Method for producing anticancer agent for bladder cancer

Abstract

To provide an anticancer agent for bladder cancer that exhibits superior activity against bladder cancer.SOLUTION: An anticancer agent for bladder cancer according to the present invention includes, as an active ingredient, at least one selected from the group consisting of inonotsusan A, inonotriol A, 21,24-cyclopenta-3β,21,25-trihydroxylanosta-8-ene, and inonotsusan B.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an anticancer agent for bladder cancer. [Background technology]

[0002] Chaga (Japanese name: Kabanoanatake) is generally known as a mushroom that parasitizes birch trees. Chaga contains functional components such as β-D-glucan, SOD (superoxide dismutase), and lignin, and is expected to have effects such as boosting immune function when ingested.

[0003] Compositions containing chaga-derived ingredients have been known in the past, such as those disclosed in Patent Documents 1 and 2. Patent Document 1 discloses an anti-glycation agent containing an extract of chaga (Irritella obliqua). Patent Document 2 discloses a functional health composition characterized by lactic acid bacteria fermentation of a mixture containing rice bran extract, rice koji extract, and an extract of chaga (Irritella obliqua) liquid culture medium. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2019-43887 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-153813 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention is based on the discovery of triterpenes in Chaga that exhibit excellent anti-bladder cancer effects. The purpose of the present invention is to provide a novel anti-bladder cancer drug. [Means for solving the problem]

[0006] As a result of research conducted by the inventors to solve the above-mentioned problems, they discovered that inonotusan A, inonotustriol A, 21,24-cyclopenta-3β,21,25-trihydroxylanost-8-ene, or inonotusan B has excellent anti-bladder cancer activity.

[0007] Various aspects for solving the above problems will be described. The anticancer agent for bladder cancer in Aspect 1 contains at least one selected from inonotusan A, inonotustriol A, 21,24-cyclopenta-3β,21,25-trihydroxylanost-8-ene, and inonotusan B as an active ingredient.

[0008] Aspect 2 is the anticancer agent for bladder cancer according to Aspect 1, wherein the active ingredient is extracted from chaga. Aspect 3 is the anticancer agent for bladder cancer according to Aspect 1 or 2, wherein the bladder cancer is muscle-invasive bladder cancer. [Effects of the Invention]

[0009] The anti-bladder cancer agent of the present invention can exert excellent anti-bladder cancer activity. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the fractionation scheme for obtaining fractions 15-9, 25-3, 25-5, and 26-2 from chaga. [Figure 2] FIG. 2 shows the results of ESI-MS (positive mode) mass spectrometry of fraction 15-9. [Figure 3] FIG. 3 shows the results of ESI-MS (positive mode) mass spectrometry of fraction 25-3. [Figure 4] FIG. 4 shows the results of ESI-MS (positive mode) mass spectrometry of fraction 25-5. [Figure 5] FIG. 5 shows the results of ESI-MS (positive mode) mass spectrometry of fraction 26-2. [Figure 6]FIG. 6 shows the results of measuring the 1H-NMR spectrum of the component (inonotusan A) in fraction 15-9. [Figure 7] FIG. 7 shows the results of measuring the 1H-NMR spectrum of the component (inonotutriol A) in fraction 25-3. [Figure 8] FIG. 8 shows the results of measuring the 1H-NMR spectrum of the component in fraction 25-5 (21,24-cyclopenta-3β,21,25-trihydroxylanost-8-ene). [Figure 9] FIG. 9 shows the results of measuring the 1H-NMR spectrum of the component (inonotusan B) in fraction 26-2. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, one embodiment of the anticancer agent for bladder cancer of the present invention will be described. The anticancer agent for bladder cancer of this embodiment contains at least one active ingredient selected from inonotusane A, inonotustriol A, 21,24-cyclopenta-3β,21,25-trihydroxylanosta-8-ene, and inonotusane B.

[0012] (active ingredient) Inonotusan A has a structure represented by the following general formula (1).

[0013] [ka] Inonottriol A has the structure shown in the following general formula (2).

[0014] [ka] 21,24-Cyclopenta-3β,21,25-trihydroxylanost-8-ene has the structure shown in the following general formula (3).

[0015] [ka] Inonotusan B has a structure represented by the following general formula (4).

