JP4928060B2 - Cytokine production enhancer derived from Izalea-type worm - Google Patents

Description

  The present invention relates to the use of an isaria-type worm as a cytokine production enhancer.

  In recent years, biological response modifiers (BRMs) and functional foods that have the effect of controlling the immune response of the body and increasing the defense against foreign substances have been used in intractable diseases such as malignant tumors and autoimmune diseases. It is attracting attention as being useful for treatment. Various treatment methods using BRM have been studied. Among them, tumor necrosis factor (TNF), interleukin (IL) s, and interferon (IFN) s released by lymphocytes that control immune responses are used for biological defense. In vivo glycoproteins collectively called cytokines have attracted attention.

  Among cytokines, for example, an increase in IL-2 and IFN-γ produced by special T cells (helper T cells) among lymphocytes induces a cellular immune response in which T cells and macrophages mainly work. When IL-4, IL-6, and IL-5 produced by cells increase, an immune response centered on antibodies by B cells is induced. Furthermore, IL-10 and GM-CSF produced by T cells have a function that IL-10 induces an immunosuppressive reaction, and GM-CSF increases the hematopoietic function.

  It is reported that the culture solution of isaria-type insects has an immunostimulatory action in the spleen (Patent Document 1 and Non-Patent Document 1), but it is produced by Peyer's patch constituent cells, which are special lymphoid organs present in the digestive tract. The effect on cytokine production is not reported.

Japanese Patent No. 2746532 F. Takano et al., Biol. Pharm. Bull., 19, p.641-643 (1996)

  The object of the present invention is to promote the production of cytokines such as interleukin, interferon, and CSF (colony stimulating factor) in the body, and to enhance the action thereof, thereby causing allergic diseases such as asthma, atopy, hay fever, urticaria, It is to provide a novel means for preventing and treating various diseases such as infectious diseases, tumors, hematopoietic dysfunction and the like.

The present invention includes the following inventions.
(1) A cytokine production enhancer containing an isaria-type worm, a culture or extract thereof, or a processed product thereof as an active ingredient, which is generated by artificially culturing a natural bacterium.
(2) The cytokine production enhancer according to (1), wherein the cytokine is at least one selected from interleukin, interferon, and CSF.
(3) The cytokine production enhancer according to (2), wherein the interleukin is interleukin-2.
(4) The cytokine production enhancer according to (2), wherein the interleukin is interleukin-6.
(5) The cytokine production enhancer according to (2), wherein the interleukin is interleukin-10.
(6) The cytokine production enhancer according to (2), wherein the interferon is interferon γ.
(7) The cytokine production enhancer according to (2), wherein the CSF is GM-CSF.
(8) The cytokine production enhancer according to any one of the above (1) to (7), which contains a culture of isaria-type insects or a processed product thereof as an active ingredient.
(9) A cytokine production enhancer containing, as an active ingredient, a mycelium, conidia or secondary metabolite of an isaria-type worm that is generated by artificially culturing natural worms.
(10) The cytokine production enhancer according to any one of the above (1) to (9), wherein the Isaria worm is Isaria japonica Yasuda or Isaria sinclairii (Berk.) Lloyd.
(11) A cytokine production enhancer comprising, as an active ingredient, a substance having an activity of enhancing cytokine production derived from an isaria type insect.
(12) The cytokine production enhancer according to any one of (1) to (11), which is added to food.
(13) A therapeutic agent for a disease in which cytokine production or activity is reduced, comprising the cytokine production enhancer according to any one of (1) to (11) as an active ingredient.

  According to the present invention, allergic diseases such as asthma, atopy, hay fever and urticaria, infections, tumors, Prevention and treatment of hematopoietic dysfunction can be performed.

  Insect fungus is a general term for fungi that parasitize live insects and grow up as nutrients from the tissues and composition of the insects to form fruiting bodies (mushroom-like parts). Call it the genus Cordyceps or insect fungus. Yes. It should be noted that worms can be divided into three parts: insect parts (parasites) that become parasitic hosts, fruit body handles, and ascidians.

Many of the worms that occur in the field are of the full-generation type, and one of the characteristics is that they form an ascollis shell on the fruit body (mushroom-like part). Bacteria called semibamboo, sanagitake, and Cordyceps sinensis are also a kind of protozoa.
Some of the worms that occur in the field are those in which spores (conidia) are attached directly to the fruit body without forming a sac in the fruit body.

