JP2020158460A - Macrophage activator - Google Patents

Abstract

To provide immunostimulants such as macrophage activators.SOLUTION: Provided is a macrophage activator containing at least one selected from the group consisting of Euglena, paramylon, processed paramylon, and β-1,3-glucan.SELECTED DRAWING: None

Description

The present invention relates to macrophage activators and the like.

Macrophages are responsible for removing pathogens and foreign substances that have entered the living body, presenting antigens, and the like. It is known that when macrophages are activated, they produce cytokines such as TNF-α, nitric oxide, and the like, and exert antibacterial effect, antiviral effect, antitumor effect, and the like. Therefore, various macrophage activators have been proposed in order to measure the expression of these effects. For example, Patent Document 1 describes a macrophage activator containing a hot water extract of bonito shavings or a dried product thereof as an active ingredient.

Euglena is a microalga belonging to the genus Euglena (= genus Euglena) and is used as a food material. Euglena extract has also been applied to the skin. In addition, paramylon is a β-1,3-glucan produced by Euglena and has been reported to be useful for wound treatment and allergy suppression. However, the relationship between Euglena and β-1,3-glucan and macrophage activation has not yet been clarified.

Japanese Unexamined Patent Publication No. 2017-075105

An object of the present invention is to provide an immunostimulant such as a macrophage activator.

The present inventor is a macrophage activator containing at least one selected from the group consisting of Euglena, paramylon, processed paramylon, and β-1,3-glucan as a result of diligent research in view of the above problems. If there is, it was found that the above problem can be solved. As a result of further research based on this finding, the present invention has been completed. That is, the present invention includes the following aspects:
Item 1. A macrophage activator containing at least one selected from the group consisting of Euglena, paramylon, processed paramylon, and β-1,3-glucan.
Item 2. Item 2. The macrophage activator according to Item 1, which contains the processed paramylon product.
Item 3. Item 2. The macrophage activator according to Item 2, wherein the processed paramylon product is fibrous paramylon.
Item 4. Item 2. The macrophage activator according to Item 2 or 3, wherein the processed paramylon is a processed product of paramylon derived from Euglena gracilis.
Item 5. Item 2. The macrophage activator according to any one of Items 2 to 4, wherein the processed paramylon is a processed paramylon derived from Euglena gracilis EOD-1 strain (accession number FERM BP-11530).
Item 6. Item 4. The macrophage activator according to any one of Items 1 to 5, which is a food composition, a food additive, a cosmetic product, a cosmetic additive, or a pharmaceutical product.
Item 7. Item 4. The macrophage activator according to any one of Items 1 to 6, which is an oral composition.
Item 8. An immunostimulant containing fibrotic paramylon.
Item 9. Item 8. The immunostimulant according to Item 8, which is a food composition, a food additive, a cosmetic product, a cosmetic additive, or a medicine.
Item 10. Item 8. The immunostimulant according to Item 8 or 9, which is an oral composition.

According to the present invention, an immunostimulant such as a macrophage activator can be provided.

An electron micrograph of fibrotic paramylon (Reference Example 3) is shown. The length of one scale on the lower right of the figure indicates 1 μm. An electron micrograph of fibrotic paramylon (Reference Example 4) is shown. The length of one scale on the lower right of the figure indicates 1 μm. The result of the nitric oxide production amount measurement test of Test Example 1 is shown. The vertical axis shows the amount of increase in nitric oxide concentration from the case where the test substance is not added. The horizontal axis shows the concentration of the test substance. The legend shows the type of test substance. Error bars indicate standard deviation. The result of the TNF-α production amount measurement test of Test Example 2 is shown. The vertical axis shows the TNF-α concentration when the test substance is not added. The horizontal axis shows the concentration of the test substance. The legend shows the type of test substance. Error bars indicate standard deviation.

In the present specification, the expressions "contains" and "contains" include the concepts of "contains", "contains", "substantially consists" and "consists of only".

In the present specification, the expression "and / or" includes the meaning when either "and" or "or" is selected. That is, the expression "A and / or B" includes both the meanings of "A or B" and "A and B".

In one aspect thereof, the present invention relates to a macrophage activator containing at least one selected from the group consisting of Euglena, paramylon, processed paramylon, and β-1,3-glucan. The present invention also relates to an immunostimulant containing fibrotic paramylon in one aspect thereof. Hereinafter, these may be collectively referred to as "the agent of the present invention". These will be described below.