[0016] [ka] The four active ingredients above all have the molecular formula C 30 H 50 O3, with a molecular weight of approximately 458. They are a group of compounds that share a common skeleton with lanostane triterpenes, differing only in the relative configurations at the 21st and 24th positions.

[0017] The above four active ingredients may be chemically synthesized, or may be crude extracts or refined products extracted from natural materials using water, hydrophilic organic solvents, etc. An example of a natural material used as an extraction raw material is chaga. Chaga belongs to the family Inonotaceae and is a type of mushroom that parasitizes birch trees. Its scientific name is Inonotus obliquus and its Japanese name is Kabanoanatake. There are no particular limitations on the raw material for chaga extraction, and any of the fruiting body, mycelium, or liquid culture of mycelium may be used.

[0018] The above four active ingredients can be extracted from chaga using known extraction methods based on purified or standard products, such as extraction methods using water, hydrophilic organic solvents, mixed solvents such as water / hydrophilic organic solvents, and supercritical extraction. The hydrophilic organic solvent used in this embodiment can be appropriately selected from lower alcohols such as ethanol, methanol, and isopropanol that dissolve in water, as well as ketones such as acetone. These hydrophilic organic solvents can be used alone or in combination of two or more. Among these, water is preferred from the perspective of extraction efficiency.

[0019] When water is used as a solvent, the volume of water used is preferably 5 to 30 times, and more preferably 10 to 25 times, the mass of the chaga raw material powder. When the volume of water used is 5 times or more, the extraction efficiency of the target component can be further improved. When the volume of water used is 30 times or less, the process time for concentration and the like can be shortened, and work efficiency can be improved.

[0020] Furthermore, when extracting using water as a solvent, it is preferable to remove impurities such as dust that may be mixed in during collection and pulverize the extract before the extraction process in order to improve the extraction efficiency of the target component. The extraction temperature is preferably 5 to 40°C. When the extraction temperature is within this range, the extraction efficiency of the target component can be improved. The extraction operation may be carried out, for example, for 4 hours or more while stirring at the extraction temperature. After the target component has been sufficiently extracted under the above extraction conditions, a crude extract can be obtained by filtration using a filter paper, diatomaceous earth, or the like.

[0021] Next, the four active ingredients of this embodiment can be separated and purified from the solvent extract using one or more chromatographies, using the purified product or a standard as an index. Known chromatographies, such as gas chromatography, liquid chromatography, supercritical fluid chromatography, and thin-layer chromatography, can be used. Liquid chromatographies include column chromatography, more specifically, high-performance liquid chromatography (HPLC) and open-column chromatography. Chromatographic supports include ion exchange chromatography, partition chromatography (normal-phase and reverse-phase chromatography), adsorption chromatography, and molecular exclusion chromatography. For partition chromatography, it is preferable to use a silica gel support or an ODS support, specifically, because of its excellent separation efficiency of the extracted components. By appropriately combining these, the target component can be purified using a known method.

[0022] (action) The above four active ingredients exert a strong anti-bladder cancer effect. More specifically, they are expected to be effective in treating, preventing, and alleviating various symptoms of bladder cancer. Therefore, for the purpose of achieving anti-bladder cancer effects, they can be used as an anti-bladder cancer drug containing at least one of the above four active ingredients.

[0023] (Usage form) As a specific formulation, the anticancer agent for bladder cancer can be preferably applied as a medicine for bladder cancer patients, or as a food or drink for alleviating or preventing various symptoms of bladder cancer.

[0024] When the anticancer agent for bladder cancer of this embodiment is used as a pharmaceutical, any administration method can be adopted, such as oral administration, intravascular administration, transdermal administration, or intraperitoneal administration. The dosage form can be appropriately selected according to the administration method, and examples include powders, powders, granules, tablets, capsules, pills, suppositories, liquids, and injections. Furthermore, if necessary, additives such as excipients, bases, emulsifiers, solvents, and stabilizers may be added.

