JP6592010B2 - Cancer cell growth inhibitory composition and cancer cell growth inhibitory method - Google Patents

Description

  The present invention relates to a cancer cell proliferation inhibiting composition and a method for inhibiting cancer cell proliferation.

  Currently, cancer is the leading cause of death and the number of patients is increasing year by year. Among them, an extract derived from a natural product having a cancer cell growth inhibitory effect has been found, and since it is safer than conventional anticancer agents, it contains a cell growth inhibitor with few side effects and an anti-cancer agent containing these components. It is expected to provide tumor agents (also called anticancer agents and anticancer agents), and health foods for preventing cancer.

  In recent years, pets such as dogs tend to be regarded as members of the family, and in the 2001 survey, 64% of the total population thought that “pets are also part of the family” and the lifestyle was It is close to humans. On the other hand, with increasing life expectancy of pets, neoplastic diseases are increasing. Especially in dogs and cats, the death rate due to cancer is high in elderly people, and the incidence of cancer may be higher than that in humans depending on the breed of dog and cat. However, pets, like humans, rarely show symptoms at an early stage even when they have cancer, and often progress only to symptoms such as loss of appetite and swelling of lymphoma. Cancer treatment methods, like humans, include surgery, chemotherapy, drug therapy, radiation therapy, and immunotherapy. Although these methods are effective if they are early treatments, they cannot be said to be effective treatments at the stage of considerable progress as described above. In addition, all of the above methods put a considerable burden on the pet, which makes it difficult for the pet and the owner.

  For this reason, drugs effective for cancer cell growth inhibition are administered to cancer patients (humans, pets) to treat cancer or alleviate cancer symptoms, or ingest food or pet food containing such substances in advance. There is a need to prevent cancer. For example, Patent Document 1 has found that Hatake shimeji mushroom extract significantly suppresses the decrease in the number of white blood cells, particularly the number of neutrophils, caused by an anticancer drug, and the use of Hatake shimeji mushroom extract to reduce the side effects caused by chemotherapy with an anticancer drug. It is disclosed that it can be used.

JP 2010-173977 A

  The present invention provides a highly safe cancer cell growth inhibitory composition (a composition for preventing or treating cancer) that can replace a conventionally known antitumor agent, and a method for suppressing cancer cell growth using the composition. The purpose is to do.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive studies on the cancer cell growth inhibitory effect using extracts of various plants that have been recognized to be safe. As a result, it was found that the combined use of rosemary extract and green tea extract at a specific mixing ratio can significantly improve the cancer cell proliferation inhibitory effect, and the present invention has been completed.

  That is, the above object is achieved by a cancer cell growth inhibitory composition comprising a green tea extract and a rosemary extract in a weight ratio of 10: 1 to 1:10 (mixed weight ratio of green tea extract: rosemary extract). it can.

  In addition, the object is to administer a green tea extract and a rosemary extract in a weight ratio of 10: 1 to 1:10 (mixed weight ratio of green tea extract: rosemary extract), for cancer cell proliferation. It can also be achieved by a suppression method.

In Examples 1-7 and Comparative Examples 1-5, it is a graph which shows the cell growth inhibitory effect with respect to leukemia cell by combined use of the green tea extract and rosemary extract in each mixing ratio. In Examples 11-17 and Comparative Examples 13-17, it is a graph which shows the cell growth inhibitory effect with respect to a colon cancer cell by combined use of the green tea extract and rosemary extract in each mixing ratio.

  Embodiments of the present invention will be described below. In addition, this invention is not limited only to the following embodiment. In this specification, “X to Y” indicating a range includes X and Y, and means “X or more and Y or less”. Unless otherwise specified, measurement of operation and physical properties is performed under conditions of room temperature (20 to 25 ° C.) / Relative humidity 40 to 50%.

(Cancer cell proliferation inhibiting composition and cancer cell proliferation inhibiting method)
The cancer cell growth inhibitory composition of the present invention comprises a green tea extract and a rosemary extract in a weight ratio of 10: 1 to 1:10 (mixed weight ratio of green tea extract: rosemary extract).

  In addition, the method for inhibiting cancer cell growth of the present invention comprises a method of treating a cancer cell with a green tea extract and a rosemary extract at a weight ratio of 10: 1 to 1:10 (mixed weight ratio of green tea extract: rosemary extract). Having administration to a subject in need of inhibiting proliferation.

  The inventors of the present invention have conducted extensive research on the cancer cell growth inhibitory effect using various plant extracts. As a result, when rosemary extract and green tea extract are used in combination at a specific mixing ratio, cancer cell growth inhibition is achieved. It has been found that the effect can be synergistically improved. In addition, the green tea extract and rosemary extract used in the cancer cell growth inhibitory composition of the present invention are both used in foods as antioxidants, spices and functional extracts, and are taken on a daily basis. And safety is recognized. For this reason, the cancer cell growth inhibitory composition of the present invention is ingested daily for the purpose of preventing cancer, in addition to the purpose of treatment after onset of cancer, as a health food, etc. or mixed with pet food. It is also possible.

  That is, according to the present invention, a safe cancer cell growth inhibitory composition (composition for cancer prevention / treatment) that can replace a conventionally known cancer cell growth inhibition (cancer prevention / treatment agent) is provided. Is done.

  The cancer cell growth inhibitory composition of the present invention comprises a green tea extract and a rosemary extract in a weight ratio of 10: 1 to 1:10 (mixed weight ratio of green tea extract: rosemary extract). When the mixing ratio of the green tea extract and the rosemary extract is out of the above range, no significant difference in the cancer cell proliferation inhibitory effect is recognized as compared with the green tea extract alone or the rosemary extract alone. Here, from the viewpoint of achieving a more remarkable synergistic effect of the cancer cell growth inhibitory effect, the cancer cell growth inhibitory composition is preferably a green tea extract and a rosemary extract, preferably 5: 1 to 1: 5, more preferably. In a weight ratio of 2: 1 to 1: 2, particularly preferably approximately 1: 1 (substantially equivalent) (green tea extract: rosemary extract mixing weight ratio). In the above mixed weight ratio, the cancer cell proliferation inhibitory effect by the combined use of the green tea extract and the rosemary extract can be achieved particularly synergistically.

  Here, the treatment target of the cancer cell growth inhibitory composition (subject who needs to suppress cancer cell growth) is not particularly limited, but is a mammal or a bird, preferably a mammal or a bird suffering from cancer. . Here, mammals include primates such as humans, monkeys, gorillas, chimpanzees, orangutans, and non-humans such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, camels, and goats. Includes both human mammals. Examples of birds include chickens, quails and pigeons. Of these, humans, hamsters, guinea pigs, rabbits, dogs, cats and pigs are preferred, and humans and pet animals such as dogs, cats and rabbits are more preferred. That is, according to a preferred embodiment of the present invention, the cancer cell growth-suppressing composition of the present invention is used orally for at least one pet animal selected from the group consisting of humans, dogs, cats, and rabbits. According to a more preferred embodiment of the present invention, the cancer cell growth inhibitory composition of the present invention is used orally for humans, dogs and cats.

  The cancer cell proliferation-suppressing composition of the present invention is effective for the treatment and / or prevention of cancer (herein, simply referred to as “cancer treatment / prevention”). For this reason, the cancer cell proliferation inhibitory composition of the present invention is also useful as an agent for treating / preventing cancer. In the present specification, “treatment / prevention of cancer” refers to suppressing the progression of cancer in a patient suffering from cancer, eliminating cancer or tumor, suppressing the growth of cancer or tumor, and onset of cancer. It means satisfying at least one of prevention of cancer and prevention of cancer recurrence. Here, “prevention” means preventing or delaying the onset of cancer or tumor, or reducing the risk of onset of cancer or tumor. Therefore, in the present specification, the “cancer treatment / prevention agent” means a drug exhibiting the above-mentioned cancer treatment / prevention effect when used in the treatment of cancer. Alternatively, the cancer cell growth inhibitory composition of the present invention can be taken daily as a health food or mixed with pet food for the purpose of preventing cancer. That is, according to a preferred embodiment of the present invention, the cancer cell growth inhibitory composition of the present invention is a human health food or pet food composition.

