Neutrophil activator

Abstract

The features of the present invention for providing a novel neutrophil activator are as follows. 1. An IL-8 production accelerator containing at least one selected from GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 22:0], GlcCer[d18:2(4E,8Z) / 24:0], and GlcCer[d18:2(4E,8Z) / 26:0] as an active ingredient. 2. An IL-8 production accelerator containing GlcCer[d18:2(4E,8E) / 24:0] as an active ingredient. 3. An IL-8 production accelerator containing at least one selected from GlcCer[t18:1(8Z) / 24:0] and GlcCer[t18:1(8Z) / 26:0] as an active ingredient. 4. An IL-8 production accelerator containing GlcCer[d18:1(4E) / 20:0] as an active ingredient. 5. An IL-8 production accelerator containing at least one selected from Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 24:0), Cer(t18:0 / 25:0), and Cer(t18:0 / 26:0) as an active ingredient. 6. An IL-8 production accelerator containing at least one selected from Cer[t18:1(8Z) / 24:0] and Cer[d18:2(4E,8Z) / 20:0] as an active ingredient. 7. An IL-8 production accelerator containing at least one selected from Oryza Ceramide A, Oryza Ceramide B, and Oryza Ceramide C as an active ingredient.

Description

Neutrophil activators

[0001] This invention relates to a neutrophil activator. This invention is widely used in pharmaceuticals, health foods, food products, and the like.

[0002] When pathogens or bacteria invade the body through cuts or mucous membranes, dendritic cells, which are distributed throughout the body, recognize and phagocytose the antigens, and antigen presentation is performed on white blood cells, lymphocytes, and other cells. Subsequently, neutrophils, macrophages, and natural killer cells phagocytose and kill the invading pathogens (innate immunity). When evaluating the IL-8 production-promoting effect, these cells are the target of the tests.

[0003] There are two main defense mechanisms against foreign substances: innate immunity and adaptive immunity. In innate immune responses, for example, immune cells such as neutrophils, dendritic cells, and macrophages are known to produce cytokines in response to innate immune-activating substances derived from bacteria and viruses, which then triggers an immune response. The innate immune system is a common infection defense mechanism shared by all living organisms, and because it is generally nonspecific, it is characterized by its rapid response and its effectiveness against a wide range of infectious agents.

[0004] Neutrophils make up about 50% of white blood cells and primarily play a role in preventing infection by phagocytosing (engulfing) and breaking down pathogens such as bacteria and fungi, as well as foreign substances that invade the body, thereby killing them. They are also characterized by their large numbers and the fastest migration to antigens. Interleukin-8 (IL-8) is an inflammatory cytokine produced by vascular endothelial cells, airway smooth muscle cells, and neutrophils, and is known as a neutrophil chemotactic, playing an important role in the immune response (see Figure 11).

[0005] The bactericidal functions of neutrophils include degranulation, release of reactive oxygen species, phagocytosis, and formation of neutrophil extracellular traps (NETs). NET formation refers to the spiderweb-like substance released from neutrophils to capture pathogens such as viruses and bacteria. Its components include potent antimicrobial substances such as DNA, myeloperoxidase, and neutrophil elastase, making it a tool that also possesses bactericidal properties (Non-Patent Literature 1). NET release is triggered by the activation of Toll-like receptors (TLRs) and macrophage-induced C-type lectin receptors (Mincle), stimulating neutrophil elastase release and downstream events of the MEK / ERK pathway (Non-Patent Literature 2) (see Figure 12).

[0006] Shoichi Iwamuro: Antibacterial action of extracellular histones. Comparative Endocrinology 2016; 42; 112-113. Sharma A. et al., Glycolipid Metabolite β-Glucosylceramide Is a Neutrophil Extracellular Trap-Inducing Ligand of Mincle Released during Bacterial Infection and Inflammation. J. Immunol. 15;209(2):391-400 (2022).

[0007] Against this backdrop, the inventors conducted extensive research on ceramides contained in rice bran and discovered that certain glucosylceramides, free ceramides, and acylated ceramides have excellent neutrophil-activating effects (see Table 2 below). For details, see GlcCer[d18:2(4E,8Z)], GlcCer[d18:2(4E,8E) / 24:0] with fatty acid chain lengths of 18, 20, 22, 24, 26; GlcCer[t18:1(8Z)], GlcCer[d18:1(4E) / 20:0] with fatty acid chain lengths of 20, 22, 24, 26; Cer(t18:0), Cer[t18:1(8Z) / 24:0], Cer[d18:2(4E,8Z) / 20:0] with fatty acid chain lengths of 22, 23, 24, 25, 26; and oryzaceramide A, B. We discovered that C has an IL-8 production-promoting effect, a neutrophil extracellular trap production-promoting effect, and a neutrophil cell migration-promoting effect, thereby activating neutrophils and enhancing innate immunity, thus completing the present invention. In other words, the present invention aims to provide a novel neutrophil activator that activates innate immunity.

[0008] The features of the present invention for solving the above problems are as follows: 1. An IL-8 production promoter comprising at least one selected from GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 22:0], GlcCer[d18:2(4E,8Z) / 24:0], and GlcCer[d18:2(4E,8Z) / 26:0] as an active ingredient. 2. An IL-8 production promoter comprising GlcCer[d18:2(4E,8E) / 24:0] as an active ingredient. 3. 1. An IL-8 production promoter containing at least one selected from GlcCer[t18:1(8Z) / 24:0] and GlcCer[t18:1(8Z) / 26:0] as an active ingredient. 4. An IL-8 production promoter containing GlcCer[d18:1(4E) / 20:0] as an active ingredient. 5. An IL-8 production promoter containing at least one selected from Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 24:0), Cer(t18:0 / 25:0), and Cer(t18:0 / 26:0) as an active ingredient. 6. An IL-8 production promoter comprising at least one selected from Cer[t18:1(8Z) / 24:0] and Cer[d18:2(4E,8Z) / 20:0] as an active ingredient. 7. An IL-8 production promoter comprising at least one selected from oryzaceramide A, oryzaceramide B, and oryzaceramide C, represented by the following chemical formulas, as an active ingredient. 8. A neutrophil extracellular trap production promoter containing at least one selected from GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 24:0], and GlcCer[d18:2(4E,8Z) / 26:0] as an active ingredient. 9. A neutrophil extracellular trap production promoter containing GlcCer[d18:2(4E,8E) / 24:0] as an active ingredient. 10. A neutrophil extracellular trap production promoter containing at least one selected from Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), and Cer(t18:0 / 26:0) as an active ingredient. 11. A neutrophil extracellular trap production promoter comprising at least one selected from oryzaceramide A, oryzaceramide B, and oryzaceramide C, represented by the following chemical formulas, as an active ingredient. 12. A neutrophil cell migration enhancer containing GlcCer[d18:2(4E,8Z) / 18:0] as the active ingredient. 13. A neutrophil cell migration enhancer containing at least one selected from GlcCer[t18:1(8Z) / 20:0] and GlcCer[t18:1(8Z) / 22:0] as the active ingredient. 14. A neutrophil activator containing any of the agents from 1. to 13. above as the active ingredient. 15. A method for activating the human immune system, comprising: (a) GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 22:0], GlcCer[d18:2(4E,8Z) / 24:0], GlcCer[d18:2(4E,8Z) / 26:0], GlcCer[d18:2(4E,8E) / 24:0], GlcCer[t18:1(8Z) / 24:0], GlcCer[t18:1(8Z) / 26:0] from rice bran extract. A step of isolating at least one selected from the group consisting of GlcCer[d18:1(4E) / 20:0], Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 24:0), Cer(t18:0 / 25:0), Cer(t18:0 / 26:0), Cer[t18:1(8Z) / 24:0], Cer[d18:2(4E,8Z) / 20:0] and oryzaceramide A, oryzaceramide B, and oryzaceramide C represented by the following chemical formulas, (b) A method comprising the step of administering an effective amount of the compound isolated in step a to a human being by oral or parenteral administration to promote the production of IL-8 in the human being. 16. A method for activating the human immune system, comprising the step of (a) isolating at least one selected from the group consisting of GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 24:0], GlcCer[d18:2(4E,8Z) / 26:0], GlcCer[d18:2(4E,8E) / 24:0], Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 26:0), and oryzaceramide A, oryzaceramide B, and oryzaceramide C represented by the following chemical formulas from an extract of rice bran, (b) A method comprising the step of administering an effective amount of the compound isolated in step a to a human being by oral or parenteral administration to promote the production of extracellular traps in human neutrophils. 17. A method for activating the human immune system, comprising the step of (a) isolating at least one compound selected from the group consisting of GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[t18:1(8Z) / 20:0], and GlcCer[t18:1(8Z) / 22:0] from an extract of rice bran, and (b) a method comprising the step of administering an effective amount of the compound isolated in step a to a human being by oral or parenteral administration to promote the cell migration ability in human neutrophils.

