JP6324551B2 - Anti-breast cancer agent, food for anti-breast cancer, method for inhibiting breast cancer, and method for producing anti-breast cancer agent - Google Patents

Description

  The present invention relates to an antitumor agent, a tumor cell growth inhibitor, an anti-breast cancer agent, and a method for producing an antitumor agent.

Euglena genus microorganism (genus name: Euglena, Japanese name: Euglena) (hereinafter referred to as "Euglena") is a representative of a mobile microalgae having flagella and photosynthesis. Since various nutrients such as vitamins, minerals, unsaturated fatty acids and β-glucan are contained, various applications are expected as functional materials (for example, Non-Patent Document 1).
As a functional material derived from Euglena, various functional searches have been conducted for paramylon, a kind of β-glucan, and now it has immunostimulatory action, antioxidant action, colon cancer suppression action, etc. Is confirmed (for example, Patent Documents 1 and 2).

Ryo Arashi, Characteristics of microalgae Euglena and its application to food and environmental fields, Photosynthesis research, 2012, 22 (1), p.33-38.

International Publication No. 2015/156339 JP2013-75869A

  However, other than paramylon, the search for Euglena-derived substances having functionality for living organisms has not progressed much, and the development of new functional substances derived from Euglena has been expected.

  The present invention has been made in view of the above problems, and an object of the present invention is to produce a novel antitumor agent derived from Euglena, a tumor cell growth inhibitor, an anti-breast cancer agent and an antitumor agent, and An object of the present invention is to provide an anti-tumor food and a method for suppressing tumors (excluding medical practice for humans).

When the present inventors tried extraction by various extraction methods using Euglena, the hot water extract of Euglena was found to have an anti-breast cancer effect and led to the present invention.
According to the present invention, the above-mentioned problems are solved by an anti-breast cancer agent, an anti-breast cancer food, and a method for producing an anti-breast cancer agent characterized by containing a hot water extract of Euglena as an active ingredient.
The object is achieved, according to the present invention, it is solved by a method for inhibiting breast cancer, characterized in that to feed a composition comprising a hot water extract of Euglena as an active ingredient (except human from a subject) .

Thus, since the hot water extract of Euglena is used, the anti- breast cancer effect which cannot be obtained with the Euglena extract using another solvent can be acquired. As a result, a novel anti- breast cancer agent and anti-breast cancer food having anti- breast cancer activity can be provided which contains Euglena hot water extract as an active ingredient.
In the present invention, no side effects have been reported so far, and a hot water extract of Euglena having a safety level consistent with the Food Sanitation Law is used as an active ingredient. Continuous administration of the period is possible.
Therefore, it can be used without side effects in place of conventional anticancer agents for chemotherapy, and can be administered or ingested for preventing recurrence after surgery for removing tumor cells.
According to the present invention, according to the present invention, a step of obtaining a dispersion by adding water to Euglena and shaking, a step of heating the dispersion, and centrifuging the heated dispersion, , it is achieved by a process for preparing an anti-breast cancer agents, which comprises a step of obtaining a hot water extract of Euglena, a.

According to the present invention, since the Euglena hot water extract is used, an anti- breast cancer effect that cannot be obtained by the Euglena extract using other solvents can be obtained. As a result, it is possible to provide a novel anti- breast cancer agent and anti-breast cancer food containing an Euglena hot water extract as an active ingredient and having an anti- breast cancer action.
In the present invention, no side effects have been reported so far, and a hot water extract of Euglena having a safety level consistent with the Food Sanitation Law is used as an active ingredient. Continuous administration of the period is possible.
Therefore, it can be used without side effects in place of conventional anticancer agents for chemotherapy, and can be administered or ingested for preventing recurrence after surgery for removing tumor cells.

It is a graph which shows the influence with respect to the proliferation of the breast cancer origin cell of the Euglena hot water extract of Example 1 of this invention. It is a graph which shows the influence with respect to the proliferation of the breast cancer origin cell of the Euglena ethanol extract of contrast 1. It is a graph which shows the influence with respect to the proliferation of the breast cancer origin cell of the Euglena hexane extract of the proportional 2.

The present invention relates to an antitumor agent, a tumor cell growth inhibitor, an anti-breast cancer agent, a method for producing an antitumor agent, an antitumor food, and a tumor for suppressing an antitumor agent containing a hot water extract of Euglena as an active ingredient. Regarding methods (excluding medical practices for humans). Hereinafter, the present invention will be described in detail.
The antitumor agent of the present invention contains a hot water extract of Euglena as an active ingredient.
<Euglena>
The “Euglena” of the present invention means a microorganism including all of the plants classified as Euglena in zoology or botany, varieties thereof, and mutants thereof.

