Interleukin-9 production inhibitory composition

Abstract

The object of the present invention is to provide a novel interleukin-9 production inhibitory composition for anti-allergic purposes. [Solution] The interleukin-9 production inhibitory composition according to the present invention contains an aqueous extract of Terminalia bellirica fruit as an active ingredient. The term "composition" as used herein includes preparations such as liquid foods, supplements and food additives, food and beverages (excluding plants and animals themselves), food and beverage compositions (including processed food and beverages), pharmaceutical compositions, and other substances that can be ingested by animals (including humans).

Description

Technical Field

[0001] The present invention relates to an interleukin-9 production inhibitory composition.

Background Art

[0002] Allergic diseases such as bronchial asthma, allergic rhinitis, and atopic dermatitis have rapidly increased in the past few decades, and currently, it is considered that at least about 1 / 2 of the population suffers from some allergic disease. Many of the current allergy treatment drugs are symptomatic, and a more effective treatment method is desired in terms of the increasing number of affected patients and side effects associated with long-term use.

[0003] In response to such demands, various anti-allergy agents and anti-allergy compositions have been proposed in the past (see, for example, JP-A-2021-080197, JP-A-2021-28344, JP-A-2019-202955, etc.).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a novel interleukin-9 production inhibitory composition for anti-allergy.

Means for Solving the Problems

[0006] To solve the above problems, the inventors diligently conducted research and found that when an aqueous extract of Terminalia bellirica fruit was administered to the target, the production of interleukin (hereinafter sometimes abbreviated as "IL")-9 was significantly suppressed, thus completing the present invention. In other words, the present invention is as follows:

[0007] (1) An IL-9 production inhibitory composition containing an aqueous extract of Terminalia bellirica fruit as an active ingredient. The term "composition" as used herein includes preparations such as liquid foods, supplements and food additives, food and beverages (excluding plants and animals themselves), food and beverage compositions (including processed food and beverages), pharmaceutical compositions, and other substances that can be ingested by animals (including humans).

[0008] Furthermore, the above-mentioned invention can also be expressed as follows. (2) Use of aqueous extract of Terminalia bellirica fruit for the production of IL-9 production inhibitory compositions.

[0009] (3) Aqueous extract of Terminalia bellirica fruit for use in the manufacture of IL-9 production inhibitory compositions.

[0010] (4) A method using an aqueous extract of Terminalia bellirica fruit as an IL-9 production inhibitor.

[0011] (5) Aqueous extract of Terminalia bellirica fruit for use as an IL-9 production inhibitor. [Effects of the Invention]

[0012] To the best of the inventors' knowledge, no IL-9 production inhibitory composition containing an aqueous extract of Terminalia bellirica fruit as an active ingredient has existed previously. Therefore, the inventors of this application provide a novel IL-9 production inhibitory composition. [Brief explanation of the drawing]

[0013] [Figure 1] This graph shows the IL-9 gene expression suppression effect of Terminalia bellirica extract confirmed in Example 1. [Figure 2] This figure shows the inhibitory effect of Terminalia bellirica extract on nuclear translocation induced by ionomycin stimulation, as confirmed in Example 1. [Modes for carrying out the invention]

[0014] The present invention relates to an IL-9 production inhibitory composition comprising an aqueous extract of Terminalia bellirica fruit as an active ingredient. While the IL-9 production inhibitory composition according to the present invention is particularly desirable for administration to patients whose allergic symptoms do not improve with antihistamines, it is also possible to use the above-mentioned composition in combination with an antihistamine, anticipating that there may be patients whose allergic symptoms do not improve with antihistamines. In such cases, it is desirable to provide the above-mentioned composition and the antihistamine as an anti-allergic combination drug set to the patient.

[0015] The Terminalia extract described above is obtained by immersing crushed Terminalia or similar material in an extraction solvent for a certain period of time. Terminalia fruits may be used as is, or they may be dried before extraction. Examples of extraction solvents include water, ethanol, organic solvents, and mixtures thereof, but water is particularly preferred among these extraction solvents. Furthermore, the extraction solvent may be heated during the extraction process to increase extraction efficiency or to extract desirable components. The extract may be used as is, or it may be concentrated, dried, powdered, etc.