  Here, the type of cancer that is the subject of the cancer cell proliferation inhibitory composition (for cancer treatment / prevention) is not particularly limited. For example, cancer of the nervous system (eg, brain tumor, cervical cancer); cancer of the digestive system (eg, oral cancer, pharyngeal cancer, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, spleen cancer, colon cancer, small intestine cancer) , Duodenal cancer, colon cancer, colon adenocarcinoma, rectal cancer, pancreatic cancer, liver cancer); musculoskeletal cancer (eg sarcoma, osteosarcoma, myeloma); urinary cancer (eg bladder cancer, renal cancer) ); Reproductive cancer (eg, breast cancer, uterine cancer, ovarian cancer, testicular cancer, prostate cancer); Respiratory cancer (eg, lung cancer); Hematopoietic cancer (eg, acute or chronic myeloid leukemia, Acute promyelocytic leukemia, leukemia such as acute or chronic lymphocytic leukemia, malignant lymphoma (lymphosarcoma), angiosarcoma, multiple myeloma, myelodysplastic syndrome, primary myelofibrosis, epivascular cell tumor); thyroid Cancer, parathyroid cancer, tongue cancer, malignant melanoma, mastocytoma, skin tissue Tumor, lipoma, follicular tumor, cutaneous papillomas, sebaceous adenoma, such as basal cell cancer. Among these, the cancer cell proliferation suppressing composition of the present invention is more effective against hematopoietic cancer, digestive cancer, genital cancer, malignant melanoma, mastocytoma, and basal cell carcinoma. Has a high effect. That is, according to a preferred embodiment of the present invention, the composition for inhibiting cancer cell proliferation of the present invention comprises hematopoietic cancer, digestive cancer, genital cancer, melanoma, mastocytoma and basal tumor. It is used for the prevention and / or treatment of at least one disease selected from the group consisting of cell carcinomas.

(Green tea extract)
The green tea extract, which is an active ingredient of the cancer cell growth inhibitory composition of the present invention, can be obtained by extracting chanoki (Camellia sinensis) (whole or part) using a suitable solvent. Here, the part of the tree that is to be extracted is not particularly limited, the whole plant body, leaves, stems, buds, flowers (including gaku, petals, pistil, stamens, etc.), woody parts, bark parts (bark), Fruit (including flesh (fruit flesh), ovary, pericarp (inner pericarp, mesocarp, outer pericarp), etc.), seeds, etc .; parts of plants such as roots, rhizomes, tubers, etc. Include. The plant part may be subjected to extraction alone or may be subjected to extraction in the form of a mixture of two or more. Among these, it is preferable to use tea leaves (tea leaves) as a raw material in consideration of the effect of suppressing / preventing cancer cell proliferation and safety. Or you may use for the extraction what heat-processed the leaf of a tea tree (tea leaf) in order to prevent fermentation. Specifically, the portion of the tea tree to be extracted is steamed, crushed and crushed, and dried while preparing the shape of the tea leaf (steaming ⇒ cooling ⇒ leaf punching ⇒ rough cocoon ⇒ sprain ⇒ moderate ⇒ fine twisting ⇒ drying ⇒ sieving and cutting ⇒ tree stem separation). In the present specification, the portion of the tea tree to be extracted is also simply referred to as “green tea material”.

  The green tea raw material may be subjected to extraction in the form as it is, but may be previously dried and / or pulverized before being subjected to extraction. Thereby, a desired active ingredient can be extracted more efficiently from a green tea raw material.

  Here, the drying conditions when drying the green tea raw material in advance are not particularly limited. Considering ease of pulverization and the like, the drying temperature is preferably 20 to 90 ° C, more preferably 25 to 80 ° C. The drying time is preferably 24 to 120 hours, more preferably 48 to 96 hours. Or you may dry a green tea raw material by methods, such as a freeze-drying powdering method, a high pressure method, and an ultrahigh pressure method. Among these, the high-pressure method is a method of crushing cells in the green tea raw material under pressure by, for example, high-pressure heat treatment at 100 to 150 ° C. for 2 to 10 hours (for example, 120 ° C. for 4 hours).

  Moreover, the pulverization conditions for pulverizing the green tea raw material in advance are not particularly limited. In consideration of extraction efficiency and the like, it is preferable that a predetermined plant part (green tea raw material) can be pulverized to a size of about 0.1 to 10 mm, more preferably a size of about 0.5 to 5 mm. Examples of the pulverization method include a method of chopping with a cutting machine, a slicer, a cutter, a peeler, a jaw crusher, a jet mill, a blender, and the like.

  Next, if necessary, the green tea raw material dried and / or crushed in advance is extracted using a suitable solvent. The solvent that can be used here is not particularly limited as long as it can extract an active ingredient from a green tea raw material, and is appropriately selected according to the plant or plant part to be used. Specifically, water (including tap water, industrial water, distilled water, reverse osmosis membrane water, filtered water, sterilized water, purified water, etc.); methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, Examples include alcohols such as 2-butanol, propylene glycol, and 1,3-butylene glycol; esters such as methyl acetate and ethyl acetate; ketones such as acetone; ethyl ether, formaldehyde, and dimethyl sulfoxide. The said solvent may be used independently or may be used with the form of 2 or more types of mixed solvents. Among these, in consideration of extraction efficiency, safety, etc., water, ethanol, or a mixture of water and ethanol is preferable as the extraction solvent. That is, in a preferred embodiment of the present invention, the extract is an extract of the green tea material from water, ethanol, or a mixture of water and ethanol.

  Further, the extraction may be performed only once or twice or more. In the latter case, any solvent may be used in each extraction step, and the extraction conditions in each step may be the same or different. Extraction with water, hot water, ethanol or a mixture of water and ethanol is preferably performed at least twice, and after extraction with water or hot water, extraction with ethanol or a mixture of water and ethanol is more preferable. It is particularly preferable to perform extraction with ethanol after extraction with hot water. By this method, the purity of the active ingredient (tea polyphenol which is an active ingredient) can be further improved.

  If necessary, the green tea raw material may be extracted under acidic or alkaline conditions, that is, an acidic or alkaline solvent may be used. Here, the acid that can be used when preparing the acidic solvent is not particularly limited, but inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, succinic acid, formic acid, propionic acid, etc. Organic acids and the like. Moreover, as alkalinity, although it does not restrict | limit especially as an alkali which can be used when preparing a solvent, Potassium hydroxide, sodium hydroxide, sodium carbonate etc. are mentioned. The pH of the solvent for extracting the green tea raw material under acidic or alkaline conditions is appropriately selected in consideration of the type of plant and plant part used, extraction conditions, and the like. Moreover, when an acidic solvent or an alkaline solvent is used, it is preferable to neutralize the extract so that it becomes neutral (pH = about 7 ± 1) after extraction.

  The addition amount of a solvent will not be restrict | limited especially if an active ingredient can be extracted from a green tea raw material. Specifically, the solvent is preferably added in an amount of 10 to 1000 ml, more preferably 50 to 500 ml, per 10 g of green tea raw material.

