Compositions for suppressing differentiation to mucosal mast cells

Abstract

To provide compositions capable of reducing the ratio of mucosal mast cells by ingestion.SOLUTION: The invention relates to a composition for suppressing the differentiation to mucosal mast cells and an antiallergic composition which comprise a pine extract as the effective ingredient, and are expected to improve, ameliorate, suppress, treat or prevent symptoms of mucosal allergy and related food allergy, hay fever, gastritis, enteritis, diarrhea, ulcerative colitis, rhinitis, bronchitis, bronchial asthma, Loeffler syndrome, stomatitis as well as autoimmune symptoms.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a composition for inhibiting differentiation of mucosal mast cells. [Background technology]

[0002] Mast cells (fat cells) are a type of blood cell derived from hematopoietic stem cells and are distributed throughout various tissues in the body, similar to other immune cells such as macrophages and dendritic cells.

[0003] Mucosal mast cells (MMCs) are known as a type of mature mast cell. While connective tissue mast cells are localized in the skin and blood vessels, mucosal mast cells are localized in mucosal tissues within the body and express IgE receptors (FcεRIα) on their cell surface, which have high affinity for IgE.

[0004] Allergens and IgE form immune complexes via cross-linking with FcεRIα, inducing various responses. The outcome of this response is, for example, food allergy, which involves mucosal mast cells in intestinal tissue.

[0005] Food allergy develops when a food-derived substance acts as an allergen, forming an immune complex with IgE and cross-linking it with FcεRIα, activating mucosal mast cells in the intestinal tissue. In other words, activation of mucosal mast cells induces an allergic reaction in the body.

[0006] Patent Document 1 discloses that fermented soybeans and fermented wheat contained in fermented foods suppress abnormal differentiation of immature cells into mucosal mast cells and maintain the proportion of mucosal mast cells at a normal value, thereby reducing the proportion of mucosal mast cells per cell, thereby reducing the number of cells on which food allergens such as soybean allergens and wheat allergens act, and may have an inhibitory effect on allergies.

[0007] Furthermore, Patent Document 2 discloses that soy sauce may have an inhibitory effect against allergies by suppressing the abnormal differentiation of immature mast cells into mucosal mast cells and by maintaining the ratio of mucosal mast cells at a normal value, thereby reducing the ratio of mucosal mast cells per cell, thereby reducing the number of cells on which food allergens such as soybean allergens and wheat allergens act.

[0008] Thus, it is known that there are foods that suppress food-related allergies by suppressing the differentiation of mucosal mast cells and reducing the ratio of mucosal mast cells per cell. However, it was not previously known that extracts from wild pine trees have the effect of suppressing the differentiation of mucosal mast cells and suppressing food-related allergies. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] JP2018-083775A [Patent Document 2] JP2018-083776A Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, an objective of the present invention is to provide a composition for inhibiting differentiation into mucosal mast cells and an anti-allergic composition, which contain a pine extract as an active ingredient. [Means for solving the problem]

[0011] The present invention relates to a composition for inhibiting differentiation into mucosal mast cells, which contains a pine extract as an active ingredient, and is characterized in that the extract is obtained from pine leaves, particularly by hot water extraction, with Ryukyu pine being preferred.

[0012] The present invention is also characterized in that the composition is a differentiation-inhibiting composition that has the effect of inhibiting differentiation from immature mast cell lines to mucosal mast cells in the presence of interleukin-3 (IL-3) and interleukin-10 (IL-10) or transforming growth factor-β (TGF-β), and is an anti-allergic composition containing this composition as an active ingredient. Effect of the Invention

[0013] According to the composition for inhibiting differentiation into mucosal mast cells or the anti-allergic composition containing the pine extract of the present invention as an active ingredient, it is possible to obtain the effects of inhibiting differentiation of mucosal mast cells and suppressing food-related allergies. For example, it is expected that the symptoms of mucosal allergies and related food allergies, hay fever, gastritis, enteritis, diarrhea, ulcerative colitis, rhinitis, bronchitis, bronchial asthma, Loeffler's syndrome, stomatitis, and the like, as well as autoimmune symptoms, can be improved, alleviated, suppressed, treated, or prevented. [Brief description of the drawings]

[0014] [Figure 1] Graph showing total cell counts for each pine extract [Diagram 2] Graph showing mast cell counts for each pine extract [Diagram 3] Graph showing the number of MMCP-1 positive cells for each pine extract [Figure 4] Graph showing the rate of MMCP-1 positive cells for each pine extract [Diagram 5] Graph showing total cell counts by molecular weight of Ryukyu pine extract [Figure 6] Graph showing the number of MMCP-1 positive cells by molecular weight of Ryukyu pine extract [Figure 7] Graph showing the MMCP-1 positive cell rate by molecular weight of Ryukyu pine extract [Figure 8] Graph showing the amount of MCPT-1 expression by Ryukyu pine extract [Figure 9]Graph showing the MCPT-1 expression rate by Ryukyu pine extract [Figure 10] Graph showing the amount of MCPT-1 expression caused by Picea glehnii extract [Figure 11] Graph showing the amount of MCPT-1 expression over time during MMC induction after addition of TGF-β [Figure 12] Graph showing mRNA expression levels of MCPT-1 and MCPT-2 for each pine extract [Figure 13] A graph showing the results of analyzing the mRNA expression levels of Smad, a TGF-β signaling factor involved in the development of allergies. [Figure 14] A graph showing the results of an analysis of the mRNA expression levels of transcriptional corepressors and ubiquitin ligases (smad degrading enzymes), which are TGF-β signaling factors involved in the onset of allergies. [Figure 15] Graph showing the results of analysis of the mRNA expression levels of TGF-β type I receptor, which is a TGF-β signal transduction factor involved in the onset of allergies, and stem cell factor SCF receptor. [Figure 16] Graph showing the results of analysis of mRNA expression levels of high affinity IgE receptor and tyrosine kinase, which are FcεRI signal transduction factors involved in the onset of allergy. [Figure 17] A graph showing the results of analysis of the mRNA expression level of phospholipase PLCγ, which is important for mast cell activation. [Figure 18] A graph showing the results of analysis of the RNA expression levels of genes that contribute to the activation of phospholipase PLCγ. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The composition for inhibiting differentiation into mucosal mast cells and the anti-allergic composition containing the pine extract according to the present invention as an active ingredient are described below.

