Immunomodulatory composition

Abstract

The purpose of the present invention is to provide an immunomodulatory composition. The present invention relates to an immunomodulatory composition characterized by containing, as an active ingredient, at least one substance selected from the group consisting of Cyclo (Val-Pro), Cyclo (Gly-Pro), and salts thereof.

Description

Technical Field

[0001] The present invention relates to compositions for immunomodulation and the like. Background Art

[0002] Macrophages are one of the important factors in the immune system. Macrophages are polarized by signals from the environment, such as cytokines and bacterial stimuli. Polarized macrophages are roughly divided into two phenotypes: the pro-inflammatory M1 type and the anti-inflammatory M2 type.

[0003] M1 macrophages (M1 macrophages) are induced by interferon gamma (IFN-γ), cytokines (e.g., TNF-α, GM-CSF), bacterial components (e.g., lipopolysaccharide (LPS)), etc. On the other hand, M2 macrophages (M2 macrophages) are induced by cytokines such as interleukin 4 (IL-4) and IL-13. M1 macrophages produce inflammatory cytokines such as TNF-α, reactive oxygen species (ROS), etc., and as part of the immune system, they have the function of defending the body against diseases such as infectious diseases. On the other hand, M1 macrophages play a negative role in chronic inflammation, autoimmune diseases, etc. M2 macrophages play a role in anti-inflammatory response, immune regulation, wound healing, pathogen clearance, tissue reconstructing, etc. M1 macrophages and M2 macrophages constantly balance each other, but once this balance collapses, it will become the cause of various diseases. For example, if the polarization to M1 macrophages is maintained excessively for a long time, inflammatory diseases will be caused.

[0004] At this time, cyclic dipeptides are reported to have various physiological activities. Patent Document 1 describes cyclic dipeptides such as Cyclo (Gln-Lys) as immunomodulatory diketopiperazines.

[0005] Patent Document 1: Japanese Patent Application Laid-Open No. 57-32272 Summary of the invention

[0006] When M1 macrophages are overactivated, they produce excessive amounts of inflammatory cytokines, reactive oxygen species, etc., causing inflammatory diseases and tissue damage. Substances with immune (immune system) regulatory effects are useful, for example, by suppressing the overactivity of the immune system and restoring the immune system to the basal level.

[0007] The object of the present invention is to provide an immunomodulatory composition.

[0008] The present inventors have conducted intensive studies on the above-mentioned subject and found that cyclic dipeptides Cyclo(Val-Pro) (cyclovalylproline) and Cyclo(Gly-Pro) (cycloglycylproline) have immunomodulatory effects.

[0009] That is, the present invention is not limited thereto, but relates to the following immunomodulatory composition and the like. [1] An immunomodulatory composition comprising, as an active ingredient, at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and salts thereof. [2] The immunomodulatory composition according to [1] above, characterized in that it contains Cyclo (Val-Pro) or a salt thereof as an active ingredient and is used to regulate immunity by regulating M1 / M2 macrophage polarization. [3] The immunomodulatory composition according to [1] or [2] above, which is used to modulate immunity by promoting defense against reactive oxygen species in macrophages. [4] The immunomodulatory composition according to any one of [1] to [3] above, wherein the composition is an oral composition. [5] The immunomodulatory composition according to any one of [1] to [4] above, wherein the composition is a food or beverage or a pharmaceutical product. [6] An application characterized in that at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and salts thereof is used to prepare an immunomodulatory composition.

