JP7045661B2 - Immunostimulatory composition and immunostimulatory food composition - Google Patents

Description

The present invention relates to an immunostimulatory composition and an immunostimulatory food composition, specifically, an immunostimulatory composition containing endo-1,3-β-D-glucanase and an immunostimulatory food composition. According to the present invention, the immune function of a living body can be activated.

The immunostimulatory composition is also used in the medical field for the treatment of various diseases. For example, in the treatment of cancer, improvement of the therapeutic effect has been recognized by using an immunopotentiator in combination with chemotherapy or radiotherapy.

On the other hand, preventive medicine that improves the immune function of a living body by administering an immunostimulatory composition, prevents infectious diseases caused by microorganisms or viruses, and eliminates cancer cells is also attracting attention. The immunostimulatory composition used in this preventive medicine is ingested on a daily basis. Therefore, there is a demand for an immunostimulatory composition that can be ingested safely and easily on a daily basis, without fear of side effects, and expected to have excellent effects.
For example, Patent Document 1 describes an immunostimulatory agent in which a polysaccharide such as D-mannose or arabinoxylan coexists with lactic acid bacteria. Further, Patent Document 2 describes an immunostimulant containing a partially decomposed product of hemicellulose obtained from corn hull as an active ingredient. However, there has been a demand for an immunostimulatory composition that is safer and more convenient and can be continuously ingested on a daily basis and has a remarkable immunostimulatory effect.

Japanese Unexamined Patent Publication No. 2005-8616 Japanese Unexamined Patent Publication No. 2001-322942

An object of the present invention is to provide an immunostimulatory composition that can be ingested on a daily basis, has no side effects, and is expected to have excellent effects.

As a result of diligent research on an immunostimulatory composition that can be ingested on a daily basis, has no side effects, and is expected to have excellent effects, the present inventor surprisingly, End-1,3-β It was found that -D-glucanase has an excellent immunostimulatory effect.
The present invention is based on these findings.
Therefore, the present invention
[1] An immunostimulatory composition comprising endo-1,3-β-D-glucanase,
[2] The immunostimulatory composition according to [1], wherein the immunostimulatory activity is at least one immunostimulatory activity selected from the group consisting of antibody production promoting activity, cytokine production promoting activity, and macrophage activation.
[3] The immunostimulatory composition according to [1] or [2], wherein the endo-1,3-β-D-glucanase is derived from a plant.
[4] An immunostimulatory food composition comprising endo-1,3-β-D-glucanase,
[5] The immunostimulatory food composition according to [4], wherein the immunostimulatory activity is at least one immunostimulatory activity selected from the group consisting of antibody production-promoting activity, cytokine production-promoting activity, and macrophage activation. And [6] the immunostimulatory food composition according to [4] or [5], wherein the endo-1,3-β-D-glucanase is derived from a plant.
Regarding.

According to the immunostimulatory composition and the immunostimulatory food composition of the present invention, the immune function can be activated. In addition, the immunostimulatory composition and the immunostimulatory food composition of the present invention can promote antibody production, cytokine production, and macrophage activation. The immunostimulatory food composition of the present invention can provide highly safe foods and drinks.

It is a graph which added the endo-1,3-β-D-glucanase to RAW264.7 cells, and measured the production amount of IL-6 and TNF-α in the culture supernatant. It is a graph which measured the cytotoxicity to RAW264.7 cells of endo-1,3-β-D-glucanase. It is a graph which showed that the cumin aqueous solvent extract containing endo-1,3-β-D-glucanase promotes IgM production of HB4C5 cells. It is a graph which showed that the cumin aqueous solvent extract containing endo-1,3-β-D-glucanase promoted the production of IL-6, and TNF-α of RAW264.7 cells. FIG. 6 is a graph showing that a cumin-aqueous solvent extract containing endo-1,3-β-D-glucanase promotes the production of IL-6 and TNF-α in primary mouse intraperitoneal macrophages (P-Mac). .. It is a graph which showed that the cumin aqueous solvent extract containing endo-1,3-β-D-glucanase promotes nitric oxide (NO) production of RAW264.7 cells. It is a graph which showed that the cumin aqueous solvent extract containing endo-1,3-β-D-glucanase promotes the phagocytic activity of RAW264.7 cells.