[0009] This figure shows the proton-carbon correlation (arrows) and proton-proton correlation (thick line) of oryzaceramide A. This figure shows the proton-carbon correlation (arrows) and proton-proton correlation (thick line) of oryzaceramide B. This figure shows the proton-carbon correlation (arrows) and proton-proton correlation (thick line) of oryzaceramide C. This is an explanatory diagram showing the method for recovering and purifying mouse peritoneal neutrophils in the test example of this embodiment. This is a graph showing the evaluation results of the IL-8 production promoting effect of rice-derived GlcCer in mouse peritoneal neutrophils. This is a graph showing the evaluation results of the IL-8 production promoting effect of oryzaceramide AC and Cer in mouse peritoneal neutrophils. This is a graph showing the NETs production promoting effect of rice-derived GlcCer in mouse peritoneal neutrophils. This is a graph showing the NETs production promoting effect of oryzaceramide AC and Cer in mouse peritoneal neutrophils. This is an explanatory diagram about the chemotaxis assay. This graph shows the effect of rice-derived GlcCer on the cell migration ability of mouse peritoneal neutrophils. This diagram illustrates the action of interleukin (IL)-8 in neutrophils. This diagram illustrates the action of neutrophil extracellular traps (NETs) in neutrophils.

[0010] The embodiments of the present invention will be described in detail below. The active ingredients for neutrophil activation are the 21 types of ceramides described below (see Table 2 below). [IL-8 production promoting effect: Glucosylceramide] The IL-8 production promoting agent of the present invention is characterized by having at least one selected from GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 22:0], GlcCer[d18:2(4E,8Z) / 24:0], and GlcCer[d18:2(4E,8Z) / 26:0] as an active ingredient.

[0011] Here, GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 22:0], GlcCer[d18:2(4E,8Z) / 24:0], GlcCer[d18:2(4E,8Z) / 26:0] are glucosylceramides represented by the following chemical formula.

[0012] Further, the IL-8 production promoter of the present invention is characterized by containing GlcCer[d18:2(4E,8E) / 24:0] as an active ingredient.

[0013] GlcCer[d18:2(4E,8E) / 24:0] is a glucosylceramide represented by the following chemical formula.

[0014] Also, the IL-8 production promoter of the present invention is characterized by containing at least one selected from GlcCer[t18:1(8Z) / 24:0] and GlcCer[t18:1(8Z) / 26:0] as an active ingredient.

[0015] GlcCer[t18:1(8Z) / 24:0] and GlcCer[t18:1(8Z) / 26:0] are glucosylceramides represented by the following chemical formula.

[0016] Furthermore, the IL-8 production promoter of the present invention is characterized by containing GlcCer[d18:1(4E) / 20:0] as an active ingredient.

[0017] GlcCer[d18:1(4E) / 20:0] is a glucosylceramide represented by the following chemical formula.

[0018] The above-mentioned glucosylceramide can be obtained by isolating it from sphingoglycolipids derived from rice, but is not limited to this method. As the sphingoglycolipids derived from rice, for example, "Oryza Ceramide®" manufactured by Oryza Oil & Fat Chemical Co., Ltd. can be used, but is not limited to this. Furthermore, while it may be used if it is isolated from sphingolipids derived from rice, a mixture of rice-derived extracts such as "Oryza Ceramide®" manufactured by Oryza Oil & Fat Chemical Co., Ltd., and concentrated sphingolipids derived from rice may also be used. Specifically, it can be obtained by the method of the examples described herein, but is not limited to this method.

[0019] [IL-8 production promoting effect: Free ceramide] Furthermore, the IL-8 production promoting agent of the present invention is characterized by containing at least one selected from Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 24:0), Cer(t18:0 / 25:0), and Cer(t18:0 / 26:0) as an active ingredient.

[0020] The above Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 24:0), Cer(t18:0 / 25:0), and Cer(t18:0 / 26:0) are free ceramides represented by the following chemical formulas.

[0021] Furthermore, the IL-8 production promoter of the present invention is characterized by containing at least one selected from Cer[t18:1(8Z) / 24:0] and Cer[d18:2(4E,8Z) / 20:0] as an active ingredient.

[0022] The above Cer[t18:1(8Z) / 24:0] and Cer[d18:2(4E,8Z) / 20:0] are free ceramides represented by the following chemical formulas.

[0023] The method for producing the free ceramide described above is not particularly limited, but it can be obtained by extracting rice bran with a polar solvent to obtain a rice bran extract, and then isolating the free ceramide from the extract.

[0024] The "rice bran extract" mentioned above is not particularly limited as long as it is produced in the process of manufacturing rice bran oil. For example, rice bran extract can be obtained by extracting rice bran with an organic solvent and then precipitating the extract, but the method is not limited to this. In addition, the fine bran extract mentioned above may be obtained by extracting a by-product produced from rice bran oil during the manufacturing process.

[0025] Here, the method for isolating free ceramide from the above rice bran extract is not particularly limited, but for example, it can be isolated by chromatography using activated clay, activated carbon, silica gel, alumina, diatomaceous earth, synthetic adsorbents, ion exchange resins, etc., or by removing components other than free ceramide by adsorption, decomposition, precipitation, filtration, dissolution, distillation, etc.

[0026] In order to separate free ceramide from components other than free ceramide, or from a fraction containing free ceramide to a fraction not containing free ceramide (fraction: separating a substance containing a mixture of multiple components into its constituent components), it is preferable to use activated clay, activated carbon, silica gel, alumina, diatomaceous earth, synthetic adsorbents, ion exchange resins, etc., as used in the purification method described above, in appropriate combination. At this time, it is preferable to prepare a "coating" by adsorbing the extract onto an adsorbent in advance.

