptus, Berith, Gnaphalium, Arctostaphylos, Centrole, Euphrasia, Lislam, etc. Examples thereof include cosmetics (for example, see Patent Document 3) characterized by blending an IgE production inhibitor composed of one or more kinds from the group of extracts and dried red wine powders.

  By the way, nigana is a perennial plant belonging to the family Asteraceae, and contains white milky milk with a bitter taste on the leaves and stems. It is vegetated normally in mountains and fields, and is often found in Okinawa. Nigana contains a component having a function (ACE activity inhibitory function) that suppresses the conversion of angiothecin I, which is a peptide present in blood, to angiophysin II by the action of an enzyme, thereby exhibiting an effect of suppressing an increase in blood pressure. It is known. Examples of using the functions of such Japanese nigana include, for example, oilseed rape, Italian Pasley leaves, udder leaves, persimmon bovine leaves, caladium leaves, peony flowers, kudu leaves flowers, shisiudo berries, sprette leaves, sorrel leaves, and dale leaves. At least one selected from the group consisting of whole grass, alligator flowers, hymedione flowers, peppermint leaves, mustard leaves, walnut leaves, cornflower leaves, yamafuji flowers, freeze-dried and ground, and the ground product is extracted with water A food additive having an inhibitory function (ACE activity inhibitory function) of conversion from angiostaticin 1 to angiostaticin 2 in the presence of angiotensin converting enzyme (see, for example, Patent Document 4) ) Etc. are known.

  In addition, the present inventors have previously made a fermented processed product production method characterized by fermenting a Japanese cabbage using lactic acid bacteria, lactic acid bacteria and yeast, lactic acid bacteria and Bacillus subtilis, or lactic acid bacteria and yeast and Bacillus subtilis (for example, patents) And a lipoxygenase inhibitor (see, for example, Patent Document 6) characterized in that it comprises as an active ingredient a Japanese algae (Crepidiastrum lanceolatum Nakai) and / or a Japanese algae fermentation product using the fermentation method. Yes.

  This lipoxygenase inhibitor suppresses the production of arachidonic acid metabolites such as 5-hydroperoxy-6,8,10,14-eicosatetraenoic acid (5-HPETE), thereby causing diseases caused by arachidonic acid metabolites For example, it can be used as a preventive / therapeutic agent for allergic diseases, inflammation, asthma and the like.

  However, although it has been known so far that it has a lipoxygenase inhibitory activity in Japanese algae and a processed powder of Japanese algae, reactive oxygen production inhibitory activity, leukotrienes inhibitory activity, histamine release inhibitory activity, IL-1β release inhibitory activity, IL- It was not known at all that it has 6 release inhibitory activity.

JP 2000-86510 A Japanese Patent Laid-Open No. 2001-27879 JP 2003-28111 A Japanese Patent Laid-Open No. 06-225723 JP 2004-73050 A Japanese Patent Laying-Open No. 2005-089385

  The object of the present invention is to make effective use of a biologically active Japanese algae and / or a processed fermentation of a Japanese algae, which is safe for the living body and has few side effects, an active oxygen production inhibitory activity, a leukotrienes production inhibitory activity, a histamine release inhibitory activity, The object is to provide a pharmaceutical composition having IL-1β release inhibitory activity and IL-6 release inhibitory activity.

  As a result of intensive research on the physiological activity of the Japanese algae plant for the purpose of effective utilization of the Japanese algae plant growing in Okinawa, the present inventors have found that the extract of the Japanese algae has an active oxygen production inhibitory activity, a leukotrienes production inhibitory activity, a histamine release inhibitory activity. Activity, IL-1β release inhibitory activity, and IL-6 release inhibitory activity, and further, it has been found that the fermented product of Japanese algae has increased active oxygen production inhibitory activity and leukotrienes production inhibitory activity as compared with Japanese greenfish. The present invention has been completed.