[1] Immunostimulatory composition The immunostimulatory composition of the present invention contains endo-1,3-β-D-glucanase.

<< End-1,3-β-D-glucanase >>
Endo-1,3-β-D-glucanase is a β-D-glucan (1 → 3) when the hydrolyzed bond is substituted at the C-3 position of the glucose residue at the reducing end. -An enzyme that hydrolyzes bonds to the end form.
The endo-1,3-β-D-glucanase contained in the composition of the present invention is not particularly limited as long as it has the endo-1,3-β-D-glucanase activity. For example, barley endo-1,3-β-D-glucanase contains three isozymes, GI, GII, and GIII, and these three isozymes are used as the active ingredient of the immunostimulatory composition of the present invention. be able to.
As one embodiment of the endo-1,3-β-D-glucanase, an endo-1,3-β-D-glucanase consisting of the amino acid sequence represented by SEQ ID NO: 1, 2, 3, 4, or 5 is used. Can be mentioned.
Further, as another embodiment of endo-1,3-β-D-glucanase, one or more amino acids are inserted in the amino acid sequence represented by SEQ ID NO: 1, 2, 3, 4, or 5. Endo-1,3-β-D-glucanase consisting of an amino acid sequence substituted or deleted, or added to one or both ends, and having endo-1,3-β-D-glucanase activity. be able to.
Further, as another embodiment of endo-1,3-β-D-glucanase, one or more amino acids are inserted in the amino acid sequence represented by SEQ ID NO: 1, 2, 3, 4, or 5. Endo-1,3-β-D-glucanase comprising an amino acid sequence substituted, deleted, or added to one or both ends, and having endo-1,3-β-D-glucanase activity. be able to.
The endo-1,3-β-D-glucanase which is the active ingredient of the composition of the present invention may be a natural endo-1,3-β-D-glucanase, but is a genetically engineered recombinant endo. -1,3-β-D-glucanase can also be used.

The origin of the endo-1,3-β-D-glucanase is also not particularly limited. For example, animal-derived or plant-derived endo-1,3-β-D-glucanase can be used, but plant-derived endo-1,3-β-D-glucanase is preferred. Examples of the animal-derived endo-1,3-β-D-glucanase include shellfish-derived endo-1,3-β-D-glucanases such as Kitanonishikigai (SEQ ID NO: 5). The plants from which endo-1,3-β-D-glucanase can be obtained are not particularly limited, but are not particularly limited, but are grains such as barley (SEQ ID NOs: 1 to 3), wheat, embaku, rye, and pearl barley, and potatoes (SEQ ID NO: 1 to 3). Examples thereof include potatoes such as 4), seaweeds such as Kumin and Combu, mushrooms, and plants such as ground clothing, but endo-1,3-β-D-glucanase derived from barley or Kumin is preferable.

《Immune activation》
Examples of the immunostimulatory activity of the immunostimulatory composition of the present invention include antibody production promoting activity, cytokine secretion promoting activity, and macrophage activation. The immunostimulatory composition of the present invention may exhibit all of the above-mentioned immunostimulatory activities, or may exhibit any one of the above-mentioned immunostimulatory activities.

(Promotion of antibody production)
Antibodies are produced by antibody-producing cells. The immunostimulatory composition of the present invention promotes the production of antibodies from antibody-producing cells. The antibody-producing cells are preferably mammalian cells (primates such as humans, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, or mice), most preferably human cells. .. Also, antibody-producing cells can be in vivo or in vitro cells. The antibody whose production is promoted is preferably an IgM, IgA, and / or IgG antibody, and most preferably an IgM antibody.