[0027] Specifically, the extract can be obtained by adsorbing it onto silica gel to create a "coating," and then fractionating this coating using medium-pressure flash chromatography.

[0028] [IL-8 production promoting effect: acylated ceramide] Furthermore, the IL-8 production promoting agent of the present invention contains at least one selected from oryzaceramide A, oryzaceramide B, and oryzaceramide C as an active ingredient.

[0029] In this application, oryzaceramide A, oryzaceramide B, and oryzaceramide C refer to acylated ceramides represented by the following chemical formulas, respectively.

[0030] The method for obtaining oryzaceramide A, B, and C is not particularly limited, but a preferred method is to isolate oryzaceramide A, B, and C from a rice bran extract, for example, because it contains oryzaceramide A, B, and C in high concentrations. The "rice bran extract" is not particularly limited as long as it is produced in the process of manufacturing rice bran oil. For example, a rice bran extract can be obtained by extracting rice bran with an organic solvent and then precipitating the extract, but the method is not limited to this. Alternatively, the above rice bran extract may be a by-product produced in the manufacturing process of rice bran oil.

[0031] Here, the method for isolating oryzaceramide A, B, and C from the extract is not particularly limited, but for example, they can be isolated by chromatography using activated clay, activated carbon, silica gel, alumina, diatomaceous earth, synthetic adsorbents, ion exchange resins, etc., or by removing components other than oryzaceramide A, B, and C by adsorption, decomposition, precipitation, filtration, dissolution, distillation, etc. After that, purification may be carried out as appropriate by HPLC or the like. Specifically, it can be manufactured according to the examples in this specification.

[0032] [Neutrophil extracellular trap production promoting effect (NETs production promoting effect)] Furthermore, the neutrophil extracellular trap production promoting agent of the present invention contains at least one selected from GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 24:0], GlcCer[d18:2(4E,8Z) / 26:0], GlcCer[d18:2(4E,8E) / 24:0], Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 26:0), oryzaceramide A, oryzaceramide B, and oryzaceramide C as an active ingredient.

[0033] [Cell migration ability promotion (cell chemotaxis) effect] Furthermore, the cell migration ability promoting agent for neutrophils of the present invention comprises at least one selected from GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[t18:1(8Z) / 20:0], and GlcCer[t18:1(8Z) / 22:0] as an active ingredient.

[0034] GlcCer[t18:1(8Z) / 20:0] and GlcCer[t18:1(8Z) / 22:0] are glucosylceramides represented by the following chemical formulas.

[0035] [Examples of Application] The IL-8 production promoter, neutrophil extracellular trap production promoter, and neutrophil cell migration ability promoter of the present invention (hereinafter simply referred to as "the neutrophil activator of the present invention") can be used as ingredients in various foods and beverages. Examples of foods and beverages include confectionery (gum, candy, caramel, chocolate, cookies, snacks, jelly, gummies, tablets, etc.), noodles (soba, udon, ramen, etc.), dairy products (milk, ice cream, yogurt, etc.), seasonings (miso, soy sauce, etc.), soups, beverages (juice, coffee, tea, carbonated drinks, sports drinks, etc.), and other general foods, as well as health foods (tablets, capsules, etc.) and nutritional supplements (nutritional drinks, etc.). The neutrophil activator of the present invention may be appropriately incorporated into these foods and beverages.

[0036] These foods and beverages can be formulated with various ingredients depending on their type. For example, they can contain food ingredients such as glucose, fructose, sucrose, maltose, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, gum arabic, carrageenan, casein, gelatin, pectin, agar, B vitamins, nicotinamide, calcium pantothenate, amino acids, calcium salts, colorants, flavorings, and preservatives.

[0037] Specifically, the neutrophil activator of the present invention can be spray-dried or freeze-dried together with powdered cellulose, and this can be easily incorporated into food and beverages (such as instant foods) by processing it into a powder, granules, tablets, or solution. Furthermore, the agent of the present invention can be dissolved in, for example, oil, fat, ethanol, glycerin, or a mixture thereof to form a liquid, which can then be added to beverages or solid foods. If necessary, it can also be mixed with a binder such as gum arabic or dextrin to form a powder or granules, which can then be added to beverages or solid foods.

[0038] When applying the neutrophil activator of the present invention to food and beverages, the amount added is preferably such that the total content of the active ingredient relative to the food and beverage is 1 to 20 wt%, since the main purpose is disease prevention and health maintenance.

[0039] The neutrophil activator of the present invention may be used as a material for pharmaceuticals (including pharmaceuticals and quasi-drugs). It can be manufactured by appropriately blending the agent of the present invention with raw materials for pharmaceutical formulations. Examples of formulation raw materials that can be incorporated into the agent of the present invention include excipients (glucose, lactose, sucrose, sodium chloride, starch, calcium carbonate, kaolin, crystalline cellulose, cocoa butter, hydrogenated vegetable oil, kaolin, talc, etc.), binders (distilled water, physiological saline, ethanol water, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.), disintegrants (sodium alginate, agar, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, starch, lactose, acacia powder, gelatin, ethanol, etc.), disintegration inhibitors (sucrose, stearin, cocoa butter, hydrogenated oil, etc.), absorption enhancers (quaternary ammonium base, sodium lauryl sulfate, etc.), adsorbents (glycerin, starch, lactose, kaolin, bentonite, silicic acid, etc.), and lubricants (purified talc, stearate, polyethylene glycol, etc.).

[0040] The neutrophil activator of the present invention can generally be administered orally in the form of tablets, pills, soft or hard capsules, granules, powders, granules, or liquids, but it may also be administered parenterally. When administered parenterally, it can be administered in solution form or with the addition of dispersants, suspensions, stabilizers, etc., by local tissue administration, intradermal, subcutaneous, intramuscular, or intravenous injection. It may also be administered in the form of suppositories or other preparations.

[0041] The dosage may vary depending on the method of administration, the patient's condition, and the patient's age, but typically, adults can be administered 0.5 to 5000 mg of the active ingredient per day, and children can be administered 0.5 to 3000 mg per day. The mixing ratio of the neutrophil activator of the present invention can be appropriately changed depending on the dosage form, but typically, when administered orally or by mucosal absorption, it should be about 0.3 to 15.0 wt%, and when administered parenterally, it should be about 0.01 to 10 wt%. Note that the dosage will vary under various conditions, so in some cases a smaller amount than the above dosage may be sufficient, and in other cases, it may be necessary to administer a dose exceeding the range.

[0042] The following describes embodiments of the present invention. The embodiments described below are provided to confirm the various actions and effects of the neutrophil activator obtained by the present invention, and the scope of the present invention is not limited to these products and manufacturing methods.