[0016] For example, Terminalia bellirica is preferred as the terminalia. Terminalia bellirica is a broad-leaved tree belonging to the genus Terminalia in the family Combretaceae. In the present invention, any part of Terminalia bellirica, such as the leaves, bark, roots, flowers, wood, fruit, or seeds, may be used, but it is preferable to use the part of the fruit excluding the fruit or the seeds (the pericarp or pulp).

[0017] When formulating the above-described composition according to the present invention as a beverage, it is desirable to use Terminalia extract, water, and a pH adjuster as raw materials. In such a case, the Terminalia extract is preferably contained in an amount of 0.001% by weight or more and 1% by weight or less, more preferably 0.001% by weight or more and 0.6% by weight or less, still more preferably 0.002% by weight or more and 0.3% by weight or less, even more preferably 0.003% by weight or more and 0.05% by weight or less, and particularly preferably in the range of 0.005% by weight or more and 0.020% by weight or less. Further, the pH adjuster is preferably contained in the range of 0.1% by weight or more and 0.15% by weight or less. Also, in such a case, the pH of the beverage is preferably in the range of 2 or more and 10 or less, more preferably in the range of 3 or more and 9 or less, still more preferably in the range of 4 or more and 7 or less, and particularly preferably in the range of 4 or more and 6 or less.

[0018] By forming the above-described composition according to the present invention into a single-packaged form in an amount appropriate for one intake, the composition according to the present invention can be appropriately and easily ingested, which is preferable from the viewpoint of usability. Although the amount appropriate for one intake varies depending on factors such as the degree of allergic reaction and individual differences, for example, it is preferably an amount such that the intake amount of Terminalia extract per day is in the range of 0.5 mg or more and 1 g or less, more preferably in the range of 1 mg or more and 500 mg or less, still more preferably in the range of 2 mg or more and 100 mg or less, even more preferably in the range of 3 mg or more and 70 mg or less, particularly preferably in the range of 5 mg or more and 50 mg, and particularly preferably in the range of 10 mg or more and 30 mg.

[0019] The "single-pack form" encompasses all forms, such as common packaging forms like containers with lids, bottles with caps, individual bags, pouches, tubes, etc. It is possible to clarify the use of the above-described composition according to the present invention by describing, on each single-pack or on the packaging containing a plurality of single-packs, explanations such as the use, efficacy, and intake method of the composition, and / or by enclosing, etc., an item (accompanying document) with such an explanation, and / or by posting, separately, an item such as a pamphlet with such an explanation.

[0020] Incidentally, it is preferable to display explanations such as the use, efficacy, function, types of active ingredients, and usage method of the above-described composition according to the present invention. The "display" referred to here includes all displays for informing consumers of the above effects. This display may be any display as long as it can remind or analogize the above-described display content, and may include all displays regardless of the purpose of the display, the content of the display, the object / media to be displayed, etc. For example, displaying the above explanation on the packaging / container of the product, displaying and exhibiting or distributing the above explanation in advertisements / price lists or transaction documents related to the product, or providing information containing these by electromagnetic (such as the Internet) methods.

[0021] When the composition according to the present invention is made into a product as an IL-9 production inhibitory composition, it is preferable that the product formed by packaging the IL-9 production inhibitory composition is labeled, for example, with an indication such as "inhibits IL-9 production".

[0022] In addition, the language used for the above display is not limited to the above examples, and it may be a language that is synonymous with such a meaning. As such a language, for example, various languages such as "suppresses the production of IL-9" and "restricts the production of IL-9" may be acceptable to consumers.

[0023] Furthermore, the above-mentioned composition according to the present invention can also be in the form of special-use foods, complete nutritional foods, nutritional supplements, foods for specified health uses, foods with functional claims, processed foods, etc. In addition, the composition can be incorporated into all kinds of food and beverages, such as drinks, liquid foods, dairy products (fermented milk), and confectionery. [Examples]

[0024] The present invention will be described in more detail below based on examples. These examples are not intended to limit the present invention.

[0025] 1. Terminalia bellirica extract (1) Terminalia bellirica extract sample For the Terminalia bellirica fruit extract (water extract) sample, we used Terminalia bellirica (manufactured by Toyo Shinyaku Co., Ltd.). This extract is obtained by water extraction from Terminalia bellirica fruit that has been harvested when ripe and dried. It is a relatively water-soluble powder (water-soluble) that exhibits a yellowish-brown to reddish-brown color.