(Promotion of cytokine production)
Cytokines whose secretion is promoted include, for example, interleukins, interferons, chemokines, phosphokines, hematopoietic factors, cell growth factors, cytotoxic factors, adipokines, and neurotrophic factors. Examples of the interleukin include IL1 to 35 and the like. Examples of interferon include IFN-α, IFN-β, IFN-γ and the like. Examples of chemokines include MIP and MCP. Examples of hematopoietic factors include SCF, GM-CSF, G-CSF, M-CSF, erythropoietin, thrombopoietin and the like. Examples of cell growth factors include epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, hepatocyte growth factor, transforming growth factor and the like. Examples of the cytotoxic factor include TNF-α and TNF-β. Examples of adipokines include leptin. Examples of the neurotrophic factor include NGF and the like. Preferred cytokines are IL-4, IL-6, TNF-α and / or IFN-γ. The cytokine whose secretion is promoted may be one of these or two or more of them.

(Activation of macrophages)
As the specific action of macrophage activation exerted by the immunostimulatory composition of the present invention, for example, one or 2 selected from promotion of phagocytic activity, promotion of active oxygen production, promotion of nitric oxide production, and enhancement of antigen presentation function. One or more can be mentioned.

The phagocytosis of macrophages is a function in which macrophages take in foreign substances such as bacteria, viruses, or dead cells and decompose them, and is also called phagocytosis or phagocytosis. The immunostimulatory composition of the present invention can promote the phagocytic action of this macrophage.

Macrophages kill or eliminate foreign substances by producing reactive oxygen species such as superoxide or hydrogen peroxide, or nitric oxide, which are highly toxic to foreign substances such as microorganisms, viruses, and cancer cells. .. The immunostimulatory composition of the present invention can promote the active oxygen production promoting action and / or the nitric oxide production promoting action of this macrophage.

Antigen presentation is a mechanism in which an antigen-presenting cell takes up an antigen such as a bacterium into the cell, decomposes it, and then presents a part of the antigen to the cell surface. Macrophages break up and decompose bacteria and the like into several fragments, bind to MHC class II molecules present in macrophages, and present them on the cell surface. The immunostimulatory composition of the present invention can promote the antigen-presenting function of this macrophage.

The dosage form of the immunostimulatory composition of the present invention is not particularly limited, and is a powder, a fine granule, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, an extract, or a pill. Oral preparations such as, or parenteral preparations can be mentioned.

The oral preparation includes, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannit, carboxymethyl cellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid ester, talc, magnesium stearate, and the like. Excipients such as polyethylene glycol, magnesium silicate, anhydrous silicate, or synthetic aluminum silicate, binders, disintegrants, surfactants, talc, fluidity enhancers, diluents, preservatives, colorants, It can be produced according to a conventional method using a fragrance, a flavoring agent, a stabilizer, a moisturizing agent, a preservative, an antioxidant or the like.

Examples of parenteral preparations include injections. In the preparation of injections, in addition to the active ingredient, for example, a water-soluble solvent such as physiological saline or Ringer's solution, a water-insoluble solvent such as vegetable oil or fatty acid ester, an tonicity agent such as glucose or sodium chloride, and a solubilizing agent. , Stabilizers, preservatives, suspending agents, emulsifiers and the like can be optionally used.

The immunostimulatory composition of the present invention contains, but is not limited to, endo-1,3-β-D-glucanase, for example, 0.01 to 500 mg / mL, preferably 0.1 to 400 mg / mL. It can be more preferably contained in an amount of 0.5 to 300 mg / mL, more preferably 1 to 200 mg / mL, still more preferably 2 to 150 mg / mL, and most preferably 1 to 100 mg / mL.

The dose of the immunostimulatory composition of the present invention can be appropriately determined according to the type of disease, the age, sex, body weight, degree of symptoms, administration method, etc. of the patient, and can be appropriately determined, for example, End-1,3-. β-D-glucanase at 0.1-1000 mg / kg body weight / day, preferably 0.5-500 mg / kg body weight / day, more preferably 1-250 mg / kg body weight / day, still more preferably 3-100 mg / day. It can be kg body weight / day, more preferably 5-50 mg / kg body weight / day, or most preferably 8-30 mg / kg body weight / day.

Of course, the above administration method is an example, and other administration methods may be used. It is desirable that the method, dose, administration period, administration interval, etc. of the immunostimulatory composition for humans be determined by a controlled clinical trial.

