WO2015156339A1 - 免疫バランス調整剤 - Google Patents
免疫バランス調整剤 Download PDFInfo
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- WO2015156339A1 WO2015156339A1 PCT/JP2015/061043 JP2015061043W WO2015156339A1 WO 2015156339 A1 WO2015156339 A1 WO 2015156339A1 JP 2015061043 W JP2015061043 W JP 2015061043W WO 2015156339 A1 WO2015156339 A1 WO 2015156339A1
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- paramylon
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- immune balance
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/10—Protozoa; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the present invention relates to a novel immune balance regulator.
- An immune system that excludes foreign substances from living organisms has a complicated mechanism involving various cells and cytokines.
- the balance between cellular immunity and humoral immunity is known.
- Cellular immunity is a reaction in which antigens are mainly eliminated by killer T cells and macrophages
- humoral immunity is a reaction in which antigens are mainly eliminated by antibodies produced by B cells. The combination of these two mechanisms eliminates antigens.
- Patent Document 1 foods and drinks that contain lactic acid bacteria or processed products thereof and have been normalized while maintaining a balance between cellular immunity and humoral immunity.
- Patent Document 1 the food / beverage product of patent document 1 can adjust the immune balance of cellular immunity and humoral immunity, since it contains the substance derived from lactic acid bacteria, there is a flavor peculiar to lactic acid bacteria, Only applicable.
- helper T cells are classified into three subtypes: type 1 helper T cell (Th1), type 2 helper T cell (Th2), and type 17 helper T cell (Th17). It has been found for mice and humans that Th2 and Th17 that induce humoral immunity control diseases related to various immune responses. Th1, Th2 and Th17 are balanced with each other, and if this balance is maintained, the risk of becoming a given disease can be suppressed, but if the balance is lost, it will lead to the onset and progression of various diseases Has been speculated. It is thought that when the Th1 / Th2 / Th17 balance in an appropriate state is biased toward the Th1 side, cellular immunity becomes too strong and autoimmune diseases such as rheumatoid arthritis may develop.
- the humoral immunity becomes too strong, and it is thought that it is easy to fall into cancer, immunodeficiency, asthma, dermatitis, allergic symptoms, nephritis, infectious diseases, stress-related diseases and the like.
- Th17 a autoimmune disease
- the immune function is not necessarily strong, but is composed of a balance of cellular immunity, humoral immunity, and Th17 immunity.
- Euglena (genus name: Euglena, Japanese name: Euglena) attracts attention as a biological resource that is expected to be used as food, feed, fuel, and the like.
- Euglena has 59 kinds of nutrients that correspond to most of the nutrients necessary for humans to live, such as vitamins, minerals, amino acids, and unsaturated fatty acids, and as a supplement to take a variety of nutrients in a balanced manner.
- the possibility of use as a food supply source in poverty areas where the necessary nutrients cannot be ingested has been proposed.
- Euglena is located at the bottom of the food chain, and it is difficult to mass culture because it is preyed by predators and the culture conditions such as light, temperature conditions, and stirring speed are difficult compared to other microorganisms.
- a mass culture technique has been established, which has opened the way for mass supply of paramylon.
- Euglena is a unique organism that possesses the animal nature of flagellar movement and at the same time has chloroplasts as a plant and performs photosynthesis, and Euglena itself and substances derived from Euglena are expected to have many functions. However, many of its functions and mechanisms of functional expression remain unknown.
- Euglena-derived substances such as Euglena and Paramylon that can be supplied in large quantities, and the mechanism of functional expression, and to develop a method for using these substances.
- processed products such as paramylon and amorphous paramylon do not impair the flavor of the base food and drink even when mixed with food and drink such as cookies, biscuits, chips, Japanese sweets, and smoothies.
- the present invention has been made in view of the above problems, and an object of the present invention is to provide a novel immune balance regulator that regulates Th1 / Th2 / Th17 immune balance of a living body. Another object of the present invention is to provide an immune balance regulator that provides a novel method of using Euglena-derived substances.
- the present inventors have produced euglena itself, paramylon, processed products of paramylon, etc., when a Euglena-derived substance such as a processed product is administered to a living body, the production of specific cytokines involved in the immune system And the production of other cytokines was suppressed. That is, the immune system of the living body is composed of various cells and cytokines having different functions such as T cells, B cells, and a large number of cytokines that interact with each other.
- Th1 / Th2 / Th17 immune balance which is a balance of immune responses induced by Th1, Th2 and Th17 in a living body, according to the immune balance regulator of the present invention, including a Euglena-derived substance.
- the immune balance regulator of the present invention including a Euglena-derived substance.
- a constitution improving agent that improves the undesirable constitution caused by the deviation of Th1 / Th2 / Th17 immune balance toward Th1, Th2, or Th17, Th1 side, Th2 or Th17 It can be used as a disease preventive agent, therapeutic agent, etc. for a disease caused by side bias.
- the Th1 / Th2 / Th17 immune balance of the living body can be adjusted to an appropriate balance that does not bias to either Th1, Th2, or Th17, healthy individuals who do not develop disease, elderly people whose immunity age has increased, etc. Although it is not used as an immune balance regulator for physical condition management or suffering from a specific disease, the period of poor physical condition persists for a certain period or more because the immune balance is biased to Th1, Th2 or Th17 It can be used as an agent for improving the physical condition of a person who is living with the disease, or as a natural remedy for a patient with a disease that develops due to the biased Th1 / Th2 / Th17 immune balance of the living body.
- the immune balance regulator of the present invention since Euglena-derived substances are used as active ingredients, even if the immune balance regulator of the present invention is configured as a food, beverage, supplement or the like containing Euglena-derived substances, the flavor may be impaired by the active ingredients. Therefore, it is possible to provide an immune balance regulator in a form that is easy to take.
- the immune balance regulator adjusts the Th1 / Th2 / Th17 immune balance in such a direction that the immune response induced by Th1 is relatively superior to the immune response induced by Th2 or Th17. May be.
- the Th1 / Th2 / Th17 immune balance may be used to improve the constitution biased to Th2. Because it is configured in this way, it is not enough to receive treatment or treatment by a doctor, but it can improve poor physical condition caused by Th1 / Th2 / Th17 immune balance being biased to Th2, and QOL (quality of life) Can be improved.
- the constitution in which the Th1 / Th2 / Th17 immune balance is biased to Th2 may be a constitution that is susceptible to infection or stress-related diseases.
- the Th1 / Th2 / Th17 immune balance may be used for prevention or treatment of a disease associated with Th2 bias. Since it is configured in this way, the present invention is not limited to treatments mainly composed of synthetic drugs, and can also be used for natural medicine for adjusting immune balance. As a disease in which the Th1 / Th2 / Th17 immune balance is biased toward Th2, it can be used for prevention or treatment of, for example, cancer, immunodeficiency, asthma, dermatitis, allergic disease, nephritis, infection and the like.
- the disease involving the Th1 / Th2 / Th17 immune balance bias to Th2 may be an infectious disease or a stress-related disease.
- the infectious disease is influenza, and may be used as an anti-influenza agent.
- the stress-related disease is a peptic ulcer and may be used as a preventive or therapeutic agent for peptic ulcer.
- the ratio of the production amount of IFN- ⁇ to the production amount of IL-4 in the living body may be increased. Further, the production of IFN- ⁇ in the living body may be promoted, and the production of IL-4, IL-5 and IL-10 may be suppressed.
- Th1 / Th2 / Th17 immune balance may be adjusted in such a way that the immune response induced by Th2 is relatively superior to the immune response induced by Th1 or Th17.
- the Th1 / Th2 / Th17 immune balance may be used to improve the constitution predisposed to Th1 and / or Th17.
- Th1 / Th2 / Th17 immune balance is biased toward Th1 and / or Th17.
- the constitution biased to Th17 may be a constitution predisposed to diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, and inflammatory bowel disease.
- diseases involving the Th1 / Th2 / Th17 immune balance deviation to Th1 and / or Th17 such as diabetes, liver injury, airway inflammation, host versus graft reaction, chronic joints It can be used for prevention or treatment of rheumatism, multiple sclerosis, arteriosclerosis, psoriasis, gastritis and the like.
- Th17 bias can be used for prevention or treatment of diseases associated with Th17 bias, such as rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease and the like. It is used for the prevention or treatment of a disease involving a Th1 / Th2 / Th17 immune balance bias to Th1 and / or Th17, and the disease may be rheumatoid arthritis.
- the Th1 / Th2 / Th17 immune balance may be administered prior to the time when the onset of a disease involving a deviation of Th1 and / or Th17 is predicted.
- the Euglena-derived substance may be paramylon or a processed product thereof.
- paramylon or a processed product thereof is used as an active ingredient, even if the immune balance regulator of the present invention is configured as a food, beverage, supplement or the like containing paramylon or a processed product thereof, It is possible to provide an immune balance regulator in a form that is easy to take without losing the flavor.
- the immune balance regulator of the present invention adjusts the Th1 / Th2 / Th17 immune balance of the living body, and thus arises due to the Th1 / Th2 / Th17 immune balance being deflected to Th1, Th2, or Th17.
- It can be used as a constitution improving agent for improving an unfavorable constitution, a disease preventive agent, a therapeutic agent, etc. for a disease caused by Th1 or Th2 or Th17 bias.
- the Th1 / Th2 / Th17 immune balance of the living body can be adjusted to an appropriate balance that does not bias to either Th1, Th2, or Th17, healthy individuals who do not develop disease, elderly people whose immunity age has increased, etc. Although it is not used as an immune balance regulator for physical condition management or suffering from a specific disease, the period of poor physical condition persists for a certain period or more because the immune balance is biased to Th1, Th2 or Th17 It can be used as an agent for improving the physical condition of a person who is living with the disease, or as a natural remedy for patients with diseases that develop due to the biased Th1 / Th2 / Th17 immune balance in the living body.
- paramylon or a processed product thereof is used as an active ingredient, even if the immune balance regulator of the present invention is configured as a food, beverage, supplement or the like containing paramylon or a processed product thereof, the active ingredient has a flavor. It is possible to provide an immune balance regulator in a form that is easy to take without being damaged.
- mice were mixed with paramylon prepared in Example 2 of the present invention, amorphous paramylon of Example 3 and Euglena of Example 1 for 2 weeks and then infected with influenza virus, and then the virus titer on the second day was measured. It is a graph which shows the result. After the mice were mixed with paramylon prepared in Example 2 of the present invention, amorphous paramylon of Example 3 and Euglena of Example 1 for 2 weeks and then infected with influenza virus, It is a graph which shows the result of having measured the titer.
- Example 2 of the present invention After the mice were mixed with paramylon prepared in Example 2 of the present invention, amorphous paramylon of Example 3 and Euglena of Example 1 for 2 weeks and then infected with influenza virus, IL was administered on days 1, 2 and 3 It is a graph which shows the result of having measured -1 (beta). After the mice were mixed with paramylon prepared in Example 2 of the present invention, amorphous paramylon of Example 3 and Euglena of Example 1 for 2 weeks and then infected with influenza virus, IL was administered on days 1, 2 and 3 It is a graph which shows the result of having measured -6.
- mice After infecting influenza mice with influenza virus after feeding the mice with paramylon prepared in Example 2 of the present invention, amorphous paramylon of Example 3 and Euglena of Example 1 for 2 weeks, IFN was observed on days 1, 2, and 3. It is a graph which shows the result of having measured - ⁇ .
- the mice were fed with paramylon prepared in Example 2 of the present invention, amorphous paramylon of Example 3 and Euglena of Example 1 for 2 weeks, and then infected with influenza virus, and then on days 1, 2, and 3 It is a graph which shows the result of having measured - ⁇ .
- Example 6 After infecting influenza mice with influenza virus after feeding the mice with paramylon prepared in Example 2 of the present invention, amorphous paramylon of Example 3 and Euglena of Example 1 for 2 weeks, IFN was observed on days 1, 2, and 3. It is a graph which shows the result of having measured - ⁇ . In Experiment 6, it is a graph which shows the food intake of 14 days when the food of each group was ingested. In Experiment 6, it is a graph which shows the body weight for 14 days when the food of each group was ingested. In Experiment 6, it is the photograph which image
- Experiment 6 it is a photograph which shows the detection band of iNOS (TM) mRNA, COX-2 (TM) mRNA and ⁇ -actin mRNA.
- TM iNOS
- COX-2 TM
- TM COX-2
- iNOS / ⁇ -actin a graph showing iNOS / ⁇ -actin.
- COX-2 / ⁇ -actin It is a graph which shows the measurement result of the arthritis score in evaluation using the collagen arthritis model of the mouse
- FIG. It is a graph which shows the measurement result of anti-collagen IgG in the evaluation using the collagen arthritis model of the mouse
- 14 is a graph showing measurement results of cytokine (IL-17) in evaluation using a mouse collagen arthritis model of Test Example 7.
- FIG. 10 is a graph showing measurement results of cytokine (IFN- ⁇ ) in evaluation using a collagen arthritis model in mice of Test Example 7. It is explanatory drawing which shows the pathological specimen preparation method of the knee joint for the evaluation of the knee joint tissue in the evaluation using the collagen arthritis model of the mouse
- FIG. It is a photograph which shows the pathological sample of the left knee joint tissue of an untreated group in the evaluation using the collagen arthritis model of the mouse
- IFN- ⁇ cytokine
- FIG. 7 It is a photograph which shows the pathological sample of the left knee joint tissue of the Euglena group in the evaluation using the collagen arthritis model of the mouse
- FIG. It is a photograph which shows the pathological sample of the left knee joint tissue of the paramylon group in the evaluation using the collagen arthritis model of the mouse
- FIG. It is a photograph which shows the pathological sample of the left knee joint tissue of the amorphous paramylon group in the evaluation using the collagen arthritis model of the mouse
- FIG. It is a graph which shows the ratio of the number of IL-17 producing cells in the CD4 positive T cell in the evaluation using the collagen arthritis model of the mouse
- Helper T cells travel through lymphoid tissues in the body and repeat contact with antigen-presenting cells until they encounter antigens such as specific viruses and microorganisms. Naive T cells that have encountered specific antigens repeatedly proliferate to become immature effector T cells (Th0). Th0 is an effector T cell, Th1 or Th2 or Th17, by cytokine stimulation or co-stimulation from antigen-presenting cells. To differentiate. Effector T cells are classified into helper type 1 (Th1), helper type 2 (Th2), and helper type 17 (Th17), which are different types depending on the type of cytokine to be produced.
