Degranulation inhibitor (-)-neoechinulin a

Abstract

To expand use of (-)-Neoechinulin A produced also by Aspergillus genus and Eurotium genus, indole alkaloid that is secondary metabolite of marine fungus, used for molding dried bonito flakes.SOLUTION: Degranulation inhibition (-)-Neoechinulin A. It can also be used for food additives. It was confirmed that (-)-Neoechinulin A does not affect the activation of Lyn, a non-receptor tyrosine kinase located upstream of degranulation signal, and suppresses the phosphorylation of Syk, a non-receptor tyrosine kinase downstream. A decrease in phosphorylation levels of Akt, a serine / threonine kinase, was also confirmed. These results suggest that (-)-Neoechinulin A inhibits extracellular Ca2+ influx and suppresses degranulation by downregulating the Syk and Akt signaling pathways.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to (-)-neoechinulin A for inhibiting degranulation, and more particularly to (-)-neoechinulin A for inhibiting degranulation as a food additive. [Background technology]

[0002] (-)-Neoechinulin A is an indole alkaloid that is a secondary metabolite of marine fungi, and has been confirmed to be produced by Aspergillus and Eurotium species, which are used for mold-fermenting bonito flakes. It is also known that neoechinulin B can be used as an antiviral drug (Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2022-545553 [Patent Document 2] Special Publication No. 2022-531556 Summary of the Invention [Problem to be solved by the invention]

[0004] However, since it is not possible to predict what specific physiological activities (-)-neoechinulin A may have other than those already known, it is possible that it may have other physiological activities that have not yet been identified. On the other hand, since there are concerns about side effects on the living body when a newly developed drug is used, even if a specific efficacy or usefulness for the living body is discovered for a newly developed drug, it takes a long time and a lot of verification work to confirm its safety. An object of the present invention is to broaden the uses of (-)-neoechinulin A obtained from safe raw materials that are conventionally used for food. [Means for solving the problem]

[0005] Means for Solving the Problems The present inventors conducted intensive research to solve the above problems and discovered that (-)-neoechinulin A can be used to inhibit degranulation, thereby completing the present invention.

[0006] That is, the present invention provides: [1] The present invention provides (-)-neoechinulin A for inhibiting degranulation. [ka]

[0007] Another aspect of the present invention is [2] The present invention provides (-)-neoechinulin A for inhibiting degranulation as described in [1] above, which is used as a food additive. Effect of the Invention

[0008] The degranulation-inhibiting (-)-neoechinulin A of the present invention has an anti-allergic effect by inhibiting degranulation. The degranulation-inhibiting (-)-neoechinulin A of the present invention is also produced by the genera Aspergillus and Eurotium, which are used for mold-making of dried bonito flakes, and has therefore been routinely ingested as a food ingredient and is therefore highly safe. Furthermore, the degranulation-inhibiting (-)-neoechinulin A of the present invention inhibits degranulation in rat basophil-like cell line RBL-2H3 cells at a concentration of 0.25 to 1.0 mM, while not exhibiting cytotoxicity, and is therefore highly safe. The degranulation-inhibiting (-)-neoechinulin A of the present invention can be widely used as a food additive in cooking applications such as soup stock, tofu, vegetables, etc., the uses for which have traditionally been used for dried bonito flakes. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram for explaining the placement of a suspension of rat basophil-like cell line RBL-2H3 cells in a 96-well culture plate when preculture was performed in an incubator. [Diagram 2]FIG. 2 is a schematic diagram illustrating a 96-well culture plate in which the cell lysate and the culture supernatant have been transferred to separate empty wells. [Diagram 3] FIG. 3 is a schematic diagram illustrating a 96-well culture plate used for four procedures (a) to (d) on the cell lysate and cell supernatant. [Figure 4] FIG. 4 is a graph showing the relative release rate of β-hexosaminidase contained in the granules based on the control. [Diagram 5] FIG. 5 is a graph showing the results of cytotoxicity evaluated by the WST-8 method. [Figure 6] FIG. 6 is a graph showing the effect of (-)-neoechinulin A on intracellular calcium ion concentration. [Figure 7] FIG. 7 is a photograph substituting a drawing showing the results of Western blotting to examine the effect of (-)-neoechinulin A on signal transduction factors. [Figure 8] FIG. 8 is a graph showing the ratio of P-Lyn (a phosphorylated non-receptor tyrosine kinase that phosphorylates Syk) to Lyn (a type of non-receptor tyrosine kinase) in the phosphorylation of Lyn. [Figure 9] FIG. 9 is a graph showing the ratio of P-Syk (phosphorylated non-receptor tyrosine kinase phosphorylated by P-Lyn) to Syk (non-receptor tyrosine kinase phosphorylated by P-Lyn) in the phosphorylation of Syk. [Figure 10] FIG. 10 is a graph showing the ratio of P-PI3K (phosphorylated lipid kinase) to PI3K (phosphoinositide 3-kinase, lipid kinase) in phosphorylation of PI3K. [Figure 11] FIG. 11 is a graph showing the ratio of P-Akt (phosphorylated serine / threonine kinase) to Akt in the phosphorylation of Akt (serine / threonine kinase). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] After sensitization of rat basophil-like cell line RBL-2H3 cells with dinitrophenyl (DNP)-specific IgE, (-)-neoechinulin A was added to the culture medium, and degranulation was induced by antigen stimulation with DNP-HSA. The effect of the compound on antigen-induced degranulation was evaluated using the release rate of β-hexosaminidase contained in the granules as an index. The cytotoxicity was evaluated using the WST-8 method, and the effect on signal transduction factors was examined using Western blotting.

[0011] We investigated the effect of (-)-neoechinulin A on antigen-induced degranulation of rat basophil-like cell line RBL-2H3 cells. (-)-Neoechinulin A significantly inhibited degranulation without showing cytotoxicity. To clarify the mechanism by which degranulation is inhibited, intracellular calcium ions (Ca 2+ ) concentration, we found that antigen-induced intracellular Ca 2+ The increase in concentration was suppressed by (-)-neoechinulin A. In addition, when we examined the effects on signal transduction factors, we found that (-)-neoechinulin A did not affect the activation of Lyn (non-receptor tyrosine kinase), which is located upstream of the degranulation signal, but inhibited the phosphorylation of Syk (non-receptor tyrosine kinase), which is located downstream of Lyn. A decrease in the phosphorylation level of Akt (serine / threonine kinase) was also confirmed.