The immunostimulatory composition of the present invention can be administered to humans, but the subject may be an animal other than humans, and pets such as dogs, cats, rabbits, hamsters, guinea pigs, and squirrels; cows. And domestic animals such as pigs; experimental animals such as mice and rats; and animals bred in zoos and the like.

The immunostimulatory composition of the present invention can be an immunostimulatory pharmaceutical composition. Examples of the target disease of the immunodeficiency pharmaceutical composition of the present invention include cancer, immunodeficiency, and infectious diseases.

Cancers include tongue cancer, gingival cancer, malignant lymphoma, malignant melanoma, maxillary cancer, nasal cancer, nasal cavity cancer, laryngeal cancer, pharyngeal cancer, glioma, meningeal carcinoma, glioma, neuroblastoma, thyroid carcinoma. Papillary adenocarcinoma, thyroid follicular cancer, thyroid medullary carcinoma, primary lung carcinoma, squamous epithelial carcinoma, adenocarcinoma, alveolar epithelial carcinoma, large undifferentiated carcinoma, small cell undifferentiated carcinoma, carcinoid, testicle tumor, prostate carcinoma , Breast cancer, breast pace jet disease, breast carcinoma, bone tumor, thyroid cancer, gastric cancer, liver cancer, acute myeloid leukemia, acute promyelinated leukemia, acute myeloid monocytic leukemia, acute monocytic leukemia, acute lymphocytic leukemia, Acute undifferentiated leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, malignant lymphoma, multiple myeloma, primary macroglobulinemia, childhood leukemia, esophageal carcinoma, gastric cancer, gastric / colon smoothing Myoma, gastric / intestinal malignant lymphoma, pancreatic / bile sac cancer, duodenal cancer, colon cancer, primary liver cancer, hepatoblastoma, intrauterine intraepithelial carcinoma, cervical squamous epithelial carcinoma, uterine adenocarcinoma, uterine gland squamous epithelial carcinoma, Uterine adenocarcinoma, uterine sarcoma, uterine carcinoma, uterine destructive miracle, uterine malignant villous epithelioma, uterine malignant melanoma, ovarian cancer, mesolobular mixed tumor, renal cancer, renal pelvis transition epithelial cancer, urinary tract Transitional epithelial carcinoma, bladder papillary carcinoma, bladder transitional epithelial carcinoma, urinary squamous epithelial carcinoma, urinary adenocarcinoma, Wilms tumor, rhizome myoma, fibrosarcoma, osteosarcoma, chondrosarcoma, synovial sarcoma, mucinosarcoma, liposarcoma, Ewing sarcoma, cutaneous squamous cell carcinoma, basal cell carcinoma of the skin, Bowen's disease of the skin, Paget's disease of the skin, malignant melanoma of the skin, malignant mesothel carcinoma, metastatic adenocarcinoma, metastatic squamous cell carcinoma, metastatic sarcoma and mesopharyngeal carcinoma Can be mentioned.

Immunodeficiency includes, for example, cancer, poor regeneration anemia, leukemia, myelofibrosis, renal failure, diabetes, immunodeficiency associated with liver disease or splenic disease, acquired immunodeficiency syndrome due to human immunodeficiency virus infection, and Primary immunodeficiency syndrome may be mentioned.

Infectious diseases include human hepatitis virus, human retrovirus, human immunodeficiency virus, human T cell leukemia virus, human T lymphotrophic virus, simple herpesvirus type 1 or 2, Epstein bar virus, cytomegalovirus, and varicella.・ Viruses caused by herpes zoster virus, human herpes virus, poliovirus, measles virus, ruin virus, Japanese encephalitis virus, mumps virus, influenza virus, cold virus, virus that causes severe acute respiratory syndrome, Ebola virus, or West Nile virus. Infectious diseases can be mentioned. Infectious diseases also include infectious diseases caused by pathogenic protozoa, bacteria, or fungi.