- Th1 helper type 1
- Th2 helper type 2
- Th17 helper type 17
- Th1, Th2 and Th17 have different types of cytokines to be produced, and activate different immune systems, that is, cellular, humoral and Th17 unique immune systems, respectively.
- cell-mediated immunity the killer T cell itself performs an immune reaction. T cells gather around the antigen, and the antigen is surrounded, attacked and destroyed.
- humoral immunity antibodies are produced by antibody-producing cells, and the antibodies undergo an immune reaction. Antibodies are in the blood and an antigen-antibody reaction occurs throughout the body. Different antibodies are produced depending on the type of antigen, and specifically bind to the antigen to suppress the action of the antigen.
- T cells directly attack the antigen
- humoral immunity creates an antibody
- the antibody specifically binds to the antigen to deactivate the antigen.
- the flow of humoral immunity is as follows.
- antigens such as pathogenic bacteria enter the body
- the antigens are processed by macrophage phagocytosis.
- Macrophages become antigen-presenting cells and present phagocytic antigen information to helper T cells.
- Helper T cells that have recognized the antigen information release cytokines and stimulate specific B cells to promote proliferation.
- Proliferated B cells become antibody-producing cells and produce antibodies.
- Helper T cells also have a role in assisting antibody production.
- the antigen causes an antigen-antibody reaction with the antibody secreted in the body fluid, and is removed by aggregation, precipitation, and dissolution.
- macrophages process antigens that have entered the body, and information on the antigens is transmitted to helper T cells.
- the helper T cell receiving the information stimulates the killer T cell. Stimulated killer T cells proliferate and react directly with the antigen to inactivate the antigen.
- Th1 produces cytokines IFN- ⁇ and IL-2 that cause the activation of cellular immunity, and enhances the activity of killer T cells and macrophages.
- IL-2 performs B cell proliferation, Th1 proliferation and activation.
- IFN- ⁇ also activates macrophages.
- TNF- ⁇ induces IFN- ⁇ production and activates macrophages and is involved in cellular immunity.
- Differentiation into Th1 requires IL-12 secreted by antigen-presenting cells, and IFN- ⁇ produced by Th1 promotes differentiation of Th0 into Th1.
- IL-10 produced by Th2 suppresses IL-12 production of macrophages, and indirectly suppresses Th0 differentiation to Th1 by suppressing IFN- ⁇ production of Th1.
- Th2 produces cytokines IL-4, IL-5, and IL-10 that cause activation of humoral immunity, and promotes B cell activation and antibody production.
- PGE 2 secreted by macrophages during antigen presentation is thought to play an important role in Th2 differentiation.
- IL-4 and IL-6 produced by Th2 promote the differentiation of Th0 into Th2.
- IFN- ⁇ produced by Th1 suppresses Th0 differentiation to Th2.
- IL-4 also activates and proliferates B cells, suppresses Th1 and macrophage activation, and proliferates Th2.
- IL-5 proliferates and differentiates B cells.
- PGE 2 promotes differentiation of Th0 into Th2 and suppresses IFN- ⁇ production.
- Th17 is a new T cell subset discovered recently and is said to be involved in the development of autoimmune diseases. Differentiation from Th0 to Th17 is induced by stimulation with TGF- ⁇ and IL-6. Th17 cells themselves cause IL-17 production through IL-23 expression. In addition, IL-2, IL-6, TNF- ⁇ , etc. are produced.
- Th1 / Th2 / Th17 immune balance refers to the balance of immune responses induced by Th1, Th2 and Th17, respectively, and the immune responses induced by Th1, Th2 and Th17 are antagonistic to each other. However, there is no deflection. On the other hand, the state in which the immune response induced by any one of the effector T cells including Th1, Th2, and Th17 is excessive with respect to other immune responses is a state in which the effector T cells are excessive.
- examples of “the immune response induced by Th1 is adjusted in a direction that is relatively superior to the immune response induced by Th2 or Th17” include an immune response induced by Th2 or Th17. To suppress the Th2 or Th17 immune response by suppressing the Th2 or Th17 immune response, or to induce a Th1 immune response that is not excessive with respect to the Th1 induced immune response. Including.
- an example of “adjusting the immune response induced by Th2 to be relatively superior to the immune response induced by Th1 or Th17” is an example where the immune response induced by Th1 or Th17 is an immune response induced by Th2.
- the state of being excessive with respect to the reaction includes making the immune response of Th1 or Th17 not excessive by suppressing the immune response of Th1 or Th17 or inducing the immune response of Th2.
- the “relative direction” means that the immune response of one effector T cell is superior to the immune response of another effector cell. Although it is not superior to the immune response of effector T cells, it also includes inducing, enhancing, or activating the immune response before adjustment.
- the “relative direction” means that the immune response of other effector T cells is suppressed, and as a result, the response of a certain effector T cell changes to a relatively dominant direction. Including. In this case, as a result, it is sufficient that the response of a certain effector T cell is changed in a relatively dominant direction, and it does not matter whether the response of a certain effector T cell is enhanced or suppressed.
- the Th1 / Th2 / Th17 immune balance is also adjusted by suppressing or promoting the differentiation of naive T cells into Th1, Th2 or Th17, respectively.
- the immune balance regulator of this embodiment contains a Euglena-derived substance, and adjusts the Th1 / Th2 / Th17 balance of immunity of the living body, that is, the balance between cellular immunity, humoral immunity and immunity by Th17. It is.
- the immune balance regulator of this embodiment adjusts the Th1 / Th2 / Th17 balance to the cellular immunity side induced by Th1 and / or the humoral immunity side induced by Th17, or Th2, preferably, either Move the biased Th1 / Th2 / Th17 balance to the proper balance and maintain it.
- Euglena-derived substances are preferably those containing paramylon, and include Euglena or Euglena dry products, paramylon, paramylon powder, processed products of paramylon, and the like. Further, a product obtained by adding paramylon or a processed product of paramylon to Euglena or a dried product of Euglena may be used.
- Euglena gracilis E. gracilis
- E. gracilis Z strain E. gracilis Z strain
- other species such as Euglena gracilis krebs, Euglena gracilis barbatillas, etc.
- SM-ZK chloroplast-deficient
- Euglena is widely distributed in fresh water such as ponds and swamps, and may be used separately from these, or any Euglena that has already been isolated may be used.
- the Euglena genus of this invention includes all the mutants. These mutant strains also include those obtained by genetic methods such as recombination, transduction, transformation and the like.
- examples of the culture solution include a culture solution to which nutrient salts such as a nitrogen source, a phosphorus source, and a mineral are added, for example, a modified Cramer-Myers medium ((NH 4 ) 2 HPO 4 1.0 g / L.
- a modified Cramer-Myers medium ((NH 4 ) 2 HPO 4 1.0 g / L.
- KH 2 PO 4 1.0 g / L, MgSO 4 .7H 2 O 0.2 g / L, CaCl 2 .2H 2 O 0.02 g / L, Fe 2 (SO 2 ) 3 7H 2 O 3 mg / L, MnCl 2 ⁇ 4H 2 O 1.8 mg / L, CoSO 4 ⁇ 7H 2 O 1.5 mg / L, ZnSO 4 ⁇ 7H 2 O 0.4 mg / L, Na 2 MoO 4 ⁇ 2H 2 O 0.2 mg / L, CuSO 4 .5H 2 O 0.02 g / L, thiamine hydrochloride (vitamin B 1 ) 0.1 mg / L, cyanocobalamin (vitamin B 12 , (pH 3.5)) can be used.
- (NH 4 ) 2 HPO 4 can also be converted to (NH 4 ) 2 SO 4 or NH 3 aq.
- a known Hutner medium or Koren-Hutner medium prepared based on the description of Euglena Physiology and Biochemistry (Edited by Shozaburo Kitaoka, Society Publishing Center, Inc.) may be used.
- the pH of the culture solution is preferably 2 or more, and the upper limit thereof is preferably 6 or less, more preferably 4.5 or less.
- the photosynthetic microorganisms can grow more dominantly than other microorganisms, so that contamination can be suppressed.
- Euglena cells may be cultured by an open pond method using sunlight directly, a light collecting method in which sunlight condensed by a light concentrator is sent through an optical fiber, etc., and irradiated in a culture tank and used for photosynthesis.
- Euglena cells can be cultured using, for example, a fed-batch method, but flask culture, fermentation using a fermentor, batch culture, semi-batch culture (fed-batch culture), continuous culture (perfusion) Any liquid culture method such as a culture method may be used.
- the culture can be performed using a known culture apparatus such as an open pond type, a raceway type, a tube type or the like, or an experimental culture vessel such as a Sakaguchi flask, an Erlenmeyer flask, or a reagent bottle. Since Euglena assimilate CO 2 , it is preferable to pass air containing 1 to 5% CO 2 through the medium when culturing using the Cramer-Myers medium, which is an autotrophic medium. Furthermore, in order to sufficiently develop the chloroplast, about 1 to 5 g of ammonium phosphate may be added per liter of the medium.
- the culture temperature is usually 20 to 34 ° C., particularly preferably 28 to 30 ° C.
- Euglena Depending on the culture conditions, Euglena usually enters a logarithmic growth phase 2 to 3 days after the start of culture, and reaches a stationary phase about 4 to 5 days. Euglena may be cultured under light irradiation (light culture) or non-irradiated (dark culture).
- Euglena cell separation can be performed, for example, by centrifugation of the culture or simple sedimentation.
- Paramylon is a high molecular weight polymer ( ⁇ -1,3-glucan) in which about 700 glucoses are polymerized by ⁇ -1,3-linkages, and is a storage polysaccharide contained in the genus Euglena.
- Paramylon particles are flat spheroid particles, and ⁇ -1,3-glucan chains are entangled in a spiral shape.
- Paramylon exists as granules in Euglena cells of all species and varieties, and the number, shape, and uniformity of the particles are characterized by species.
- Paramylon consists only of glucose, and the average degree of polymerization of paramylon obtained from a wild strain of E. gracilis Z and a chloroplast-deficient strain SM-ZK is about 700 in glucose units.
- Paramylon is insoluble in water and hot water, but is soluble in dilute alkali, concentrated acid, dimethyl sulfoxide, formaldehyde, and formic acid.
- the average density of paramylon is 1.53 for E. gracilis Z and 1.63 for E. gracilis var. Bacillaris SM-L1.
- Paramylon has a gentle helical structure in which three linear ⁇ -glucans are twisted like a right-handed rope according to X-ray analysis using a powder pattern method. Several of these glucan molecules gather to form paramylon granules. Paramylon granules are very high in crystal structure and occupy about 90%, and are the compounds with the highest crystal structure ratio among polysaccharides. Paramylon is hard to contain water (Euglena Physiology and Biochemistry (Edited by Shozaburo Kitaoka, Society Publishing Center, Inc.)). The particle size distribution of Paramylon (manufactured by Euglena Co., Ltd.) has a median diameter of 1.5 to 2.5 ⁇ m when measured with a laser diffraction / scattering particle size distribution analyzer.
- Paramylon particles are isolated from the cultured Euglena genus by any appropriate method and purified to a fine particle, and are usually provided as a powder.
- paramylon particles can be (1) cultured Euglena cells in any suitable medium; (2) separation of Euglena cells from the medium; (3) isolation of paramylon from isolated Euglena cells; 4) purification of isolated paramylon; and (5) cooling and subsequent lyophilization if necessary.
- Paramylon isolation can be performed, for example, using non-ionic or anionic surfactants of the type that are largely biodegradable.
- the purification of paramylon can be performed substantially simultaneously with the isolation.
- processed paramylon examples include amorphous paramylon and emulsion paramylon.
- Amorphous paramylon is a substance obtained by making crystalline paramylon derived from Euglena amorphous.
- the amorphous paramylon used in the present embodiment has a relative crystallinity of 1 to 20% with respect to the crystalline paramylon produced by a known method from Euglena. However, this relative crystallinity was determined by the method described in Japanese Patent No. 5612875.
- Amorphous paramylon used in the present embodiment is prepared by subjecting crystalline paramylon powder to alkali treatment after neutralization with acid, followed by washing, moisture removal steps, and drying according to the method described in Japanese Patent No. 5612875. Is done.
- Other processed products of paramylon include water-soluble paramylon obtained by chemically or physically treating paramylon by various known methods, sulfated paramylon, and paramylon derivatives.
- Emulsion paramylon is a substance called emulsion paramylon because its processing method and physical properties are similar to those of emulsions, and a fluid obtained by adding water to paramylon is ejected from a pore nozzle at ultra high pressure. It is a processed paramylon that is obtained by performing a collision treatment to collide with an object to be collided and is swollen by being combined with water four times or more.
- Emulsion paramylon is a known physical property reforming apparatus (for example, Japanese Patent Application Laid-Open No. 2011-88108) in which a slurry obtained by adding a water-soluble solvent to a solid such as powder is ejected from a pore nozzle at an ultrahigh pressure to collide with a collision target.
- Emulsion paramylon has a median diameter of at least 5 times that of paramylon when measured with a laser diffraction / scattering particle size distribution analyzer, and is at least 7 ⁇ m. It is observed that it adheres, and swells by combining with water four times or more than paramylon. Slurry mixed with raw material paramylon and water is a free-flowing fluid, but emulsion paramylon is dispersed in water molecules, the viscosity increases and becomes viscous, and when touched, it adheres to the hand.