The taxonomic position of insects is as follows.
ASCOMYTINA
Nucleus mycelia: Pyrenomycetes
Ergotaceae (Cannibus): Claviciptaceae
Nomushitake: Cordyceps

1． Complete generation type: Insect fungus that has an ascomb sac and repeats generation in ascospores (1) Cordyceps (Cordyceps = semi-bamboo shoots, bamboo shoots, cordyceps, etc.)
(2) Torbiela (Torrubiella = coral spider parasite on arthropod spider, scale insect parasite on scale insect)
(3) Podonectrioides (Podonectrioides)
The full-generation type of Bacillus subtilis has many of the above three genus types and is considered as a representative.

2. Incomplete generation type: insect fungus that does not have ascolic shells and repeats generations in conidia (1) Isaria (Isaria = Japanese bamboo shoot, Japanese bamboo shoot, etc.)
(2) Gibellula (Gibellula)
(3) Hymenostilbe (Yamatake, etc. parasitic on dragonflies)
(4) Polycephalomyces (Polycephalomyces)
The incomplete generation type is typified by the above-mentioned four genera types, but in the field, the most common isaria type fungus.

  When artificially cultivating all generations of Cordyceps, Torviera, and Podecteria spp. Collected from the field on an agar medium that does not contain parasite components, fruit bodies with all incomplete generation type conidia attached to them It is customary to generate it constantly. Most of them are Isaria-type protozoa, and except for some species, no full-generation protozoa with a cyst is generated.

The agar medium can be prepared, for example, as follows.
Dry yeast 0.6% (W / V), plant protein provider (Kyowa Hakko) 1.5% (W / V), peptone 0.2% (W / V), agar powder 2.2% (W / V) V) was dissolved in distilled water, autoclaved (120 ° C., 20 minutes), cooled to around 40 ° C., filtered and sterilized with a membrane filter (pore size 0.2 μm), and glucose 1.5% (W / V) and an inosine 0.01% (W / V) aqueous solution are added. Dispense 20 ml each of the sterilized culture solution into a conical flask (100 ml) sterilized by autoclaving and solidify to prepare an agar medium.

  The Isaria-type insects used in the present invention are conidia when cultivating Cordyceps insects that develop in the field or Isaria insects that form conidial stalks on an agar medium that does not contain parasite components. There is no particular limitation as long as it is a worm that forms the fruiting body of the stalk, and examples of the insect fungus used to obtain the isaria-type worm include those shown in Table 1, and preferably, the flower of Isaria japonica Yasuda), Isaria sinclairii (Berk.) Lloyd, and Cordyceps formicarum Kobayashi.

  In the present invention, the Izaria type worm is an artificial culture of a mycelium lyophilized product, a processed product such as a pulverized product, a cultivated product or an extract thereof, or a processed product thereof, preferably a natural fungus. It is used as a mycelium, conidia or secondary metabolite of the Izalea-type insects that are generated in this way.

  In the present invention, the substance derived from isaria-type worms and having cytokine production enhancing activity is obtained from isaria-type worms, and is not particularly limited as long as it has activity to enhance cytokine production. In addition, it refers to a culture or extract of an Izalea-type worm, or one obtained by subjecting these to at least one of various treatments such as separation, purification, and isolation, and retaining cytokine production enhancing activity.

  Examples of the extraction solvent include water; alcohols such as methanol, ethanol, propanol and butanol; esters such as acetate such as ethyl acetate; ethers such as ethyl ether and dioxane; ketones such as acetone and the like. When the extract is once solvent-removed and used as a dried product, any of the above-described solvents can be used alone or in combination. On the other hand, when the extract is used in a state dissolved in a solvent, it is necessary to use a solvent that does not have a harmful effect on the human body. In this case, it is preferable to use water, ethanol, or a mixture thereof. . In the extraction, the mycelium or the culture generated after the cultivation of the Izaria worm can be used as it is, or it can be crushed or pulverized after drying to enhance the contact with the solvent.

  Extract with 1 to 3 L of solvent per kg of mycelium or culture of Izaria type worms. The extraction temperature is preferably in the range of room temperature to the boiling point of the solvent under normal pressure, and the extraction time varies depending on the extraction temperature and the like, but is preferably 50 to 70 ° C.