  Moreover, extraction conditions will not be restrict | limited especially if an active ingredient can be extracted from a green tea raw material. Specifically, the extraction temperature is preferably 25 to 100 ° C, more preferably 40 to 100 ° C. The extraction time is preferably 30 minutes to 6 hours, more preferably 1 to 4 hours. Under such conditions, the active ingredient can be efficiently extracted from the green tea raw material.

  After the extraction step, the solid (green tea raw material residue) is removed from the mixed liquid of the green tea raw material and the solvent, and the extract is separated. Here, the separation method is not particularly limited, and examples thereof include filtration and centrifugation. Further, this extract may be used as it is, but if necessary, diluted form by diluent, extract by concentration, paste or solid form, frozen form by freezing, dry powder form by freeze drying, etc. You may convert into various forms (extract). Here, the conversion method may be applied alone or in combination of two or more. Preferably, the extract is concentrated to an appropriate concentration (for example, concentrated under reduced pressure), and the resulting concentrate is frozen and then freeze-dried or the extract is made into a fine mist, which is then heated in hot air. A method of spraying and obtaining a powdery dried product instantaneously is preferably used.

  Moreover, you may further refine | purify an extract or an extract. Here, the purification method is not particularly limited, and a known purification method can be used. Specifically, a quaternary ammonium salt such as cetylpyridinium chloride is added to obtain a precipitate, and this precipitate is washed with an appropriate solvent (eg, water, alcohol), anion exchange ion exchange chromatography, Cation exchange ion exchange chromatography, phosphocellulose chromatography, chromatography using hydrophobic reaction (hydrophobic interaction), affinity chromatography, chromatography using hydroxyapatite chromatography, lectin chromatography, etc., recrystallization Law.

  If caffeine is given to a pet animal (for example, a dog), symptoms such as tachycardia, tremors / convulsions, arrhythmia, excessive excitement, and diarrhea may occur. For this reason, when using the composition of this invention for pet food (for example, for dogs), it is preferable that a composition does not contain caffeine as much as possible. Specifically, the content of caffeine in the green tea extract is preferably 5% by weight or less, more preferably 2.5% by weight or less, still more preferably 1% by weight or less, particularly preferably 0.5% by weight. % Or less (lower limit: 0% by weight). For this reason, caffeine may be reduced or removed before and after extraction from the green tea raw material as described above. Here, the method for reducing or removing caffeine is not particularly limited, and a known method can be used similarly or appropriately modified. Specifically, publicly known methods such as JP 2014-140351 A, JP 2013-209428 A, JP 2008-21024 A and the like can be mentioned.

  Alternatively, the green tea extract may be a commercially available product. Commercially available products include green tea extract powder (Matsuura Pharmaceutical Co., Ltd.), Sun Food (registered trademark) powder, Sun Food (registered trademark) 100, Sun Food (registered trademark) CD, Sun Food (registered trademark) aqueous, Sunfood (registered trademark) oil-based sunfood (registered trademark) series (all manufactured by Mitsubishi Chemical Foods), green tea extract MF (manufactured by Maruzen Pharmaceutical), Sunphenon (registered trademark) 30S-OP, Sunphenon (registered trademark) 30LB-OP, Sunphenon (registered trademark) EGCG-OP, Sunphenon (registered trademark) BG-3, Sunphenon (registered trademark) 90LB-OP, Sunphenon (registered trademark) 90S, Sunphenon (registered trademark) 90MB- OP, Sunphenon (registered trademark) CF-T-OP, Sunphenon (registered trademark) NB-60, etc. (All manufactured by Taiyo Kagaku Co., Ltd.), polyphenone (registered trademark) series such as polyphenone G and polyphenone 70A, sancatechin aqueous F, sancatechin oily E, GTA-MNK-1 (all manufactured by Mitsui Norin Co., Ltd.) ), Tea extract-40, tea extract-70, tea extract (decaffeinated product) (all manufactured by Tama Seikagaku Co., Ltd.), and the like.

  The green tea extract may be used alone or in the form of a mixture of two or more.

(Rosemary extract)
The rosemary extract, which is an active ingredient of the cancer cell growth inhibitory composition of the present invention, can be obtained by extracting rosemary (Rosmarinus officinalis L.) (whole or part) using a suitable solvent. Here, the part of the rosemary to be extracted is not particularly limited, and the whole plant body, leaves, stems, buds, flowers (including gaku, petals, pistil, stamens, etc.), woody parts, bark parts (bark) , Fruits (including flower buds (fruit flesh), ovary, pericarp (inner pericarp, mesocarps, outer pericarp), etc.), seeds, etc .; parts of plants such as roots, rhizomes, tubers, etc. Is included. The plant part may be subjected to extraction alone or may be subjected to extraction in the form of a mixture of two or more. Among these, it is preferable to use rosemary leaves as a raw material in view of cancer cell growth suppression / prevention effect, safety, and the like. In the present specification, the portion of rosemary to be extracted is also simply referred to as “rosemary raw material”.

  The rosemary raw material may be subjected to extraction in the form as it is, but may be previously dried and / or pulverized before being subjected to extraction. Thereby, a desired active ingredient can be extracted more efficiently from a rosemary raw material.

  Here, the drying conditions when the rosemary raw material is previously dried are not particularly limited. Considering ease of pulverization and the like, the drying temperature is preferably 20 to 90 ° C, more preferably 25 to 80 ° C. The drying time is preferably 24 to 120 hours, more preferably 48 to 96 hours. Or you may dry a rosemary raw material by methods, such as a freeze-drying powdering method, a high pressure method, and an ultrahigh pressure method. Among these, the high pressure method is a method in which cells in a rosemary raw material are crushed under pressure by, for example, high pressure heat treatment at 100 to 150 ° C. for 2 to 10 hours (for example, 120 ° C. for 4 hours).

  Also, the pulverization conditions when the rosemary raw material is pulverized in advance are not particularly limited. In consideration of extraction efficiency and the like, it is preferable that a predetermined plant part (rosemary raw material) can be pulverized to a size of about 0.1 to 10 mm, more preferably a size of about 0.5 to 5 mm. Examples of the pulverization method include a method of chopping with a cutting machine, a slicer, a cutter, a peeler, a jaw crusher, a jet mill, a blender, and the like.

  Next, the rosemary raw material previously dried and / or pulverized if necessary is extracted using a suitable solvent. The solvent that can be used here is not particularly limited as long as it can extract an active ingredient from a rosemary raw material, and is appropriately selected according to the plant or plant part to be used. Specifically, water (including tap water, industrial water, distilled water, reverse osmosis membrane water, filtered water, sterilized water, purified water, etc.); methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, Examples include alcohols such as 2-butanol, propylene glycol, and 1,3-butylene glycol; esters such as methyl acetate and ethyl acetate; ketones such as acetone; ethyl ether, formaldehyde, and dimethyl sulfoxide. The said solvent may be used independently or may be used with the form of 2 or more types of mixed solvents. Among these, in consideration of extraction efficiency, safety, etc., water, ethanol, or a mixture of water and ethanol is preferable as the extraction solvent. The fat-soluble fraction contains a larger amount of a cancer cell growth inhibitory component. For this reason, it is more preferable to extract from a rosemary raw material by ethanol or the liquid mixture of water and ethanol from a viewpoint that the cancer cell proliferation inhibitory effect can be improved more. That is, in a preferred embodiment of the present invention, the extract is an extract of the rosemary raw material by ethanol or a mixed solution of water and ethanol.

