Immunomodulators

Abstract

The present application provides an immunomodulator, which uses a cereal as an effective ingredient. It is preferable that the cereal is one or more selected from the group consisting of buckwheat, pearl barley, long-grain barley, barnyard grass, green pea, and wheat germ. It is also preferable that the immunomodulation is the modulation of cytokine production. It is further preferable that the cytokine is one or more selected from the group consisting of IL-6, TNF-α, IL-1β, IL-8, and IL-10. It is also preferable that the cereal is an ethanol extract of a cereal. It is particularly preferable that the immunomodulator is an ethanol extract of one or more selected from the group consisting of buckwheat, pearl barley, long-grain barley, barnyard grass, green pea, and wheat germ.

Description

Technical Field

[0001] This disclosure relates to an immunomodulator. Background Technology

[0002] The immune system is the system that protects an organism from microorganisms such as bacteria and viruses, or substances not originally present in the body, often referred to as foreign substances. The immune system comprises the innate immune system, which functions through the action of white blood cells such as neutrophils and macrophages, and the acquired immune system, which specifically recognizes and remembers pathogens or foreign substances, thus establishing an effective response against the same pathogen. It is known that the immune capacity responsible for this organism's defense function peaks around age 20, and declines with age, particularly due to the aging of the acquired immune system. The aging of the immune system involves a decline in both its ability to suppress infection and its control function. Specifically, this decline refers to the immune system's inability to maintain control, resulting in immune system dysregulation. It is known that when the immune system's control function is reduced, persistent low-level inflammation leads to tissue damage, and this tissue impairment is believed to influence disease pathogenesis and the progression of aging. Therefore, it is important to use components that can suppress inflammatory responses as immunomodulators.

[0003] Lipopolysaccharides (LPS) activate macrophages, a type of immune cell, inducing the production of interleukin-6 (IL-6) and interleukin-10 (IL-10) within them. Furthermore, the balance between inflammatory cytokines, represented by IL-6, and anti-inflammatory cytokines, represented by IL-10, is known to be important for the recovery of patients with inflammatory diseases (Non-Patent Literature 1 and 2).

[0004] For example, Patent Document 1 describes that buckwheat leaf extract can protect lymphatic endothelial cells. Existing technical documents Patent documents

[0005] Patent Document 1: US2017 / 340692A1 Non-patent literature

[0006] Non-patent literature 1: Mediat. Inflamm. 2014, 2014, 561459. Non-patent literature 2: J. Psychiatry 2011, 33, 268-274. Summary of the Invention

[0007] As a method to prevent and activate immune function during aging, it is recommended to limit calorie intake, engage in moderate exercise, increase antioxidants, and consume vitamins that regulate endocrine function. Of particular importance is not treatment starting from the onset of the disease, but prevention based on daily habits. That is, it is necessary to discover components that can be ingested daily and have immunomodulatory effects.

[0008] The inventors conducted in-depth research and, by exploring foods with immune-regulating effects using cereals as the subject, discovered that cereals, especially certain types of cereals, play an excellent role. Attached Figure Description

[0009] Figure 1 This is a graph showing the real-time monitoring results of IL-10 and IL-6 gene expression when buckwheat and LPS are added. Figure 1 (a) is a diagram showing the gene expression of IL-10. Figure 1 (b) is a diagram showing the gene expression of IL-6. Figure 2 This is a graph showing the real-time monitoring results of IL-10 and IL-6 gene expression when pearl millet and LPS are added. Figure 2 (a) is a diagram showing the gene expression of IL-10. Figure 2 (b) is a diagram showing the gene expression of IL-6. Figure 3 This is a graph showing the real-time monitoring results of IL-10 and IL-6 gene expression when dragon claw millet was added and when LPS was added. Figure 3 (a) is a diagram showing the gene expression of IL-10. Figure 3 (b) is a diagram showing the gene expression of IL-6. Figure 4 This is a graph showing the real-time monitoring results of IL-10 and IL-6 gene expression when barnyardgrass and LPS are added. Figure 4 (a) is a diagram showing the gene expression of IL-10. Figure 4 (b) is a diagram showing the gene expression of IL-6. Figure 5 This is a graph showing the real-time monitoring results of IL-10 and IL-6 gene expression when green peas and LPS were added. Figure 5 (a) is a diagram showing the gene expression of IL-10. Figure 5 (b) is a diagram showing the gene expression of IL-6. Figure 6 This is a graph showing the real-time monitoring results of IL-10 gene expression when wheat germ and LPS are added, which is the result of observing IL-10 gene expression. Figure 7 The results were obtained by ELISA analysis of the expression level of IL-6 when wheat germ was added to the culture medium of macrophage-like cells along with LPS. Figure 8 The results were obtained by ELISA analysis of the expression level of IL-10 when wheat germ was added to the culture medium of macrophage-like cells along with LPS. Figure 9 The results were obtained by ELISA analysis of the expression level of TNF-α when wheat germ was added to the culture medium of macrophage-like cells along with LPS. Figure 10 The results were obtained by ELISA analysis of the expression level of IL-1β when wheat germ was added to the culture medium of macrophage-like cells along with LPS. Figure 11 The results were obtained by ELISA analysis of the expression level of IL-8 when wheat germ was added to the culture medium of macrophage-like cells along with LPS. Detailed Implementation