In the present invention, it is preferable to use a culture of Isaria worms or a processed product thereof, such as a dry powder or an extract.
There are no particular limitations on the method for cultivating the Izaria worm, and a known method such as stationary liquid culture (for example, Japanese Patent Publication No. 61-53033 and Japanese Patent No. 2746532) can be used.

  The extract or culture solution obtained as described above is used after removing insoluble matter, medium insoluble matter, impurities, etc. by filtering with a cloth, stainless steel filter, filter paper, filter for filter sterilization, etc., if necessary. May be. Moreover, you may give processes, such as a spray-dry process, a freeze-dry process, a supercritical process, to the extract or culture solution after filtration.

  The extract, culture or treated product thus obtained can be used as it is as an active ingredient of the cytokine production enhancer, therapeutic agent and food of the present invention. In addition, the extract, culture, etc. may be treated by various purification means such as ion exchange chromatography, gel filtration chromatography, dialysis, etc., and used as a treated product with further enhanced activity.

  The cytokine production enhancer and therapeutic agent of the present invention can be formulated by combining a culture, extract, or processed product of Izaria worms with a known pharmaceutical carrier. The dosage form is not particularly limited and is appropriately selected as necessary. In general, tablets, capsules, granules, fine granules, powders, solutions, syrups, suspensions, emulsions, elixirs, etc. It is used as oral preparations or parenteral preparations such as injections, drops, suppositories, inhalants, transdermal absorbents, transmucosal absorbents, patches, ointments and the like.

  The dosage of the cytokine production enhancer and therapeutic agent of the present invention varies depending on the patient's age, weight, degree of disease, route of administration, but for oral administration, as a dry powder of the culture or extract of the Izalea-type worm, Usually, it is 5 to 500 mg per day, and the frequency of administration is usually 1 to 3 times per day for oral administration.

Oral preparations are produced according to a conventional method using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts.
In this type of preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be appropriately used in addition to the above-mentioned excipients.

  Specific examples of the binder include crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carmellose sodium , Ethylcellulose, carboxymethylethylcellulose, hydroxyethylcellulose, wheat starch, rice starch, corn starch, potato starch, dextrin, pregelatinized starch, partially pregelatinized starch, hydroxypropyl starch, pullulan, polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, aminoalkyl METAKU Rate copolymer RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylamino acetate, polyvinyl alcohol, gum arabic, gum arabic powder, agar, gelatin, white shellac, tragacanth, purified sucrose, macrogol.

  Specific examples of disintegrants include crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, and partially pregelatinized. Starch, hydroxypropyl starch, sodium carboxymethyl starch, tragacanth can be mentioned.

  Specific examples of surfactants include soybean lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate, sorbitan monostearate Sorbitan monopalmitate, sorbitan monolaurate, polysorbate, glyceryl monostearate, sodium lauryl sulfate, lauromacrogol.

  Specific examples of lubricants include wheat starch, rice starch, corn starch, stearic acid, calcium stearate, magnesium stearate, hydrous silicon dioxide, light anhydrous silicic acid, synthetic aluminum silicate, dry aluminum hydroxide gel, talc, Examples thereof include magnesium aluminate metasilicate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, sucrose fatty acid ester, waxes, hydrogenated vegetable oil, and polyethylene glycol.

Specific examples of the fluidity promoter include hydrous silicon dioxide, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.
In addition, the cytokine production enhancer and therapeutic agent of the present invention may contain a flavoring agent and a coloring agent when administered as a liquid, syrup, suspension, emulsion, or elixir.

  The cytokine production enhancer of the present invention is a cytokine production agent such as interleukin (eg, interleukin-2, interleukin-6, interleukin-10), interferon (eg, interferon γ), CSF (eg, GM-CSF). Has activity to enhance production.

  Accordingly, the cytokine production enhancer of the present invention can be expected to have preventive and therapeutic effects on tumors, allergic diseases and infectious diseases by enhancing the production of interleukin-2, and by enhancing the production of interleukin-6. It can be expected to prevent and treat thrombocytopenia, oral mucosa, periodontal tissue, tooth structure, enamel, swallowing and other oral environment, and failure of oral function, and promote various tissue repair after inflammation. By enhancing the production of interleukin-10, it can be expected to prevent and treat Crohn's disease, nodular purpura, type I diabetes, collagen disease, rheumatism, arthritis, hepatitis, and enhance the production of interferon γ Can be expected to prevent and treat malignant tumors, allergic diseases, infectious diseases and malaria, and to enhance the production of GM-CSF. Ri, it is possible to particularly recover hematopoietic function of leukocytes system is effective to opportunistic infections and liver diseases, blood disorders and platelet production failure that occurs when bone marrow function has decreased by long-term use or radiotherapy anticancer agents.