[0016] <<Experiment 1>> In order to investigate the inhibitory effect of a hot water extract of pine tree on the differentiation of mucosal mast cells, the following experiment was carried out.

[0017] Mouse mucosal mast cells (MMCs) express the surface marker molecules FcεRIα and c-Kit of mast cells, and also express a protease called MMCP-1 intracellularly. Mouse bone marrow-derived mast cells (BMMCs) express FcεRIα and c-Kit, but do not express MMCP-1, a marker for mucosal mast cells (MMCs). Therefore, the following experiment was conducted to investigate the inhibitory effect of hot water extract of pine tree on differentiation of mucosal mast cells (MMCP-1 positive cells = MMC) from mouse bone marrow-derived mast cells (BMMC).

[0018] <Experimental Method> 1. Preparation of Medium for Cell Culture The medium was prepared by adding reagents to GIBCO (registered trademark) RPMI1640 medium (Thermo Fisher Scientific) to the following final concentrations. 10% (w / v) FBS 1%(w / v)Penicilline / Streptomycin 2mM L-glutamine 0.05mM 2-mercaptoethanol

[0019] 2. Preparation of Mouse Bone Marrow-derived Mast Cell (BMMC) Suspension (1) Bone marrow was collected from mouse femurs, mixed with culture medium, and seeded. The cells were then cultured with medium changes approximately twice a week, and the cells were harvested at the 5th passage (P5). (2) The collected cells were washed once with medium, suspended in trypan blue (Gibco), and the cell number was counted using a hemocytometer. (3) Then, 5 × 10 6 A cell suspension was prepared by suspending the cells in medium at 100 cells / mL.

[0020] 3. Preparation of Cytokine Solutions Mouse recombinant protein IL-3 (200 μg / mL; Biolegend) and mouse recombinant protein IL-10 (200 μg / mL; Biolegend) were diluted with medium to 120 ng / mL, respectively, to prepare an IL-3 solution and an IL-10 solution.

[0021] 4.Culture method The "pine extract content" and medium shown in Table 1 were added to each well of a 96-well flat-bottom plate (manufactured by BDFALCON), and the initial cell number was increased to 5.0 × 10 5 cells / well and cultured for 3 days in the presence of 30 ng / mL of IL-3 and IL-10.

[0022] 5. Test Sample (1) The following pine trees were used as test samples. Akamatsu (produced in Nagano and Shizuoka prefectures) Black pine (Shizuoka Prefecture) Ryukyu pine (Okinawa Prefecture) (2) Each of the pine leaves (approximately 0.5 g) in (1) above, which were test samples, was soaked in 50 ml of hot water maintained at 98 to 105° C. for approximately 5 minutes to obtain an extract (pine extract). (3) The extract at room temperature was used as the original solution, which was diluted as shown in Table 1 and used in the prepared medium.

[0023] [Table 1]

[0024] 6. FACS Aria analysis for cell counting (1) After culturing, the cells were collected and washed twice with 1×PBS / 2% FBS, and then reacted with Fc block (BD Bioscience). (2) After the reaction, the cells were reacted with PE-labeled anti-mouse FcεRIα (Biolegend) and PE / cy7-labeled anti-mouse CD117 (c-Kit) (Biolegend). (3) After the reaction, the cells were washed once with 1×PBS and then reacted with Fixable Viability Dyee Fluor 450 (Ebioscience). (4) After the reaction, the cells were washed once with 1×PBS / 2% FBS and then fixed using IC buffer (Ebioscience). (5) The fixed cells were washed with Permeabilization buffer (Ebioscience), and then reacted with Alexa647-labeled anti-MMCP-1 antibody (purified anti-mouse MCPT-1 (mMCP-1) (Ebioscience) labeled with Alexa Flour 647 Antibody Labeling Kit (Life Technology)) to perform intracellular labeling. (6) The labeled cells were washed with Permeabilization buffer (Ebioscience), suspended in 200 μL of 1×PBS / 2% FBS, and then analyzed using a FACS Aria.

