immunomodulator

Abstract

The present invention provides an immunomodulator that contains a cereal as an active ingredient. It is preferred that the cereal comprises one or more kinds of cereals selected from buckwheat, pearl millet, finger millet, Japanese barnyard millet, green pea, and wheat germ. It is also preferred that the immunomodulation is the modulation of the production of a cytokine. It is also preferred that the cytokine comprises one or more cytokines selected from IL-6, TNF-α, IL-1β, IL-8, and IL-10. It is also preferred that the cereal is in the form of an ethanol extract from the cereal. It is particularly preferred that the immunomodulator comprises one or more ethanol extracts selected from ethanol extracts from buckwheat, pearl millet, finger millet, Japanese barnyard millet, green pea, and wheat germ.

Description

immunomodulators

[0001] The present disclosure relates to immunomodulatory agents.

[0002] The immune system defends the body against microorganisms such as bacteria and viruses, as well as foreign substances that are not naturally present in the body. The immune system consists of the innate immune system, in which white blood cells such as neutrophils and macrophages function to attack, and the adaptive immune system, which specifically recognizes and memorizes infectious pathogens and foreign substances to establish an effective response against the same pathogen. It is known that the immune capacity that controls this biological defense function peaks around the 20s, and declines with age, particularly with the adaptive immune system. Immune aging is characterized by a decline in the ability to suppress infection and a decline in the immune system's regulatory function. The decline in immune system regulatory function specifically refers to the phenomenon in which the immune system becomes uncontrollable and goes out of control. It is known that a decline in immune system regulatory function leads to tissue damage due to sustained low-level inflammatory responses, and this tissue damage is thought to affect the onset of disease and the progression of aging. Therefore, the use of immunomodulators that can suppress inflammatory responses is important.

[0003] Lipopolysaccharide (LPS) activates macrophages, a type of immune cell, and induces the production of interleukin-6 (IL-6) and interleukin-10 (IL-10) in macrophages. It is known that the balance between pro-inflammatory cytokines such as IL-6 and anti-inflammatory cytokines such as IL-10 is important for the recovery of patients suffering from inflammatory diseases (Non-Patent Documents 1 and 2).

[0004] For example, Patent Document 1 describes that a buckwheat leaf extract protects lymphatic endothelial cells.

[0005] US2017 / 340692A1

[0006] Mediat. Inflamm. 2014, 2014, 561459.J. Psychiatry 2011, 33, 268-274.

[0007] Methods proposed for preventing and revitalizing immune function include restricting calorie intake, moderate exercise, increasing antioxidants, and taking vitamins to regulate endocrine function. Prevention through daily lifestyle habits, rather than treatment after the onset of the disease, is particularly important. Therefore, there is a need to find components that can be taken daily and have immunomodulatory effects.

[0008] As a result of extensive research, the present inventors have searched for grain foods that have immune-regulating effects, and have found that grains, particularly specific grains, have excellent effects.

[0009] Figure 1 is a graph showing the results of real-time monitoring of IL-10 and IL-6 gene expression upon LPS addition when buckwheat was added. Figure 1(a) shows the observation of IL-10 gene expression, and Figure 1(b) shows the observation of IL-6 gene expression. Figure 2 is a graph showing the results of real-time monitoring of IL-10 and IL-6 gene expression upon LPS addition when pearl millet was added. Figure 2(a) shows the observation of IL-10 gene expression, and Figure 2(b) shows the observation of IL-6 gene expression. Figure 3 is a graph showing the results of real-time monitoring of IL-10 and IL-6 gene expression upon LPS addition when finger millet was added. Figure 3(a) shows the observation of IL-10 gene expression, and Figure 3(b) shows the observation of IL-6 gene expression. Figure 4 is a graph showing the results of real-time monitoring of IL-10 and IL-6 gene expression upon LPS addition when barnyard millet was added. Figure 4(a) shows the results of observing IL-10 gene expression, and Figure 4(b) shows the results of observing IL-6 gene expression. Figure 5 is a graph showing the results of real-time monitoring of IL-10 and IL-6 gene expression upon addition of LPS when green peas were added. Figure 5(a) shows the results of observing IL-10 gene expression, and Figure 5(b) shows the results of observing IL-6 gene expression. Figure 6 is a graph showing the results of real-time monitoring of IL-10 gene expression upon addition of LPS when wheat germ was added, and shows the results of observing IL-10 gene expression. Figure 7 shows the results of measuring the expression level of IL-6 by ELISA when wheat germ was added together with LPS to a macrophage-like cell culture medium. Figure 8 shows the results of measuring the expression level of IL-10 by ELISA when wheat germ was added together with LPS to a macrophage-like cell culture medium. Figure 9 shows the results of measuring the expression level of TNF-α by ELISA when wheat germ was added together with LPS to the culture medium of macrophage-like cells. Figure 10 shows the results of measuring the expression level of IL-1β by ELISA when wheat germ was added together with LPS to the culture medium of macrophage-like cells.FIG. 11 shows the results of ELISA analysis of the expression level of IL-8 when wheat germ was added together with LPS to a culture medium for macrophage-like cells.

