Immunostimulants

Abstract

To provide a novel immunostimulator.SOLUTION: The present invention provides (1) a neutrophil chemotaxis enhancer comprising a monoacyldigalactosylglycerol as an active ingredient. (2) a promoter of interleukin-8 (IL-8) gene expression in neutrophils, comprising a monoacyldigalactosylglycerol as an active ingredient, and (3) the agent set forth in (1) or (2), wherein the monoacyldigalactosylglycerol is 1-O-palmitoyl-3-O-[α-D-galactopyranosyl(16)-β-D-galactopyranosyl]-sn-glycerol.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an immunostimulant. The present invention is widely used in pharmaceuticals, health foods, foods, etc.

Background Art

[0002] When pathogens or bacteria invade the living body through cuts or mucous membranes, dendritic cells distributed throughout the body recognize and phagocytize the antigen, and antigen presentation is carried out to white blood cells, lymphocytes, etc. Thereafter, neutrophils, macrophages and natural killer cells phagocytize and kill the pathogens that have invaded the living body (innate immunity). When evaluating the immunostimulatory effect, these cells are tested.

[0003] As defense reactions against foreign substances, there are innate immunity and acquired immunity. In the innate immune response, for example, it is known that immune cells such as neutrophils, dendritic cells and macrophages produce cytokines in response to innate immunostimulatory substances derived from bacteria or viruses, and subsequent immune reactions occur. The innate immune mechanism is an infection defense mechanism common to organisms, and is generally non-specific, so it is characterized by a rapid reaction and effective functioning against many infectious agents.

[0004] Neutrophils account for about 50% of white blood cells, and mainly play a role in preventing infection by phagocytizing (swallowing) and decomposing pathogenic bacteria and foreign substances such as bacteria and fungi that have invaded the living body, and performing sterilization. Also, they are numerous and are characterized by the fastest migration to antigens. In addition, interleukin-8 (IL-8) is an inflammatory cytokine produced by vascular endothelial cells, airway smooth muscle cells and neutrophils, etc., is known as a chemotactic factor for neutrophils, and plays an important role in the immune response.

[0005] Macrophages possess stronger phagocytic capabilities compared to neutrophils. By phagocytizing, they release antigen-presenting and inflammatory cytokines, which act on other cells and enhance the immune response. While there are various inflammatory cytokines, it is known that LPS stimulation releases IL-6 (interleukin-6) and TNF-α, which in turn activates dendritic cells.

[0006] Furthermore, when pathogens invade the body, neutrophils, macrophages, natural killer (NK) cells, and dendritic cells kill or engulf the pathogens. NK cells are known to release interferon-gamma (IFN-γ), which has the effect of activating macrophages. [Disclosure of the Invention] [Problems that the invention aims to solve]

[0007] Against this background, the inventors discovered that monoacyldigalactosylglycerol improves the migratory ability of neutrophils, promotes IL-8 gene expression in migratory neutrophils, and thereby enhances innate immunity, thus completing the present invention. In other words, the present invention aims to provide a novel immunostimulant that activates innate immunity. [Means for solving the problem]

[0008] The features of the present invention for solving the above problems are as follows. 1. A neutrophil migration enhancer containing monoacyldigalactosylglycerol as the active ingredient. 2. A gene expression enhancer for interleukin-8 (IL-8) in neutrophils, containing monoacyldigalactosylglycerol as the active ingredient. 3. The agent according to 1. or 2. above, characterized in that monoacyldigalactosylglycerol is 1-O-palmitoyl-3-O-[α-D-galactopyranosyl(16)-β-D-galactopyranosyl]-sn-glycerol. [Effects of the Invention]

[0009] This invention suggests that monoacyldigalactosylglycerol enhances the migration ability of neutrophils and increases the gene expression function of interleukin-8 (IL-8) in neutrophils, thereby enhancing the chemotactic function of neutrophils. This improves the ability to phagocytose (engulf) and decompose pathogenic bacteria, fungi, and other foreign substances that invade the body, thereby enhancing the ability to kill them. This can improve innate immunity. [Brief explanation of the drawing]

[0010] [Figure 1] This graph shows the effect on IL-8 gene expression levels in HL-60 cells (neutrophils). [Modes for carrying out the invention]

[0011] The immunostimulant of the present invention is characterized by containing monoacyldigalactosylglycerol as an active ingredient. Here, "monoacyldigalactosylglycerol" in this embodiment refers to the compound shown in the following chemical formula (1).

