TW200400195A - Apoptosis inducer - Google Patents

Abstract

Apoptosis inducer, carcinostatic agent and carcinogenic preventive agent which comprise fucose sulfuric acid-containing polysaccharide and/or degradation material thereof and an apoptosis induction method which uses fucose sulfuric acid-containing polysaccharide and/or degradation material thereof as an active ingredient are provided. The digestive enzyme used in the process for production of degradation material of fucose sulfuric acid-containing polysaccharide is also provided.

Description

200400195 (1) Description of the invention: [Technical field to which the invention belongs] The present invention relates to a cell autoinactivation inducer, a carcinostatic agent, and a carcinogenic preventive agent that can be used as pharmaceuticals. In addition, the present invention provides a method for inducing cell self-destruction useful for clarification of cell self-destruction mechanism and screening of inhibitors for inducing cell self-destruction. Furthermore, the fucose-containing sulfated polysaccharide purified according to the present invention and its decomposed matter are provided, and the production of fucose-containing sulfated polysaccharide decomposed matter is provided, and the fucose-containing sulfated polysaccharide decomposing enzyme useful in structural investigation is provided. [Prior technology] In recent years, the death of cell tissues has focused on the so-called cell death (apoptosis, also known as phagocytosis; self-violence or cell self-destruction). This cell self-destruction, unlike pathological cell death, is considered to be the death of the cell's own genes that were originally recombined. That is to say, the main cause of any external or internal cause is the activation of the gene for planning the self-destruction of cells, and the use of this gene will biosynthesize the dead gene protein, and the generated dead protein will The cell itself breaks down and dies | 死. If this kind of cell self-destruction can make it appear in the desired tissues and cells, it can make unnecessary or pathogenic cells be eliminated from the organism in a natural form, which is of profound significance. An object of the present invention is to develop a compound having high safety in inducing cell self-destructive effects, and to provide a cell self-destructive inducer, a carcinostatic agent, a carcinogenic preventive agent containing the compound, and a method for inducing self-destructive cells using the compound as an active ingredient. The present invention also provides a compound-decomposing enzyme for use in the production of a decomposed product of the present invention. [Summary of the Invention] If the present invention is described, the first invention of the present invention relates to a cell self-killing inducer, which is characterized by containing a fucose-containing sulfated polysaccharide and / or a degradation product thereof. The second invention of the present invention relates to a method for inducing cell self-destruction, which is characterized in that the fucose-containing sulfated polysaccharide and / or a decomposed product thereof are used as an active ingredient. The third invention of the present invention relates to a carcinostatic agent, which contains the fucose-containing sulfated polysaccharide and / or a degradation product thereof of the fifth or sixth invention of the present invention. The fourth invention of the present invention relates to a carcinogen preventive agent, which contains a fucose-containing sulfated polysaccharide and / or a degradation product thereof. The fifth invention of the present invention relates to a fucose-containing sulfated polysaccharide having the following physical and chemical properties. (1) Constituting sugar; containing uronic acid. 910069) produced fucoidan, which decomposes enzymes and reduces their molecular weight, and produces at least one selected from the compounds represented by the following formulae (丨), (Z; [), (ΠI) Of compounds. 8 200400195 ch2 on

OH

200400195

on

10 200400195 The sixth invention of the present invention relates to fucose-containing sulfated polysaccharide having the following physical and chemical properties. (1) Structural sugar: It does not substantially contain uronic acid. (2) The fucoidan-degrading enzyme produced by Flavobacterium sp. SA-0082 (CCRC 9 1 0069) cannot be substantially reduced in molecular weight. The seventh invention of the present invention relates to a method for preparing fucose-containing sulfated polysaccharide according to the fifth invention of the present invention, which is characterized in that the fucose-containing sulfated polysaccharide mixture is treated with an agent having an acidic polysaccharide aggregation ability in the presence of salts. And remove the sedimentation process. The eighth invention of the present invention relates to a method for producing fucose-containing sulfated polysaccharide according to the fifth invention of the present invention, which is characterized in that the fucose-containing sulfated polysaccharide mixture is treated with an anion exchange resin in the presence of a mixture of divalent cations and The process of collecting the target polysaccharide. The ninth invention of the present invention is a method for producing a fucose-containing sulfated polysaccharide according to the fifth invention of the present invention, which is characterized by including a coexisting coloring substance when producing the fucose-containing sulfated polysaccharide of the fifth invention. Process for removing polysaccharide substance or substance having anion exchange group. The tenth invention of the present invention is a method for preparing fucose-containing sulfated polysaccharides according to the sixth invention of the present invention, which is characterized by including a mixture of fucose-containing sulfated polysaccharides to decompose fucose-containing sulfated polysaccharides Capable degrading enzyme 'or a process for treating and collecting the target polysaccharide with a microorganism having the degrading enzyme. The eleventh invention of the present invention is a method for preparing fucose-containing sulfate polysaccharide 11 200400195 according to the sixth invention of the present invention, which is characterized by including a fucose-containing sulfate polysaccharide mixture in the presence of salt to have an acidic polysaccharide aggregation ability The process of precipitating the target polysaccharide. The twelfth invention of the present invention is a method for preparing a fucose-containing sulfated polysaccharide according to the sixth invention of the present invention, which is characterized in that the fucose-containing sulfated polysaccharide mixture is treated with an anion exchange resin in the presence of a mixture of divalent cations and The process of collecting the target polysaccharide. The thirteenth invention of the present invention relates to a method for producing fucose-containing sulfated polysaccharide according to the sixth invention of the present invention, which comprises using a polysaccharide that coexists as a coloring substance when producing the fucose-containing sulfated polysaccharide of the sixth invention of the present invention. Removal of sexual substances or substances with anion exchange groups. The fourteenth invention of the present invention is a method for preparing a fucose-containing sulfated polysaccharide mixture, which is characterized in that the fucose-containing sulfated polysaccharide mixture used in the seventh, eight, ten, eleven, or twelfth invention of the present invention is extracted from seaweed At that time, acetate ions and calcium ions are allowed to coexist. The fifteenth invention of the present invention relates to an end-type fucose-containing sulfated polysaccharide-decomposing enzyme having the following physical and chemical properties as its characteristics. (i) Action: It acts on the fucose-containing sulfated polysaccharide having the following physical and chemical properties, and makes the fucose-containing sulfated polysaccharide low-molecular. (a) Constituting sugar: It does not substantially contain uronic acid. (b) The fucoidan-degrading enzyme produced by Flavobacterium sp. SA-0082 (CCRC 9 1 0069) cannot be reduced in molecular quality. It has no effect on fucose-containing sulfate polysaccharide having the following physical and chemical properties. (c) Structural sugar: contains uronic acid. 12 200400195 (d) The fucoidan-degrading enzyme produced by Flavobacterium sp. SA-0082 (CCRC 9 1 00 69) is reduced in molecular weight and is produced by at least the following formula (I), ( II), (III) One or more selected compounds.