[0012] The terms used in the description of the present invention have the following meanings. [Table 1]

[0013] The present invention will now be described in detail with reference to examples, but the present invention is not limited to the following examples in any way. EXAMPLES

[0014] [Summary of the experiment to inhibit degranulation in rat basophil-like cell line RBL-2H3 cells] After sensitizing rat basophil-like cell line RBL-2H3 cells with dinitrophenyl (DNP)-specific IgE, (-)-neoechinulin A dissolved in dimethylsulfoxide (DMSO) was added to the culture medium at concentrations of 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM, and degranulation was induced by antigen stimulation with DNP-HSA. The effect of the compound on antigen-induced degranulation was evaluated using the release rate of β-hexosaminidase contained in the granules as an index. Cytotoxicity was evaluated using the WST-8 method. The effect of (-)-neoechinulin A on antigen-induced degranulation of rat basophil-like cell line RBL-2H3 cells was examined, and it was found that (-)-neoechinulin A significantly inhibited degranulation at concentrations of 0.25 to 1.0 mM without showing cytotoxicity.

[0015] [Preparation of sterile Tyrode's buffer (pH 7.4)] 4.77 g of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 7.89 g of NaCl, 0.37 g of KCl, 0.26 g of CaCl2·2H2O, 0.20 g of MgCl2·6H2O, 1.01 g of glucose, and 0.50 g of BSA were dissolved in distilled water, and the pH was adjusted to 7.4 with sodium hydroxide. The volume was adjusted to 1 L with distilled water to prepare Tyrode's buffer (20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.05% BSA). The solution was then sterilized by filtration using a membrane filter with a pore size of 0.22 μm, and stored in a refrigerator before use.

[0016] [Rat basophil-like cell line RBL-2H3 cells] The rat basophil-like cell line RBL-2H3 cells used in Example 1 were sold by American Type Culture Collection (ATCC) under the product number "CRL-2256". The RBL-2H3 cells (Rat basophilic leukemia cell line) are a rat basophil-like cell line used to evaluate the degranulation inhibitory effect. The rat basophil-like cell line RBL-2H3 cells were cultured in 10% FBS-DMEM medium at 37°C and 5% CO2. FBS (Fetal Bovine Serum) is fetal bovine serum, and is used as a growth additive for cell culture medium. DMEM (Dulbecco's Modified Eagle Medium) is Dulbecco's modified Eagle medium, and is used for culturing RBL-2H3 cells.

[0017] [Seeding and IgE sensitization] Rat basophil-like cell line RBL-2H3 cells were cultured at 4.0 × 10 4 10 ml of the cell suspension was added to a 100 mm dish to give a cell concentration of 4.0 × 10 5 The rat basophil-like cell line RBL-2H3 cells were cultured at 37°C and 5% CO2 in a dish of 10 cells / dish. After 48 hours, the rat basophil-like cell line RBL-2H3 cells had changed to their initial state (4.0 × 10 5 It was possible to visually confirm that the cells had proliferated by 70 to 80% in a 100 mm dish from the total number of cells / dish. Mixture A was prepared by adding 1 mL of fetal bovine serum (FBS, Sigma-Aldrich, product name: non-USA origin, sterile-filtered, suitable for cell culture) and 7 mL of Dulbecco's modified Eagle medium (DMEM) to a sterile 15 mL centrifuge tube. DMEM medium was prepared by mixing 13.4 g of DMEM (Sigma-Aldrich, product number D5648) and 3.7 g of NaHCO3 (Fujifilm Wako Pure Chemical Industries, product number 191-01305). The solution was prepared by dissolving 0.1 g of streptomycin (Sigma-Aldrich, product number S9137) and 0.0625 g of penicillin (Sigma-Aldrich, product number P7794) in 1 L of distilled water, followed by filtration sterilization using a pressure-sterilized housing.

[0018] After aspirating and removing the cell supernatant of rat basophil-like cell line RBL-2H3 cells that had proliferated to 70-80% in a 100 mm dish, 10 mL of sterilized phosphate buffer solution (sterile PBS) was added and the dish was gently shaken to wash the rat basophil-like cell line RBL-2H3 cells. PBS (Phosphate-buffered saline) stands for phosphate-buffered saline, and is the buffer solution used in this test to wash the RBL-2H3 cells.

[0019] The sterile PBS was removed by aspiration, and 1 mL of the mixed aqueous solution (0.25% trypsin, 0.02% ethylenediaminetetraacetic acid) and 1 mL of sterile PBS were added to the rat basophil-like cell line RBL-2H3 cells and incubated for 2 minutes at 37°C and 5% CO2.

[0020] Mixture A was added to the rat basophil-like cell line RBL-2H3 cells after the 2-minute incubation, and the rat basophil-like cell line RBL-2H3 cells adhered to the 100 mm dish were detached by repeatedly sucking up and injecting the cells with a pipette, to obtain a suspension of rat basophil-like cell line RBL-2H3 cells.

[0021] The above rat basophil-like cell line RBL-2H3 cell suspension was collected in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes. rpm (revolutions per minute) is the number of rotations per minute of the centrifuge.

[0022] After centrifugation, aspirate the supernatant from the 15 mL centrifuge tube and count 4.0 x 10 cells using a hemocytometer or cell counter. 5 A 10% FBS-DMEM solution was added to give a cell concentration of 100 cells / mL to prepare a suspension of rat basophil-like cell line RBL-2H3 cells.

[0023] A cell suspension of rat basophil-like cell line RBL-2H3 was seeded at 100 μL / well in a 96-well culture plate.

[0024] A 500 μg / mL monoclonal anti-dinitrophenyl (DNP)-IgE solution (Sigma-Aldrich, product number D8406) was diluted 5000-fold with 10% FBS-DMEM solution to make 100 ng / mL, and added to the above-mentioned seeded 96-well culture plate at 100 μL / well. DNP (Dinitrophenyl) is an incomplete antigen used to induce degranulation. It binds to the partial antigen antibody monoclonal anti-dinitrophenyl (DNP)-IgE, but due to its small molecular weight, it is a substance that does not exhibit the activity of inducing antibody production by itself.

[0025] IgE (Immunoglobulin E) is one of the major classes of immunoglobulins, and is present mainly on the cell surface of mast cells and basophils. It induces allergic responses by binding to allergens. In this study, RBL-2H3 cells were sensitized with monoclonal anti-dinitrophenyl (DNP)-IgE to make them sensitive to DNP-HSA (Dinitrophenyl-Human Serum Albumin), and used to induce degranulation.

[0026] DNP-HSA is a complete antigen consisting of dinitrophenyl and human serum albumin, which is used to stimulate RBL-2H3 cells with an antigen and induce degranulation.

[0027] The final concentration of monoclonal anti-dinitrophenyl (DNP)-IgE solution in the medium was 50 ng / mL, and the rat basophil-like cell line RBL-2H3 cells were 4.0 × 10 4 cells / well.

[0028] Next, pre-culture was carried out in an incubator at 37°C and 5% CO2 for 18 to 24 hours.