1. 1. Euglena Euglena is a microalga belonging to the genus Euglena (= genus Euglena), and is not particularly limited as long as it is. As Euglena, specifically, for example, Euglena gracilis (Euglena gracilis), Euglena longa, Euglena caudata, Euglena oxyuris, Euglena tripteris, Euglena proxima, Euglena viridis, Euglena sociabilis, Euglena ehrenbergii, Euglena deses, Euglena pisciformis, Euglena spirogyra, Euglena acus , Euglena geniculata , Euglena intermedia , Euglena mutabilis , Euglena sanguinea , Euglena stellata , Euglena terricola , Euglena klebsi , Euglena rubra , Euglena cyclopicola and the like. Among these, from the viewpoint of the effect of the present invention, Euglena gracilis is preferably mentioned, and more preferably Euglena gracilis EOD-1 strain [As of June 28, 2013, National Institute of Technology and Evaluation Patent Organism Deposited Center {NITE-IPOD (Postal code 292-0818, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan, Room 120)} is listed as a deposit number FERM BP-11530 under the provisions of the Budapest Treaty] Be done.

The morphology of Euglena is not particularly limited as long as it contains most of Euglena's cell body or its components. Examples of the form of Euglena include a dry powder form of Euglena, a suspension of Euglena, a Euglena extract and the like, and among them, a dry powder form of Euglena is preferable.

The paramylon content of Euglena in the dry state is not particularly limited. The content is, for example, 20% or more, preferably 30% or more, more preferably 40% or more, still more preferably 50% or more, still more preferably 60% or more, and particularly preferably 70% or more.

Euglena may be a single species or a combination of two or more species.

2. If β-1,3-glucan and paramylon β-1,3-glucan have one sugar chain (or sugar chain structure) in which glucose is linked only by β1,3 bonds as the main chain. There are no particular restrictions. β-1,3-glucan is not limited to linear ones, but also includes those having branched chains.

The weight average molecular weight of the β-1,3-glucan derivative is not particularly limited, but is, for example, 1 × 10 ⁴ to 2 × 10 ⁶ , preferably 5 × 10 ⁴ to 1 × 10 ⁶ , and more preferably 1 × 10 ⁵ to 1. There are 1 x 10 ⁶ . The weight average molecular weight can be measured by the GPC method.

The β-1,3-glucan may be obtained by chemical synthesis, but from the viewpoint of availability and the like, natural β-1,3-glucan produced by various organisms is preferable. Examples of the natural β-1,3-glucan include paramylon, curdlan, laminarin, callose, lentinan, schizophyllan and the like. Among these, paramylon is preferably mentioned from the viewpoint of the effect of the present invention. Hereinafter, paramylon will be described.

Paramylon is a β-1,3-glucan derived from Euglena, and is not particularly limited as long as it is.

The Euglena from which paramylon is derived is the same as the explanation in "1. Euglena" above.

The mass average molecular weight of paramylon is not particularly limited, but is, for example, 1 × 10 ^{4 to} 5 × 10 ⁶ , preferably 2 × 10 ⁴ to 1 × 10 ⁶ , more preferably 5 × 10 ⁴ to 1 × 10 ⁶ , and even more preferably. Is 1 × 10 ^{5 to} 5 × 10 ⁵ .

The mass average molecular weight can be measured by SEC-MALS analysis under the following conditions:
SEC equipment: LC-10AVP system (Shimadzu Co., Japan),
Column used: KD-806M (shodex., Japan),
MALS detector: DAWN HELEOSII (wyatt Technologies., USA),
Eluent: 1% LiCl / DMI,
Flow rate: 0.5 mL / min.

Paramylon exists in Euglena cells as paramylon particles in which triple helix structures formed by β-1,3-glucan chains are highly integrated under a certain regularity.

The shape of the paramylon particles is not particularly limited, but is usually a flat spheroid.

The particle size distribution of the paramylon particles is not particularly limited, but is, for example, 0.5 to 15 μm, preferably 1 to 6 μm. The average particle size of the paramylon particles is also not particularly limited, but is, for example, 1 to 10 μm, preferably 2 to 4 μm.

The form of paramylon is not particularly limited as long as it contains paramylon. Examples of the form of Euglena include a dry powder form of paramylon, a suspension of paramylon, and the like, and among them, a dry powder form of paramylon is preferable.

Paramylon may be used alone or in combination of two or more.

3. 3. Method for Producing Euglena and Paramylon Euglena can be prepared in large quantities by a method including a step of culturing Euglena contained in a liquid (culture step). The culturing step can be carried out according to, for example, a known method (for example, the method described in Japanese Patent No. 5883532). In the culture step, Euglena microalgae are typically cultured under aerobic conditions while stirring a liquid (culture solution) containing water, Euglena, and nutrients available to Euglena.