[0043] [Example: Preparation of Ceramides] (1) Preparation of Glucosylceramide Oryza ceramide (manufactured by Oryza Oil & Fat Chemical Co., Ltd.) was crudely fractionated by medium-pressure preparative liquid chromatography. The fractionation conditions were as follows, using a Yamazen Corporation Universal Silica Gel Premium column, and sequential elution was performed under the conditions of hexane:ethyl acetate (9:1 → 8:2 → 7:3 → 5:5) → ethyl acetate → chloroform:methanol (9:1 → 8:2 → 7:3 → 5:5) → methanol. Next, the chloroform:methanol (8:2) fraction was subjected to reverse-phase HPLC (COSMOSIL C18 MS-II, methanol) to purify the following 11 types of other glucosylceramides (hereinafter referred to as "GlcCer"). The purified GlcCer species were found to have the following properties by comparing their NMR and MS spectra with the reference values ​​1-8 below: GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 22:0], GlcCer[d18:2(4E,8Z) / 24:0], GlcCer[d18:2(4E,8Z) / 26:0], and GlcCer[d18:2(4E,8E) / 24:0]. They were identified as GlcCer[t18:1(8Z) / 20:0], GlcCer[t18:1(8Z) / 22:0], GlcCer[t18:1(8Z) / 24:0], GlcCer[t18:1(8Z) / 26:0], and GlcCer[d18:1(4E) / 20:0].1. Inagaki. et al., Chem. Pharm. Bull., 52(11), 1307-1311 (2004) 2. Jung JH et al., J. Nat. Prod., 59, 319-322 (1996) 3. Liu H. et al., Phytochemistry., 49(8), 2403-2408 (1998) 4. Ryu J. et al., Arch. Pharm. Res., 26(2), 138-142 (2003) 5. Pittaya T. et al., Chem. Pharm. Bull., 52(1), 27-32 (2004) 6. Kang SS et al., Chem. Pharm. Bull., 49, 321-323 (2001) 7. Luo Y. et al., Lipids, 39(9), 907-914 (2004) 8. Zhang WK ​​et al., Chem. Phys. Lipids., 148, 77-83 (2007).

[0044] (2) Preparation of Free Ceramide An ethanol extract of rice bran was used as the starting material. The extract (10 g) was adsorbed onto silica gel (22 g) to prepare a "coating". This was fractionated by medium-pressure flash chromatography (Yamazen Corporation). Specifically, the "coating" was attached to a medium-pressure silica gel flash column (universal column silica gel, 3 L) and fractionated sequentially with the following solvents (1. Hexane:ethyl acetate, 2. Chloroform:methanol). An ELSD detector was used for detection.

[0045]

[0046] Fractions 17 and 18 were concentrated from the fractions eluted with chloroform:methanol (90:10 → 80:20) for 5-10 minutes to obtain crude fractions. These were repeatedly purified by preparative HPLC (C18, Osaka Soda, CAPCELL PAK C18, 20×250 mm) using THF:methanol (1:9) as the solvent to obtain S1 (10 mg), S2 (5 mg), and S3 (5 mg). By comparing the respective proton and carbon-NMR spectra and mass spectra with the literature values ​​S1 (Teinkela JEM et al. Fitoterapia, 112, 65-73, 2016, 2. Huang Q. et al. Chem. Pharm. Bull., 43, 1035-1038, 1995), S2, S3 (Muralidhar P. et al. chem, pham. Bull., 51, 1193-1195, 2003), we obtained Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 24:0), Cer(t18:0 / 25:0), Cer(t18:0 / 26:0), and Cer[t18:1(8Z) / 24:0]. It was determined to be Cer[d18:2(4E,8Z) / 20:0].

[0047] (3) Preparation of acylated ceramides (oryzaceramide A, B, and C) (3)-1. Method for preparing oryzaceramide A A fraction (15.4 g) in which glucosylceramide (GlcCer) was concentrated from a by-product obtained in the rice bran oil manufacturing process was subjected to medium-pressure silica gel chromatography (column: Universal Column Premium, 3L, Yamazen Corporation), washed with ethyl acetate for 20 minutes, and then sequentially eluted with a chloroform:methanol gradient (95:5 → 50:50, v / v, 50 minutes). The eluate from the 95:5 → 90:10 gradient was concentrated and dried to obtain a fraction (580 mg). This fraction was crudely purified by HPLC [column: CAPCELLPAK C18, 250 mm × inner diameter 20 mm (Shiseido), eluent: methanol:THF = 90:10, detection: RI, flow rate 9 mL / min], and then further purified by HPLC [column: Sunrise C30, 250 mm × inner diameter 20 mm (Chromanic Technologies), eluent: methanol:THF = 90:10, detection: RI, flow rate 9 mL / min] to obtain the white powder component oryzaceramide A (8.0 mg).

[0048] (3)-2. Preparation method of oryzaceramide B and C A fraction (15.4 g) containing a high concentration of glucosylceramide (GlcCer) was obtained from the by-products of the rice bran oil manufacturing process and subjected to medium-pressure silica gel chromatography (column: Universal Column Premium, 3L, Yamazen Corporation). After washing with ethyl acetate for 20 minutes, it was sequentially eluted using a chloroform:methanol gradient (95:5 → 50:50, v / v, 50 minutes). The eluate from the 95:5 → 90:10 gradient was concentrated and dried to obtain a fraction (580 mg). This fraction was crudely purified by HPLC [column: CAPCELLPAK C18, 250 mm × inner diameter 20 mm (Shiseido), eluent: methanol:THF = 90:10, detection: RI, flow rate 9 mL / min], and then further purified by HPLC [column: Sunrise C30, 250 mm × inner diameter 20 mm (Chromanic Technologies), eluent: methanol:THF = 90:10, detection: RI, flow rate 9 mL / min] to obtain the white powder components (oryzaceramide B, 15.2 mg) and (oryzaceramide C, 12.8 mg).