[0012] [ka]

[0013] The above-mentioned "monoacyldigalactosylglycerol" is not particularly limited, but is preferably, for example, the one shown in the following chemical formula (2), i.e., 1-O-palmitoyl-3-O-[α-D-galactopyranosyl(16)-β-D-galactopyranosyl]-sn-glycerol. [ka]

[0014] The method for producing the above-mentioned monoacyl digalactosyl glycerol 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 monoacyl digalactosyl glycerol from the above extract.

[0015] The above-mentioned "rice bran extract" is not particularly limited as long as it is produced in the process of producing rice bran oil. For example, the rice bran extract can be obtained by extracting rice bran with an organic solvent and then precipitating the extract, but it is not limited to this method. Also, the above rice bran extract may be extracted from by-products produced in the manufacturing process from rice bran oil.

[0016] Here, the method for isolating monoacyl digalactosyl glycerol from the above rice bran extract is not particularly limited. For example, it can be isolated by chromatography using activated clay, activated carbon, silica gel, alumina, diatomaceous earth, synthetic adsorbent, ion exchange resin, etc., or by removing components other than free ceramide by adsorption, decomposition, precipitation, filtration, dissolution, distillation, etc. Specifically, for example, it can be obtained by the method of the examples in this specification, but it is not limited to this method.

[0017] The immunostimulant of the present invention can be used as a raw material for various foods and drinks. Examples of foods and drinks include general foods such as confectioneries (gum, candy, caramel, chocolate, cookie, snack, jelly, gummy, tablet confectionery, etc.), noodles (buckwheat noodles, udon, ramen, etc.), dairy products (milk, ice cream, yogurt, etc.), seasonings (miso, soy sauce, etc.), soups, beverages (juice, coffee, black tea, tea, carbonated beverage, sports drink, etc.), as well as health foods (tablets, capsules, etc.), dietary supplements (nutritional drinks, etc.). The agent of the present invention may be appropriately formulated in these foods and drinks.

[0018] These food and drink products can be formulated with various ingredients according to their types. For example, 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, vitamin Bs, nicotinamide, calcium pantothenate, amino acids, calcium salts, pigments, flavors, preservatives and other food materials can be used.

[0019] As a specific production method, the agent of the present invention can be spray - dried or freeze - dried together with powdered cellulose, and then made into powder, granules, tablets or solutions, and can be easily incorporated into food and drink products (such as instant foods). Also, the agent of the present invention can be dissolved in, for example, oils and fats, ethanol, glycerin or mixtures thereof to make it liquid, and can be added to beverages or solid foods. If necessary, it can be mixed with binders such as gum arabic and dextrin to make it powdery or granular, and then added to beverages or solid foods.

[0020] When applying the agent of the present invention to food and drink products, the addition amount is preferably such that the total content of the active ingredients is 1 - 20 wt% or less with respect to the food and drink products, since the main purposes are disease prevention and health maintenance.

[0021] The immunostimulant 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 ingredients 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.).

[0022] The immunostimulant 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, etc. Furthermore, it can be administered as eye drops.

[0023] 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 usually be administered 0.5 to 3000 mg. The mixing ratio of immunostimulants can be adjusted as appropriate depending on the dosage form, but generally, it is appropriate to use approximately 0.3 to 15.0 wt% when administered orally or via mucosal absorption, and approximately 0.01 to 10 wt% when administered parenterally. Note that the dosage varies depending on various conditions, so in some cases a smaller amount than the above may be sufficient, and in other cases it may be necessary to administer a larger amount. [Examples]

[0024] The following describes examples of the present invention. These examples are provided to confirm the various actions and effects of the agent obtained by the present invention, and the scope of the present invention is not limited to these products and manufacturing methods.