Nutrients include sugars (monosaccharides such as glucose (dextrose) and fructose (fruit sugar)) and minerals (eg sodium, potassium, magnesium, calcium, iron, zinc, molybdenum, copper, phosphorus, nitrogen, sulfur, or boron. , Vitamin Bs (eg, vitamin B1 (thiamine), vitamin B2 (riboflavin), niacin, pantothenic acid, vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine), vitamin B12 (cyanocobalamine), folic acid, biotin, etc.) Be done. The concentration of nutrients in the culture solution is not particularly limited as long as Euglena can survive, proliferate, and the like.

The light conditions of the culturing step are not particularly limited, and the culturing step may be carried out under either light or dark conditions. When culturing in heterotrophic culture, it is cultivated under dark conditions. As a bright condition, normal light intensity for growing algae can be adopted. Dark conditions include, for example, less than 10 μmol / m ² / s, preferably complete dark conditions with no light.

The culture temperature in the culture step is not particularly limited as long as it is a temperature at which Euglena can grow. As the culture temperature (temperature of the culture solution), for example, 20 ° C to 35 ° C is adopted.

The pH of the liquid in the culturing step is not particularly limited as long as it is a pH at which Euglena can grow. For example, 3.0 to 5.5 is adopted as the pH at which Euglena can grow.

After the culturing step, it is preferable to concentrate Euglena by centrifugation or gravity separation of the liquid. The obtained Euglena can be subjected to additional treatments (eg, suspension in liquid, dispersion in water or oil, extract extraction, dry powdering, etc.), depending on the desired form.

Paramylon particles can be produced by separating, isolating, or purifying Euglena according to or according to known methods (eg, methods described in Japanese Patent No. 5883532). Paramylon particles can be easily obtained, for example, by recovering the cell content component obtained by destroying the cell membrane of Euglena. In addition, paramylon particles may be purified if necessary. Various types of purification of paramylon particles are known (for example, Japanese Patent No. 5883532), and the purification can be carried out according to these methods. Examples of the purification step include a surfactant treatment step and a cleaning step. The resulting Euglena can be subjected to additional treatments (eg, suspension in liquid, dispersion in water or oil, dry powdering, etc.), depending on the desired form.

Four. Processed paramylon The processed paramylon is obtained by processing paramylon, for example, physical treatment, chemical treatment, or the like, and is not particularly limited as long as it is. Examples of the paramylon compound include fibrous paramylon and amorphous paramylon. Amorphal paramylon can be obtained by chemically treating according to or according to a known method, for example, using the method described in JP-A-2011-184592.

As the paramylon compound, fibrous paramylon is preferable. The fibrous paramylon will be described below.

The fibrous paramylon is a β-1,3-glucan derived from Euglena, and is not particularly limited as long as it is in a fibrous form. Amorphous paramylon obtained by chemically treating paramylon particles (alkaline treatment, etc.) has been reported so far, but this is not recognized as fibrosis when observed with an electron microscope, and its shape and size are Since it is an amorphous mass, it is not included in fibrotic paramylon.

The weight average molecular weight of the fibrous paramylon is not particularly limited, but is, for example, 1 × 10 ⁴ to 2 × 10 ⁷ , preferably 1 × 10 ^{5 to} 5 × 10 ⁵ .

The weight average molecular weight can be measured by the following method by SEC-MALS analysis: SEC device: LC-10ADvp system (Shimadzu Co., Japan),
Column used: KD-806M (shodex., Japan),
MALS detector: DAWN HELEOSII (wyatt Technologies., USA),
Eluent: 1% LiCl / DMI,
Flow rate: 0.5 mL / min.

The fiber diameter of the fibrous paramylon is not particularly limited, but is, for example, 10 to 500 nm, preferably 20 to 300 nm, and more preferably 50 to 200 nm. The fiber diameter of the fibrous paramylon can usually be measured based on an electron micrograph of the fibrous paramylon.

The settling volume of the fibrous paramylon in water is not particularly limited, but is, for example, 30 to 300 mL / g, preferably 50 to 250 mL / g, and more preferably 70 to 200 mL / g.

The submerged volume can be measured according to or according to the following method:
Measure according to the method described in "Supervised by the Dietary Fiber Society of Japan, Editorial Committee of the Dietary Fiber Society of Japan (2008) Dietary Fiber-Basics and Applications-Third Edition, p.111 First Publishing, Tokyo" .. Specifically, it is as follows. Weigh 125 mg of the sample slurry test sample into a 25 mL volume plastic tube in terms of dry weight, and shake the plastic tube vigorously by hand to stir the contents. Then transfer the contents to a 25 mL volume graduated cylinder and add pure water to 25 mL. After stirring the liquid in the measuring cylinder, leave it at 37 ° C for 24 hours. As a result, the sample precipitates, and two layers (a layer mainly containing the precipitated sample (lower layer) and a layer mainly containing water (upper layer)) are formed. The volume of the lower layer is obtained from the scale of the measuring cylinder, and the obtained volume is divided by the sample mass (dry mass) to calculate the submerged volume (mL / g).