[0010] This disclosure relates to an immunomodulator that uses cereals as the active ingredient. In this specification, "immunomodulator" refers to an immunomodulatory effect obtained by reducing the expression of inflammatory cytokines and / or increasing the expression of anti-inflammatory cytokines, and may be selected from one or more of the following: inhibiting or controlling abnormal immune function, enhancing normal immune function, repairing reduced immune function, and providing a desired immune response. In particular, the immunomodulators disclosed herein can exert excellent anti-inflammatory or anti-inflammatory effects. The immune function targeted for regulation includes one or more selected from humoral (antibody-mediated) immunity, cellular immunity, and innate immunity.

[0011] In this specification, "regulation" is preferably the regulation of cytokine production. Regulation of cytokine production preferably refers to an increase, decrease, or equilibrium in the amount and / or rate of cytokine production. Equilibrium, as referred to herein, can be exemplified by adjusting to a constant concentration.

[0012] Cytokines are molecules that regulate cell-cell interactions in immune responses or inflammatory reactions, and belong to the category of secreted polypeptides. Examples of inflammatory cytokines in this disclosure include at least one selected from IL-6, TNF-α, IL-1 (IL-1α, IL-1β), and IL-8, preferably one or more selected from IL-6, TNF-α, IL-1β, and IL-8.

[0013] IL-6 induces B cells to differentiate into antibody-producing cells (plasma cells), playing a role including inflammatory responses. TNF-α not only induces expression in vascular endothelial cells, but also enhances leukocyte adhesion molecules that stimulate immune cell infiltration and promotes lymphocyte infiltration to the site of infection. IL-1β is a potent inflammatory cytokine; IL-1β stimulates CD4+. + The cells were induced to differentiate into Th17 cells. IL-8 induces chemotaxis of neutrophils or other granulocytes toward the site of infection, and when they arrive, IL-8 further induces phagocytosis. It is expected that reducing the production of these inflammatory cytokines can suppress various inflammatory symptoms.

[0014] In this invention, the inflammatory cytokine to be regulated particularly preferably includes IL-6. Excessive production of IL-6 is associated not only with various age-related inflammatory symptoms such as cardiovascular disease, osteoporosis, arthritis, type 2 diabetes, cancer, periodontal disease, weakness, and functional decline, but also with insulin resistance, hyperlipidemia, hyperglycemia, and obesity. Therefore, immunomodulatory agents capable of inhibiting IL-6 expression can be expected to exhibit excellent anti-inflammatory effects and other immunomodulatory effects. Specifically, in this invention, it is preferable to regulate the production of IL-6 and one or more inflammatory cytokines selected from TNF-α, IL-1β, and IL-8, and most preferably, the production of IL-6, TNF-α, IL-1β, and IL-8.

[0015] In addition, at least one selected from IL-10 and TGF-β can be cited as anti-inflammatory cytokines, with IL-10 being particularly preferred. IL-10 is known to inhibit the expression of IL-6 in LPS-stimulated macrophages. Previously, the balance of IL-6 / IL-10 activation has been considered an indicator of anti-inflammation. By increasing the production of IL-10, it is expected to suppress various inflammatory symptoms. In this invention, it is preferred to regulate (e.g., increase) the production of IL-10, or to regulate (e.g., decrease) the production of one or more cytokines selected from IL-6, TNF-α, IL-1β, and IL-8, more preferably to regulate the production of IL-10, and to regulate the production of one or more cytokines selected from IL-6, TNF-α, IL-1β, and IL-8, particularly preferably to regulate the production of IL-10 and IL-6.