[0029] FIG. 1 is a schematic diagram for explaining the placement of a suspension of rat basophil-like cell line RBL-2H3 cells in a 96-well culture plate when preculture was performed in an incubator. As illustrated in Fig. 1, the rat basophil-like cell line RBL-2H3 cell suspension was actually placed in eight vertical rows from A to H and six horizontal columns from 1 to 6 of a 96-well culture plate designated by reference numeral 100. The rest of the procedure was as described above.

[0030] [From antigen stimulation to degranulation evaluation] After carrying out the pre-culture, the supernatant of each of the rat basophil-like cell line RBL-2H3 cells in the 96-well culture plate was removed by aspiration. The previously prepared sterile Tyrode's buffer was slowly added to each well of the 96-well culture plate at 200 μL / well to wash each of the rat basophil-like cell line RBL-2H3 cells. This wash was repeated twice.

[0031] (-)-Neoechinulin A was dissolved in dimethylsulfoxide (DMSO) to a concentration of 200 mM. 0 2 in DMSO as 1 , 2 2 , and 2 3 The neoechinulin A 200 mM solution was diluted 2 times. 0 , 2 1 , 2 2 , and 2 3 Doubling dilutions were prepared in sterile Tyrode's buffer at 0.5% to give concentrations of 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM. After removing the sterile Tyrode's buffer from the 96-well culture plate by suction, solutions of 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM were added to each well in triplicate at 120 μL / well. For the blank (no antigen stimulation with DNP-HSA) and control (with antigen stimulation with DNP-HSA), a solution of 0.5% DMSO in sterile Tyrode's buffer was added to each well in triplicate at 120 μL / well.

[0032] Then, each 96-well culture plate was used and cultured in an incubator at 37° C. and 5% CO 2 for 10 minutes.

[0033] During this incubation, a 10 mg / mL dinitrophenyl (DNP)-human serum albumin (HSA) solution (hereinafter referred to as "DNP-HSA solution") was diluted 16,000-fold (0.625 μg / mL) with sterile Tyrode's buffer.

[0034] After 10 minutes, the above 16,000-fold diluted (0.625 μg / mL) DNP-HSA solution was added at 10 μL / well to the 96-well culture plate after the 10 minutes of incubation so that the final DNP-HSA solution concentration in the medium was 48.1 ng / mL. Using this 96-well culture plate, the cells were cultured in an incubator at 37° C. and 5% CO 2 for 30 minutes. The total amount of solution in the well was 130 μL.

[0035] All subsequent operations were carried out outside the clean bench.

[0036] During the above incubation, the following reagents were prepared. (1) Preparation of 0.1% Triton X-100-Tyrode's buffer 4.77 g of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 7.89 g of NaCl, 0.37 g of KCl, 0.26 g of CaCl2·2H2O, 0.20 g of MgCl2·6H2O, 1.01 g of glucose, and 0.50 g of BSA were dissolved in distilled water, and the pH was adjusted to 7.4 with sodium hydroxide. The volume was adjusted to 1 L with distilled water to prepare Tyrode's buffer (20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.05% BSA). 10 mg of Triton X-100 (Sigma-Aldrich, product number T9284) was diluted in 10 mL of Tyrode's buffer to prepare 0.1% Triton X-100-Tyrode's buffer.

[0037] (2) Substrate solution As a substrate, p-nitrophenyl-2-acetoamid-2deoxy-β-D-glucopyranoside (Nacalai Tesque, Inc., product number 24937-81) was dissolved in 100 mM citrate buffer (pH 4.5) to obtain a 3.3 mM substrate solution (11.3 mg of substrate was dissolved in 10 mL of 100 mM citrate buffer).

[0038] The 96-well culture plate, which had been cultured in the incubator at 37°C and 5% CO2 for 30 minutes and had a total solution volume of 130 μL in each well, was removed from the incubator and placed on ice for 10 minutes to stop the degranulation reaction.

[0039] After standing on ice for 10 minutes, 130 μL of each culture supernatant was transferred to another empty well or another 96-well culture plate.

[0040] FIG. 2 is a schematic diagram illustrating a 96-well culture plate in which the cell lysate and the culture supernatant have been transferred to separate empty wells. 2, the cell lysate is placed in eight vertical rows from A to H and six horizontal columns from 1 to 6 of the 96-well culture plate designated by reference numeral 120, and the culture supernatant transferred thereto is placed in eight vertical rows from A to H and six horizontal columns from 7 to 12 of the 96-well culture plate designated by reference numeral 140. The rest of the operations are as described above.

[0041] After removing the culture supernatant, the previously prepared 0.1% Triton X-100-Tyrode's buffer solution was added to the rat basophil-like cell line RBL-2H3 cells at a ratio of 130 μL / well. Next, rat basophil-like cell line RBL-2H3 cells in 0.1% Triton X-100-Tyrode's buffer solution (130 μL / well) were sonicated while cooling on ice to disrupt the rat basophil-like cell line RBL-2H3 cells. This was used as a cell lysate. The sonication time per well is 5 seconds.

[0042] In a newly prepared clean 96-well culture plate, 24 wells (4 rows and 6 columns) were used as one unit, and 2 units of culture supernatant and 2 units of cell lysate (50 μL each) were added, and the plates were incubated at 37°C for 5 minutes in a constant temperature room.

[0043] Next, 2M glycine buffer was prepared as a reaction stop solution, and the substrate solution prepared in (2) above was used. FIG. 3 is a schematic diagram illustrating a 96-well culture plate used for four procedures (a) to (d) on the cell lysate and cell supernatant. In FIG. 3, the upper left is reference symbol (a), the upper right is reference symbol (b), the lower left is reference symbol (c), and the lower right is reference symbol (d). The above 24 wells in 4 columns and 6 rows were treated as one unit, and the following four operations (a) to (d) were carried out on each well of the four units.

[0044] (a) Add 100 μL / well of substrate solution to cell lysate (50 μL). (b) Add 100 μL / well of reaction stop solution to the cell lysate (50 μL). (c) Add 100 μL / well of substrate solution to cell supernatant (50 μL). (d) Add 100 μL / well of reaction stop solution to the cell supernatant (50 μL). These (a) to (d) were kept in a thermostatic chamber at a temperature of 37° C. for 25 minutes.

[0045] Next, 100 μL / well of a substrate solution or a reaction stop solution was added to each of (a) to (d) above as described below to terminate the enzyme reaction. (a) Add 100 μL / well of reaction stop solution (b) Add 100 μL / well of substrate solution (c) Add 100 μL / well of reaction stop solution (d) Add 100 μL / well of substrate solution

[0046] Since (-)-neoechinulin A may have an absorption region at 415 nm during absorbance analysis, the absorbance was measured under conditions (b) and (d), in which the substrate solution was added after the reaction stop solution was added, and this was used as a blank for the enzyme reaction (an absorbance value used to correct for the effects of other reagents such as neoechinulin A in order to calculate the release rate of β-hexosaminidase).

