Method for producing cancer cell proliferation inhibitor, cancer cell proliferation inhibitor, and method for treating cancer

Abstract

Provided are a method for producing a cancer cell proliferation inhibitor, a cancer cell proliferation inhibitor obtained by the production method, and a method for treating cancer. The present invention relates to, for example, a method for producing a cancer cell proliferation inhibitor, the method including: a mixing step for mixing worm castings and water; and a recovery step for recovering vaporized water generated from the mixture obtained in the mixing step to obtain a liquid. In the mixing step, it is preferable to further mix an organic substance together with the worm castings and the water.

Description

Method for producing a cancer cell proliferation inhibitor, cancer cell proliferation inhibitor, and method for treating cancer

[0001] The present invention relates to a method for producing a cancer cell proliferation inhibitor, a cancer cell proliferation inhibitor obtained by the said production method, and a method for treating cancer.

[0002] Conventionally, much research has been conducted on methods to suppress the proliferation of cancer cells for the purpose of treating cancer (for example, Patent Documents 1-3). In order to expand the treatment options for cancer, there is a need for cancer cell proliferation inhibitors that are different from those used in the past.

[0003] Patent No. 6814483 Patent No. 7549338 Patent No. 7566012

[0004] Therefore, the object of the present invention is to provide a method for producing a cancer cell proliferation inhibitor, a cancer cell proliferation inhibitor obtained by the said production method, and a method for treating cancer.

[0005] As a result of diligent research to solve the above problems, the inventors of the present invention discovered that a liquid obtained by recovering the vaporized water produced when earthworm castings and water are mixed suppresses the proliferation of cancer cells, and thus completed the present invention.

[0006] In other words, the method for producing the cancer cell proliferation inhibitor of the present invention is characterized by including a mixing step of mixing earthworm excrement and water, and a recovery step of recovering vaporized water produced from the mixture obtained in the mixing step to obtain a liquid.

[0007] In the method for producing the cancer cell proliferation inhibitor of the present invention, it is preferable to further mix organic matter with the earthworm castings and water in the mixing step.

[0008] In the method for producing the cancer cell proliferation inhibitor of the present invention, it is preferable that the organic material is wood.

[0009] The method for producing the cancer cell proliferation inhibitor of the present invention preferably further includes a dilution step of diluting the liquid obtained by recovering the vaporized water in the recovery step with water.

[0010] The cancer cell proliferation inhibitor of the present invention is characterized by being obtained by the method for producing the cancer cell proliferation inhibitor described above.

[0011] The cancer cell growth inhibitor of the present invention is preferably for the treatment of cancer.

[0012] The cancer cell growth inhibitor of the present invention preferably has the cancer being lung cancer, colorectal cancer, gastric cancer, liver cancer, breast cancer, cervical cancer, neuroblastoma, or malignant lymphoma.

[0013] The cancer treatment method of the present invention is characterized by administering the cancer cell growth inhibitor to a patient. The use of the present invention is to use the cancer cell growth inhibitor for the treatment of cancer.

[0014] The present invention can provide a method for producing a cancer cell growth inhibitor, the cancer cell growth inhibitor obtained by the production method, and a cancer treatment method.

[0015] It is a graph showing the cell viability (%) of human cervical cancer cells HeLa using the cancer cell growth inhibitor prepared in the examples as a test specimen. The control group is sterilized water. n = 6. ＃＃ p < 0.01 (vs control), ＊ p < 0.05, ＊＊ It is a graph showing the cell viability (%) of human lung cancer cells A549 using the cancer cell growth inhibitor prepared in the examples as a test specimen. The control group is sterilized water. n = 6. ＃ p < 0.05, ＃＃ p < 0.01 (vs control), ＊＊ It is a graph showing the cell viability (%) of human colorectal cancer cells Caco-2 using the cancer cell growth inhibitor prepared in the examples as a test specimen. The control group is sterilized water. n = 6. ＃ p < 0.05, ＃＃ p < 0.01 (vs control), ＊ p < 0.05, ＊＊ It is a graph showing the cell viability (%) of human neuroblastoma SH-SY5Y using the cancer cell growth inhibitor prepared in the examples as a test specimen. The control group is sterilized water. n = 6. ＃＃ p < 0.01 (vs control), ＊＊ p < 0.01