[0010] The present disclosure relates to an immunomodulator that uses a grain as an active ingredient. As used herein, the term "immunomodulator" refers to an immunomodulatory effect achieved by reducing the expression of inflammatory cytokines and / or increasing the expression of anti-inflammatory cytokines, and includes one or more selected from the following: suppressing or controlling abnormal immune function; promoting normal immune function; restoring impaired immune function; and providing a desired immune response. In particular, the immunomodulator of the present disclosure may exhibit excellent anti-inflammatory or prophylactic effects. The immune function to be regulated includes one or more selected from humoral (antibody-mediated) immunity, cellular immunity, and innate immunity.

[0011] As used herein, "modulation" preferably refers to the modulation of cytokine production. Modulation of cytokine production preferably refers to an increase, decrease, or balance in the amount and / or rate of cytokine production. Balance in this context includes adjustment to a constant concentration.

[0012] Cytokines are secreted polypeptides that regulate cell-cell interactions in immune or inflammatory responses. Examples of inflammatory cytokines in the present disclosure include at least one selected from IL-6, TNF-α, IL-1 (IL-1α, IL-1β), and IL-8, with one or more selected from IL-6, TNF-α, IL-1β, and IL-8 being preferred.

[0013] IL-6 induces the differentiation of B cells into antibody-producing cells (plasma cells) and plays a role in the inflammatory response. TNF-α not only induces the expression of vascular endothelial cells, but also enhances leukocyte adhesion molecules that stimulate immune cell infiltration, promoting lymphocyte infiltration into the infected site. IL-1β is a potent inflammatory cytokine, and IL-1β acts on CD4 ＋It stimulates cells to differentiate into Th17 cells. IL-8 induces chemotaxis of neutrophils and other granulocytes to the site of infection, and once they arrive, IL-8 further induces phagocytosis. Reducing the production of these inflammatory cytokines is expected to suppress various inflammatory symptoms.

[0014] In particular, in the present invention, it is particularly preferred that the inflammatory cytokine to be regulated includes IL-6. Overproduction of IL-6 is associated with various inflammatory symptoms associated with aging, such as cardiovascular disease, osteoporosis, arthritis, type 2 diabetes, cancer, periodontal disease, frailty, and functional decline, as well as insulin resistance, hyperlipidemia, hyperglycemia, and obesity. Therefore, immunomodulators capable of suppressing IL-6 expression are expected to exhibit excellent immunomodulatory effects, such as anti-inflammatory effects. In particular, in the present invention, it is preferred to regulate the production of IL-6 and one or more inflammatory cytokines selected from TNF-α, IL-1β, and IL-8, and it is most preferred to regulate the production of IL-6, TNF-α, IL-1β, and IL-8.

[0015] Furthermore, examples of anti-inflammatory cytokines include at least one selected from IL-10 and TGF-β, with IL-10 being particularly preferred. IL-10 is known to inhibit the expression of IL-6 in macrophages stimulated with LPS. The balance between IL-6 and IL-10 activation has traditionally been considered an indicator of anti-inflammatory properties. Enhanced IL-10 production is expected to suppress various inflammatory symptoms. In the present invention, it is preferable to regulate (e.g., enhance) the production of IL-10 or to regulate (e.g., reduce) the production of one or more cytokines selected from IL-6, TNF-α, IL-1β, and IL-8. It is more preferable to regulate the production of IL-10 and one or more cytokines selected from IL-6, TNF-α, IL-1β, and IL-8, with IL-10 and IL-6 being particularly preferred.

[0016] The immunomodulators of the present disclosure are useful for modulating an immune response in an individual. The immunomodulators of the present disclosure are useful for reducing an undesirable immune response in an individual. The immunomodulators of the present disclosure are useful for reducing inflammation in an individual. The present disclosure provides a method for modulating an immune response in an individual, comprising administering to the individual an immunomodulator comprising a grain. The present disclosure provides a method for suppressing inflammation in an individual, comprising administering to the individual an immunomodulator comprising a grain. These methods may be for non-medical purposes, such as cosmetic purposes. Furthermore, these methods may be intended for humans or animals, such as non-human mammals.