[0016] The immunomodulators disclosed herein are useful for modulating immune responses in an individual. The immunomodulators disclosed herein are useful for reducing unwanted immune responses in an individual. The immunomodulators disclosed herein are useful for reducing inflammation in an individual. This disclosure provides a method for modulating an immune response in an individual, comprising administering an immunomodulator containing cereal to the individual. This disclosure provides a method for suppressing inflammation in an individual, comprising administering an immunomodulator containing cereal to the individual. These methods can be for non-medical purposes such as cosmetic purposes. Furthermore, these methods can be used on humans or on animals such as mammals other than humans.

[0017] This disclosure relates to the use of cereals as preparations for achieving prevention or improvement in immunomodulatory biology. The immunomodulators of this disclosure (hereinafter, sometimes referred to as "the preparations of this disclosure") use cereals as their active ingredient. Therefore, plant seeds are used in this disclosure. In this disclosure, the seed is any seed harvested at the time of maturity as a cereal and before germination. Sometimes the seed coat is removed, sometimes it is not. In this disclosure, the cereal can be the seed itself (endosperm and plumule) or a portion of the seed, such as the plumule. Furthermore, the plumule refers to the portion within the seed that subsequently develops into a bud. Germinated seedlings are not considered cereals as described in this specification. The immunomodulators of this disclosure may contain, individually or separately, seeds containing the aforementioned plumule portion or plumules separated from the seed, or any two or more may be used together.

[0018] In this disclosure, from the perspective of superior effects in reducing the expression of inflammatory cytokines and / or increasing the expression of anti-inflammatory cytokines, one or more of the following selected cereals are preferred as active ingredients: buckwheat, pearl millet, dragon claw millet, barnyard millet, green pea, and wheat germ. Hereinafter, when referred to as "cereals," it means a substance containing one or more of these.

[0019] Buckwheat refers to an annual herb belonging to the genus *Fagopyrum* of the family Polygonaceae, including species such as sweet buckwheat (*Fagopyrum esculentum*) and bitter buckwheat (*Fagopyrum tataricum*). Buckwheat seeds comprise the seed coat, pericarp, embryo, and endosperm (outer, middle, and inner starch layers). This disclosure may include any of these species, but for buckwheat, seeds containing both the embryo and endosperm are preferred.

[0020] Pearl millet refers to plants belonging to the genus *Pennisetum* of the family Poaceae, such as *Pennisetum glaucum*. As a widely cultivated grain, it is heat- and drought-resistant and nutritionally excellent. There are white cultivated varieties and black natural varieties. For the pearl millet used in this disclosure, seeds containing not only the embryo but also the endosperm are preferred.

[0021] Furthermore, *Eleusine esculenta* is a plant belonging to the genus *Eleusine* of the family Poaceae, and *Eleusine esculenta* is an example. *Eleusine esculenta* is consumed in parts of India and East Africa. In this disclosure, seeds containing not only the embryo but also the endosperm are preferred as *Eleusine esculenta*.

[0022] Barnyard grass (Echinochloa sculenta) is a plant belonging to the genus Echinochloa in the family Poaceae. Cultivated barnyard grass (Echinochloa esculenta) is an example. Barnyard grass is also cultivated in Japan and consumed in parts of India and East Africa. It is divided into two main groups: a group of varieties cultivated primarily in Northeast Asia, including the Japanese archipelago, the Korean Peninsula, and Northeast China; and a group of varieties cultivated primarily in Yunnan Province, China, specifically the Lijiang barnyard grass. In this disclosure, seeds containing not only the plumule but also the endosperm are preferred when using barnyard grass.

[0023] Green peas are also known as blue peas or green beans. The scientific name for peas is *Pisum sativum*. Among peas, there are subspecies: red peas with hard pods and red-flowered seeds, and green peas with soft pods and white-flowered seeds. Green pea seeds are green, while red pea seeds are reddish-brown. In this invention, from the perspective of excellent immunomodulatory effects, seeds containing not only the embryo but also the endosperm are preferred when using green peas.

[0024] Wheat germ refers to the embryonic portion contained in wheat seeds that becomes the young root and cotyledon upon germination. In the case of the immunomodulator disclosed herein containing wheat germ, the wheat seed containing the embryonic portion may be used directly, but it is preferable to contain the embryo isolated from the wheat seed. As the wheat germ, any type of defatted wheat germ (with oil components removed) or wheat germ (with oil components not removed, also known as "full-fat wheat germ") may be used. As the raw material for the wheat germ, any wheat variety can be used, as long as it is available. Preferred examples include common wheat, durum wheat, high-spike wheat, spelt wheat, emmer wheat, etc. (all belonging to the genus *Triticum* of the Poaceae family), and *Tetranychus timofievanifolia* and *Aegilops spp.* (all belonging to the genus *Aegilops* of the Poaceae family).