[0016] The present invention provides a method for producing a cancer cell proliferation inhibitor, characterized by comprising a mixing step of mixing earthworm castings and water, and a recovery step of recovering vaporized water produced from the mixture obtained in the mixing step. In the mixing step, it is preferable to further mix organic matter with the earthworm castings and water. Although the detailed mechanism is not clear, it is thought that microorganisms such as bacteria contained in the earthworm castings decompose and ferment the organic matter contained in the earthworm castings and separately mixed organic matter, and that the liquid obtained by recovering the vaporized water is effective in inhibiting the proliferation of cancer cells.

[0017] The method for producing the cancer cell proliferation inhibitor of the present invention, and the cancer cell proliferation inhibitor itself, will be described in detail below.

[0018] [Method for producing cancer cell proliferation inhibitors] (Mixing step) The mixing step is the process of mixing earthworm castings and water.

[0019] Earthworm castings are not particularly limited and include, for example, red earthworm (Lumbricus rubellus), LT earthworm (Lumbricus terrestris), striped earthworm (Eisenia foetida), brown earthworm (Allolobophora caliginosa), purple earthworm (Dendrobaena octaedra), cherry earthworm (Allolobophora japonica Michaelsen), hatta earthworm (Drawida hattamimizu Hatai), black-backed earthworm (Pheretima divergens Michaelsen), and common earthworm (Pheretima) Castile soil from earthworms such as *Pheretima*, *Pheretima agrestis*, *Pheretima sieboldi Horst*, *Pheretima hilgendorfi*, *Pontodrillus matsushimensis Iizuka*, *Tubifex hattai Nomura*, and *Limnodrilus gotoi Hatai* (L. Socialis Stephenson) can be used.

[0020] The water used to mix with the earthworm castings is not particularly limited, and tap water or distilled water can be used. The water may also be purified using filter media, reverse osmosis membranes, etc. From the viewpoint of removing microorganisms such as bacteria, it is preferable to use fine-pore filter media or reverse osmosis membranes, for example, activated carbon or SPG (shirasu porous glass) permeable membranes.

[0021] The mixing ratio of earthworm castings to water is preferably 0.05 to 20 L of water per 1 kg of earthworm castings, more preferably 0.1 to 10 L, even more preferably 0.2 to 5 L, and especially preferably 0.5 to 2 L.

[0022] In the mixing process, it is preferable to further mix organic matter with the earthworm castings and water. Mixing organic matter also allows for adjustment of the pH of the cancer cell proliferation inhibitor. The organic matter is not particularly limited as long as it is organic matter that decomposes when mixed with earthworm castings; for example, organic matter of biological origin such as animals, plants, fungi, and protozoa can be used. Examples of plant-derived organic matter include wood chips, sawdust, rice hulls, and mushroom growing substrates. Mixing wood as organic matter is preferable because it facilitates the recovery of an odorless liquid in the recovery process. Wood chips are preferred as the type of wood used. Furthermore, the type of wood is not particularly limited and can be any coniferous tree such as Japanese yew, Japanese nutmeg, cypress, Japanese cypress, Japanese cedar, Japanese umbrella pine, pine, Japanese fir, spruce, fir, hemlock, redwood, or broad-leaved tree such as cherry, sawtooth oak, zelkova, beech, birch, oak, maple, alder, linden, hornbeam, or bamboo. Among the woods, pine wood can be preferably used. More preferably, wood from the cypress family can be used, even more preferably, wood from the cypress subfamily can be used, and particularly preferably, wood from the genus Cryptomeria can be used.

[0023] The mixing ratio of earthworm castings to organic matter is preferably 0.05 to 20 kg, more preferably 0.1 to 10 kg, even more preferably 0.2 to 5 kg, and particularly preferably 0.5 to 2 kg of organic matter per 1 kg of earthworm castings.