  The cytokine production enhancer of the present invention can be added to foods, chewing gums, beverages, etc. to make so-called foods for specified health use (for example, antiallergic foods, intestinal preparations, nourishing tonics, immunoregulatory functional foods) and the like. .

  EXAMPLES The present invention will be specifically described below with reference to examples and the like, but the present invention is not limited to these. In the following, “%” means “% by weight” unless otherwise specified.

(Production Example 1) Production of culture dry powder Glucose 0.1-2.0% of the protozoa of Isaria japonica Yasuda or Isaria sinclairii (Berk.) Lloyd, which is an example of Isaria-type insects, Inoculated into a medium consisting of 0.1 to 2.0% dry yeast (Iwaki Pharmaceutical Co., Ltd.), 0.01 to 0.1% inosine, and 1.0% agar, and slanted and separated in a test tube. . As a result of culturing at 10 to 30 ° C. for 14 to 21 days, the fruiting bodies obtained were the same as those in the natural world both morphologically and microscopically. The fruiting body obtained here was used as the inoculum for inoculation for population culture.

  Mass culture liquid medium is aseptically inoculated with a bacterial culture for inoculation in a liquid medium containing 0.1-2.0% glucose and 0.1-2.0% dry yeast, and cultured at 20 ° C. for 20 days It was. As a result, mycelium was formed in the liquid medium. The liquid medium at the start of the culture was a yellow cloudy liquid, but gradually became a slightly yellow transparent liquid with the lapse of time for culturing the bacteria. When the culture of the fungus is completed, the fruiting bodies (conidial bundles) and mycelium are completely removed using a filter sterilization membrane filter having a pore size of 0.2 μm, and a sterile culture solution (secondary culture) Metabolic fluid) was obtained. This culture solution (culture secondary metabolite) was freeze-dried to obtain a dry powder of the culture.

Example 1
Experiments were carried out on Peyer's plates of special lymphoid tissues in the digestive tract using freeze-dried powders of the cultures of Isaria japonica Yasuda or Isaria sinclairii (Berk.) Lloyd obtained in Production Example 1. It was.

Experiment A (Experiment to examine cytokine production by adding directly to Peyer's patch constituent cells):
Peyer's patch was extracted from the small intestine of male C57BL / 6J mice (8-9 weeks old), and connective tissue was degraded with collagenase, and then Peyer's patch lymphocytes were collected. Next, Peyer's patch cells were suspended in a medium (RPMI 1640 medium containing 5% fetal calf serum) at 2 × 10 ⁶ cells / mL and spread on a 24-well plastic plate. To this was added freeze-dried powder of the cultivated bamboo cultivated bamboo or bamboo shoot prepared so as to have a concentration of 10 or 100 μg / mL, and concanavalin A which is a T cell activating protein at a concentration of 5 μg / mL was added or The cells were cultured for 96 hours without addition. Note that lymphocytes present on Peyer's patches do not cause cytokine production unless there is a condition that stimulates T cells. Therefore, Concanavalin A is added to the culture system at the same time as the specimen (Cultivation edulis or Takutoku Hoshitake). Experimental conditions were also added to promote cytokine production.

  After completion of the culture, the culture supernatant was collected, and the amount of cytokine, which is an in vivo immunoregulatory substance produced by Peyer's plate constituent cells of the mouse, was measured in this culture by ELISA. The cytokines measured were IL (interleukin) -2, IL-4, IL-5, IL-6, IL-10, GM-CSF and IFN-γ (interferon γ). Production of IL-2 and IFN-γ Increasing the amount induces a T-dependent cellular immune response, and increasing IL-4, IL-6, and IL-5 production induces an immune response by the antibody. In IL-10 and GM-CSF (granulocyte-macrophage colony stimulating factor) produced by Peyer's patch constituent cells, IL-10 induces an immunosuppressive reaction, and GM-CSF has a hematopoietic function of leukocytes. There is work to rise.