  Here, Euglena microorganisms are, in zoology, the protozoan Protozoa (Mastigophorea), Phytomastigophorea (Euglenida) Euglenoidina subgenus It is a microorganism belonging to the eye (Euglenoidina). On the other hand, microorganisms belonging to the genus Euglena belong to the order of Euglenales belonging to Euglenophyceae in Euglenophyta in botany.

Specific examples of Euglena microorganisms include Euglena acus, Euglena caudata, Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena intermedia, Euglena mutabilis, Euglena oxyuris, Euglena proxima, Euglena proxima, Euglena proxima, Euglena proxima, , Euglena intermedia, Euglena piride and the like. Of these, Euglena gracilis, which is widely used for research, is particularly suitable. In particular, the Euglena gracilis Z strain is mentioned.
In addition, mutant strains of Euglena gracilis Z strain SM-ZK strain (chloroplast-deficient strain), variant var. Bacillaris, chloroplast mutant strains of these species, etc. May be used. In addition, other Euglena species such as Astaia longa may be used.
Euglena is widely distributed in fresh water such as ponds and swamps, and may be used separately from these, or any Euglena that has already been isolated may be used.
The Euglena genus of this invention includes all the mutants. These mutant strains also include those obtained by genetic methods such as recombination, transduction, transformation and the like.

In the culture of Euglena cells, examples of the culture solution include a culture solution to which nutrient salts such as a nitrogen source, a phosphorus source, and a mineral are added, for example, a modified Cramer-Myers medium ((NH ₄ ) ₂ HPO ₄ 1.0 g / L. , KH ₂ PO ₄ 1.0 g / L, MgSO ₄ .7H ₂ O 0.2 g / L, CaCl ₂ .2H ₂ O 0.02 g / L, Fe ₂ (SO ₂ ) ₃ 7H ₂ O 3 mg / L, MnCl ₂ · 4H ₂ O 1.8 mg / L, CoSO ₄ · 7H ₂ O 1.5 mg / L, ZnSO ₄ · 7H ₂ O 0.4 mg / L, Na ₂ MoO ₄ · 2H ₂ O 0.2 mg / L, CuSO ₄ .5H ₂ O 0.02 g / L, thiamine hydrochloride (vitamin B ₁ ) 0.1 mg / L, cyanocobalamin (vitamin B ₁₂ , (pH 3.5)) can be used. Note that (NH ₄ ) ₂ HPO ₄ can also be converted to (NH ₄ ) ₂ SO ₄ or NH ₃ aq. In addition, a known Hutner medium or Koren-Hutner medium prepared based on the description of Euglena Physiology and Biochemistry (Edited by Shozaburo Kitaoka, Society Publishing Center, Inc.) may be used. You may use the AY culture medium etc. which are autotrophic culture media remove | excluding heterotrophic components, such as glucose, malic acid, and an amino acid, from the Koren-Hutner culture medium generally used as a Euglena heterotrophic culture medium.

The pH of the culture solution is preferably 2 or more, more preferably 3 or more, still more preferably 3.5 or more, and the upper limit thereof is preferably 7 or less, more preferably 6.5 or less, still more preferably 4. 5 or less. By setting the pH to the acidic side, the photosynthetic microorganisms can grow more dominantly than other microorganisms, so that contamination can be suppressed.
Euglena cells may be cultured by an open pond method using sunlight directly, a light collecting method in which sunlight condensed by a light concentrator is sent through an optical fiber, etc., and irradiated in a culture tank and used for photosynthesis.
Euglena cells can be cultured using, for example, a fed-batch method, but flask culture, fermentation using a fermentor, batch culture, semi-batch culture (fed-batch culture), continuous culture (perfusion) Any liquid culture method such as a culture method may be used.

The culture can be performed using a known culture apparatus such as an open pond type, a raceway type, a tube type or the like, or an experimental culture vessel such as a Sakaguchi flask, an Erlenmeyer flask, or a reagent bottle. Since Euglena to assimilate CO _2, when cultured using the Cramer-Myers medium is autotrophic medium is preferably be passed through into the medium air containing 1 to 5% CO _2. Further, in order to sufficiently develop the chloroplast, about 1 to 5 g of ammonium phosphate may be added per liter of the medium. The culture temperature is usually 20 to 34 ° C., particularly preferably 28 to 30 ° C. Depending on the culture conditions, Euglena usually enters a logarithmic growth phase 2 to 3 days after the start of the culture, and reaches a stationary phase about 4 to 5 days.
Euglena may be cultured under light irradiation (light culture) or non-irradiated (dark culture).