ch2 on

OH

13 200400195

ch2〇h

OH

/ OH 〇 (111)

OH 14 200400195 (ii) Optimum pH: The optimum pH of this enzyme is around 7-8. (ill) Optimum temperature: The optimum temperature of this enzyme is around 30-35 ° C. A sixteenth invention of the present invention relates to an enzyme containing a calcium source and a terminal fucose-containing sulfate polysaccharide decomposing enzyme of the fifteenth invention of the present invention. The seventeenth invention of the present invention is a method for producing a terminal fucose-containing sulfate polysaccharide-decomposing enzyme according to the fifteenth invention of the present invention, which is characterized in that The productivity-promoting cells of the genus Zymomonas are cultured, and the enzyme is collected from the culture. The eighteenth invention of the present invention is a low molecular weight compound of fucose-containing sulfated polysaccharide, which is characterized in that the terminal fucose-containing sulfated polysaccharide decomposing enzyme of the fifteenth invention of the present invention acts on the Acquired from fucose sulfate polysaccharide. The present inventors have succeeded in obtaining various purified fucose-containing sulfated polysaccharides and their decomposition products, and then reviewed their biological activities, and found that these substances cause cancer cells to induce cell self-destruction and show strong carcinogenesis. It was also found that the fucose-containing sulfated polysaccharide and / or its decomposed product showed a strong carcinogenic effect ®. Furthermore, the fucose-containing sulfated polysaccharide-decomposing enzyme useful for preparing the decomposed product of the present invention was successfully isolated, and thus the present invention was completed. [Embodiment] Hereinafter, the present invention will be specifically described. The fucose-containing sulfated polysaccharide used in the present invention is not particularly limited, and for example, a substance derived from 桧 华 Fork, a substance derived from GAGOME kelp, a substance derived from true kelp, and a substance derived from wakame In addition to substances, 15 200400195 all substances derived from brown algae plants can also be used. In addition, it is known that fucose-containing sulfated polysaccharides are also present on the body wall of sea cucumbers. Therefore, fucose-containing sulfated polysaccharides from sea cucumbers are also used in the present invention. In the present invention, a decomposed product of a fucose-containing sulfuric acid polysaccharide can also be used. Examples of the method for decomposing fucose-containing sulfated polysaccharide include chemical decomposition methods such as acid treatment, physical decomposition methods such as ultrasonic treatment, and enzyme decomposition methods. The present inventors have found that when various fucose-containing sulfated polysaccharides and / or their degradation products are added to the culture solution of cancer cells as described above, cell self-destruction is caused 1 to several days after the addition. In addition, it was also confirmed that it does not show toxicity to normal cells. In the present invention, the so-called fucose-containing sulfuric acid polysaccharide is a polysaccharide containing fucose sulfuric acid in the molecule, and is not particularly limited. For example, it is contained in brown algae plants, sea cucumbers, etc. Kyoritsu Publishing Co., Ltd., published on December 15, Showa 30, Polysaccharide Chemistry, p. 319, p. 321].岩 Fucose-containing sulfated polysaccharides from brown algae plants are commonly referred to as fucoidan, fucoidan, and fucoidan, and several molecular species are known but they are collectively referred to as fucoidan. For example, it has been reported that fucoidan manufactured by the commercial SIGMA company can be divided into 13 molecular types [Carbohydrate research, Vol. 255, pp. 213-224 (1994)], among which fucose is the main component One group, and the constituent sugars containing a few% of uronic acid mostly contain molecular groups of fucose and mannose. Various cases of enhanced macrophage activity, cancer metastasis inhibition, anticoagulation, etc. in its biological activity have been reported, but because of the molecular species in the fucose-containing sulfated polysaccharide, in order to investigate which molecule the activity itself is in Species 16 200400195, it is necessary to separate and refine fucose-containing sulfated polysaccharides and investigate. Among the fucose-containing sulfated polysaccharides, fucose is the main component of the sugar that does not substantially contain uronic acid, and fucose and mannose are contained in the constituent sugar that contains several percent of the uronic acid. Hereinafter, those who do not substantially contain uronic acid will be referred to as fucose-containing sulfated polysaccharide-F, and those who contain uronic acid will be referred to as fucose-containing sulfated polysaccharide · U. This mixture is described as a fucose-containing sulfated polysaccharide mixture. The methods known so far for separating fucose-containing sulfated polysaccharide-F and fucose-containing sulfated polysaccharide-U are separated according to molecular weight classification and anion exchange resin. Due to insufficient separation, it is difficult to prepare a large amount for pharmaceutical and functional properties. food. In addition, it is known that it is difficult to completely remove a coloring substance from a fucose-containing sulfated polysaccharide, and a commercially available fucoidan and the like also contain a coloring substance. Generally, this coloring substance is a substance polymerized by polyphenol, which inhibits various enzyme reactions and hinders cell growth with extremely strong reactivity, and, for