- the obtained processed paramylon is referred to as emulsion paramylon in this specification, but it is unclear whether it is emulsified, and the state where paramylon is combined with water and swells. It is.
- the immune balance regulator of this embodiment can be used as a composition such as a pharmaceutical composition, a food composition, or a cosmetic composition containing the immune balance regulator.
- the immune balance regulator of this embodiment can be used for the improvement of a constitution in which Th1 / Th2 / Th17 immune balance is biased to Th2, and the prevention or treatment of a disease involving a bias of Th1 / Th2 / Th17 immune balance to Th2. it can.
- an allergic disease refers to a disease in which an immune reaction occurs excessively with respect to a specific antigen
- the immune balance regulator of this embodiment includes atopic dermatitis, pollinosis, allergic rhinitis, allergic conjunctivitis, etc. It is applied to various allergic diseases such as type I to type IV allergy.
- the immune balance regulator of the present embodiment is used for the prevention or treatment of infectious diseases by adjusting the immune balance, and for fine adjustment of the auxiliary physical condition after treatment, for example, for viral diseases such as influenza. It can be used as an antiviral agent or an anti-influenza agent for prevention and treatment of infection.
- the immune balance regulator of the present embodiment has an effect of suppressing infection and onset when sensitized to a virus such as influenza virus.
- the immune balance regulator of the present embodiment is applied to B-type and C-type as well as A-type influenza including H1N1, H2N2, and H3N2.
- Antiviral agents and anti-influenza agents containing Euglena-derived substances have not been conventionally known. Some infectious diseases, such as influenza, become more severe when affected. In such infectious diseases, prevention by vaccines and administration of therapeutic drugs according to the infectious diseases are performed, but some vaccines and therapeutic drugs have side effects. There has been a need for an infectious disease treatment and prevention agent with no side effects. Anti-viral or anti-influenza agents that are easy to produce, process, handle, and ingest are possible by using substances derived from Euglena that can be ingested as food and can be mass-produced as anti-viral or anti-influenza agents. It becomes.
- an anti-influenza agent that can be ingested for a long time can be provided by using a Euglena-derived substance that can be ingested as food and has no side effects. Therefore, it can be taken all year round, and as a result, a high anti-influenza action can be constantly obtained while enhancing the immunity of the living body itself.
- the immune balance regulator of this embodiment can also be used for the prevention and treatment of stress-related diseases, such as peptic ulcers such as gastric ulcer and duodenal ulcer, and for fine adjustment of auxiliary physical condition after treatment.
- stress-related diseases such as peptic ulcers such as gastric ulcer and duodenal ulcer
- a peptic ulcer refers to an ulcer in which a partial defect of epithelial tissue extends deeply in the mucosal layer of the digestive tract.
- causes of onset include gastric acid, pepsin, stress, Helicobacter pylori (hereinafter referred to as “H.
- NSAID non-steroidal anti-inflammatory drugs
- gastrointestinal mucosa The basic theory is the balance theory that the functions of protective factors, that is, the mucus / mucosal barrier, blood flow / microcirculation, growth factors, and prostaglandins are lost in balance, resulting in ulcers.
- Gastric ulcers are mainly caused by weakened defense mechanisms of the gastric mucosa.
- Helicobacter pylori infection, NSAID and stress weaken the defense mechanism and cause damage to the gastric mucosa, which progresses to ulcers.
- Duodenal ulcers result from high gastric acid secretion, which damages the duodenal mucosa, which is less resistant to gastric acid attack.
- H. pylori infection also weakens the duodenal mucosa.
- a diet high in fat leads to an increase in gastric acid secretion.
- Three major causes of gastric and duodenal ulcers are H. pylori infection, non-steroidal anti-inflammatory drugs (NSAIDs), and stress.
- the immune balance adjusting agent of the present embodiment is a person who has a high risk of suffering from peptic ulcer, for example, a person who has psychogenic stress, a person who has completed the treatment of peptic ulcer, and pylori sterilization treatment. It can be administered to a person who has performed the treatment, a person who has failed to disinfect H. pylori, or the like.
- the immune balance regulator of this embodiment is useful for people in an environment that is prone to psychological and social stress, such as a workplace that is prone to psychological stress, a person in a living environment, and a person preparing for a test. Can be administered continuously for a period of time.
- the immune balance adjusting agent of the present embodiment is administered so that the amount of paramylon or a processed product of paramylon is 0.05 g or more, preferably 1 g or more per day for a person having a body weight of 40 to 90 kg. Is done.
- the immune balance regulator of this embodiment since the immune balance regulator of this embodiment has a greater effect of returning the Th1 / Th2 immune balance to the Th1 side after administration for 8 weeks than after administration for 4 weeks, A balance adjustment effect can be obtained.
- the immune balance regulator of this embodiment By continuously administering the immune balance regulator of this embodiment from a week ago, preferably from a year ago, the immune balance of the person is improved and the basic immunity is improved. It is improved, and even if it touches some antigens, it becomes difficult to develop allergic symptoms. In addition, unpleasant symptoms such as cough, runny nose, sneezing, headache, etc., the cause is not specified, but in fact it was allergic constitution, such as due to the Th1 / Th2 / Th17 immune balance biased to Th2, In such a case, it can be used as a remedy for an unpleasant symptom whose cause is not specified.
- the immune balance regulator of the present embodiment suppresses the onset of infectious diseases by adjusting the immune balance, and also reduces the symptoms when an infectious disease develops. You may continue to administer as an anti-influenza agent from before. For example, continuous intake from one week to one year before the influenza epidemic improves immunity through adjustment of the immune balance, making it less susceptible to infection even when exposed to some viruses.
- the immune balance regulator of this embodiment is being administered during psychological stress such as natural disasters, large-scale fires, accidents, crimes, wartime, and non-steroidal anti-inflammatory drugs (NSAIDs). For example, it may be continuously administered as a preventive agent for peptic ulcer.
- NSAIDs non-steroidal anti-inflammatory drugs
- the immune balance regulator of the present embodiment is related to the improvement of constitution that Th1 / Th2 / Th17 immune balance is biased to Th1 and / or Th17, and the Th1 / Th2 / Th17 immune balance is biased to Th1 and / or Th17. It can be used for prevention or treatment of diseases.
- Th1 / Th2 / Th17 Immunity balance involving Th1 and / or Th17 bias involves diseases such as diabetes, liver damage, airway inflammation, host versus graft reaction, rheumatoid arthritis, multiple sclerosis, arteriosclerosis, It can be used for prevention or treatment of psoriasis, gastritis, etc., and for auxiliary fine adjustment of physical condition after treatment.
- Th17 bias as a disease associated with Th17 bias, for example, it can be used for the prevention or treatment of rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, etc., and auxiliary fine adjustment of physical condition after treatment.
- Rheumatoid arthritis is a disease in which the joints of the limbs are swollen or damaged by self-immunity affecting the joints of the limbs. As it progresses, bones and cartilage are broken and the joints cannot move, greatly limiting daily life.
- rheumatoid arthritis drugs that relieve rheumatoid inflammation, suppress the process of joint destruction, have no immediate effect, and some have strong side effects and some have weak side effects. Are used together.
- conventional anti-rheumatic drugs generally have large individual differences in effects, and may be effective in some cases but ineffective in other cases.
- the incidence of side effects is high, and the onset of effects takes about 2 weeks to 3 months.
- Anti-rheumatic drugs cannot be administered until a definitive diagnosis of rheumatoid arthritis because of the high incidence of side effects. Moreover, more than 50% of patients with rheumatoid arthritis require more than 3 months to make a definitive diagnosis, and 80% of those who require more than half a year from the onset of subjective symptoms to the start of administration of antirheumatic drugs. Results of a field survey showing that it is superfluous (Pfizer Co., Ltd. “Survey of 500 patients with rheumatoid arthritis”, November 24, 2011), manifesting effects of anti-rheumatic drugs from manifestation of subjective symptoms In many cases, it takes half a year to more than one year. In addition, anti-rheumatic drugs, starting with a small amount of administration, and confirming the presence of effects and side effects are also factors that delay the onset of anti-rheumatic drugs.
- Anti-rheumatic and anti-rheumatic drugs that are easy to produce, process, handle, and ingest by using substances derived from Euglena that can be ingested as food and that can be mass-produced as anti-rheumatic drugs and anti-rheumatic drugs Can provide.
- the immune balance regulator of this embodiment can be administered as a rheumatoid arthritis inhibitor, a rheumatoid arthritis preventive agent and a rheumatoid arthritis therapeutic agent which has no side effects and can normalize immune abnormalities of rheumatoid arthritis. It can also be administered as a rheumatoid arthritis inhibitor, a rheumatoid arthritis preventive agent and a rheumatoid arthritis therapeutic agent that can be administered even before the onset or diagnosis of rheumatoid arthritis.
- a pharmaceutical composition having an immune balance adjusting action is provided by combining a pharmaceutically acceptable carrier or additive together with an amount of Euglena-derived substance capable of effectively exerting an immune balance adjusting action.
- the pharmaceutical composition may be a pharmaceutical or a quasi drug.
- the pharmaceutical composition may be applied internally or externally. Therefore, the pharmaceutical composition is used in a pharmaceutical form such as an internal preparation, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and / or intraperitoneal injection, transmucosal application agent, transdermal application agent, etc. be able to.
- the dosage form of the pharmaceutical composition can be appropriately set depending on the form of application, for example, solid preparations such as tablets, granules, capsules, powders, powders, etc .; liquid preparations such as liquids and suspensions And semi-solid agents such as ointments and gels.
- a food composition having an immune balance adjusting action can be provided by incorporating an effective amount of Euglena-derived substance capable of exerting an immune balance adjusting action in vivo as a food material into various foods. . That is, the present invention can provide a food composition that is displayed for adjusting the immune balance in the field of food.
- the food composition include foods for specified health use, nutritional supplements, functional foods, foods for hospital patients, supplements and the like in addition to general foods. It can also be used as a food additive.
- Examples of the food composition include seasonings, processed meat products, processed agricultural products, beverages (soft drinks, alcoholic beverages, carbonated beverages, milk beverages, fruit juice beverages, tea, coffee, nutritional drinks, etc.), powdered beverages (powder) Juice, powdered soup, etc.), concentrated beverages, confectionery (candy, cookies, biscuits, gum, gummi, chocolate, etc.), bread, cereals and the like.
- beverages soft drinks, alcoholic beverages, carbonated beverages, milk beverages, fruit juice beverages, tea, coffee, nutritional drinks, etc.
- powdered beverages powdered beverages (powder) Juice, powdered soup, etc.)
- concentrated beverages confectionery (candy, cookies, biscuits, gum, gummi, chocolate, etc.), bread, cereals and the like.
- dietary supplement, functional food, etc. it may be in the form of capsule, troche, syrup, granule, powder or the like.
- Example 1 Euglena gracilis powder (manufactured by Euglena Co., Ltd.) was used as Euglena in Example 1.
- Example 2 Crystalline paramylon was prepared by the following procedure. That is, Euglena gracilis powder of Example 1 (manufactured by Euglena Co., Ltd.) was placed in distilled water and stirred at room temperature for 2 days. This was sonicated to break the cell membrane, and crude paramylon particles were recovered by centrifugation. The collected paramylon particles are dispersed in a 1% sodium dodecyl sulfate aqueous solution and treated at 95 ° C. for 2 hours.
- paramylon particles collected by centrifugation again are dispersed in a 0.1% sodium dodecyl sodium sulfate aqueous solution and treated at 50 ° C. for 30 minutes. did. Lipids and proteins were removed by this operation, then washed with acetone and ether, and then dried at 50 ° C. to obtain purified paramylon particles. 1 g of the prepared paramylon was stored in a known capsule to prepare the immune balance regulator of Example 2.
- Example 3 Using paramylon prepared in Example 2, amorphous paramylon was prepared according to the method described in Japanese Patent No. 5612875. That is, the crystalline paramylon powder prepared in Example 2 was dissolved in 1N aqueous sodium hydroxide solution by adding 5% (w / v) of paramylon powder, and stirred with a stirrer for 1 to 2 hours. Processed. Thereafter, 1N hydrochloric acid was added dropwise to a 1N sodium hydroxide aqueous solution in which paramylon powder was dissolved to neutralize it. After centrifuging, the supernatant was discarded and the step of washing the precipitate with distilled water was repeated. The precipitated gel was collected, frozen, and lyophilized with a freeze dryer to obtain amorphous paramylon of Example 3.
- Example 4 Using the paramylon prepared in Example 2, emulsion paramylon was prepared by the following procedure. Ion exchange water was added to crystalline paramylon powder (manufactured by Euglena Co., Ltd., median diameter 2.591 ⁇ m) to obtain a paramylon slurry having a paramylon concentration of 10 wt%. The inside of the apparatus circuit of the wet atomizer (Starburst 18KW medium size machine, manufactured by Sugino Machine Co., Ltd., oblique collision chamber) was replaced with ion-exchanged water. The nozzle of the apparatus was pressurized, paramylon slurry was supplied into the circuit, and the initial discharge liquid was discarded as a dead volume in the circuit.
- Ion exchange water was added to crystalline paramylon powder (manufactured by Euglena Co., Ltd., median diameter 2.591 ⁇ m) to obtain a paramylon slurry having a paramylon concentration of 10 wt%.
- a jet of paramylon slurry was jetted from a pair of nozzles facing each other at an angle, and jet jet collision by oblique collision was performed.
- the slurry processed product was collected from the outflow passage to make one pass.
- the processing pressure at this time was 245 MPa
- the slurry processing amount was 240 mL
- the nozzle diameter was 0.17 mm.
- This process was repeated three times to perform a three-pass process, and the emulsion paramylon of Example 4 was obtained.
