Immunostimulating composition

Abstract

To provide a novel immunostimulatory composition.SOLUTION: An immunostimulatory composition contains Agrimol B as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an immunostimulatory composition. [Background technology]

[0002] Currently, plant extracts are used for various medical purposes, and agrimony extract has been attracting attention in recent years. Agrimonia pilosa (scientific name: Agrimonia pilosa) is a perennial plant of the Rosaceae family, used in herbal medicine. Its herbal name is Senkakusou. The stems and leaves of the aboveground part of Agrimonia in flowering season contain essential oils and tannins, the main component of which is known to have an astringent effect that tightens cell tissue. In addition, an aqueous extract boiled in water is said to have a choleretic effect that helps the gallbladder function. The roots are known to contain tannins as well as phenolic glycosides, agrimonolide, phytosterol, vanillic acid, taxifolin, etc.

[0003] Various pharmacological effects of an extract from agrimony are known. Patent Document 1 describes a composition for activating nerves that contains an extract from agrimony as an active ingredient. Patent Document 2 describes a skin whitening agent, an anti-aging agent, and a skin cosmetic material that contain an extract of agrimony. Patent Document 3 describes an α-glucosidase inhibitor containing an extract of agrimony. Patent Document 4 describes an external skin preparation and an allergen activator that contain an extract of agrimony as an active ingredient. Patent Document 5 describes that a 70% ethyl alcohol extract of agrimony is useful as an arachidonic acid metabolism inhibitor. In an in vitro test using a rat basophilic leukemia cell line (RBL-1), this extract suppressed the production of leukotriene C, an inflammation-causing substance. 4 It has been reported that it has an inhibitory effect on the production of In addition, Non-Patent Document 1 describes that a methanol extract of agrimony exhibits antitumor properties via activation of macrophages and cytotoxic lymphocytes. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2018-8888 A [Patent Document 2] JP 2008-174529 A [Patent Document 3] Japanese Patent Application Publication No. 08-176003 [Patent Document 4] Japanese Patent Application Publication No. 11-335256 [Patent Document 5] JP 2006-16388 A [Non-patent literature]

[0005] [Non-Patent Document 1] Gifu Medical University Bulletin, Volume 35, Pages 36-44, 1986 Summary of the Invention [Problem to be solved by the invention]

[0006] In the course of studying the physiological effects of an extract from agrimony, the present inventors discovered that the extract from agrimony promotes the phagocytic activity of phagocytes such as macrophages, and found that one of the active ingredients is agrimol B. The present invention is based on this finding. That is, an objective of the present invention is to provide a novel composition for immunostimulation. [Means for solving the problem]

[0007] The present invention is configured as follows. (1) An immunostimulating composition containing Agrimole B as an active ingredient. (2) An immunostimulating composition containing a plant extract including Agrimol B as an active ingredient. (3) An immunostimulating composition according to (2), in which the plant extract containing Agrimole B contains at least 0.4% by mass of Agrimole B per total amount of dry matter. (4) The immunostimulating composition according to (2) or (3), wherein the plant containing Agrimole B is Agrimony. Effect of the Invention

[0008] The present invention provides a novel immunostimulatory composition. The immunostimulatory composition of the present invention can stimulate the phagocytosis of macrophages and activate the initial immune response to activate the immune function of the living body, and can therefore be used to treat infectious diseases and the like. [Brief description of the drawings]

[0009] [Figure 1] 1 is a graph showing the results of a test conducted in the examples to measure the phagocytic activity-promoting effects of Agrimole B, agrimony extract, and concentrated agrimony extract. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] In this specification, the term "composition" refers to medicines, quasi-drugs, and foods. In addition, in the present specification, the term "agrimony extract" refers to an extract obtained by extracting arimony with ethyl alcohol or aqueous ethyl alcohol that contains water. Furthermore, the extract obtained by washing the above-mentioned agrimony extract with 0 to 40% ethyl alcohol to remove 0 to 40% ethyl alcohol soluble components is referred to as a "concentrated agrimony extract" in the present specification.