[0010] According to the present invention, there is provided an immunomodulatory composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 middle, Figure 1 A. Figure 1 B and Figure 1 C is a graph showing the results of examining the mRNA expression of the M1 macrophage marker in RAW264.7 cells treated with Cyclo (Val-Pro) under lipopolysaccharide (LPS) stimulation. Figure 1 A represents the relative mRNA expression of TNF-α, Figure 1 B represents the relative mRNA expression of IL-6, Figure 1 C represents the relative mRNA expression level of IL-1β. Figure 1 A. Figure 1 B and Figure 1 In C, C represents the control group, LPS represents the LPS-treated group, and CVP represents the LPS and Cyclo (Val-Pro)-treated group. Figure 2 middle, Figure 2 A. Figure 2 B and Figure 2C is a graph showing the results of examining the mRNA expression of M2 macrophage markers in RAW264.7 cells treated with Cyclo (Val-Pro) under interleukin 4 (IL-4) stimulation. Figure 2 A represents the relative mRNA expression of CD206, Figure 2 B represents the relative mRNA expression of Arg1, Figure 2 C represents the relative mRNA expression level of Mgl2. Figure 2 A. Figure 2 B and Figure 2 In C, C represents the control group, IL-4 represents the IL-4-treated group, and CVP represents the IL-4 and Cyclo (Val-Pro)-treated group. Figure 3 middle, Figure 3 A. Figure 3 B and Figure 3 C is a graph showing the results of examining the mRNA expression of antioxidant enzymes in RAW264.7 cells treated with Cyclo (Val-Pro) or Cyclo (Gly-Pro) under LPS stimulation. Figure 3 A represents the relative mRNA expression of SOD1, Figure 3 B represents the relative mRNA expression of CAT, Figure 3 C represents the relative mRNA expression level of Gpx. Figure 3 A. Figure 3 B and Figure 3 In C, C represents the control group, LPS represents the LPS-treated group, CGP represents the LPS and Cyclo (Gly-Pro)-treated group, and CVP represents the LPS and Cyclo (Val-Pro)-treated group. DETAILED DESCRIPTION

[0012] The immunomodulatory composition of the present invention contains at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts as an active ingredient. In this specification, the immunomodulatory composition of the present invention is sometimes referred to as the composition of the present invention. The composition of the present invention is used to regulate immunity (immune system). The composition of the present invention may contain one of the above-mentioned compounds as an active ingredient, or may contain two or more as an active ingredient.

[0013] Cyclo(Val-Pro) (cyclovalylproline) is a cyclic dipeptide having a structure formed by condensation of amino acids valine and proline. Cyclo(Gly-Pro) (cycloglycylproline) is a cyclic dipeptide having a structure formed by condensation of amino acids glycine and proline. In this specification, the so-called "cyclic dipeptide" refers to a compound characterized by having amino acids as constituent units and having a diketopiperazine structure generated by dehydration condensation of the amino group of the N-terminal amino acid and the carboxyl group of the C-terminal amino acid. In addition, in this specification, when the amino acid composition of the cyclic dipeptides is the same, it does not matter which order they are recorded in. For example, Cyclo (Val-Pro) and Cyclo (Pro-Val) (cycloprolyl valine) represent the same cyclic dipeptide. Cyclo (Gly-Pro) and Cyclo (Pro-Gly) (cycloprolyl glycine) represent the same cyclic dipeptide.

[0014] As a salt of Cyclo (Val-Pro) or Cyclo (Gly-Pro), there is no particular limitation as long as it is a pharmacologically permitted salt or a salt permitted in food and beverages, and it may be any of an acidic salt and an alkaline salt. As an acidic salt, for example, inorganic acid salts such as hydrochloride, sulfate, nitrate, and phosphate can be listed; organic acid salts such as acetate, citrate, maleate, malate, oxalate, lactate, succinate, fumarate, and propionate can be listed. As an alkaline salt, for example, alkali metal salts such as sodium salt and potassium salt can be listed; alkaline earth metal salts such as calcium salt and magnesium salt can be listed. The salt of the cyclic dipeptide can be simply prepared by a technician in the field by any method known in the art.

[0015] There is no particular limitation on the source and production method of Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts. Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be produced according to known methods. Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be derived from natural products, or they can be artificially synthesized, or they can be produced by enzymatic methods or microbial fermentation methods, or they can be synthesized by dehydrating and cyclizing linear dipeptides. For example, by heating protein hydrolysates such as collagen hydrolysates, a peptide heat-treated product rich in cyclic dipeptides such as Cyclo (Val-Pro) and Cyclo (Gly-Pro) can be obtained. Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be formulated in the composition using protein hydrolysates or heat-treated products thereof, or can be formulated in the composition using concentrates, dry powders or substances obtained by increasing the degree of purification of protein hydrolysates or heat-treated products thereof. The composition of the present invention, for example, contains protein hydrolysates or heat-treated products thereof, and at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can also be a part of protein hydrolysates or heat-treated products thereof. Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can also use commercially available products.