Euglena cells are separated by, for example, centrifugation or simple sedimentation of a culture solution, membrane filtration, or the like.
The dried Euglena alga body is prepared by washing the separated Euglena cells and then freeze-drying them in a known manner. However, adjustment of the dried alga body may be performed by spray drying, heating vacuum drying, or the like.
In the antitumor agent of the present invention, Euglena alga bodies separated from the culture solution by centrifugation, sedimentation, membrane filtration or the like may be used as Euglena, or dried alga bodies may be used. Moreover, you may use the dry powder of Euglena which grind | pulverized the algal body dried material with the well-known grinder.

<Heuglena hot water extract>
The Euglena hot water extract was prepared by adding water to the Euglena alga body dry powder and shaking to disperse uniformly, and then heating and separating the supernatant obtained by centrifugation. Is.

<Anti-tumor agent>
Antitumor agents are used to suppress the generation, proliferation, and recurrence of cancer cells or tumor cells by being administered to cancer patients or tumor patients.
In the present specification, tumors include both benign tumors that have a slow growth rate, proliferate in an expandable manner, and do not cause metastasis, and malignant tumors that have a fast growth rate, proliferate invasively, and cause metastasis. The present invention is mainly applied to malignant tumors. In the present specification, the term “cancer” is used synonymously with “malignant tumor”.
Malignant tumors include squamous cell carcinoma that occurs in the esophagus, lung, cervix, etc., adenocarcinoma that occurs in the stomach, large intestine, etc., epithelial malignant tumors such as transitional cell carcinoma that occurs in the bladder, etc. Hematopoiesis such as non-epithelial malignancies such as fibrosarcoma, osteosarcoma, liposarcoma, hemangiosarcoma, rhabdomyosarcoma, lymphoma in the whole body, lymphoma in the lymph nodes, leukemia in the blood Includes tissue tumors. Benign tumors include epithelial benign tumors and non-epithelial benign tumors.

  The antitumor agent of the present invention is particularly suitably used as an anti-breast cancer agent used for breast cancer patients. No side effects have been reported so far, and Euglena's hot water extract with a level of safety consistent with the Food Sanitation Law is used, so there are no side effects like conventional anticancer drugs used in chemotherapy. . Therefore, it can be suitably used as a substitute for conventional anti-cancer drugs for chemotherapy. In addition, for the purpose of preventing recurrence after tumor removal surgery, it can be continuously administered to patients after surgery.

Further, the Euglena hot water extract has a function of suppressing the growth of tumor cells, as is apparent from the results of Examples described later, and the antitumor agent of the present invention has an action of suppressing the growth of tumor cells. By exhibiting antitumor action.
Euglena hot water extract can be used not only as an antitumor agent, but also as a tumor cell growth inhibitor that suppresses or inhibits the growth of tumor cells.

The antitumor agent of this embodiment can be used as a composition such as a pharmaceutical composition or a food composition containing the antitumor agent.
(Pharmaceutical composition)
In the field of medicine, pharmaceuticals are used together with an amount of Euglena hot water extract that can effectively exert the action of suppressing the progression of cancer or tumor, that is, the action of suppressing the generation, proliferation, metastasis, recurrence, etc. of cancer cells or tumor cells The pharmaceutical composition which has the said effect | action is provided by mix | blending an acceptable carrier and additive. The pharmaceutical composition may be a pharmaceutical or a quasi drug.
The pharmaceutical composition is used in a pharmaceutical form such as an internal preparation, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection and / or intraperitoneal injection, transmucosal application agent, transdermal application agent, etc. However, it may be an oral preparation for oral administration or an injection for intravenous injection.
The dosage form of the pharmaceutical composition can be appropriately set depending on the form of application, for example, solid preparations such as tablets, granules, capsules, powders, powders, liquid preparations such as liquids and suspensions, Semi-solid preparations such as ointments or gels are mentioned.

(Food composition)
In the field of food, an effective amount of hot water extract of Euglena capable of exerting an antitumor action or an anticancer action, or an action of suppressing the generation, proliferation, metastasis, recurrence, etc. of cancer cells or tumor cells in vivo Can be provided in various foods as a food material to provide a food composition having the action.
That is, the present invention provides a food composition labeled as anti-cancer, anti-tumor, or for inhibiting the generation, proliferation, metastasis, recurrence, etc. of cancer cells or tumor cells in the field of food. Can do. Examples of the food composition include foods for specified health use, functional nutritional foods, functional labeling foods, hospital patient foods, and supplements in addition to general foods. It can also be used as a food additive.
Examples of the food composition include seasonings, processed meat products, processed agricultural products, beverages (soft drinks, alcoholic beverages, carbonated beverages, milk beverages, fruit juice beverages, tea, coffee, nutritional drinks, etc.), powdered beverages (powder) Juice, powdered soup, etc.), concentrated beverages, confectionery (candy, cookies, biscuits, gum, gummi, chocolate, etc.), bread, cereals and the like. Moreover, in the case of food for specified health use, nutritional functional food, functional indication food, etc., it may be in the form of capsule, troche, syrup, granule, powder or the like.