example, irreversibly adsorbs resin and resinous containers in contact. Therefore, in order to accurately investigate the biological activity of fucose-containing sulfated polysaccharides, and to prevent contamination of containers and resins, it is necessary to remove highly reactive colored substances from fucose-containing sulfated polysaccharides. Also, it is known that when fucose-containing sulfated polysaccharide mixture is extracted from brown algae or brown algae's ethanol washing residue, etc., 'the use of soluble barium acetate, barium chloride, and calcium chloride can inhibit the mixing of alginic acid', which is beneficial to it. After purification, but the soluble barium salt is not easy to be used in waste liquid treatment, etc., and because calcium chloride is mixed with seaweed 17 200400195, the pH will change, and the pH of the fucose-containing sulfated polysaccharide containing non-decomposable succinate is § weekly Is necessary. At pH gjf, because the bath powder is sticky and aggregates, the subsequent extraction efficiency decreases and the filtration of the separated liquid is difficult. In other words, although the industrial usefulness of fucose-containing sulfated polysaccharides is currently expected, not only are commercially available products of fucose-containing sulfated polysaccharides-F and fucose-containing sulfated polysaccharides-U that are not sufficiently classified by molecular species, but also There are no reports on its efficiency laws. Furthermore, the commercially available fucose-containing sulfated polysaccharide contains a reactive coloring substance as described above. · Although the fucose-containing sulfated polysaccharide has various activities, as mentioned above, due to its difficulty in grading and preparation, substantially no fucose-containing sulfated polysaccharide-F and fucose-containing sulfated polysaccharide-U have been obtained. However, according to the present invention, a substantially purified fucose-containing sulfated polysaccharide-U, a simple extraction direction thereof, and a method for preparing fucose-containing sulfated polysaccharide-U can be provided. According to the present invention, there is provided a fucose-containing sulfated polysaccharide-u, which is a highly reactive colored substance that is generally difficult to remove from fucose-containing sulfated polysaccharides and causes enzyme reaction inhibition and resin pollution. Furthermore, according to the present invention, a fucose-containing sulfated polysaccharide-F that is substantially purified, a simple extraction method thereof, and a method for producing fucose-containing sulfated polysaccharide-F are provided. According to the present invention, fucose-containing sulfated polysaccharide-F, which is generally difficult to remove and has a highly reactive colored substance that causes enzyme reaction inhibition and resin pollution, is provided. The fucose-containing sulfated polysaccharide used in the present invention may be a fucose-containing sulfated polysaccharide containing a brown algae plant, 18 200400195 sea cucumber, and the like. For example, the fucose-containing sulfated polysaccharide may be dried, pulverized, and used. An extract containing rock bath sugar sulfate polysaccharide containing sugar sulfate polysaccharide and a refined product from the extract may also be used. The method for preparing the fucose-containing sulfuric acid polysaccharide extraction solution may be performed by a known method, and the extraction method is not particularly limited. Also, the so-called fucose-containing sulfated polysaccharide decomposition product used in the present invention is a substance obtained by decomposing the fucose-containing sulfated polysaccharide by an enzyme chemical method, a chemical method, or a physical method, and a known enzyme chemistry can be used. Sexual methods, chemical methods, physical methods. Furthermore, the fucose-containing sulfated polysaccharide and the fucose-containing sulfated polysaccharide decomposed product used in the present invention include pharmacologically acceptable salts thereof. Brown algae plants containing fucose-containing sulfated polysaccharides are, for example, Yamasuke Yukio, Segawa Soyoshi, Conservation Agency, Japanese color seaweed illustrated book published in 1982, and brown algae plants described in pages 22 to 52, for example Fucus evanescens, Kjellmaniella crassifolia, Laminaria japonica, and Undaria pinnatifida can be used to prepare fucose-containing sulfated polysaccharides. For example, a sea cucumber containing rock bath sugar sulfate polysaccharide is, for example, a sea cucumber of Japanese Patent Application Laid-Open No. 4-91027 5 Tiger Gongcheng 05 years. Mother (51; 丨 (: 11 port 118 japonicus), Hollothuria leucospilota), etc., and can prepare fucose-containing sulfated polysaccharides according to the method described in the bulletin. Fucose-containing sulfated polysaccharides are in The molecule has a sulfuric acid group which forms a reaction salt with various bases. These fucose-containing sulfated polysaccharides and their decomposition products are stable in the state of 19 200400195 salt, usually in the form of salts such as sodium and / or potassium The salt of these substances is treated with cation exchange resins such as Dowex 50W to free fucose-containing sulfated polysaccharides, and may lead to the decomposition of the fucose-free sulfates. Moreover, it is also possible to perform well-known salt exchange, if necessary, Exchange with various salts as desired. Salts containing fucose sulfate polysaccharides and their decomposition products can be used as pharmaceutically acceptable salts. Examples include alkali metal salts such as potassium, sodium, and alkaline earth metal salts such as calcium, magnesium, and barium. Salts of organic bases and ammonium salts