JP2006306897A - Mozuku (edible seaweed)-originating fucoidan including agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new anti-gastric cancer agent that originates from natural product and can be used not only as a medicine but also as a beverage or food. <P>SOLUTION: The gastric cancer cell proliferation suppressing agent employs the fucoidan originating from Mozuku seaweed as an effective component. As a fucoidan originating from Mozuku, can be used a hot water extract of Mozuku seaweeds, for example, Ito mozudu (Nemacystus decipieus), Okinawa mozuku (Cladosiphon okamuranus) or Futo mozuku (Tinocladia crassa) or the like or a purified product thereof obtained by treating the extract with a quaternary ammonium salt. The purified product is used to develop excellent gastric cancer cell proliferation suppressive action with no problem on the safety because it originates from natural product. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to effective use of mozuku-derived fucoidan for gastric cells.

Mozuku is mainly used for food as vinegared food in Japan, but it is not used as other processed foods or resources. Mozuku contains polysaccharides, but the effective use of these polysaccharides has not been fully elucidated.
"General Food Encyclopedia (new edition)" Tokyo Dobunin, March 15, 1971, p. 637

  The present invention has been made for the purpose of newly developing effective use of mozuku in view of the current state of the art.

  In order to achieve the above-mentioned object, the present inventors have been studying various aspects of mozuku in recent years as a major cause of death among Japanese adult diseases, so-called lifestyle-related diseases. It is the number one cause of death among Japanese, and gastric cancer is one of the most common cancers that has been ranked first in mortality by region. Further attention and further research on the safety and functionality of mozuku foods that have been performed in the past will be carried out. From Okinawa mozuku and Ito mozuku to fucoidan (heterosulfate polysaccharides mainly composed of fucose with a molecular weight of several hundred thousand) In the course of studying the effects on human gastric cells, the novel and useful physiological activity of mozuku-derived fucoidan on human gastric cells was confirmed for the first time.

  More specifically, the present inventors extracted and purified fucoidan, which is a physiologically active component, from the Okinawa mozuku Cladosiphon okamuranus and the Imozuku nemacystus decipiens, which are mainly eaten as mozuku, and examined the effect on human gastric cells. As a model for the action of mozuku on human gastric cells, the effect on proliferation of cultured human gastric cancer cells and cultured human normal gastric cells was examined. In addition, as a model experiment of the effect of mozuku food on human gastric cells when using anticancer drugs, anti-cancer drug 5-fluorouracil (hereinafter referred to as 5-FU) against human gastric cancer cells and normal human gastric cells in mozuku-derived fucoidan-added medium. To evaluate the effect of

  As a result, as will be apparent from the following, a number of useful physiological actions of mozuku-derived fucoidan as exemplified below were confirmed for the first time: gastric cell protective action; gastric cancer prevention / treatment action; , An action that enhances the action of an anticancer drug, and / or an action that prevents or suppresses a decrease in the action of an anticancer drug; Anti-cancer agent, anti-ulcer agent, antipyretic agent, cold agent, gastrointestinal agent, antibiotics, antiviral agent, immunopotentiator, immunosuppressant, sedative agent, and other agents alleviate, suppress or stop side effects Other.

  The present invention is based on the above-described useful new findings, particularly the preventive / therapeutic action of gastric cancer, and has been further researched and examined, and is characterized by comprising mozuku-derived fucoidan, Specifically, the present invention provides an agent that suppresses or stops the growth of gastric cancer cells.

  According to the present invention, mozuku-derived fucoidan can be extracted and purified, and in particular, Okinawa mozuku-derived fucoidan has a high growth inhibitory effect on human gastric cancer cells. Further, normal human gastric cells did not show an effect of suppressing proliferation. And it turned out that it does not inhibit the anticancer action of the anticancer agent 5-FU on human gastric cancer cells and suppresses the influence of the anticancer agent 5-FU on normal human cells.

  Therefore, according to the present invention, it is possible to provide a growth inhibitory agent for gastric cancer cells characterized by containing mozuku-derived fucoidan.