[0016] In the present invention, so-called immunomodulation refers to adjusting the immune response from an abnormal state to a normal state, and suppressing the immune response from a normal state to an abnormal state. The abnormal state of the immune response includes the state of excessive immune response. Immunomodulation includes suppressing excessive immune response. The composition of the present invention is preferably used to adjust the state of excessive immune response to a normal state, or to suppress the immune response from a normal state to an excessive state. The composition of the present invention is preferably used to suppress excessive immune response. For example, immunity can be regulated by regulating M1 / M2 macrophage polarization. In addition, promoting defense against reactive oxygen species in macrophages is also effective for immunomodulation. In one embodiment, the composition of the present invention can be preferably used to regulate immunity by regulating M1 / M2 macrophage polarization, and / or promoting defense against reactive oxygen species in macrophages.

[0017] Macrophages can be polarized (differentiated) into either M1 macrophages or M2 macrophages. M1 macrophages are characterized by the production of large amounts of inflammation-inducing cytokines such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α), reactive oxygen species (ROS), and the like. M2 macrophages can be characterized by the expression of macrophage mannose receptor type C 1 (CD206), arginase-1 (Arg1), and macrophage galactose C-type lectin 2 (Mgl2). In the present invention, the regulation of M1 / M2 macrophage polarization is the promotion of polarization toward M2 macrophages (increase of M2 macrophages) and the inhibition of polarization toward M1 macrophages (increase of M1 macrophages). By regulating the polarization of M1 / M2 macrophages, the effects of maintaining normal immune function (immune system), wound healing, etc. can be obtained.

[0018] Cyclo (Val-Pro) and its salts have the effect of promoting polarization toward M2 macrophages (increasing M2 macrophages) and inhibiting polarization toward M1 macrophages (increasing M1 macrophages). In one embodiment, the composition of the present invention preferably contains Cyclo (Val-Pro) or its salt as an active ingredient. In one embodiment, the composition of the present invention contains Cyclo (Val-Pro) or its salt as an active ingredient, and is preferably used to regulate immunity by regulating M1 / M2 macrophage polarization. In one embodiment, the composition of the present invention can be preferably used to regulate M1 / M2 macrophage polarization. M2 macrophages have anti-inflammatory effects, etc. Therefore, by promoting polarization toward M2 macrophages and inhibiting polarization toward M1 macrophages, excessive immune responses can be regulated. Promoting polarization toward M2 macrophages and inhibiting polarization toward M1 macrophages are useful, for example, for maintaining normal immune function (immune system) by inhibiting excessive immune responses, inhibiting excessive inflammation and oxidative stress.

[0019] Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts have the effect of promoting the expression of antioxidant enzymes (e.g., superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase (Gpx)) in macrophages. Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be used to promote the expression of antioxidant enzymes in macrophages. Antioxidant enzymes have the effect of removing reactive oxygen species. By promoting the expression of antioxidant enzymes, the effect of promoting the removal of reactive oxygen species can be expected.

[0020] By promoting the expression of antioxidant enzymes, the defense against reactive oxygen species in macrophages can be promoted. By promoting the defense against reactive oxygen species in macrophages, for example, excessive inflammation or excessive immune response can be suppressed. In one embodiment, the composition of the present invention is preferably used to regulate immunity by promoting the defense against reactive oxygen species in macrophages.

[0021] The composition of the present invention has an immunomodulatory effect, and therefore, for example, is useful for preventing or improving a state or disease related to an immunomodulatory disorder, an immunomodulatory disorder (for example, a state or disease caused by an immunomodulatory disorder), or a state or disease caused by an excessive immune response. As such a state or disease, inflammatory diseases can be cited. The composition of the present invention, for example, can be used for preventing or improving inflammatory diseases such as asthma, irritable bowel syndrome, arthritis, rheumatoid arthritis, eczema, allergies, etc. In one embodiment, the composition of the present invention can be used to prevent or improve an excessive state of an immune response. In this specification, prevention of a condition or disease includes: preventing onset, delaying onset, reducing the incidence rate, reducing the risk of onset, etc. Improvement of a condition or disease includes: recovering the subject from the condition or disease, alleviating the symptoms of the condition or disease, improving the symptoms of the condition or disease, delaying or preventing the development of the condition or disease, etc.

[0022] The composition of the present invention can be used for either therapeutic purposes (medical use) or non-therapeutic purposes (non-medical use). The term "non-therapeutic" refers to a concept that does not include medical actions, that is, human surgery, treatment or diagnosis. As an example, the immunomodulatory composition of the present invention can be provided in the form of a dose, but is not limited to this form. The dose can be provided directly as a composition, or as a composition containing the dose. In one form, the immunomodulatory composition of the present invention can also be referred to as an immunomodulator.