  That is, the present invention has at least one of (1) active oxygen production inhibitory activity, leukotrienes production inhibitory activity, histamine release inhibitory activity, IL-1β release inhibitory activity, and IL-6 release inhibitory activity. , An active oxygen production inhibitor, a leukotrienes production inhibitor, a histamine release inhibitor, an IL-1β release inhibitor, or an IL-6 release, which contains an extract of Japanese red crab (Crepidiastrum lanceolatum Nakai) and / or its fermented product. An inhibitor or a composite active agent having two or more activities, or (2) 2 to 10 parts per 1 part by weight of a pulverized product after pulverizing the leaves of Japanese algae to a particle size of 0.1 to 3.0 mm. The active acid according to (1) above, comprising an extract obtained by adding a part by weight of water and further fermenting by adding a microorganism group and extracting the fermented product with a solvent. Production inhibitor, leukotrienes production inhibitor, histamine release inhibitor, IL-1β release inhibitor, or IL-6 release inhibitor, or a combined active agent having two or more of the above activities, or (3) fermentation of Japanese algae The treated product is obtained by fermenting with lactic acid bacteria, lactic acid bacteria and yeast, lactic acid bacteria and Bacillus subtilis, or lactic acid bacteria and yeast and Bacillus subtilis, the active oxygen production inhibitor according to (1) or (2) above, Leukotrienes production inhibitor, histamine release inhibitor, IL-1β release inhibitor, or IL-6 release inhibitor, or a composite active agent having two or more activities described above, and (4) The active oxygen production inhibitor, leukotrienes production inhibitor, histamine release inhibitor, IL-1β migration according to any one of the above (1) to (3), wherein 10 to 10% by weight of a microorganism group is added A release inhibitor, an IL-6 release inhibitor, or a combined active agent having two or more of the above activities, or (5) at least one of methanol, ethanol, ethyl acetate, or a mixed solvent thereof as an extraction solvent. The active oxygen production inhibitor, leukotrienes production inhibitor, or histamine according to any one of the above (1) to (4), characterized by containing an extract of a Japanese cabbage obtained by use and / or a fermented product thereof The present invention relates to a release inhibitor, an IL-1β release inhibitor, or an IL-6 release inhibitor, or a combined active agent having two or more activities.

  The present invention also has at least one of (6) active oxygen production inhibitory activity, leukotrienes production inhibitory activity, histamine release inhibitory activity, IL-1β release inhibitory activity, and IL-6 release inhibitory activity. , Characterized by containing extract of Japanese red crab (Crepidiastrum lanceolatum Nakai) and / or fermented products thereof, and a function with an indication that it is used to improve inflammation and allergic diseases due to these activities After pulverizing foods and (7) Nigara leaves to a particle size of 0.1 to 3.0 mm, 2 to 10 parts by weight of water is added to 1 part by weight of the pulverized product, and a microorganism group is added. The functional food according to (6) above, which contains an extract obtained by performing fermentation treatment and extracting the fermentation treatment product with a solvent, and yeast It is obtained by fermentation with lactic acid bacteria and Bacillus subtilis, or lactic acid bacteria, yeast and Bacillus subtilis, and the functional food according to (6) or (7) above, and (9) The functional food according to any one of (6) to (8) above, wherein 10% by weight of a microorganism group is added, and (10) methanol, ethanol, ethyl acetate or a mixture thereof as an extraction solvent The functional food according to any one of (6) to (9) above, which contains an extract of a Japanese cabbage obtained by using at least one solvent or a fermented product thereof.

  According to the present invention, the active oxygen production inhibitory activity, leukotrienes production inhibitory activity, histamine release inhibitory activity, IL-1β release inhibitory activity, IL-6 release inhibitory activity can be obtained by containing the Japanese charcoal and / or the fermented product of Japanese charcoal. Therefore, it is possible to provide a safe preventive / therapeutic agent for allergic diseases, inflammatory diseases, etc., and a functional food for the prevention / treatment of allergic diseases, inflammatory diseases, etc.

  The present invention relates to nigana having at least one of active oxygen production inhibitory activity, leukotrienes production inhibitory activity, histamine release inhibitory activity, IL-1β release inhibitory activity, and IL-6 release inhibitory activity. Or an active oxygen production inhibitor, a leukotrienes production inhibitor, a histamine release inhibitor, an IL-1β release inhibitor, or an IL-6 release inhibitor, or an activity of two or more of the above, containing an extract of the fermented product thereof The active agent is not particularly limited as long as it is a composite activator with a specific activity. Specifically, it contains an active oxygen production inhibitor containing nigarana and / or its fermented product, nigarana and / or its fermented product. Leukotrienes production inhibitor, histamine release inhibitor containing nigana and / or fermented product thereof, IL containing nigana and / or fermented product thereof IL-6 release inhibitor containing -1β release inhibitor, nega and / or its fermented product, nega and / or combined active agent for inhibiting active oxygen production / leukotrienes production containing fermented product thereof, And / or the active oxygen production inhibition containing the fermented product thereof, leukotrienes production inhibition, histamine release inhibition combined active agent, niobana and / or the fermented product containing active oxygen production inhibition, leukotrienes production inhibition, histamine Complex active agents such as complex activators of release suppression, IL-1β release inhibition, and IL-6 release inhibition (these may be collectively referred to as “pharmaceutical compositions”) can be exemplified. In addition, the functional food of the present invention includes at least one of active oxygen production inhibitory activity, leukotrienes production inhibitory activity, histamine release inhibitory activity, IL-1β release inhibitory activity, and IL-6 release inhibitory activity. It is particularly limited if it is a food that has an activity and contains a Japanese charcoal and / or fermented product thereof and is labeled for use in improving inflammation and allergic diseases due to these activities. It is not something.