[0017] The present disclosure relates to the use of grains as agents for biologically preventing or improving immunomodulation. The immunomodulator of the present disclosure (hereinafter sometimes referred to as the "agent of the present disclosure") uses grains as an active ingredient. Accordingly, the present disclosure uses plant seeds. The seeds referred to in the present disclosure may be those harvested at the time of harvest as grains after ripening and before germination. The seeds may or may not have their outer shells removed. The grains used in the present disclosure may be the seeds themselves (endosperm and germ), or parts of the seeds, such as the germ. The germ refers to the part of the seed that will eventually grow into a sprout. Already germinated sprouts are not considered grains in this specification. The immunomodulator of the present disclosure may contain either the above-mentioned seeds containing the germ portion or the germ separated from the seed, either alone or in combination.

[0018] In the present disclosure, the active ingredient of the grain is preferably one or more selected from buckwheat, pearl millet, finger millet, barnyard millet, green pea, and wheat germ, because they are effective in reducing the expression of inflammatory cytokines and / or increasing the expression of anti-inflammatory cytokines. Hereinafter, the term "grain" is intended to include one or more of these grains.

[0019] Buckwheat refers to an annual plant belonging to the genus Fagopyrum in the Polygonaceae family, and examples thereof include Fagopyrum esculentum and Fagopyrum tataricum. Buckwheat seeds include the seed coat, pericarp, germ, and endosperm (outer flour, middle flour, and inner flour). While the present disclosure may include any of these, it is preferable to use buckwheat seeds that include not only the germ but also the endosperm.

[0020] Pearl millet is a plant belonging to the genus Pennisetum in the family Poaceae, including Pennisetum glaucum. It is a widely cultivated grain known for its heat and drought tolerance and excellent nutritional value. It comes in white cultivated varieties and black natural varieties. It is preferable to use pearl millet seeds containing not only the germ but also the endosperm in this disclosure.

[0021] Finger millet is a plant belonging to the genus Eleusine in the family Poaceae, including Eleusine Esculenta. Finger millet is eaten in parts of India and East Africa. In the present disclosure, it is preferable to use seeds containing not only the germ but also the endosperm as finger millet.

[0022] Barnyard millet is a plant belonging to the genus Echinochloa in the family Poaceae, and examples thereof include Echinochloa esculenta. Barnyard millet is cultivated in Japan and eaten in parts of India and East Africa. Barnyard millet is divided into two major varieties: a group of varieties cultivated mainly in Northeast Asia, such as the Japanese archipelago, the Korean peninsula, and northeastern China, and Lijiang barnyard millet, cultivated mainly in Yunnan Province, China. In the present disclosure, it is preferable to use seeds containing not only the germ but also the endosperm as barnyard millet.

[0023] Green peas are also called green peas or green peas. The scientific name for peas is Pisum sativum. There are two subspecies of peas: red peas, which have hard pods and safflower-like seeds, and blue peas, which have soft pods and white-flower-like seeds. Green peas have green seeds, while red peas have reddish-brown seeds. In the present invention, it is preferable to use seeds containing not only the germ but also the endosperm as the green peas, as these have excellent immunomodulatory effects.

[0024] Wheat germ refers to the germ portion contained in wheat seeds that becomes the root and cotyledons upon germination. When the immunomodulator of the present disclosure contains wheat germ, wheat seeds containing the germ portion may be contained as they are, but it is preferable that the germ be separated from the wheat seeds. As wheat germ, either defatted wheat germ from which fats and oils have been removed or unremoved wheat germ (also referred to as "full-fat wheat germ") can be used. Any available wheat can be used as the source of wheat germ. Suitable examples include bread wheat, durum wheat, club wheat, spelt wheat, emmer wheat, etc. of the genus Triticum of the family Poaceae, and Aegilops styloides and wedge wheat, etc. of the genus Aegilops of the family Poaceae.

[0025] As shown in the examples below, when the immunomodulator of the present invention contains one or more active ingredients selected from buckwheat, pearl millet, finger millet, barnyard millet, green pea, and wheat germ, it can simultaneously promote the production of anti-inflammatory cytokines by IL-10 under inflammatory conditions and reduce the production of one or more pro-inflammatory cytokines selected from L-6, TNF-α, IL-1β, and IL-8, particularly IL-6. This action allows the immunomodulator of the present invention to suppress inflammation and promote tissue repair, potentially contributing to the resolution of post-infection inflammation. When wheat germ is used as an active ingredient, the immunomodulator of the present invention is preferred in that it effectively regulates the production of IL-10, L-6, TNF-α, IL-1β, and IL-8.