[0025] As described in the embodiments below, the immunomodulator of the present invention, when using one or more of the following selected ingredients—buckwheat, pearl millet, dragon claw millet, barnyard millet, green pea, and wheat germ—as active ingredients, can simultaneously promote the production of IL-10-based anti-inflammatory cytokines in an inflammatory state and reduce the production of one or more selected ingredients—IL-6, TNF-α, IL-1β, and IL-8, particularly IL-6. Through this action, the immunomodulator of the present invention can inhibit inflammation, promote tissue repair, and contribute to the termination of post-infectious inflammation. From the perspective of effectively regulating the production of IL-10, IL-6, TNF-α, IL-1β and IL-8, the immunomodulator of the present invention preferably uses wheat germ as the active ingredient.

[0026] There are no particular limitations on the method for separating the embryo from seeds such as wheat; conventional methods can be used. For example, by applying mechanical force to the seeds, a mixture containing embryo, endosperm fragments, and seed coat fragments is obtained. The endosperm fragments and seed coat fragments are then removed from this mixture to obtain a crude embryo component (a mixture mainly composed of embryo, containing endosperm fragments and seed coat fragments). The force applied to the seeds only needs to be strong enough to separate the embryo from the wheat seeds. One method of applying mechanical force to the seeds is a pulverizing process using a known pulverizing device. The degree of pulverization can be appropriately selected according to the size of the embryo. Next, a known grading device, such as a sieve, is used to obtain the crude embryo component from the obtained seed pulverization. If necessary, the obtained crude embryo component can be flattened by rolling and then sieved to remove the seed coat, endosperm, impurities, etc. Alternatively, wind power or electrostatic force can be used to remove the seed coat, endosperm, impurities, etc., contained in the obtained crude embryo component. Alternatively, crude embryo fractions can be obtained by utilizing the density differences between the embryo, seed coat, and endosperm, such as through heavy liquid separation. Further, as needed, embryos can be separated from the obtained crude embryo fraction using methods such as visual inspection or color sorting machines, and then washed.

[0027] The formulation disclosed herein may directly contain the cereals listed above, or may contain cereals that have undergone further treatment selected from pulverization, extraction, drying, or dry heat treatment, or may be an extract. Examples of extracts mentioned here include solvent extracts. Cereals may be used directly as the extraction raw material, or the cereals may be treated with methods such as drying, pulverization, or dry heat treatment. From the perspective of improving extraction efficiency, pulverization is preferred.

[0028] When using solvent extraction of cereals, organic solvents are preferred from the perspective of obtaining superior immunomodulatory properties. Examples of organic solvents include polar and non-polar organic solvents. Examples of suitable polar organic solvents include lower aliphatic alcohols with 1 to 5 carbon atoms such as methanol, ethanol, propanol, and isopropanol; polyols with 2 to 5 carbon atoms such as 1,3-butanediol, propylene glycol, and glycerol; and lower aliphatic ketones such as acetone and methyl ethyl ketone. Examples of non-polar organic solvents include straight-chain, branched, or cyclic saturated hydrocarbons such as hexane, heptane, octane, nonane, decane, 2-ethylhexane, and cyclohexane; and straight-chain or branched alcohols with 6 or more carbon atoms. The inventors believe that organic solvent extracts are more advantageous for the purposes of this invention compared to water-based extraction solvents.

[0029] In this disclosure, from the perspective of excellent immunomodulatory effects and ease of food processing, polar solvents are preferred. Among them, lower aliphatic alcohols with 1 to 5 carbon atoms are particularly preferred, lower aliphatic alcohols with 2 to 3 carbon atoms are more preferred, and ethanol is most preferred.

[0030] In this specification, alcoholic extracts, such as ethanol extracts, include not only those using pure alcohol as the extraction solvent, but also those using mixed solvents with alcohol as the main component. Here, "alcohol as the main component" means that the extraction solvent contains 60% or more by mass of alcohol, more preferably 70% or more by mass, even more preferably 80% or more by mass, and even more preferably 90% or more by mass. Extracts obtained using extraction solvents with alcohol (e.g., ethanol) as the main component are also referred to as alcoholic extracts (e.g., ethanol extracts). It should be noted that pure alcohol refers to alcohol with a purity of 98% or more by mass, preferably 99% or more by mass.