  The cruciferous plant used in the present invention is a plant belonging to the Asteraceae family, and the use site may be an underground part such as a root or an above-ground part such as a stem, a leaf, or a flower. A mixture of the above or whole grass may be used. Of these, leaves are particularly preferred. As the treatment of the fermented product of the Japanese algae, it is preferable to pulverize the Japanese apricot before the fermentation because a ground contact area with the fermenting bacteria can be sufficiently secured and the fermentation can be effectively advanced. Specifically, a treatment of pulverizing to a particle size of 3 mm or less, preferably 0.5 mm to 1.0 mm is preferable.

Further, in the above fermentation treatment, it is preferable to add 2 to 10 parts by weight, particularly about 5 parts by weight, of water to 1 part by weight of dry matter in order to promote the progress of fermentation to the ground crushed product. Microorganisms are added to the pulverized product. The microorganism is preferably mixed in advance after culturing and before adding to the medium, and added in an amount of 1 to 10% by weight based on the weight of the plant in the case of a dry body. The fermentation is performed at a temperature of 20 to 50 ° C., preferably 40 ° C., and the fermentation time can be appropriately selected depending on the progress of the fermentation depending on the conditions such as pH and the number of bacteria. For example, the pH is 4 to 5 and the number of bacteria is 10 If it is ⁶ or more, it can be about 72 hours. At the time of fermentation treatment, aeration or deoxygenation treatment can be performed as necessary, but fermentation can be performed in static culture after the deoxygenation treatment. The fermentation format is preferably solid culture rather than liquid culture.

  As the microorganism used for the fermentation treatment, lactic acid bacteria, yeast, Bacillus subtilis can be used, and these can be used alone or in combination of two or more, but lactic acid bacteria, lactic acid bacteria and yeast, lactic acid bacteria and Bacillus subtilis, or A combination of lactic acid bacteria, yeast and Bacillus subtilis is particularly preferred.

Examples of the lactic acid bacteria used in the fermentation treatment include Streptococcus, Lactobacillus, Leuconostoc, Pediococcus, Bifidobacterium, and Tetrageno. Bacteria belonging to any of the genera Tetragenococcus are preferred, and Lactobacillus is particularly preferred. The bacterium belonging to the genus Streptococcus is preferably Streptococcus thermophilus (S. thermophilus), and specific examples include Streptococcus thermophilus IFO13957 strain. Moreover, as a bacterium belonging to the genus Lactobacillus, either Lactobacillus plantarim (L. plantrum), Lactobacillus delbruckii (L. delbruckii), Lactobacillus pentosus (L. pentosus) or Lactobacillus casei (L. casei) It is preferable that the bacterium belongs, and among these bacteria, Lactobacillus plantarim is particularly preferable. Specific examples of such Lactobacillus plantarim include IFO14712 and IFO14713 strains, Lactobacillus delbriqui as IFO13953 strain, Lactobacillus pentosus as IFO12011 strain, and Lactobacillus casei as IFO15883 strain. Moreover, as a microbe which belongs to Tetragenococcus genus, it is preferable that it is Tetrageno halophyllus (T. halophilus), and tetrageno halophyllus IFO12172 strain can be illustrated concretely. These lactic acid bacteria are preferably used in an amount of usually 10 ³ to 10 ⁷ , especially 10 ⁶ to 10 ⁷ , per 1 g of dried dry matter of the plant.

The yeast used for the fermentation treatment is preferably a bacterium belonging to the genus Candida or Saccharomyces. As such a bacterium belonging to the genus Candida, Candida versatilis is preferable, and IFO10038 strain can be specifically exemplified as Candida versatilis. The bacterium belonging to the genus Saccharomyces is preferably S. cerevisiae, and IFO0555 strain can be specifically exemplified as Saccharomyces cerevisiae. It is preferable to use 10 ³ to 10 ⁷ , especially 10 ⁶ to 10 ^{7 of} these yeasts per 1 g of dried dry matter of the plant.

Further, Bacillus subtilis (B. subtilis) IFO3013 strain can be specifically exemplified as the Bacillus subtilis used in the fermentation treatment. These Bacillus subtilis are preferably used in an amount of usually 10 ³ to 10 ⁷ , especially 10 ⁶ to 10 ⁷ , per 1 g of dried dry matter of the Japanese algae plant.

  In the above fermentation treatment, the microorganism group preferably used is a microorganism group including lactic acid bacteria, yeast and Bacillus subtilis, and among these microorganism groups, a mixed bacteria of Lactobacillus plantarim, Streptococcus thermophilus, Bacillus subtilis. It is preferable to use the same number as the number of bacteria with respect to the dry matter of the Japanese algae plant. By using the bacteria in such a combination of the number of bacteria, the fermentation time can be shortened, and thus the propagation of various bacteria can be suppressed.