  Further, as described above, according to the present invention, it is possible to provide a growth inhibitor against gastric cancer cells that can be used as a preventive / therapeutic agent for gastric cancer, as well as drugs such as anticancer agents, antiulcer agents, antibiotics, It is also possible to provide an agent that reduces, suppresses or stops at least one side effect selected from an antipyretic agent, a common cold agent, a gastrointestinal agent, an antiviral agent, an immunopotentiating agent, an immunosuppressive agent, and a sedative agent.

  Furthermore, according to the present invention, it contains a mozuku-derived fucoidan, an agent for enhancing the action of a drug against gastric cancer cells, particularly an anticancer agent, and / or a mozuku-derived fucoidan. It is also possible to provide an agent that suppresses a decrease in the action of an agent against gastric cancer cells, particularly an anticancer agent.

  One of the uses of fucoidan derived from mozuku is functional foods with added health promotion and cancer prevention functions, foods that can be ingested by cooking, and other uses. It can be used as a side effect inhibitor that suppresses side effects or as a hospital food.

  According to the present invention, mozuku-derived fucoidan is used directly as a drug (that is, a gastric cancer cell growth inhibitor or anti-gastric cancer drug) and, if desired, does not directly act as a drug but is used in combination with it. It is possible to reduce or suppress the side effects of drugs, or to enhance the action of anti-gastric cancer drugs or to suppress the reduction of the action, and not to have a direct pharmacological action, but to use in combination It has very unique characteristics in that it enhances the action of the drug and also has an indirect pharmacological action.

  In the latter case, in the present invention, the mozuku-derived fucoidan and the drug can be formulated into one agent by mixing them, and both are prepared separately and used at the same time during use. Alternatively, it may be used after a certain time, that is, by shifting the time.

  Examples of drugs used in combination with mozuku-derived fucoidan include the above-mentioned drugs, and anti-cancer drugs include 5-FU, various pyrimidine antagonists, purine antagonists; antibiotics such as bleomycin; vinblastine and vincristine Plant components such as: hormones and other known anticancer agents.

  In the present invention, mozuku-derived fucoidan is used. Examples of the mozuku used in the present invention include Ito mozuku (fine mozuku) (Nemocytus depipius), Okinawa mozuku (Nagamatsu Mosaka), and various types of tuna clas.

  As mozuku-derived fucoidan, pure fucoidan extracted, isolated and purified from mozuku can be used (commercially available), mozuku hot water extract and / or processed product thereof can also be used It is.

  In order to extract the extract of the present invention from mozuku, mozuku (cut, pasted, dried, frozen) may be used for 30 minutes to 3 hours using hot water of 60 ° C. or higher, preferably 80 ° C. or higher. After extraction (for example, after treatment at 100 ° C. for 45 minutes to 1 hour 30 minutes in an autoclave), the obtained extract is desalted by electrodialysis. The hot water extract of the present invention includes an extract obtained by simply extracting mozuku with hot water as described above, its filtrate, and an extract obtained by further desalting the above (etc.). A purified product purified by quaternary ammonium salt treatment, alcohol precipitation, ion exchange resin chromatography, or gel filtration chromatography is also included.

  In the present invention, a processed product of mozuku hot water extract can also be used. Examples of the treated product include concentrates, pasted products, dried products (spray dried products, freeze dried products, vacuum dried products, drum dried products, etc.), emulsions, liquid products, and diluted products.

  The agent according to the present invention contains fucoidan derived from mozuku, its content, for example, hot water extract of mozuku, and at least one of its processed products, as it is or other food and drink and its It can be formulated and processed into various dosage forms and forms together with the raw materials, and it can be used as an active ingredient to make food and drink, or as a pharmaceutical product by using it as an active ingredient. Can be used freely.

  The blended amount or the amount used as an active ingredient may be appropriately determined according to the purpose of use (prevention, health or treatment), patient age, administration method, dosage form, etc. It is appropriate to orally administer 1 mg to 5 g (dry matter) / day in terms of However, when ingested for the purpose of health maintenance or health maintenance over a long period of time, the amount may be smaller than the above range, and this active ingredient may affect the drug efficacy of the combined drug. There is no problem with regard to safety, so even if it is used in a larger amount than the above range, there is no problem. As a result of a 10-day acute toxicity test using mice, no deaths were observed even after oral administration of 100 mg / kg.