[0025] <Experimental Results> 1. Comparative Example (1) When IL-3 was used alone, little differentiation of mouse bone marrow-derived mast cells (BMMCs) into MMCP-1-positive cells was observed, and the rate of MMCP-1-positive cells was extremely low (Comparative Example 1 in Table 1). (2) In the coexistence of IL-3 and IL-10, differentiation of mouse bone marrow-derived mast cells (BMMCs) into MMCP-1-positive cells progressed, and the rate of MMCP-1-positive cells was high (Comparative Example 2 in Table 1). (3) From the results of these comparative examples, it was confirmed that this example is an experimental system capable of evaluating the induction of differentiation from mouse bone marrow-derived mast cells (BMMCs) to MMCP-1 positive cells.

[0026] 2. Red pine extract (Examples 1 to 3 in Table 1) (1) In the case of the Akamatsu extract, a remarkable inhibitory effect on the total cell count was observed in the 5-fold diluted solution, and the total cell count (including cells that had not yet differentiated from mouse bone marrow-derived mast cells (BMMC) into MMCP-1-positive cells (hereinafter referred to as "immature mast cells")) was approximately 37% compared to Comparative Example 2 (Figure 1). On the other hand, the 25-fold diluted solution was equivalent to Comparative Example 2, and the 125-fold diluted solution showed a slight increase over Comparative Example 2 (Figure 1). (2) Regarding the differentiation of mouse bone marrow-derived mast cells (BMMCs) into MMCP-1-positive cells, both the number of MMCP-1-positive cells and the MMCP-1-positive cell differentiation rate were lower in all of Examples 1 to 3 than in Comparative Example 2, and in particular, the MMCP-1-positive cell differentiation rate was concentration-dependently lower (Figures 3 and 4). (3) These results confirmed that the red pine extract has an inhibitory effect on the differentiation of mouse bone marrow-derived mast cells (BMMCs) into MMCP-1 positive cells.

[0027] 3. Black Pine Extract (Examples 4 to 6 in Table 1) (1) In the case of the Kuromatsu extract, a remarkable increase in the total cell count (including immature mast cells) was observed in the 5-fold diluted solution, which was an increase of about 63% compared to Comparative Example 2. On the other hand, in the 25-fold diluted solution, the result was equivalent to that in Comparative Example 2, and in the 125-fold diluted solution, a slight decrease was confirmed compared to Comparative Example 2 (Figure 1). (2) Similarly, a significant increase in the number of mucosal mast cells (MMC) was observed in the 5-fold diluted solution, which was an increase of about 110% in the number of mucosal mast cells (MMC) compared to Comparative Example 2. On the other hand, the number of MMC was similar to that in Comparative Example 2 in the 25-fold diluted solution, and a slight decrease was confirmed in the 125-fold diluted solution compared to Comparative Example 2 (Figure 2). (3) Regarding the differentiation of mouse bone marrow-derived mast cells (BMMCs) into MMCP-1-positive cells, both the number of MMCP-1-positive cells and the MMCP-1-positive cell differentiation rate were lower in all of Examples 4 to 6 than in Comparative Example 2, and in particular, the MMCP-1-positive cell differentiation rate was concentration-dependently low (Figures 3 and 4). (4) These results confirmed that the black pine extract has an inhibitory effect on the differentiation of mouse bone marrow-derived mast cells (BMMCs) into MMCP-1 positive cells.

[0028] 4. Ryukyu Pine Extract (Examples 7 to 9 in Table 1) (1) In the case of the Ryukyu pine extract, no change was observed in the total cell number (including immature mast cells) in any of Examples 7 to 9, and the number was similar to that in Comparative Example 2 (FIG. 1). (2) The number of mucosal mast cells (MMC) in the 5-fold diluted solution was increased by about 30% compared to Comparative Example 2. On the other hand, the numbers in the 25-fold and 125-fold diluted solutions were similar to those in Comparative Example 2 (Figure 2). (3) Regarding the differentiation of mouse bone marrow-derived mast cells (BMMCs) into MMCP-1-positive cells, both the number of MMCP-1-positive cells and the MMCP-1-positive cell differentiation rate were lower in all of Examples 7 to 9 than in Comparative Example 2, and in particular, the MMCP-1-positive cell differentiation rate was concentration-dependently lower (Figures 3 and 4). (4) These results confirmed that Ryukyu pine extract has the effect of inhibiting the differentiation of mouse bone marrow-derived mast cells (BMMCs) into MMCP-1 positive cells.

[0029] <<Experiment 2>> The following experiment was conducted to fractionate Ryukyu pine extract according to molecular weight and to investigate the effects of each component.

[0030] <Experimental Method> 1. Test Sample (1) The Ryukyu pine leaves were placed in a ziplock bag and stored in a freezer at -30°C overnight to freeze. (2) After freezing, the samples were dried in a vacuum freeze dryer (Tokyo Rikakikai Co., Ltd.) for more than 24 hours and dehydrated, and then crushed in a multi-beads shocker (Yasui Kikai Co., Ltd., 3000 rpm, 60 sec). (3) The crushed material was added to a tube containing 50 mL of boiled distilled water and mixed by inversion until the whole was homogenized. Two tubes were prepared. (4) The crushed material was autoclaved together with the tube (105°C, 5 min), and then allowed to stand and return to room temperature. (5) The tube was centrifuged (10,000 rpm, 4°C, 20 min), and the supernatant was sterilized by filtration using a syringe with a 0.22 µm filter and then transferred to a 50 mL tube. (6) In a safety cabinet, 12 mL of the hot water extract sample was added to an Amicon Ultra-15 centrifugal filter unit (membrane NMWL 10,000; this molecular weight fractionation filter can separate samples into those with molecular weights of 10 kDa or more and those below 10 kDa), and four tubes were prepared. (7) The filter unit of (6) was centrifuged in a fixed-angle rotor centrifuge (5000 G, 25° C., 20 min). (8) After centrifugation, the liquid with a molecular weight of less than 10 kDa was added to two Amicon Ultra-15 centrifugal filter units (membrane NMWL 3,000; this molecular weight cutoff filter can separate samples into those with molecular weights of 3 kDa or more and those with molecular weights of less than 3 kDa), and two of these were prepared. The liquid with a molecular weight of 10 kDa or more was frozen and stored. (9) The filter unit of (8) was further centrifuged in a fixed-angle rotor centrifuge (5000G, 25°C, 25 min), and after centrifugation, the 3 to 10 kDa sample and the less than 3 kDa sample were frozen and stored. (10) These samples were diluted as shown in Table 2.