  In addition, it is preferable to dry so that a moisture value may be 10 weight% or less after completion | finish of fermentation, as a drying method, it can be by heat drying or freeze-drying, and in the case of heat drying, product temperature is It is preferable to be performed at 100 ° C. or lower because the deactivation of the physiologically active component can be prevented. After drying, if necessary, sterilization is carried out by a known method such as heating, and the pharmaceutical composition of the present invention (reactive oxygen production inhibitor, leukotrienes production inhibitor, histamine release, containing nigana and / or its fermented product) Inhibitors, IL-1β release inhibitors, IL-6 release inhibitors, combined active agents for inhibiting active oxygen production / leukotrienes production, active oxygen production inhibiting / leukotrienes production inhibiting / histamine release inhibiting composite active agents, activity A fermented product of Japanese charcoal that can be suitably used for oxygen production inhibition, leukotrienes production inhibition, histamine release inhibition, IL-1β release inhibition, and IL-6 release inhibition).

  The nigana and / or fermented product thereof of the present invention can also be an extract of nigana and / or fermented product thereof obtained using an extraction solvent, and the extraction solvent includes water, methyl alcohol, and ethyl alcohol. 1 type or 2 or more types of lower monohydric alcohols, such as liquid polyhydric alcohols, such as glycerol, propylene glycol, and 1, 3- butylene glycol, nonpolar solvents, such as hexane, and polar solvents, such as ethyl acetate. Can do. Among these, methanol, ethanol, and ethyl acetate are preferable.

  The pharmaceutical composition of the present invention includes a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a diluent, a pH buffer, a solubilizing agent, as necessary. An isotonic agent or the like can be added to prepare a solid preparation such as a tablet, granule, powder, powder or capsule, or a liquid preparation such as a normal solution, suspension or emulsion. These preparations can be administered orally or parenterally. That is, it can be administered orally in commonly used dosage forms, such as powders, granules, capsules, syrups, suspensions, etc., or, for example, in dosage forms such as solutions, emulsions, suspensions, etc. These can be administered parenterally in the form of injections or can be administered intranasally in the form of sprays. The dose can be appropriately selected depending on the type of disease, the weight of the patient, the dosage form, and the like. The dosage of such a preparation is appropriately set according to the preparation form, administration method, purpose of use and age, weight, and symptoms of the patient applied thereto, and is not constant, but in general, the active ingredient contained in the preparation The amount is 10 μg to 200 mg / kg per adult day. Since the dose varies depending on various conditions, an amount smaller than the above dose may be sufficient or may be necessary beyond the range.

  Since the pharmaceutical composition of the present invention has an active oxygen production inhibitory activity, a leukotrienes production inhibitory activity, a histamine release inhibitory activity, an IL-1β release inhibitory activity, and an IL-6 release inhibitory activity, the prevention of diseases involving them. And can be used as a therapeutic agent. Specifically, since it has an active oxygen production inhibitory activity, various pathologies and diseases involving active oxygen such as arteriosclerosis, cerebral infarction, cerebral ischemia-reperfusion injury, ulcer, malignant tumor, diabetes, epilepsy, It can be used as a preventive and therapeutic agent for aging, inflammation, etc. and has leukotrienes production inhibitory activity, so it exhibits therapeutic effects on inflammation and other diseases caused by an increase in leukotrienes, and has histamine release inhibitory activity. Therefore, it can be used as a preventive and therapeutic agent for diseases associated with histamine release, such as hay fever, asthma, hay fever, rhinitis, urticaria and drug allergy. Furthermore, since it has IL-1β release inhibitory activity and IL-6 release inhibitory activity, prevention and treatment of inflammatory bowel diseases such as cancer, arteriosclerosis, chronic lymphadenitis, rheumatoid arthritis, colitis, Crohn's disease, etc. It can be used as an agent.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Adjustment of fermented negatives]
30 g of dried Japanese algae leaves were pulverized to a particle size of 0.1 to 3 mm and placed in a container. 150 g of water, 0.9 g of molasses, and 0.9 g of rice bran were added to the container containing the nigana. After culturing each strain of Lactobacillus plantarim (IFO14712 strain, IFO14713 strain), Streptococcus thermophilus (IFO13957 strain), and Bacillus subtilis (IFO3013 strain) in a container containing the pulverized Japanese algae, the number of bacteria was 1: 1. The mixture was mixed at a ratio of 1: 10% by weight with respect to the weight of the nigana, the container was sealed, and fermentation was performed by stationary culture. The fermentation temperature was 40 ° C. and the fermentation time was 72 hours. Then, after drying at 60 degreeC until the moisture value became 10 weight% or less with a dryer, the sterilization process (130 degreeC steam, 5 to 15 second) was performed, and 31 g of fermented Japanese charcoal was obtained.