  When this drug is not used as a medicine, it can be used as a food or drink. When used as a food and drink product type, this active ingredient (mozuku-derived fucoidan and drug as desired) is mixed and used as is, or used in combination with other foods or food ingredients as appropriate. Therefore it can be used. Foods and drinks formulated with this active ingredient may be solid (powder, granule, etc.), paste, liquid or suspension, but are commonly used for the production of sweeteners, acidulants, vitamins and other drinks. It is preferable to formulate a health drink using the various ingredients. It is also suitable as a supplement.

  That is, the foods and drinks according to the present invention include those that can be eaten immediately as they are, those that are cooked and eaten, premixed materials for food production, and the like. The solid product may be any of powder, granule, and solid, for example, confectionery such as biscuits, cookies, cakes, snacks, rice crackers, bread, powdered beverages (powdered coffee, cocoa, etc.) included. Examples of liquids, emulsions, and pastes include various beverages such as juices, carbonated beverages, and lactic acid bacteria beverages. Among these, in the present invention, as described above, beverages are particularly preferable.

  When used as a pharmaceutical type, the active ingredient (mozuku-derived fucoidan and optionally a drug) is administered in various forms. Examples of the dosage form include oral administration using tablets, capsules, granules, powders, syrups and the like. These various preparations are usually used in the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. Can be formulated using known adjuvants.

  In the present invention, it is possible to use a mozuku-derived fucoidan and a drug in combination as desired, but in that case, in both cases of the pharmaceutical type and the food and drink type, as described above, the mozuku-derived fucoidan and In addition to being formulated as a single drug, it is also possible to separate the mozuku-derived fucoidan from the drug and formulate it into a dosage form for use at the time of use so that both can be used together.

  In this case as well, it may be formulated as described above. In this case, however, mozuku-derived fucoidan and the drug are prepared separately without mixing and used together at the time of use. For example, both may be administered at the same time or may be administered at different times.

  In the present invention, a growth inhibitor of gastric cancer cells comprising mozuku-derived fucoidan as an active ingredient is provided. In addition to using the active ingredient alone as described above, the active ingredient may be combined with other drugs as desired. It can also be used in combination, but even in that case, it exhibits various excellent effects, and since the effect lasts for a long period of time, it can be used as a medicine, and it can be used as a food or drink or other food or drink. Can be used together.

  In addition, mozuku-derived fucoidan, which is one of the active ingredients of the present invention, is originally a food or derived from food, so there is no problem with safety, and it also has various wide-ranging actions. Therefore, it can be used not only as a specialized drug as an agent based on each of the above-mentioned individual actions, but also as a home-use medicine for the purpose of protecting the stomach. As a health care agent for patients or postoperative patients (for example, patients after surgery for gastric cancer, etc.), preventive and / or therapeutic agents for cancer and other diseases, cancer recurrence prevention agents, etc. It can be used in the form of pharmaceuticals or foods and drinks (including foods for specified health use).

  Examples of the present invention and reference examples will be described below.

Example 1
(1) Production of mozuku-derived fucoidan 100 ml of distilled water was added to 100 g of wet mozuku alga bodies (Cladosiphon okamuranus) and Imozuku (Namocytus decipiens) from which foreign substances adhering to the surface were removed. Was heated for 1 hour at 100 ° C. The residue was removed by filtration, and the extract was desalted by electrodialysis.The obtained hot water extract can be used as it is. In this example, however, the powder was made into a powder by freeze-drying, that is, the obtained hot water extract was frozen to spread on the side of the container, depressurized at room temperature (about 20 ° C.) to remove moisture for one day, A water extract was obtained as mozuku-derived crude fucoidan, which means that in this way mozuku 100 g Or to give Cladosiphon okamuranus from crude fucoidan 1.3g, the Itomozuku from crude fucoidan 0.99g respectively.