[0031] [Table 2]

[0032] 2. Other experimental methods excluding test samples The experiment was carried out in the same manner as in Experiment 1.

[0033] <Experimental Results> 1. Comparative Example (1) As in Experimental Example 1, when IL-3 was used alone, little differentiation of mouse bone marrow-derived mast cells (BMMCs) into MMCP-1-positive cells was observed, and the rate of MMCP-1-positive cells was extremely low (Comparative Example 3 in Table 2). (2) In the coexistence of IL-3 and IL-10, differentiation of mouse bone marrow-derived mast cells (BMMCs) into MMCP-1-positive cells progressed, and the rate of MMCP-1-positive cells was also high (Comparative Example 4 in Table 2). (3) From the results of these comparative examples, it was confirmed that this example is an experimental system capable of evaluating the induction of differentiation from mouse bone marrow-derived mast cells (BMMCs) to MMCP-1 positive cells.

[0034] 2. Ryukyu pine extract, 3 kDa or less (Examples 10 to 12 in Table 2) At 3 kDa or less, no significant change was observed in the total cell number (including immature mast cells), but a concentration-dependent decrease in the number of MMCP-1-positive cells and the differentiation rate of MMCP-1-positive cells was observed (Figures 5 to 7).

[0035] 3. Ryukyu pine extract, 3-10 kDa (Examples 13 to 15 in Table 2) Although no significant change in the total cell number (including immature mast cells) was observed with 3 to 10 kDa, a concentration-dependent decrease in the number of MMCP-1 positive cells and the differentiation rate of MMCP-1 positive cells was observed (Figures 5 to 7).

[0036] 4. Ryukyu pine extract, 10 kDa or more (Examples 16 to 18 in Table 2) (1) The 5-fold diluted solution showed a significant decrease in the total cell count (including immature mast cells) (Figure 5). In addition, the number of MMCP-1 positive cells was also significantly decreased (Figure 6). (2) The 25-fold diluted solution also showed a significant decrease in the number of MMCP-1 positive cells (Figure 6). However, the MMCP-1 positive cell rate was equivalent to that of the 25-fold diluted solution of 3 kDa or less (Example 11 in Table 2) and the 25-fold diluted solution of 3 to 10 kDa (Example 14 in Table 2).

[0037] 5. Summary These results indicate that the use of Ryukyu pine extract fractions of 10 kDa or more resulted in a significant decrease in the total cell number (including immature mast cells), and that even the use of fractions of less than 10 kDa resulted in a concentration-dependent decrease in the number of MMCP-1 positive cells and the differentiation rate of MMCP-1 positive cells. As shown in the results of Experiment 1, even with pine extracts before fractionation (red pine, black pine, and Ryukyu pine), a concentration-dependent decrease in the number of MMCP-1-positive cells and the differentiation rate of MMCP-1-positive cells was observed.

[0038] <<Experiment 3>> The following experiment was carried out to investigate the inhibitory effect of Ryukyu pine extract on the expression of MCPT-1, an activation marker of mucosal mast cells.

[0039] <Experimental Method> 1. Extract (1) The test samples, Ryukyu pine, were provided by a company in Okinawa Prefecture. (2) Ryukyu pine leaves were dried and dehydrated for 24 hours using a vacuum freeze dryer (Tokyo Rikakikai Co., Ltd.), and then crushed using a multi-beads shocker (Yasui Kikai Co., Ltd., 3000 rpm, 60 sec). (3) The following extracts were obtained from the crushed material: Organic solvent extract: 2 g of the ground material was added to 40 ml of 70% ethanol, and then the mixture was shaken for 24 hours using a shaker (manufactured by AS ONE Co., Ltd.) to obtain an extract. Hot water extract: 2 g of the crushed material was added to 40 ml of boiling water, autoclaved (105°C, 5 min), allowed to stand and return to room temperature, then centrifuged (10,000 rpm, 20 min) to obtain the supernatant as an extract. (4) Each of the extracts from (3) above was filtered through a 0.22 μm filter and used in the experiments.