[Sample preparation]
(i) 0.1 to 3 mm crushed and dried leaves of nigana, fermented nigana obtained in Example 1, 10 ml of methanol per 1 g, extracted by standing for 12 hours, and after filtration, 10 ml of methanol was added to the residue The same extraction was performed to obtain a methanol extract. A sample with a final concentration of 25 μg / mL was prepared by diluting the methanol extract with dimethyl sulfoxide (DMSO).
(ii) 0.1 to 3 mm crushed and dried leaves of nigana and 10 ml of ethyl acetate per 1 g of fermented nigana obtained in Example 1 and extracted by standing for 12 hours. After filtration, ethyl acetate was added to the residue. 10 ml was added and extracted in the same manner to obtain an ethyl acetate extract. A sample with a final concentration of 25 μg / mL was prepared by diluting the ethyl acetate extract with dimethyl sulfoxide (DMSO).

[Evaluation of active oxygen production inhibitory activity of Japanese and Japanese broths]
The active oxygen production amount was measured about the 0.1-3 mm crushed dried leaf of Japanese agar and the extract of the fermented Japanese agar obtained in Example 2, and the active oxygen production inhibitory activity was evaluated. Adjust the concentration of HL-60 cells to 4 × 10 ⁵ cells / ml and add dimethyl sulfoxide to a final concentration of 1.25% in a CO ₂ incubator at 37 ° C. for 4 days. The cells were cultured. Cells differentiated into neutrophilic granulocytes were collected, washed with HBSS (Hanks' Balanced Salt Solution), resuspended, and adjusted to 1 × 10 ⁶ cells / ml. The sample prepared in Example 2 (final concentration: 25 μg / ml) or 1 μl of dimethyl sulfoxide (control) and 1 ml of the cell suspension were mixed and incubated in a 37 ° C. water bath for 15 minutes. After washing with HBSS, it was resuspended in 1 ml and incubated in a 37 ° C. water bath for 5 minutes. TPA (Phorbol 12-Myristate 13-Acetate) was added so that the final concentration was 2 μM (EtOH dilution), and after 90 seconds, 50 μl of Cytochrome C (20 mg / ml) was added (final concentration: 76 μM). . After incubating in a 37 ° C. water bath for 15 minutes, the mixture was allowed to stand in ice for 5 minutes, centrifuged (6600 rpm, 5 minutes), and the absorbance of the supernatant at 550 nm was measured with a spectrophotometer (UV-2200AI: manufactured by Shimadzu Corporation). It was measured. The difference of the absorbance TPA whether the A ₅₅₀ in each sample by the following equation (Meth.Enzymol :, 105,358-365,1984), superoxide anion radicals _- was calculated amount of ^(· O ^{2). ·} In methanol extract and ethyl acetate extract O ₂ ^- shows the production of results in FIGS. 1 and 2, respectively.

_{^{· O 2 - (nmol / ml}} ) = 47.7 × A 550
Both the methanol extract and ethyl acetate showed an active oxygen production inhibitory activity. In both cases, the active oxygen production inhibitory activity was enhanced after fermentation. From this, it became clear that Japanese algae and / or its fermented product has an active oxygen production inhibitory activity.

[Measurement of leukotrienes production inhibitory activity of Japanese and fermented Japanese teas]
The leukotriene (LT) production inhibitory activity was measured for 0.1 to 3 mm pulverized and dried leaves of Japanese algae and the fermented Japanese algae extract obtained in Example 2 (ii).

[reagent]
(1) Preparation of 10 × PBS After 0.2M NaH ₂ PO ₄ and 0.2M Na ₂ HPO ₄ are mixed and adjusted to pH 7.0, 3.0M NaCl is dissolved, and the volume is increased to 2 times. The mixture was autoclaved at 120 ° C. for 20 minutes to prepare 0.1 M PBS (phosphate buffer containing 0.15 M NaCl) (10 × PBS).
(2) Preparation of 1 × PBS A 10 × PBS solution was diluted 10 times and autoclaved at 120 ° C. for 20 minutes to prepare 0.01M PBS (1 × PBS).
(3) Preparation of CaCl ₂ solution 1.47 g of CaCl ₂ · 2H ₂ O was dissolved in 100 ml of 1 × PBS so that the final concentration was 1 mM, and autoclaved at 120 ° C. for 20 minutes to prepare a CaCl ₂ solution. . This CaCl ₂ solution was stored at 4 ° C.
(4) Preparation of calcium ionophore A23187 382 μl of methanol was added to 1 mg of A23187 (manufactured by Sigma) to form a 5 mM stock solution and stored at −20 ° C. This stock solution was diluted 10-fold with 1 × PBS and prepared at the time of use so that the final concentration was 5 μM.
(5) Preparation of 0.4% dichitonin solution 400 mg of dichitonin, 400 mg of tritron X-100, and 100 ml of MillQ water were mixed to prepare a 0.4% dichitonin solution.