[2] Immunostimulated food composition The immunostimulated food composition of the present invention contains endo-1,3-β-D-glucanase.
As used herein, the immunostimulatory food composition means one comprising endo-1,3-β-D-glucanase and a food or beverage. As the endo-1,3-β-D-glucanase, the endo-1,3-β-D-glucanase described in the above-mentioned "[1] immunostimulatory composition" can be used without limitation.

Specific foods include fresh cooked foods such as salads; cooked foods such as steaks, pizzas and hamburgers; fried cooked foods such as fried vegetables; tomatoes, peppers, celery, niggauri, carrots, potatoes and asparagus. Vegetables such as and cooked products processed from these vegetables; cookies, bread, biscuits, dried bread, cakes, rice cakes, sheep rice, pudding, jelly, ice creams, chewing gum, crackers, chips, chocolate and sweets such as candy; udon, Noodles such as pasta and buckwheat; fish paste products such as kamaboko, ham, and fish sausage; dairy products such as cheese, cream, and butter; miso, soy sauce, dressing, ketchup, mayonnaise, soup base, noodle soup, curry flour , Mirin, ru, seasoning spices and other seasonings; soybean foods such as tofu; konjac; and supplements. Foods preferably include roux (eg, for curry or stew), seasoning spices, or supplements.

Beverages include, for example, coffee beverages; cocoa beverages; vegetable juices obtained from the above vegetables; fruit juice beverages such as grapefruit juice, orange juice, grape juice, and lemon juice; tea beverages such as green tea, tea, roasted tea, and oolong tea; Alcoholic beverages such as beer, wine (red wine, white wine, or sparkling wine, etc.), sake, plum wine, sparkling liquor, whiskey, brandy, shochu, lamb, gin, and liqueurs; milk beverages; soy milk beverages; liquid foods; and Sports drinks and the like can be mentioned. Beverages preferably include spice teas (eg herbal teas or chai).

The food or beverage herein includes feed for animals. Examples of the target animal include primates such as humans, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like.

These foods or beverages may include antioxidants, fragrances, acidulants, colorants, emulsifiers, preservatives, seasonings, sweeteners, spices, pH regulators, stabilizers, antioxidants, vegetable oils, animal oils, if desired. , Sugar and sugar alcohols, vitamins, organic acids, fruit juice extracts, vegetable extracts, grains, beans, vegetables, meats, seafood and other food additives and food ingredients alone or in combination of two or more. be able to. The blending amount of these food materials and food additives can be appropriately determined within a range that does not impair the object of the present invention.

These foods or beverages are commonly sterilized, for example, heat and pressure sterilization such as retort and autoclave, batch sterilization, plate sterilization, energization heat sterilization, microwave heat sterilization, and steam sterilization such as injection and infusion. Processing can be performed.

Foods and beverages include functional foods (beverages) and health foods (beverages). In the present specification, the "health food (beverage)" means a food or beverage that has or can be expected to have some effect on health, and the "functional food (beverage)" is described above. Among "health foods (beverages)", it means foods or beverages designed and processed so as to sufficiently express a bioregulatory function (that is, an immunostimulatory function). Functional foods and health foods can be in the form of granules, solids, liquids, capsules, gels or tablets.

The cumin extract contains endo-1,3-β-D-glucanase, which is an active ingredient of the immunostimulatory composition of the present invention and the immunostimulatory food composition. Oral administration of a cumin extract containing this endo-1,3-β-D-glucanase to mice promotes the production of antibodies such as IgM in the living body of mice. It also promotes the production of cytokines such as IL-4 or IL-6. The immunostimulatory composition and the immunostimulatory food composition of the present invention can exhibit immunostimulatory activity by oral administration.

Hereinafter, the present invention will be specifically described with reference to Examples, but these do not limit the scope of the present invention.

<< Example 1 >>
In this example, the immunostimulatory activity of endo-1,3-β-D-glucanase was analyzed.
(Preparation of endo-1,3-β-D-glucanase solution)
Barley-derived endo-1,3-β-D-glucanase (Megazyme) was diluted with sterile water to 2 mg / mL and sterilized by filtration through a 0.45 μm sterile filter. As a control, a solution of endo-1,3-β-D-glucanase (0.02% (w / v) sodium azide-50% (v / v) glycerol aqueous solution) was prepared and 0.45 μm. The one sterilized by filtration with a filter was used.