[0025] Example: Preparation of monoacyldigalactosylglycerol Oryza ceramide (manufactured by Oryza Oil & Fat Chemical Co., Ltd.) was subjected to coarse fractionation by medium-pressure preparative liquid chromatography. The fractionation conditions were as follows: using a Yamazen Corporation Universal Silica Gel Premium column, elution was performed sequentially under chloroform:methanol (9:1 → 8:2 → 7:3 → 5:5) → methanol conditions. Next, the chloroform:methanol (5:5) fraction was subjected to reverse-phase HPLC (CAPCELLPAK C18 SG-120, methanol) to purify three types of monoacyldigalactosylglycerol (MADG): MADG Fraction 1, MADG Fraction 2, and MADG Fraction 3. Of the purified MADG Fraction 1, MADG Fraction 2, and MADG Fraction 3, MADG Fraction 3 was identified as 1-O-palmitoyl-3-O-[α-D-galactopyranosyl(16)-β-D-galactopyranosyl]-sn-glycerol, represented by the above chemical formula (2), by comparing its NMR and MS spectra with those of reference 1 below. While MADG Fraction 1 and MADG Fraction 2 were clearly identified as MADG from their NMR spectra, the types of constituent fatty acids could not be determined. Reference 1. Arthur Millius, Orion D. Weiner, Chemotaxis in Neutrophil-Like HL-60 Cells, Methods Mol Biol, 571, 167-77 (2009).

[0026] Test Example 1: Evaluation of the effect of monoacyldigalactosylglycerol (MADG) on the cell migration ability of differentiated HL-60 cells (neutrophils). (1) Method for culturing human promyelocytic leukemia-derived cells (HL-60 cells) Human promyelocytic leukemia-derived HL-60 (JCRB 0085) cells purchased from the JCRB Cell Bank were cultured and used in the experiment. The culture medium used was Roswell Park Memorial Institute 1640 (RPMI 1640) supplemented with 10 (v / v) % FBS (Fetal bovine serum), 100 units / mL penicillin G, and 100 μg / mL streptomycin. Cell culture was performed on 25 cm³. 2 The procedure was carried out in a culture flask at 37°C in the presence of 5% CO2. Since the cells were suspension cells, the cultured cells were centrifuged (100 g, 5 min) before being used in the experiment.

[0027] (2) Method for evaluating the effect on the cell migration ability of differentiated HL-60 cells (neutrophils) HL-60 cells, a leukemia cell line, were prepared according to the method of Millius et al. (see Reference 1 below) for 25 cm 2 Seed cells in a flask (1.5 × 10 6 Neutrophils were differentiated by culturing them for one week in RPMI1640 medium containing 1.3% (v / v) DMSO (cells / flask). Differentiation was confirmed by Meiglinwald staining. Using a differentiated HL-60 cell suspension, Cytoselect TM The effect of the test substance on cell migration ability was measured according to the method described in the 96-well Cell Migration Assay kit. The cell seeding density was 1.0 × 10⁻⁶. 5The final concentration of DMSO obtained by dissolving the test substance in cells / well / 100 μL was 0.5% (v / v). Furthermore, RPMI1640 without FBS was used as the culture medium. The results are shown in Table 1 below. In Table 1, MADG Fraction 1 and MADG Fraction 2 are monoacyldigalactosylglycerol (MADG), and compound 1 is the compound represented by the above chemical formula (2), which is 1-O-palmitoyl-3-O-[α-D-galactopyranosyl(16)-β-D-galactopyranosyl]-sn-glycerol. (Reference 1) Arthur Millius, Orion D. Weiner, Chemotaxis in Neutrophil-Like HL-60 Cells, Methods Mol Biol, 571, 167-77 (2009).

[0028] [Table 1]

[0029] (3) Results and the effects of the example in Test Example 1 As shown in Table 1, significant activity was observed for MADG Fraction 1, MADG Fraction 2, and Compound 1, with particularly strong activity observed for Compound 1. Based on the above, it has been confirmed that MADG Fraction 1, MADG Fraction 2, and Compound 1, particularly Compound 1, are useful as neutrophil migration enhancers. This improves the function of phagocytosis (engulfing) and decomposition of pathogenic bacteria, fungi, and other foreign substances that invade the body, thereby enhancing innate immunity.