- the emulsion paramylon of Example 4 was not separated from the ion exchange water added at the time of slurry preparation, but was combined with water and swollen.
- the median diameter of the emulsion paramylon of Example 4 was 27.127 ⁇ m (measured by a laser diffraction / scattering particle size distribution analyzer, Horiba, LA-960).
- Blood was collected from each subject immediately before the start of the test (week 0), 4 weeks and 8 weeks after the start of the test.
- a test for detecting the production amount of each cytokine in the supernatant of PMA (Phorbol 12-myristate 13-acetate) + Ionomycin-stimulated culture was performed by a known method. Specifically, 8 mL of peripheral blood was collected into a mononuclear cell separation blood collection tube (Becton Dickinson, 362761), then centrifuged at 3000 rpm for 20 minutes, and the cell layer on the gel barrier was collected in a 50 mL tube.
- the culture supernatant was stored at ⁇ 80 ° C. until measurement.
- the amount of cytokine in the culture supernatant was quantitatively analyzed according to the procedure of the cytokine measurement kit (Flowcytomix, eBioscience, BMS810FFRTU).
- the monocyte content was also measured. Specifically, peripheral blood from each subject was collected with an EDTA-2K-added blood collection tube, and then a flow cytometer (Beckman-Coluter, Navios) analysis was performed using various fluorescently labeled antibodies. At the time of measurement, data were collected and analyzed for FSC, SSC and CD45 antibody positive lymphocyte populations. The antibodies used were used in the following combinations (all antibodies were manufactured by Beckman-Coluter).
- PC7 labeled anti-CD45 antibody PE labeled anti CD3 antibody, FITC labeled anti CD20 antibody, APC labeled anti CD56 antibody, PC5 labeled anti CD16 antibody
- PC7 labeled anti CD45 antibody FITC labeled anti CD8 antibody
- PC5 labeled anti-CD28 antibody ECD labeled anti-CD45RA antibody
- FIGS. 1 and 2 The measurement results are shown in FIGS. 1 and 2, IFN- ⁇ increased significantly (p ⁇ 0.05 by t-test) and IL-4 significantly decreased (p ⁇ 0.01 by t-test) during the 8-week administration period. Moreover, when the ratio of the production amount of IFN- ⁇ to the production amount of IL-4, that is, IFN- ⁇ / IL-4 was calculated using the data of FIG. 1 and FIG. 2, as shown in FIG. There was a significant increase (p ⁇ 0.01 by t test). Therefore, cellular immunity is more significant than humoral immunity, and cellular immunity (Th1-induced immune response) against humoral immunity (Th2-induced immune response) in the administration period of 8 weeks. It turns out that the advantage is increasing.
- IL-6 that promotes differentiation of Th0 into Th2 also significantly decreased (p ⁇ 0.01 by t test) in the administration period of 8 weeks, and over time, It was decreasing.
- IL-12 that promotes the differentiation of Th0 into Th1 tended to increase over time in the 8-week administration period.
- IL-10 which suppresses the production of IFN- ⁇ and IL-12, decreased significantly (p ⁇ 0.01 by t test) during the 8-week administration period, and decreased with the passage of time.
- IL-5 involved in humoral immunity, which proliferates and differentiates B cells, significantly decreases (p ⁇ 0.01 by t test) during the 8-week administration period. It was decreasing according to.
- Test Examples 2 to 5 the antiviral effect by ingesting Euglena, paramylon, and amorphous paramylon, the anti-infection effect at the time of viral infection, and the influenza symptoms alleviation effect were confirmed.
- Test Example 2 Survival confirmation test of influenza virus-infected mice
- Mice that were ingested with paramylon prepared in Example 2, amorphous paramylon in Example 3 and Euglena gracilis powder in Example 1 (manufactured by Euglena Co., Ltd.) were infected with influenza virus.
- a test was conducted to confirm the action.
- BALB / c Cr Slc (SPF) male mice (Japan SLC, Inc.) were used. Feed and drinking water (distilled water) were ad libitum. The mice were divided into a control group, a paramylon group, an amorphous paramylon group, and a Euglena group.
- the test was performed twice.
- the paramylon prepared in Example 2 and the amorphous paramylon of Example 3 were applied to the feed of the paramylon group, the amorphous paramylon group, and the Euglena group for one week before virus infection.
- 1 Euglena gracilis powder manufactured by Euglena Co., Ltd.
- food was similarly fed for 2 weeks before virus infection.
- the number of n in each group was 7, and in the second test, the number of n in each group was 15.
- each group of 6-week-old mice was administered Influenza virus A / PR / 8/34 (H1N1) intranasally at 1000 PFU to cause nasal infection.
- life or death was determined until 10 days after infection.
- the results of the first test are shown in FIG. 9, and the results of the second test are shown in FIG.
- the virus titer was then measured by plaque method. That is, on the day before virus infection, 5 ⁇ 10 5 cells of MDCK cells suspended in E'MEM (Eagle MEM medium “Nissui”: Nissui Pharmaceutical) supplemented with 10% FBS (fetal bovine serum) were cultured. It seed
- E'MEM Eagle MEM medium “Nissui”: Nissui Pharmaceutical
- FBS fetal bovine serum
- the virus strength values were 69.2% in the Euglena 1 week group, 83.3% in the Paramylon 1 week group, 32.0% in the amorphous Paramylon 1 week group, and Euglena 2. It decreased to 59.1% in the weekly group, 57.6% in the 2-week group of paramylon, and 54.5% in the 2-week group of amorphous paramylon.
- Ingestion of Euglena, paramylon, and amorphous paramylon before influenza virus infection The Dunnett test showed that influenza infection was significantly suppressed.
- mice were infected with Influenza virus A / PR / 8/34 (H1N1) by nasal infection in the same procedure as the test mixed for 2 weeks before infection in Test Example 2, and the test example The virus titer in the lungs of each group of mice was measured on the first, second, and third days after infection in the same manner as in FIG. The results are shown in Table 2 and FIG.
- the virus titer was 46.4% in the Euglena group on the first day of infection, 42.7% in the paramylon group, 20.9% in the amorphous paramylon group, and Euglena on the second day of infection. 58.6% in the group, 34.3% in the paramylon group, 30.0% in the amorphous paramylon group, 55.1% in the Euglena group on the third day of infection, 42.4% in the paramylon group, and 33 in the amorphous paramylon group It was found that when the Euglena, paramylon and amorphous paramylon were ingested before the influenza virus infection, the Dunnett test significantly suppressed the influenza infection.
- Example 5 Cytokine measurement test in lungs of influenza virus-infected mice
- Mice that were ingested with paramylon prepared in Example 2, amorphous paramylon in Example 3 and Euglena gracilis powder in Example 1 (manufactured by Euglena) were infected with influenza virus, and cytokines in the lung (IL-1 ⁇ , IL -6, IL-10, IL-12 (p70), IFN- ⁇ , TNF- ⁇ , IFN- ⁇ ) were measured.
- cytokines in the lung IL-1 ⁇ , IL -6, IL-10, IL-12 (p70), IFN- ⁇ , TNF- ⁇ , IFN- ⁇
- Example 1 After acclimatization of 4-week-old BALB / c Cr Slc (SPF) male mice (Japan SLC Co., Ltd.) for 1 week, the Euglena gracilis powder of Example 1 (Euglena group) was added to the purified feed (control group) or purified feed. ), Feed containing 2% each of the paramylon (paramylon group) prepared in Example 2 and the amorphous substance amorphous paramylon (amorphous paramylon group) in Example 3 was allowed to freely ingest from 2 weeks before virus inoculation until laparotomy .
- 6-week-old mice that received the test substance for 2 weeks from 4 weeks of age were inoculated nasally with an LD50 value (1000 PFU) of influenza virus A / PR / 8/34 (H1N1).
- LD50 value 1000 PFU
- influenza virus A / PR / 8/34 H1N1
- the abdomen was opened and the lungs were removed.
- Various cytokines were measured from lung homogenization. Measurement was performed by ELISA for IFN- ⁇ and Bio-Plex for other cytokines.
- mice that were bred in the same manner as in the control group for 2 weeks and did not receive the virus were laparotomized at the same timing as the first day after the virus inoculation in the other groups, and each cytokine was measured. This data is shown as a normal group.
- the lung cytokines on the first day, the second day, and the third day after the virus inoculation are IL-6 and TNF- ⁇ , which are inflammatory cytokines, on the first day after the virus inoculation, The values were significantly higher in the paramylon group and the amorphous paramylon group.
- IL-10 significantly increased in the paramylon group and the amorphous paramylon group. From this result, the release of inflammatory cytokines in the early stage of infection causes infection defense by inflammation, and the inflammation is suppressed by the release of IL-10, which contributes to the increase in survival rate. Seem.
- IL-12 levels were significantly higher in the paramylon group and the amorphous paramylon group. Thereafter, the IFN- ⁇ value was significantly higher in the paramylon group and the amorphous paramylon group. From this result, it was found that IL-12 production activates NK cells and induces IFN- ⁇ .
- IFN- ⁇ which is a cytokine showing antiviral action
- the value of IFN- ⁇ was significantly higher in the amorphous paramylon group on the second day. 14 to 20, a difference in behavior was observed between the Euglena group, the paramylon group, and the amorphous paramylon group. From this difference in behavior, Euglena was found to be less effective because the amount of paramylon contained was relatively small, and it was found that the active ingredient in immunity was paramylon.
- mice infected with influenza die due to strong inflammation in the lungs. From the results of Test Examples 2 to 5, mice in the Euglena group, paramylon group, and amorphous paramylon group were infected with influenza virus compared to the control group. Later survival rates were significantly higher, and each virus titer decreased. This is because mice in the Euglena group, paramylon group, and amorphous paramylon group are protected against infection by releasing inflammatory cytokines at the early stage of infection, and the inflammation generated in the process is suppressed by the release of IL-10. Therefore, it is considered that an increase in the survival rate and a decrease in the viral titer in the lung occurred. From the above, it has been found that Euglena, Paramylon, and Amorphous Paramylon have an effect of alleviating influenza symptoms.
- the Euglena group of Example 2, the paramylon group of Example 2, and the amorphous paramylon group of Example 3 were fed the diet of Table 3 for 14 days.
- the diet of Example 1 group was adjusted by adding 3% of Euglena to the total amount after reducing each formulation of the control group to 97%.
- the amount of cellulose in the control group was reduced by 3% and adjusted by adding 3% of paramylon or amorphous paramylon.
- Paramylon or amorphous paramylon is a glucan, so it can nutritionally replace cellulose, whereas Euglena has various nutrients in addition to glucan, so it replaces 3% of each dietary composition. It is.
- each group was fed the diet of Table 3 for 14 days and then fasted overnight. Thereafter, each group of rats was placed in a restraint stress cage and immersed in water to the chest for 18 hours. Thereafter, the rats were dissected and gastric ulcers were confirmed. After measuring the body weight of each group of rats, the kidney, spleen, duodenum and epididymal adipose tissue of each group were removed and weighed, and the relative weight was calculated by calculating the weight of each organ relative to the body weight of the rat. It was. As a result, compared with the control group, the organ relative weight was not particularly changed in the organs excluding the duodenum.
- FIG. 23 A photograph of a gastric ulcer of a representative example of each group is shown in FIG. 23, and a measurement result of the ulcer area is shown in FIG.
- the gastric ulcer portion in the control group, the gastric ulcer portion (inside the ellipse) where blood was blurred and blackened was clearly observed, whereas Examples 1 to 3 groups (Euglena group, paramylon group, amorphous paramylon) Group), the gastric ulcer portion was clearly smaller compared to the control group.
- the gastric ulcer portion was remarkably reduced. Further, as shown in FIG.
- the area of gastric ulcer was significantly reduced in the Euglena group (Example 1) as compared to the control group (p ⁇ 0.05 by Tukey-Kramer test). Moreover, the tendency which also fell in the paramylon group (Example 2) and the amorphous paramylon group (Example 3) was seen. Furthermore, as shown in Table 5, since the relative weight of the duodenum increases in the Euglena group (Example 1) and the paramylon group (Example 2), there may be an action mechanism that protects the digestive organs from stress. It was.
- iNOS is a kind of nitric oxide synthase (NOS) that generates nitric oxide from L-arginine and oxygen by an oxidation reaction.
- NOS nitric oxide synthase
- i-conducting nitrogen NOS is classified into neural type (type I, 1neutronal NOS: nNOS), vascular endothelial type (type III, endothlial NOS: eNOS), and inductive type (type II, inducible NOS: iNOS).
- iNOS is naturally associated with calmodulin and calcium and does not require an increase in intracellular free calcium. Induced by cytokines and endotoxins, it is known to be involved in inflammatory conditions.
- Nitric oxide derived from iNOS exhibits antiviral and antibacterial actions in the host defense system and plays an important protective role in infections, but also causes exacerbations of inflammation (Med. Bull, Fukuoka Univ.): 29 (4), 247-255,2002).
- COX-2 is a kind of cyclooxygenase (COX).
- COX is a rate-limiting enzyme for prostaglandin (PG) biosynthesis, and there are two isozymes COX-1 and COX-2.
- COX-2 is an inducible enzyme that is associated with pathologies such as inflammation and tumorigenesis, and is mainly present in the nuclear membrane in cells.
- COX expressed at the site of inflammation is mainly COX-2, and PG synthesized by the expression of COX-2 at the site of inflammation exacerbates inflammation.
- FIGS. 25 The results of the analysis are shown in FIGS. As shown in FIG. 25, bands were observed at positions of 434 bp, 253 bp and 162 bp, and iNOS mRNA, COX-2 mRNA and ⁇ -actin mRNA were detected as PCR products, respectively. iNOS and COX-2 were corrected with ⁇ -actin, and the relative index of each group when the control was 1.0 was shown in the figure.