[0031] The amount of the extraction solvent is not particularly limited, but it is generally preferred to be 2 parts by mass or more, more preferably 3 parts by mass or more, and even more preferably 4 parts by mass or more, relative to 1 part by mass of cereal. Furthermore, it is generally preferred to be 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] Furthermore, the extraction temperature is generally preferably above 0°C, more preferably above 10°C, and even more preferably above 20°C. Additionally, it is generally preferably below 100°C, more preferably below 70°C, and even more preferably below 50°C.

[0033] Furthermore, the extraction time is generally preferably 1 minute or more, and generally preferably 72 hours or less. More preferably, it is 10 minutes or more and 48 hours or less, and even more preferably 1 hour or more and 24 hours or less.

[0034] The extract obtained through the extraction process can be used directly or after dilution, concentration, solvent removal, etc., and then prepared into powder or paste as needed. Solvent removal can be carried out by known methods such as vacuum distillation, vacuum drying, freeze drying, and spray drying. Alternatively, the obtained extract can be further purified. The extract may contain solvent components; alternatively, only the solvent components may be removed to obtain an aqueous extract.

[0035] Furthermore, the above-mentioned extracts can be prepared into any dosage form, such as powder, granules, or liquid, using any pharmaceutically acceptable carriers such as dextrin and cyclodextrin, according to conventional methods. In this case, excipients, stabilizers, and odorants can be used as adjuvants.

[0036] The formulations disclosed herein can be made into pharmaceuticals, quasi-drugs, or food supplements for animals, including humans, or used in the manufacture of such products. The formulations disclosed herein can be made into pharmaceuticals, quasi-drugs, or food supplements for direct administration to animals, including humans, or for ingestion. They can also be added to or formulated into animal feeds such as food supplements or pet food. In the latter case, there are no particular limitations on the method of adding or formulating cereals into food supplements or animal feeds. For example, cereals can be directly incorporated into raw materials before the manufacture of food supplements or animal feeds, added during the manufacturing process of food supplements or animal feeds, or added to finished food supplements or animal feeds. The term "food and beverage products" refers to substances that can be ingested by humans as food. In addition to general food and beverage products, including so-called health foods, examples include specific health foods, nutritional functional foods, and nutritional supplements as stipulated in the Ministry of Health, Labour and Welfare's Health Functional Foods Regulations. The term "animal feed" refers to substances provided as feed to animals other than humans (animals raised by humans), such as livestock feed and pet food.

[0037] When the formulation disclosed herein is used as a pharmaceutical or quasi-drug product, it may contain cereal as an active ingredient alone, or it may further contain a pharmaceutically acceptable carrier, or it may further contain other active ingredients or pharmacological components without impairing the cereal-based effects. Examples of such carriers include excipients, coating agents, binders, expanders, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure regulators, pH regulators, emulsifiers, preservatives, stabilizers, antioxidants, colorants, ultraviolet absorbers, humectants, thickeners, activity enhancers, bactericides, flavoring agents, and odorants.

[0038] When the formulation disclosed herein is used as a pharmaceutical or quasi-drug product, it can be administered by any method of administration. The method of administration can be oral or non-oral. Examples of oral administration include solid dosage forms such as tablets, coated tablets, granules, powders, and capsules, as well as liquid dosage forms such as elixirs, syrups, and suspensions. Examples of non-oral administration include injection, infusion, transdermal, transmucosal, nasal, enteral, inhalation, suppositories, boluses, and patches. Oral administration is preferred.

[0039] In the formulation disclosed herein, the amount of cereals can be arbitrary, as long as it constitutes an effective ingredient. In the solid components of the formulation, cereals can account for 5% or more by mass, or 30% or more by mass, or 50% or more by mass, or 70% or more by mass. It should be noted that the solid components referred to here are the total amount after removing the solvent. Examples of solvents include various solvents listed as extraction solvents, as well as commonly used organic solvents.

[0040] When the formulations disclosed herein are used in food or beverage production, they may contain cereals as the active ingredient alone, or they may further contain various additives used in the manufacture of the food or beverage, to the extent that the immunomodulatory effects based on cereals are not impaired. Examples of such additives include various oils, crude drugs, amino acids, polyols, natural polymers, vitamins, dietary fiber, surfactants, purified water, excipients, stabilizers, pH adjusters, antioxidants, sweeteners, flavoring agents, acidulants such as organic acids, stabilizers, flavoring agents, coloring agents, and flavorings.