[0049] (3)-3. Structural Identification of Oryzaceramide A. Optical rotation, mass spectrometry, infrared absorption spectroscopy, and 1H and 13C-NMR spectroscopy were performed on the obtained components. 1) Physicochemical Data: White powder. [α]D25 199.1° (c=0.15, MeOH). High resolution ESI-MS Calcd for C 60 H 113 NO 10Na (M+Na)+: ; Found: 1030.8243, IR (KBr, cm-1): 3410, 2918, 2843, 1732, 1653, 1527, 1467, 1078. 1H-NMR (C5D5N, 800 MHz) δ: 0.88 (3H×2, t, J=7.3 Hz 18, 20'-H), 0.89 (3H, t, J=7.3 Hz 16'''-H), 1.27 (2H×3, m, 16, 18', 14'''-H), 1.28 (2H×3, m, 17, 19', 15'''-H), 1.28-1.31 (m, 11-15, 5'-17', 4'''-13'''-H) 1.65 (2H, tt, J=7.6, 7.8 Hz, 3'''-H), 1.72 (1H, m, 4'a-H), 1.80 (1H, m, 4'b-H), 2.00 (1H, m, 3'a-H), 2.10 (2H, m, 10-H), 2.20 (2H, m, 6-H), 2.20 (1H, m, 3'b-H), 2.22 (2H, m, 7-H), 2.36 (2H, m, 2'''-H), 3.98 (1H, ddd, J=2.1, 5.7, 9.2 Hz, 5''-H), 4.00 (1H, dd, J=7.8, 8.9 Hz, 2''-H), 4.04 (1H, dd, J=8.9, 9.2 Hz, 4''-H), 4.16 (1H, dd, J=8.7, 8.7 Hz, 3''-H), 4.26 (1H, dd, J=3.9, 10.7 Hz, 1a-H), 4.55 (1H, m, 2'-H), 4.71 (1H, dd, J=6.2, 10.7 Hz, 1b-H),4.73 (1H, m, 3-H), 4.75 (1H, dd, J=5.7, 11.7 Hz, 6''a-H), 4.78 (1H, m, 2-H), 4.88 (1H, d, J=7.8 Hz, 1''-H), 4.96 (1H, dd, J=2.1, 11.7 Hz, 6''b-H), 5.50 (2H, m, 8,9-H), 5.95 (1H, dd, J=15.6, 6.4 Hz, 5-H), 6.00 (1H, dd, J=6.2, 15.6 Hz, 4-H), 8.29 (1H, d, J=8.9 Hz, NH). 13C-NMR (C5D5N, 200 MHz) δC: 70.2 (C-1), 54.6 (C-2), 72.5 (C-3), 132.2 (C-4), 132.1 (C-5), 32.9 (C-6), 27.4 (C-7), 129.5 (C-8), 130.7 (C-9), 27.6 (C-10), 29.5, 29.6, 29.8, 29.9, 30.0, 30.1 (C-11-15), 32.1 (C-16), 22.9 (C-17), 14.3 (C-18), 175.7 (C-1'), 72.5 (C-2'), 34.4 (C-3'), 25.9 (C-4'), 29.5, 29.6, 29.8, 29.9, 30.0, 30.1 (C-5'-17'), 32.1 (C-18'), 22.9 (C-19'), 14.3 (C-20'), 105.5 (C-1''), 75.4 (C-2''), 78.3 (C-3''), 71.5 (C-4''), 75.0 (C-5''), 64.6 (C-6''), 173.7 (C-1''), 34.4 (C-2''), 25.3 (C-3''), 29.5, 29.6, 29.8, 29.9, 30.0, 30.1 (C-4'''-13'''), 32.1 (C-14'''), 22.9 (C-15'''), 14.3 (C-16'''). Positive-ion ESI-MS: m / z 1030 (M+Na)+.

[0050] 2) Analysis of two-dimensional NMR HMQC (1H-detected multiple quantum coherence spectrum), HMBC (1H-detected multi-bond heteronuclear multiple quantum coherence spectrum), and 1H-1H COSY (correlation spectroscopy) revealed proton-carbon correlations as shown by the arrows in Figure 1 and proton-proton correlations as shown by the thick lines.

[0051] (3)-4. Identification of the structure of oryzaceramide B For the obtained components, specific rotation, mass spectrometry, infrared absorption spectrum, 1 H and 13 C-NMR spectra were measured. 1) Physicochemical data White powder. [α] D 25 ₂₈.₀° (c = 0.2, MeOH). High resolution ESI-MS Calcd for C 62 H 115 NO 10 Na (M+Na) + : ; Found: 1056.8419, IR (KBr, cm -1 ⁻¹): 3379, 2920, 2851, 1740, 1624, 1516, 1467, 1080. 1H-NMR (C5D5N, 800 MHz) δ: 0.88 (3H×2, t, J=7.1 Hz 18, 20'-H), 0.89 (3H, t, J=7.1 Hz 18'''-H), 1.25 (2H×3, m, 16, 18', 16'''-H), 1.29 (2H×3, m, 17, 19', 17'''-H), 1.25-1.32 (m, 11-15, 6'-17', 4'''-7''', 12'''-15'''-H), 1.64 (2H, tt, J=7.6, 7.8 Hz, 3'''-H), 1.72 (1H, m, 4'a-H), 1.79 (1H, m, 4'b-H), 1.99 (1H, m, 3'a-H), 2.10 (2H, m, 10, 8''', 11'''-H), 2.20 (2H, m, 6-H), 2.20 (1H, m, 3'b-H), 2.22 (2H, m, 7-H), 2.35 (2H, m, 2'''-H), 3.98 (1H, ddd, J=2.1, 6.2, 9.4 Hz, 5''-H), 3.99 (1H, dd, J=7.8, 8.9 Hz, 2''-H), 4.03 (1H, dd, J=9.2, 9.2 Hz, 4''-H), 4.16 (1H, dd, J=8.9, 8.9 Hz, 3''-H), 4.25 (1H, dd, J=3.7, 10.5 Hz, 1a-H), 4.54 (1H, m, 2'-H), 4.70 (1H, m, 1b-H), 4.71 (1H, m, 3-H), 4.76 (1H, dd, J=6.0, 11.7 Hz, 6''a-H), 4.79 (1H, m, 2-H), 4.88 (1H, d, J=7.8 Hz, 1''-H), 4.96 (1H, dd, J=1.8, 11.7 Hz, 6''b-H), 5.50 (2H×2, m, 8,9; 9''', 10'''-H), 5.94 (1H, dd, J=5.7, 15.3 Hz, 5-H), 5.99 (1H, dd, J=5.7, 15.5 Hz, 4-H), 8.28 (1H, d, J=8.9 Hz, NH). 13 C-NMR (C5D5N, 200 MHz) δ C: 70.2 (C-1), 54.6 (C-2), 72.5 (C-3), 132.2 (C-4), 132.1 (C-5), 32.9 (C-6), 27.4 (C-7), 129.5 (C-8), 130.7 (C-9), 27.7 (C-10), 29.6, 29.9, 30.0, 30.1 (C-11-15), 32.1 (C-16), 22.9 (C-17), 14.3 (C-18), 175.7 (C-1'), 72.5 (C-2'), 35.7 (C-3'), 25.9 (C-4'), 30.1 (C-5'), 29.6, 29.9, 30.0, 30.1 (C-6'-17'), 32.1 (C-18'), 22.9 (C-19'), 14.3 (C-20'), 105.5 (C-1''), 75.4 (C-2''), 78.3 (C-3''), 71.5 (C-4''), 75.0 (C-5''), 64.6 (C-6''), 173.7 (C-1'''), 34.4 (C-2'''), 25.3 (C-3'''), 29.4 (C-4'''), 29.4 (C-5'''), 29.5 (C-6'''), 29.6 (C-7'''), 27.6 (C-8'''), 130.3 (C-9'''), 130.3 (C-10'''), 27.6 (C-11'''), 29.8 (C-12'''), 29.6 (C-3'''), 29.6 (C-14'''), 29.6 (C-15'''), 32.1 (C-16'''), 22.9 (C-17'''), 14.3 (C-18'''). Positive-ion ESI-MS: m / z 1030 (M+Na) + .

[0052] 2) Two-dimensional NMR, HMQC ( 1 H-detected multiple quantum coherence spectrum), HMBC ( 1 H-detected multi-bond heteronuclear multiple quantum coherence spectrum) and 1 H- 1Analysis using H-COSY (Correlation Spectroscopy) revealed proton-carbon correlations (indicated by the arrows in Figure 2) and proton-proton correlations (indicated by the thick lines).