(Cytokine production)
Mouse macrophage cell line RAW264.7 cells ⁽ 3.0 × 105 cells / mL) were seeded on 96-well plates at 200 μL / well and precultured for 12 hours. After preculture, the medium was removed and cultured in 10% FBS-DMEM supplemented with controls and endo-1,3-β-D-glucanase at each concentration. After culturing for 12 hours, the culture supernatant was collected, and the amount of IL-6 and TNF-α produced in the culture supernatant was measured by the enzyme antibody method (Mouse IL-6 ELISA MAX Standard, manufactured by BioLegend; Mouse TNF-α ELISA Ready-). Quantified by set-go (manufactured by eBioscience). The results are shown in FIG.

As shown in FIG. 1, the addition of endo-1,3-β-D-glucanase promoted IL-6 and TNF-α production in the low concentration region. On the other hand, a decrease in the production promoting effect was observed at an addition concentration of 40 μg / mL or more for IL-6 and 200 μg / mL or more for TNF-α.

The toxicity of endo-1,3-β-D-glucanase to RAW264.7 cells was evaluated. After culturing at various concentrations of endo-1,3-β-D-glucanase, the survival rate was evaluated by the WST-8 method. As a result, as shown in FIG. 2, a significant decrease in survival rate was observed at 1000 μg / mL. Therefore, the decrease in IL-6 and TNF-α production promoting effect in the high concentration range is due to cytotoxicity. It was inferred.

<< Preparation Example 1 >>
In this adjusted example, a cumin water-soluble extract was prepared from cumin seeds and the content of endo-1,3-β-D-glucanase was analyzed.
Cumin seed powder was suspended in 10 mM NaPB (phosphate buffer) to 0.1 g / mL, and extracted by stirring at 15 times / min for 24 hours. After centrifuging (1,2000 rpm) for 20 minutes under the condition of 4 ° C., the supernatant was collected. After centrifuging (70,000 rpm) for 30 minutes under the condition of 4 ° C., the supernatant was collected. A dialysis membrane having a molecular weight cut off of 14 kHz was used to dialyze 10 mM NaPB overnight. The pH was adjusted to 7.4 using 1M NaOH, and the mixture was filtered and sterilized with a filter having a pore size of 0.45 μm to obtain a cumin aqueous solvent extract (CCE).

The obtained cumin aqueous solvent extract was analyzed by MS / MS. The sample was desalted and eluted with acetonitrile / trifluoroacetic acid. The eluted sample was air-dried and analyzed by mass spectrometer. When the database was searched from the mass value of the peak of the obtained mass spectrum, the cumin aqueous solvent extract contained endo-1,3-β-D-glucanase.

<< Example 2 >>
In this example, the antibody-producing effect of a cumin aqueous solvent extract containing endo-1,3-β-D-glucanase on human hybridoma HB4C5 cells was investigated.
Cumin-liquid solvent extract was added to ITES-ERDF medium at several concentrations. Human hybridoma HB4C5 cells were seeded at 5.0 × 10 ⁴ cells / mL, cultured for 6 hours, and then the culture supernatant was collected. Using anti-human IgM antibody (manufactured by Cappel), HRP-labeled anti-human IgM antibody (manufactured by Abcam), and ABTS (manufactured by Wako Junyaku Co., Ltd.), which is a color-developing substrate, the amount of IgM antibody in the culture solution was determined by the ELISA method. It was measured. 10 mM NaPB (phosphate buffer) was used as a control. The results are shown in FIG. In the antibody production of HB4C5 cells, the cumin aqueous solvent extract containing endo-1,3-β-D-glucanase showed an IgM production promoting effect on the control, and the effect was about 2.2 times at the highest point. rice field.