[0023] The composition of the present invention may be any of oral or non-oral. The composition of the present invention is preferably an oral composition. The composition of the present invention may be made into forms such as food and drink, medicine, quasi-drug, feed, etc., preferably food and drink or medicine. The composition of the present invention may also be added to food and drink, medicine, quasi-drug, feed, etc. for use. The form of the composition of the present invention is not particularly limited, and may be any of solid (e.g., powder, granular, tablet, etc.), liquid, paste, etc.

[0024] The composition of the present invention contains at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts, and may further contain various diluents, acidulants, antioxidants, stabilizers, preservatives, flavors, emulsifiers, pigments, flavoring agents, pH adjusters, nutritional enhancers, etc. that are permitted as additives to foods, beverages, medicines, etc.

[0025] For example, when the composition of the present invention is made into food and drink, various food and drink can be prepared by mixing ingredients (e.g., food materials, food additives used as needed, etc.) that can be used in food and drink selected from at least one of Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts. Food and drink are not particularly limited, and for example, general food and drink, health food, functional labeled food, specific health food, health supplement food, and patient food and drink can be listed. The above-mentioned health food, functional labeled food, specific health food, health supplement food, etc. can be used in various preparation forms such as liquid, fine granules, tablets, granules, powders, capsules, chewables, syrups, and liquid food.

[0026] When the composition of the present invention is made into a pharmaceutical or quasi-drug, at least one pharmacologically permitted carrier selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be prepared, and additives added as needed can be made into pharmaceuticals or quasi-drugs of various dosage forms. Such carriers, additives, etc., can be substances that can be used for pharmaceuticals or quasi-drugs. For example, one or more of excipients, binders, disintegrants, lubricants, antioxidants, colorants, etc. can be listed. As the mode of administration (intake) of pharmaceuticals or quasi-drugs, oral or non-oral (dermal, mucosal, enteral, injection, etc.) administration can be listed. When the composition of the present invention is made into a pharmaceutical or quasi-drug, it is preferably made into an oral pharmaceutical or quasi-drug. As the dosage form for oral administration, liquid, tablets, powders, fine granules, granules, sugar-coated tablets, capsules, suspensions, emulsions, chewables, etc. can be listed. Examples of dosage forms for parenteral administration include injections, drips, ointments, lotions, patches, suppositories, nasal preparations, pulmonary preparations (inhalants), etc. The pharmaceutical product may be a pharmaceutical product for non-human animals.

[0027] When the composition of the present invention is made into a feed, at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and salts thereof may be mixed into the feed. The feed also includes feed additives. Examples of the feed include feed for livestock such as cattle, pigs, chickens, sheep, and horses; feed for small animals such as rabbits, rats, and mice; and pet food for dogs, cats, and birds.

[0028] When the composition of the present invention is made into, for example, food and beverages, pharmaceuticals, quasi-drugs, feeds, etc., the production method is not particularly limited and can be produced by a general method using at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts.

[0029] In one embodiment, the composition of the present invention is preferably a liquid composition, and more preferably a beverage. The beverage may be, for example, a functional beverage. The form of the beverage is not particularly limited, and may be a container-packed beverage. The container of the container-packed beverage is not particularly limited, and any container of any form and material may be used, for example, a metal container such as an aluminum can, a steel can, a resin container such as a PET bottle, a paper container such as a paper box, a glass container such as a glass bottle, a wooden container such as a wooden barrel, or any commonly used container. By filling the beverage into such a container and sealing it, a container-packed beverage may be obtained.

[0030] The content of at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and salts thereof contained in the composition of the present invention is not particularly limited and can be appropriately set depending on the form and the like. In one embodiment, the total content of Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts in the composition of the present invention is, for example, preferably 0.0000001% by weight or more, more preferably 0.000001% by weight or more, and preferably 99% by weight or less, more preferably 90% by weight or less. In one embodiment, the total content of Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts is, for example, preferably 0.0000001 to 99% by weight, more preferably 0.000001 to 90% by weight in the composition of the present invention. Cyclo(Val-Pro), Cyclo(Gly-Pro) and salts thereof can be quantified by a known method, for example, by liquid chromatography-mass spectrometry (LC / MS).

[0031] The composition of the present invention is preferably taken orally (oral administration). The dosage (also referred to as intake) of the composition of the present invention is not particularly limited. The dosage of the composition of the present invention can be appropriately set according to the administration form, administration method, weight of the subject, etc. as long as it is an amount that can obtain an immunomodulatory effect.