  If necessary, the rosemary raw material may be extracted under acidic or alkaline conditions, that is, an acidic or alkaline solvent may be used. Here, the acid that can be used when preparing the acidic solvent is not particularly limited, but inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, succinic acid, formic acid, propionic acid, etc. Organic acids and the like. Moreover, as alkalinity, although it does not restrict | limit especially as an alkali which can be used when preparing a solvent, Potassium hydroxide, sodium hydroxide, sodium carbonate etc. are mentioned. The pH of the solvent when the rosemary raw material is extracted under acidic or alkaline conditions is appropriately selected in consideration of the types of plants and plant parts used, extraction conditions, and the like. Moreover, when an acidic solvent or an alkaline solvent is used, it is preferable to neutralize the extract so that it becomes neutral (pH = about 7 ± 1) after extraction.

  The amount of the solvent added is not particularly limited as long as the active ingredient can be extracted from the rosemary raw material. Specifically, the solvent is preferably added in an amount of 10 to 1000 ml, more preferably 50 to 500 ml, with respect to 10 g of the rosemary raw material.

  Also, the extraction conditions are not particularly limited as long as the active ingredient can be extracted from the rosemary raw material. Specifically, the extraction temperature is preferably 25 to 100 ° C, more preferably 40 to 100 ° C. The extraction time is preferably 30 minutes to 6 hours, more preferably 1 to 4 hours. Under such conditions, the active ingredient can be efficiently extracted from the rosemary raw material.

  After the said extraction process, a solid (rosemary raw material residue) is removed from the liquid mixture of a rosemary raw material and a solvent, and an extract is isolate | separated. Here, the separation method is not particularly limited, and examples thereof include filtration and centrifugation. Further, this extract may be used as it is, but if necessary, diluted form by diluent, extract by concentration, paste or solid form, frozen form by freezing, dry powder form by freeze drying, etc. You may convert into various forms (extract). Here, the conversion method may be applied alone or in combination of two or more. Preferably, the extract is concentrated to an appropriate concentration (for example, concentrated under reduced pressure), and the resulting concentrate is frozen and then freeze-dried or the extract is made into a fine mist, which is then heated in hot air. A method of spraying and obtaining a powdery dried product instantaneously is preferably used.

  Moreover, you may further refine | purify an extract or an extract. Here, the purification method is not particularly limited, and a known purification method can be used. Specifically, a quaternary ammonium salt such as cetylpyridinium chloride is added to obtain a precipitate, and this precipitate is washed with an appropriate solvent (eg, water, alcohol), anion exchange ion exchange chromatography, Cation exchange ion exchange chromatography, phosphocellulose chromatography, chromatography using hydrophobic reaction (hydrophobic interaction), affinity chromatography, chromatography using hydroxyapatite chromatography, lectin chromatography, etc., recrystallization Law.

  Alternatively, the rosemary extract may be a commercially available product. Commercially available products include RM such as Rosemary Extract (Matsuura Pharmaceutical Co., Ltd.), Rosemary Extract MF (Maruzen Pharmaceutical Co., Ltd.), RM Keeper MP, RM Keeper SF, RM Keeper OS, and RM Keeper OSE. Keeper series (all manufactured by Mitsubishi Chemical Foods), RM-21A, RM-21S, RM-21A base, RM-21B base and other RM-21 series (all manufactured by Mitsubishi Chemical Foods), Mulka (Rosemary extract, manufactured by Asama Kasei Co., Ltd.).

  The rosemary extract may be used alone or in the form of a mixture of two or more.

(Use of cancer cell growth inhibitory composition)
The cancer cell growth inhibitory composition of the present invention may be used for medical purposes (therapeutic or preventive purposes) or as a health food, except that it contains the cancer cell proliferation inhibitory composition of the present invention as an active ingredient. , And can be used in the same dosage form as before. That is, the cancer cell growth inhibitory composition of the present invention is mixed with a pharmaceutically acceptable additive such as an excipient, and is suitable for parenteral, oral or external administration. It can be used in the form of a thing.

  Here, when the cancer cell proliferation inhibitory composition of the present invention is used in the form of a food composition, the cancer cell proliferation inhibitory composition can be used as a food such as an oil product, an emulsified product, a soft drink, It can be added and used as a health food. Here, the “food composition” means a composition intended for consumption by mammals (including pets) such as humans. For example, a pet food composition is a food composition intended for consumption by a pet. Food compositions are widely known in the art. A pet food composition may or may not be nutritionally balanced, and in addition to supplements (eg, food), it is a nutritionally balanced composition suitable for a daily diet. May be. Here, “nutritionally balanced” means that the composition of the present invention has known nutrients necessary to maintain life in an appropriate amount and proportion in the pet nutrition field. To do.

  Especially when the cancer cell proliferation inhibitory composition of the present invention is added to and used in human health food (health food for humans) and pet food, the amount (content) of the cancer cell proliferation inhibitory composition is particularly limited. Although not, the amount is preferably 0.01 to 30% by weight, more preferably about 0.05 to 20% by weight with respect to health food and pet food (in terms of solid content). Alternatively, the amount is such that the cancer cell growth inhibitory composition of the present invention is administered at a total weight of 0.1 to 1000 mg / kg body weight, more preferably about 1 to 500 mg / kg body weight per day. With such an amount, a sufficient cancer preventive effect can be achieved. In addition, if the amount is as described above, the pet will not disturb the pet's taste and the pet will consume the entire amount of the given pet food.

  As described above, when caffeine is given to a pet animal (for example, a dog), symptoms such as tachycardia, tremors / convulsions, arrhythmia, excessive excitement, and diarrhea may occur. For this reason, when using the composition of this invention for pet food (for example, for dogs), it is preferable that a composition does not contain caffeine as much as possible. Specifically, the content of caffeine in the cancer cell growth inhibitory composition is preferably 10% by weight or less, more preferably 5% by weight or less, still more preferably 2% by weight or less, and particularly preferably 1% by weight. The following (lower limit: 0% by weight).

  The pet food contains the same components as in the prior art except that it contains the cancer cell growth inhibitory composition of the present invention. For example, the pet food is a monosaccharide, oligosaccharide, polysaccharide, dietary fiber, starch (for example, waxy corn starch, corn starch, wheat starch, rice starch, glutinous rice, in addition to the cancer cell growth inhibitory composition of the present invention. Starch, potato starch, honeydew starch, tapioca starch, sago starch, chemically treated or chemically modified processed starch) and cereals (eg, corn, barley, wheat, rye, sorghum, rice, mushroom, awa Carbohydrate sources such as cattle, pigs, sheep, rabbits, kangaroos, by-products and processed products, chicken, turkey, quail and other meats, by-products and household goods , Fish, white fish, etc., by-products and processed products, meat meal, meat bones, chicken meal, porter meal, fish Animal protein sources such as lettering such as schmir; vegetable protein sources such as soy protein, wheat protein, wheat gluten, corn gluten; α-sitosterol, β-sitosterol, stigmasterol, campesterol, α-sit Plant sterols such as free forms such as stanol, β-sitostanol, stigmasteranol, campestanol, and cycloartenol, and esters such as these fatty acid esters, ferulic acid esters, and cinnamic acid esters; bran such as rice bran and bran And soy bean paste, vegetables such as vegetable extract, vitamins A, B1, B2, D, E, niacin, pantothenic acid, carotene and other vitamins. In addition to the above, other additives such as a gelling agent, a shape-retaining agent, a pH adjuster, a seasoning, an antiseptic, and a nutrient reinforcing agent that are generally used in pet foods may be contained. In addition to or in place of the other additives, an antioxidant such as butylhydroxyanisole (BHA), dibutylhydroxytoluene (BHT), ethoxyquin, tert-butylhydroquinone (TBHQ), propyl gallate, etc. It can also be used in combination. The addition amount (content) of each component described above is not particularly limited, and the same amount as conventional can be applied.