[0040] 2. Induction of mucosal mast cells from bone marrow cells (1) Preparation of mucosal mast cell culture medium (MC / 9) The following reagents were added to RPMI 1640 (Corning) medium to the following final concentrations. 10% (w / v) FBS (HyClone) 1%(w / v) Penicilline / Streptomycin (manufactured by GIBCO) 4mM L-glutamine (GIBCO) 0.05mM 2-mercaptoethanol (GIBCO) 25mM and 1M HEPES (ECACC) 1 mM sodium pyruvate (GIBCO) 1 × non-essential amino acids (GIBCO) (2) Preparation of mouse bone marrow cells Femurs harvested from BALB / c mice (Charles River Biosciences Japan) were placed in 5 ml / well of MC / 9 medium in a 6-well plate, washed, and then transferred to new 5 ml / well. Both ends of the washed femur were cut with autoclaved (121°C, 20 min) dissection scissors, and a 100 µm cell strainer (Falcon) was placed in one well of a 6-well plate. MC / 9 medium was aspirated from 10 ml / well using a 10 ml syringe, and a 26 G injection needle was attached. The femur was grasped with tweezers, an injection needle was inserted into one end, and the bone marrow was pushed into a 100 μm cell strainer using MC / 9 medium. Once all femoral bone marrow had been extruded, the plug of the 1 ml syringe was removed and the bone marrow remains were ground in a 100 μm cell strainer with a black rubber tip. A 100 μm cell strainer was attached to a 50 ml tube, and the entire cell suspension suspended with a pipette aid was aspirated into a 10 ml syringe and passed through the 100 μm cell strainer and transferred into the 50 ml tube. (3) Induction of cultured mast cells derived from bone marrow The bone marrow cell suspension prepared in (2) above was centrifuged (300×g, 4°C, 5 min), the supernatant was discarded, and the cells were placed in MC / 9 medium (without IL-3), after which IL-3 (Biolegend) was added to a concentration of 30 ng / ml. Thereafter, the cells were cultured at 37°C and 5% CO2. The medium was changed twice a week, and after one week, the cells were cultured at 1.0 × 10 6 The concentration was adjusted to cells / mL. Further, the same culture was carried out for one week. (4) Induction of mucosal mast cells The cultured mast cells derived from the bone marrow in (3) above were centrifuged, the supernatant was discarded, and the cells were suspended in MC / 9 medium (without IL-3) to a cell concentration of 5 × 10 5 ~10 6 Prepared at 50 ml / ml. The cells were plated at 50 μl / well in a 96-well plate. To the seeded cells, 50 μl of medium containing 150 ng / ml IL-3 ("IL-3" in the graphs of Figures 8 and 9 showing "Inhibitory effect of hot water extract of Ryukyu pine on MCPT-1 expression"), 50 μl of medium containing 150 ng / ml IL-10 (Biolegend) ("IL3 / IL10" in the graph of Figure 9 showing "Inhibitory effect of hot water extract of Ryukyu pine on MCPT-1 expression"), and 100 μl of medium (without IL-3) were added. As a positive control, a well plate to which only IL-3 was added was prepared. To this well plate, 150 μl of MC / 9 medium (without IL-3) was added without adding IL-10. Furthermore, as samples, 50 μl of diluted solutions (5x, 25x, 50x, 500x) of the Ryukyu pine extract mentioned above was added at each dilution ratio ("x5", "x50", and "x500" in the graph of Figure 8 showing the "MCPT-1 expression inhibitory effect of Ryukyu pine hot water extract"). Thereafter, the cells were cultured at 37°C and 5% CO2 for 3 days to induce the cells. Table 3 shows the conditions for creating each of the legends for the data series in Figures 8 and 9 (the final liquid volume of each sample was 250 μl / well).

[0041] [Table 3]

[0042] 3. ELISA Measurement Using the induced mucosal mast cells described in 2(4) above, the expression level of mast cell protease-1 (MCPT-1), a protein produced in a tissue-specific manner by mucosal mast cells, was quantified by ELISA using the MCPT-1 Mouse Uncoated ELISA kit with Plates (Thrombophila Fisher).

[0043] 4. FACS Measurement (1) After induction, the cells were collected by centrifugation and suspended in 100 μl of medium containing 100-fold diluted purified anti-mouse CD16 / 32 antibody (biolegend). (2) The cell solution was reacted on ice for 10 minutes, and 1 μl each of BV421 anti-mouse CD117 antibody (biolegend) and PE anti-mouse FcεRIα antibody (biolegend) was added and stirred. (3) 1 ml of PBS was added, followed by centrifugation (1300 rpm, 5 min, 25° C.), the supernatant was discarded, and 100 μl of PBS containing 400-fold diluted Zombie Green (biolegend) was added and suspended. (4) Then, the plate was kept in the dark at room temperature for 15 minutes. (5) 1 ml of medium was added and centrifuged (1300 rpm, 5 min, 25° C.), the supernatant was discarded, and the mixture was stirred. (6) After washing, the supernatant was discarded, and intracellular staining of MCPT-1 was performed according to the eBioscience (registered trademark) Intracellular Fixation & Permeabilization Buffe Set (Thermo Fisher). (7) MCPT-1 antibody was stained using Zip Alexa Fluor 488 Rapid Antibody Labeling Kit. (8) 10 FACS Accadrop Beads in each tube 6 300 μl of Permeabilization Buffer containing 10 μl of PBS was added. (9) The cells were transferred to BD Trucount tubes and analyzed using BD FACS Aria III. (10) Data analysis included calculating the total number of live cells and the number of MCPT-1-expressing cells.