Here, food for specified health use is a food containing health functional ingredients that affect physiological functions, etc., and can be used to indicate that it is suitable for specific health use with the permission of the Commissioner of the Consumer Affairs Agency. It is. In the present invention, as a specific health use, it is a food that is marketed and sold as antitumor or anticancer, or for suppressing the occurrence, proliferation, metastasis, recurrence, etc. of cancer cells or tumor cells.
The nutritional functional food is a food used for supplementing nutritional components (vitamins and minerals) and displays the function of the nutritional components. In order to sell as a functional nutritional food, the amount of nutrients contained in the daily intake standard amount must be within the specified upper and lower limits. You also need to.
In addition, the functional labeling food is a food that displays the functionality based on the scientific basis at the responsibility of the operator. Information on safety and functionality grounds was reported to the Commissioner of the Consumer Affairs Agency before sales.
In the above, the present invention comprises a hot water extract of Euglena as an active ingredient, a cancer or tumor patient, a specific health food for antitumor intended for animals other than humans suffering from cancer or tumor, and antitumor nutrition It can be used as a functional food or a functional display food for antitumor.
The present invention also includes a specific health food for an anti-tumor comprising a hot water extract of Euglena as an active ingredient and intended for patients or non-human animals after undergoing surgery or radiation therapy for tumors or cancer, It can be used as an anti-tumor functional food or an anti-tumor functional food.
The composition containing the hot water extract of Euglena of the present embodiment as an active ingredient is orally ingested by humans or animals other than humans (excluding medical practices for humans) to suppress tumors. be able to.

(Usage and dosage)
As the usage of the antitumor agent of the present embodiment, when administered by oral administration or intravenous injection to tumor patients or cancer patients, patients after undergoing surgery or radiation treatment of tumors or cancer, or animals other than humans, etc. good.

<Method for producing antitumor agent>
The antitumor agent of this embodiment is manufactured by the following method.
First, a step of adding water to Euglena and shaking to obtain a dispersion is performed. In this step, Euglena powder may be used as Euglena.
Next, the dispersion is heated and extracted using a high-pressure steam sterilizer or the like.
Thereafter, the heated dispersion is centrifuged to obtain a supernatant as a hot water extract of Euglena.
The hot water extract of Euglena obtained as described above is used for the antitumor agent of the present invention.

EXAMPLES Hereinafter, although this invention is demonstrated concretely based on a specific Example, this invention is not limited to these.
In the following test examples, the effects of Euglena hot water extract (Example 1), ethanol extract (Comparative 1), and hexane extract (Comparative 2) on the growth of breast cancer-derived cells were confirmed. The extraction method of Euglena which increases the cell growth inhibitory effect was examined.
<Sample solution adjustment>
Example 1
10 mL of water was added to 0.5 g of Euglena powder (EU-1593, Euglena Co., Ltd.), and the mixture was shaken and dispersed uniformly, followed by heat extraction (100 ° C., 10 minutes) using a high-pressure steam sterilizer. The supernatant obtained by centrifugation (10,000 rpm, 10 minutes, 25 ° C) was collected, aliquoted in 1 mL portions, and stored frozen at -30 ° C until the cell test (sample solution concentration 50 mg / mL). The sample solution of Example 1 was used.
・ Proportional 1
To 0.5 g of Euglena powder (EU-1593, Euglena Co., Ltd.), 10 mL of 50% ethanol was added and dispersed by shaking. The supernatant obtained by centrifugation (10,000 rpm, 10 minutes, 25 ° C) was collected, aliquoted in 1 mL portions, and stored frozen at -30 ° C until the cell test (sample solution concentration 50 mg / mL). A sample solution with a proportionality of 1 was obtained.
・ Comparison 2
Euglena powder (EU-1593, Euglena Co., Ltd.) 0.5g, 5mL of hexane and 5mL of PBS (phosphate buffered saline) were added and dispersed by shaking, then centrifuged (10,000rpm, 10 minutes, 25 ° C). 1 mL of the hexane phase was dried, DMSO (dimethyl sulfoxide) was added, and the sample was frozen and stored at −30 ° C. until the cell test (sample solution concentration 200 mg / mL). did. Moreover, in order to confirm the influence of DMSO alone, DMSO alone was also used as a solvent control.