[0030] Test Example 2: Evaluation of the effect on IL-8 (interleukin 8) gene expression levels in HL-60 cells. (1) Test method HL-60 cells were seeded in a 12-well plate (1.0 × 10⁶). 5Cells (500 μL / well) and culture medium containing the test substance (500 μL / well) were added. After 24 hours of incubation, RNA was extracted from the cells and gene expression levels were measured according to real-time RT-PCR. The results are shown in Figure 1. In Figure 1, MADG Fraction 1 and MADG Fraction 2 are monoacyldigalactosylglycerol (MADG), and compound 1 is the compound shown in chemical formula (2) above, which is 1-O-palmitoyl-3-O-[α-D-galactopyranosyl(16)-β-D-galactopyranosyl]-sn-glycerol.

[0031] (2) Results and the effects of the example in Test Example 2 When MADG Fraction 1, MADG Fraction 2, and Compound 1 (1-O-palmitoyl-3-O-[α-D-galactopyranosyl(16)-β-D-galactopyranosyl]-sn-glycerol) were added, an increasing trend was observed in MADG Fraction 1, and a significant increase in IL-8 gene expression was confirmed in MADG Fraction 2 and Compound 1. Therefore, MADG Fraction 1, MADG Fraction 2, and Compound 1 (1-O-palmitoyl-3-O-[α-D-galactopyranosyl(16)-β-D-galactopyranosyl]-sn-glycerol), particularly MADG Fraction 2 and Compound 1, are useful as gene expression promoters. This has been shown to enhance the chemotactic factors and function of neutrophils, thereby improving their ability to phagocytose (engulf) and break down pathogens and foreign substances such as bacteria and fungi that invade the body, and to kill them, thereby improving innate immunity.

[0032] The following are examples of formulations of the agent (monoacyldigalactosylglycerol) according to the present invention. Note that the following formulation examples are not intended to limit the scope of the present invention. Example formulation 1: Chewing gum Sugar 53.45 wt% Gum base 20.0 Glucose 10.0 Starch syrup 16.0 Fragrance 0.5 Monoacyldigalactosylglycerol 0.05 100.0 wt%

[0033] Formula example 2: Gummy Reduced starch syrup 40.9 wt% Granulated sugar 20.0 Glucose 20.0 Gelatin 4.7 Wednesday 9.68 Yuzu juice 4.0 Yuzu flavor 0.6 Dye 0.02 Monoacyldigalactosylglycerol 0.1 100.0 wt%

[0034] Formula example 3: Candy Sugar 50.36 wt% Starch syrup 33.0 Wednesday 14.4 organic acid 2.0 Fragrance 0.2 Monoacyldigalactosylglycerol 0.04 100.0 wt%

[0035] Example of formulation 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 Monoacyldigalactosylglycerol 0.04 fragrance trace amount water residue 100.0 wt%

[0036] Formulation example 5: Soft drink Fructose-glucose liquid sugar 30.0 wt% Emulsifier 0.5 Monoacyldigalactosylglycerol 0.03 Fragrance (appropriate amount) Purified water remainder 100.0 wt%

[0037] Formulation example 6: Tablet confectionery Sugar 76.4 wt% Glucose 19.0 Sucrose fatty acid ester 0.2 Monoacyldigalactosylglycerol 0.05 Purified water 3.9 100.0 wt%

[0038] Formulation Example 7: Soft Capsules Brown rice germ oil 47.9 wt% Yuzu seed oil 40.0 Emulsifier 12.0 Monoacyldigalactosylglycerol 0.1 100.0 wt%

[0039] Formulation example 8: Tablets Lactose 54.9wt% Crystalline cellulose 30.0 Starch decomposition product 10.0 Glycerin fatty acid ester 5.0 Monoacyldigalactosylglycerol 0.1 100.0 wt% [Industrial applicability]

[0040] Based on the above, the present invention can provide an immunostimulant containing a novel component as an active ingredient.

Claims

1. A neutrophil migration enhancer containing monoacyldigalactosylglycerol as its active ingredient.

2. A gene expression enhancer for interleukin-8 (IL-8) in neutrophils, containing monoacyldigalactosylglycerol as the active ingredient.

3. The agent according to claim 1 or 2, characterized in that monoacyldigalactosylglycerol is 1-O-palmitoyl-3-O-[α-D-galactopyranosyl(16)-β-D-galactopyranosyl]-sn-glycerol.

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