[0044] <Experimental Results> 1. By acting on bone marrow cells with interleukin 3 (IL-3), differentiation and proliferation into monocytes / macrophages is stimulated. Furthermore, by adding interleukin 10 (IL-10) and stimulating them, they differentiate into "mucosal mast cells." It is believed that the activation of these mucosal mast cells causes food allergies. 2. From the graphs in Figures 8 and 9, it is clear that stimulation with IL-3 alone hardly results in the expression of MCPT-1, an activation marker for mucosal mast cells, but further stimulation with IL-10 leads to differentiation into "mucosal mast cells," resulting in a dramatically increased concentration and expression of MCPT-1. 3. However, the results of the samples to which the hot water extract of Ryukyu pine was added, diluted in three different concentrations, revealed that the hot water extract of Ryukyu pine had the effect of suppressing the expression of MCPT-1 in a concentration-dependent manner. 4. These results indicate that the hot water extract of Ryukyu pine, an endemic species of Okinawa, has the effect of suppressing the expression of MCPT-1, in other words, inactivating mucosal mast cells and suppressing the onset of food allergies.

[0045] <<Experiment 4>> The following experiment was carried out to investigate the inhibitory effect of Picea glehnii extract on the expression of MCPT-1, an activation marker of mucosal mast cells.

[0046] <Experimental Method> 1. Extract (1) The test sample, red spruce, was provided by Asahikawa National College of Technology, National Institute of Technology, Independent Administrative Institution. (2) Picea glehnii leaves were dried and dehydrated for 24 hours using a vacuum freeze dryer (Tokyo Rikakikai Co., Ltd.), and then crushed using a multi-beads shocker (Yasui Kikai Co., Ltd., 3000 rpm, 60 sec). (3) 2 g of the crushed material was added to 40 ml of boiling water, autoclaved (105°C, 5 min), allowed to stand and return to room temperature, then centrifuged (10,000 rpm, 4 min) to obtain an extract. (4) The extract from (3) above was filtered through a 0.22 μm filter and used in the experiment.

[0047] 2. Induction of mucosal mast cells from bone marrow cells (1) Preparation of mucosal mast cell culture medium (MC / 9) The following reagents were added to RPMI 1640 (Corning) medium to the following final concentrations. 10% (w / v) FBS (HyClone) 1%(w / v) Penicilline / Streptomycin (manufactured by GIBCO) 4mM L-glutamine (GIBCO) 0.05mM 2-mercaptoethanol (GIBCO) 25mM and 1M HEPES (ECACC) 1 mM sodium pyruvate (GIBCO) 1 × non-essential amino acids (GIBCO) (2) Preparation of mouse bone marrow cells Femurs harvested from BALB / c mice (Jackson Laboratory Japan, Inc.) were placed in 5 ml / well of MC / 9 medium in a 6-well plate, washed, and then transferred to new 5 ml / well. Both ends of the washed femur were cut with autoclaved (121° C., 20 min) dissection scissors, and a 100 μm Cell strainer (CORNING) was placed in one well of a 6-well plate. MC / 9 medium was aspirated from 10 ml / well using a 10 ml syringe, and a 28G injection needle was attached. The femur was grasped with tweezers, an injection needle was inserted into one end, and the bone marrow was pushed into a 100 μm cell strainer using MC / 9 medium. Once all femoral bone marrow had been extruded, the plug of the 1 ml syringe was removed and the bone marrow remains were ground in a 100 μm cell strainer with a black rubber tip. A 100 μm cell strainer was attached to a 50 ml tube, and the entire cell suspension suspended with a pipette aid was aspirated into a 10 ml syringe and passed through the 100 μm cell strainer and transferred into the 50 ml tube. (3) Induction of cultured mast cells derived from bone marrow The bone marrow cell suspension prepared in (2) above was centrifuged (300×g, 4°C, 5 min), the supernatant was discarded, and the cells were placed in MC / 9 medium (without IL-3), after which IL-3 (Biolegend) was added to a concentration of 30 ng / ml. Thereafter, the cells were cultured at 37°C and 5% CO2. The medium was changed twice a week, and after one week, the cells were cultured at 1.0 × 10 6 The concentration was adjusted to cells / mL. The same culture was continued for another 3 weeks. (4) Induction of mucosal mast cells The bone marrow-derived cultured mast cells in (3) above were centrifuged, the supernatant was discarded, and the cell concentration was 1×10 5 The cells were prepared in MC / 9 medium so that the number of cells per well was 100. 1 x 10 in a 96-well plate 5 The bone marrow cell suspension (2) above was seeded at 100 cells / well. To the 96-well plate seeded with the bone marrow cell suspension (2) above, 1 μl each of IL-3 adjusted to 7.5 ng / μl ("IL-3" in the graph in Figure 10 showing "Inhibitory effect of the hot water extract of Picea glehnii on MCPT-1 expression") and IL-10 adjusted to 15 ng / μl ("IL-3+10" in the graph in Figure 10 showing "Inhibitory effect of the hot water extract of Picea glehnii on MCPT-1 expression") was added. As a positive control, a well plate to which only IL-3 was added was prepared. To this well plate, MC / 9 medium (without IL-3) was added without adding IL-10. Furthermore, as samples, 50 μl of each of the diluted solutions (5 times, 25 times, and 50 times) of the Picea glehnii extract of the above 1 was added at each dilution ratio. Thereafter, the cells were cultured at 37°C and 5% CO2 for 3 days to induce the cells. The conditions for preparing each of the data series in FIG. 10 are shown in Table 4 (the amount of MC / 9 medium (without IL-3) added to all samples was adjusted so that the final volume was 250 μl / well).