[0041] As a food and beverage, its uses include, for example, oral care products (chewing gum, candy, etc.), processed aquatic products such as fish cakes and fish rolls, livestock products such as sausages and ham, bread, Western-style pastries, Japanese sweets, fresh noodles, Chinese noodles, lo mein, buckwheat noodles, sauces, soy sauce, condiments, sugar, honey, powdered syrups, water syrups, spices such as curry powder, mustard powder, and pepper, jams, citrus marmalades, chocolate spreads, pickles, ready-made dishes, rice seasonings, or various canned or bottled processed vegetables and fruits, dairy products such as cheese, butter, and yogurt, miso soup, soup, fruit juice, vegetable juice, whey drinks, soft drinks, alcoholic beverages, and health foods, as well as other common food and beverage uses.

[0042] This disclosure includes a package comprising a packaging body and the immunomodulator of the present disclosure described above, or a food or animal feed containing the immunomodulator, contained within the packaging body. The packaging body only needs to be able to contain the disclosed preparations, food and beverages, or animal feed, and be able to print their ingredient displays, etc., and there are no particular restrictions on its shape and material. Examples of packaging body shapes include boxes and bags. Examples of raw materials for the packaging body include paper, plastic, fabric, and metal. The packaging clearly displays various information, such as the content of cereals in the preparations, food, or animal feed contained within the packaging. There are no particular limitations on the method of displaying this information on the packaging; for example, it can be displayed in the following ways: 1) printed on the outer or inner surface of the packaging; 2) printed on printing media such as printing paper inside the packaging along with the food, beverage, or animal feed; 3) a URL for the Internet is recorded on the packaging or the printing media inside it, allowing access to that URL.

[0043] As shown in the examples described later, the formulations disclosed herein can effectively reduce the expression of inflammation-inducible IL-6 and / or increase the expression of anti-inflammatory IL-10 in LPS-activated macrophage-like cells. Therefore, the formulation disclosed herein balances the production of inflammatory-inducing and anti-inflammatory cytokines in immune cells such as macrophages, adjusting and / or regulating the inflammatory state in an individual, specifically, inhibiting and / or preventing inflammation.

[0044] Examples of inflammatory conditions include diarrheal diseases, mucositis, gastroenteritis, cystitis, obesity-related inflammation, appendicitis, bacterial infections, viral infections, fungal infections, cancer-related inflammation, genitourinary diseases, bacterial vaginosis, surgical trauma, sepsis, ulcers, wound healing, kidney disease, liver disease, liver fibrosis, alcoholic hepatitis, pulmonary fibrosis, renal fibrosis, idiopathic pulmonary fibrosis and other fibrotic diseases, acne, unintended respiratory inflammatory activity, inflammation-related cancers, inflammation-related organ damage and injury (lungs, liver, kidneys, heart, gastrointestinal tract, brain, etc.), autoinflammatory diseases (TNF receptor-related periodic fever syndrome, Dubin-Johnson syndrome, Behçet's disease, etc.), allergies, rheumatoid arthritis, asthma, diabetes, systemic lupus erythematosus (SLE), atherosclerosis, multiple sclerosis, schizophrenia, hypopituitarism, neurodegenerative disorders, cardiovascular diseases, psoriasis, and metabolic diseases. By modulating immune mechanisms (including immune cells, cytokines, antibodies, etc.) through the immunomodulators disclosed herein, abnormal immune regulation can be prevented and / or inhibited.

[0045] Furthermore, in the immunomodulatory method using cereals of the present invention, it is preferable to include the following method: ingesting cereals by a person requiring immunomodulation for non-medical purposes such as cosmetics or health foods, or for purposes other than medical purposes. Additionally, the present invention includes the use of cereals as a dietary food for persons requiring immunomodulation, the use of cereals in the manufacture of preventative or ameliorative (therapeutic) agents for states (symptoms) requiring immunomodulation, and the use of dietary foods.

[0046] The formulation disclosed herein has high safety and can be taken continuously over a long period of time because its active ingredients are derived from natural plants. For example, when a specific cereal is used for immunomodulation in the case of a solid extract of cereal, the preferred daily intake for an adult (60 kg) is 1 mg to 15 g, more preferably 150 mg to 10 g. Furthermore, when used for immunomodulation, the preferred daily intake for an adult (60 kg) is 10 mg to 100 g, more preferably 1 g to 50 g, based on the amount of cereal itself (not the extract). Example

[0047] (Example 1) [Material] Using multiple cereals as subjects, the anti-inflammatory effects were confirmed based on whether they affected the expression levels of IL-6 and IL-10. The experiment was conducted using the following method.