RBL2H3 cells (obtained from Kitasato University, Faculty of Pharmaceutical Sciences, Hygienic Chemistry) were seeded in a 6-well plate (medium 3 ml / well) at 1.0 × 10 ⁵ cells / ml and cultured for 2 days. The medium was removed, and the sample prepared in Example 2 (ii) was added to 1 ml of fresh medium to a final concentration of 25 μg / ml. Thereafter, the cells were incubated at 37 ° C. for 1 hour, the medium was removed, and the cells were washed twice with 1 ml of 1 × PBS. 1 ml of 1 × PBS was newly added and 10 μl of CaCl ₂ solution was added (final concentration 1 mM), followed by incubation at 37 ° C. for 5 minutes. Subsequently, 10 μl of A23187 (final concentration 5 μM) was added, and the mixture was incubated at 37 ° C. for 5 minutes for stimulation. Immediately after the reaction, the culture supernatant was collected in an eppen and centrifuged (4 ° C., 2000 rpm, 3 minutes) to remove floating cells. The supernatant was collected from the LTC ₄ EIA kit (Cayman) and Cysteinyl-LT EIA. A kit (Cayman) was used. Further, 0.4% dichitonin solution was added to the cells after collecting the supernatant, shaken at 37 ° C. for 10 minutes, collected in an eppen, and centrifuged (4 ° C., 5000 rpm, 5 minutes). Using the supernatant, proteins were quantified by the BCA method shown below. The results obtained with the EIA kit were corrected as values per protein amount, and the amounts of LTC4 and Cysteinyl-LTs were calculated.

[Protein quantification by BCA method]
The collected cell samples were dispensed in triplicate at 10 μl in a 96-well microplate. As a calibration curve, 0.2 mg / ml to 1.0 mg / ml BSA was similarly added. BCA Protein Assay Reagent A (manufactured by Pierce) and BCA Protein Assay Reagent B (manufactured by Pierce) were mixed at a ratio of 50: 1, and 195 μl was quickly added to the microplate. After incubating at 37 ° C. for 30 minutes, the absorbance at 562 nm was measured. A calibration curve was created and the protein concentration of the sample was calculated.

  FIGS. 3 and 4 show the amounts of LTC4 and Cysteinyl-LTs in the ethyl acetate extract (diluted with DMSO) of Japanese algae and its fermented product. The case where 1 μl of DMSO was used in place of the ethyl acetate extract of Japanese algae and its fermented product was used as a control. The amount of LTC4 and Cysteinyl-LTs was suppressed for both of the extract of Nagana and its fermented product. In particular, the inhibitory activity of the fermented product was remarkable. From this, it became clear that Japanese algae and / or its fermented processed product has leukotrienes production inhibitory activity.

[Evaluation of inhibition of histamine release by Japanese and fermented Japanese tea]
Histamine release inhibitory activity was measured for each of the extract of Japanese agar and fermented Japanese agar obtained in (i) of Example 2.

[Adjustment of Tyrode buffer (pH 7.2)]
Ultrapure water containing 1.6 g of 137 mM sodium chloride (NaCl), 40.3 mg of 2.7 mM potassium chloride, 40.7 mg of 1.8 mM magnesium chloride (MgCl ₂ ), and 201.8 mg of 5.6 mM glucose. Dissolved in. The solution was adjusted to pH 7.2 with 201.6 mg of 12 mM sodium hydrogen carbonate (NaHCO 3) or 28.7 mg of 0.4 mM sodium dihydrogen phosphate (NaH ₂ PO ₄ ), and Tyrode buffer (pH 7. 2) was adjusted. This buffer was stored at room temperature.

KU812 cells (cultured cells derived from human basophils) were obtained from Research Resource Bank, Human Science Foundation. KU812 cells were cultured in 10% FBS RPMI-1640 medium. KU812 cells (5 × 10 ⁶ cells / ml) were seeded in a 35 mm dish, and the sample prepared in Example 2 (i) was treated as a test substance for 30 minutes. The cells were collected in an Eppendorf tube and then washed twice with PBS. After removing the supernatant and suspending the cells with 200 μl of Tyrode buffer, 10 μl of CRA-1 (1 mg / ml) was added and incubated at 37 ° C. for 30 minutes. The reaction was stopped by cooling, centrifuged (4 ° C., 300 g, 5 minutes), and the amount of histamine released in 100 μl of the supernatant was quantified by fluorescence measurement (E _m ; 355 nm, E _x ; 460 nm). The measurement scheme of histamine release amount and the results of histamine release amount are shown in FIG.