[0047] Next, the absorbance at 415 nm of each of the above wells (a) to (d) was measured using a microplate reader (manufactured by Bio-Rad, product name: iMark microplate reader, model number: 1681135JA).

[0048] The well conditions and the absorbance values ​​used in the calculation of the β-hexosaminidase release rate are shown in the following table. [Table 2]

[0049] The release rate of β-hexosaminidase was calculated according to the following formula.

number

[0050] The results are shown in Figure 4. FIG. 4 is a graph showing the relative release rate of β-hexosaminidase contained in the granules based on the control. Reference number 10 is the case where (-)-neoechinulin A was used, reference number 20 is the control, and reference number 30 is the blank. As shown in FIG. 4, (-)-neoechinulin A was shown to inhibit degranulation in rat basophil-like cell line RBL-2H3 cells at concentrations of 0.25 to 1.0 mM.

[0051] [Cytotoxicity test] The following equipment was used in the cytotoxicity test: Sterile 15 mL centrifuge tubes were used for centrifugation, and 100 mm dishes and 96-well culture plates for adhesion.

[0052] The reagents used were DMEM, FBS, sterile PBS, a mixed aqueous solution of trypsin and ethylenediaminetetraacetic acid (trypsin 0.25%, ethylenediaminetetraacetic acid 0.02%), monoclonal anti-dinitrophenyl (DNP)-IgE solution, DNP-HSA solution, and sterile Tyrode's buffer, each of which was the same as those exemplified above. The cell counting reagent used was Cell Count Reagent SF (Nacalai Tesque, Inc.).

[0053] [Preparation of sterile Tyrode's buffer (pH 7.4)] The preparation of sterile Tyrode's buffer (pH 7.4) used in the cytotoxicity tests was similar to that described above.

[0054] [Seeding and IgE sensitization] Rat basophil-like cell line RBL-2H3 cells were cultured at 4.0 × 104 10 ml of the cell suspension was added to a 100 mm dish to give a cell concentration of 4.0 × 10 5 The rat basophil-like cell line RBL-2H3 cells were cultured at 37°C and 5% CO2 in a dish of 10 cells / dish. After 48 hours, the rat basophil-like cell line RBL-2H3 cells had changed to their initial state (4.0 × 10 5 It was possible to visually confirm that the cells had proliferated by 70 to 80% in a 100 mm dish from the total number of cells / dish. Mixture A was prepared by adding 1 mL of fetal bovine serum (FBS, Sigma-Aldrich, product name: non-USA origin, sterile-filtered, suitable for cell culture) and 7 mL of Dulbecco's modified Eagle medium (DMEM) to a sterile 15 mL centrifuge tube. DMEM medium was prepared by dissolving 13.4 g of DMEM (Sigma-Aldrich, product number D5648), 3.7 g of NaHCO3 (Fujifilm Wako Pure Chemical Industries, Ltd., product number 191-01305), 0.1 g of streptomycin (Sigma-Aldrich, product number S9137), and 0.0625 g of penicillin (Sigma-Aldrich, product number P7794) in 1 L of distilled water, followed by filtration sterilization using a pressure-sterilized housing.

[0055] The cell supernatant of rat basophil-like cell line RBL-2H3 cells that had grown to 70-80% in a 100 mm dish was aspirated off, and then 10 mL of sterilized phosphate buffer solution (sterile PBS) was added and the rat basophil-like cell line RBL-2H3 cells were washed by gently shaking the dish.

[0056] The sterile PBS was removed by aspiration, and 1 mL of the mixed aqueous solution (0.25% trypsin, 0.02% ethylenediaminetetraacetic acid) and 1 mL of sterile PBS were added to the rat basophil-like cell line RBL-2H3 cells and incubated for 2 minutes at 37°C and 5% CO2.

[0057] Mixture A was added to the rat basophil-like cell line RBL-2H3 cells after the 2-minute incubation, and the rat basophil-like cell line RBL-2H3 cells adhered to the 100 mm dish were detached by repeatedly sucking up and injecting the cells with a pipette, to obtain a suspension of rat basophil-like cell line RBL-2H3 cells.

[0058] The above-mentioned rat basophil-like cell line RBL-2H3 cell suspension was collected in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes.

[0059] After centrifugation, aspirate the supernatant from the 15 mL centrifuge tube and count 4.0 x 10 cells using a hemocytometer or cell counter. 5 A 10% FBS-DMEM solution was added to give a cell concentration of 100 cells / mL to prepare a suspension of rat basophil-like cell line RBL-2H3 cells.

[0060] A cell suspension of rat basophil-like cell line RBL-2H3 was seeded at 100 μL / well in a 96-well culture plate.

[0061] A 500 μg / mL monoclonal anti-dinitrophenyl (DNP)-IgE solution (Sigma-Aldrich, product number D8406) was diluted 5000-fold with 10% FBS-DMEM solution to make 100 ng / mL, and added to the above-mentioned seeded 96-well culture plate at 100 μL / well. The final concentration of monoclonal anti-dinitrophenyl (DNP)-IgE solution in the medium was 50 ng / mL, and the rat basophil-like cell line RBL-2H3 cells were 4.0 × 10 4 cells / well.

[0062] Next, pre-culture was carried out in an incubator at 37°C and 5% CO2 for 18 to 24 hours.

[0063] [From antigen stimulation to degranulation evaluation] After carrying out the pre-culture, the supernatant of each of the rat basophil-like cell line RBL-2H3 cells in the 96-well culture plate was removed by aspiration. The previously prepared sterile Tyrode's buffer was slowly added to each well of the 96-well culture plate at 200 μL / well to wash each of the rat basophil-like cell line RBL-2H3 cells. This wash was repeated twice.

[0064] (-)-Neoechinulin A was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 200 mM. 0 2 in DMSO as 1 , 2 2 , and 2 3 The neoechinulin A 200 mM solution was diluted 2 times. 0 , 2 1 , 2 2 , and 2 3 Doubling dilutions were prepared in sterile Tyrode's buffer at 0.5% to give concentrations of 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM. After removing the sterile Tyrode's buffer from the 96-well culture plate by suction, solutions of 0.125 mM, 0.25 mM, 0.5 mM, and 1 mM were added to each well in triplicate at 120 μL / well. For the blank (no antigen stimulation with DNP-HSA) and control (with antigen stimulation with DNP-HSA), a solution of 0.5% DMSO in sterile Tyrode's buffer was added to each well in triplicate at 120 μL / well.