  Pet food is manufactured by a conventionally well-known method. For example, it can be produced by mixing the cancer cell growth inhibitory composition of the present invention and the above-mentioned necessary components into a desired form. As an example, citric acid and citrate are mixed together with the cancer cell growth inhibitory composition of the present invention, cereal, meat meal, and mineral components such as iron and copper, and after sufficient mixing, water and water vapor are added. While extruding with an extruder. Thereafter, it is preferably dried with hot air until the water content becomes 10% or less, to produce a pet food. In addition, when pet food contains the fats and oils which have the fatty acid which has two or more double bonds, it is desirable to coat after drying with hot air.

The pet food may be in any form such as dry type, wet type, semi-moist type, jerky type, biscuits type, gum type, granular, powdery, soup, etc. It is preferable from the property. Examples of dry-type pet foods include kibble shapes, flat plate shapes, and bone shapes. From the standpoint of obtaining pets that are easy to chew and handle, the bulk density is 100 kg / m ³ or more, preferably 300 kg / m ³ or more, and 900 kg / m ³ or less, preferably 700 kg / m ³ or less. Or 100 to 900 kg / m ³ , particularly 300 to 700 kg / m ³ is preferable. In addition, the pet food can be provided in the form of bagging, boxing, packing, canning, and retort pouch.

  Moreover, the cancer cell proliferation inhibiting composition of the present invention can also be used as a cancer treatment / prevention agent for medical use (for treatment or prevention). That is, the present invention has a weight ratio of 10: 1 to 1:10 (mixed weight ratio of green tea extract: rosemary extract) that requires the green tea extract and rosemary extract to suppress cancer cell growth. There is also provided a method for inhibiting cancer cell growth comprising administering to a tester (including a patient suffering from a disease). Here, the green tea extract and rosemary extract may be administered in the form of one agent containing both of these active ingredients, or may be administered simultaneously in the form of two agents containing these active ingredients separately. Good. Considering the improvement of synergistic effect by these active ingredients and ease of administration, it is preferable to administer in the form of one agent containing both the green tea extract and the rosemary extract.

  In this embodiment, the cancer cell growth inhibitory composition can be added to oral preparations, external preparations, injections, inhalants, nasal drops, eye drops, etc., and tablets, liquids, injections can be used depending on their usage. , Ointments, creams, lotions, aerosols, suppositories and the like. Moreover, an excipient | filler, a base, an emulsifier, a stabilizer, a solubilizing agent, a corrigent, a preservative, a fragrance | flavor, a coloring agent, a coating agent, etc. can be suitably mix | blended as needed. For quasi-drugs and cosmetics, it can be added to lotions, emulsions, creams, etc., if necessary, oil, moisturizer, UV absorber, water-soluble polymer, antioxidant, surfactant, metal An ion sequestering agent, an antibacterial preservative and the like can be blended.

  When the cancer cell growth inhibitory composition of the present invention is used as a pharmaceutical, the dosage is determined by the route of administration; the nature of the patient's disease; the patient's size, weight, surface area, age and sex; other drugs to be administered; It depends on the judgment. A suitable dose (total amount of green tea extract and rosemary extract (in terms of dry weight)) is 1 to 500 mg / kg body weight per day. Given the different available compositions and the different effectiveness of the different routes of administration, it is expected that the required dosage can vary widely. These dosage level variations can be adjusted using standard empirical procedures for optimality known in the art. In particular for oral delivery, encapsulation of the composition in a suitable delivery vehicle (eg, polymer microparticles or implantable devices) increases delivery efficiency. The dose may be divided once or multiple times a day. Or, in some cases, it may be administered less frequently (eg, weekly or monthly). In addition, even in the same patient, the dose may vary depending on the patient's symptoms and severity.

  For use in pharmaceuticals (cancer treatment / prevention agents), a therapeutically effective amount of a cancer cell growth inhibitory composition is formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. The That is, the cancer cell growth inhibitory composition of the present invention can be provided in the form of a pharmaceutical composition further containing a pharmaceutically acceptable additive (eg, excipient, carrier, etc.). The cancer cell proliferation inhibiting composition of the present invention in this form can be specifically formulated for administration in solid or liquid as will be described in detail below. As oral administration, for example, liquid medicine (aqueous solution or non-aqueous solution or suspension), tablet, bolus, powder medicine, granule, paste for application to the tongue can be exemplified. For parenteral administration, for example, as a sterile solution or suspension, for example, a formulation for subcutaneous, intramuscular or intravenous injection, or as a topical, for example as a cream, ointment or spray applied to the skin, or It can be formulated in the vagina or rectum, for example, as a vaginal suppository, cream or foam.

  As used herein, “therapeutically effective amount” as used herein is effective to produce any desired therapeutic effect at a reasonable benefit / risk ratio applicable to any medical treatment. Means the amount of the active agent or composition. For example, although the dose of the cancer cell growth inhibitory composition of the present invention varies depending on the target disease, administration subject, administration route, etc., the cancer cell growth inhibitory composition of the present invention is orally administered for the purpose of cancer treatment / prevention. In this case, the volume can be easily changed depending on conditions such as the weight of the subject person, and can be appropriately selected by those skilled in the art. In the end, the attending physician will make an appropriate selection in consideration of the patient's symptoms and severity.

  As used herein, “pharmaceutically acceptable” means that within a reasonable medical judgment, commensurate with a reasonable benefit / risk ratio, without problems or complications such as excessive toxicity, irritation, and allergic reaction, Used to refer to compounds, materials, compositions, and / or dosage forms suitable for use in contact with tissue of a subject to be treated (human, mammal, etc.).

  A pharmaceutically acceptable carrier is a liquid or solid filler involved in transporting or transporting the cancer cell growth-inhibiting composition of the present invention from one organ or part of the body to another organ or part of the body, By a pharmaceutically acceptable material, composition or excipient, such as a diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the dosage form and not injurious to the patient. Pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; Powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; Polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; magnesium hydroxide and water Buffers such as aluminum; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; and other adaptations non-toxic substance which is used in the drug formulation and the like. In some embodiments, the drug formulation is non-pyrogenic. That is, the thing which does not raise a patient's body temperature is desirable. In addition, wetting agents such as sodium lauryl sulfate and magnesium stearate, emulsifiers and lubricants, as well as colorants, release agents, coating agents, sweeteners, flavors and fragrances, preservatives and antioxidants are cancer cells of the present invention. It may be contained in a growth inhibitory composition (cancer treatment / prevention agent).

  Pharmaceutically acceptable antioxidants include, but are not limited to, water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium disulfite, sodium sulfite and the like; ascorbyl palmitate, butylhydroxyanisole ( Oil-soluble antioxidants such as BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol and the like; and citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like A metal chelating agent is mentioned.

  The cancer cell growth inhibitory composition of the present invention can be used in various dosage forms such as oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The dosage form may be conveniently presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of compound that produces a therapeutic effect, but generally the amount of active ingredient (green tea extract and rosemary extract) is The cancer cell growth-suppressing composition is about 0.1 to about 99 parts by weight, preferably about 1 to about 70 parts by weight, more preferably about 5 to about 50 parts by weight per 100 parts by weight of the composition. Parts by weight.

  Methods for preparing these dosage forms or compositions comprise the step of combining the cancer cell growth inhibitory composition of the present invention with a carrier and optionally one or more accessory ingredients. In general, dosage forms are prepared by uniformly and intimately bringing into association one or more agents of the present invention with liquid carriers or finely divided solid carriers or both and, if necessary, shaping the product.

  For example, dosage forms of the invention suitable for oral administration are in the form of capsules, cachets, pills, tablets, lozenges (with seasoned active ingredients, usually sucrose and gum arabic or tragacanth), powders, granules Or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or pastilles (such as gelatin and glycerin, or sucrose and gum arabic) And / or a gargle and the like, each containing a predetermined amount of the compound of the present invention as an active ingredient. The agent of the present invention may be administered as a bolus, electuary or paste.