- FIG. 26 shows iNOS / ⁇ -actin
- FIG. 27 shows COX-2 / ⁇ -actin. From the results in FIG. 26, suppression of iNOS expression was observed in the Euglena group, the paramylon group, and the amorphous paramylon group as compared with the control group. In particular, it was significantly suppressed in the paramylon group and the amorphous paramylon group (p ⁇ 0.05 by Turkey-Kramer test).
- FIG. 27 shows the COX-2 / ⁇ -actin in FIG.
- COX-2 expression was significantly suppressed in the Euglena group and the paramylon group (p ⁇ 0.05 by the Turkey-Kramer test). Therefore, since the suppression of iNOS and COX-2 expression was observed by ingestion of Euglena, paramylon and amorphous paramylon, it was considered that gastric ulcer was suppressed by reducing oxidative damage caused by stress.
- Euglena, Paramylon and Amorphous Paramylon are effective in suppressing the expression of iNOS, which has an exacerbating effect on inflammation, and / or the expression of COX-2, the rate-limiting enzyme for biosynthesis of PG, which has an exacerbating effect on inflammation. It was found to exert an inflammatory effect. Further, it was shown that Euglena, paramylon and amorphous paramylon of this example had an iNOS expression inhibitory action and / or a COX-2 expression inhibitory action. Therefore, it was found that Euglena, paramylon and amorphous paramylon of this example can be used as an iNOS expression inhibitor, a COX-2 expression inhibitor and an anti-inflammatory agent.
- Test Example 7 Evaluation of effects on rheumatoid arthritis using a mouse collagen arthritis model
- mice DBA / 1J Jms Slc (SPF), 6-week-old male, Nippon SLC Co., Ltd.
- mice were used as experimental animals.
- Solid feed CE-2 (Nippon Claire Co., Ltd.) was mixed with 2% of each of the test substances of Examples 1 to 4, and each mouse in the Euglena group, the paramylon group, the amorphous paramylon group, and the emulsion paramylon group was boosted 5 days later. From day to day, they were allowed to take a free oral dose.
- the measurement result of the arthritis score is shown in FIG.
- all the Euglena, Paramylon, Amorphous Paramylon, and Emulsion Paramylon groups had significantly lower values compared to the control group, and suppression of joint inflammation by continuous intake of the test substance was observed. It was.
- the emulsion paramylon group had the same value as the control group, but the Euglena group, the paramylon group, and the amorphous paramylon group showed lower values than the control group.
- lymphocytes were divided into three equal parts and cultured in a medium supplemented with anti-CD3 antibody. About 48 hours after the start of culture, the culture supernatant was collected, and the amount of cytokine (IL-17A, IFN- ⁇ ) secreted from the culture supernatant was measured with a multiplex suspension array.
- the measurement results are shown in FIGS. Regarding the measured cytokines IL-17A and IFN- ⁇ , the Euglena group, the paramylon group, the amorphous paramylon group, and the emulsion paramylon group all showed lower values than the control group.
- 30 and 31 confirm that IL-17A and IFN- ⁇ , which are Th1 / Th17 cytokines, were suppressed in the Euglena group, the paramylon group, the amorphous paramylon group, and the emulsion paramylon group. Adjust the Th2 / Th17 immune balance in such a way that the immune response induced by Th1 or Th17 is suppressed, that is, the immune response induced by Th2 is relatively superior to the immune response induced by Th1 or Th17. Was confirmed.
- the left knee joint after excision was fixed with 10% neutral buffered formalin, decalcified with 10% formic acid formalin demineralized solution, then cut out at section A at the femoral pulley groove in FIG. After the preparation, HE staining was performed.
- the evaluation was performed according to the following knee joint tissue evaluation method.
- FIGS. 33 Photographs of pathological specimens of the left knee joint tissues of representative examples in each group are shown in FIGS.
- FIG. 33 in the non-treated group, the findings considered to be caused by arthritis were not observed in the synovial membrane and the femoral pulley groove.
- FIG. 34 synovial edema, inflammation, granulation tissue formation, fibrosis, and exudate were observed from slight (score 1) to moderate (score 2).
- score 1 As for the femoral pulley groove, pannus formation and cartilage destruction were slightly observed (rating 2).
- FIG. 39 shows the ratio of the number of IL-17-producing cells in the CD4-positive T cells obtained by analysis using a flow cytometer.
- the paramylon group and the amorphous paramylon group were decreased as compared with the control group. In the emulsion paramylon group, there was a significant decrease compared to the control group.
- the arthritis score increased daily and the serum IgG concentration increased. Histopathological examination of the knee joint showed mild to moderate pannus formation and cartilage destruction for synovial inflammation, granulation tissue formation, fibrosis, exudate, and femoral pulley groove.
- the Euglena group, the paramylon group, the amorphous paramylon group, and the emulsion paramylon group had a lower gross score than the control group, and a statistically significant difference was also observed.
- serum IgG concentration a low value was observed in the amorphous paramylon group.
- no findings were observed in the Euglena group and the amorphous paramylon group, except for the formation of synovial granulation tissue.
- synovial inflammation, granulation tissue formation, fibrosis, pannus formation of the femoral pulley groove, and cartilage destruction were observed, but the degree was not significant compared to the control group.
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Abstract
Description
免疫のメカニズムの一つとして、細胞性免疫と液性免疫のバランスが知られている。細胞性免疫は、キラーT細胞やマクロファージが主体となって抗原を排除する反応で、液性免疫は、B細胞が産生する抗体が主体となって抗原を排除する反応のことである。この2つの仕組みが合わさることで、抗原を排除している。
しかし、特許文献1の飲食品は、細胞性免疫と液性免疫の免疫バランスを調整できるものの、乳酸菌由来の物質を含むため、乳酸菌特有の風味があり、乳酸菌特有の風味に合った飲食品にしか適用できない。
適正な状態のTh1/Th2/Th17バランスよりもTh1側に偏向すると、細胞性免疫が強くなりすぎて、関節炎リウマチなどの自己免疫疾患が発症することもあると考えられている。一方、Th2側に偏向すると、液性免疫が強くなりすぎて、がん、免疫不全、喘息、皮膚炎、アレルギー症状、腎炎、感染症、ストレス性の疾患等に陥りやすいと考えられている。一方、Th17側に偏向した場合も、関節炎リウマチなどの自己免疫疾患等に陥りやすいと考えられている。
つまり、免疫機能はいずれかが強いことがよいのではなく、細胞性免疫,液性免疫及びTh17による免疫のバランスで成り立っていることが分かってきた。
ユーグレナは、ビタミン,ミネラル,アミノ酸,不飽和脂肪酸など、人間が生きていくために必要な栄養素の大半に該当する59種類もの栄養素を備え、多種類の栄養素をバランスよく摂取するためのサプリメントとしての利用や、必要な栄養素を摂取できない貧困地域での食糧供給源としての利用の可能性が提案されている。
ユーグレナは、鞭毛運動をする動物的性質をもちながら、同時に植物として葉緑体を持ち光合成を行うユニークな生物であり、ユーグレナ自体やユーグレナ由来の物質に、多くの機能性があることが期待されているが、その機能や、機能性発現のメカニズムの多くが未知のままである。
そのため、大量供給可能となったユーグレナ及びパラミロン等のユーグレナ由来物質の機能や、機能性発現のメカニズムの解明、ひいては、これらの物質の利用法等の開発が望まれている。
更に、ユーグレナ由来物質の中でもパラミロンやアモルファスパラミロンのような加工品は、クッキー,ビスケットやチップス類,和菓子,スムージー等の飲食品に混ぜ込んでも、基体となる飲食品の風味を損なうことがない。
本発明の他の目的は、ユーグレナ由来物質の新規な利用法となる免疫バランス調整剤を提供することにある。