[0065] Then, each 96-well culture plate was used and cultured in an incubator at 37° C. and 5% CO 2 for 10 minutes.

[0066] During this incubation, a 10 mg / mL dinitrophenyl (DNP)-human serum albumin (HSA) solution (hereinafter referred to as "DNP-HSA solution") was diluted 16,000-fold (0.625 μg / mL) with sterile Tyrode's buffer.

[0067] After 10 minutes, the above 16,000-fold diluted (0.625 μg / mL) DNP-HSA solution was added at 10 μL / well to the 96-well culture plate after the 10 minutes of incubation so that the final DNP-HSA solution concentration in the medium was 48.1 ng / mL. Using this 96-well culture plate, the cells were cultured in an incubator at 37° C. and 5% CO 2 for 30 minutes. The total amount of solution in the well was 130 μL.

[0068] During the above culture, a DMEM solution containing 10% Cell Count Reagent SF (manufactured by Nacalai Tesque, Inc., product name: WST-8) was prepared as a cell counting reagent (referred to as 10% WST-8-DMEM). WST (water-soluble tetrazolium) is a water-soluble tetrazolium that is used to measure cell viability. In this test, WST-8 is used to measure the cell viability of RBL-2H3 cells and evaluate cytotoxicity.

[0069] After culturing for 30 minutes at 37°C and 5% CO2 in a 96-well culture plate, the cell supernatant was aspirated and DMEM was slowly added to each well of the 96-well culture plate at 200 μL / well to wash each rat basophil-like cell line RBL-2H3 cell.

[0070] After removing the DMEM from each well of the 96-well culture plate by suction, the previously prepared 10% WST-8-DMEM was added to each well of the 96-well culture plate at 100 μL / well, and the plates were cultured in an incubator at 37°C and 5% CO2 for 20 minutes.

[0071] As in the previous case, the absorbance was measured at 450 nm (reference: 655 nm) using a microplate reader.

[0072] The results are shown in Figure 5. FIG. 5 is a graph showing the results of cytotoxicity evaluated by the WST-8 method. Reference number 12 is the case where (-)-neoechinulin A was used, reference number 22 is the control, and reference number 32 is the blank. Here, blank refers to conditions in which degranulation of RBL-2H3 cells was not induced without antigen stimulation with DNP-HSA. Control refers to conditions in which degranulation of RBL-2H3 cells was induced with antigen stimulation with DNP-HSA. The case in which (-)-neoechinulin A was used refers to the case in which antigen stimulation with DNP-HSA was present. (-)-Neoechinulin A was added under conditions in which degranulation of RBL-2H3 cells was induced, and the β-hexosaminidase release rates of the blank and control were used as a control for comparison to evaluate whether degranulation was inhibited. As shown in FIG. 5, (-)-neoechinulin A was shown to have no toxicity to the rat basophil-like cell line RBL-2H3 cells at concentrations of 0.25 to 1.0 mM.

[0073] [Summary of intracellular calcium ion concentration measurement results] Next, to clarify the mechanism by which (-)-neoechinulin A inhibits degranulation, we investigated the intracellular calcium ion (Ca 2+ The effect of chlorine on the concentration of chlorine was examined. Addition of (-)-neoechinulin A at a concentration of 1 mM significantly suppressed antigen-induced intracellular Ca 2+ The increase in the concentration of was suppressed by (-)-neoechinulin A.

[0074] [Intracellular calcium ion concentration measurement] The following instruments were used to measure intracellular calcium ion concentrations: For centrifugation, a sterile 15 mL centrifuge tube was used, and for adhesion, a 100 mm dish and a 96-well culture plate were used. The 96-well culture plate was black.

[0075] The reagents used were DMEM, FBS, sterile PBS, a mixed aqueous solution of trypsin and ethylenediaminetetraacetic acid (trypsin 0.25%, ethylenediaminetetraacetic acid 0.02%), monoclonal anti-dinitrophenyl (DNP)-IgE solution (Sigma-Aldrich), and DNP-HSA solution (Sigma-Aldrich), each of which was the same as those exemplified above.

[0076] In addition, Fluo 3-AM (1-[2-Amino-5-(2,7-dichloro-6-acetoxymethoxy-3-oxo-9-xanthenyl)phenoxy]-2-(2-amino-5methylphenoxy)ethane-N,N,N',N'-tetraacetic acid,tetra(acetoxymethyl)ester)) (Dojindo Laboratories), 2x recording medium (see [Preparation of 2x recording medium] below), probenecid (Fujifilm Wako Pure Chemical Industries, Ltd., product number 165-15472), and Cremophor EL (Nacalai Tesque, Inc.) were used as reagents for measuring intracellular calcium ions.

[0077] [Preparation of 2x Recording Medium] 2x recording medium (40 mM HEPES, 230 mM NaCl, 1.6 mM MgCl2, 10.8 mM KCl, 3.6 mM CaCl2, and 27.6 mM glucose) was prepared by dissolving 9.53 g HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 13.44 g NaCl, 0.81 g KCl, 0.53 g CaCl2·2H2O, 0.33 g MgCl2·6H2O, and 4.972 g glucose in distilled water and adjusting the volume to 1 L with distilled water. To prepare 2x recording medium, we referred to the Dojindo Chemical Laboratory website (Dojindo, https: / / www.dojindo.co.jp / ).

[0078] [Seeding and IgE sensitization] Rat basophil-like cell line RBL-2H3 cells were cultured at 4.0 × 10 4 10 ml of the cell suspension was added to a 100 mm dish to give a cell concentration of 4.0 × 10 5 The cells were cultured in a single dish at 37°C and 5% CO2. After 48 hours, the rat basophil-like cell line RBL-2H3 cells were in their initial state (4.0 × 10 5 It was possible to visually confirm that the cells had proliferated by 70 to 80% in a 100 mm dish from the total number of cells / dish. Mixture A was prepared by adding 1 mL of fetal bovine serum (FBS, Sigma-Aldrich, product name: non-USA origin, sterile-filtered, suitable for cell culture) and 7 mL of Dulbecco's modified Eagle medium (DMEM) to a sterile 15 mL centrifuge tube. DMEM medium: 13.4 g of DMEM (Sigma-Aldrich, product number D5648) The solution was prepared by dissolving 3.7 g of NaHCO3 (Fujifilm Wako Pure Chemical Industries, Ltd., product number 191-01305), 0.1 g of streptomycin (Sigma-Aldrich Corporation, product number S9137), and 0.0625 g of penicillin (Sigma-Aldrich Corporation, product number P7794) in 1 L of distilled water, followed by filtration sterilization using a pressure-sterilized housing.