  As shown in FIG. 5, it was clarified that nigana and / or its fermented product exhibited histamine release inhibition in a concentration-dependent manner.

[Evaluation of inhibition of inflammatory cytokine IL-1b production by Japanese and fermented Japanese monkeys in mouse peritoneal macrophages stimulated with dextran sulfate (DSS)]
IL-1b production in monocyte-derived peritoneal macrophages of mice stimulated with dextran sulfate (DSS) was measured for 0.1 to 3 mm ground and dried leaves of Japanese nigga, the Japanese algae obtained in Example 1, and the fermented Japanese agar.

Monocyte-derived peritoneal macrophages were prepared as described in the article (Kwon 2002). Dulbecco supplemented with 10% fetal bovine serum (FBS; manufactured by Gibco BRL), penicillin (100 U / ml), and streptomycin (100 μg / ml) so that the peritoneal exudate cells were in a 96-well plate at 1 × 10 ⁶ cells / well. The cells were seeded in a modified Eagle medium (DMEM; manufactured by Gibco BRL) and incubated at 37 ° C. under 5% CO ₂ . After 24 hours of incubation, peritoneal macrophages were stimulated with 10 mg / ml DSS and treated with various concentrations of Japanese algae and fermented Japanese algae in DMSO (4 mg / ml to 100 mg / ml) for 24 hours as test substances. Thereafter, the mixture was centrifuged (1900 g, 4 ° C., 15 minutes), and the concentrations of IL-1β and IL-6 in the supernatant (dilution factor = 20) were measured using an ELISA kit according to the manufacturer's instructions.

  As shown in FIG. 6, the negative and fermented negative obtained in Example 1 showed an inhibitory effect on IL-1b production in mouse peritoneal macrophages.

[Evaluation of inhibition of production of inflammatory cytokines IL-1β and IL-6 by dextran sulfate (DSS) in the mucosa of the large intestine of mice]
About dextran sulfate (DSS) about 0.1-3 mm ground dried leaves of Japanese algae, fermented Japanese algae obtained in Example 1 and luteolin (LT) and luteolin-6-glucoside (LT-G) contained therein IL-1b and IL-6 production in the large intestine mucosa of mice administered with the above was measured.

  Induction of colitis in mice was performed as shown in FIG. That is, a mouse (Group 1) to which drinking water and food (MF pellet; manufactured by Oriental Yeast Co., Ltd.) can be freely given was used as a control mouse. Normal drinking water is given for the first 7 days, drinking water to which 3% (w / v) DSS is added for the next 7 days, and food is freely given to mice (Group 2). A model mouse was used. Normal drinking water is given for the first 7 days, drinking water to which 3% (w / v) DSS is added is given for the following 7 days, and the food is supplemented with nigana or fermented nigana as a test substance. Mice (Group 3 or 4) given from the beginning to the end of the study (14 days) were used as study mice.

  The colons without appendix were removed from the mice of groups 1 to 4 (each group n = 3) after the protocol for development of colitis. After washing with ice-cold PBS, the specimen was placed on filter paper, and the length of the large intestine was measured (see FIG. 8). The large intestine was opened with surgical scissors and the contents were removed. The colonic mucosa was rubbed off with a razor while the specimen was frozen with liquid nitrogen. The colonic mucosa exfoliation specimen was finely chopped with surgical scissors, and the specimen was crushed in ice-cold PBS using a homogenizer. To obtain the supernatant, the homogenized tissue was centrifuged (1900 g, 4 ° C., 15 minutes). The concentrations of IL-1β and IL-6 in the supernatant (dilution factor = 20) obtained by centrifugation from the large intestine exfoliated mucosa were measured using an ELISA kit according to the manufacturer's instructions. The result was corrected as a value per protein amount. The total protein concentration in the tissue supernatant was determined by using DC Protein Assay kit (manufactured by Bio-Rad Laboratories) using γ-globulin as an internal standard according to the manufacturer's instructions. The results are shown in FIGS.

  From these results, the length of the large intestine in group 3 mice (nigana) and group 4 mice (fermented nigana) was less changed compared to group 2 mice (colitis model mice; DSS). Close to mouse (control; CT). In addition, the production amounts of IL-1β and IL-6 in group 3 mice (nigana) and group 4 mice (fermented nigana) are the same as IL-1β and IL-6 in group 2 mice (colitis model mice; DSS). It was found that the amount of -6 and 6 was less than that of -6, and even in vivo, the intestinal membrane mucosa such as IL-1β and IL-6 showed an inhibitory effect on the production of inflammatory cytokines.