[0026] (2) Preparation of Terminalia bellirica extract solution Water (Milli-Q water) was added to the above powdered extract sample to prepare Terminalia bellirica extract solutions of different concentrations: 25 mg / mL, 50 mg / mL, 100 mg / mL, and 200 mg / mL.

[0027] 2. Verification of the anti-allergic effect of Terminalia bellirica extract (1)Cell culture RBL-2H3 cells were cultured in MEM medium supplemented with 10% FBS and an antibiotic (antibiotic-antimycotic, manufactured by Nacalai Tesque Co., Ltd.). The cell culture was performed at approximately 80% confluence.

[0028] (2) Treatment with Terminalia bellirica extract solution RBL-2H3 cells seeded in 6-well plates were treated for 24 hours with each of the Terminalia bellirica extract solutions described above (added to final concentrations of Terminalia bellirica extract at 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL) and with a blank. After treatment, the RBL-2H3 cells were stimulated with 1 μM ionomycin for 2 hours. After stimulation, the RBL-2H3 cells were scraped off, and the IL-9 mRNA in the RBL-2H3 cells was quantified using real-time RT-PCR. Specifically, the results are as follows.

[0029] (2-1) Real-time RT-PCR (2-1-1) Extraction of total RNA from cells RBL-2H3 cells, Ca 2+ Mg 2+The RBL-2H3 cells were washed twice with phosphate buffer (hereinafter referred to as "PBS(-)"), and 700 μL of RNAiso Plus (manufactured by Takara Bio Inc.) was added to the washed RBL-2H3 cells, after which the RBL-2H3 cells were scraped off. The scraped RBL-2H3 cells were allowed to stand for 5 minutes, and then 140 μL of chloroform was added to the RBL-2H3 cells. Next, the chloroform containing the RBL-2H3 cells was shaken vigorously for 30 seconds to separate it into two phases, and then allowed to stand at room temperature for 5 minutes. Subsequently, the two-phase separated chloroform containing the RBL-2H3 cells was centrifuged at 15,000 rpm for 15 minutes at a temperature of 4°C. Then, the upper phase (aqueous phase) containing RNA was collected, the same amount of isopropanol was added to the upper phase, and the upper phase was shaken vigorously for 30 seconds, and then allowed to stand at room temperature for 10 minutes. Subsequently, the upper phase was centrifuged at 15,000 rpm for 15 minutes at a temperature of 4°C to obtain a pellet of RNA. To wash this pellet of RNA, 0.3 mL of 75% ethanol cooled to -30°C was added. The RNA-containing ethanol was then centrifuged at 15,000 rpm for 15 minutes at a temperature of 4°C to remove the ethanol. Diethyl pyrocarbonate-treated water (hereinafter referred to as "DEPC water") was added to the obtained pellet of RNA to prepare the RNA solution. The absorbance of the RNA solution at wavelengths of 260 nm and 280 nm was measured using a spectrophotometer (Thermo Fisher Scientific Nanodrop ND-1000). The total RNA concentration and purity in each RNA solution were calculated by hypothesis testing of the absorbance at 260 nm and the ratio of the two wavelengths.

[0030] (2-1-2) cDNA synthesis RNA solution and DEPC water were added to a sample tube to make an RNA solution equivalent to 1.0 μg of total RNA, bringing the total volume to 5 μL. A PrimeScript® RT reagent Kit was added to this to prepare a sample (see Table 1 for composition), and a reverse transcription reaction was performed on this sample using a thermal cycler (Biometra T3000 thermocycler) according to the program shown in Table 2 below.

[0031] [Table 1]

[0032] [Table 2]

[0033] (2-1-3) Real-time PCR The above-mentioned samples were mixed with reagents having the compositions shown in Table 3 to prepare 18 μL of reagent per well of a CFX Qualification Plate (BioRad). A PCR reaction was performed on the reagent using a Sequence Detector (BioRad CFX96 Touch Sequence Detection System), and the amplification curve of the PCR product was detected in real time. The detection results were analyzed using Sequence Detection software to quantify the mRNA in each RNA solution.