[0079] The cell supernatant of rat basophil-like cell line RBL-2H3 cells that had grown to 70-80% in a 100 mm dish was aspirated off, and then 10 mL of sterilized phosphate buffer solution (sterile PBS) was added and the rat basophil-like cell line RBL-2H3 cells were washed by gently shaking the dish.

[0080] The sterile PBS was removed by aspiration, and 1 mL of the mixed aqueous solution (0.25% trypsin, 0.02% ethylenediaminetetraacetic acid) and 1 mL of sterile PBS were added to the rat basophil-like cell line RBL-2H3 cells and incubated for 2 minutes at 37°C and 5% CO2.

[0081] Mixture A was added to the rat basophil-like cell line RBL-2H3 cells after the 2-minute incubation, and the rat basophil-like cell line RBL-2H3 cells adhered to the 100 mm dish were detached by repeatedly sucking up and injecting the cells with a pipette, to obtain a suspension of rat basophil-like cell line RBL-2H3 cells.

[0082] The above-mentioned rat basophil-like cell line RBL-2H3 cell suspension was collected in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes.

[0083] After centrifugation, aspirate the supernatant from the 15 mL centrifuge tube and count 4.0 x 10 cells using a hemocytometer or cell counter. 5 A 10% FBS-DMEM solution was added to give a cell concentration of 100 cells / mL to prepare a suspension of rat basophil-like cell line RBL-2H3 cells.

[0084] A cell suspension of rat basophil-like cell line RBL-2H3 was seeded at 100 μL / well in a 96-well culture plate.

[0085] A 500 μg / mL monoclonal anti-dinitrophenyl (DNP)-IgE solution (Sigma-Aldrich, product number D8406) was diluted 5000-fold with 10% FBS-DMEM solution to make 100 ng / mL, and added to the above-mentioned seeded 96-well culture plate at 100 μL / well. The final concentration of monoclonal anti-dinitrophenyl (DNP)-IgE solution in the medium was 50 ng / mL, and the rat basophil-like cell line RBL-2H3 cells were 4.0 × 10 4 cells / well.

[0086] Next, pre-culture was carried out in an incubator at 37°C and 5% CO2 for 18 to 24 hours.

[0087] [From antigen stimulation to intracellular calcium ion concentration measurement] Loading buffer and recording medium were prepared according to the compositions in the table below. (1) Loading buffer [Table 3]

[0088] (2) Recording Medium [Table 4]

[0089] [From antigen stimulation to degranulation evaluation] After carrying out the pre-culture, the supernatant of each of the rat basophil-like cell line RBL-2H3 cells in the 96-well culture plate was removed by aspiration. The previously prepared 2x recording medium was diluted 2-fold with sterilized distilled water to prepare 1x recording medium. 200 μL / well of 1x recording medium was slowly added to each well of the 96-well culture plate to wash each rat basophil-like cell line RBL-2H3 cell. This wash was repeated twice.

[0090] After removing the 1x recording medium by aspiration, 100 μL / well of loading buffer was added to each well of the 96-well culture plate, and the cells were cultured in an incubator at 37°C and 5% CO2 for 1 hour.

[0091] During this incubation, a 10 mg / mL monoclonal anti-dinitrophenyl (DNP)-human serum albumin (HSA) solution (hereafter referred to as "DNP-HSA solution") was diluted 16,000-fold (0.625 μg / mL) in 1x recording medium.

[0092] After 1 hour, the supernatant of the loading buffer solution in each well of the 96-well culture plate that had been cultured in an incubator for 1 hour was removed by aspiration. Next, the previously prepared 1x recording medium was slowly added to each well of the 96-well culture plate at 200 μL / well to wash each rat basophil-like cell line RBL-2H3 cell. This wash was repeated twice.

[0093] (-)-Neoechinulin A was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 200 mM to prepare a 200 mM solution of (-)-neoechinulin A. The 200 mM solution of (-)-neoechinulin A was added at 0.5% to the recording medium prepared according to the formulation in Table 4 to prepare a 1 mM (-)-neoechinulin A solution. After removing the 1x recording medium from each well in the 96-well culture plate by aspiration, 100 μL / well of (-)-neoechinulin A prepared to a concentration of 1 mM was added to each well in the 96-well culture plate. For the blank (condition without antigen stimulation with DNP-HSA) and control (condition with antigen stimulation with DNP-HSA), a solution containing 0.5% DMSO added to the recording medium prepared in Table 4 was added at 100 μL / well. The plate was incubated at 37°C for 10 minutes using a fluorescent plate reader (Tecan, product name: fluorescent microplate reader Infinite F200 Pro).

[0094] Next, the fluorescence intensity before antigen stimulation (0 seconds) was measured (λex = 485 nm, λem = 535 nm) using a fluorescence plate reader (manufactured by Tecan, product name: fluorescence microplate reader Infinite F200 Pro).

[0095] The previously prepared DNP-HSA solution was added to each well of the 96-well culture plate at 10 μL / well, and measurements were taken with a fluorescent plate reader for 10 seconds at a time up to 60 seconds. 10 μL / well of 1x recording medium was added to blank wells (conditions without antigen stimulation with DNP-HSA) instead of DNP-HSA solution.

[0096] The results are shown in Figure 6. FIG. 6 is a graph showing the effect of (-)-neoechinulin A on intracellular calcium ion concentration. Reference number 14 is the case where (-)-neoechinulin A was used, reference number 24 is the control, and reference number 34 is the blank. Here, blank refers to conditions without antigen stimulation with DNP-HSA that do not induce an increase in intracellular calcium ion concentration in RBL-2H3 cells. Control refers to conditions with antigen stimulation with DNP-HSA that induce an increase in intracellular calcium ion concentration in RBL-2H3 cells. The case where (-)-neoechinulin A was used refers to the case where antigen stimulation with DNP-HSA was present. (-)-Neoechinulin A was added under conditions that increase the intracellular calcium ion concentration in RBL-2H3 cells, and the calcium ion concentrations (evaluated by relative fluorescence intensity) of the blank and control were used as comparative controls to evaluate whether the increase in calcium ion concentration was suppressed. The vertical axis of Fig. 6 represents the relative fluorescence intensity, and the horizontal axis represents the reaction time (seconds). As shown in Fig. 6, it was confirmed that (-)-neoechinulin A suppressed the increase in intracellular calcium ion concentration in rat basophil-like cell line RBL-2H3 cells.

[0097] [Outline of Western blotting method] The effect of (-)-neoechinulin A on signal transduction factors was examined by Western blotting. (-)-Neoechinulin A was added at a concentration of 1 mM.

[0098] [Western blotting method] The following instruments were used for the Western blotting method: For centrifugation, use sterile 15 mL centrifuge tubes, 100 mm dishes and Six-well culture plates were used.