[0053] (3)-5. Structural determination of oryzaceramide C. For the obtained components, optical rotation, mass spectrometry and infrared absorption spectrum, 1 H and 13 ¹¹C-NMR spectra were measured. 1) Physicochemical data: White powder. [α] D 25 27.9° (c=0.17, MeOH). High resolution ESI-MS Calcd for C 62 H 113 NO 10 Na (M+Na) + : ; Found: 1054.8246, IR (KBr, cm -1 ): 3374, 2957, 2891, 1740, 1628, 1522, 1468, 1080. 1H-NMR (C5D5N, 800 MHz) δ: 0.87 (3H, t, J=7.0 Hz 18 or 20'-H), δ: 0.88 (3H, t, J=7.0 Hz 18 or 20'-H), 0.89 (3H, t, J=6.9 Hz 16'''-H), 1.25 (2H×2, m, 16, 18', 16'''-H), 1.29 (2H×3, m, 17, 19', 17'''-H), 1.26-1.32 (m, 11-15, 5'-17', 4'''-7''', 15'''-H), 1.64 (2H, tt, J=7.3, 7.6 Hz, 3'''-H), 1.72 (1H, m, 4'a-H), 1.79 (1H, m, 4'b-H), 2.01 (1H, m, 3'a-H), 2.11 (2H×3, m, 10, 8''', 14'''-H), 2.20 (2H, m, 6-H), 2.20 (1H, m, 3'b-H), 2.22 (2H, m, 7-H), 2.35 (2H, m, 2'''-H), 2.92 (2H, dd, J=5.7, 6.0 Hz, 11'''-H), 3.98 (1H, ddd, J=2.1, 6.0, 9.2 Hz, 5''-H), 4.00 (1H, dd, J=7.8, 8.9 Hz, 2''-H), 4.04 (1H, dd, J=8.9, 9.2 Hz, 4''-H), 4.16 (1H, dd, J=8.9, 8.9 Hz, 3''-H), 4.25 (1H, dd, J=3.7, 10.5 Hz, 1a-H), 4.55 (1H, m, 2'-H), 4.71 (1H, dd, J=6.2, 10.5 Hz, 1b-H), 4.73 (1H, m, 3-H), 4.76 (1H, dd, J=6.0, 11.7 Hz, 6''a-H), 4.79 (1H, m, 2-H), 4.89 (1H, d, J=7.8 Hz, 1''-H), 4.96 (1H, dd, J=2.1, 11.7 Hz, 6''b-H), 5.50 (2H×3, m, 8,9; 9''', 10'''; 12''', 13''' -H), 5.95 (1H, dt, J=15.6, 6.0 Hz, 5-H), 5.99 (1H, dd, J=6.2, 15.6 Hz, 4-H), 8.29 (1H, d, J=8.9 Hz, NH). . 13 C-NMR (C5D5N, 200 MHz) δ C : 70.2 (C-1), 54.6 (C-2), 72.5 (C-3), 132.2 (C-4), 132.1 (C-5), 33.0 (C-6), 27.4 (C-7), 129.5 (C-8), 130.7 (C-9), 27.6 (C-10), 29.6, 29.9, 30.0, 30.1 (C-11-15), 32.1 (C-16), 22.9 (C-17), 14.3 (C-18), 175.7 (C-1'), 72.5 (C-2'), 35.7 (C-3'), 25.9 (C-4'), 30.1 (C-5'), 29.6, 29.9, 30.0, 30 .1 (C-6'-17'), 32.1 (C-18'), 22.9 (C-19'), 14.3 (C-20'), 105.5 (C- 1''), 75.4 (C-2''), 78.3 (C-3''), 71.5 (C-4''), 75.0 (C-5''), 64.6 (C-6''), 173.7 (C-1'''), 34.4 (C-2'''), 25.3 (C-3'''), 29.4 (C-4'''), 29.4 (C-5'''), 29.5 (C-6'''), 29.6 (C-7'''), 27.6 (C-8'''), 128.5 (C-9'''), 128.5 (C-10'''), 26.1 (C-11'''), 130.5 (C-12'''), 130.5 (C-3'''), 27.5 (C-14'''), 29.9 (C-15'''), 31.7 (C-16'''), 22.8 (C-17'''), 14.2 (C-18'''). Positive-ion ESI-MS: m / z 1054 (M+Na) + .

[0054] 2) Two-dimensional NMR HMQC ( 1 H-detected multiple quantum coherence spectrum), HMBC ( 1(H-detected multi-bond heteronuclear multiple quantum coherence spectrum) and 1 H- 1 Analysis using H-COSY (Correlation Spectroscopy) revealed proton-carbon correlations (indicated by the arrows in Figure 3) and proton-proton correlations (indicated by the thick lines).

[0055] (3)-6. Identification of Acyl Groups and GlcCer Molecular Isles 1) For oryzaceramide A, the identification of the acyl group by solvolysis was performed as follows: A novel component (1.0 mg) was dissolved in 0.5% sodium methoxide / methanol solution (500 μL) and reacted on ice for 3 hours, after which 1% acetic acid / methanol solution (500 μL) was added to the reaction mixture. The acyl group was identified by subjecting the reaction mixture to reverse-phase HPLC [column: CAPCELLPAK C18 SG-120, 250 mm × inner diameter 4.6 mm (Shiseido), eluent: acetonitrile:methanol:water = 75:11:14, detection: CAD, flow rate 1 mL / min; column temperature: 40 °C], and the retention time of the fatty acid methyl ester of the derivative was compared to the retention time of the standard (t R The acyl group was identified as palmitic acid by comparing it with the retention times of the standard (methyl palmitate; 25.4 min, methyl oleate; 26.3 min, methyl linoleate; 13.8 min). The GlcCer molecular species was identified by subjecting the reaction mixture to a column [Column: CAPCELLPAK C18 SG-120, 250 mm × inner diameter 4.6 mm (Shiseido), Eluent: methanol, Detection: CAD, Flow rate 1 mL / min; Column temperature: 40 °C], and the retention time of the GlcCer of the derivative was compared with the retention time of the standard (t RBy comparing it with GlcCer[d18:2(4E,8Z / 20:0)]; 7.1 min and GlcCer[d18:2(4E,8E / 20:0)]; 7.3 min), the GlcCer molecular species was identified as GlcCer[d18:2(4E,8Z / 20:0)]. From the above physicochemical data, it was found that this novel component is 6"-O-palmitoyl-GlcCer[d18:2(4E,8Z / 20:0)] (oryzaceramide A), in which palmitic acid is ester-bonded to the 6" position of glucose in GlcCer[d18:2(4E,8Z / 20:0)].