つまり、生体の免疫系は、T細胞、B細胞や、多数のサイトカインなど、機能の異なる種々の細胞やサイトカインが、相互に作用して、全体として成り立っているものであるところ、本発明者らは、生体にユーグレナ、パラミロン又はパラミロン加工品等のユーグレナ由来物質を投与すると、生体のTh1/Th2/Th17免疫バランスが、Th1又はTh2又はTh17側に移行して適正なバランスに調整することを見出して、本発明をするに至った。
このように構成しているため、Th1/Th2/Th17免疫バランスのTh1側又はTh2側又はTh17側への偏向が関与して生ずる好ましくない体質を改善する体質改善剤や、Th1側又はTh2又はTh17側への偏向が関与して生ずる疾患の疾患予防剤、治療剤等に用いることができる。
また、有効成分として、ユーグレナ由来物質を用いているため、本発明の免疫バランス調整剤を、ユーグレナ由来物質を含む食品、飲料、サプリメント等として構成しても、有効成分によって風味が損なわれることがなく、服用し易い形態の免疫バランス調整剤の提供が可能となる。
このように構成しているため、医師の治療や治療を受けるほどではないが、Th1/Th2/Th17免疫バランスがTh2に偏向することにより生ずる体調不良の改善ができ、QOL(quality of life)を向上できる。
Th1/Th2/Th17免疫バランスがTh2に偏向した体質は、感染症又はストレス性の疾患を罹患しやすい体質であってもよい。
このように構成しているため、合成薬を主体とした治療に限られず、免疫バランスを整える自然医療にも利用可能となる。
Th1/Th2/Th17免疫バランスのTh2への偏向が関与する疾患として、例えば、がん、免疫不全、喘息、皮膚炎、アレルギー疾患、腎炎、感染症等の予防又は治療に用いることができる。
このように構成しているため、予想される疾患の発症自体を、事前に防ぐことができる。
Th1/Th2/Th17免疫バランスのTh2への偏向が関与する疾患は、感染症又はストレス性の疾患であってもよい。
また、感染症は、インフルエンザであって、抗インフルエンザ剤として用いられてもよい。前記ストレス性の疾患は、消化性潰瘍であって、消化性潰瘍予防又は治療剤として用いられてもよい。
このように構成しているため、例えば、Th1/Th2/Th17免疫バランスのTh1及び/又はTh17への偏向が関与する疾患、例えば、糖尿病、肝障害、気道炎症、宿主対移植片反応、慢性関節リウマチ、多発性硬化症、動脈硬化、乾癬、胃炎等の予防又は治療に用いることができる。特にTh17への偏向が関与した疾患、例えば、慢性関節炎リウマチ、多発性硬化症、乾癬、炎症性腸疾患等の予防又は治療に用いることができる。
Th1/Th2/Th17免疫バランスのTh1及び/又はTh17への偏向が関与する疾患の予防又は治療に用いられ、疾患は、関節リウマチであってもよい。Th1/Th2/Th17免疫バランスのTh1及び/又はTh17への偏向が関与する疾患の発症が予測される時期よりも前に投与されてもよい。
このように、有効成分として、パラミロン又はその加工品を用いているため、本発明の免疫バランス調整剤を、パラミロン又はその加工品を含む食品、飲料、サプリメント等として構成しても、有効成分によって風味が損なわれることがなく、服用し易い形態の免疫バランス調整剤の提供が可能となる。
また、有効成分として、パラミロン又はその加工品を用いているため、本発明の免疫バランス調整剤を、パラミロン又はその加工品を含む食品、飲料、サプリメント等として構成しても、有効成分によって風味が損なわれることがなく、服用し易い形態の免疫バランス調整剤の提供が可能となる。
<<細胞性免疫と液性免疫のメカニズム>>
ヘルパーT細胞(ナイーブT細胞)は、体内のリンパ組織を巡り、特異的なウイルスや微生物などの抗原と出会うまで抗原提示細胞と接触を繰り返す。特異的な抗原と出会ったナイーブT細胞は増殖を繰り返し未成熟エフェクターT細胞(Th0)になり、Th0はサイトカインの刺激や抗原提示細胞からの補助刺激によって、エフェクターT細胞であるTh1またはTh2またはTh17に分化する。エフェクターT細胞は、産生するサイトカインの種類により異なる種類であるヘルパー1型(Th1)とヘルパー2型(Th2)とヘルパー17型(Th17)に分類される。
細胞性免疫では、キラーT細胞そのものが免疫反応を行う。抗原の周りにT細胞が集まり、抗原はT細胞に囲まれ、攻撃、破壊される。
液性免疫では、抗体産生細胞によって抗体が作られ、抗体が免疫反応を行う。抗体は血液中にあり、全身で抗原抗体反応が起こる。抗原の種類に応じて異なる抗体が作られ、抗原と特異的に結合して抗原の働きを抑制する。
つまり、細胞性免疫はT細胞が直接抗原を攻撃し、液性免疫は抗体を作り、抗体が抗原と特異的に結合して抗原を失活させる。
一方、細胞性免疫の流れとしては、体内に侵入した抗原をマクロファージが処理をして、ヘルパーT細胞にその抗原の情報を伝える。その情報を受けたヘルパーT細胞はキラーT細胞を刺激する。刺激を受けたキラーT細胞は増殖し、抗原と直接反応して、抗原を不活性化させる。
Th1への分化には抗原提示細胞の分泌するIL-12が必要で、Th1などが産生するIFN-γはTh0のTh1への分化を促進する。またTh2が産生するIL-10はマクロファージのIL-12産生を抑制し、Th1のIFN-γ産生を抑制することで間接的にTh0のTh1への分化を抑制している。
本明細書において、Th1/Th2/Th17免疫バランスとは、Th1,Th2及びTh17がそれぞれ誘導する免疫反応のバランスをいい、Th1,Th2及びTh17がそれぞれ誘導する免疫反応が、互いに拮抗している状態が、偏向のない状態である。
一方、Th1,Th2及びTh17を含むエフェクターT細胞のうちいずれかが誘導する免疫反応が、他の免疫反応に対して過剰である状態が、過剰であるエフェクターT細胞に偏向した状態である。
また、本明細書において、「Th1が誘導する免疫反応がTh2又はTh17が誘導する免疫反応に対して相対的に優位になる方向に調整する」例には、Th2又はTh17が誘導する免疫反応がTh1が誘導する免疫反応に対して過剰となった状態を、Th2又はTh17の免疫反応を抑制又は、Th1の免疫反応を誘導することにより、Th2又はTh17の免疫反応が過剰でない状態にすることを含む。
また、「相対的に優位になる方向」とは、あるエフェクターT細胞の免疫反応を、他のエフェクター細胞の免疫反応よりも優位にする場合のほか、あるエフェクターT細胞の免疫反応を、他のエフェクターT細胞の免疫反応よりも優位ではないが、調整前の免疫反応よりも誘導,亢進,活性化された状態にすることも含む。
更に、「相対的に優位になる方向」とは、他のエフェクターT細胞の免疫反応が抑制されることより、結果として、あるエフェクターT細胞の反応が相対的に優位になる方向に変化する場合も含む。この場合、結果として、あるエフェクターT細胞の反応が相対的に優位になる方向に変化していればよく、あるエフェクターT細胞の反応が亢進,抑制されているか否かは、問わない。
本実施形態の免疫バランス調整剤は、ユーグレナ由来物質を含み、生体の免疫のTh1/Th2/Th17バランス、つまり、細胞性免疫,液性免疫とTh17による免疫とのバランスを調整する免疫バランス調整剤である。
本実施形態の免疫バランス調整剤は、Th1/Th2/Th17バランスを、Th1が誘導する細胞性免疫側及び/又はTh17、又はTh2が誘導する液性免疫側に調整し、好ましくは、いずれかに偏っているTh1/Th2/Th17バランスを、適正なバランスに移動させて維持させる。
ユーグレナ由来物質としては、パラミロンを含むものが好ましく、ユーグレナ又はユーグレナの乾燥品のほか、パラミロン、パラミロン粉末や、パラミロンの加工品等が含まれる。また、ユーグレナ又はユーグレナの乾燥品に、パラミロン又はパラミロン加工品が添加されたものでもよい。
ユーグレナ属は、池や沼などの淡水中に広く分布しており、これらから分離して使用してもよく、また、すでに単離されている任意のユーグレナ属を使用してもよい。
本発明のユーグレナ属は、その全ての変異株を包含する。また、これらの変異株の中には、遺伝的方法、たとえば組換え,形質導入,形質転換等により得られたものも含有される。
ユーグレナ細胞の培養は、太陽光を直接利用するオープンポンド方式、集光装置で集光した太陽光を光ファイバー等で送り、培養槽で照射させ光合成に利用する集光方式等により行ってもよい。
また、ユーグレナ細胞の培養は、例えば供給バッチ法を用いて行われ得るが、フラスコ培養や発酵槽を用いた培養,回分培養法,半回分培養法(流加培養法),連続培養法(灌流培養法)等、いずれの液体培養法により行ってもよい。
ユーグレナは、光照射下で培養(明培養)されてもよく、無照射で培養(暗培養)されてもよい。
パラミロン(Paramylon)は、約700個のグルコースがβ-1,3-結合により重合した高分子体(β-1,3-グルカン)であり、ユーグレナ属が含有する貯蔵多糖である。パラミロン粒子は、扁平な回転楕円体粒子であり、β-1,3-グルカン鎖がらせん状に絡まりあって形成されている。
パラミロンは、グルコースのみからなり、E. gracilis Zの野生株と葉緑体欠損株SM-ZKから得られたパラミロンの平均重合度は、グルコース単位で約700である。
パラミロンは、水,熱水には不溶性であるが、希アルカリ,濃い酸,ジメチルスルホキシド,ホルムアルデヒド,ギ酸に溶ける。
パラミロンの平均密度は、E. gracilis Zでは、1.53、E. gracilis var. bacillaris SM-L1では、1.63である。
なお、パラミロン((株)ユーグレナ製)の粒度分布は、レーザ回折/散乱式粒度分布測定装置で測定したときのメジアン径が、1.5~2.5μmである。
例えば、パラミロン粒子は、(1)任意の適切な培地中でのユーグレナ細胞の培養;(2)当該培地からのユーグレナ細胞の分離;(3)分離されたユーグレナ細胞からのパラミロンの単離;(4)単離されたパラミロンの精製;および必要に応じて(5)冷却およびその後の凍結乾燥により得ることができる。
パラミロンの単離は、例えば、大部分が生物分解される種類の非イオン性または陰イオン性の界面活性剤を用いて行われ得る。パラミロンの精製は、実質的には単離と同時に行われ得る。
アモルファスパラミロンとは、ユーグレナ由来の結晶性パラミロンをアモルファス化した物質である。
本実施形態で用いるアモルファスパラミロンは、ユーグレナから公知の方法で生成された結晶性のパラミロンに対する相対結晶度が、1~20%である。
但し、この相対結晶度は、特許第5612875号記載の方法により求めたものである。
つまり、アモルファスパラミロン及びパラミロンを、それぞれ、粉砕機(Retsh社製ボールミルMM400)にて、振動数20回/秒で5分間粉砕後、X線回折装置(スペクトリス社製H’PertPRO)を用い、管電圧45KV、管電流40mAにて、2θが5°乃至30°の範囲でスキャンを行い、パラミロンとアモルファスパラミロンの2θ=20°の付近の回折ピークPc,Paを得る。
このPc,Paの値を用い、アモルファスパラミロンの相対結晶度を、
アモルファスパラミロンの相対結晶度=Pa/Pc×100(%)
により算出する。
パラミロンの加工品には、その他、公知の種々の方法によりパラミロンを化学的又は物理的に処理して得た水溶性パラミロン、硫酸化パラミロン等や、パラミロン誘導体も含まれる。
エマルジョンパラミロンは、粉体等の固体に水溶性溶媒を加えたスラリーを、細孔ノズルから超高圧で噴出させて被衝突物に衝突させる公知の物性改質装置(例えば、特開2011-88108号公報、特開平6-47264号公報記載の装置)で、噴出時のノズル圧力245MPaで、1回以上衝突処理を行うことにより得ることができる。
エマルジョンパラミロンは、レーザ回折/散乱式粒度分布測定装置で粒度を測定したときのメジアン径が、パラミロンの5倍以上であり、7μm以上であって、光学電子顕微鏡により、粒子が、隣接する粒子と付着していることが観察され、パラミロンに対して4倍以上の水と結合して膨潤している。
原料パラミロンと水を混合したスラリーは、さらさらした流体であるが、エマルジョンパラミロンは、パラミロンが水分子中に分散して、粘度が増加して粘性を有し、触ったときに手に付着するような粘着性と、弾力性を有し、糊のような触感を備えている。
なお、その処理方法と物性から、得られた加工パラミロンを本明細書においてエマルジョンパラミロンと呼んでいるが、エマルジョン化しているか否かは不明であり、パラミロンが水と結合して膨潤している状態である。
本実施形態の免疫バランス調整剤は、Th1/Th2/Th17免疫バランスがTh2に偏向した体質の改善、Th1/Th2/Th17免疫バランスのTh2への偏向が関与する疾患の予防又は治療に用いることができる。Th1/Th2/Th17免疫バランスのTh2への偏向が関与する疾患として、例えば、がん、免疫不全、喘息、皮膚炎、アレルギー疾患、腎炎、感染症等の予防又は治療、治療後の補助的な体調の微調整に用いることができる。
また、本実施形態の免疫バランス調整剤は、免疫バランスを調整することにより、感染症の予防又は治療、治療後の補助的な体調の微調整に用いられ、例えば、インフルエンザ等のウイルス性疾患の感染の予防及び治療用の抗ウイルス剤、抗インフルエンザ剤として用いることができる。本実施形態の免疫バランス調整剤は、インフルエンザウイルス等のウイルスに感作したときの感染、発症を抑制する効果を備える。本実施形態の免疫バランス調整剤は、H1N1,H2N2,H3N2を初めとするA型インフルエンザのほか、B型,C型にも適用される。
感染症の中には、インフルエンザなど、罹患すると重症化するものがある。このような感染症では、ワクチンによる予防や、感染症に応じた治療薬の投与などが行われるが、ワクチンや治療薬には副作用のあるものがある。副作用のない感染症治療剤、予防剤が求められていた。
食品としても摂取可能で、大量生産が可能なユーグレナに由来する物質を、抗ウイルス剤又は抗インフルエンザ剤として用いることにより、生産、加工、取り扱い及び摂取のし易い抗ウイルス剤又は抗インフルエンザ剤が可能となる。
更に、抗インフルエンザ剤として、食品としても摂取可能で、副作用のないユーグレナ由来物質を用いることにより、長期摂取が可能な抗インフルエンザ剤を提供できる。従って、通年摂取することも可能となり、結果として、生体自体の免疫力を上げながら、高い抗インフルエンザ作用を常時獲得可能となる。
消化性潰瘍とは、消化管の粘膜層において上皮組織の部分的欠損が深部に及んでいる潰瘍を指す。発症の原因としては、胃酸、ペプシン、ストレス、ヘリコバクターピロリ(以下、「ピロリ菌」という。)または非ステロイド性抗炎症薬(以下、「NSAID」という。)等の攻撃因子と、消化管の粘膜防御因子、すなわち、粘液・粘膜関門や血流・微小循環、増殖因子、プロスタグランジンの機能の平衡がくずれて潰瘍が生じるというバランス説が基本的な考えとなっている。
胃潰瘍・十二指腸潰瘍の三大原因は、ピロリ菌感染、非ステロイド性抗炎症薬(NSAID)、ストレスである。
本実施形態の免疫バランス調整剤は、心理・社会的ストレスのかかり易い環境にある人、例えば、心因性ストレスのかかりやすい職場,住環境にある人や試験等の準備中の人に、長期間継続投与できる。
また、本実施形態の免疫バランス調整剤は、4週間投与後よりも8週間投与後の方が、Th1/Th2免疫バランスをTh1側に戻す効果が大きいため、より長い期間の投与により、高い免疫バランス調整効果を得ることができる。
また、咳、鼻水、くしゃみ、頭痛等の不快な症状が、原因が特定されないが、実は、アレルギー体質であったなど、Th1/Th2/Th17免疫バランスのTh2への偏向に起因する場合もあり、このような場合には、原因の特定されない不快な症状の改善薬として利用可能である。