[0026] The method for separating germs from seeds such as wheat is not particularly limited and can be performed by conventional methods. For example, a mixture containing germs, crushed endosperm, and crushed seed coats is obtained by applying mechanical force to the seeds, and then the crushed endosperm, crushed seed coat, etc. are removed from the mixture to obtain a crude germ fraction (a mixture containing germ as the main component and containing crushed endosperm and crushed seed coat). The force applied to the seeds may be strong enough to separate the germs from the wheat seeds. Examples of methods for applying mechanical force to seeds include a crushing process using a known crushing device. The degree of crushing may be selected appropriately depending on the size of the germ. Next, a crude germ fraction is obtained from the crushed seeds using a known classification device, such as a sieve. If necessary, the obtained crude germ fraction may be rolled to compress it, and then sieved to remove the seed coat, endosperm, debris, etc. Alternatively, the seed coat, endosperm, debris, etc. contained in the obtained crude germ fraction may be removed using wind power or electrostatic force. Alternatively, a crude germ fraction can be obtained by a method utilizing the difference in specific gravity between the germ, the seed coat, and the endosperm, for example, by heavy liquid sorting. If necessary, the germ can be sorted from the obtained crude germ fraction by visual inspection or using a color sorter, and then washed.

[0027] The agent of the present disclosure may contain the above-mentioned grains as they are, or may further contain grains that have been treated by a method selected from crushing, extraction, drying, and dry heat, and may be an extract.The extract referred to here may be a solvent extract.The grains may be used as they are as extraction raw materials, but may also be treated by a method selected from drying, crushing, heating such as dry heat, and crushing is preferred in terms of improving extraction efficiency.

[0028] The solvent used for the solvent extraction of grains is preferably an organic solvent, as it provides superior immunomodulatory properties. Examples of organic solvents include polar and nonpolar organic solvents. Examples of organic solvents that can be used as polar organic solvents include alcohols such as lower aliphatic alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol, and polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin; and lower aliphatic ketones, such as acetone and methyl ethyl ketone. Examples of nonpolar organic solvents include linear, branched, or cyclic saturated hydrocarbons, such as hexane, heptane, octane, nonane, decane, 2-ethylhexane, and cyclohexane; and alcohols, such as linear or branched alcohols having 6 or more carbon atoms. The inventors believe that organic solvent extracts are more advantageous for the effects of the present invention than aqueous extractants.

[0029] In the present disclosure, it is preferable to use a polar solvent because of its excellent immunomodulatory effect and ease of handling as a food product. Among these, it is particularly preferable to use a lower aliphatic alcohol having 1 to 5 carbon atoms, more preferably a lower aliphatic alcohol having 2 to 3 carbon atoms, and most preferably to use ethanol.

[0030] As used herein, alcoholic extracts, such as ethanolic extracts, include not only those using pure alcohol as the extraction solvent, but also those extracted with a mixed solvent primarily containing alcohol. Here, "mainly alcoholic" means that the extraction solvent contains 60% by mass or more of alcohol, more preferably 70% by mass or more, more preferably 80% by mass or more, and even more preferably 90% by mass or more. Such an extract using an extraction solvent primarily containing alcohol (e.g., ethanol) is also referred to as an alcoholic extract (e.g., ethanol extract). Note that pure alcohol refers to alcohol with a purity of 98% by mass or more, preferably 99% by mass or more.

[0031] The amount of the extraction solvent used is not particularly limited, but is usually preferably 2 parts by mass or more, more preferably 3 parts by mass or more, and even more preferably 4 parts by mass or more, per part by mass of grain, and is usually preferably 100 parts by mass or less, more preferably 50 parts by mass or less, and even more preferably 20 parts by mass or less.

[0032] The extraction temperature is preferably 0° C. or higher, more preferably 10° C. or higher, and even more preferably 20° C. or higher. The extraction temperature is preferably 100° C. or lower, more preferably 70° C. or lower, and even more preferably 50° C. or lower.

[0033] The extraction time is preferably 1 minute or longer and 72 hours or shorter, more preferably 10 minutes to 48 hours, and even more preferably 1 hour to 24 hours.