with pyridine, etc. Brown algae plants and sea cucumbers containing fucose-containing sulfated polysaccharides are dried and pulverized to prepare fucose-containing sulfated polysaccharide powders. sulfuric acid The polysaccharide powder is subjected to hot water extraction. The dilute acid extraction can be used to prepare the fucose-containing sulfated polysaccharide extract. The fucose-containing sulfated polysaccharide content can be extracted at a temperature of 0 ~ 200 ° C for 1 ~ 360 minutes. The range can be selected according to the purpose, usually 10 ~ 150 ° C, 5 ~ 240 minutes, preferably 50 ~ 130 ° C, 10 ~ 180 minutes. It is better to perform fucose. Methods for purifying sulphuric acid-containing polysaccharide extracts include grading methods using fucose-containing sulfated polysaccharides such as calcium chloride and barium acetate, and fucose-containing sulfated polysaccharides using acid polysaccharide agglutinating agents such as cetylpyridinium chloride. The classification method uses a fucose-containing sulfated polysaccharide classification method using an acidic polysaccharide aggregating agent in the presence of salts, gel filtration, ion exchange chromatography, etc., and can be combined for purification if necessary. The method for decomposing sulfated polysaccharides can be the method of using fucose-containing sulfated polysaccharides to decompose enzymes, the method of acid decomposition, the method of ultrasonic treatment, and the method of decomposing fucose-containing sulfated polysaccharides. The method and the purification of the decomposed product can be performed by the above-mentioned method. Generally, there are many kinds of fucose-containing sulfated polysaccharides in brown algae, but the type of brown algae used in the present invention is not particularly limited. From high fruit beauty kelp, from true kelp 'from wakame, others from all brown algae. For the production of fucose-containing sulfated polysaccharides, first use an aqueous solvent of brown algae to obtain an extract. Also, for extraction Even if the seaweed is raw seaweed, if the brown algae is dried before making the extraction solution, it is made into a dry powder, washed with 60 to 100% ethanol and acetone, and immersed in a solution containing formazan, ethylacetate and glutaraldehyde. In aqueous solutions such as ammonia and ammonia, it is advantageous to greatly reduce the mixing of colored substances into fucose-containing sulfated polysaccharides. When fucose-containing sulfated polysaccharides are extracted from brown algae or brown algae ethanol-washed residues, the use of soluble barium acetate, barium chloride, or calcium chloride is advantageous because it can suppress the incorporation of alginic acid. For the above reasons, when extracting for the reason described above, it is better to extract with a calcium acetate solution of about 1 mM to 1 M at 50 to 130 ° C. In the case where the seaweed is thick and the powder (particles) is large, because the initial use of calcium acetate above 0.2M makes the extraction efficiency worse, it is first extracted with water, and then calcium acetate is added to remove the generated alginic acid precipitate. can. However, in the case where the fucose-containing sulfated polysaccharide and alginic acid are to be extracted simultaneously, and the case where a certain degree of decomposition is to be obtained during extraction, the solvent and extraction conditions are not particularly limited. 'Water or table salt can be used, Neutral salt aqueous solutions of various concentrations such as magnesium chloride, acidic aqueous solutions of various concentrations such as citric acid, phosphoric acid, and hydrochloric acid, acidic aqueous solutions of various concentrations such as citric acid, phosphoric acid, and hydrochloric acid, alkaline solutions of various concentrations such as sodium hydroxide and potassium hydroxide Aqueous solution, and buffers and preservatives can also be added. The pH, extraction temperature, and extraction time of the extraction solution are also not particularly limited. Generally, fucose-containing sulfated polysaccharides are weak to acids and bases, so it is easy to reduce the molecular weight when using acidic and alkaline solutions. Arbitrary decomposition products can be prepared by adjusting the heating temperature, time, pH, etc. For example, the average molecular weight and molecular weight distribution of the decomposition products can be adjusted by gel filtration treatment, molecular weight classification membrane treatment, and the like. That is, the molecular weight and sugar composition of the fucose-containing sulfated polysaccharide-U and fucose-containing sulfated polysaccharide-F according to the present invention are based on the harvesting time of the fucose-containing sulfated polysaccharide raw material, the drying method of the raw material, and the storage method of the raw material. It varies according to the heating conditions and pH conditions during fucose-containing sulfated polysaccharide extraction. For example, the fucose-containing sulfated polysaccharide is hydrolyzed with an acid, and is desorbed from the uronic acid under alkaline conditions, thereby reducing the molecular weight. Therefore, the molecular weight distribution and molecular weight distribution of fucose-containing sulfated polysaccharide-U and fucose-containing sulfated polysaccharide-F described in this specification are only examples, and can be easily changed according to the processing conditions of fucose-containing sulfated polysaccharide. Its molecular weight and molecular weight distribution. For example, when desalting at 100 t under mild alkaline and heating for 1 hour, if a molecular sieve membrane with a pore size of 300 is used, the fucose-containing sulfated polysaccharide-U and fucose-containing sulfated polysaccharide having a molecular weight distribution of about 1,000 to 10,000 can be adjusted. -F, and the fucose-containing sulfuric acid according to the present invention, which has an arbitrary molecular weight and molecular weight distribution, can be adjusted according to the use conditions. 