[0056] 2) The identification of acyl groups in oryzaceramide B and C by solvolysis was performed as follows: A novel component (oryzaceramide B, 2.1 mg) or (oryzaceramide C, 2.0 mg) was dissolved in 0.5% sodium methoxide / methanol solution (500 μL) and reacted on ice for 3 hours. Then, 1% acetic acid / methanol solution (500 μL) was added to the reaction mixture. The identification of acyl groups was performed by subjecting the reaction mixture to reverse-phase HPLC [column: CAPCELLPAK C18 SG-120, 250 mm × inner diameter 4.6 mm (Shiseido), eluent: acetonitrile:methanol:water = 75:11:14, detection: CAD, flow rate 1 mL / min; column temperature: 40 °C], and the retention time of the fatty acid methyl ester of the derivative was compared to the retention time of the standard (t R The acyl group of novel component (2) was identified as oleic acid and the acyl group of novel component (3) as linoleic acid by comparing it with the following: methyl palmitate; 25.4 min, methyl oleate; 26.3 min, methyl linoleate; 13.8 min). For the identification of GlcCer molecular species, the reaction solution was subjected to a column [Column: CAPCELLPAK C18 SG-120, 250 mm × inner diameter 4.6 mm (Shiseido), Eluent: methanol, Detection: CAD, Flow rate 1 mL / min; Column temperature: 40 °C], and the retention time of GlcCer of the derivative was compared with the retention time of the standard (t RThe GlcCer molecular species was identified as GlcCer[d18:2(4E,8Z / 20:0)] by comparing it with GlcCer[d18:2(4E,8Z / 20:0)]; 7.1 min and GlcCer[d18:2(4E,8Z / 20:0)]; 7.3 min). Based on the above physicochemical data, it was found that the novel component (oryzaceramide B) is 6"-O-oleoyl-GlcCer[d18:2(4E,8Z / 20:0)], in which oleic acid is ester-bonded to the 6" position of glucose in GlcCer[d18:2(4E,8Z / 20:0)], and the novel component (oryzaceramide C) is 6"-O-linoleoyl-GlcCer[d18:2(4E,8Z / 20:0)], in which linoleic acid is ester-bonded to the 6" position of glucose in GlcCer[d18:2(4E,8Z / 20:0)].

[0057] [Test Example 1: Evaluation of the effect on the IL-8 production capacity of mouse peritoneal neutrophils] (1) Method of recovery and purification of mouse peritoneal neutrophils Six-week-old male ICR mice purchased from CLEA Japan were used in the experiment. The mice were intraperitoneally administered a 7.5% casein solution (1 mL / mice) and reared for 24 hours. The mice were then sacrificed, and the cells that had infiltrated the peritoneal cavity due to casein administration were recovered in PBS(-) containing 1% bovine serum albumin and centrifuged. The pellet was hemolyzed to obtain cells that had infiltrated the peritoneal cavity (1.0 × 10^7 cells per mouse). The cell suspension was processed by Percoll® density gradient centrifugation to obtain a neutrophil fraction (neutrophil purity of 90% or more). Furthermore, 1 mL of the cell suspension was layered on top of 5 mL of Percoll® 63% solution and centrifuged (1,500 g, 20 min, 4°C). Neutrophil purity testing was performed using a flow cytometer to identify and confirm neutrophils as Ly6G and CD11b-positive cells (see Figure 4).

[0058] (2) Effect on IL-8 production capacity of mouse peritoneal neutrophils The neutrophil fraction obtained by the above method (2.0 × 10^5 cells / well) was seeded in a 96-well plate and culture medium containing the test substance was added (final concentration of DMSO: 0.1%). After incubation at 37°C in the presence of 5% CO2 for 24 hours, IL-8 in the culture supernatant was quantified by ELISA. The test substances were trehalose-6,6-dibehenate (TDB) at a final concentration of 100 μM as a positive control, a high-concentration fraction of glucosylceramide (GlcCer) isolated in this example at a final concentration of 10 μg / mL, various GlcCer, acylated GlcCer (oryzaceramides A~C), and free ceramide (Cer) at a final concentration of 10 μM.

[0059] (3) Measurement results and effects of the example in Test Example 1 The amount of IL-8 produced was evaluated, and the high-concentration fractions of GlcCer, GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 22:0], GlcCer[d18:2(4E,8Z) / 24:0], GlcCer[d18:2(4E,8Z) / 26:0], GlcCer[d18:2(4E,8E) / 24:0], GlcCer[t18:1(8Z) / 24:0], GlcCer[t18:1(8Z) / 26:0], and GlcCer[d18:1(4E) / 20:0] showed a significant IL-8 production-promoting effect (Figure 5). Furthermore, oryzaceramide AC, Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 24:0), Cer(t18:0 / 25:0), and Cer(t18:0 / 26:0), as well as Cer[t18:1(8Z) / 24:0] and Cer[d18:2(4E,8Z) / 20:0] also showed significant IL-8 production-promoting effects (Figure 6). This confirms that the above-mentioned glucosylceramide, free ceramide, and acylated GlcCer are useful as IL-8 production promoters.

[0060] [Test Example 2: Effects on NETs production capacity of mouse peritoneal neutrophils] (1) Test method Neutrophil fraction (1.0 × 10^5 cells / well) was seeded in a 96-well plate (black), and the test substance and SYTOX TM A culture medium containing Green Nucleic Acid Stain (Thermo Fisher Scientific) was added (DMSO final concentration: 0.1%, SYTOX TM Green: 200 nM). After incubation at 37°C in the presence of 5% CO2 for 2 hours, the fluorescence was measured using a fluorescence plate reader (excitation wavelength: 480 nm, fluorescence wavelength: 530 nm). (Non-patent document 3) For positive controls, trehalose-6,6-dibehenate (TDB) and phorbol myristate acetate (PMA) at a final concentration of 100 μM were used. The test substances were high-concentration fractions of GlcCer at a final concentration of 10 μg / mL, various GlcCer, oryzaceramide AC, and Cer at a final concentration of 10 μM. Tong M. et al., Visualization and Quantification of Neutrophil Extracellular Traps. Methods Mol Biol. 2255:87-95 (2021).

[0061] (2) Results and Effects of the Examples in Test Example 2 Evaluation of NETs production revealed that GlcCer[d18:2(4E,8Z)] and GlcCer[d18:2(4E,8E) / 24:0] with fatty acid chain lengths of 20, 24, and 26 showed a significant NETs production-promoting effect (Figure 7). Furthermore, oryzaceramide AC and Cer(t18:0) with fatty acid chain lengths of 22, 23, and 26 also showed a significant NETs production-promoting effect (Figure 8).

[0062] [Test Example 3: Effects on Cell Migration Ability of Mouse Peritoneal Neutrophils] (1) Test Method Cell migration ability was evaluated using the CytoSelect™ 96-Well Cell Migration Assay (CELL BIOLABS), which was prepared in accordance with the cell chemotaxis assay shown in Figure 9. In this kit, migratory cells attracted by chemotactic factors pass through the membrane and adhere to the bottom surface of the membrane, while non-migratory cells do not pass through the membrane and remain there. This kit is suitable for screening substances that affect cell migration. Neutrophil fraction (1.0 × 10^5 cells / well) was seeded on the 96-well plate (upper side of the membrane) included with the above kit, and the culture medium containing the test substance was added to the lower side of the membrane (final concentration of DMSO: 0.1%). After culturing at 37°C in the presence of 5% CO2 for 4 hours, the cells attached to the membrane were detached, and the cells that passed through the membrane (lower side) were collected. The collected cells were stained, and migrating cells were quantified using a fluorescence plate reader (excitation wavelength: 480 nm, fluorescence wavelength: 530 nm). N-Formylmethionine-leucyl-phenylalanine (FMLP) at a final concentration of 1 μM was used as a positive control. The test substances were high-concentration fractions of GlcCer at a final concentration of 10 μg / mL and various GlcCer at a final concentration of 10 μM.

[0063] (2) Results and Effects of the Examples in Test Example 3 As a result of evaluating the cell migration ability, a significant cell migration-promoting (cell chemotaxis) effect was observed in neutrophils with high-concentration GlcCer fraction, GlcCer[d18:2(4E,8Z) / 18:0] and GlcCer[t18:1(8Z)] with fatty acid chain lengths of 20 and 22 (Figure 10).