Fibrotic paramylon has a relatively high resistance to enzymatic degradation. For example, the amount of monomer (glucose) produced by the decomposition of β-glucanase is, for example, 0.1 to 50 mg, preferably 1 to 10 mg per 1 g of fibrotic paramylon.

This amount can be measured according to or according to the following method:
Reaction solution [Test substance 30 mg (dry weight), buffer solution (Tokyo Kasei Kogyo Co., Ltd. B0156, potassium hydrogen phthalate-sodium hydroxide buffer (pH 4.0)) 5 mL, enzyme solution (Nippon Biocon Co., Ltd. endo- 1,3-β-Glucanase (enzyme content: 50 units / mL)) 0.1 mL, pure water, reaction solution volume 10 mL] is prepared, and shaken horizontally at 40 ° C. for 24 hours at 45 rpm. Immediately after shaking, cryopreserve and lyophilize for concentration. After lyophilization, add 0.5 mL of pure water to each sample and stir (concentrate 20 times). Centrifuge (10000G, 5 minutes, 4 ° C) and collect the supernatant twice. The glucose concentration in the recovered supernatant is measured using a measurement kit (Fujifilm Wako Pure Chemical Industries, Ltd., Glucose CII-Test Wako). Based on the measured values, the amount of glucose produced per 1 g of the test substance (mg) is calculated.

Fibrotic paramylon has relatively low solubility in alkaline solutions. For example, fibrotic paramylon is insoluble in 0.1-0.3 M aqueous sodium hydroxide solution. Here, "insoluble" means that, for example, the absorbance (660 nm) of the solution after suspending the fibrotic paramylon in the aqueous solution (for example, immediately after 1 hour) is, for example, 0.1 or more, preferably 1.0. It means that it is the above.

Solubility can be measured according to or according to the following methods:
Suspend 250 mg (dry weight) of the test substance in 10 mL of the test solution (0.1 M NaOH aqueous solution, 0.3 M NaOH aqueous solution) in the vial. After shaking the vial vigorously by hand for 20 seconds and shaking at 80 rpm for 1 hour on a shaker, measure the absorbance of the liquid in the vial at 660 nm. The absorbance is measured using a spectrophotometer V-730 manufactured by JASCO Corporation.

The relative value of the crystallinity of the fibrous paramylon with respect to the granular paramylon (crystallinity of the fibrous paramylon / crystallinity of the granular paramylon) is, for example, 0.60 to 0.90, preferably 0.65 to 0.80.

Crystallinity can be measured according to or according to the following method:
XRD measure the test substance. The conditions are as follows. Equipment: PANalytical X'Pert3 Powder, tube voltage: 45kV, tube current: 40mA, measurement range: 5.005 to 50.018 °, measurement interval: 0.013 °, analysis software: High Score. The crystallinity is analyzed by the ratio of the strength of the amorphous part to the strength of the crystal part at 2θ = 5 to 80 °. The analysis is performed after removing the background by the device from each measurement (background setting Auto, venting factor 0, granularity 100), and the amorphous part is determined by the tangent line passing through 2θ = 14, 29 °. To do. The pending factor and granularity conditions that determine each amorphous part are 0/20.

The fibrous paramylon may be in a form dispersed in a solvent such as water, or may be in a dry form. The fibrous paramylon can be redispersed in water, even in dry form.

In addition, in this specification, a "dry form" means that a water content is 15% by mass or less, preferably 10% by mass or less, and more preferably 5% by mass or less.

As the fibrous paramylon, preferably, a defibrated product of paramylon particles obtained by physically defibrating the paramylon particles can be used. In addition, Euglena's defibrated product obtained by applying this defibration treatment to Euglena can also be used as fibrillated paramylon.

The defibration treatment hardly cleaves the hydrogen bonds of β-1,3 glucan present in the paramylon particles (for example, 10% or less, 5% or less, 2% of the hydrogen bonds of β-1,3-glucan). Below, a process that can be defibrated (by cutting only 1% or less), or a part or all of the β-1,3-glucan chain existing in the paramylon particles or the triple spiral structure formed by the chain. There are no particular restrictions as long as the processing can be performed. It is preferable that the β-1,3-glucan present in the paramylon particles is defibrated with almost no breakage to form a fibrous form. A known treatment capable of grinding (shearing) or crushing (preferably grinding (shearing)) fine particles such as paramylon particles can be adopted as the defibration treatment.

The defibration treatment can be performed using a known grinder (shearing machine), a crusher, or the like. Examples of the apparatus used for the defibration treatment include a stone mill type grinder, a jet mill, a twin-screw kneader, a high-pressure homogenizer, a high-pressure emulsifier, a twin-screw extruder, and a bead mill. Among these, a stone mill type grinder and a bead mill are preferable.