22 200400195 Sugar-u and fucose-containing sulfated polysaccharide_F. In order to remove alginic acid and neutral sugars from the aforementioned brown algae extract, for example, in the presence of a salt having a concentration of 0.2 to 0.6 M of table salt, etc., cetylpyridinium chloride and the like can be added without causing precipitation. Acidic polysaccharide agglutinating agent, and the precipitation can be set. If necessary, the precipitate is washed with a salt solution such as common salt at a concentration of 0.2 to 0.6 M, and the cetylpyridinium chloride in the precipitate is washed with salt-saturated ethanol to obtain a fucose-containing sulfated polysaccharide mixture. In order to remove the pigment from the fucose-containing sulfuric acid polysaccharide mixture obtained by this treatment, the precipitate can be dissolved and then treated with an anion exchange resin and a polysaccharide resin for ultrafiltration. Dry samples can also be obtained by freeze-drying after desalting. The inventors have found that in the presence of 0.6 to 3 ^ 5 of one or more salts, the fucose-containing sulfated polysaccharide-F of the present invention and the fucose-containing sulfated polysaccharide-U of the present invention are acidic. Polysaccharide agglutinants show completely different behaviors. For example, using the method of the present invention, the fucose-containing sulfated polysaccharide_U of the present invention can be separated from an aqueous solution of the fucose-containing sulfated polysaccharide mixture. First, one or two or more salts are added to the aqueous solution containing the fucose sulfate polysaccharide mixture and the total concentration is 0.6 to 2M. The added salts are not particularly limited, such as sodium chloride and chlorinated fish. Usually, when the fucose-containing sulfated polysaccharide-F of the present invention is separated from the fucose-containing sulfated polysaccharide-U of the present invention, the purpose can be achieved with a salt concentration of about 1.5M (refer to the description of FIG. 1 described later). For example, after the salt concentration of the above salts is adjusted to 1.5M, if acidic polysaccharide agglutinating agents such as cetylpyridinium chloride are added so that precipitation will not occur again 23 200400195, the fucose-containing sulfated polysaccharide-F is formed. Precipitation, so if the precipitate is removed, the fucose-containing sulfated polysaccharide-U solution of the present invention is obtained. After concentrating the solution as needed, the fucose-containing sulfated polysaccharide-U in the solution is precipitated with 4 times the amount of ethanol, etc., and the cetylpyridinium chloride in the precipitate is washed out with saturated salt ethanol to obtain the present invention. Fucose-containing sulfated polysaccharide-U. In order to remove the pigment from the fucose-containing sulfated polysaccharide-U obtained by this treatment, the precipitate may be dissolved and then subjected to ultrafiltration and the like. Also, dry samples can be obtained by freeze-drying after desalting. It is also possible to add a preservative to the process. Secondly, in the case where only the fucose-containing sulfated polysaccharide-F of the present invention is to be efficiently produced, when agglutinating with cetylpyridinium chloride or the like, the salt concentration of 0.2 to 0.6 N is not used, However, if the salt concentration is 2M, for example, the fucose-containing sulfated polysaccharide-F of the present invention may be contained only. The present inventors have also found that when the fucose-containing sulfated polysaccharide is refined with an anion exchange resin, if there are divalent cations coexisting, the amount of fucose-containing sulfated polysaccharide adsorbed per unit of resin increases and the fucose-containing sulfated polysaccharide is separated Get better. That is, when the fucose-containing sulfated polysaccharide-U of the present invention is produced by the method of the present invention, it is preferable that the medicine which is a source of divalent cations in the fucose-containing sulfated polysaccharide mixture is preferably added at 1 mM or more. Next, the anion exchange resin is equilibrated with a liquid containing divalent cations of more than ImM, and the fucose-containing sulfated polysaccharide mixture is adsorbed. After thoroughly washing the anion exchange resin with a balanced solution, the fucose-containing sulfated polysaccharide is dissolved out, for example, with a sodium chloride gradient. When using this method, the concentration of the divalent cation to be added may be 1 mM or more. However, when the fucose-containing sulfated polysaccharide-U of the present invention is adsorbed on the column 24 200400195, it is expected to be less than 0.5. Μ. In addition, in this method, the medicine used as a source of divalent cations is particularly excellent in the effects of calcium salts and barium salts, but it is not particularly limited, and magnesium sulfate, manganese chloride, and the like can also be used. In addition, if a fucose-containing sulfated polysaccharide mixture is produced from brown algae by a common method, a highly reactive coloring substance is mixed as described above, which not only pollutes the resin and the resinous container in contact, but also hinders the enzyme reaction and cell growth. . It was found that this coloring substance can be easily removed if it is bound or adsorbed to a polysaccharide substance or a substance having