[0032] In one embodiment, when the composition of the present invention is ingested or administered to a human (adult), the dosage of Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts (in terms of cyclic dipeptide) is preferably 0.001 mg or more per day, more preferably 0.01 mg or more, and even more preferably 0.1 mg or more, and preferably 50000 mg or less, more preferably 10000 mg or less, and even more preferably 1000 mg or less. In one embodiment, when the composition of the present invention is ingested or administered to a human (adult), the dosage of Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts (in terms of cyclic dipeptide) is preferably 0.001 to 50000 mg per day, more preferably 0.01 to 10000 mg, and even more preferably 0.1 to 1000 mg. The above amount is preferably taken or administered once or more per day, for example, once or more than once per day (for example, 2 to 3 times). In one embodiment, the above amount of at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts is preferably taken orally or administered. In the case of humans (adults), the above amount of at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts is preferably taken or administered per 60 kg of body weight per day. In one embodiment, the composition of the present invention may be an oral composition for humans to take or administer to humans at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts per 60 kg of body weight per day. In this specification, the amount of cyclic dipeptide conversion or similar expressions, when it is a cyclic dipeptide (Cyclo (Val-Pro) or Cyclo (Gly-Pro)), refers to the amount of the cyclic dipeptide. When the cyclic dipeptide is in the form of a salt, it refers to the value obtained by multiplying the molar value of the salt by the molecular weight of the corresponding cyclic dipeptide.

[0033] The composition of the present invention is preferably taken or administered continuously. A higher effect can be expected by continuously taking or administering at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts. In one embodiment, the composition of the present invention is preferably taken or administered continuously for more than 1 week, more preferably taken or administered continuously for more than 4 weeks, and further preferably taken or administered continuously for more than 8 weeks. Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be taken as food and drink, and from the perspective of safety, it is believed that there are few problems even if they are taken for a long time.

[0034] The subject to which the composition of the present invention is ingested or administered (may also be referred to as the administration subject) is not particularly limited, but is preferably a human or a non-human mammal, and more preferably a human. In one embodiment, as the subject of administration of the composition of the present invention, it is preferred that the subject needs or desires immunoregulation (regulation of the immune system), the subject needs or desires the prevention or improvement of immunoregulatory disorders, the subject needs or desires the prevention or improvement of states or diseases related to immunoregulatory disorders, etc. In one embodiment, as the subject of administration in the present invention, patients with inflammatory diseases such as asthma, irritable bowel syndrome, arthritis, rheumatoid arthritis, eczema, allergies, etc. are preferred. As the preferred subject of administration in the present invention, the subject of prevention or improvement of excessive immune response can also be listed. In one embodiment, as the subject of administration in the present invention, middle-aged and elderly people can be listed. Middle-aged and elderly people include elderly people. Middle-aged and elderly people, for example, can be humans over 40 years old. In one embodiment, among middle-aged and elderly people, elderly people are preferred as the subject. Elderly people can be, for example, humans over 60 years old or over 65 years old. In one embodiment, the subject of administration of the composition of the present invention can also be a healthy person. For example, it can also be used for healthy people for the purpose of preventing immunoregulatory disorders, preventing states or diseases related to immunoregulatory disorders, preventing excessive immune responses, etc.

[0035] The composition of the present invention may also be accompanied by a label indicating the function exerted by regulating the immune system. Such a label is also referred to as a functional label. The above label is not particularly limited. Examples of such labels include "improving immunity", "strengthening immune function", "preventing inflammatory diseases", "preventing allergies", "promoting wound healing", "promoting tissue repair or regeneration", "improving pathogen clearance" and labels or functional labels that can be considered equivalent thereto. In one embodiment of the present invention, the composition of the present invention is preferably a food and beverage product with 1 or more than 2 above-mentioned indications. In addition, the above-mentioned indications may also be indications of the meaning of using the above-mentioned composition for obtaining the above-mentioned functions. The above-mentioned indications may be attached to the composition itself, or to a container or a package of the composition.

[0036] The present invention also includes the following methods and applications. An immunomodulatory method, characterized in that at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and salts thereof is administered. An application characterized in that at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and salts thereof is used for immunomodulation. The above method may be a therapeutic method or a non-therapeutic method. The above application may be a therapeutic application or a non-therapeutic application.