[0024] The mixing method in the mixing process is not particularly limited, but it is preferable to thoroughly mix the mixture by stirring or other means. The order of mixing is not particularly limited; for example, the earthworm castings and organic matter may be placed in the container and then water may be added, or the organic matter may be placed in the container, water may be added, and then the earthworm castings may be added.

[0025] It is not necessary to mix the entire amount of each component at once; water, earthworm castings, and / or organic matter can be added and mixed in multiple stages. Adding in stages is preferable because it allows for the continuous recovery of vaporized water while replenishing the water lost through evaporation and the earthworm castings and organic matter lost through decomposition.

[0026] Furthermore, since fermentation becomes more stable as time passes after the initial mixing, if the vaporized water is collected after, for example, about a day, a liquid that is more effective in suppressing the proliferation of cancer cells can be obtained. From this viewpoint as well, it is preferable to continuously collect the vaporized water by adding more as needed.

[0027] The mixture generates heat through the fermentation of earthworm castings and other added organic matter, but depending on the ambient temperature, it is preferable to heat the mixture while mixing. When heating, for example, the temperature should be between 20 and 60°C.

[0028] (Recovery Process) The recovery process involves recovering the vaporized water produced from the mixture obtained in the mixing process to obtain a liquid (hereinafter also referred to as "organic matter decomposition water"). The recovery process may be carried out while the mixing process is being performed.

[0029] In the recovery process, it is sufficient to recover the vaporized water generated in the temperature range that rises due to the fermentation heat (reaction heat) produced by the fermentation of the mixture; there is no need to heat it to the boiling point. Depending on the ambient temperature, it is preferable to mix while heating, and if heating is required, it is appropriate to heat it to, for example, 20 to 60°C.

[0030] The method of recovery is not particularly limited as long as the vaporized water can be recovered; for example, the vaporized water can be recovered using a dehumidifier. For example, cooling-type or compression-type dehumidifiers can be used. Furthermore, it is preferable to recover the vaporized water without boiling the mixture.

[0031] The method for converting the recovered vaporized water back into liquid is not particularly limited; for example, by using a dehumidifier, organic matter decomposition water can be obtained. The dehumidifier is not particularly limited as long as it can recover the vaporized water as liquid, but for example, cooling type or compression type dehumidifiers can be used.

[0032] The pH of the liquid obtained by recovering the vaporized water is preferably 5 to 9, more preferably 6 to 8, and even more preferably 6.5 to 7.5.

[0033] (Dilution step) The organic matter decomposition water obtained by recovering the vaporized water in the recovery step can be used as is as a cancer cell proliferation inhibitor, but it is preferable to use it after dilution. Water can be used as the solvent for dilution, and tap water or distilled water can be used. The water used for dilution may also be purified using a filter material, reverse osmosis membrane, etc. From the viewpoint of removing microorganisms such as bacteria, it is preferable to use a filter material with fine pores or a reverse osmosis membrane, for example, it is preferable to use an SPG (shirasu porous glass) permeable membrane.

[0034] When diluting, for example, it is fine to dilute it 1.5 to 10 times, preferably 4 to 6 times, and more preferably 4.5 to 5.5 times.

[0035] [Cancer Cell Proliferation Inhibitor] The cancer cell proliferation inhibitor of the present invention is characterized by being obtained by the method for producing the cancer cell proliferation inhibitor of the present invention. The cancer cell proliferation inhibitor of the present invention is obtained by the method for producing the cancer cell proliferation inhibitor of the present invention and is not particularly limited as long as it is effective in inhibiting the proliferation of cancer cells, but it is preferably in liquid form. Furthermore, the liquid cancer cell proliferation inhibitor may be used by spraying it in the form of a mist or by evaporation. In addition, it may be processed into a solid form such as a gel, powder, or granules by mixing it with an excipient, for example.