[0025] When the anticancer agent for bladder cancer of this embodiment is applied to food and beverage products, it can be appropriately incorporated into anti-bladder cancer food and beverage products. The anticancer agent for bladder cancer of this embodiment can be used by adding it to various food or beverage ingredients. The form of the food and beverage product is not particularly limited, and may be any of liquid, powder, gel, solid, etc., and the dosage form may be any of tablets, capsules, granules, and drinks. Among these, capsules are preferred because of their reduced hygroscopicity. The food and beverage product may also contain other ingredients, such as gelling agent-containing foods, sugars, flavorings, sweeteners, oils and fats, base materials, excipients, food additives, secondary ingredients, and bulking agents.

[0026] (Effects of this embodiment) The effects of the anticancer agent for bladder cancer of this embodiment will be described. (1) The anticancer agent for bladder cancer of this embodiment contains, as an active ingredient, at least one selected from inonotusan A, inonotustriol A, 21,24-cyclopenta-3β,21,25-trihydroxylanost-8-ene, and inonotusan B. These active ingredients can exhibit excellent anti-bladder cancer effects that have not been reported before.

[0027] (2) Muscle-invasive bladder cancer (MIBC) is known to have a poor prognosis due to its high stem cell potential, metastatic potential, and treatment resistance. The anticancer agent for bladder cancer of this embodiment is expected to exert an anti-bladder cancer effect against such muscle-invasive bladder cancer.

[0028] (Example of change) The above embodiment may be modified as follows: The above embodiment and the following modifications may be combined with each other within the scope of technical compatibility.

[0029] The anticancer agent for bladder cancer in the above embodiment can be applied not only to humans but also to pets such as dogs, livestock, and other domestic animals. [Example]

[0030] The present invention will be described in more detail below with reference to examples. Note that the present invention is not limited to the following examples, and various modifications can be made without departing from the spirit of the present invention.

[0031] <Test Example 1: Isolation of components with anti-bladder cancer activity> Using the fractionation scheme shown in Figure 1, chaga powder was used as the extraction raw material, and after water extraction, various silica gels were used as separation carriers for fractionation. Using cytotoxic activity against canine bladder cancer organoids as an indicator, fractions with such activity and a single peak (fractions 15-9, 25-3, 25-5, and 26-2) were obtained.

[0032] (1) Test method for evaluating cytotoxicity against canine bladder cancer organoids Since canine bladder cancer has been confirmed to share similarities with human bladder cancer in etiology, pathology, symptoms, genetic profile, and treatment, we screened for anticancer activity by assessing cytotoxicity using canine bladder cancer organoid cells (DBCO), as described below.

[0033] Organoid cells were harvested by dissolving the gel in the culture medium for canine bladder cancer organoid cells with 5mM EDTA / PBS. They were treated with 0.05% trypsin for 5 minutes at 37°C. They were then passed through a 70µm cell strainer and counted. Cells were mixed with 10µL of gel and seeded into a 96-well plate (2000 cells / well). The gel solidified in an incubator, and 100µL of culture medium was added. The samples were processed the next day, and 72 hours later, 10µL of Presto Blue was added. Fluorescence intensity (570nm) was measured after 4 hours, and the blank was subtracted to calculate the relative survival rate, with the control survival rate set at 1.

[0034] (2) Fraction 15-9 100g of chaga powder was suspended in 2.5L of water and extracted at room temperature. The supernatant was obtained by centrifugation.

[0035] Next, the obtained supernatant was fractionated by column chromatography under the following separation condition 1 to obtain a total of seven fractions (fractions 1-1 to 1-7). (Separation condition 1) Column: OSD flash CC (inner diameter 5 x 5 cm) (YMC) Mobile phase: H2O, 20%, 50%, 70%, 100% MeOH, CHCl3 / MeOH / H2O (6:4:1), 100% MeOH + 0.05% TFA Column temperature: room temperature Fraction 1-5 (86.9 mg) of 100% MeOH obtained under separation condition 1 was further fractionated under separation condition 2 of column chromatography described below to obtain a total of seven fractions (fractions 3-1 to 3-7).

[0036] (Separation condition 2) Column: SiO2open CC (inner diameter 2 × 10 cm) (Fuji Silysia Chemical Ltd.) Mobile phase: n-hexane / EtOAc (7:3, 6:4, 5:5), CHCl3, CHCl3 / MeOH (19:1, 9:1), CHCl3 / MeOH / H2O (6:4:1) Column temperature: room temperature Fraction 3-3 (12.2 mg) was further fractionated under the following column chromatography separation condition 3 to obtain a total of 12 fractions (fractions 15-1 to 15-12).