[0011] Agrimonia pilosa (scientific name: Agrimonia pilosa) is a perennial plant of the genus Agrimonia in the family Rosaceae, and grows naturally on forest edges, in fields, and by roadsides in Honshu, Shikoku, Kyushu, and other areas. It is also known as dragon-tooth grass. In the present invention, the dried Agrimonia pilosa that is commercially available as a raw material for Chinese herbal medicines, folk remedies, and health foods (herbal tea) can be used. The dried whole plant is commercially available under the herbal name Senkakusou. In addition, the whole plant, either naturally growing or cultivated, may be collected and naturally or heat-dried for use. The agrimony is cut into small pieces and immersed in about 10 times the amount of water or 0-99.5% (v / v) ethyl alcohol, preferably 1-50% ethyl alcohol, particularly preferably 5-10% ethyl alcohol, for 3-5 days to extract at room temperature, or extracted at 50-80°C for 5-24 hours using a reflux condenser, and then filtered to recover an agrimony extract. Water and ethyl alcohol are removed from the extract using a reduced pressure vacuum drying device such as a rotary evaporator, or a freeze-drying device, to obtain an agrimony extract. In the present specification, the agrimony extract is also referred to as agrimony extract. This agrimony extract (solid agrimony extract) is washed with 0 to 40% ethanol, particularly preferably with 10 to 30% ethanol, and components soluble in 0 to 40% ethanol, particularly preferably with 10 to 30% ethanol, are removed by means of filtration or the like, and then the filtration residue is dried using a reduced pressure vacuum drying apparatus such as a rotary evaporator or a freeze-drying apparatus to remove water and ethyl alcohol, thereby obtaining a concentrated agrimony extract. The concentrated agrimony extract can be redissolved in 50 to 99.5% ethanol, particularly preferably 70 to 90% ethanol, and then the excipients used in formulation are added and freeze-dried to obtain a powder composition containing the concentrated agrimony extract of the desired concentration.

[0012] The above-mentioned agrimony extract and concentrated agrimony extract contain Agrimole B. Agrimol B is a compound identified by the following chemical formula 1.

[0013] [ka]

[0014] Agrimol B can be synthesized using phloroglucinol or the like as a starting material. For example, it can be synthesized by the method described in Acta Pharmaceutica Sinica 1989, 24(6): pp. 431-437 or the method disclosed in Patent Document 4.

[0015] In addition, there are two types of Agrimol B that can be obtained by synthesis: (S)-(+)-Agrimol B and its optical isomer, (R)-(-)-Agrimol B. In this specification, "Agrimol B" may refer to a single compound consisting of one of these types, or a mixture consisting of two types.

[0016] Agrimol B obtained by chemical synthesis or extraction, separation and purification from natural products is used in the immunostimulating composition of the present invention. Specifically, plant extracts, crude products and the like containing Agrimol at a certain concentration obtained when extracting, separating and purifying Agrimol B from the above-mentioned natural products can be used in the immunostimulating composition of the present invention.

[0017] The immunostimulating composition containing Agrimole B or a plant extract containing Agrimole B as an active ingredient can be administered to animals and humans as a pharmaceutical composition with Agrimole B or the extract as is, or together with a conventional pharmaceutical formulation carrier. The dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected as needed. For example, oral preparations such as tablets, capsules, granules, fine granules, and powders, and parenteral preparations such as injections and suppositories can be included. The dosage of oral preparations is usually formulated to contain 1 to 1000 mg of Agrimole B in terms of the dry mass of the extract for adults, and it is appropriate to take this in several divided doses per day. When it is made into a food product, Agrimole B or an Agrimole B-containing plant extract can be dispersed in an appropriate diluting agent and then mixed with food ingredients to obtain a desired form. When it is added to food, it is mixed so that Agrimole B is contained at 1 to 1000 mg per 100 g of food.

[0018] In the present invention, oral preparations such as tablets, capsules, granules, fine granules, and powders containing Agrimole B or a plant extract containing Agrimole B are prepared according to conventional methods using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts. In addition to the composition of the present invention, oral preparations may appropriately contain binders, disintegrants, surfactants, lubricants, flow enhancers, flavorings, colorants, and flavors.

[0019] The following substances can be used as ingredients to be blended with Agrimol B or a plant extract containing Agrimol B. Examples of binders include starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, sodium methylcellulose, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, polyvinylpyrrolidone, and macrogol. Examples of disintegrants include starch, hydroxypropyl starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, and low-substituted hydroxypropyl cellulose. Examples of surfactants include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, and polyoxyethylene sorbitan fatty acid ester. Examples of lubricants include talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, and polyethylene glycol. Examples of flow promoters include light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate. The present invention will be described in further detail below with reference to test examples which confirmed the effects of the composition of the present invention. EXAMPLES

[0020] <Test on the phagocytosis-promoting effect of Agrimole B, agrimony extract, or concentrated agrimony extract on macrophage cells> 1. Test Method (1) Preparation of test samples An example of a plant extract containing Agrimole B is an extract of Agrimony. Preparation of agrimony extract and concentrated agrimony extract 100g of agrimony (Sengakuso, Fukuda Ryu Co., Ltd.) was mixed with 10 times the amount of 90% ethyl alcohol (1 kg) and reflux extraction was repeated twice. After completion, solid-liquid separation was performed to obtain an extract. The obtained extract was treated with activated charcoal and filtered through diatomaceous earth in the usual manner, then concentrated and freeze-dried to obtain a crude extract (solid amount 9.28g, yield 9.28% (vs. raw material)). This crude extract was used in this test as "Agrimony Extract". 8.51 g of this dried agrimony extract was dissolved and suspended (10% solids) in a 20% ethyl alcohol solution heated to 60°C, and the residue was recovered by diatomaceous earth filtration in a conventional manner. The residue was again dissolved in 90% ethyl alcohol, and filtered through diatomaceous earth in a conventional manner, and then concentrated and freeze-dried again to obtain a concentrate (0.59 g solids, 6.93% yield (relative to agrimony extract)). This was designated as the concentrated agrimony extract. This concentrated agrimony extract was obtained by removing 20% ​​ethyl alcohol soluble components from the agrimony extract, and fat-soluble components including Agrimole B were concentrated in it.