[0034] The extract obtained in the extraction step can be used as is, or after dilution, concentration, solvent removal, etc., it can be prepared into a powder or paste as needed. Solvent removal can be carried out by known methods such as reduced pressure distillation, reduced pressure / vacuum drying, freeze drying, spray drying, etc. The obtained extract may also be subjected to further purification treatment. The extract may contain the solvent, or an aqueous solution can be obtained by removing only the solvent.

[0035] The extract can be formulated into any dosage form, such as powder, granules, or liquid, using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin, or any other auxiliary agent, according to a conventional method. In this case, examples of the auxiliary agent that can be used include excipients, stabilizers, and flavoring agents.

[0036] The agent of the present disclosure can be used as a pharmaceutical, quasi-drug, or food or beverage product for animals, including humans, or for the production thereof. The agent of the present disclosure can be administered or ingested directly to animals, including humans, as a pharmaceutical, quasi-drug, or food or beverage product, or can be added or blended into food, beverage, or animal feed, such as pet food, to be used as food, beverage, or animal feed. In the latter case, the method of adding or blending grains into food, beverage, or animal feed is not particularly limited. For example, grains can be directly blended with raw materials or ingredients before the production of the food, beverage, or animal feed, added during the production process of the food, beverage, or animal feed, or added to the produced food, beverage, or animal feed. The term "food or beverage" refers to substances that can be consumed by humans, including general foods and beverages, including so-called health foods, as well as health functional foods such as foods for specified health uses and foods with nutrient functions, as defined by the Ministry of Health, Labor, and Welfare's Health Function Food System, and supplements. The term "animal feed" refers to substances fed to non-human animals (animals kept by humans), such as livestock, poultry, and farmed fish, including livestock feed and pet food.

[0037] When the agent of the present disclosure is used as a pharmaceutical or quasi-drug, it may contain the active ingredient, cereal, alone, or may further contain a pharmaceutically acceptable carrier, or may further contain other active ingredients or pharmacological ingredients to the extent that the effect of the cereal is not impaired. Examples of such carriers include excipients, coating agents, binders, fillers, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure adjusters, pH adjusters, emulsifiers, preservatives, stabilizers, antioxidants, colorants, UV absorbers, moisturizers, thickeners, activity enhancers, disinfectants, flavorings, and odor enhancers.

[0038] When the agent of the present disclosure is used as a pharmaceutical or quasi-drug, it can be administered in any dosage form.The dosage form may be oral or parenteral.For example, oral dosage forms include solid dosage forms such as tablets, coated tablets, granules, powders, and capsules, and liquid dosage forms such as elixirs, syrups, and suspensions.Non-oral dosage forms include injections, infusions, transdermal, transmucosal, nasal, enteral, inhalation, suppositories, boluses, patches, etc.Among these, oral dosage forms are preferred.

[0039] The amount of grain in the agent of the present disclosure is arbitrary as long as it is an amount that can serve as an active ingredient, and the grain may account for 5% by mass or more, 30% by mass or more, 50% by mass or more, or 70% by mass or more of the solid content of the agent. Note that the solid content here refers to the total amount excluding the solvent, and examples of the solvent include the various solvents listed above as extraction solvents and organic solvents that are commonly used as solvents.

[0040] When the agent of the present disclosure is used as a food or beverage, it may contain only the grain as an active ingredient, or may further contain various additives used in the production of foods and beverages, as long as the immunomodulatory effect of the grain is not impaired. Examples of such additives include various oils and fats, herbal medicines, amino acids, polyhydric alcohols, natural polymers, vitamins, dietary fiber, surfactants, purified water, excipients, stabilizers, pH adjusters, antioxidants, sweeteners, taste components, acidulants such as organic acids, stabilizers, flavors, colorants, fragrances, etc.

[0041] Examples of foods and beverages include oral preparations (gum, candy, etc.), processed seafood paste products such as kamaboko (fish cake) and chikuwa (fish cake), livestock products such as sausages and ham, bread, Western confectionery, Japanese confectionery, noodles such as fresh noodles, Chinese noodles, boiled noodles, and buckwheat, seasonings such as sauces, soy sauce, dressings, sugar, honey, powdered sugar, and starch syrup, spices such as curry powder, mustard powder, and pepper powder, jam, marmalade, chocolate spread, pickles, pickled vegetables, sprinkles, and various canned and bottled vegetables and fruits, dairy products such as cheese, butter, and yogurt, beverages such as miso soup, soup, fruit juice, vegetable juice, whey drinks, soft drinks, and alcoholic beverages, and other general foods and beverages such as health foods.