[0036] In the cancer cell growth inhibitor of the present invention, it may contain a pharmaceutically acceptable carrier, and can be administered orally or parenterally (for example, intravenous administration, direct administration to the affected area, etc.) in the form of, for example, tablets, granules, powders, capsules, soft capsules, liquids, injections, suppositories, sustained-release agents, etc. As the pharmaceutically acceptable carrier, excipients, binders, disintegrants, fluidizing agents, lubricants, coating agents, suspending agents, coloring agents, sweeteners or surfactants, etc. can be used, and it can be made into the form of a general pharmaceutical preparation according to a known method. Further, it may contain other therapeutic or prophylactic components and pharmaceutically acceptable additives.

[0037] Also, in the cancer cell growth inhibitor of the present invention, it may contain additives usually used in foods. For example, excipients, binders, disintegrants, fluidizing agents, lubricants, coating agents, suspending agents, coloring agents, sweeteners or surfactants, etc. can be used, and it can be made into the form of a general food composition according to a known method. Further, it may contain other foods or food-derived components.

[0038] The blending amount of the liquid recovered in the recovery step in the cancer cell growth inhibitor of the present invention may be an effective amount according to the purpose. The appropriate blending amount depends on various factors such as the purpose, administration route and form, manufacturing conditions, etc. For example, in the case of a liquid cancer cell growth inhibitor, the blending amount of the liquid recovered in the recovery step may be 5% by volume or more, preferably 5 to 50% by volume, more preferably 10 to 40% by volume, and even more preferably 15 to 30% by volume.

[0039] Also, the amount of use of the cancer cell growth inhibitor of the present invention may also be an effective amount according to the purpose. The appropriate amount of use depends on various factors such as the purpose, administration route and form, manufacturing conditions, etc. For example, in the case of a liquid cancer cell growth inhibitor, 500 to 3000 mL may be taken per day. The intake amount as the liquid recovered in the recovery step may be, for example, 100 to 500 mL taken per day.

[0040] The cancer cell proliferation inhibitor of the present invention is preferably used to inhibit the proliferation of cancer cells and is suitably used in the treatment of cancer. Examples of cancers include carcinoma, sarcoma, blastoma, germ cell tumor, lymphoma, and leukemia. Specific cancers include lung cancer, colorectal cancer, small intestine cancer, stomach cancer, esophageal cancer, liver cancer, pancreatic cancer, kidney cancer, breast cancer, prostate cancer, bladder cancer, uterine cancer, cervical cancer, vaginal cancer, testicular cancer, ovarian cancer, cervical cancer, laryngeal cancer, pharyngeal cancer, oral cancer (tongue cancer, oral floor cancer, gum cancer), nasal cavity and paranasal sinus cancer, salivary gland cancer, thyroid cancer and other head and neck cancers, brain tumors, mesothelioma, skin cancer, adenocarcinoma, myelodysplastic syndrome, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, bone tissue, cartilage tissue, adipose tissue, and muscle tissue. Examples of cancers include cancers of tissue, vascular tissue and hematopoietic tissue, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant Schwannoma, osteosarcoma, soft tissue sarcoma, glioblastoma, glioblastoma multiforme, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary sarcoma, retinoblastoma, malignant lymphomas such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, and T-cell lymphoma, as well as multiple myeloma. Cancer cells include cancer cells of the above-mentioned cancers. Animal species of cancer cells include mammals, such as primates (humans, monkeys, etc.), canids, felids, equids, bovids, pigs, mice, rats, and guinea pigs. The cancer is preferably lung cancer, colorectal cancer, stomach cancer, liver cancer, breast cancer, cervical cancer, neuroblastoma, or malignant lymphoma.

[0041] The cancer cell proliferation inhibitor of the present invention may contain other active ingredients or known and commonly used additives for cancer cell proliferation inhibitors, such as colorants, fragrances, antioxidants, UV absorbers, chelating agents, surfactants, viscosity modifiers, pH adjusters, thickeners, defoaming agents, preservatives, bactericidal / antibacterial agents, dispersants, organic solvents, etc., to the extent that they do not impair the effects of the present invention.