It is a figure which shows the result of having measured the amount of active oxygen production in the methanol extract of the Japanese red crab and fermented Japanese char. It is a figure which shows the result of having measured the amount of active oxygen production in the ethyl acetate extract of the Japanese algae and fermented Japanese agar. It is a figure which shows the result of having measured the amount of LTC4 in the ethyl acetate extract of the Japanese algae of this invention and fermented Japanese agar. It is a figure which shows the result of having measured the amount of Cysteinyl-LTs in the ethyl acetate extract of the Japanese leek of this invention, and fermented Japanese agar. It is a figure which shows the measurement scheme of the result of having measured the amount of histamine release in the Japanese leek of this invention, and fermentation Japanese agar and the amount of histamine release. It is a figure which shows the result of having measured the production amount of IL-1 (beta) in the mouse | mouth peritoneal macrophage which stimulated with DSS in the Japanese algae and fermented Japanese agar of this invention. It is a figure which shows the colon inflammation generation | occurrence | production protocol of the DSS administration mouse | mouth in the Japanese algae of this invention and fermented Japanese agar. It is a figure which shows the result of large intestine length change of the DSS administration mouse | mouth in the naganas and fermented nigana of this invention. It is a figure which shows the result of the IL- (beta) production amount in the large intestine mucosa of the DSS administration mouse | mouth in the naganas and fermented nigana of this invention. It is a figure which shows the result of the production amount of IL-6 in the large intestine mucosa of the DSS administration mouse | mouth in the naganas of this invention and fermented nigana.

Claims (10)

Japanese red crab (Crepidiastrum lanceolatum Nakai) having at least one of active oxygen production inhibitory activity, leukotrienes production inhibitory activity, histamine release inhibitory activity, IL-1β release inhibitory activity, and IL-6 release inhibitory activity, and Or an active oxygen production inhibitor, leukotrienes production inhibitor, histamine release inhibitor, IL-1β release inhibitor, or IL-6 release inhibitor, or an extract of the fermented product thereof, or two or more of the above A complex active agent with activity. After pulverizing the leaves of Japanese green leaf to a particle size of 0.1 to 3.0 mm, 2 to 10 parts by weight of water is added to 1 part by weight of the pulverized product, and further a microorganism group is added for fermentation treatment. An active oxygen production inhibitor, a leukotrienes production inhibitor, a histamine release inhibitor, an IL-1β release inhibitor, or an IL according to claim 1, comprising an extract obtained by extracting the fermentation product with a solvent. -6 release inhibitor, or a combined active agent having two or more activities. The active oxygen production inhibitor according to claim 1 or 2, wherein the fermented product of Japanese algae is obtained by fermentation with lactic acid bacteria, lactic acid bacteria and yeast, lactic acid bacteria and Bacillus subtilis, or lactic acid bacteria, yeast and Bacillus subtilis, A leukotriene generation inhibitor, a histamine release inhibitor, an IL-1β release inhibitor, or an IL-6 release inhibitor, or a combined active agent having two or more of the above activities. The active oxygen production inhibitor, leukotrienes production inhibitor, histamine release inhibitor according to any one of claims 1 to 3, wherein 1 to 10% by weight of a microorganism group is added to the weight of the nigana. IL-1β release inhibitor, IL-6 release inhibitor, or a combined active agent having two or more activities. The extract solvent contains nigana obtained by using at least one or more of methanol, ethanol, ethyl acetate or a mixed solvent thereof and / or an extract of the fermented product thereof. An active oxygen production inhibitor, a leukotrienes production inhibitor, a histamine release inhibitor, an IL-1β release inhibitor, or an IL-6 release inhibitor according to any one of the above, or a composite active agent having two or more activities. Japanese red crab (Crepidiastrum lanceolatum Nakai) having at least one of active oxygen production inhibitory activity, leukotrienes production inhibitory activity, histamine release inhibitory activity, IL-1β release inhibitory activity, and IL-6 release inhibitory activity, and A functional food containing an extract of a fermented product thereof and / or a label indicating that it is used for improving inflammation and allergic diseases due to these activities. After pulverizing the leaves of Japanese green leaf to a particle size of 0.1 to 3.0 mm, 2 to 10 parts by weight of water is added to 1 part by weight of the pulverized product, and further a microorganism group is added for fermentation treatment. The functional food according to claim 6, further comprising an extract obtained by extracting the fermentation product with a solvent. The functional food according to claim 6 or 7, wherein the fermented product of Japanese algae is obtained by fermentation with lactic acid bacteria, lactic acid bacteria and yeast, lactic acid bacteria and Bacillus subtilis, or lactic acid bacteria, yeast and Bacillus subtilis. The functional food according to any one of claims 6 to 8, wherein 1 to 10% by weight of a microbial group is added to the weight of the nigana. The extract solvent contains nigana obtained by using at least one of methanol, ethanol, ethyl acetate or a mixed solvent thereof and / or an extract of the fermented product thereof. The functional food in any one.