[0064] [Summary of Neutrophil Activation Effects] Table 2 shows the neutrophil activation effects of various ceramides as revealed by Test Examples 1-3. In Table 2, "○" indicates that an effect was observed, and "◎" indicates that a particularly excellent effect was observed.

[0065] [Formulation Examples] The following are formulation examples of the neutrophil activator of the present invention (GlcCer[d18:2(4E,8Z) / 18:0]). Note that the following formulation examples are not limiting to the present invention. While GlcCer[d18:2(4E,8Z) / 18:0] is shown in these formulation examples, other ceramide compounds can be used in a similar manner. Not only these ceramide compounds individually, but also compositions, such as plant extracts containing them, can be used. Formulation Example 1: Chewing Gum Sugar 53.0 wt% Gum Base 20.0 Glucose 10.0 Starch Syrup 16.0 Flavoring 0.5 Neutrophil Activator of the Present Invention 0.5 100.0 wt%

[0066] Formula Example 2: Gummy Reduced Starch Syrup 40.0 wt% Granulated Sugar 20.0g Glucose 20.0g Gelatin 4.7g Water 9.6g Yuzu Juice 4.0g Yuzu Flavor 0.6g Coloring 0.02g Neutrophil Activator of the Present Invention 1.0g 100.0 wt%

[0067] Formula Example 3: Candy Sugar 50.0 wt%, Starch Syrup 33.0%, Water 14.4%, Organic Acid 2.0%, Flavoring 0.2%, Neutrophil Activator of the Present Invention 0.4%, 100.0 wt%

[0068] Formula Example 4: Yogurt (Hard / Soft) Milk 41.5 wt% Skim milk powder 5.8 Sugar 8.0 Agar 0.15 Gelatin 0.1 Lactic acid bacteria 0.005 Neutrophil activator of the present invention 0.4 Flavoring traces Water residue 100.0 wt%

[0069] Formula Example 5: Soft drink, fructose-glucose liquid sugar 30.0 wt%, emulsifier 0.5%, neutrophil activator of the present invention 0.3%, appropriate amount of flavoring, purified water, residue 100.0 wt%

[0070] Formulation Example 6: Tablet Confectionery Sugar 76.4 wt% Glucose 19.0 Sucrose Fatty Acid Ester 0.2 Neutrophil Activator of the Present Invention 0.5 Purified Water 3.9 100.0 wt%

[0071] Formulation Example 7: Soft capsules containing 47.0 wt% brown rice germ oil, 40.0 wt% yuzu seed oil, 12.0 wt% emulsifier, and 1.0 wt% neutrophil activator of the present invention.

[0072] Formulation Example 8: Tablet Lactose 54.0 wt%, Crystalline Cellulose 30.0%, Starch Hydrolysate 10.0%, Glycerin Fatty Acid Ester 5.0%, Neutrophil Activator of the Present Invention 1.0%, 100.0 wt%

[0073] Based on the above, the present invention can provide a novel neutrophil activator.

Claims

1. An IL-8 production promoter comprising at least one selected from GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 22:0], GlcCer[d18:2(4E,8Z) / 24:0], and GlcCer[d18:2(4E,8Z) / 26:0] as an active ingredient.

2. An IL-8 production promoter containing GlcCer[d18:2(4E,8E) / 24:0] as the active ingredient.

3. An IL-8 production promoter comprising at least one selected from GlcCer[t18:1(8Z) / 24:0] and GlcCer[t18:1(8Z) / 26:0] as an active ingredient.

4. An IL-8 production promoter containing GlcCer[d18:1(4E) / 20:0] as the active ingredient.

5. An IL-8 production promoter comprising at least one selected from Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 24:0), Cer(t18:0 / 25:0), and Cer(t18:0 / 26:0) as an active ingredient.

6. An IL-8 production promoter comprising at least one selected from Cer[t18:1(8Z) / 24:0] and Cer[d18:2(4E,8Z) / 20:0] as an active ingredient.

7. An IL-8 production promoter comprising at least one selected from oryzaceramide A, oryzaceramide B, and oryzaceramide C, represented by the following chemical formulas, as an active ingredient.

8. A neutrophil extracellular trap production promoter comprising at least one selected from GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 24:0], and GlcCer[d18:2(4E,8Z) / 26:0] as an active ingredient.

9. A neutrophil extracellular trap production promoter containing GlcCer[d18:2(4E,8E) / 24:0] as the active ingredient.

10. A neutrophil extracellular trap production promoter comprising at least one selected from Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), and Cer(t18:0 / 26:0) as an active ingredient.

11. A neutrophil extracellular trap production promoter comprising at least one selected from oryzaceramide A, oryzaceramide B, and oryzaceramide C, represented by the following chemical formulas, as an active ingredient.

12. A neutrophil cell migration enhancer containing GlcCer[d18:2(4E,8Z) / 18:0] as the active ingredient.

13. A neutrophil cell migration enhancer comprising at least one selected from GlcCer[t18:1(8Z) / 20:0] and GlcCer[t18:1(8Z) / 22:0] as an active ingredient.

14. A neutrophil activator comprising any of the agents specified in claims 1 to 13 as an active ingredient.

15. A method for activating the human immune system, comprising: (a) GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 22:0], GlcCer[d18:2(4E,8Z) / 24:0], GlcCer[d18:2(4E,8Z) / 26:0], GlcCer[d18:2(4E,8E) / 24:0], GlcCer[t18:1(8Z) / 24:0], GlcCer[t18:1(8Z) / 26:0] from rice bran extract. A step of isolating at least one selected from the group consisting of GlcCer[d18:1(4E) / 20:0], Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 24:0), Cer(t18:0 / 25:0), Cer(t18:0 / 26:0), Cer[t18:1(8Z) / 24:0], Cer[d18:2(4E,8Z) / 20:0] and oryzaceramide A, oryzaceramide B, and oryzaceramide C represented by the following chemical formulas, (b) A method comprising the step of administering an effective amount of the compound isolated in step a to a human being by oral or parenteral administration to promote IL-8 production in the human being.

16. A method for activating the human immune system, comprising: (a) isolating at least one selected from the group consisting of GlcCer[d18:2(4E,8Z) / 20:0], GlcCer[d18:2(4E,8Z) / 24:0], GlcCer[d18:2(4E,8Z) / 26:0], GlcCer[d18:2(4E,8E) / 24:0], Cer(t18:0 / 22:0), Cer(t18:0 / 23:0), Cer(t18:0 / 26:0), and oryzaceramide A, oryzaceramide B, and oryzaceramide C represented by the following chemical formulas, (b) A method comprising the step of administering an effective amount of the compound isolated in step a to a human being by oral or parenteral administration to promote the production of extracellular traps in neutrophils of the human being.

17. A method for activating the human immune system, comprising: (a) isolating at least one compound selected from the group consisting of GlcCer[d18:2(4E,8Z) / 18:0], GlcCer[t18:1(8Z) / 20:0], and GlcCer[t18:1(8Z) / 22:0] from an extract of rice bran; and (b) administering an effective amount of the compound isolated in step a to a human being by oral or parenteral administration to promote cell migration in the human neutrophils.

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