  In solid dosage forms of the present invention (capsules, tablets, pills, dragees, powders, granules, etc.) for oral administration, the active ingredients (green tea extract and rosemary extract) are sodium citrate or diphosphate. One or more pharmaceutically acceptable carriers such as calcium and / or mixed with any of the following: fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid. A binder such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or gum arabic; a humectant such as glycerol; agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicic acids; Disintegrants such as salt and sodium carbonate Solubilizers such as paraffin; absorption enhancers such as quaternary ammonium compounds; wetting agents such as cetyl alcohol and glycerol monostearate; absorbents such as kaolin and bentonite clay; talc, calcium stearate, magnesium stearate Lubricants such as solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and colorants. In the case of capsules, tablets and pills, the cancer cell growth inhibitory composition may include a buffer. Similar types of solid compositions can also be used as fillers in soft and hard-filled gelatin capsules with excipients such as lactose or lactose and high molecular weight polyethylene glycols and the like.

  A tablet may also be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be binders (eg, gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (eg, sodium glycolate starch or crosslinked sodium carboxymethylcellulose), surfactants Alternatively, it can be prepared using a dispersant. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

  Solid dosage forms such as tablets of the cancer cell growth-inhibiting composition of the present invention, such as sugar-coated tablets, capsules, pills and granules, are optionally nicked or such as enteric coatings well known in the pharmaceutical dispensing arts. It may be prepared using a coating and shell. They are controlled or controlled release of the internal active ingredient using, for example, hydroxypropyl methylcellulose, other polymer matrices, liposomes and / or microspheres in various ratios to provide the desired release profile. It may be formulated to provide release. They may be sterilized, for example, by filtration through a bacteria retaining filter, or by incorporating a sterilant in the form of a sterile solid composition that can be dissolved in a sterile injectable medium such as sterile water immediately before use. . These compositions may optionally contain opacifiers, in compositions that release one or more active ingredients only in certain parts of the gastrointestinal tract or preferentially there, optionally in a delayed manner. There may be. Examples of embedding compositions that can be used are polymeric substances and waxes. The active ingredient may be in microencapsulated form, if appropriate, with one or more of the above-described excipients.

  Liquid dosage forms for oral administration of the cancer cell growth inhibitory composition of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms include, in addition to the active ingredient, inert diluents commonly used in the art, such as water and other solvents, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzoic acid Like fatty acids esters of benzyl, propylene glycol, 1,3-butadiene glycol, oils (especially cottonseed oil, peanut oil, corn oil, embryo oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and sorbitan Solubilizers and emulsifiers, and mixtures thereof. In addition to inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, flavoring and preserving agents. Suspensions may be suspended in addition to the active compound, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof. A turbidity agent may be included.

  The dosage form of the cancer cell growth inhibitory composition of the present invention for rectal or vaginal administration may be presented as a suppository. This suppository comprises mixing one or more agents of the present invention with one or more suitable nonirritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository waxes or salicylates. Which is solid at room temperature but liquid at body temperature, it will melt in the rectum or vaginal cavity and release the active compound. Dosage forms suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray dosage forms containing carriers as known to be suitable in the art.

  Dosage forms for topical or transdermal administration of one or more cancer cell growth inhibitory compositions of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active ingredients (green tea extract and rosemary extract) may be mixed under sterile conditions with a pharmaceutically acceptable base material and, if necessary, preservatives, buffers, or propellants. Ointments, pastes, creams and gels contain animal or vegetable fat, oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite in addition to the active ingredients (green tea extract and rosemary extract) May include excipients such as silicic acid, talc and zinc oxide, or mixtures thereof.

  Powders and sprays can be supplemented with active ingredients (green tea extract and rosemary extract) as well as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances May contain medicine. The spray may further comprise customary high pressure gases such as chlorofluorinated hydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

  Transdermal patches have the additional advantage of delivering the cancer cell growth inhibitory composition of the present invention to the body in a controlled manner. Such a dosage form can be obtained by dissolving or dispersing the cancer cell growth inhibitory composition of the present invention in an appropriate medium. It is also possible to increase the flux of the substance containing the cancer cell proliferation inhibiting composition of the present invention across the skin using an absorption enhancer. The speed of such flux can be controlled by either providing a speed control membrane or by dispersing the compound in a polymer matrix or gel.

  The cancer cell growth inhibitory composition of the present invention suitable for parenteral administration is used with one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions or emulsions, or uses with the composition. Includes sterile powders that can be reconstituted in sterile injectable solutions or dispersions immediately before, for example, solutes or suspensions that make the antioxidants, buffers, bacteriostats, preparations isotonic with the blood of the intended recipient. Can include turbidity or thickening agents.

  Examples of suitable aqueous and non-aqueous carriers that can be used in the cancer cell growth inhibitory composition of the present invention include water, ethanol, polyol (eg, glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof. There are vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The inherent fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersants and by the use of surfactants.

  The cancer cell growth inhibitory composition of the present invention may contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the activity of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol and phenol sorbate. It may be desirable to include isotonic agents such as sugars, sodium chloride in the composition. In addition, prolonged absorption of the injectable drug form can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

  Moreover, the cancer cell proliferation inhibitory composition of this invention can be utilized also as a foodstuff (foodstuff composition). At this time, the cancer cell proliferation inhibiting composition of the present invention may be used as it is, and it may be used as a liquid, gel or solid food such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding. , Sprinkles, infant formula, cake mix, powdered or liquid dairy products, bread, cookies, etc., pellets, tablets with excipients such as dextrin, lactose, starch, flavorings, pigments, etc. It can be processed into granules, etc., or coated with gelatin and molded into capsules for use as health foods or dietary supplements. The amount of the cancer cell proliferation-suppressing composition of the present invention in these foods or edible compositions is difficult to define uniformly depending on the type or state of the food or composition, but with respect to the total weight of the food, 0.01 to 90% by weight, more preferably 0.1 to 80% by weight. If it is such a compounding quantity, the effect by the cancer cell proliferation inhibitory composition can fully be exhibited, without impairing flavor. Moreover, food can be easily prepared.

  Furthermore, the cancer cell growth inhibitory composition of the present invention can also be used as a cosmetic (cosmetic composition). In this case, the form of the cosmetic (cosmetic composition) includes lotion, milky lotion, cream, powder and the like, but is not particularly limited thereto. Such a cosmetic (cosmetic composition) containing the cancer cell growth-inhibiting composition of the present invention can be produced using techniques known to those skilled in the art. Examples of the form in which the cancer cell growth inhibitory composition of the present invention is used as a cosmetic (cosmetic composition) include lotions, emulsions, creams, powders and the like, but are not particularly limited thereto. Such a cosmetic composition containing the cancer cell proliferation-inhibiting composition of the present invention can be produced using techniques known to those skilled in the art.

  The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples. In the following examples, the operation was performed at room temperature (25 ° C.) unless otherwise specified. Unless otherwise specified, “%” and “part” mean “% by weight” and “part by weight”, respectively.