(2) Purification of mozuku-derived fucoidan Purified fucoidan was obtained by a purification method using a quaternary ammonium salt. A quaternary ammonium salt, cetyltrimethylammonium bromide, and an acidic polysaccharide formed a complex and precipitated, and the precipitate was fractionated by the difference in solubility of the salt concentration. As a result, 990 mg of Okinawa mozuku-derived purified fucoidan and Itomozuku-derived purified fucoidan were divided into two fractions per 100 g of mozuku to obtain <1>: 160 mg and <2>: 110 mg.

(3) Qualitative and quantitative analysis of mozuku-derived fucoidan The molecular weight distribution of the obtained mozuku-derived fucoidan was analyzed by size exclusion chromatography, the sulfate group content was analyzed by the Dodgson method, the constituent sugar analysis was hydrolyzed by fucoidan, and developed on a silica gel TLC plate. analyzed. The content of the peak detected in the molecular weight distribution was determined from the peak area ratio, with fraction 1 having a small retention time (high molecular weight) as fraction 1 and large fraction (low molecular weight) as fraction 2. The analysis results of the obtained polysaccharide are shown in Table 1. As a result, the refined fucoidan derived from Okinawa Mozuku has a molecular weight of about 3.2 million and a sulfate group content of 9.8%, and the purified Imozuku derived fucoidan has a molecular weight of about 21.50 million and a sulfate group content of 26.9%. Another fraction <2> had a molecular weight of about 2.5 million and a sulfate group content of 27.8%. In addition, Okinawa mozuku-derived purified fucoidan is L-fucose or D-mannose, one fraction <1> of Itomozuku-derived purified fucoidan is L-fucose, and another fraction <2> is L-fucose and D-galactose. It was thought to be a constituent sugar.

(Table 1)
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Sample Fraction 1-Molecular weight * Fraction 2-Molecular weight * Sulfate content
(Content) (Content) (SO ₄ )
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Sigma Fucoidan 10.4 × 10 ⁴ (80%) 1700 (20%) 18.8%
Sodium alginate 70.0 × 10 ⁴ (94%) 1730 (6%) 0%
Okinawa Mozuku Coarse Fucoidan 308 × 10 ⁴ (65%) 1670 (35%) 5.6%

Ito Mozuku Coarse Fucoidan 246 × 10 ⁴ (39%) 1680 (61%) 17.0%
Okinawa Mozuku Refined Fucoidan 319 × 10 ⁴ (94%) 1740 (6%) 9.8%
Ito Mozuku Refined Fucoidan <1> 215 × 10 ⁴ (100%) − 26.9%
Ito Mozuku Refined Fucoidan <2> 250 × 10 ⁴ (98%) 1680 (2%) 27.8%
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* Equivalent molecular weight based on pullulan molecular weight

(Example 2)
The effect of mozuku-derived fucoidan on cultured human gastric cells was confirmed by the following.

(1) GT3TKB (distributed from Riken Genebank, Cell Number: RCB0885) and MKN45 (distributed from Riken Genebank, Cell Number: RCB1001) as normal cell lines, human normal stomach cells Hs677. MTT assay was performed using St (dispensed from ATCC, ATCC Number: CRL-7407).

(2) In the MTT assay, when yellow MTT, which is a tetrazolium salt, is taken into living cells for a certain period of time, it is converted into a blue formazan product by the action of succinate dehydrogenase in the mitochondria, and the absorbance is an indicator of the number of living cells. Is a method for measuring the number of living cells. Each sample solution was added at 10 ml / well, and PBS (-) was added at 10 ml / well to the control well. In each growth medium, 90 ml of a cell suspension adjusted to 2 × 10 ³ cells / well for GT3TKB and 5 × 10 ³ cells / well for MKN45 was added, and GT3TKB was maintained at 37 ° C. in a 5% CO ₂ incubator for 3 days. MKN45 was cultured for 4 days. Thereafter, the MTT solution was added at 10 ml / well, cultured in a CO ₂ incubator at 37 ° C. for 4 days, added with 100 ml of 10% SDS solution, and allowed to stand in the CO ₂ incubator for 1 day, and the absorbance at 570 nm was measured at a target wavelength of 650 nm. . The fucoidan used as a control was Fucus vesiculosus derived from Fucus vesiculosus, a kind of hibermata. The absorbance was 0% in the medium containing neither the cell nor the sample, and the absorbance in the cell cultured in the growth medium not containing the sample. The survival rate was calculated as 100%.