[0037] At least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and salts thereof can be used to promote defense against reactive oxygen species in macrophages. At least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and salts thereof can be used to regulate immunity by promoting defense against reactive oxygen species in macrophages.

[0038] In one embodiment, in the above method, Cyclo (Val-Pro) or a salt thereof is preferably administered. In the above application, Cyclo (Val-Pro) or a salt thereof is preferably used. In one embodiment, Cyclo (Val-Pro) or a salt thereof can be used to regulate M1 / M2 macrophage polarization. Cyclo (Val-Pro) or a salt thereof can be preferably used to regulate immunity by regulating M1 / M2 macrophage polarization. Cyclo (Val-Pro) or a salt thereof can be preferably used to promote polarization toward M2 macrophages and inhibit polarization toward M1 macrophages.

[0039] In the above methods and applications, it is preferred that at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts is ingested or administered to the subject once or more than once a day, for example, once to multiple times (for example, 2 to 3 times) a day. The above application is preferably applied to humans or non-human mammals, more preferably humans. In one embodiment, at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be preferably used to prevent or improve immunoregulatory disorders by regulating the immune system, and to prevent or improve states or diseases associated with immunoregulatory disorders. The present invention also includes a method for preventing or improving immunoregulatory disorders or states or diseases associated with immunoregulatory disorders by administering at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts. In one embodiment, at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be preferably used to prevent or improve excessive states of immune response. In one embodiment, at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and salts thereof is preferably used for preventing or ameliorating inflammatory diseases (eg, asthma, irritable bowel syndrome, arthritis, rheumatoid arthritis, eczema, allergies, etc.).

[0040] In the above methods and applications, at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be used in an amount (also called an effective amount) that can obtain an immunomodulatory effect. In one embodiment, an effective amount of Cyclo (Val-Pro) or its salt is preferably used. In one embodiment, it is preferred to use an amount of Cyclo (Val-Pro) or its salt that can promote polarization to M2 macrophages and inhibit polarization to M1 macrophages. The preferred dosage, method of administration, and subject of administration of Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts are the same as those of the above-mentioned composition of the present invention. At least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be directly ingested or administered, or can be ingested or administered as a composition containing it. For example, the composition of the present invention can also be ingested or administered.

[0041] At least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be used to manufacture food, medicine, quasi-drug, feed, etc. for immunomodulation. In one embodiment, the present invention also includes the use of at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts in the manufacture of immunomodulatory compositions. At least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and their salts can be preferably used for the manufacture of compositions for the prevention or improvement of immunomodulatory disorders or for the prevention or improvement of conditions or diseases related to immunomodulatory disorders.

[0042] In this specification, the numerical range represented by the lower limit and the upper limit, that is, "lower limit - upper limit" includes these lower limit and upper limit. For example, the range represented by "1 to 2" means 1 or more and 2 or less, and includes 1 and 2. In this specification, the upper limit and the lower limit can be set as a range based on any combination. Example

[0043] Hereinafter, the present invention will be described in further detail by way of examples, but the scope of the present invention is not limited thereto.

[0044] <Example 1> In the examples, the following cyclic dipeptides were used. Cyclo (Val-Pro) (Bachem, USA) Cyclo(Gly-Pro)(Bachem, USA)

[0045] Macrophages were stimulated with lipopolysaccharide (LPS) or interleukin 4 (IL-4) (both Sigma-Aldrich, USA) to investigate the effect of Cyclo (Val-Pro) on macrophage polarization. In addition, macrophages were stimulated with LPS to investigate the effects of Cyclo (Val-Pro) and Cyclo (Gly-Pro) on the expression of antioxidant enzymes in macrophages.

[0046] (Cell Culture and Treatment) Mouse macrophage Raw264.7 cells (ChuanQiu Biotechnology Co., Ltd., Shanghai, China) were cultured in DMEM containing 10% (vol / vol) fetal bovine serum (HAKATA, ChuanQiu Biotechnology Co., Ltd., Shanghai, China) and supplemented with 1% penicillin-streptomycin solution (Gibco, USA) in a humidified environment (5% CO2, 95% air, 37°C). Then, the cells were cultured under LPS or IL-4 stimulation as follows.