[0042] The present invention will be described in more detail below with reference to examples. The present invention is not limited in any way by the following examples. In the following, "%" refers to mass unless otherwise specified.

[0043] (Method for producing a cancer cell proliferation inhibitor) 40 kg of earthworm castor soil and 15 kg of wood (cedar) (5 kg of wood chips and 10 kg of a blend of sawdust and rice hulls) were placed in a reaction tank. 40 L of water was added, and while stirring, the vaporized water was collected over approximately one day using a dehumidifier (Nakatom DM-30) attached to the reaction tank, yielding 15-20 L of liquid. Another 10-20 L of water was added to the reaction tank, and 10 kg of wood (cedar) (5 kg of wood chips and 5 kg of a blend of sawdust and rice hulls) was added. The vaporized water was similarly collected over approximately one day using a dehumidifier, yielding 15-20 L of liquid. Approximately 40 L of liquid (pH 8.71) was obtained by mixing the obtained liquids as organic matter decomposition water. Furthermore, in the production of the above-mentioned organic matter decomposition water, the water used was tap water that had been purified using an SPG (shirasu porous glass) permeable membrane (manufactured by SPG Techno Co., Ltd.) and activated carbon.

[0044] The organic matter decomposition water obtained above was diluted five times with water. The water used for dilution was tap water that had been purified using an SPG (shirasu porous glass) permeable membrane (manufactured by SPG Techno Co., Ltd.) and activated carbon. The organic matter decomposition water diluted above was passed through a 0.2 μm filter to obtain a cancer cell proliferation inhibitor.

[0045] [Evaluation Test 1] - Test sample: Cancer cell proliferation inhibitor obtained above

[0046] • Test cells: Human cervical cancer cells (HeLa: obtained from RIKEN BioResearch Center), Human lung cancer cells (A549: obtained from RIKEN BioResearch Center), Human colorectal cancer cells (CACO-2: obtained from RIKEN BioResearch Center), Human neuroblastoma cells (SH-SY5Y: obtained from RIKEN BioResearch Center)

[0047] - For cell culture, various cancer cells were cultured using DMEM medium supplemented with 10% FBS by volume, CO2 ２ Incubator (5% CO2 by volume) ２The cells were cultured at 37°C with medium changes twice a week. Preparation of DMEM medium with 10% FBS added: 500 mL of DMEM medium was mixed with 5 mL of penicillin-streptomycin solution and 50 mL of FBS.

[0048] • Test culture media were prepared by adding the test sample to the prepared liquid culture medium in proportions of 0, 3, 6, 12, 25, and 50 volume percent. A control group was prepared by adding sterile water in the same proportions.

[0049] - Measurement of cell viability: Various cancer cells were grown in a growth medium in 2 × 10⁶ units. ４ Prepare the solution to cells / mL and dispense 100 μL into a 96-well plate (2 × 10⁶). ３ (cells / well). The next day, the culture medium was removed and replaced with 100 μL of prepared test medium. After incubation for 2 days, a photograph was taken with a stereomicroscope, and then 10 μL of MTT reagent was added per well. 37°C, 5 vol% CO2 ２ After incubation for 2 hours, the absorbance of each well was measured using a microplate reader (measurement wavelength: 570 nm). Three wells were used for each concentration (n=3). The MTT reagent is used to quantify cell proliferation capacity and cell viability by colorimetric measurement. In this reagent, tetrazolium salt (MTT) is degraded by mitochondrial dehydration enzymes in viable cells, producing formazan dye. In other words, it utilizes the fact that the number of viable cells is proportional to the amount of formazan dye.