  Freeze-dried powder of the Japanese bamboo bud and bamboo shoots increased IL-2 and IFN-γ production (FIGS. 1 (a) and 2 (a)), with an effect on IL-4 and IL-5 production (FIG. 3A and FIG. 4A). Interestingly, the lyophilized powder of the Japanese bamboo bud and the bamboo shoot culture also increased the production of IL-6, IL-10 and GM-CSF (FIGS. 5-7). The IL-2, IL-6, IL-10, and GM-CSF production-enhancing effects of freeze-dried powders of the cultivated bamboo shoots and the bamboo shoots are the same as those obtained without adding concanavalin, which is a T cell stimulating factor. The effect was also observed with the lyophilized powder alone of the culture.

Experiment B (Experiment to examine cytokine production by taking Peyer's patches after oral administration to mice):
Male C57BL / 6J mice (8-9 weeks old) were treated with saline alone (control group), lyophilized powder (10 mg / kg / day), or lyophilized powder (10 mg / kg / day). kg / day) was orally administered for 7 days, Peyer's patches were excised from the small intestine, connective tissues were decomposed with collagenase, and Peyer's patch lymphocytes were collected. Next, Peyer's patch cells were suspended in a medium (RPMI 1640 medium containing 5% fetal calf serum) at 2 × 10 ⁶ cells / mL and spread on a 24-well plastic plate. The cells were cultured for 96 hours with or without the addition of concanavalin A, a T cell activation protein at a concentration of 5 μg / mL.

  After completion of the culture, the culture supernatant was collected, and the amount of cytokine, which is an in vivo immunoregulatory substance produced by the mouse Peyer's board constituent cells, in this culture was measured by ELISA. The cytokines measured are IL-2, IL-4, IL-5 and IFN-γ.

  In the presence of T cell stimulating factors, when lyophilized powders of hanasanagitake culture are orally administered to mice, IL-2, IFN-γ, and IL-4 and IL-5 produced by Peyer's patch constituent cells are produced. The same effect as described above was obtained (FIG. 1B, FIG. 2B, FIG. 3B and FIG. 4B). When studying the effects of natural products, the experimental results in vitro (when the effect is examined by sprinkling a sample directly on a cell) often do not match the results obtained by drinking the animal. However, these results confirmed the sufficient immunomodulatory effect even by oral ingestion.

  Among the Japanese bamboo shoots and the Japanese bamboo shoot, especially the freeze-dried powder of the Japanese bamboo shoots enhances the production of IL-2 and IFN-γ even when the mice are swallowed, but the production of IL-4 and IL-5 is affected. Therefore, the effect of selectively raising the T cell-dependent immune response can be expected. In tumor immunity, there are experimental results and clinical reports (eg IL-2 and LAK (Lymphokine-activated killer) therapy) that can effectively eliminate tumor cells by activating T cell-dependent immune responses. From these results, it is expected that such effects are expected in the cultures of hanasanagitake and tsukutokuboushitake, and IL-2 and IFN-γ also have a function of suppressing the humoral immune response. Indirect antiallergic effects can also be expected.

  In addition, when the mice are swallowed, the ratio of T cells and B cells in Peyer's patch cells and the ratio of helper T cells and killer T cells among T cells are completely affected. In the absence of this (not shown), the bamboo shoot culture does not alter the number of T cells, but has a unique effect of activating individual T cells (increasing cytokine production).

  Natural products with such a pharmacological action on Peyer's patch lymphocytes include Juzentaihoto, a Chinese herbal medicine and an insured medicine (Matsumoto T et al., Phytomedicien Vol. 6: p.425- 430, 2000), and this Chinese herbal medicine suppresses IL-2 production, enhances IFN-γ production, has no effect on IL-4 production, and has an effect of enhancing IL-5 production. However, it is known about the effect of selectively increasing IL-2 and IFN-γ production and the effect on GM-CSF, IL-6, and IL-10 production, as in the case of the bamboo bud and the bamboo shoot culture. Not. In addition, research results using Peyer's patch cells (Coh J.-H., et al., Biosci. Biotechnol. Biochem., Vol. 66: “Cordyceps sinensis” in China) p407-411, 2002), and only GM-CSF and IL-6 have been published to enhance their production. However, as in the Japanese bamboo shoot culture in the present invention, IL-2, IFN-γ and IL- 4. The effect on IL-5 has not been investigated. Therefore, from the results of the above experiments, isn't it found in Japanese cypress culture that Juzentaihoto and Chinese cordyceps that selectively regulate the amount of cytokine produced by T cells (especially helper T cells)? Or it can be positioned as a natural product with completely new pharmacological activity that is unknown or unknown.