[0047] (Cultivation under LPS stimulation) RAW264.7 cells were cultured in a medium supplemented with a cyclic dipeptide (Cyclo(Val-Pro) or Cyclo(Gly-Pro)) and LPS (final concentration of LPS in the medium: 1 μg / mL) for 16 hours. After culture, the amount of mRNA was measured by the method described below. Cells cultured in a medium without the addition of cyclic dipeptide and LPS were used as a control group. In addition, cells cultured in a medium without the addition of cyclic dipeptide but with the addition of LPS (final concentration in the medium: 1 μg / mL) were used as an LPS-treated group.

[0048] (Cultivation under IL-4 stimulation) RAW264.7 cells were cultured in a medium supplemented with Cyclo (Val-Pro) and IL-4 (final concentration of IL-4 in the medium: 10 ng / mL) for 16 hours. After culture, the amount of mRNA was measured by the method described below. Cells cultured in a medium without Cyclo (Val-Pro) and IL-4 were used as a control group. In addition, cells cultured in a medium without Cyclo (Val-Pro) but with IL-4 (final concentration in the medium: 10 ng / mL) were used as an IL-4 treatment group.

[0049] During the above culture, Cyclo (Val-Pro) was added to the medium to a final concentration of 1.68 μg / mL, and Cyclo (Gly-Pro) was added to the medium to a final concentration of 3.525 μg / mL. All experiments were performed in replicates of 6 times.

[0050] (Quantitative Real-Time Polymerase Chain Reaction) Total RNA was extracted from cells using Biozol reagent (Biomiga Biochemicals, USA), RNA concentration was assessed using Biotek ND5000 instrument (BioTeke, Beijing, China), and cDNA was synthesized using high-capacity cDNA reverse transcriptase kit (Tsingke Co., Ltd., Beijing, China). As previously described, quantitative real-time PCR was performed using SYBR Green Real-time PCR Master Mix (Tsingke Co., Ltd., Beijing, China) with CFX Connect Real-Time PCR System (Bio-rad, USA) (Ma et al., "Bifidobacterium animalis subsp. lactis lkm512 Attenuates Obesity-Associated Inflammation and Insulin Resistance Through the Modification of Gut Microbiota in High-Fat Diet-Induced Obese Mice.", Mol Nutr Food Res, 2022, 66(3), e2100639.doi: 10.1002 / mnfr.202100639). The transcript of β-actin was used as a housekeeping gene for data normalization.

[0051] The mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) as markers of M1 macrophages were investigated. The mRNA expression levels of macrophage mannose receptor C-type 1 (CD206), arginase-1 (Arg1), and macrophage galactose C-type lectin 2 (Mgl2) as markers of M2 macrophages were investigated. The mRNA expression levels of superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase (Gpx) as antioxidant enzymes were investigated. The sequences of the primers are shown in Table 1.

[0052] [Table 1]

[0053] (Statistical Analysis) All data are presented as mean ± SEM. Comparisons among different groups were analyzed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test. P values ​​< 0.05 or 0.01 were considered statistically significant.

[0054] (result) Polarization to M1 macrophages is induced by stimulating macrophages with LPS. Polarization to M2 macrophages is induced by stimulating macrophages with IL-4. Figure 1 and Figure 2 2 shows the results of investigating the effect of Cyclo (Val-Pro) on macrophage polarization in vitro. Figure 3 The results of the investigation of the effects of Cyclo (Val-Pro) and Cyclo (Gly-Pro) on the expression of antioxidant enzymes in macrophages are shown in Figure 1. The mRNA expression levels of the marker genes are expressed as relative amounts to the mRNA expression level of β-actin (relative mRNA expression levels).

[0055] Figure 1 A. Figure 1 B and Figure 1 C is a graph showing the results of examining the mRNA expression of the M1 macrophage marker in RAW264.7 cells treated with Cyclo (Val-Pro) after 16 hours of LPS stimulation. Figure 1 A represents the relative mRNA expression of TNF-α, Figure 1 B represents the relative mRNA expression of IL-6, Figure 1 C represents the relative mRNA expression level of IL-1β. Figure 1 A. Figure 1 B and Figure 1 In C, C represents the control group, LPS represents the LPS-treated group, and CVP represents the LPS and Cyclo (Val-Pro)-treated group.

[0056] Figure 2 A. Figure 2 B and Figure 2 C is a graph showing the results of examining the mRNA expression of the M2 macrophage marker in RAW264.7 cells treated with Cyclo (Val-Pro) after 16 hours of interleukin-4 (IL-4) stimulation. Figure 2A represents the relative mRNA expression of CD206, Figure 2 B represents the relative mRNA expression of Arg1, Figure 2 C represents the relative mRNA expression level of Mgl2. Figure 2 A. Figure 2 B and Figure 2 In C, C represents the control group, IL-4 represents the IL-4-treated group, and CVP represents the IL-4 and Cyclo (Val-Pro)-treated group.