[0099] The reagents used were DMEM, FBS, sterile PBS, a mixed aqueous solution of trypsin and ethylenediaminetetraacetic acid (trypsin 0.25%, ethylenediaminetetraacetic acid 0.02%), monoclonal anti-dinitrophenyl (DNP)-IgE solution, DNP-HSA solution, and sterile Tyrode's buffer, each of which was the same as those exemplified above.

[0100] The following antibodies were used: CelLutic NuCLEAR Extraction Kit (Sigma-Aldrich) as a nuclear protein extraction kit; Mini Protean TGX Gel (Bio-Rad) as a gel for separating intracellular proteins; Trans-Blot Turbo Transfer Pack PVDF (Bio-Rad) as a protein membrane transfer kit; Precision Plus Protein Prestained Standard (Bio-Rad) as an electrophoresis molecular weight confirmation marker; Blocking One (Nacalai Tesque) as a blocking agent; primary antibodies (Cell Signaling Technology) as antibodies; and anti-rabbit IgG-HRP conjugated antibody (Cell Signaling Technology) as a secondary antibody.

[0101] [Preparation of sterile Tyrode's buffer (pH 7.4)] The preparation of sterile Tyrode's buffer (pH 7.4) used in the cytotoxicity tests was similar to that described above.

[0102] [Seeding and IgE sensitization] Rat basophil-like cell line RBL-2H3 cells were cultured at 4.0 × 10 4 10 ml of the cell suspension was added to a 100 mm dish to give a cell concentration of 4.0 × 10 5 The rat basophil-like cell line RBL-2H3 cells were cultured at 37°C and 5% CO2 in a dish of 10 cells / dish. After 48 hours, the rat basophil-like cell line RBL-2H3 cells had changed to their initial state (4.0 × 10 5 It was possible to visually confirm that the cells had proliferated by 70 to 80% in a 100 mm dish from the total number of cells / dish. Mixture A was prepared by adding 1 mL of fetal bovine serum (FBS, Sigma-Aldrich, product name: non-USA origin, sterile-filtered, suitable for cell culture) and 7 mL of Dulbecco's modified Eagle medium (DMEM) to a sterile 15 mL centrifuge tube. DMEM medium was prepared by dissolving 13.4 g of DMEM (Sigma-Aldrich, product number D5648), 3.7 g of NaHCO3 (Fujifilm Wako Pure Chemical Industries, Ltd., product number 191-01305), 0.1 g of streptomycin (Sigma-Aldrich, product number S9137), and 0.0625 g of penicillin (Sigma-Aldrich, product number P7794) in 1 L of distilled water, followed by filtration sterilization using a pressure-sterilized housing.

[0103] The cell supernatant of rat basophil-like cell line RBL-2H3 cells that had grown to 70-80% in a 100 mm dish was aspirated off, and then 10 mL of sterilized phosphate buffer solution (sterile PBS) was added and the rat basophil-like cell line RBL-2H3 cells were washed by gently shaking the dish.

[0104] The sterile PBS was removed by aspiration, and 1 mL of the mixed aqueous solution (0.25% trypsin, 0.02% ethylenediaminetetraacetic acid) and 1 mL of sterile PBS were added to the rat basophil-like cell line RBL-2H3 cells and incubated for 2 minutes at 37°C and 5% CO2.

[0105] Mixture A was added to the rat basophil-like cell line RBL-2H3 cells after the 2-minute incubation, and the rat basophil-like cell line RBL-2H3 cells adhered to the 100 mm dish were detached by repeatedly sucking up and injecting the cells with a pipette, to obtain a suspension of rat basophil-like cell line RBL-2H3 cells.

[0106] The above-mentioned rat basophil-like cell line RBL-2H3 cell suspension was collected in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes.

[0107] After centrifugation, aspirate the supernatant from the 15 mL centrifuge tube and count 4.0 x 10 cells using a hemocytometer or cell counter. 5 A 10% FBS-DMEM solution was added to give a cell concentration of 100 cells / mL to prepare a suspension of rat basophil-like cell line RBL-2H3 cells.

[0108] A cell suspension of rat basophil-like cell line RBL-2H3 was seeded at 1 mL / well in a 6-well culture plate.

[0109] A 500 μg / mL monoclonal anti-dinitrophenyl (DNP)-IgE solution (Sigma-Aldrich, product number D8406) was diluted 5000-fold with 10% FBS-DMEM solution to make it 100 ng / mL, and added to each of the seeded 6-well culture plates at 1 mL / well. The final concentration of monoclonal anti-dinitrophenyl (DNP)-IgE solution in the medium was 50 ng / mL, and the rat basophil-like cell line RBL-2H3 cells were 4.0 × 10 5 cells / well.

[0110] Next, pre-culture was carried out in an incubator at 37°C and 5% CO2 for 18 to 24 hours.

[0111] [From antigen stimulation to protein extraction] After carrying out the pre-culture, the supernatant of each of the rat basophil-like cell line RBL-2H3 cells in the 6-well culture plate was removed by aspiration. The previously prepared sterile Tyrode's buffer was slowly added to each well of the 6-well culture plate at 2 mL / well to wash each of the rat basophil-like cell line RBL-2H3 cells. This wash was repeated twice.

[0112] (-)-Neoechinulin A was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 200 mM to prepare a 200 mM solution of (-)-neoechinulin A. The 200 mM solution of (-)-neoechinulin A was added to sterile Tyrode's buffer at 0.5% to prepare a 1 mM (-)-neoechinulin A solution. After aspirating and removing the sterile Tyrode's buffer, 1 mM (-)-neoechinulin A solution dissolved in sterile Tyrode's buffer was added to each well of the 6-well culture plate at 1.9 mL / well. For the blank (condition without antigen stimulation with DNP-HSA) and control (condition with antigen stimulation with DNP-HSA), a solution of 0.5% DMSO added to sterile Tyrode's buffer was added to each well of the 6-well culture plate at 1.9 mL / well.

[0113] Next, each of the 6-well culture plates was used and cultured in an incubator at 37° C. and 5% CO 2 for 10 minutes.

[0114] During this incubation, a 10 mg / mL dinitrophenyl (DNP)-human serum albumin (HSA) solution (hereinafter referred to as "DNP-HSA solution") was diluted 10,000-fold (1 μg / mL) with sterile Tyrode's buffer.

[0115] After 10 minutes, the above 10,000-fold diluted (1 μg / mL) DNP-HSA solution was added at 0.1 mL / well to the 6-well culture plate after the 10 minutes of incubation so that the final DNP-HSA solution concentration in the medium was 50 ng / mL. Using this 6-well culture plate, the cells were cultured in an incubator at 37° C. and 5% CO 2 for 5 minutes. The total amount of solution in the well was 2 mL.