[0048] 〔cereal〕 Regarding the cereals, the dried cereals were crushed using a mixer for 120 seconds. 25 mL of 99.5% ethanol was added to 5 g of the crushed cereals, and the mixture was allowed to stand at room temperature (20–28°C) for 24 hours. The resulting extract was filtered through defatted cotton for solid-liquid separation, and the solvent was dried using a rotary evaporator to obtain the ethanol extract. Ethanol (99.5% purity) was added to redissolve the extract at a concentration of 10 mg / mL relative to the remaining solid components, i.e., the extract, to obtain a liquid ethanol extract (hereinafter also referred to as "cereal extract"). On the other hand, as a control, ethanol (99.5% purity) was used directly.

[0049] (Preparation of RAW 264.7 cells) 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 literature, the plasmid containing the IL-6 or IL-10 reporter was introduced into the macrophage cell line (RAW 264.7 cells) along with the reference gaph reporter for transformation. Specifically, the transformation uses sequences obtained by amplifying the promoter regions of human IL-6 (nt-1000 to +121, +1 being the putative transcription start site) and the promoter regions of IL-10 (nt-1000 to +59) from the human genome. In DNA purified from human lymphocytes, the gene sequences of IL-6 and IL-10 were amplified by PCR using the primer pairs described in Reference 1. The transformation was performed as described in Reference 1, using the commercially available vector pELuc(PEST)Test(TOYOBO). Restriction enzymes HindIII, XbaI, and the hygromycin B resistance gene Hyg were used to link the promoter fragments upstream of the PEST fusion Eluc gene in pELuc-PEST-test / Hyg. Through these steps, plasmids containing IL-6 or IL-10 reporters were constructed. In addition, the gapdh-SLR reporter plasmid was constructed using the method described in reference 2 (Toxicological Sciences, 124(2)(2011)p359-369). Following the steps described in Reference 1, the plasmid constructed above was used to transform the macrophage cell line (RAW 264.7 cells) obtained from RIKEN. Specifically, as follows. RAW 264.7 cells (1.4 × 10⁻⁶) were used. 6 Cells were seeded in 35 mm diameter culture dishes containing DMEM medium and incubated under standard conditions for 24 hours. Using Hilymax transfection reagent (Dojindo Molecular Technologies), the plasmids containing IL-6 or IL-10 reporters and gapdh-SLR reporter plasmids obtained above were transformed according to the reagent's instructions.

[0050] Following transfection, RAW264.7 cells transfected with IL-6 / gapdh or IL-10 / gapdh were diluted and treated with hygromycin B for selection. A selection of cells was then treated with 1 μg / mL of LPS. LPS derived from *E. coli* O26:B6 (Sigma-Aldrich Corporation) was used as the LPS. The relative expression levels of IL-6 / gapdh and IL-10 / gapdh were then measured, and cells exhibiting 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] Using the stable cells described above, the expression of IL-6 and IL-10 was evaluated according to the method described in Reference 3. The stable cells obtained above were seeded into 96-well black plates with clear bottoms (Wallac Oy, Turku). 24 hours after seeding, the culture medium was replaced with DMEM medium containing 10% FBS, 0.1 mM D-fluorescein potassium salt, 25 mM hepes, and the cereal extract obtained above. The amount of cereal extract added to the DMEM medium was as follows: 25–75 μg / mL of solid cereal extract per 100 parts by weight of the DMEM medium containing the above components. Figures 1-6 In this example, the concentration was 75 μg / mL. After 30 minutes, LPS was added to DMEM medium. The concentration of LPS in DMEM medium was 100 ng / mL. Bioluminescence was recorded in real-time for 48 hours, with the time point of LPS addition taken as 0 hours, using a multicolor system. Specifically, bioluminescence was recorded in real-time at 37°C for 48 hours using a luminescence detector (ATTO, TWSL-1565 Kronos HT), with recordings every 10 seconds in the presence of an R60 long-pass filter (HOYA). The shift in the ratio of 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 was calculated and shown in the figure. Figures 1-6 (Solid line). In Figures 1-6 The same ratio (dashed line) is shown when ethanol is added as a control.