an anion exchange group. That is, to the solution containing the fucose-containing sulfated polysaccharide, for example, Cellulofine, GCL-2000 (manufactured by Biochemical Industry Co., Ltd.), Sephacryl S-500, Sephadex G-200, Sepharose CL-2B (also manufactured by PHARMAICA) are added. Polysaccharide resin, or DEAE-Cel lulofine A-800 (manufactured by Biochemical Industry Co., Ltd.), DEAE-Sepharose FF, DEAE-Sephadex A-50, QAE-Sephadex A-50, DEAE-Sephace 1 (also the PHARMA CIA company) (Manufactured), TSK-gel DEAE-Toyopearl 6 50, TSK-gel DEAEToyopearl 5 50 (manufactured by TOS〇), Amberlite anion exchange resin (sold by ORGAN0) Kitopearl anion exchange resin (Fuji Industries) This product is easily removed by stirring the anion-exchange-containing substance, or passing the solution containing fucose-containing sulfated polysaccharide through the column filled with it. However, in the case of anion exchange resin, since fucose-containing sulfated polysaccharide can also be combined, it is preferable that the salt concentration is about 2M when the colored substance is adsorbed. The fucose-containing sulfated polysaccharide-U of the present invention can be prepared, for example, as described in Example 6 at 2 $ 200400195. The physical and chemical properties of the fucose-containing sulfated polysaccharide-u are shown below, but the fucose-containing sulfated polysaccharide-u of the present invention is not limited to this example. The fucose-containing sulfated polysaccharide-U of the present invention and the fucose-containing sulfated polysaccharide-F of the present invention obtained in Example 8 are precipitated in the presence of excess cetylpyridinium chloride at each sodium chloride concentration. The properties are shown in Figure 1. The vertical axis in Fig. 1 represents the precipitation formation rate (%), and the horizontal axis represents the sodium chloride concentration (M). In the figure, solid lines and white circles indicate the precipitation formation rate of the fucose-containing sulfated polysaccharide-U of the present invention at various sodium chloride concentrations, and the dotted lines and white triangles in the figure represent the fucose-containing fucose of the present invention. Precipitation formation rate of sulfated polysaccharide-F at each sodium chloride concentration (M). The precipitation formation rate was measured as follows at a solvent temperature of 37 ° C. The fucose-containing sulfated polysaccharide-U and the fucose-containing sulfated polysaccharide-F of the present invention were respectively dissolved in water and 4M sodium chloride at a concentration of 2%, which were prepared by mixing in various proportions and dissolved in various concentrations of chlorine. 125 microliters each of the fucose-containing sulfated polysaccharide-U and fucose-containing sulfated polysaccharide-F solutions in sodium sulfide. Next, cetylpyridinium chloride was dissolved in water and 4M sodium chloride at a concentration of 2.5%, and 1.2% of cetyl chloride dissolved in various concentrations of sodium chloride was prepared by mixing. Pyridine solution. It takes 3.2 times to make 2% of the fucose-containing sulfated polysaccharide-U and fucose-containing sulfated polysaccharide-F dissolved in water completely dissolved in 1.2% of cetyl chloride D solution. capacity. Therefore, with respect to 125 microliters each of 2% fucose-containing sulfate polysaccharide-U and fucose-containing sulfate polysaccharide-F dissolved in 2% sodium chloride, After adding 4 0 0 26 200400195 micropyridyl solution, stir thoroughly, leave it for 30 minutes, centrifuge and separate the sugar content of the supernatant solution according to the phenol-sulfuric acid method [Analytical Chemistry, Volume 28, page 3 50 (1 9 5 6)] Measurement can calculate the precipitation formation rate of each fucose-containing sulfated polysaccharide at each sodium chloride concentration. The molecular weight distribution of the obtained fucose-containing sulfated polysaccharide-U of the present invention, when calculated using a gel filtration method of Sepacry 1 S-500, shows a molecular weight distribution centered at about 19,000 (refer to FIG. 2). ). A. In Fig. 2, the vertical axis is the sugar content in the sample measured by the phenol-sulfuric acid method, and the abscissa is 480 nm. The horizontal axis is the number of the dissociated fraction. Alas, the conditions for gel filtration are shown below. Column size: 3.08X162.5 cm Solvent: containing 0.2 M sodium chloride and 10 mM sodium phosphate buffer 10 mM (p Η 6 · 0) flow rate: 1.5 ml / min sample concentration: 0. 25% Sample liquid amount: 20 ml molecular weight standard material: Shodex STANDARD P-82 (manufactured by Showa Denko Corporation) Next, the obtained fucose-containing sulfated polysaccharide-U of the present invention was analyzed. First, the amount of fucose was quantified as described in Journal of Biological Chemistry, Vol. 175, p. 595 (1948). Secondly, the obtained dried sample of fucose-containing sulfated polysaccharide-U was dissolved in 0.5% hydrochloric acid at a concentration of 0.5%, and treated at 110 ° C for 2 hours. 27 200400195 the constituent monosaccharide was hydrolyzed . Next, the reducing end of the monosaccharide obtained by hydrolysis using Glyco TAG and Glyco TAG reagent kit (also manufactured by Takara Shuzo Co., Ltd.) was pyridyl- (2) -amination (PA), and the constituent sugars were investigated by HPLC. The ratio. A. The conditions of HPLC are as follows. Device: L-6200 (manufactured by Hitachi) Column: PERPACK type A (4.6_xi50mm: manufactured by Takara Shuzo Co., Ltd.) Eluent: 700 mM boric acid buffer (pH 9.0): acetonitrile = 9: 1 Detection: using a fluorescence detector F-1 1 5 0 (manufactured by Hitachi, Ltd.) at an excitation wavelength of 310 nm and a fluorescence wavelength of 3 80 nm. Flow rate: 0.3 ml / min