The defibration treatment can be performed wet or dry. Wet defibration treatment is preferable because it enables more efficient dispersion of fibrous paramylon in the solution. The solvent used in a wet manner is not particularly limited as long as it is a solvent in which fibrous paramylon can be dispersed, and water can be preferably used.

The defibration treatment may be performed by one type alone or in combination of two or more types. Further, a part of the paramylon may be defibrated, and it is the object of the present invention as long as the defibrated paramylon is included.

Five. Applications Euglena, paramylon, processed paramylon, and β-1,3-glucan are composed of euglena, paramylon, processed paramylon, and β-1,3-glucan because they have immunostimulant effects such as macrophage activation. At least one selected from the group can be utilized as an active ingredient of a macrophage activator and an immunostimulant.

As the active ingredient, a processed paramylon product is preferable from the viewpoint of the effect of the present invention. Among them, fibrotic paramylon is particularly preferable as an active ingredient because it has an immunostimulatory action such as a particularly high macrophage activating action.

Since the active ingredient of the present invention or the agent of the present invention promotes the production of activation markers such as TNF-α and nitric oxide, it can also be used as an M1 macrophage induction promoter.

Since the active ingredient of the present invention or the agent of the present invention promotes the production of activation markers such as TNF-α and nitrogen monoxide, applications based on these, such as antibacterial effect, antiviral effect, and antitumor effect, are used. It can also be used as various uses based on the above (for example, antibacterial activity improving agent, antiviral ability improving agent, antitumor ability stabilizing agent, etc.). In one aspect of the present invention, it can be used for a comprehensive use including a plurality of (two or more, three or more) uses among these uses.

Furthermore, the uses, purposes, and targets listed below are:
It can also be used for (a) immunopotentiation (b) improvement of natural healing power (c) prevention of infectious diseases.

The applicable bacteria are not particularly limited, and examples thereof include Gram-positive bacteria and Gram-negative bacteria regardless of the type. Gram-positive bacteria to be applied include, for example, staphylococci (eg, yellow cocci, epidermis staphylococci), enterococci (eg, Enterococcus), streptococci (eg, diplococci, quadruple, octagonal). Etc., pneumoniae, hemolytic streptococcus), Bacillus (eg, Enterococcus, Bacillus subtilis), Clostridium (eg, Enterococcus, Botulinum), Corinebacterium (eg, Difteria), Listeria, Lactobacillus Examples include spp., Bifidobacterium spp., Propionibacterium spp. (Eg, Acne cocci that cause acne), and Streptococci. Applicable Gram-negative bacteria include, for example, Escherichia (eg, Escherichia coli), Salmonella, Pseudomonas (eg, Pseudomonas aeruginosa), Helicobacter, Haemophilus influenzae, Neisseria (eg, Neisseria gonorrhoeae, Meningococcus). Bacteria).

The target virus is not particularly limited, but for example, influenza virus (for example, type A, type B, etc.), ruin virus, Ebola virus, corona virus, measles virus, varicella / herpes zoster virus, simple herpes virus, mumps virus, arbovirus. , RS virus, SARS virus, hepatitis virus (eg, hepatitis B virus, hepatitis C virus, etc.), yellow fever virus, AIDS virus, mad dog disease virus, hunter virus, dengue virus, nipavirus, lissavirus, etc. Virus); Non-enveloped viruses (viruses without envelope) such as adenovirus, norovirus, rotavirus, human papillomavirus, poliovirus, enterovirus, coxsackie virus, human parvovirus, encephalomyelitis virus, poliovirus, rhinovirus, etc. Can be mentioned.

The target tumor is not particularly limited, and is, for example, lung cancer, stomach cancer, liver cancer, esophageal cancer, pancreatic cancer, colon cancer, biliary tract cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer. , Breast cancer, prostate cancer, testicular cancer, skin cancer, leukemia, bone tumor, osteosarcoma, soft tumor, multiple myeloma, malignant lymphoma, pharyngeal cancer, head and neck cancer, childhood cancer, etc. Be done.

In various fields, the agent of the present invention includes, for example, food compositions (including health foods, health enhancers, dietary supplements (supplements, etc.)), food additives, cosmetics, cosmetic additives, pharmaceuticals, reagents, feeds, etc. Can be used as. The agent of the present invention is preferably an oral composition.

The form of the agent of the present invention is not particularly limited, and can take a form usually used in each application depending on the application.

As the form of the agent of the present invention, when the use is a food composition, liquid, gel or solid foods such as juices, soft drinks, teas, soups, soymilk and other beverages, salad oils, dressings, yogurts and jellies. , Pudding, sprinkle, milk powder for childcare, cake mix, dairy products (eg, powder, liquid, gel, solid, etc.), bread, confectionery (eg, cookies, etc.) and the like.