[0034] [Table 3]

[0035] The base sequences of the primers shown in Table 3 were as follows. IL-9 forward primer: 5'-GACGACCCATCATCAAAATGC-3' IL-9 reverse primer: 5'-CTGTGACATTCCCTCCTGGAA-3' IL-9 probe: FAM-TTGTGCCTCCCCATCCCATCTGAT-TAMRA

[0036] Furthermore, the PCR reaction was performed using the program shown in Table 4 below. [Table 4]

[0037] Furthermore, GAPDH (Glycelaldehyde-3-phosphate dehydrogenase) mRNA was used as an internal control gene to correct for differences in RNA purity and reverse transcription efficiency, which are major factors in the variability of quantitative RT-PCR. GAPDH mRNA was quantified using TaqMan rodent GAPDH Control Reagents (Thermo Fisher Scientific).

[0038] (2-1-4) Statistical analysis and discussion of results The experimental data were presented as mean value ± SEM, and statistical analysis was performed using Dunnett's multiple comparison test, with significance defined as P<0.05. The experimental data were plotted as a bar graph in Figure 1 (note that "control" in Figure 1 refers to RBL-2H3 cells that were not stimulated with 1 μM ionomycin). As is clear from Figure 1, Terminalia bellirica extract significantly suppressed IL-9 mRNA expression induced by ionomycin (also written as io or IO). Furthermore, it was found that this effect increased with increasing amounts of Terminalia bellirica extract added.

[0039] (3) Effects of Terminalia bellirica extract on the localization of NFATC1 and NFATC2 (3-1) Cell culture BHK21 cells constitutively expressing NFATc1-GFP fusion protein and NFATc2-GFP fusion protein were provided by Professor Osamu Kaminuma of Hiroshima University. These BHK21 cells were cultured in Dulbecco's Modified Eagle Medium, high glucose (DMEM, high glucose) containing 10% FBS, antibiotics (antibiotic-antimycotic), and puromycin (5 μg / mL, Nacalai Tesque Co., Ltd.) to maintain the expression of the cells.

[0040] (3-2) Observation of intracellular localization BHK21 cells were cultured in a 35 mm glass petri dish (IWAKI), and then treated with a 100 μg / mL Terminalia bellirica extract solution for 24 hours. After treatment, the BHK21 cells were stimulated with 1 μM ionomycin for 30 minutes. After stimulation, the BHK21 cells were washed twice with PBS(-), and the glass petri dish was filled with 4% paraformaldehyde (Nacalai Tesque Co., Ltd.) and fixed at 4°C for 15 minutes. Subsequently, the BHK21 cells were washed with PBS(-) for 5 minutes, and then the glass petri dish was filled with a solution of propidium iodide (PI) diluted with 1% BSA and 0.1% Tween 20 PBS(-) (final concentration 2.5 μg / mL), and allowed to stand for 10 minutes before nuclear staining of the BHK21 cells. After nuclear staining, BHK21 cells were washed with 0.1% Tween20 PBS(-) solution, then washed twice more with PBS(-) for 10 minutes each. Fluoromount (Diagnostic BioSystems) was then added to the washed BHK21 cells, and the cells were mounted on coverslips. After mounting, the BHK21 cells were observed using a confocal laser scanning microscope, yielding the image shown in Figure 2. In Figure 2, NFAT is shown in green, and the nuclei were stained orange. This indicates that Terminalia bellirica extract inhibited the nuclear translocation of both NFATs in response to ionomycin stimulation. Therefore, it was revealed that Terminalia bellirica extract exerts anti-allergic effects by inhibiting the calcineurin / NFAT signaling pathway and thereby suppressing the upregulation of IL-9 gene expression. [Industrial applicability]

[0041] The IL-9 production-inhibiting composition according to the present invention is presumed to exert an anti-allergic effect by suppressing the calcineurin / NFAT signaling pathway and thereby inhibiting the upregulation of IL-9 gene expression, and is useful for patients whose allergic symptoms do not improve with antihistamines.

Claims

[Claim 1] An interleukin-9 production inhibitory composition containing an aqueous extract of Terminalia bellirica fruit as an active ingredient.

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