[0048] [Table 4]

[0049] 3. ELISA Measurement Using the induced mucosal mast cells described in 2(4) above, the expression level of mast cell protease-1 (MCPT-1), a protein produced in a tissue-specific manner by mucosal mast cells, was quantified by ELISA using the MCPT-1 Mouse Uncoated ELISA kit with Plates (Thrombophila Fisher).

[0050] <Experimental Results> 1. By acting on bone marrow cells with interleukin 3 (IL-3), differentiation and proliferation into monocytes / macrophages is stimulated. Furthermore, by adding interleukin 10 (IL-10) and stimulating them, they differentiate into "mucosal mast cells." It is believed that the activation of these mucosal mast cells causes food allergies. 2. The graph in Figure 10 shows that stimulation with IL-3 alone hardly results in the expression of MCPT-1, an activation marker for mucosal mast cells. However, further stimulation with IL-10 leads to differentiation into "mucosal mast cells," resulting in a dramatically increased concentration and expression of MCPT-1. 3. However, the results of the samples to which hot water extract of Picea glehnii was added, diluted in three different concentrations, revealed that the hot water extract of Picea glehnii had the effect of suppressing the expression of MCPT-1 at a certain concentration. 4. These results indicate that the hot water extract of Picea glehnii, an endemic species of Hokkaido, also has the effect of suppressing the expression of MCPT-1, in other words, inactivating mucosal mast cells and suppressing the onset of food allergies. <<Experiment 5>> In order to comprehensively investigate the effects of extracts of Ryukyu pine and Picea glehnii on the gene group involved in the onset of allergies, the following experiment was carried out.

[0051] <Experimental Method> 1. Extract (1) The test samples, Ryukyu pine and red spruce, were provided by a company in Okinawa Prefecture and Asahikawa National College of Technology, an Independent Administrative Institution, National Institute of Technology, respectively. (2) Each of the pine leaves from (1) above was dried and dehydrated in a vacuum freeze dryer (Tokyo Rikakikai Co., Ltd.) for 24 hours, and then crushed in a multi-beads shocker (Yasui Kikai Co., Ltd., 3000 rpm, 60 sec). (3) 2 g of crushed pine leaves from each of the above (1) were added to 40 ml of boiling water, autoclaved (105°C, 5 min), allowed to stand and then allowed to return to room temperature. Then, the mixture was centrifuged (10,000 rpm, 4 min) to obtain each extract. (4) Each of the extracts from (3) above was filtered through a 0.22 μm filter and used in the experiments.

[0052] 2. Induction of mucosal mast cells from bone marrow cells (1) Preparation of mucosal mast cell culture medium (MC / 9) The following reagents were added to RPMI 1640 (Corning) medium to the following final concentrations. 10% (w / v) FBS (HyClone) 1%(w / v) Penicilline / Streptomycin (manufactured by GIBCO) 4mM L-glutamine (GIBCO) 0.05mM 2-mercaptoethanol (GIBCO) 25mM and 1M HEPES (ECACC) 1 mM sodium pyruvate (GIBCO) 1 × non-essential amino acids (GIBCO) (2) Preparation of mouse bone marrow cells Femurs harvested from BALB / c mice (Jackson Laboratory Japan, Inc.) were placed in 5 ml / well of MC / 9 medium in a 6-well plate, washed, and then transferred to new 5 ml / well. Both ends of the washed femur were cut with autoclaved (121° C., 20 min) dissection scissors, and a 100 μm Cell strainer (CORNING) was placed in one well of a 6-well plate. MC / 9 medium was aspirated from 10 ml / well using a 10 ml syringe, and a 28G injection needle was attached. The femur was grasped with tweezers, an injection needle was inserted into one end, and the bone marrow was pushed into a 100 μm cell strainer using MC / 9 medium. Once all femoral bone marrow had been extruded, the plug of the 1 ml syringe was removed and the bone marrow remains were ground in a 100 μm cell strainer with a black rubber tip. A 100 μm cell strainer was attached to a 50 ml tube, and the entire cell suspension suspended with a pipette aid was aspirated into a 10 ml syringe and passed through the 100 μm cell strainer and transferred into the 50 ml tube. (3) Induction of cultured mast cells derived from bone marrow The bone marrow cell suspension from (2) above was centrifuged (300×g, 4° C., 5 min), the supernatant was discarded, and the cells were placed in MC / 9 medium (without IL-3), after which IL-3 (Biolegend) was added to a concentration of 30 ng / ml. Thereafter, the cells were cultured at 37°C and 5% CO2. The medium was changed twice a week, and after one week, the cells were cultured at 1.0 × 10 6The concentration was adjusted to cells / mL. The same culture was continued for another 3 weeks. (4) Induction of mucosal mast cells The bone marrow-derived cultured mast cells (BMMCs) from (3) above were centrifuged, the supernatant was discarded, and the cell concentration was 1×10 5 The cells were prepared in MC / 9 medium so that the number of cells per well was 100. 1 x 10 in a 96-well plate 5 The bone marrow cell suspension (2) above was seeded at 100 cells / well. To the 96-well plate seeded with the bone marrow cell suspension described above in (2), four well plates were prepared in which 50 μl / well of each of the following four patterns was added: IL-3 only, IL-3 and TGF-β only, and IL-3 and TGF-β plus each of the pine extracts described above in 1.