As the form of the agent of the present invention, when the use is cosmetics, for example, milky lotion, cosmetic liquid, face cream, hand cream, lotion, body soap, shampoo, rinse, cosmetic gel, pack, foundation, lip balm, face wash. Agents and the like can be mentioned.

As the form of the agent of the present invention, when the use is pharmaceutical, for example, an ointment, an external liquid agent (liniment agent, lotion agent, etc.), a spray agent (external aerosol agent, pump spray agent, etc.), a cream agent, a gel agent, etc. , Patches (plasters, tapes such as plasters (reservoir type, matrix type, etc.), paps, patches, microneedles, etc.), eye drops, eye ointments, nasal drops, suppositories, half for rectal Formulations suitable for parenteral ingestion of solids, enema, etc. (particularly topical formulations); tablets (including medially disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.), Formulations suitable for oral ingestion such as pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), jelly, etc. Oral formulation form).

As the form of the agent of the present invention, when the use is an additive, a health promoter, a dietary supplement (supplement, etc.), for example, a tablet (intraoral disintegrating tablet, chewable tablet, effervescent tablet, troche agent, jelly) (Including pills and the like), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including suspensions and syrups), jelly and the like.

The agent of the present invention may further contain other components, if necessary. Other ingredients include ingredients that can be incorporated into food compositions (including health foods, health enhancers, dietary supplements (supplements, etc.)), food additives, cosmetics, cosmetic additives, pharmaceuticals, reagents, feeds, etc. As long as it is, for example, a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, and a coloring agent. Examples include foodstuffs, fragrances, and chelating agents.

The content of the active ingredient in the agent of the present invention depends on the intended use, usage mode, state of application, etc., and is not limited, but is, for example, 0.0001 to 100% by mass, preferably 0.001 to 50% by mass. can do.

The amount of the agent of the present invention applied (for example, administration, ingestion, inoculation, etc.) is not particularly limited as long as it is an effective amount that exhibits an immunostimulatory effect such as macrophage activating effect, and is usually used as the dry weight of the active ingredient. , Generally 0.1-10000 mg / kg body weight per day. The above application amount is preferably applied once or more once a day (for example, 1 to 3 times), and can be appropriately increased or decreased depending on the age, pathological condition, and symptoms.

Hereinafter, the present invention will be described in detail based on Examples, but the present invention is not limited to these Examples.

Reference example 1
As Euglena, Euglena Gracilis EOD-1 strain (NITE-IPOD, National Institute of Technology and Evaluation) dry powder (manufactured by Kobelco Eco-Solutions, paramylon content of 70% or more) was prepared.

Reference example 2
Paramylon was prepared as follows.

The prepared Euglena gracilis EOD-1 strain (after culturing and before drying) was collected for 5 flasks, and the collected liquid was centrifuged (500 × g, 4 minutes, room temperature) in a centrifuge tube. .. The supernatant in the centrifuge tube was once removed and collected. The collected supernatant was placed in a centrifuge tube to disperse the precipitate in the centrifuge tube, and the whole was transferred to a graduated cylinder having a volume of 100 mL. Further, the collected supernatant was added to the measuring cylinder, and the size was increased to 90 mL.

[Enzyme treatment process]
The pH of the liquid was adjusted to 3 by transferring the liquid prepared to 90 mL to a 200 mL beaker and adding an aqueous hydrochloric acid solution with stirring. A proteolytic enzyme (acidic protease product name "Protease YP-SS" manufactured by Yakult Pharmaceutical Co., Ltd., optimum pH 2.5 to 3.0) was added to the liquid to a concentration of 5 g / L. The liquid was treated with an enzyme at 50 ° C. for 2 hours while stirring.

[Surfactant treatment process]
An aqueous solution of sodium dodecyl sulfate was added to the liquid that had undergone the enzyme treatment step so that the concentration of sodium dodecyl sulfate was 3.0% by mass / volume (w / v)%. The pH of the liquid was adjusted to 3 by adding an aqueous hydrochloric acid solution while stirring the liquid containing sodium dodecyl sulfate. Further, the liquid was stirred with a propeller stirrer (rotation speed 200 rpm) at 60 ° C. for 30 minutes.

[Separation process]
Paramylon was precipitated by centrifugation (1000 × g, 2 minutes, room temperature), and paramylon was separated from the liquid that had undergone the surfactant treatment step. A surfactant treatment step was further carried out in the same manner except that the concentration of sodium dodecyl sulfate was changed to 1.0% by mass / volume% and the pH was not adjusted. Then, the separation step was performed in the same manner as described above. In this way, the surfactant treatment step and the separation step were performed three times each.