[0042] The present disclosure encompasses a package comprising a packaging body and the immunomodulatory agent of the present disclosure, or a food, beverage, or animal feed containing the same, contained in the packaging body. The packaging body is not particularly limited in shape or material, as long as it can contain the agent, food, beverage, or animal feed of the present disclosure and can print ingredient information, etc. Examples of the packaging body shape include a box shape, a bag shape, etc. Examples of the packaging body material include paper, plastic, paper, woven fabric, metal, etc. The packaging body clearly displays various information, such as the grain content of the agent, food, beverage, or animal feed of the present disclosure contained in the packaging body. The method of displaying information on such packaging body is not particularly limited, and for example, 1) the information may be printed on the exterior or interior surface of the packaging body, 2) the immunomodulatory use may be printed on a printing medium such as printing paper contained inside the packaging body together with the food, beverage, or animal feed, or 3) an internet URL may be written on the packaging body or on a printing medium contained therein, and the information may be displayed by accessing the URL.

[0043] As shown in the Examples below, the agent of the present disclosure can effectively reduce the expression of pro-inflammatory IL-6 and / or enhance the expression of anti-inflammatory IL-10 in macrophage-like cells activated with LPS. Thus, the agent of the present disclosure can balance the production amounts of pro-inflammatory cytokines and anti-inflammatory cytokines in immune cells such as macrophages, thereby adjusting and / or regulating the inflammatory state in an individual, and specifically, can suppress and / or prevent inflammation.

[0044] The inflammatory conditions include diarrheal diseases, mucositis, gastroenteritis, pouchitis, obesity-related inflammation, appendicitis, bacterial infection, viral infection, fungal infection, cancer-related inflammation, urogenital diseases, bacterial vaginosis, surgery-related trauma, sepsis, ulcers, wound healing, fibrotic diseases such as kidney disease, liver disease, liver fibrosis, alcoholic hepatitis, pulmonary fibrosis, renal fibrosis, and idiopathic pulmonary fibrosis, acne, unwanted respiratory inflammatory activity, inflammation-associated cancer, inflammation-associated organ damage and injury (e.g., lung, liver, kidney, heart, gastrointestinal tract, brain), autoinflammatory diseases (e.g., TNF receptor-associated periodic fever syndrome, Dubin-Johnson syndrome, Behcet's disease), allergies, rheumatoid arthritis, asthma, diabetes, systemic lupus erythematosus (SLE), atherosclerosis, multiple sclerosis, schizophrenia, anterior pituitary gland dysfunction, neurodegenerative disorders, cardiovascular disease, psoriasis, and metabolic diseases. By modulating immune mechanisms (including immune cells, cytokines, antibodies, etc.) through the immunomodulatory agents of the present disclosure, immune dysregulation can be prevented and / or suppressed.

[0045] Furthermore, the immunomodulation method using the grain of the present invention includes a method of having a subject who requires immunomodulation ingest the grain, preferably for non-medical purposes such as beauty or health foods, or for purposes other than human medical purposes. The present invention also includes the use of the grain as a food or beverage for a subject who requires immunomodulation, and the use of the grain to produce an agent or food or beverage for preventing or ameliorating (treating) a condition (symptom) requiring immunomodulation.

[0046] The agent of the present disclosure has a high level of safety and can be taken continuously over a long period of time because its active ingredients are derived from natural plants. For example, when the grain is a solid extract and the specific grain is used for immunomodulation, the daily intake for an adult (body weight 60 kg) is preferably 1 mg to 15 g, and more preferably 150 mg to 10 g. Furthermore, when used for immunomodulation, the amount of the grain itself, not the extract, is preferably 10 mg to 100 g, and more preferably 1 g to 50 g, per day for an adult (body weight 60 kg).

[0047] (Example 1) [Materials] The anti-inflammatory effects of several grains were confirmed based on their influence on the expression levels of IL-6 and IL-10. The experiment was carried out as follows.

[0048] [Grains] Dry grains were crushed in a mixer for 120 seconds. 25 mL of 99.5% ethanol was added to 5 g of crushed grains and allowed to stand at room temperature (20-28°C) for 24 hours. The resulting extract was separated into solid and liquid by filtration through absorbent cotton, and the solvent was evaporated to dryness using a rotary evaporator to obtain an ethanol extract. The remaining solid extract was redissolved in ethanol (99.5% purity) to a concentration of 10 mg / mL, yielding a liquid ethanol extract (hereinafter also referred to as "grain extract"). Ethanol (99.5% purity) was used as a control.