Examples 1 to 7 and Comparative Examples 1 to 5: Evaluation of cell growth inhibitory effect on leukemia cells 1
Rosemary extract (Mitsubishi Chemical Foods Co., Ltd., RM-21B base) and green tea extract (Mitsui Norin Co., Ltd., Polyphenon (registered trademark) 70A, caffeine content: 0.5% or less) The weight ratio of rosemary extract to green tea extract (mixed weight ratio of rosemary extract: green tea extract) is 20: 1 (Comparative Example 1), 10: 1 (Example 1), 5: 1 (Examples) 2) 2: 1 (Example 3), 1: 1 (Example 4), 1: 2 (Example 5), 1: 5 (Example 6), 1:10 (Example 7) and 1: 20 (Comparative Example 2) was prepared with dimethyl sulfoxide (DMSO) (concentration (total concentration) of a mixture of rosemary extract and green tea extract: 10 mg / ml). For comparison, rosemary extract (Comparative Example 3) and green tea extract (Comparative Example 4) were each adjusted to 10 mg / ml with DMSO.

Human chronic myeloid leukemia-derived K562 cells were cultured at 37 ° C. in a 5% by volume CO ₂ atmosphere using 10% FBS-containing RPMI1640 medium. After inoculating 1 ml of this cultured cell in a 24-well plate at 5 × 10 ⁴ cells per well, the solution of the mixture of each extract prepared above was adjusted to a final concentration of 10 μg / ml. 1 μl was added to the medium. Further, as a comparison target, 1 μl of rosemary extract and green tea extract were added to the medium so as to be 10 μg / ml each. As a control (Comparative Example 5), only 1 μl of the solvent DMSO was added.

Forty-eight hours after adding each extract solution or DMSO, K562 cells were cultured at 37 ° C. in a 5% by volume CO ₂ atmosphere. After culturing for a predetermined time, the number of viable cells was measured by trypan blue staining, and the cell growth rate (%) of each sample was calculated by the following formula (1). In the following formula (1), “the number of living cells of each sample” indicates the number of living cells of K562 cells when each extract solution (sample) is added and cultured, and “the number of living cells of control” is The number of viable cells of K562 cells when DMSO is added and cultured is shown. In addition, the said operation was performed about each 4 samples.

  The results are shown in FIG. In FIG. 1, each value is represented as an average value + standard deviation (n = 4). In addition, “*” indicates that there is a particularly significant difference (p <0.05) when compared with the rosemary extract alone and the green tea extract alone.

  As is clear from FIG. 1, the compositions of Examples 1 to 7 are rosemary extract alone (Comparative Example 3), green tea extract alone (Comparative Example 4), rosemary extract and green tea extract 20: It can be seen that the cancer cell growth rate is significantly lower as compared with the case of containing 1 (Comparative Example 1) and the case of containing rosemary extract and green tea extract 1:20 (Comparative Example 2). From the results, it is considered that the cancer cell proliferation inhibitory effect can be synergistically improved by using the composition of the present invention.

  Moreover, from FIG. 1, the composition (Examples 2-6) which contains a rosemary extract and a green tea extract by the weight ratio (mixed weight ratio of rosemary extract: green tea extract) of 5: 1 to 1: 5. Then, it is shown that a synergistic cancer cell proliferation inhibitory effect can be exhibited more remarkably.

  In this example, the cancer cell proliferation inhibitory effect was evaluated for human cells, but it is presumed that similar results can be obtained for pet animals such as dogs and cats.

Examples 8 to 10 and Comparative Examples 6 to 12: Evaluation of cell growth inhibitory effect on leukemia cells 2
Prepared with DMSO so that the weight ratio of rosemary extract to green tea extract (mixture weight ratio of rosemary extract: green tea extract) was 1: 1 (concentration of the mixture of rosemary extract and green tea extract) (Total concentration): 5 mg / ml, 10 mg / ml and 15 mg / ml). For comparison, rosemary extract and green tea extract were prepared in DMSO to 5 mg / ml, 10 mg / ml, and 15 mg / ml, respectively. In this example, RM-21B base manufactured by Mitsubishi Chemical Foods Co., Ltd. was used as the rosemary extract, and polyphenone (registered trademark) 70A (caffeine) manufactured by Mitsui Norin Co., Ltd. was used as the green tea extract. Content: 0.5% or less) was used.

Human chronic myeloid leukemia-derived K562 cells were cultured at 37 ° C. in a 5% by volume CO ₂ atmosphere using 10% FBS-containing RPMI1640 medium. After inoculating 1 ml of the cultured cells in a 24-well plate at 5 × 10 ⁴ cells per well, a final concentration of 5 μg / ml (Example 8) ) 1 μl was added to the medium so as to be 10 μg / ml (Example 9) and 15 μg / ml (Example 10). In addition, as a comparison object, rosemary extract and green tea extract were each independently 5 μg / ml (rosemary extract: comparative example 6, green tea extract: comparative example 7), 10 μg / ml (rosemary extract: 1 μl was added to the medium so as to be Comparative Example 8, Green Tea Extract: Comparative Example 9) and 15 μg / ml (Rosemary Extract: Comparative Example 10, Green Tea Extract: Comparative Example 11). As a control (Comparative Example 12), 1 μl of only DMSO as a solvent was added.

  For each sample prepared as described above, the cell proliferation rate (%) was calculated in the same manner as in Example 1, and according to the following method, 30% inhibitory concentration (IC30) (μg / ml), 40% inhibitory concentration (IC40) (μg / ml) and 50% inhibitory concentration (IC50) (μg / ml). Further, from each inhibitory concentration value, a combination index (CI) at a mixing weight ratio of 1: 1 was calculated by the following method.

  The results are shown in Table 1 below. In Table 1, when the combination coefficient (CI) is less than 1 (CI <1) at each inhibitory concentration, it is determined that there is a synergistic effect, and the smaller the combination coefficient (CI), the higher the synergistic effect. Judge.

(Measurement of inhibitory concentration)
The calculation is performed from the cell viability of the two kinds of concentration solutions sandwiching the viability 50% by the following formula (2).

  IC30 and IC40 are also calculated according to the above method.

(Measurement of combination coefficient (CI))
When the rosemary extract and the green tea extract were each used alone, the concentrations when the cancer cell growth inhibition rate reached x% were (Dx) ₁ and (Dx) ₂ , and the cancer cell growth inhibition rate was x The concentration of the rosemary extract and green tea extract in the mixture when reaching% is D ₁ and D _2, and is calculated from the calculation formula shown in the following formula (3).

  As can be seen from Table 1, the composition comprising rosemary extract and green tea extract in a ratio of 1: 1 was in any concentration (dose) compared to rosemary extract alone and green tea extract alone, It is shown that cell proliferation can be significantly suppressed. In addition, it can be seen from the combination factor in Table 1 that the composition of the present invention has a surprisingly high synergistic effect with respect to the rosemary extract alone and the green tea extract alone.

  From the above results, it is considered that the composition of the present invention can be used for the treatment of hematopoietic cancer such as leukemia. In addition, the green tea extract and rosemary extract constituting the composition of the present invention are both used in foods as antioxidants, spices and functional extracts, and safety is recognized. Therefore, foods and pet foods containing the composition of the present invention are expected to be useful as health foods for preventing hematopoietic cancers such as leukemia.

Examples 11 to 17 and Comparative Examples 13 to 17: Evaluation 1 of cell growth inhibitory effect on colon cancer cells 1
Rosemary extract (Mitsubishi Chemical Foods Co., Ltd., RM-21B base) and green tea extract (Mitsui Norin Co., Ltd., Polyphenon (registered trademark) 70A), Rosemary extract and green tea extract weight ratio (Mixed weight ratio of rosemary extract: green tea extract) 20: 1 (Comparative Example 13), 10: 1 (Example 11), 5: 1 (Example 12), 2: 1 (Example 13) Dimethyl to be 1: 1 (Example 14), 1: 2 (Example 15), 1: 5 (Example 16), 1:10 (Example 17) and 1:20 (Comparative Example 14). Prepared with sulfoxide (DMSO) (concentration of the mixture of rosemary extract and green tea extract (total concentration): 10 mg / ml). For comparison, rosemary extract (Comparative Example 15) and green tea extract (Comparative Example 16) were each adjusted to 10 mg / ml with DMSO.