[Washing process]
Paramylon precipitated by centrifugation in the separation step was suspended in pure water and allowed to stand at 40 ° C. for 10 minutes. Paramylon was then precipitated by centrifugation (1000 xg, 2 minutes, room temperature). Such an operation was performed a total of three times.

[Drying process]
Paramylon precipitated by centrifugation in the washing step was dried at 50 ° C. to obtain paramylon particles. To the obtained paramylon particles, twice the amount of an excipient (Nippon Kagaku: Amicol No-3L) was added and used as paramylon in the following test examples.

Reference example 3
The fibrous paramylon was prepared as follows.

Shear force was applied to the paramylon particles obtained in Reference Example 2 by a bead mill to fibrate the paramylon particles to produce a slurry (liquid) containing the fibrous paramylon. The defibration treatment with a bead mill was performed under the general operating conditions used for submicron pulverization. The raw material liquid containing 10% by mass of paramylon particles was defibrated by a bead mill.

To the obtained slurry, add twice the amount of excipient (Nichijo Kagaku: Amicol No-3L) to the dry weight of the raw material paramylon particles, mix well, and dry the obtained mixture to evaporate the water content. , Used as fibrotic paramylon in the following test examples.

The obtained fibrotic paramylon was observed with an electron microscope. The observation image is shown in FIG. As shown in FIG. 1, it was found that the structure was branched, a large number of fibrous substances had a network structure, and a plurality of fibrous substances were intertwined and gathered together. It was.

Reference example 4
The fibrous paramylon was prepared as follows.

The paramylon particles obtained in Reference Example 2 were used as a starting material. A raw material solution containing the paramylon particles was prepared by mixing the paramylon particles and water so that the concentration of the paramylon particles was 5% by mass. Using a stone mill type grinder (manufactured by Masuyuki Sangyo Co., Ltd., product name "Super Mascoroider"), put the raw material liquid between the first member (grinding stone) and the second member (grinding stone), and put the raw material liquid between the first member (grindstone) and the first member (grindstone). By sliding the two members against each other, a shearing force was applied to the paramylon particles to fiberize the paramylon particles, and a slurry containing the fibrous paramylon was produced.

The above-mentioned wet defibration treatment in the shearing step using a millstone grinder was performed under the following conditions.
[Defibering process]
・ Grinder type: MKGC type ・ Clearance (grindstone gap): -100 μm
・ Grindstone rotation speed: 1200 rpm
The slurry obtained by the stone mill type grinder is recovered, and the recovered slurry is subjected to defibration treatment again to obtain a slurry, and the same operation is repeated 20 times in total (20 passes) to obtain a slurry containing fibrous paramylon. Got

To the obtained slurry, add twice the amount of excipient (Nichijo Kagaku: Amicol No-3L) to the dry weight of the raw material paramylon particles, mix well, and dry the obtained mixture to evaporate the water content. , Used as fibrotic paramylon in the following test examples.

The obtained fibrotic paramylon was observed with an electron microscope. The observation image is shown in FIG. As shown in FIG. 2, it was found that the structure was branched, a large number of fibrous substances had a network structure, and a plurality of fibrous substances were intertwined and gathered together. It was.

Test example 1. Evaluation test of macrophage activation 1
Activated macrophages are known to produce nitric oxide (NO). In this test, macrophage activation was evaluated by allowing a test substance to act on macrophages and measuring the amount of nitric oxide produced. Specifically, it was carried out as follows.

As cells, rat alveolar-derived macrophages (NR8383, ATCC CRL-2192) were used. In addition, HAM's F12K (Thirmo Fisher) + 15% FBS (Gibco performance grade, deactivated at 56 ° C for 30 minutes) was used as the medium. The cells were cultured in this medium and passaged twice a week as a guide. The medium was changed once every two days as a guide. The culture was carried out in an incubator at 37 ° C. and 5% CO ₂ . In the following tests, an antibiotic (100 × Antibiotic Antimycotic Solution Sigma) was added to the above medium so as to be 1 ×.

The cultured cells were peeled off using pipetting and a cell scraper, and centrifuged (300 × g, 5 minutes) to submerge the cells, and the supernatant was removed. Suspend the cells in a medium containing antibiotics, take a part of the suspension, stain the dead cells with trypan blue (# 207-17081, Wako Pure Chemical Industries, Ltd.), and then use a hemocytometer. The viable cell concentration was measured. The cell concentration was adjusted to 1.6 × 10 ⁶ cell / ml, 100 μl was inoculated on a 96-well plate, and the cells were cultured for 3 hours. 100 μl of a medium in which the test substance (paramylon (Reference Example 2) or fibrotic paramylon (Reference Example 3 or 4)) was uniformly suspended was added. The concentration after mixing was adjusted to 0, 10, 30, 90 μg / ml as paramylon or fibrous paramylon. In addition, polymyxin B was added to a final concentration of 10 μg / ml in order to prevent activation of macrophage cells due to endotoxin contamination. After the addition of the test substance and polymyxin B, the cells were further cultured for 24 hours.