[0049] (Preparation of transformed RAW 264.7 cells) Transgenic RAW 264.7 cells were prepared using the method described in Biochemical and Biophysical Research Communications 505, (2018), pp. 885-890 (hereinafter also referred to as "Reference 1"). As described in this document, a macrophage cell line (RAW 264.7 cells) was transformed by introducing a plasmid containing an IL-6 or IL-10 reporter together with a reference gapdh reporter. Specifically, for transformation, the human IL-6 promoter region (nt -1000 to +121, +1 being the putative transcription start site) and the IL-10 promoter region (nt -1000 to +59) were amplified from the human genome. DNA purified from human lymphocytes was amplified by PCR using the primer pairs described in Reference 1 for IL-6 and IL-10. For transformation, as described in Reference 1, the commercially available vector pELuc(PEST)Test (TOYOBO) was used. From this vector, the respective promoter fragments were ligated upstream of the PEST-fused Eluc gene of pELuc-PEST-test / Hyg using restriction enzymes HindIII, XbaI, and the hygromycin B resistance gene Hyg. Using the above procedure, a plasmid containing an IL-6 or IL-10 reporter was constructed. Separately, a gapdh-SLR reporter plasmid was constructed using the method described in Reference 2 (Toxicological Sciences, 124(2) (2011) pp. 359-369). Using the procedure described in Reference 1, a macrophage cell line (RAW 264.7 cells) obtained from RIKEN was transformed with the constructed plasmid. Specifically, the following procedure was performed. RAW 264.7 cells (1.4 × 10 6The cells were seeded onto 35-mm Petri dishes containing DMEM medium and incubated under standard conditions for 24 hours. The obtained IL-6 or IL-10 reporter plasmid and the gapdh-SLR reporter plasmid were transfected using Hilymax transfection reagent (Dojindo Molecular Technologies) according to the manufacturer's instructions.

[0050] After transfection, RAW264.7 cells transfected with IL-6 / gapdh or IL-10 / gapdh were diluted and treated with hygromycin B for selection. Some cells were selected and treated with 1 μg / mL LPS (Sigma-Aldrich Corporation) derived from Escherichia coli O26:B6. The relative expression levels of IL-6 / gapdh and IL-10 / gapdh were then measured, and cells with stable and strong IL-6 and IL-10 expression were preserved as stable cells (Reference 3: International Journal of Molecular Sciences, 2019, 20, 4620).

[0051] The stable cells were used to evaluate IL-6 and IL-10 expression as described in Reference 3. The stable cells obtained above were seeded into 96-well black plates with clear bottoms (Wallac Oy, Turku, Germany). Twenty-four hours after seeding, the plate medium was replaced with DMEM medium containing 10% FBS, 0.1 mM D-luciferin potassium salt, 25 mM Hepes, and the cereal extract obtained above. The amount of cereal extract added to the DMEM medium was 25–75 μg / mL (75 μg / mL in the examples shown in Figures 1–6) based on the solid cereal extract per 100 parts by mass of the DMEM medium containing the aforementioned components. Thirty minutes later, LPS was added to the DMEM medium at a concentration of 100 ng / mL. Bioluminescence was recorded in real time for 48 hours using a multicolor system, starting from time 0 after LPS addition. Specifically, bioluminescence was recorded in real time every 10 seconds using a luminometer (ATTO, TWSL-1565 Kronos HT) in the presence of an R60 long-pass filter (HOYA) at 37°C for 48 hours. The ratios of the luminescence intensity corresponding to the expression levels of IL-6 and IL-10 to the luminescence intensity corresponding to the expression level of the gapdh gene were calculated over time and are shown in Figures 1 to 6 (solid lines). Figures 1 to 6 also show the same ratios when ethanol was added as a control (dotted lines).

[0052] In the above test, 33 types of grains were evaluated, and six types - buckwheat, pearl millet, barnyard millet, finger millet, and green pea (all seeds), as well as wheat germ - were found to be excellent in terms of the effect of reducing IL-6 expression and / or the effect of enhancing IL-10 expression. The results for buckwheat, pearl millet, green pea, barnyard millet, finger millet, and wheat germ are shown in Figures 1 to 6. For buckwheat, pearl millet, barnyard millet, finger millet, and green pea, mature seeds that are commercially available for consumption were used. Of the buckwheat, pearl millet, barnyard millet, finger millet, and green pea, the husk was removed for buckwheat. Furthermore, for wheat germ, wheat germ that is commercially available for consumption was used.