[0037] (Separation condition 3) Column: COSMOSIL 5C 18 AR-II (inner diameter 10 x 250 mm) (Nacalai Tesque) Solvent: 60% water / acetonitrile (0-6 min) 60% to 90% water / acetonitrile gradient (6 to 56 min) 90% acetonitrile (56-100 min) Flow rate: 2mL / min Detection wavelength: UV 220 nm Column temperature: room temperature A single peak was obtained for fraction 15-9 (0.6 mg) with an elution time of 76.8 to 79.2 minutes. Furthermore, the viability of canine bladder cancer organoid cells was measured using fraction 15-9 (vs. control), and was found to be 0.651 at a concentration of 10 μg / mL. The anticancer effect of fraction 15-9 on canine bladder cancer organoid cells was confirmed.

[0038] (3) Collection of Fraction 25-3 and Fraction 25-5 Fraction 15-6 (1.9 mg) with an elution time of 62.4 to 65.4 minutes obtained by fractionation under the above separation condition 3 was further fractionated under the following column chromatography separation condition 4 to obtain a total of 6 fractions (fractions 25-1 to 25-6).

[0039] (Separation condition 4) Column: COSMOSIL 5C 18 AR-II (inner diameter 10 x 250 mm) (Nacalai Tesque) Solvent: methanol:water = 84:16 Flow rate: 2mL / min Detection wavelength: UV 220 nm Column temperature: room temperature A single peak was obtained for fraction 25-3 (0.7 mg) with an elution time of 47.4 to 49.8 minutes. Furthermore, the viability of canine bladder cancer organoid cells (vs. control) was measured and found to be 0.697 at a concentration of 10 μg / mL. The anticancer effect of fraction 25-3 on canine bladder cancer organoid cells was confirmed.

[0040] A single peak was obtained for fraction 25-5 (0.4 mg) with an elution time of 60 to 63.5 minutes. In addition, the viability of canine bladder cancer organoid cells (vs. control) was measured and found to be 0.280 at a concentration of 10 μg / mL, demonstrating the anti-cancer effect of fraction 25-5 on canine bladder cancer organoid cells.

[0041] (4) Collection of fraction 26-2 Fraction 3-5 (6.2 mg) obtained by fractionation under the above separation condition 2 was further fractionated under the following column chromatography separation condition 5 to obtain a total of 11 fractions (fractions 19-1 to 19-11).

[0042] (Separation condition 5) Column: COSMOSIL 5C 18 AR-II (inner diameter 10 x 250 mm) (Nacalai Tesque) Solvent: 50% water / acetonitrile (0-6 min) 50% to 80% water / acetonitrile gradient (6 to 56 min) 80% acetonitrile (56-76 min) Flow rate: 2mL / min Detection wavelength: UV 220 nm Column temperature: room temperature Fraction 19-9 (0.6 mg) with an elution time of 63.6 to 64.8 was further fractionated under the following column chromatography separation condition 6 to obtain a total of four fractions (fractions 26-1 to 26-4).

[0043] (Separation condition 6) Column: COSMOSIL 5C 18 AR-II (inner diameter 10 x 250 mm) (Nacalai Tesque) Solvent: methanol:water = 84:16 Flow rate: 2mL / min Detection wavelength: UV 220 nm Column temperature: room temperature A single peak was obtained for fraction 26-2 (0.5 mg) with an elution time of 42 to 44.5 minutes.

[0044] In addition, the viability of canine bladder cancer organoid cells (vs. control) was measured and found to be 0.745 at a concentration of 10 μg / mL, demonstrating the anti-cancer effect of fraction 26-2 on canine bladder cancer organoid cells.

[0045] <Test Example 2: Structural analysis of fractions 15-9, 25-3, 25-5, and 26-2> (1) Measurement of molecular weight by mass spectrometry The molecular weights of fractions 15-9, 25-3, 25-5, and 26-2 were measured using a mass spectrometer (Compact QTOF, Bruker). Figures 2 to 5 show the extracted ion chromatograms (XICs) of the intensities of specific m / z values ​​extracted from the MS scan data of fractions 15-9, 25-3, 25-5, and 26-2, respectively.