80 μl of the supernatant was transferred to a plate for NO assay. When diluting, it was appropriately diluted with the buffer of the kit described later. The NO concentration was measured using Nitrate / Nitrite Assay Kit, Colorimetric (2 × 96 well, 3 plates) Funakoshi 780001. The usage method followed the attached instructions. It was calculated how much the measured NO concentration increased based on the time when the test substance was not added. The test was performed with n = 3 and the average value was graphed.

The results are shown in FIG. Paramylon and fibrotic paramylon increased nitric oxide concentrations, which were found to have macrophage activating effects. Among them, fibrotic paramylon was found to have an extremely high macrophage activating effect as compared with paramylon. The test was independently performed three times, and it was confirmed that the same result was obtained (Fig. 3 shows the result of one of them).

Test example 2. Evaluation test of macrophage activation 2
Activated macrophages are known to produce TNF-α. In this test, macrophage activation was evaluated by allowing a test substance to act on macrophages and measuring the amount of TNF-α produced. Specifically, it was carried out as follows.

As cells, rat alveolar-derived macrophages (NR8383, ATCC CRL-2192) were used. In addition, as a medium, F-12K nutrient mixture medium (# 21127-022, manufactured by Life Technologies Japan, Inc. + 15% FBS (Batch no.70301115, MOREGATE, deactivated at 56 ° C for 30 minutes)), penicillin-streptomycin -Glutamine (100x, liquid: # 10378-01, manufactured by Life Technologies Japan) was added to make 1x.

The cells precultured in 15 T25 culture flasks were peeled off the wall by pipetting, and the obtained cell suspension was transferred to a 50 mL conical tube. The tube was centrifuged at room temperature (1000 rpm, 5 minutes), the supernatant was decanted and cells were collected. After loosening the cells by tapping, 5 mL of the culture solution was added, and the cells were uniformly suspended by pipetting. After transferring 11 μL to another tube and adding 11 μL of 0.5% trypan blue, the cell suspension was transferred to a blood calculation plate and the cell number and viability were measured. The survival rate was 93.6%, which was 90% or more (the cell survival rate of 90% or more is the standard for cells that can be used in the test), so the residual fluid was used for the test.

Based on the measured number of cells were prepared and the number of cells so that the residual liquid in the culture was diluted by adding 1.6 × 10 ⁵ cells / mL. 100 μL of this cell suspension was added to each well of a 96 well flat bottom plate. They were transferred to an incubator and pre-cultured for 5 hours until the cells adhered to the bottom of the well and extended.

At the end of preculture, the plate was removed and 100 μL of the test solution was added to the wells at a double concentration. After adding each sample, the cells were cultured in an incubator for 48 hours. After culturing, 50 μL of the supernatant was collected on a 96-well flat bottom plate and used for TNF-α measurement.

The measurement was performed according to the operation procedure described in the instruction manual of the ELISA measurement kit (ELISA MAX® Deluxe Sets Rat TNF-α: # 438204, BioLegend). Each sample was measured with n = 1. The culture supernatant diluted 20-fold with Assay Diluent was used for measurement.

The results are shown in FIG. Paramylon and fibrotic paramylon increased TNF-α concentrations, which were found to have macrophage activating effects. Among them, fibrotic paramylon was found to have an extremely high macrophage activating effect as compared with paramylon.

Claims (10)

A macrophage activator containing at least one selected from the group consisting of Euglena, paramylon, processed paramylon, and β-1,3-glucan. The macrophage activator according to claim 1, which contains the processed paramylon product. The macrophage activator according to claim 2, wherein the processed paramylon product is fibrous paramylon. The macrophage activator according to claim 2 or 3, wherein the processed paramylon is a processed product of paramylon derived from Euglena gracilis. The macrophage activator according to any one of claims 2 to 4, wherein the processed paramylon is a processed paramylon derived from Euglena gracilis EOD-1 strain (accession number FERM BP-11530). The macrophage activator according to any one of claims 1 to 5, which is a food composition, a food additive, a cosmetic product, a cosmetic additive, or a pharmaceutical product. The macrophage activator according to any one of claims 1 to 6, which is an oral composition. An immunostimulant containing fibrotic paramylon. The immunostimulant according to claim 8, which is a food composition, a food additive, a cosmetic product, a cosmetic additive, or a medicine. The immunostimulant according to claim 8 or 9, which is an oral composition.