[0116] After 5 minutes, the supernatant in each well of the cultured 6-well culture plate was aspirated, and sterilized PBS was slowly added at 2 mL / well to wash each rat basophil-like cell line RBL-2H3 cell.

[0117] Intracellular proteins were extracted according to the procedure specified for the CelLutic NuCLEAR extraction kit (Sigma-Aldrich).

[0118] 3 μL of an electrophoresis molecular weight confirmation marker and 10 μL of each cell protein were added to a 4-15% mini-PROTEAN TGX gel (15 wells), and electrophoresis was performed at a constant voltage of 150 V for about 1 hour.

[0119] After electrophoresis, intracellular proteins were transferred to a PVDF membrane using a Trans-Blot Turbo Transfer System (Bio-Rad, product number 1704150J1). Each PVDF membrane used as a transfer membrane was blocked with 10 mL of a blocking agent (Blocking One, manufactured by Nacalai Tesque, Inc.) for 30 minutes at room temperature.

[0120] [Preparation method of 0.1% Tween-20-TBS] 24.2 g of Tris Base and 80 g of NaCl were dissolved in distilled water, adjusted to pH 7.6 with HCl, and then adjusted to 1 L of volume with distilled water to prepare 10x TBS (Tris-buffered saline). 10x TBS was diluted 10-fold with distilled water, and Tween-20 was added to a concentration of 0.1% to prepare 0.1% Tween-20-TBS. 0.1% Tween-20-TBS (t-TBS) is the buffer used for Western blotting in this study. 0.1% Tween-20-TBS was slowly added at 10 mL / membrane and washed for 3 minutes. This procedure was repeated three times.

[0121] The transfer membrane was immersed in each primary antibody diluted 1000-fold with 5% BSA-t-TBS, and reacted overnight at 4° C. with shaking.

[0122] [Table 5]

[0123] After the primary antibody reaction, 0.1% Tween-20-TBS was slowly added at 10 mL per membrane, and the membrane was washed for 3 minutes. This procedure was repeated three times.

[0124] The transfer membrane was immersed in anti-rabbit IgG-HRP-conjugated antibody (Cell Signaling Technology) diluted 1000-fold with 5% skim milk-t-TBS as a secondary antibody, and reacted with shaking at room temperature for 1 hour.

[0125] [Table 6]

[0126] After the reaction of the secondary antibody, 0.1% Tween-20-TBS was slowly added at 10 mL per membrane, and the membrane was washed for 3 minutes. This procedure was repeated three times.

[0127] The PVDF membrane was placed in contact with the luminescence solution of Immunostar-LD (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with the protein-binding surface facing down, and allowed to react for 30 seconds.

[0128] Luminescence from the target protein was confirmed using a ChemiDoc XRS system (Bio-Rad).

[0129] The results are shown in Figure 7. FIG. 7 is a photograph substituting a drawing showing the results of Western blotting to examine the effect of (-)-neoechinulin A on signal transduction factors. The reference symbol Blank indicates a blank, the reference symbol Control indicates a control, and the reference symbol NeoA indicates the case containing (-)-neoechinulin A. The same applies below. Here, the blank refers to a condition in which phosphorylation of kinases involved in degranulation induction (Lyn, Sky, PI3Kp38, and Akt) is not induced without antigen stimulation with DNP-HSA. The control refers to a condition in which phosphorylation of kinases involved in degranulation induction (Lyn, Sky, PI3Kp38, and Akt) is induced with antigen stimulation with DNP-HSA. The case in which (-)-neoechinulin A was used refers to a case in which antigen stimulation with DNP-HSA is present. (-)-neoechinulin A was added under conditions in which phosphorylation of kinases involved in degranulation induction (Lyn, Sky, PI3Kp38, and Akt) is induced, and the phosphorylation rates of Lyn, Sky, PI3Kp38, and Akt in the blank and control were used as controls to evaluate whether (-)-neoechinulin A inhibits kinase phosphorylation. This relationship is similar to the cases of Figures 8 to 11 below. In addition, in FIG. 7, Lyn represents non-receptor tyrosine kinase, p-Lyn represents phosphorylated non-receptor tyrosine kinase, Syk represents non-receptor tyrosine kinase, and p-Syk represents phosphorylated non-receptor tyrosine kinase, respectively. PI3K p85 refers to the lipid kinase subunit, and p-PI3K p85 refers to the phosphorylated lipid kinase subunit. Akt refers to serine / threonine kinase, p-Akt refers to phosphorylated serine / threonine kinase, and Actin refers to the protein actin. The same applies to the following cases:

[0130] FIG. 8 is a graph showing the ratio of P-Lyn (a phosphorylated non-receptor tyrosine kinase that phosphorylates Syk) to Lyn (a type of non-receptor tyrosine kinase) in the phosphorylation of Lyn. FIG. 9 is a graph showing the ratio of P-Syk (phosphorylated non-receptor tyrosine kinase phosphorylated by P-Lyn) to Syk (non-receptor tyrosine kinase phosphorylated by P-Lyn) in the phosphorylation of Syk. FIG. 10 is a graph showing the ratio of P-PI3K (phosphorylated lipid kinase) to PI3K (phosphoinositide 3-kinase, lipid kinase) in phosphorylation of PI3K. FIG. 11 is a graph showing the ratio of P-Akt (phosphorylated serine / threonine kinase) to Akt in the phosphorylation of Akt (serine / threonine kinase).

[0131] As a result, (-)-neoechinulin A was confirmed to have no effect on the activation of Lyn, a non-receptor tyrosine kinase located upstream of the degranulation signal, but inhibited the phosphorylation of Syk, a non-receptor tyrosine kinase located downstream of Lyn, and to have decreased the phosphorylation level of Akt, a serine / threonine kinase. These results suggest that (-)-neoechinulin A downregulates the Syk and Akt signaling pathways, thereby inhibiting extracellular Ca 2+ It was suggested that this inhibitor inhibits the influx and suppresses degranulation. [Industrial Applicability]

[0132] The degranulation-inhibiting (-)-neoechinulin A of the present invention is also produced by the Aspergillus and Eurotium genera used for mold-fermenting bonito flakes, and because it is highly safe, it can be widely used for degranulation inhibition to suppress allergies. In addition, it can also be used as a food additive for Japanese food, etc., a purpose that has traditionally been used for dried bonito. [Explanation of symbols]

[0133] 10,12,14,NeoA (-)-Neoechinulin A 20, 22, 24, Control 30, 32, 34, Blank

Claims

1. (-)-Neoechinulin A for inhibiting degranulation, represented by the following chemical formula. 【Chemistry 1】

2. The (-)-neoechinulin A for inhibiting degranulation according to claim 1, which is used as a food additive.

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