(3) In this way, the growth inhibitory effect confirmation test of mozuku-derived crude fucoidan on human gastric cancer cells was suppressed, and the results are shown in FIG. In the figure, A shows human gastric cancer cell GT3TKB, B shows human gastric cancer cell MKN45, and in each bar graph, the left side shows the concentration of 0.5 mg / ml of each sample such as fucoidan and the right side shows the same concentration of 1.0 mg / ml. Each is shown.

  As is apparent from the above results, the effect on human gastric cancer cells is that Sigma Fucoidan used as a control inhibits growth of two types of cancer cells in a concentration-dependent manner, and sodium alginate grows against MKN45. The effect was not seen, but Okinawa mozuku-derived crude fucoidan acted in a concentration-dependent manner, and at a concentration of 1.0 mg / ml against MKN45, an effect equivalent to that of Sigma Fucoidan was seen. In addition, it showed an excellent growth inhibitory action against GT3TKB (FIG. 1).

(4) In the same manner as described above, a growth inhibitory effect confirmation test of mozuku-derived purified fucoidan (1.0 mg / ml) on human gastric cancer cells was performed, and the results are shown in FIG. In the figure, A represents human gastric cancer cell GT3TKB, and B represents human gastric cancer cell MKN45. For comparison purposes, data for coarse fucoidan is also shown.

  As is clear from the above results, the effects of mozuku-derived purified fucoidan on human gastric cancer cells were all higher than those of mozuku-derived crude fucoidan (FIG. 2). 2 and 3, the circled numbers 1 and 2 represent <1> and <2>.

(5) In the same manner as described above, a test for confirming the influence of mozuku-derived fucoidan on human normal stomach cells was performed, and the results are shown in FIG.

That is, the effect on normal cells was determined by adding 10 ml / well of each sample solution to a final concentration of 1.0 mg / ml as in the above experiment, and adding Hs677. St. 90 ml of a cell suspension adjusted to 2 × 10 ³ cells / well was added and cultured in a 37 ° C., 5% CO ₂ incubator for 4 days, and MTT assay was performed. As a result, human normal stomach cells Hs677. None of the control sigma fucoidan, sodium alginate, or mozuku-derived fucoidan affected the growth of St (FIG. 3). Thus, it was confirmed that mozuku-derived fucoidan is not a cytotoxic agent for normal cells.

(Reference Example 1)
The effect of mozuku-derived fucoidan on the action of the anticancer agent 5-FU on cultured human gastric cells was confirmed by the following.

(1) The effect of mozuku-derived crude fucoidan on the action of the anticancer agent 5-FU on human gastric cancer cell lines was examined. That is, 10 ml / well of each sample solution was added to the medium and cultured for 1 hour in a polysaccharide-added medium made 0.5 or 1.0 mg / ml, 5-FU 10 ml / well of each concentration was added, and cultured for several days. The assay was performed.

  The results are shown in FIGS. In the figure, A indicates a sample of 0.5 mg / ml, B indicates a sample of 1.0 mg / ml, and the bar graph indicates 5-FU concentrations of 10 μg / ml, 25 μg / ml, and 50 μg / ml sequentially from the left side. As is clear from the above results, Sigma Fucoidan and Okinawa Mozuku-derived crude fucoidan were used in combination with 5-FU rather than the survival rate of gastric cancer cells in a medium (PBS) supplemented only with 5-FU. However, in both cases, it was confirmed that the survival rate of gastric cancer cells was low.

(2) The effect of mozuku-derived crude fucoidan on the action of the anticancer agent 5-FU was similarly examined on normal cells. The experiment was performed with a sample of 1.0 mg / ml and 5-FU of 50 μg / ml. As a result, the survival rate of the combined use of sigmafucoidan, sodium alginate and 5-FU was almost unchanged from the survival rate of 5-FU alone (PBS). However, the survival rate using Okinawa mozuku crude fucoidan, Itomozuku crude fucoidan in combination with 5-FU is higher than the survival rate of adding only 5-FU, and is similar to or higher than that of the control (FIG. 6). It has been demonstrated that mozuku-derived fucoidan protects normal gastric cells from the action of 5-FU and prevents or reduces the side effects of anticancer agents.