また、本実施形態の免疫バランス調整剤を、自然災害,大規模火災,事故,犯罪等発生時や戦時のように心因性ストレスが掛かる時期や、非ステロイド性抗炎症薬(NSAID)投与中などに、消化性潰瘍予防剤として、継続投与してもよい。消化性潰瘍発症可能性が高いこのような時期に、継続摂取することにより、免疫バランスの調整を通じて、免疫力が向上し、消化性潰瘍に感染しにくくなる。
本実施形態の免疫バランス調整剤を通年摂取し続けることで、免疫バランスの調整を通じて、免疫力が通年で向上し、体質改善されるため、多少疲れていても、インフルエンザやその他の風邪に罹患しにくくなる。
現状、関節リウマチの薬剤では、リウマチ炎症の寛解、関節破壊の経過の抑制、即効性を併せ持つものがなく、また、副作用の強いものも弱いものもあるため、薬物療法では、これらの薬剤が相補的に併用されている。しかし、従来の抗リウマチ薬は、一般的に、効果に個体差が大きく、ある症例に有効でも、他の症例には無効な場合がある。
また、副作用発現率が高く、かつ、効果発現に2週間~3か月程度かかる効果発現の遅効性がある。抗リウマチ薬は、副作用発現率が高いために関節リウマチの確定診断までは投与できない。しかも、関節リウマチの患者のうち、確定診断までに3か月以上を要している人が5割超、自覚症状発現から抗リウマチ薬投与開始までに半年以上を要している人が8割超であるとの実態調査結果も発行されており(ファイザー株式会社 「関節リウマチ患者さん500名を対象とした実態調査」,2011年11月24日)、自覚症状発現から抗リウマチ薬の効果発現までに、半年~1年以上を要する場合が少なくない。合わせて、抗リウマチ薬は、少量の投与から始めて、効果や副作用の有無を確認することも、抗リウマチ薬の効果発現を遅らせる要因となっている。
食品としても摂取可能で、大量生産が可能なユーグレナに由来する物質を、抗リウマチ薬及び抗リウマチ予防薬として用いることにより、生産、加工、取扱い及び摂取のしやすい抗リウマチ薬及び抗リウマチ予防薬を提供できる。
よって、本実施形態の免疫バランス調整剤は、副作用がなく、関節リウマチの免疫異常を正常化可能な関節リウマチ抑制剤,関節リウマチ予防剤及び関節リウマチ治療剤として投与できる。また、関節リウマチの罹患又は診断の前でも投与可能な関節リウマチ抑制剤,関節リウマチ予防剤及び関節リウマチ治療剤としても投与できる。
医薬の分野では、免疫バランス調整作用を有効に発揮できる量のユーグレナ由来物質とともに、薬学的に許容される担体や添加剤を配合することにより、免疫バランス調整作用を有する医薬組成物が提供される。当該医薬組成物は、医薬品であっても、医薬部外品であってもよい。
当該医薬組成物は、内用的に適用されても、また、外用的に適用されてもよい。したがって、当該医薬組成物は、内服剤、静脈注射、皮下注射、皮内注射、筋肉注射及び/または腹腔内注射等の注射剤、経粘膜適用剤、経皮適用剤等の製剤形態で使用することができる。
当該医薬組成物の剤型としては、適用の形態により、適当に設定できるが、例えば、錠剤、顆粒剤、カプセル剤、粉末剤、散剤、等の固形製剤;液剤、懸濁剤等の液状製剤、軟膏剤、ゲル剤等の半固形剤があげられる。
食品の分野では、免疫バランス調整作用を生体内で発揮できる有効な量のユーグレナ由来物質を食品素材として、各種食品に配合することにより、免疫バランス調整作用を有する食品組成物を提供することができる。すなわち、本発明は、食品の分野において、免疫バランス調整用と表示された食品組成物を提供することができる。当該食品組成物としては、一般の食品の他、特定保健用食品、栄養補助食品、機能性食品、病院患者用食品、サプリメント等をあげることができる。また、食品添加物として用いることもできる。
当該食品組成物としては、例えば、調味料、畜肉加工品、農産加工品、飲料(清涼飲料、アルコール飲料、炭酸飲料、乳飲料、果汁飲料、茶、コーヒー、栄養ドリンク等)、粉末飲料(粉末ジュース、粉末スープ等)、濃縮飲料、菓子類(キャンディ、クッキー、ビスケット、ガム、グミ、チョコレート等)、パン、シリアル等をあげることができる。また、特定保健用食品、栄養補助食品、機能性食品等の場合、カプセル、トローチ、シロップ、顆粒、粉末等の形状であってもよい。
ユーグレナグラシリス粉末((株)ユーグレナ製)を、実施例1のユーグレナとした。
(実施例2)
結晶性のパラミロンを、以下の手順により調製した。
つまり、実施例1のユーグレナグラシリス粉末((株)ユーグレナ製)を蒸留水に入れ、室温で2日間撹拌した。これを超音波処理して細胞膜を破壊し、遠心分離により粗製パラミロン粒子を回収した。回収したパラミロン粒子を1%ドデシル硫酸ナトリウム水溶液に分散し、95℃で2時間処理し、再度遠心分離により回収したパラミロン粒子を0.1%ドデシル硫酸ナトリウム水溶液に分散して50℃で30分間処理した。当該操作により脂質やタンパク質を除去し、その後アセトンおよびエーテルで洗浄した後、50℃で乾燥して、精製パラミロン粒子を得た。
調製したパラミロン1gを、公知のカプセルに格納して、実施例2の免疫バランス調整剤を調製した。
実施例2で調製したパラミロンを用いて、特許第5612875号記載の方法に従い、アモルファスパラミロンを調製した。
つまり、実施例2で調製した結晶性のパラミロン粉末を、1Nの水酸化ナトリウム水溶液に、パラミロン粉末を5%(w/v)添加して溶解させ、1~2時間スターラで撹拌して、アルカリ処理した。その後、1Nの塩酸を、パラミロン粉末が溶解した1N水酸化ナトリウム水溶液に滴下して中和した。遠心分離の後上澄みを捨て、沈殿を蒸留水で洗浄する工程を繰り返した後、沈殿したゲルを回収し、凍結させた後凍結乾燥機で凍結乾燥し、実施例3のアモルファスパラミロンを得た。
実施例2で調製したパラミロンを用いて、以下の手順により、エマルジョンパラミロンを調製した。
結晶性のパラミロン粉末((株)ユーグレナ製,メジアン径2.591μm)にイオン交換水を加え、パラミロン濃度が10wt%のパラミロンスラリーを得た。
湿式微粒化装置(スターバースト18KW中型機,株式会社スギノマシン製,斜向衝突チャンバ-)の装置回路内をイオン交換水に置換した。装置のノズルを加圧して、パラミロンスラリーを回路内に供給し、初期吐出液を、回路内デッドボリュームとして廃棄した。その後、相互に角度を持って対向する一対のノズルからパラミロンスラリーの噴流をそれぞれ噴出させて、相互に衝突させる斜向衝突による噴流衝突を行った。流出路からスラリー処理物を回収して、1パスとした。このときの処理圧力は、245MPa,スラリー処理量は、240mL,ノズル径は、0.17mmとした。
この処理を3回繰り返して3パスの処理を行い、実施例4のエマルジョンパラミロンを得た。
実施例4のエマルジョンパラミロンは、スラリー調製時に加えたイオン交換水と分離せず、水と結合して膨潤していた。実施例4のエマルジョンパラミロンのメジアン径は、27.127μm(レーザ回折/散乱式粒度分布測定装置,堀場製作所,LA-960にて測定)であった。
実施例2の免疫バランス調整剤を用いて、免疫バランス調整剤8週間継続摂取による免疫バランス調整効果のヒト臨床試験を実施した。
本試験の被験者は、60~65歳の健常な前期高齢者10名(男女各5名)であり、平均年齢は62.80歳、試験開始時測定の平均体重は58.93kgであった。
被験者に、実施例2の免疫バランス調整剤を毎日、一日一回、1つずつ食後に摂取させた。但し、摂取の時間帯は問わなかった。摂取は、8週間継続した。
具体的には、末梢血を単核球分離用採血管(Becton Dickinson, 362761)に8mL採血後、3000rpmで20分間の遠心を行い、ゲルバリア上の細胞層を50mLチューブに回収した。回収した細胞に生理食塩水30mL加え、1500rpmで10分間遠心した。上清を除き、生理食塩水を10mL加え、1200rpmで5分間遠心した。RPMI-1640細胞培養液(Gibco,11875-093)に再浮遊させた。1×106個の単核球を最終濃度、10%FBS、50ng/mLLPMA(Phorbol 12-Myristate 13-Acetate, Sigma,P1585)、500ng/mL Ionomycin(Sigma,I9657)添加細胞培養液にて48時間培養し、培養上清を回収した。培養上清は測定時まで-80℃にて保存した。培養上清中のサイトカイン量をサイトカイン測定キット(Flowcytomix, eBioscience,BMS810FFRTU)の手順に従い定量分析を行った。
使用した抗体は下記の組み合わせで用いた(抗体は総てBeckman-Coluter社製)。
(1) PC7標識抗CD45抗体,PE標識抗CD3抗体,FITC標識抗CD20抗体,APC標識抗CD56抗体,PC5標識抗CD16抗体
(2) PC7標識抗CD45抗体,FITC標識抗CD8抗体,APC標識抗CD4抗体,PC5標識抗CD28抗体,ECD標識抗CD45RA抗体
図1,図2より、8週間の投与期間において、IFN-γは有意に増加(t検定によりp<0.05)し、IL-4は有意に減少(t検定によりp<0.01)していた。
また、図1,図2のデータを用いて、IL-4の産生量に対するIFN-γの産生量の比、つまり、IFN-γ/IL-4を算出したところ、図3に示すように、有意に増加していた(t検定によりp<0.01)。よって、液性免疫よりも細胞性免疫が有意になっており、かつ、8週間の投与期間において、液性免疫(Th2が誘導する免疫反応)に対する細胞性免疫(Th1が誘導する免疫反応)の優位性が増加していることが分かった。
一方、図5に示すように、Th0のTh1への分化を促進するIL-12は、8週間の投与期間において、時間の経過に従い、増加傾向にあった。
図6に示すように、IFN-γ及びIL-12の産生を抑制するIL-10は、8週間の投与期間において、有意に減少(t検定によりp<0.01)し、時間の経過に従い、減少していた。
図7に示すように、B細胞の増殖、分化を行い、液性免疫に関与するIL-5は、8週間の投与期間において、有意に減少(t検定によりp<0.01)し、時間の経過に従い、減少していた。
また、単球の試験結果を図8に示す。
図8の結果より、感染に対する免疫の開始に重要な役割を果たし、細胞性免疫において産生が促進される単球が、有意に増加していた。
試験例1の結果より、60~65歳の健常な前期高齢者に対する8週間の実施例2の免疫バランス調整剤の投与により、Th1への分化促進を行うIFN-γが有意に増加し、Th1への分化促進を行うIL-12が増加傾向にあった。
また、Th2への分化促進,B細胞の活性化、増殖、Th1への分化促進の抑制、Th2の増殖、マクロファージの活性化抑制を行うIL-4、B細胞の増殖、分化を行うIL-5、INF-γ及びIL-12産生の抑制を行うIL-10が、有意に減少していた。
以上より、実施例2の免疫バランス調整剤は、液性免疫の活性化及び細胞性免疫活性の抑制を行うサイトカインIL-4,IL-5,IL-10の産生を抑制し、かつ、細胞性免疫を活性化するIFN-γの産生を促進すること、また、感染に対する免疫の開始に重要な役割を果たす単球の産生を促進することを通じて、60~65歳の健常な前期高齢者の免疫バランスを調整することが分かった。
(試験例2 インフルエンザウイルス感染マウスの生存率確認試験)
実施例2で調製したパラミロン、実施例3のアモルファスパラミロン、実施例1のユーグレナグラシリス粉末((株)ユーグレナ製)を摂取させたマウスをインフルエンザウイルスに感染させ、パラミロン、アモルファスパラミロン、ユーグレナの抗インフルエンザ作用を確認する試験を行った。
試験には、BALB/c Cr Slc(SPF)雄性マウス(日本エスエルシー株式会社)を用いた。飼料と飲料水(蒸留水)は自由摂取とした。
マウスは、コントロール群、パラミロン群、アモルファスパラミロン群、ユーグレナ群に分けた。
1回目の試験では各群のn数を7匹、2回目の試験では各群のn数を15匹とした。
カイ2乗検定において、感染10日目においてコントロール群とユーグレナ群で有意差が認められた(p=0.0464)。また、感染10日目においてコントロール群とパラミロン群およびアモルファスパラミロン群で有意差が認められた(p=0.0201)。
また、インフルエンザウイルス感染前2週間の間、実施例2のパラミロン、実施例3のアモルファスパラミロン、実施例1のユーグレナを経口投与することにより、インフルエンザウイルス感染による致死が有意に抑制されることが分かった。 パラミロン、アモルファスパラミロン、ユーグレナの経口摂取により、Th1が誘導する細胞免疫の機能が向上したため、細胞免疫の機能が向上した後に感染されたインフルエンザウイルスに対して、インフルエンザウイルス感染症の発症が抑制され、又は、発症したインフルエンザ感染症の症状が弱められたものである。
また、試験例2の感染前1,2週間混餌した1,2回目の試験と同様の手順で、各群(n=3)のマウスに、Influenza virus A /PR/8/34(H1N1)を点鼻感染させ、感染後2日目に、各群のマウスの肺中のウイルス力価の測定を行った。
ウイルス力価の測定では、まず、感染後2日目の各群のマウスの肺を摘出し、摘出した肺を1mLのPBS(-)で肺ホモジネートを作製した。
E’MEMでMDCK細胞を洗浄後、E’MEMで10倍段階希釈した肺ホモジネートを500μL接種した。5%CO2条件下35℃で1時間吸着後,ウイルス液を除去した。43℃に保温した0.75%Agarose1600(和光純薬),0.0015%DEAE-dextran(Pharmacia Biotech),3μg/mL acetyl trypsin(SIGMA,T-6763)加E’MEMを各ウェルに2mL重層し、完全に凝固するまで室温で静置した。凝固後、プレートを5%CO2条件下35℃にて3日間培養した。培養終了後10%ホルマリンで固定した。固定後培地を除去し、0.5%アミドブラックを用いて染色した。
結果を、表1,図11,12に示す。
また、試験例2の感染前2週間混餌した試験と同様の手順で、各群(n=5)のマウスに、Influenza virus A/PR/8/34(H1N1)を点鼻感染させ、試験例3と同様の手順で、感染後1,2,3日目に、各群のマウスの肺中のウイルス力価の測定を行った。
結果を、表2,図13に示す。
実施例2で調製したパラミロン、実施例3のアモルファスパラミロン、実施例1のユーグレナグラシリス粉末((株)ユーグレナ製)を摂取させたマウスをインフルエンザウイルスに感染させ、肺中サイトカイン(IL-1β、IL-6、IL-10、IL-12(p70)、IFN-γ、TNF-α、IFN-β)を測定する試験を行った。
4週齢のBALB/c Cr Slc(SPF)雄性マウス(日本エスエルシー株式会社)を1週間の順化後、精製飼料(コントロール群)あるいは精製飼料に、実施例1のユーグレナグラシリス粉末(ユーグレナ群)、実施例2で調製したパラミロン(パラミロン群)、実施例3の非結晶体物質アモルファスパラミロン(アモルファスパラミロン群)をそれぞれ2%添加した飼料をウイルス接種の2週間前から開腹まで自由摂取させた。4週齢より2週間被験物質を摂取した6週齢のマウスにインフルエンザウイルスA/PR/8/34(H1N1)のLD50値(1000PFU)の点鼻接種を行った。
ウイルス接種後1日目、2日目、3日目に開腹し、肺を摘出した。肺のホモジナイズから、各種サイトカインを測定した。測定は、IFN-βについてはELISAで、それ以外のサイトカインについてはBio-Plexで行った。