<< Example 3 >>
In this example, the effect of producing cytokines on cumin aqueous solvent extract on mouse macrophage-derived RAW264.7 cells was examined.
Mouse-derived macrophage cell line RAW264.7 cells (3 × 10 ⁵ cells / mL) were cultured for 12 hours in 10% FBS-DMEM medium supplemented with diluted cumin aqueous solvent extract, and then ELISA method (Mouse IL-6) was performed. The amounts of IL-6 and TNF-α in the culture medium were measured using ELISA MAX Standard, manufactured by BioLegend; Mouse TNF-α ELISA Ready-set-go, manufactured by eBioscience). 10 mM NaPB was used as a control. The results are shown in FIG. The cumin aqueous solvent extract containing endo-1,3-β-D-glucanase showed the effect of promoting the production of IL-6 and TNF-α.

<< Example 4 >>
In this example, the cytokine-producing effect of cumin-aqueous solvent extract on primary mouse intraperitoneal macrophages (P-Mac) was investigated.
Macrophages (P-Mac) were recovered from the abdomen of BALB / c mice and P-Mac (3 × 10 ⁶ cells / mL) was cultured for 12 hours in 10% FBS-RPMI1640 medium supplemented with diluted cumin aqueous solvent extract. After that, the amount of IL-6 and TNF-α in the culture medium was measured using the ELISA method. The same kit as in Example 3 was used for the ELISA method. 10 mM NaPB was used as a control. The results are shown in FIG. Cumin water-soluble extracts containing endo-1,3-β-D-glucanase were found to promote the production of IL-6 and TNF-α in a concentration-dependent manner. At the highest production concentration, IL-6 production was about 75 times higher and TNF-α production was about 6 times higher than the control.

<< Example 5 >>
In this example, the effect of cumin aqueous solvent extract on nitric oxide (NO) production on mouse macrophage-derived RAW264.7 cells was examined. Nitric oxide (NO) is a type of reactive oxygen species, and macrophages produce nitric oxide to treat bacteria. In this example, the effect of cumin-aqueous solvent extract on NO production in RAW264.7 cells was investigated.
RAW264.7 cells (3 × 10 ⁵ cells / mL) were cultured for 12 hours in 10% FBS-DMEM supplemented with diluted Cumin aqueous solvent extract, and then the NO concentration in the culture medium was measured by the Griess method. 10 mM NaPB was used as a control. The results are shown in FIG. Cumin aqueous solvent extract containing endo-1,3-β-D-glucanase was found to promote nitric oxide production in RAW264.7 cells.

<< Example 6 >>
In this example, the effect of cumin-liquid solvent extract on the phagocytic activity of mouse macrophage-derived RAW264.7 cells was investigated.
RAW264.7 cells (3 × 105 cells / mL) were cultured for ⁶ hours in 10% FBS-DMEM medium supplemented with Kumin aqueous solvent extract to a total protein concentration of 1000 μg / mL, and then labeled with Texas Red. After adding Zymosan A and culturing for 1 hour, its phagocytic activity was measured using a flow cytometer. 10 mM NaPB was used as a control. The results are shown in FIG. It was revealed that the cumin aqueous solvent extract containing endo-1,3-β-D-glucanase significantly promoted the phagocytic activity of RAW264.7 cells.

The immunostimulatory composition and the immunostimulatory food composition of the present invention can activate the immune action. Further, the immunostimulatory food composition of the present invention can provide foods and drinks having an immunostimulatory action and high safety.

Claims (6)

An immunostimulatory composition comprising only endo-1,3-β-D-glucanase as an active ingredient. The immunostimulatory composition according to claim 1, wherein the immunostimulatory activity is at least one immunostimulatory activity selected from the group consisting of antibody production-promoting activity, cytokine production-promoting activity, and macrophage activation. The immunostimulatory composition according to claim 1 or 2, wherein the endo-1,3-β-D-glucanase is derived from a plant. An immunostimulatory food composition comprising only endo-1,3-β-D-glucanase as an active ingredient. The immunostimulatory food composition according to claim 4, wherein the immunostimulatory activity is at least one immunostimulatory activity selected from the group consisting of antibody production-promoting activity, cytokine production-promoting activity, and macrophage activation. The immunostimulatory food composition according to claim 4 or 5, wherein the endo-1,3-β-D-glucanase is derived from a plant.

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