Column temperature: 65 ° C Second: Quantify the amount of uronic acid as described in Analytical Biochemistry, Volume 4, page 330 (1962). Secondly, the sulfuric acid content was quantified as described in Biochemical Jouorna Vol. 84, p. 106 (1962). As a result, the constituent sugars of the fucose-containing sulfated polysaccharide U are fucose, mannose, galactose, glucose, rhamnose, xylose, and uronic acid. Other neutral sugars are not substantially contained. In addition, the main component of fucose is mannose: galactose: uronic acid. The molar ratio of sulfate group is about 1 0: 7: 4: 5: 20 °. Second, fucose-containing sulfate polysaccharide-U calcium The IR spectrum of the salt was measured by a Fourier transform infrared spectrophotometer; [IR-DIAMOND 20 (manufactured by Japan Electronics Co., Ltd.) was measured to obtain the spectrum shown in FIG. 3. Alas, Figure 3 28 200400195 The vertical axis represents the transmittance (%), and the horizontal axis represents the wave number (c m-1). Next, when the NMR spectrum of the fucose-containing sulfated polysaccharide calcium salt of the present invention was measured with a 500 MHz nuclear magnetic resonance apparatus, NM_α 500 nuclear magnetic resonance apparatus (manufactured by Japan Electronics Corporation), the spectrum shown in FIG. 4 was obtained. In Fig. 4, the vertical axis represents the signal strength and the horizontal axis represents the chemical shift 値 (ppm). The chemical shift 値 of 1 'in 1H-NMR is represented by the chemical shift h of hOD as 4.65 ppm. 1H-NMR (D20) 55.27 (η at the 1st position of mannose), 5.07 (Η at the fucose position), 4.49 (Η at the fucose position 3), 4.37 (glucuron at the 1 position position) , 4.04 (Fourose at the 4th position), 3.82 (Fucose at the 2nd position), 3.54 (Furonic acid at the 3rd position), 3.28 (Glucuronic acid at the 2nd position) Η), 1.09 (Fucose 5th CH3 Η) The specific optical rotation of the freeze-dried product of the fucose-containing sulfated polysaccharide-U according to the present invention was measured with a high-sensitivity polarimeter SEPA-300 (manufactured by Horiba) At -53 · 6 degrees. The inventors of the present invention determined the structure of the fucose-containing sulfated polysaccharide-U of the present invention as described below. The fucose-containing sulfated polysaccharide-U is decomposed and refined by a decomposing enzyme having the ability to decompose the fucose-containing sulfated polysaccharide-U. The refined fucose-containing sulfated polysaccharide-U was subjected to the action of the following terminal fucoidan-decomposing enzyme to refine the decomposed product. That is, 16 ml of a 1% fucose-containing sulfated polysaccharide-U solution, 50 ml of 29 200400195 phosphate buffer solution (pH 8 · 0), 12 ml of 4 ml of sodium chloride, and 4 ml of terminal type rocks of 32 mU / ml 8 ml of Fucoidan-degrading enzyme solution was mixed and reacted at 25 ° C for 48 hours. It was confirmed that the absorbance at 2 to 30 nm increased as the reaction proceeded, and it was determined that the fucose-containing sulfated polysaccharide-U was decomposed by the enzyme. This reaction solution was desalted with MICR0ACILIZER-G3 (manufactured by Asahi Kasei Corporation), and 3 dissolved fractions (a), (b), and (c) were separated and refined with DEAE-Sepharose FF. The degrading enzyme is prepared by the following method. The strain used in the production of the terminal fucoidan-degrading enzyme may be any strain as long as it has a production capacity of the terminal fucoidan-degrading enzyme. Specific examples include, for example, Xanthophytes (Flavobacterium) sp. SA-0082 strain (CCRC 910069). This strain is a strain newly retrieved from the seawater of Aomori Prefecture by the present inventors and others, and its mycological properties are as follows. 1. Flavobacterium sp. SA-0082 strain a. Morphological properties (1) The bacterium is Brevibacterium brevis wide 0.8 ~ 1 · 0 # m length 1.0 ~ 1.2 μm (2) of spores Presence or absence (3) Gram-staining negative b. Physiological properties 30 200400195 (1) Growth temperature range is above 37 ° C. The appropriate growth temperature is 15 ~ 28 ° C. (2) Attitude to oxygen Aerobic (3) Hydrogen peroxide 18 positive (4) Oxidative 18. Positive (5) ΐ, 18. Weak positive (6) Acid production