[0053] (Example 2) In the same manner as in Example 1, a liquid ethanol extract of cereals was obtained.

[0054] (Measurement of cytokine expression levels) Wheat germ was used as the grain. THP-1 cells (human monocytic leukemia cells) (JCRB Cell Bank) were cultured in a growth medium (RPMI1640 (Sigma-Aldrich) + 10% FBS (Sigma-Aldrich)), and then cultured at 6.3 × 10 cells / mL in a macrophage-like differentiation induction medium (RPMI1640 (Sigma-Aldrich) + 10% FBS (Sigma-Aldrich) + 0.5 μg / mL Phorbol 12-myristate 13-acetate (Fujifilm Wako Pure Chemical Industries)). 4 The cells were seeded in a 24-well plate at 0.5 mL / well under conditions of 37°C and 5% CO ２ After confirming that the cells had differentiated into macrophage-like cells, the test medium was replaced with a test medium (10% FBS, 100 units / mL penicillin, 100 units / mL streptomycin-RPMI1640 medium) containing the test substance at each concentration shown in Tables 7 to 11, and the medium was incubated at 37°C, 5% CO ２After culturing for 1 hour under these conditions, the medium was replaced with a test medium containing LPS (Lipopolysaccharides from Escherichia coli 055:B5, Sigma-Aldrich) and the test substance at the concentrations shown in Tables 7 to 11, and the cells were cultured for 24 hours. LPS was added at a concentration of 1 μg / mL. As a control, the culture was performed in the same manner, except that the medium contained 1% DMSO (purity ≥ 99.5%) instead of the test substance. The culture supernatant from each well was transferred to a 1.5 mL microtube with a lid and centrifuged at 3,000 × g for 5 minutes. The precipitate-free supernatant after centrifugation was used for ELISA analysis, and the contents of IL-1β, IL-6, IL-8, IL-10, and TNF-α were measured. Measurements were performed in triplicate for each cytokine, and the average values ​​were calculated. ELISA measurements were performed using a Varioskan LUX absorbance, fluorescence, and luminescence plate reader (ThermoFisher). In the ELISA analysis, the following reagents were used to measure the amount of each cytokine: IL-1β: IL-1β ELISA Kit, Human (ProteinTech). IL-6: Levis (registered trademark) Human IL-6 ELISA Kit (Fujifilm Wako Shibayagi). IL-8: Levis (registered trademark) Human IL-8 ELISA Kit (Fujifilm Wako Shibayagi). IL-10: AuthentiKine (trademark) IL-10 ELISA Kit, Human (ProteinTech). TNF-α: AuthentiKine (trademark) TNF-alpha ELISA Kit, Human (ProteinTech). The average values ​​and standard deviations are shown as bar graphs in Figures 7 to 11.

[0055] As shown in Figures 7 to 11, the addition of wheat germ extract to macrophage-like cells promoted the production of IL-10, an anti-inflammatory cytokine, and suppressed the production of TNF-α, IL-1β, and IL-8, which are inflammatory cytokines, upon stimulation with LPS.

[0056] According to the present disclosure, an immunomodulator having excellent immunomodulatory effects can be provided. The immunomodulator of the present disclosure can exert excellent immunoregulatory action.

Claims

1. An immunomodulator with grain as its active ingredient.

2. The immunomodulator according to claim 1, wherein the cereal is one or more selected from the group consisting of buckwheat, pearl millet, finger millet, barnyard millet, green peas, and wheat germ.

3. The immunomodulator according to claim 1, wherein the immunomodulation of the immunomodulator is regulation of cytokine production.

4. The immunoregulator according to claim 3, wherein the cytokine is one or more selected from IL-6, TNF-α, IL-1β, IL-8 and IL-10.

5. The immunomodulator of claim 1, wherein the cereal is an ethanol extract of cereal.

6. The immunomodulator according to claim 2, wherein one or more selected from buckwheat, pearl millet, finger millet, barnyard millet, green pea and wheat germ are ethanol extracts.

7. Immunomodulatory methods using grains.

8. The method according to claim 7, wherein the cereal is one or more selected from buckwheat, pearl millet, finger millet, barnyard millet, green pea and wheat germ.

9. The method of claim 7, wherein the immunomodulation of said immunomodulatory method is modulation of cytokine production.

10. The method of claim 9, wherein the cytokine is one or more selected from IL-6, TNF-α, IL-1β, IL-8, and IL-10.

11. The method according to any one of claims 7 to 10, wherein the grain is an ethanol extract of a grain.

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