(3) Therefore, in the combined use with 5-FU, even if Mozuku-derived fucoidan is not particularly marked in its direct anticancer effect as compared with other polysaccharides, As described above, since it excels in the protective action of normal gastric cells, 5-FU can be administered in a large amount and / or over a long period of time. As a result, the anti-cancer action of 5-FU can be improved. It has been found that enhancement and / or its side effects are suppressed.

(Example 3)
As mozuku-derived fucoidan, 70 parts by weight of mozuku hot water extract (Example 1: dry powder), 20 parts by weight of lactose, and 10 parts by weight of corn starch were mixed, and the resulting mixture was fluidized with 5% aqueous solution of hydroxypropylmethylcellulose. Granules were obtained.

Example 4
Mozuku hot water extract (Example 1) 100g, sugar 150g, honey 15g, ascorbic acid 1g, citric acid 0.5g, fragrance appropriate amount of water to 1kg, sterilized at 95 ° C for 20 minutes Then, 100 ml each was aseptically filled into a bottle to produce a food and drink type health drink.

(Example 5)
As mozuku-derived fucoidan, add 20 g of 20% aqueous solution of mozuku hot water extract (freeze-dried powder), 5 g of tocopherol acetate, 10 g of thiamine nitrate, 20 g of nicotinic acid amide, 50 g of anhydrous caffeine, benzoate and perfume to the appropriate amount. After sterilization, each bottle was aseptically filled in 30 ml bottles to produce a health drink as a pharmaceutical product.

(Example 6)
Mix 8 parts of shortening with 18 parts of sugar, then 42 parts of weak wheat flour, 7.5 parts of mozuku hot water extract as mozuku-derived fucoidan (Example 1), 0.8 part of baking powder, 16 parts of egg, glucose 1 part and 25 parts of water were added and stirred to make a dough. This dough is rolled to a thickness of 5 mm, die-cut so that one piece becomes 16 to 17 g, and baked in an oven at 90 ° C. for 32 to 36 minutes. As a result, about 12 g of biscuit was obtained. It is calculated to contain 1 g of mozuku-derived fucoidan in 12 g of biscuits. These are examples of formulation of mozuku-derived fucoidan in the case of the pre-use formulation type, but in the same manner, according to a conventional method, an agent containing both components simultaneously mixed with 5-FU Manufactured.

1 shows the growth inhibitory effect of mozuku-derived crude fucoidan on human gastric cancer cells. The growth inhibitory effect of the mozuku origin refinement | purification fucoidan (1.0 mg / ml) with respect to a human stomach cancer cell is shown. Normal human stomach cells Hs677. The influence of mozuku-derived fucoidan on St is shown. The influence of the mozuku origin fucoidan with respect to the effect | action of the anticancer agent 5-FU in human gastric cancer cell GT3TKB is shown. The influence of the mozuku origin fucoidan with respect to the effect | action of the anticancer agent 5-FU in the human stomach cancer cell MKN45 is shown. Normal human stomach cells Hs677. The effect of mozuku-derived fucoidan on the action of the anticancer agent 5-FU on St is shown.

Claims (6)

  A growth inhibitor for gastric cancer cells, characterized by comprising mozuku-derived fucoidan as an active ingredient.   The agent according to claim 1, wherein the mozuku-derived fucoidan is a hot water extract of mozuku and / or a processed product thereof.   The agent according to claim 2, wherein the hot water extract of mozuku is a liquid obtained by heating a mixture of mozuku and water and then filtering the mixture and desalting the resulting filtrate.   The agent characterized in that the fucoidan derived from mozuku is a purified product obtained by further purifying the hot water extract of mozuku according to claim 2 or 3 and / or a processed product thereof.   The agent according to any one of claims 1 to 4, wherein the treated product is at least one selected from a concentrate, a paste, a dried product, an emulsion, a liquid, and a diluted product.   The agent according to any one of claims 1 to 5, wherein the mozuku is at least one of a yarn mozuku, a thick mozuku, and an Okinawa mozuku.

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