<Cell test>
Human breast cancer cell line MCF7 (RCB1904, RIKEN BioResource Center) in RPMI 1640 medium (186-02155, Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (FBS) (10437-028, Gibco) Passage was maintained every 2-3 days. The cell suspension (10 ⁵ cells / mL), the sample solution of Comparative Example 1, 2 (0.16 to 10 mg / mL) and DMSO (0.31 v / v%, 0.63), both of which were diluted with a medium. 50 μL each of v / v%, 1.25 v / v%, 2.50 v / v%) was dispensed into 96-well plates and cultured for 72 hours in a CO ₂ incubator. After removing the culture by decantation, add 100 μL of WST-1 reaction reagent (500 μmol / L WST-1, 20 μmol / L 1-Methoxy-PMS, Dojindo Laboratories Co., Ltd.) to each well at 37 ° C. Incubated for 1 hour. Using a microplate reader (Emax, Molecular Devices), the absorbance at 450 nm / 650 nm was measured, and the cell proliferation rate was determined as a relative value with respect to a control not containing the sample solution (n = 6).
As a positive control for growth inhibition, 10 mg of fucoidan (F5631, Sigma), which is a polysaccharide derived from seaweed, was dissolved in 1 mL of water, sterilized by filtration through a 0.2 μm membrane filter, and stored frozen at −30 ° C. At the time of use, it was diluted to 0.4 mg / mL with a medium and used. 17β-estradiol 40 nmol / L was used as a positive control for promoting growth.

<Result>
The result of the hot water extract of Euglena in Example 1 is shown in FIG. As shown in FIG. 1, the Euglena hot-water extract of Example 1 had a MCF7 cell growth rate of less than 10%, a cell growth rate of 5 mg / mL in well, 28% and 2.5% at 2.5 mg / mL. In the case of mg / mL, up to 58% each showed a clear inhibitory effect, and the concentration (IC50 value) showing 50% inhibition was 1.62 mg / mL. Euglena hot water extract showed a concentration-dependent inhibitory effect on human breast cancer-derived cell MCF7, and almost completely inhibited its growth at 5 mg / mL (positive control fucoidan was 0.2 mg / mL).

  The results of the ethanol extract with proportional 1 and the hexane extract with proportional 2 are shown in FIGS. The ethanol extract of comparative 1 and the hexane extract of comparative 2 showed a growth promoting action, not an inhibitory action, on the human breast cancer-derived cell MCF7 on the low concentration side.

Considering the functional component extraction conditions from Euglena powder in this experiment, heating at 100 ° C for 10 min, and subsequent centrifugation at room temperature, the functional component extracted into the supernatant solution is heat denaturation. It is unlikely that it will be a protein or lipid with low water solubility. Therefore, there may be a possibility of a water-soluble low molecule originally present, or a polysaccharide that is fragmented by heating and has increased solubility in water.
Assuming that polysaccharides are candidates for the active ingredient, Euglena contains abundant paramylon as a storage polysaccharide, which is a linear β-glucan in which glucose is linked by β-1,3-linkages. As β-glucan, those that are actually applied clinically as anticancer agents, such as Shiitake lentinan and Suehirotake Shizofiran, are known. These mushroom β-glucans are characterized by a branched structure containing β-1,6-links in addition to β-1,3-linkages, and the relationship between this steric structure and immunostimulatory activity is presumed. However, these action mechanisms are considered to be activation of the immune response of the living body to the last, and do not directly inhibit the growth of tumor cells as shown in this experiment. In the case of mushrooms, since the content of the active ingredient varies greatly depending on the cultivation conditions and the individual bodies, problems in quality control of the preparation have been pointed out. In this regard, it can be said that Euglena is more advantageous because it is easy to obtain a uniform and large-scale culture.

Claims (5)

  An anti-breast cancer agent comprising a hot water extract of Euglena as an active ingredient.   The anti-breast cancer agent according to claim 1, wherein the concentration of the Euglena hot water extract is 1.25 mg / mL or more.   A food for anti-breast cancer comprising Euglena hot water extract as an active ingredient. Methods for inhibiting breast cancer, characterized in that to feed a pharmaceutical composition comprising a hot water extract of Euglena as an active ingredient (except human from a subject). A step of adding water to Euglena and shaking to obtain a dispersion;
Heating the dispersion;
Centrifuge the heated dispersion to obtain a supernatant as a hot water extract of Euglena, and a method for producing an anti-breast cancer agent.

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