[0052] In the above experiment, 33 cereals were evaluated. Buckwheat, pearl millet, barnyard millet, dragon claw millet, green pea (all seeds), and wheat germ showed excellent effects in reducing IL-6 expression and / or enhancing IL-10 expression. The results for buckwheat, pearl millet, green pea, barnyard millet, dragon claw millet, and wheat germ are recorded as follows: Figures 1-6For buckwheat, pearl millet, barnyard millet, dragon claw millet, and green peas, mature seeds that are commercially available for consumption are used. Among buckwheat, pearl millet, barnyard millet, dragon claw millet, and green peas, the outer husk of the buckwheat has been removed. Additionally, commercially available wheat germ for consumption is used.

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

[0054] (Measurement of cytokine expression levels) As a cereal, wheat germ is used. THP-1 cells (human monocytic leukemia cells) (JCRB cell bank) were cultured in proliferation medium (RPMI 1640 (Sigma-Aldrich) + 10% FBS (Sigma-Aldrich)) and then inducing macrophage-like differentiation induction medium (RPMI 1640 (Sigma-Aldrich) + 10% FBS (Sigma-Aldrich) + 0.5 μg / mL phorbol 12-myristate 13-acetate (Fujifilm and Kodenpaku)) to reach a cell size of 6.3 × 10⁻⁶. 4 Cells were seeded at a rate of 0.5 mL / well in 24-well plates. After culturing at 37°C and 5% CO2 for 4 days to confirm differentiation into macrophage-like cells, the medium was replaced with the test medium (10% FBS, 100 Units / mL penicillin, 100 Units / mL streptomycin-RPMI 1640 medium) containing the various concentrations of the test substances shown in Tables 7-11. After culturing at 37°C and 5% CO2 for 1 hour, the medium was replaced with test medium containing LPS (lipopolysaccharide derived from E. coli O55:B5, Sigma-Aldrich) and the various concentrations of the test substances shown in Tables 7-11, and cultured for 24 hours. The concentration of LPS in the medium was 1 μg / mL. On the other hand, as a control, a culture medium containing 1% DMSO (with a purity of 99.5% or higher) was used instead of the test substance, and the culture was carried out in the same manner otherwise. Transfer the culture supernatant from each well to a 1.5 mL capped microtube and centrifuge at 3,000 × g for 5 minutes. Use the supernatant, free of precipitate, for ELISA analysis to determine the levels of IL-1β, IL-6, IL-8, IL-10, and TNF-α. For each cytokine, measurements were performed with n=3, and the average values ​​were calculated. The ELISA assay was performed using a Varioskan LUX (ThermoFisher) absorbance-fluorescence-luminescence plate reader / writer. The following reagents were used in the ELISA analysis to measure the levels of the aforementioned cytokines. IL-1β was measured using the IL-1β ELISA kit, Human (ProteinTech). IL-6 is supplied using the Revis (registered trademark) Human IL-6 ELISA kit (Fujifilm Wako Shibayagi). IL-8 was measured using the Revis (registered trademark) Human IL-8 ELISA kit (Fujifilm Wako Shibayagi). IL-10 was measured using the AuthentiKine (trademark) IL-10 ELISA kit, Human (ProteinTech). TNF-α was measured using the AuthentiKine (trademark) TNF-alpha ELISA kit, Human (ProteinTech). The mean and standard deviation are plotted as a bar chart. Figures 7-11 .

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

[0056] According to this disclosure, immunomodulators with excellent immunomodulatory effects can be provided. The immunomodulators of this disclosure can exert excellent immune control effects.

Claims

1. An immunomodulatory agent, wherein cereals are the active ingredient.

2. The immunomodulator according to claim 1, wherein, The cereals are selected from one or more of the following: buckwheat, pearl millet, dragon claw millet, barnyard millet, green peas, and wheat germ.

3. The immunomodulatory agent according to claim 1, wherein, The immunomodulatory agent modulates the immune response by regulating cytokine production.

4. The immunomodulator according to claim 3, wherein, The cytokines are selected from one or more of IL-6, TNF-α, IL-1β, IL-8 and IL-10.

5. The immunomodulator according to claim 1, wherein, The cereals mentioned are ethanol extracts of cereals.

6. The immunomodulatory agent according to claim 2, wherein, The extract is an ethanol extract selected from one or more of the following: buckwheat, pearl millet, dragon claw millet, barnyard millet, green pea, and wheat germ.

7. An immunomodulatory method that uses cereals.

8. The method according to claim 7, wherein, The cereals are selected from one or more of the following: buckwheat, pearl millet, dragon claw millet, barnyard millet, green peas, and wheat germ.

9. The method according to claim 7, wherein, The immune regulation method described herein involves the regulation of cytokine production.

10. The method according to claim 9, wherein, The cytokines are selected from one or more of 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 cereal is used as an ethanol extract.

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