[0046] As a result, each fraction had m / z: 481.3617 [M+Na] + Each fraction had a molecular weight of 458.33061 and a molecular formula of C 30 H 50 It was estimated to be O3.

[0047] (2) Structural determination of fraction 15-9 using nuclear magnetic resonance (NMR) The component with a molecular weight of 458 in fraction 15-9 was analyzed using a nuclear magnetic resonance spectrometer (Avance 600 MHz, manufactured by Bruker). 1H-NMR and 13 The structure was analyzed by measuring C-NMR (hereinafter the same). The sample was dissolved in CDCl3 deuterium chloroform. Nuclear magnetic resonance spectrum 1 The results of H-NMR measurements are shown in Figure 6. Nuclear magnetic resonance spectrum 1 H-NMR and 13 The results of C-NMR measurements are shown in Table 1.

[0048] In determining the structure of each fraction, we also referred to the following publications: "21,24-Cyclolanostanes revisited: Structural revision and biological evaluation" Fitoterapia, Volume 156, January 2022, 105101; and "Triterpenoids from Inonotus obliquus and their antitumor activities" Fitoterapia, March 2015, 101:34-40.

[0049] [Table 1] As a result, the compound in fraction 15-9 was found to be inonotusane A (molecular weight 458, molecular formula: C) shown in the above chemical formula (1). 30 H 50 O3).

[0050] (3) Structural determination of fraction 25-3 using nuclear magnetic resonance (NMR) Regarding the component with a molecular weight of 458 in fraction 25-3, 1 H-NMR and 13 The structure was analyzed by measuring C-NMR. The sample was dissolved in CDCl3 deuterated chloroform. Nuclear magnetic resonance spectrum 1 The results of H-NMR measurements are shown in Figure 7. Nuclear magnetic resonance spectrum 1 H-NMR and 13 The results of C-NMR measurements are shown in Table 2.

[0051] [Table 2] As a result, the compound in fraction 25-3 was found to be inonottriol A (molecular weight 458, molecular formula: C) shown in the above chemical formula (2). 30 H 50 O3).

[0052] (4) Structural determination of fraction 25-5 using nuclear magnetic resonance (NMR) Regarding the component with a molecular weight of 458 in fraction 25-5, 1 H-NMR and 13 The structure was analyzed by measuring C-NMR. The sample was dissolved in CDCl3 deuterated chloroform. Nuclear magnetic resonance spectrum 1 The results of H-NMR measurements are shown in Figure 8. Nuclear magnetic resonance spectrum 1 H-NMR and 13 The results of C-NMR measurements are shown in Table 3.

[0053] [Table 3] As a result, the compound in fraction 25-5 was 21,24-cyclopenta-3β,21,25-trihydroxylanost-8-ene (21,24-cyclopenta-3β,21,25-trihydroxylanost-8-ene, molecular weight 458, molecular formula: C 30 H 50 O3).

[0054] (5) Structural determination of fraction 26-2 using nuclear magnetic resonance (NMR) Regarding the component with a molecular weight of 458 in fraction 26-2, 1 H-NMR and 13 The structure was analyzed by measuring C-NMR. The sample was dissolved in CDCl3 deuterated chloroform. Nuclear magnetic resonance spectrum 1 The results of H-NMR measurement are shown in Figure 9. Nuclear magnetic resonance spectrum 1 H-NMR and 13 The results of C-NMR measurements are shown in Table 4.

[0055] [Table 4] As a result, the compound in the fraction 26-2 was found to be inonotusane B (molecular weight 458, molecular formula: C) represented by the chemical formula (4). 30 H 50 O3).

[0056] The compounds of the above chemical formulas (1) to (4) have not been known to have anti-bladder cancer activity until now, and this test revealed a novel action effect.

Claims

1. An anti-cancer agent for bladder cancer, comprising at least one active ingredient selected from inonotusan A, inonotustriol A, 21,24-cyclopenta-3β,21,25-trihydroxylanost-8-ene, and inonotusan B.

2. The anticancer agent for bladder cancer according to claim 1, wherein the active ingredient is extracted from chaga.

3. The anticancer agent for bladder cancer according to claim 1 or 2, wherein the bladder cancer is muscle-invasive bladder cancer.

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