[0057] Figure 3 A. Figure 3 B and Figure 3 C is a graph showing the results of examining the mRNA expression of antioxidant enzymes in RAW264.7 cells treated with Cyclo (Val-Pro) or Cyclo (Gly-Pro) under 16 hours of LPS stimulation. Figure 3 A represents the relative mRNA expression of SOD1, Figure 3 B represents the relative mRNA expression of CAT, Figure 3 C represents the relative mRNA expression level of Gpx. Figure 3 A. Figure 3 B and Figure 3 In C, C represents the control group, LPS represents the LPS-treated group, CGP represents the LPS and Cyclo (Gly-Pro)-treated group, and CVP represents the LPS and Cyclo (Val-Pro)-treated group.

[0058] exist Figure 1 A. Figure 1 B and Figure 1 C. Figure 2 A. Figure 2 B and Figure 2 C. Figure 3 A. Figure 3 B and Figure 3 In C, the data are expressed as mean ± SEM. * and ** indicate significant differences from the control group (*: P < 0.05, **: P < 0.01, vs. control group). Figure 1 A. Figure 1 B and Figure 1 C and Figure 3 A. Figure 3 B and Figure 3 In C, # and ## indicate significant differences compared with the LPS-treated group (#: P < 0.05, ##: P < 0.01, vs. LPS-treated group). Figure 2 A. Figure 2 B and Figure 2In C, # and ## indicate significant differences compared with the IL-4-treated group (#: P < 0.05, ##: P < 0.01, vs IL-4-treated group).

[0059] To evaluate the intervention of cyclic dipeptide on macrophage polarization, cells were co-treated with LPS and Cyclo(Val-Pro) or IL-4 and Cyclo(Val-Pro). In LPS-treated cells, the mRNA expression of M1 inflammatory macrophage markers (TNF-α, IL-6, IL-1β) was significantly reduced by Cyclo(Val-Pro) ( Figure 1 A. Figure 1 B and Figure 1 C). On the other hand, in IL-4 treated cells, the mRNA expression of M2 anti-inflammatory macrophage markers (Arg1, CD206 and Mgl2) was significantly increased by Cyclo (Val-Pro) ( Figure 2 A. Figure 2 B and Figure 2 C) Cyclo (Val-Pro) promotes polarization toward M2 macrophages (increase in M2 macrophages) and inhibits polarization toward M1 macrophages (increase in M1 macrophages).

[0060] Depend on Figure 3 A. Figure 3 B and Figure 3 C It can be seen that Cyclo (Val-Pro) and Cyclo (Gly-Pro) increase the expression of antioxidant enzymes (SOD1, CAT, Gpx) in M1 macrophages. When the expression of antioxidant enzymes in M1 macrophages increases, the defense against reactive oxygen species in macrophages is promoted. Thus, excessive immune response can be suppressed. Cyclo (Val-Pro) treatment and Cyclo (Gly-Pro) treatment alleviate the inflammation of RAW264.7 cells induced by LPS.

[0061] The above results show that Cyclo (Val-Pro) and Cyclo (Gly-Pro) exert strong anti-inflammatory and anti-oxidative effects in macrophages during immune response. These cyclic dipeptides and their salts have immunomodulatory effects.

Claims

1. An immunomodulatory composition, characterized in that The invention contains at least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and salts thereof as an active ingredient.

2. The immunomodulatory composition according to claim 1, characterized in that The invention contains the Cyclo (Val-Pro) or a salt thereof as an effective ingredient and is used for regulating immunity by regulating M1 / M2 macrophage polarization.

3. The immunomodulatory composition according to claim 1 or 2, characterized in that: Used to modulate immunity by promoting defense against reactive oxygen species in macrophages.

4. The immunomodulatory composition according to claim 1 or 2, characterized in that: The composition is an oral composition.

5. The immunomodulatory composition according to claim 1 or 2, characterized in that: The composition is a food or beverage or a medicine.

6. An application, characterized in that: At least one selected from Cyclo (Val-Pro), Cyclo (Gly-Pro) and salts thereof is used for producing an immunomodulatory composition.

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