[0053] 3.RNA-seq Analysis In the plate to which TGF-β was added, MCPT-1 expression was most active 8 to 10 hours after the addition of TGF-β (Figure 11). Therefore, the RNA expression levels of mucosal mast cells in each plate in 2(4) above at these time points were comprehensively analyzed using a next-generation sequencer.

[0054] <Experimental Results> 1. Gene expression analysis of MMC-differentiated cells 9 hours after addition of TGF-β showed that both extracts of Picea glehnii and Ryukyu pine suppressed the RNA expression levels of MCPT-1 and MCPT-2 (Figure 12).

[0055] 2. The results of comprehensive analysis of the RNA expression levels of genes involved in TGF-β signal transduction are as follows. (1) Of the TGF-β signal transduction factors (receptors) Smad3, Smad4, and Smad6, the extract of Picea glehnii significantly reduced the expression of Smad6, and the extract of Ryukyu pine significantly reduced the expression of both Smad3 and Smad6 (Figure 13). (2) Extracts of Picea glehnii significantly reduced the expression of the ubiquitin ligases Smurf1 and Smurf2, which are Smad degrading enzymes, and extracts of Ryukyu pine significantly reduced the expression of the transcriptional corepressor TRIM3 (Figure 14). (3) The extract of Picea glehnii significantly reduced the expression of the TGF-β type I receptor TGF-βRI and the SCF receptor c-kit, and the extract of Ryukyu pine significantly reduced the expression of the SCF receptor c-kit, which is a stem cell factor (Figure 15). From these results, the inhibitory effect of extracts of Picea glehnii and Ryukyu pine on MMC differentiation can be summarized as shown in the following table.

[0056] [Table 5]

[0057] 3. The results of analysis of the RNA expression levels of genes involved in FcεRI signal transduction are as follows. (1) Extracts from both Picea glehnii and Ryukyu pine suppressed the expression of the high-affinity IgE receptor FcεRI and the tyrosine kinase Syk, and inhibited FcεRI signal transduction (Figure 16). (2) Extracts of Picea glehnii significantly reduced the expression of phospholipase PLCγ (Plcg1 and Plcg2), and extracts of Ryukyu pine significantly reduced the expression of phospholipase PLCγ (Plcg2) (Figure 17). (3) Both extracts of Picea glehnii and Ryukyu pine significantly reduced the expression of genes contributing to the activation of phospholipase PLCγ (Plcg2) (Figure 18). From these results, the FcεRI signaling inhibitory effects of extracts of Picea glehnii and Ryukyu pine can be summarized as shown in the following table.

[0058] [Table 6]

[0059] 4. Discussion From the above results, it was found that both extracts of Picea glehnii and Ryukyu pine had the effect of inhibiting MMC differentiation, but there were differences in the MMC differentiation inhibitory effects of each extract as follows. (1) Inhibition of MMC differentiation in Picea glehnii R-Smad is inactivated by suppressing the expression of TGF-βRI, the TGF-β type I receptor. It promotes the degradation of Smad by increasing the expression of the ubiquitin ligases Smurf1 and Smurf2, which are Smad degrading enzymes. It inhibits SCF binding by reducing the expression of the SCF receptor c-kit. (2) Inhibition of MMC differentiation in Ryukyu pine It suppresses signal transduction by decreasing the expression of R-Smad (Smad3). It inhibits the regulation of R-Smad expression by decreasing the expression of the transcriptional corepressor TRIM33. It inhibits SCF binding by reducing the expression of the SCF receptor c-kit.

[0060] In addition, it was found that both extracts of Picea glehnii and Ryukyu pine inhibited the activation of mucosal mast cells and the production of effector substances by inhibiting FcεRI signal transduction through the following actions. In other words, the expression of the high-affinity IgE receptor FcεRI was suppressed, and FcεRI signaling was inhibited, resulting in decreased expression of the downstream tyrosine kinase Syk, suppression of activation of phospholipase PLCγ, and suppression of production of the second messenger (IP3). This inhibits degranulation of effector substances in mucosal mast cells, thereby suppressing the onset of allergies.

Claims

1. A composition for inhibiting differentiation into mucosal mast cells, comprising a pine extract as an active ingredient.

2. The composition for inhibiting differentiation into mucosal mast cells according to claim 1 , wherein the extract is an extract obtained from pine leaves.

3. The composition for inhibiting differentiation into mucosal mast cells according to claim 1 or 2, wherein the extract is obtained by hot water extraction.

4. 4. The composition for inhibiting differentiation into mucosal mast cells according to claim 1, wherein the extract is an extract obtained from Ryukyu pine.

5. 4. The composition for inhibiting differentiation into mucosal mast cells according to claim 1, wherein the extract is an extract obtained from Picea glehnii.

6. The composition for inhibiting differentiation into mucosal mast cells according to any one of claims 1 to 5, which has the effect of inhibiting differentiation of immature mast cells into mucosal mast cells in the presence of interleukin-3 and interleukin-10 or Transforming Growth Factor-β (TGF-β).

7. An antiallergic composition comprising the composition according to any one of claims 1 to 6 as an active ingredient.

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