Human colon adenocarcinoma-derived DLD-1 cells were cultured at 37 ° C. in a 5% by volume CO ₂ atmosphere using 10% FBS-containing RPMI1640 medium. 1 ml of this cultured cell was seeded on a 12-well plate so that the number of cells was 1 × 10 ⁴ per well. After culturing DLD-1 cells for 24 hours at 37 ° C. in an atmosphere of 5% by volume CO ₂ , the solution of each extract mixture prepared above was added to the medium so that the final concentration was 10 μg / ml. 1 μl was added. For comparison, rosemary extract and green tea extract were each added in an amount of 1 μl to the medium at 10 μg / ml. As a control (Comparative Example 17), only 1 μl of the solvent DMSO was added.

Forty-eight hours after adding each extract solution or DMSO, DLD-1 cells were cultured at 37 ° C. in a 5% by volume CO ₂ atmosphere. After culturing for a predetermined time, the number of viable cells was measured by trypan blue staining, and the cell growth rate (%) of each sample was calculated by the following formula (4). In the following formula (4), “the number of viable cells of each sample” indicates the number of viable cells of DLD-1 cells when each extract solution (sample) is added and cultured, and “the number of viable cells of control”. Indicates the number of viable cells of DLD-1 cells when DMSO is added and cultured. In addition, the said operation was performed about each 4 samples.

  The results are shown in FIG. In FIG. 2, each value is expressed as an average value + standard deviation (n = 4). In addition, “*” indicates that there is a particularly significant difference (p <0.05) when compared with the rosemary extract alone and the green tea extract alone.

  As can be seen from FIG. 2, the compositions of Examples 11 to 17 are rosemary extract alone (Comparative Example 13), green tea extract alone (Comparative Example 14), rosemary extract and green tea extract 20: It can be seen that the cancer cell growth rate is significantly lower as compared with the case of containing 1 (Comparative Example 11) and the case of containing rosemary extract and green tea extract at 1:20 (Comparative Example 12). From the results, it is considered that the cancer cell proliferation inhibitory effect can be synergistically improved by using the composition of the present invention.

  Moreover, from FIG. 2, the composition (Examples 12-16) which contains a rosemary extract and a green tea extract by the weight ratio (mixture weight ratio of rosemary extract: green tea extract) of 5: 1 to 1: 5. Then, it is shown that a synergistic cancer cell proliferation inhibitory effect can be exhibited more remarkably.

Examples 18 to 20 and Comparative Examples 18 to 24: Evaluation 2 of cell growth inhibitory effect on colon cancer cells 2
Prepared with DMSO so that the weight ratio of rosemary extract to green tea extract (mixture weight ratio of rosemary extract: green tea extract) was 1: 1 (concentration of the mixture of rosemary extract and green tea extract) (Total concentration): 5 mg / ml, 10 mg / ml and 15 mg / ml). For comparison, rosemary extract and green tea extract were prepared in DMSO to 5 mg / ml, 10 mg / ml, and 15 mg / ml, respectively. In this example, RM-21B base manufactured by Mitsubishi Chemical Foods Co., Ltd. was used as the rosemary extract, and polyphenone (registered trademark) 70A (caffeine) manufactured by Mitsui Norin Co., Ltd. was used as the green tea extract. Content: 0.5% or less) was used.

Human colon adenocarcinoma-derived DLD-1 cells were cultured at 37 ° C. in a 5% by volume CO ₂ atmosphere using 10% FBS-containing RPMI1640 medium. After 1 ml of the cultured cells were seeded at 1 × 10 ⁴ per well in a 12-well plate, a solution of the mixture of each extract prepared above was added to a final concentration of 5 μg / ml (Example 18). ) 1 μl was added to the medium so as to be 10 μg / ml (Example 19) and 15 μg / ml (Example 20). In addition, as a comparison object, rosemary extract and green tea extract were each independently 5 μg / ml (rosemary extract: comparative example 18, green tea extract: comparative example 19), 10 μg / ml (rosemary extract: 1 μl was added to the medium so as to be Comparative Example 20, Green Tea Extract: Comparative Example 21) and 15 μg / ml (Rosemary Extract: Comparative Example 22, Green Tea Extract: Comparative Example 23). As a control (Comparative Example 24), 1 μl of only DMSO as a solvent was added.

  For each sample prepared as described above, the cell growth rate (%) was calculated in the same manner as in Example 1, and in the same manner as in Example 8, a 30% inhibitory concentration (IC30 ) (Μg / ml), 40% inhibitory concentration (IC40) (μg / ml) and 50% inhibitory concentration (IC50) (μg / ml) did. Further, in the same manner as in Example 8, a combination index (CI) at a mixing weight ratio of 1: 1 was calculated from each inhibitory concentration value.

  The results are shown in Table 2 below. In Table 2, when the combination coefficient (CI) is less than 1 (CI <1) at each inhibitory concentration, it is determined that there is a synergistic effect. The smaller the combination coefficient (CI), the higher the synergistic effect. Judge.

  As can be seen from Table 2, the composition comprising rosemary extract and green tea extract at a ratio of 1: 1 was in any concentration (dose) compared to rosemary extract alone and green tea extract alone, It is shown that the proliferation of cancer cells can be significantly suppressed. In particular, since a significant effect of suppressing cancer cell growth suppression is observed for colon cancer at a low concentration (low dose) of 5 to 10 μg / ml, the composition of the present invention is a digestive cancer at a low dose. It is considered to have a therapeutic / preventive effect on

  In addition, it can be seen from the combination coefficient shown in Table 2 that the composition of the present invention has a surprisingly high synergistic effect with respect to the rosemary extract alone and the green tea extract alone.

  From the above results, it is considered that the composition of the present invention can be used for the treatment of cancers of the digestive system such as colon cancer. Moreover, the green tea extract and the rosemary extract which comprise the composition of this invention are used for food as an antioxidant, a spice, and a functional extract, and safety is recognized. Therefore, foods and pet foods containing the composition of the present invention are expected to be useful as health foods for the prevention of cancers of the digestive system such as colon cancer.

  This application is based on Japanese Patent Application No. 2014-258968 filed on December 22, 2014, the disclosure of which is referenced and incorporated in its entirety.

Claims (7)

A composition for inhibiting colon adenocarcinoma cell proliferation, comprising a green tea extract and a rosemary extract in a weight ratio of 10: 1 to 1:10 (mixed weight ratio of green tea extract: rosemary extract). The composition for inhibiting colon adenocarcinoma cell proliferation according to claim 1, comprising a green tea extract and a rosemary extract in a weight ratio of 5: 1 to 1: 5 (mixed weight ratio of green tea extract: rosemary extract). object. The composition for inhibiting colon adenocarcinoma cell proliferation according to claim 2, comprising a green tea extract and a rosemary extract in a weight ratio of 2: 1 to 1: 2 (mixed weight ratio of green tea extract: rosemary extract). object. The composition for inhibiting colon adenocarcinoma cell growth according to claim 3, comprising a green tea extract and a rosemary extract in a weight ratio of 1: 1. The composition for inhibiting colon adenocarcinoma growth according to any one of claims 1 to 4 , which is used for prevention and / or treatment of colon adenocarcinoma . The composition for inhibiting colon adenocarcinoma growth according to any one of claims 1 to 5 , which is used orally for at least one pet animal selected from the group consisting of humans, dogs, cats and rabbits. The composition for suppressing colon adenocarcinoma growth according to any one of claims 1 to 6 , which is a human health food or pet food composition.