[0050] - Test results: Using test media prepared at 0, 3, 6, 12, 25, and 50 volume percent concentrations, cell viability was measured for four types of cancer cells using the MTT reagent as an indicator of proliferation inhibition (n=6; the above measurement was performed twice to confirm reproducibility). The results are shown in Figures 1-4 and Tables 1-4. In human cervical cancer cells HeLa, cell morphology deteriorated and cell viability decreased at a concentration of 6 volume percent. In human lung cancer cells A549, cell morphology deteriorated and cell viability decreased at a concentration of 3 volume percent. In human colorectal cancer cells CACO-2, cell morphology deteriorated and cell viability decreased at a concentration of 25 volume percent. In human neuroblastoma SH-SY5Y, cell morphology deteriorated at a concentration of 6 volume percent and cell viability decreased at a concentration of 50 volume percent. From the above, it can be seen that a cancer cell proliferation inhibitor can be manufactured by recovering vaporized water produced from a mixture of earthworm castings and water.

[0051]

[0052]

[0053]

[0054]

[0055] [Evaluation Test 2] The changes in CEA values ​​(ng / mL) in tumor marker tests (normal value is 5.0 ng / mL or less) were examined when the cancer cell proliferation inhibitor obtained above was orally ingested at a dose of 1 to 2 L per day.

[0056] Male, 69 years old. Diagnosis: Gastric cancer (stage 4), metastasis to lymph nodes. Treatment started... CEA level (252) 1 month later... CEA level (126) 2 months later... CEA level (14) 3 months later... CEA level (7.7) 4 months later... CEA level (4.6) 5 months later... CEA level (4.1) 6 months later... CEA level (2.4)

[0057] Male, 60 years old, Diagnosis: Liver cancer (stage 2) Treatment started: CEA level (68) 1 month later: CEA level (31) 2 months later: CEA level (6) 3 months later: CEA level (2.9)

[0058] Male, 55 years old, Diagnosis: Liver cancer and colorectal cancer (stage 3) Treatment started: CEA level (198) 1 month later: CEA level (78) 2 months later: CEA level (18) 3 months later: CEA level (8.1) 4 months later: CEA level (4.5) 5 months later: CEA level (3.1) 6 months later: CEA level (2.2)

[0059] Female, 52 years old, Diagnosis: Breast cancer (stage 3), metastasis to lymph nodes. Treatment started... CEA level (201) 1 month later... CEA level (110) 2 months later... CEA level (15) 3 months later... CEA level (8) 4 months later... CEA level (5.5) 5 months later... CEA level (3.2) 6 months later... CEA level (2.5) 7 months later... CEA level (2.1)

[0060] Female, 60 years old, Diagnosis: Lung cancer (stage 3), metastasis to lymph nodes. Treatment started... CEA level (211) 1 month later... CEA level (101) 2 months later... CEA level (25) 3 months later... CEA level (15) 4 months later... CEA level (7.8) 5 months later... CEA level (5.6) 6 months later... CEA level (3.8) 7 months later... CEA level (2.2)

Claims

1. A method for producing a cancer cell proliferation inhibitor, comprising a mixing step of mixing earthworm castings and water, and a recovery step of recovering vaporized water produced from the mixture obtained in the mixing step to obtain a liquid.

2. The method for producing a cancer cell proliferation inhibitor according to claim 1, wherein in the mixing step, organic matter is further mixed together with the earthworm excrement and the water.

3. The method for producing a cancer cell proliferation inhibitor according to claim 2, wherein the organic material is wood.

4. A method for producing a cancer cell proliferation inhibitor according to any one of claims 1 to 3, further comprising a dilution step of diluting the liquid obtained by recovering the vaporized water in the recovery step with water.

5. A cancer cell proliferation inhibitor characterized by being obtained by a method for producing a cancer cell proliferation inhibitor according to any one of claims 1 to 4.

6. The cancer cell proliferation inhibitor according to claim 5, for the treatment of cancer.

7. The cancer cell proliferation inhibitor according to claim 6, wherein the cancer is lung cancer, colorectal cancer, gastric cancer, liver cancer, breast cancer, cervical cancer, neuroblastoma, or malignant lymphoma.

8. A method for treating cancer, characterized by administering the cancer cell proliferation inhibitor described in claim 6 to a patient.

9. Use of the cancer cell proliferation inhibitor according to claim 6 for the treatment of cancer.

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