[0021] Determination of the content of Agrimole B in agrimony extract and concentrated agrimony extract The content of Agrimole B in the test samples used in this example was measured by a conventional method. The content was 0.036% by mass in the agrimony extract and 0.447% by mass in the concentrated agrimony extract. The content of Agrimole B in the test samples used in this example was measured by HPLC under the following conditions. Column: Wakosil-ll5C18 AR 4.6mm x 150mm Mobile phase: distilled water: acetonitrile: phosphoric acid = 100:900:1 Flow rate: 1mL / min Detection: UV288nm Injection volume: 10μL

[0022] Agrimole B Purified Agrimole B (Biorbyt) was prepared as a stock solution at 10 mM with dimethyl sulfoxide (DMSO). This was used in the following tests.

[0023] (2) Test method The test was carried out according to the following procedure. 1)Cell culture Macrophage cell line RAW264.7 (obtained from the European Collection of Authenticated Cell Cultures (ECACC)) established from mouse ascites-derived cells was cultured at a cell density of 3 × 10 4 The cells were seeded at 100 cells / well in a 24-well plate and cultured for 24 hours in DMEM (high glucose: Invitrogen) medium.

[0024] 2) Addition of Agrimole B, agrimony extract, and concentrated agrimony extract After removing the culture supernatant, the samples were dissolved in new medium, and 400 μl of this medium was added and cultured for 24 hours. The final concentrations of each test sample were adjusted to 1.25, 2.5, and 5 μM for Agrimole B, 12.5, 25, and 50 μg / ml for the agrimony extract, and 12.5, 25, and 50 μg / ml for the concentrated agrimony extract. As a positive control (macrophage activation promoter), lipopolysaccharide (LPS: manufactured by Sigma-Aldrich) was added to a final concentration of 1 μg / ml.

[0025] 3) Cytochalasin D (Cyt D) treatment The culture supernatant was removed, and cytochalasin D was added to the medium to a final concentration of 10 μM, followed by incubation at 37° C. for 1 hour.

[0026] 4) Measurement of phagocytosis rate Killed bacteria (Escherichia coli (K-12 strain) BioParticles™, Alexa Fluor™ 488 conjugate) were added to each well at 15,000 cells / well / 100 μl. The plate was then left to stand at room temperature for 20 minutes to allow the cells to phagocytose the killed bacteria. After that, the entire culture supernatant was removed, and the plate was washed with 1 ml of FACS buffer. After washing, 300 μl of fresh FACS buffer was added, the cells were collected with a cell scraper, placed in a tube with a cell strainer, and subjected to FACS analysis. The ratio of FACS-separated cells to total cells was defined as the phagocytosis rate.

[0027] 5) Statistical analysis The phagocytosis rate of each test sample was calculated by calculating the average value and standard deviation, and the significance test against the control (Cont) that did not contain the sample was performed by Dunnett's method. The significance level was set to p<0.05 (*) and p<0.01 (**).

[0028] 2. Test Results The results of the phagocytosis rate measurement are shown in Table 1 and FIG.

[0029] [Table 1]

[0030] When no additive was added (Cont), the phagocytosis rate was 18%. On the other hand, Agrimole B increased the phagocytosis rate in a concentration-dependent manner. The phagocytosis rate was 20% at 1.25μM, 21.5% at 2.5μM, and 27.7% at 5μM, showing a significant increase in the phagocytosis rate. Concentrated agrimony extract also increased the phagocytosis rate in a concentration-dependent manner, showing 19.3% at 12.5μg / ml, 20.1% at 25μg / ml, and 24.4% at 50μg / ml. The agrimony extract also slightly increased the levels compared to the control. Therefore, it was demonstrated that Agrimole B and concentrated Agrimony extract are effective as immunostimulating compositions since they increase the macrophage phagocytosis rate. In addition, the results of measuring the macrophage phagocytosis rate of agrimony extract and concentrated agrimony extract suggested that a content of Agrimole B of 0.4 mass% or more per total amount of dry matter of the plant extract is necessary to stimulate the immune system.

Claims

1. A food, medicine, or quasi-drug containing Agrimole B as an active ingredient for immunostimulation by improving macrophage phagocytic activity (excluding those containing naringin, hesperidin, strychnine, brudin, wogonin, and protocatechuic acid).

2. A food, medicine or quasi-drug containing as an active ingredient an extract of agrimor B containing at least 0.4% by mass or more of Agrimole B per total amount of dry matter, and for enhancing the phagocytic activity of macrophages to stimulate the immune system (excluding those containing naringin, hesperidin, strychnine, brudin, wogonin and protocatechuic acid).

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