  The cytokine production enhancer of the present invention is used in the fields of pharmaceuticals and health foods.

It is a figure which shows the amount of IL-2 production when the freeze-dried powder of the culture of Japanese bamboo shoots and the black bamboo shoot is directly added to Peyer's patch constituent cells (a) and when orally administered (b). It is a figure which shows the amount of IFN- (gamma) production when the freeze-dried powder of the culture of Japanese bamboo shoots and Takutokuboushitake is directly added to Peyer's board constituent cells (a) and when orally administered (b). It is a figure which shows the IL-4 production amount when (a) and the oral administration (b) when the freeze-dried powder of the culture of Japanese bamboo shoots and Takutokuboushitake is directly added to Peyer's board constituent cells. It is a figure which shows the amount of IL-5 production when the freeze-dried powder of the culture of Japanese bamboo shoots and Takutokuboushitake is directly added to Peyer's patch constituent cells (a) and when orally administered (b). It is a figure which shows the amount of IL-6 production at the time of adding the freeze-dried powder of the culture of Japanese bamboo shoots and Takutokuboushitake directly to Peyer's board constituent cells. It is a figure which shows the IL-10 production amount at the time of adding the freeze-dried powder of the culture of a hanasanagitake and a bamboo shoot bamboo directly to a Peyer's board constituent cell. It is a figure which shows the amount of GM-CSF production at the time of adding the freeze-dried powder of the culture of Japanese bamboo shoots and a bamboo shoot bamboo directly to Peyer's board constituent cells.

Explanation of symbols

* Indicates that a significant difference was observed at a risk rate of less than 5% compared to the value obtained when culturing without adding anything to the culture system.
It shows that a significant difference was not recognized as compared with the value when cultured without adding anything to the NS culture system.

Claims (14)

Included as an active ingredient, Isaria sinclairii (Berk.) Lloyd , a culture or extract thereof, or a processed product thereof as an active ingredient, interleukin-2, interleukin-6, interleukin-10, interferon γ and GM -A cytokine production enhancer for enhancing production of cytokines selected from CSF by Peyer's patch constituent cells.   The cytokine production enhancer according to claim 1, which is used for enhancing production of interleukin-2, interleukin-6, interleukin-10, interferon γ and GM-CSF by Peyer's patch constituent cells.   The cytokine production enhancer according to claim 1, wherein the cytokine is interleukin-2.   The cytokine production enhancer according to claim 1, wherein the cytokine is interleukin-6.   The cytokine production enhancer according to claim 1, wherein the cytokine is interleukin-10.   The cytokine production enhancer according to claim 1, wherein the cytokine is interferon γ. A GM-CSF Peyer's plate containing, as an active ingredient, an Isaria-type insect grass selected from Isaria japonica Yasuda or Isaria sinclairii (Berk.) Lloyd, a culture or extract thereof, or a processed product thereof A cytokine production enhancer for enhancing production by constituent cells . The cytokine production enhancer according to claim 7 , which contains, as an active ingredient, a culture of Isaria-type worms selected from Isaria japonica Yasuda or Isaria sinclairii (Berk.) Lloyd, or a processed product thereof. Containing a mycelium, conidia or secondary metabolite of Isaria-type insects selected from Isaria japonica Yasuda or Isaria sinclairii (Berk.) Lloyd as an active ingredient, and a Peyer's patch composition of GM-CSF A cytokine production enhancer for enhancing production by cells. The cytokine production enhancer according to any one of claims 7 to 9, wherein the Isaria-type worm is Isaria japonica Yasuda and is for oral administration. The cytokine production enhancer according to any one of claims 7 to 9, wherein the Isaria worm is Isaria sinclairii (Berk.) Lloyd. A substance derived from Isaria sinclairii (Berk.) Lloyd and having a cytokine production enhancing activity as an active ingredient, selected from interleukin-2, interleukin-6, interleukin-10, interferon γ and GM-CSF A cytokine production enhancer for enhancing the production of cytokines by Peyer's patch constituent cells.   The cytokine production enhancer according to any one of claims 1 to 12, which is added to food.   The therapeutic agent of the disease in which the production or activity of cytokine fell including the cytokine production enhancer of any one of Claims 1-12 as an active ingredient.

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