図14~20の結果において、ウイルス接種後1日目、2日目、3日目の肺中サイトカインは、炎症性サイトカインであるIL-6、TNF-αでは、ウイルス接種後1日目に、パラミロン群、アモルファスパラミロン群で有意に値が高値となった。ウイルス接種後3日目になると、IL-10でパラミロン群、アモルファスパラミロン群で有意に値が高値となった。この結果より、感染初期に炎症性サイトカインが放出されることで、炎症による感染防御が起こり、その炎症がIL-10の放出で抑制されることで、生存率の上昇に寄与しているものと思われる。
この結果より、IL-12の産生がNK細胞を活性化させ、IFN-γを誘導するという作用機序であることが分かった。
図14~20では、ユーグレナ群と、パラミロン群及びアモルファスパラミロン群との間に、挙動の違いが観察された。この挙動の違いより、ユーグレナはパラミロンの含まれている量が相対的に少なかったので、効果が薄かったものと考えられ、免疫における有効成分はパラミロンであることが分かった。
以上のことから、ユーグレナ、パラミロン、アモルファスパラミロンは、インフルエンザ症状緩和効果を備えることが分かった。
ラットを用いた水浸拘束ストレス試験において、実施例1のユーグレナ、実施例2のパラミロン、実施例3のアモルファスパラミロンを摂取させ、実施例1~3によるストレス性疾患の一例である胃潰瘍の抑制作用を確認する試験を行った。
6週齢の雄性ラット(Wistar)を用い、試験開始4日前から馴化のための食餌(CLEA Rodent Diet CE-2;日本クレア株式会社)により、予備飼育を行った後、コントロール群、実施例1のユーグレナ群、実施例2のパラミロン群、実施例3のアモルファスパラミロン群に、14日間、表3の食餌を与えた。
表3において、実施例1群の食餌は、コントロール群の各配合を、それぞれ97%の量に減らした上で、全量の3%量のユーグレナを添加して調整した。実施例2,3群の食餌では、コントロール群のセルロース量を3%減量し、パラミロン又はアモルファスパラミロンを3%添加して調整した。パラミロン又はアモルファスパラミロンは、グルカンであるため、栄養的にセルロースを代替できるのに対し、ユーグレナは、グルカンのみでなく種々の栄養素を備えているため、食餌の各配合の3%ずつと代替したものである。
その結果、各群の食餌における三大栄養素のエネルギー比率及びエネルギー密度は、表4の通りとなり、栄養のバランスが各群でほぼ同等となった。
各群の食餌を摂取させた14日間の食餌摂取量を図21に、体重を図22に示す。
その後、各群のラットを拘束用ストレスケージに入れ、18時間、胸部まで水中に浸した。その後、ラットを解剖し、胃潰瘍を確認した。
各群のラットの体重を測定後、各群の腎臓、脾臓、十二指腸、精巣上体脂肪組織を摘出して重量を測定して、ラットの体重に対する各臓器の重量を算出して相対重量を求めた。結果、コントロール群と比較し、十二指腸を除く臓器においては、特に臓器相対重量に変化はみられなかった。しかしながら、十二指腸のみユーグレナ群(実施例1)及びパラミロン群(実施例2)において有意に増加を示した(Tukey - Kramer検定によりp<0.05)。このことから、本発明は消化器を増大するよう作用することが考えられる。十二指腸の相対重量を表5に示す。
各群の代表例の胃潰瘍の写真を図23に、潰瘍面積の測定結果を図24に示す。
図23に示すように、コントロール群では、血液が滲んで黒化した胃潰瘍部分(楕円の内部)が明瞭に観察されたのに対し、実施例1~3群(ユーグレナ群、パラミロン群、アモルファスパラミロン群)では、胃潰瘍部分が、コントロール群に対比して明らかに小さくなっていた。特に、ユーグレナ群(実施例1),パラミロン群(実施例2)では、胃潰瘍部分が顕著に小さくなっていた。
また、図24に示すように、コントロール群と比較し、ユーグレナ群(実施例1)において、胃潰瘍の面積が有意に低下していた(Tukey - Kramer検定によりp<0.05)。また、パラミロン群(実施例2)及びアモルファスパラミロン群(実施例3)においても低下する傾向がみられた。さらに表5で示すように、ユーグレナ群(実施例1)及びパラミロン群(実施例2)で十二指腸の相対重量が増加することから、消化器をストレスから防御する作用機序があることが考えられた。
図26にはiNOS/β‐アクチンを、図27には、COX-2/β‐アクチンを示した。図26の結果より、コントロール群と比較して、ユーグレナ群、パラミロン群及びアモルファスパラミロン群において、iNOSの発現の抑制が認められた。特にパラミロン群、アモルファスパラミロン群では有意に抑制していた(Turkey-Kramer検定により、p<0.05)。
よって、ユーグレナ,パラミロン及びアモルファスパラミロンの摂取により、iNOS及びCOX-2の発現の抑制が認められていることから、ストレスによる酸化障害を軽減することで胃潰瘍の抑制をしたことが考えられた。
つまり、ユーグレナ,パラミロン及びアモルファスパラミロンは、炎症の増悪作用を有するiNOSの発現の抑制、及び/又は炎症の増悪作用を有するPGの生合成の律速酵素であるCOX-2の発現の抑制を通して、抗炎症作用を発揮することが分かった。
また、本実施例のユーグレナ,パラミロン及びアモルファスパラミロンは、iNOSの発現抑制作用及び/又はCOX-2の発現抑制作用を有することが示された。従って、本実施例のユーグレナ,パラミロン及びアモルファスパラミロンは、iNOS発現抑制剤,COX-2発現抑制剤及び抗炎症剤として用いることができることが分かった。
実施例1~4の被験物質の関節リウマチに対する作用について、マウスのコラーゲン関節炎モデルを用いて評価を行った。
実験用の動物には、マウス(DBA/1J Jms Slc(SPF),6週齢雄,日本エスエルシー株式会社)を使用した。
ニワトリII型コラーゲン(SIGMA)を0.01 M 酢酸水溶液に2 mg/mLとなるように溶解し,これに等量のFreund’s complete adjuvant(Difco)を加えて作製したエマルジョン(コラーゲン1 mg/mL)を、イソフルランによる吸入麻酔下で,マウスの尾根部に0.1 mL(コラーゲン量として0.1 mg)皮内投与し、コラーゲンに感作させた。また、3週間後,同様に投与を行い、ブーストとした。また、無処置動物には、感作及びブーストは行わなかった。
スコア付は、垣本ら(新生化学実験講座12,分子免疫学II,東京化学同人:360-372,1989.)のスコアに準じて、表6の関節炎スコアに従って、3回/週(月・水・金)の頻度で評価を行い、四肢のスコアの合計値を算出した。
スコア測定最終日において、対照群と比較してユーグレナ群、パラミロン群、アモルファスパラミロン群、エマルジョンパラミロン群すべての群において、有意に低値となり、被験物質を継続摂取することによる関節炎症状の抑制が認められた。
抗コラーゲンIgGの測定結果を、図29に示す。
エマルジョンパラミロン群については、対照群と同程度の値であったが、ユーグレナ群、パラミロン群、アモルファスパラミロン群では、対照群と比較して、低値を示していた。
鼠径部リンパ節については、リンパ球を分画後3等分し、抗CD3抗体を添加した培地でそれぞれ培養を行った。培養開始後約48時間後に培養上清を採取し、培養上清のサイトカイン(IL-17A,IFN-γ)分泌量を、マルチプレックス サスペンションアレイにて測定した。
測定したサイトカインIL-17A,IFN-γについて、ユーグレナ群、パラミロン群、アモルファスパラミロン群、エマルジョンパラミロン群いずれも、対照群と比較して、低値を示していた。
図30,図31の結果より、ユーグレナ群、パラミロン群、アモルファスパラミロン群、エマルジョンパラミロン群において、Th1/Th17系のサイトカインであるIL-17A,IFN-γが抑制されたことが確認され、Th1/Th2/Th17免疫バランスを、Th1又はTh17が誘導する免疫反応が抑制される方向,つまり、Th2が誘導する免疫反応がTh1又はTh17が誘導する免疫反応に対して相対的に優位になる方向に調整されたことが確認された。
評価は、次の膝関節組織の評価法に従った。即ち、滑膜の組織については、浮腫,炎症性細胞浸潤,滑膜細胞増生,肉芽組織形成,線維化,関節腔内滲出物、大腿骨滑車溝の組織については、パンヌス形成,関節軟骨の破壊(変性・線維化),骨破壊(吸収),骨棘形成(反応性類骨形成)について、認められた所見を、変化なし(評点0),軽微(評点1),軽度(評点2),中等度(評点3),高度(評点4)の5段階で評価した。
なお、各群の代表例の選択については、四肢の合計スコアの平均値の近い動物を指標に選択した。無処置群と対照群は2例,被験物質摂取群は3例を選択した。
結果を、表7,表8に示す。
図33に示すように、無処置群では、滑膜及び大腿骨滑車溝に、関節炎に起因すると考えられる所見は認められなかった。
図34の対照群では、滑膜の浮腫,炎症,肉芽組織形成,線維化,滲出物が軽微(評点1)から中等度(評点2)に認められた。大腿骨滑車溝については,パンヌス形成及び軟骨破壊が軽度(評点2)に認められた。
図35のユーグレナ群及び図37のアモルファスパラミロン群については、滑膜の肉芽組織形成が軽微(評点1)に認められた以外は、所見は認められなかった。
図36のパラミロン群では、滑膜の炎症,肉芽組織形成,線維化及び大腿骨滑車溝のパンヌス形成,軟骨破壊が、軽微(評点1)から軽度(評点2)に認められた。
図38のエマルジョンパラミロン群については、対照群とほぼ同様で、滑膜の浮腫・炎症、肉芽組織形成、線維化、滲出物が軽微(評点1)に認められた。大腿骨滑車溝については、パンヌス形成及び軟骨破壊が軽度(評点2)に認められた。
フローサイトメーターを用いた解析で得られたCD4陽性T細胞中のIL-17産生細胞数の割合を、図39に示す。
図39のIL-17A産生細胞数について、パラミロン群、アモルファスパラミロン群については、対照群よりも減少していた。エマルジョンパラミロン群では、対照群と比較して有意に減少していた。
病理組織学的検査では、ユーグレナ群及びアモルファスパラミロン群で、滑膜の肉芽組織形成が軽微に認められた以外は、所見は認められなかった。パラミロン群については、滑膜の炎症,肉芽組織形成,線維化及び大腿骨滑車溝のパンヌス形成,軟骨破壊が認められたものの、その程度は、対照群と比較して顕著ではなかった。
リンパの培養上清中のサイトカインについては、Anti-CD3刺激により、ユーグレナ群,パラミロン群,アモルファスパラミロン群及びエマルジョンパラミロン群で、いずれも対照群と比較して分泌の低下が認められた。特に、ユーグレナ群及びアモルファスパラミロン群で、その低下は顕著であった。
以上より、マウスのコラーゲン関節炎モデルを用いてユーグレナ由来物質の自己免疫疾患に対する作用について検討を行った結果、ユーグレナ群,パラミロン群,アモルファスパラミロン群及びエマルジョンパラミロン群で、関節炎の発症抑制作用が認められた。特に、ユーグレナ群及びアモルファスパラミロン群では、その作用が顕著であり、組織学的にも顕著な抑制が認められた。
b 脛骨
c 膝蓋骨
d 後十字靭帯
e 半月板
f 前十字靭帯
Claims (14)
- ユーグレナ由来物質を含み、生体においてTh1,Th2及びTh17がそれぞれ誘導する免疫反応のバランスであるTh1/Th2/Th17免疫バランスを調整することを特徴とする免疫バランス調整剤。
- 前記Th1/Th2/Th17免疫バランスを、Th1が誘導する免疫反応がTh2又はTh17が誘導する免疫反応に対して相対的に優位になる方向に調整することを特徴とする請求項1記載の免疫バランス調整剤。
- 前記Th1/Th2/Th17免疫バランスがTh2に偏向した体質の改善に用いられることを特徴とする請求項1又は2記載の免疫バランス調整剤。
- 前記Th1/Th2/Th17免疫バランスがTh2に偏向した体質は、感染症又はストレス性の疾患を罹患しやすい体質であることを特徴とする請求項3記載の免疫バランス調整剤。
- 前記Th1/Th2/Th17免疫バランスのTh2への偏向が関与する疾患の予防又は治療に用いられることを特徴とする請求項1又は2記載の免疫バランス調整剤。
- 前記Th1/Th2/Th17免疫バランスのTh2への偏向が関与する疾患の発症が予測される時期よりも前に投与されることを特徴とする請求項4記載の免疫バランス調整剤。
- 前記Th1/Th2/Th17免疫バランスのTh2への偏向が関与する疾患は、感染症又はストレス性の疾患であることを特徴とする請求項5又は6記載の免疫バランス調整剤。
- 前記感染症は、インフルエンザであって、抗インフルエンザ剤として用いられることを特徴とする請求項7記載の免疫バランス調整剤。
- 前記ストレス性の疾患は、消化性潰瘍であって、消化性潰瘍予防又は治療剤として用いられることを特徴とする請求項7記載の免疫バランス調整剤。
- 前記生体におけるIL-4の産生量に対するIFN-γの産生量の比率を上昇させることを特徴とする請求項1又は2記載の免疫バランス調整剤。
- 前記生体におけるIFN-γの産生を促進し、IL-4、IL-5及びIL-10の産生を抑制することを特徴とする請求項1又は2記載の免疫バランス調整剤。
- 前記Th1/Th2/Th17免疫バランスを、Th2が誘導する免疫反応がTh1又はTh17が誘導する免疫反応に対して相対的に優位になる方向に調整することを特徴とする請求項1記載の免疫バランス調整剤。
- 前記Th1/Th2/Th17免疫バランスのTh1及び/又はTh17への偏向が関与する疾患の予防又は治療に用いられ、
前記疾患は、関節リウマチであることを特徴とする請求項1又は12記載の免疫バランス調整剤。 - 前記ユーグレナ由来物質は、パラミロン又はその加工品であることを特徴とする請求項1乃至13いずれか記載の免疫バランス調整剤。
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Cited By (17)
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WO2016072507A1 (ja) * | 2014-11-07 | 2016-05-12 | 株式会社ユーグレナ | 消化性潰瘍の予防又は治療剤,及び予防又は治療のための食品添加物,iNOS発現抑制剤及びCOX-2発現抑制剤 |
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WO2018183560A1 (en) * | 2017-03-28 | 2018-10-04 | Kemin Industries, Inc. | Method of promoting immune health using the water-soluble component from genus euglena organism |
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Also Published As
Publication number | Publication date |
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CN106163533A (zh) | 2016-11-23 |
CN106163533B (zh) | 2021-08-20 |
MY173162A (en) | 2019-12-31 |
KR20160142831A (ko) | 2016-12-13 |
JP6307153B2 (ja) | 2018-04-04 |
US20170020939A1 (en) | 2017-01-26 |
SG11201608207XA (en) | 2016-11-29 |
JPWO2015156339A1 (ja) | 2017-04-13 |
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