D-glucose positive lactose positive maltose positive D-mannitol positive sucrose negative trehalose negative (7) hydrolyzed starch negative

Gelatin-positive casein-negative dagger tree 22. Positive (8) Nitrate reduction negative (9) Indole production negative (1 0) Hydrogen sulfide production negative (1 1) Milk coagulation negative (1 2) Sodium Requirement positive 200 400 195 (1 3) Salts required growth negative in 0% common salt medium Growth negative in 1% common salt medium Negative growth positive in seawater medium (1 4) quinone menaquinone 6 (15 ) GC content of DNA in bacteria 32% (16) 〇F-assay 〇 (17) The hue of the colony is yellow (18) Mobility is not (19) Sliding is not This strain is considered as Be r gey's Manual of Systematic Bacteriology, Volume 1 (1984), and Bergey, s Manual of Systematic Bacteriology, Volume 9 (1994), contains Flavobacterium aquatile and Flavobacterium meningosepticum It is similar to the former, but it is incompatible with the former in terms of not forming an acid using sucrose, incapable of decomposing casein, in terms of decomposing dagger leaf glycosides, in terms of liquefying gelatin, and in terms of positive enzyme Different and later They are different in terms of inability to break down casein and slow growth at 37 ° C. Therefore, this strain was identified as a bacterium belonging to the genus Xanthomonas and named as a genus Xanthomonas sp. SA-0082. Alas, the above-mentioned strains are expressed as Xanthomonas sp. SA-0082, and are at the Institute of Biotechnology and Industrial Technology, Industrial Technology Research Institute, Ministry of International Trade and Industry [No. 3, No. 1, No. 1-Chome, Tsukuba City, Tanagi Prefecture, Japan (Post No. 3 0 5 )] From 2004 to March 2004 200400195 Deposited under FERM P-14872, and in the aforementioned Institute of Industrial Technology, Ministry of International Trade and Industry ’s Institute of Biotechnology and Industrial Technology as FERM BP-5402 (application for transfer of international deposit Date: February 15, 2008) Deposited, and deposited in CCRC No. 910069 on December 24, 1996 in the strain preservation and research center of the Food Industry Development Research Institute of your country. If the nutrient source added to the culture medium of the strain is available, the strain can be used and a terminal type fucoidan-decomposing enzyme can be generated. The carbon source can be, for example, fucoidan, seaweed powder, alginic acid, Fucose, glucose, mannitol, glycerol, sucrose, maltose, lactose, starch, etc., nitrogen source is yeast extract, protein gland, casein amino acid, corn pulp, meat extract, defatted soybean, ammonium sulfate, Ammonium chloride and the like are suitable. Others, such as sodium salt, phosphate salt, potassium salt, magnesium salt, zinc salt, and metal salts may be added. When cultivating this terminal fucoidan-producing enzyme-producing bacteria, although the production amount varies depending on the culture conditions, the culture temperature is generally 15 ° C ~ 30 ° C, and the pH of the culture medium is 5 ~ 9. Good, under 5 ~ 7 2 hours aeration and agitation culture, the production amount of this terminal fucoidan-degrading enzyme reached the highest. The culture conditions naturally depend on the strain used, the composition of the culture medium, etc., and the production amount of the fucoidan-degrading enzyme of the terminal type is set to the maximum. The end-type fucoidan-degrading enzyme is present in the bacterial cells and also in the mash on the culture. If the above-mentioned Xanthomonas sp. SA- 00 82 strain is cultured in an appropriate culture medium, and the bacterial cells are collected, the bacterial cells are broken by a conventional cell destruction method such as ultrasonic treatment, etc. Cell extract. 33 200400195 Secondly, from this extract, purified enzyme samples can be obtained by the usual purification methods. For example, purification by salting out, ion-exchange column chromatography, hydrophobic bond column chromatography, gel filtration, etc., can obtain purified fumatosan decomposed without other fucoidan-decomposing enzymes. Enzymes. In addition, since the present enzyme (extracellular enzyme) is also present in a large amount on the culture solution of the culture medium from which the bacterial cells are removed from the above-mentioned culture solution, it can be purified by the same purification means as the enzyme in the bacterial cells. A purification example of a terminal fucoidan-decomposing enzyme is shown. Flavobacterium sp. SA- 0082 (CCRC 9 1 0069) was inoculated to an artificial seawater (manufactured by Germaline Laboratory) ρΗ7.5, containing 0.25% glucose, 1.0% protein gland, and 0.05% yeast extract. The medium was 600 ml and sterilized (120 ° C, 20 minutes) in a 2 liter Erlenmeyer flask, and cultured at 24 ° C for 24 hours to prepare a seed culture solution. Artificial seawater (manufactured by Germalin Laboratory) pH 7.0 containing 0.25% of glucose, 1.0% of protein glands, 0.05% of yeast extract, and 0.05% of antifoaming agent (KM70 manufactured by Shin-Etsu Chemical Co., Ltd.) 20 liters of the culture medium was put into a 30 liter capacity fermentation tank and sterilized at 120 ° C for 20 minutes. After cooling, 600 ml of the above-mentioned culture solution was inoculated, and cultured at 24 ° C for 24 hours, with a ventilation volume of 10 liters per minute and a stirring speed of 125 rpm. After the end of the culture, the culture solution was centrifuged to obtain bacterial cells. This bacterial cell was suspended in a 20 μm acetic acid-phosphate buffer solution (ρ 7.5) containing 200 μM sodium chloride, and after being crushed with an ultrasonic wave, centrifugation was performed to obtain a bacterial cell extract. When measuring the activity of the terminal fucoidan-degrading enzyme 34 200400195 in this bacterial cell extract, an activity of 5 mU was detected in 1 ml of the culture. To this extract, add saturated ammonium sulfate with a final concentration of 90%, stir and dissolve it, centrifuge, and suspend the precipitate in the same buffer as the above bacterial cell extract, and buffer it with 20 mM acetic acid-phosphate containing 50 mM common salt. Solution (PH7. 5). The precipitate produced by dialysis was removed by centrifugation, and then adsorbed to a DEAE-Sepharose FF column equilibrated with 20 mM acetic acid-phosphate buffer (PH7.5) containing 50 mM common salt, and the adsorbate was subjected to the same buffer. After sufficient washing, it was dissolved in a gradient of 50 mM to 600 mM sodium chloride, and the active fraction was collected. Second, add 4M table salt to this active dissolution fraction to adsorb to a phenyl-Sepharose CL-4B column that was previously equilibrated with a 20mM dish acid buffer solution (ρΗ8 · 0) containing 4M table salt. After the adsorbate was sufficiently washed with the same buffer solution, it was dissolved out with a salt gradient of 4M to 1M, and the live soluble fraction was collected. Next, the active fraction was concentrated by an ultrafilter, followed by gel filtration with Sephacryl S-300 equilibrated with a 10 mM phosphate buffer containing 50 mM common salt in advance, and the active dissolution was collected. The molecular weight of this enzyme is calculated from Sephaph a c r y 1 S-3 0 0 retention time Xin is about 460,000. The active fraction was dialyzed against a 10 mM phosphate buffer (pH 7) containing 250 mM common salt. This enzyme solution was adsorbed on a MonoQHR 5/5 column equilibrated with a 10 mM dish acid buffer (pH 7) containing 250 mM common salt, and the adsorbate was thoroughly washed with the same buffer, and then the solution was washed with 2 5 mM to 4 5 OmM salt gradient to dissolve it, and collect active dissociation to obtain refined enzyme. The above refining processes are shown in Table 1. 35 200400195

Total engineering protein (mg) Total activity (milli units) Specific activity (milli units / mg) Yield (%) Cell extract 61,900 101,000 1.63 100 Ammonium sulfate salting out 33,800 88,600 2.62 87.7 DEAE-Sepharose FF 2,190 40,400 18.4 40.0 Phenyl-Sepharose CL-4B 48.2 29,000 601 28.7 Sephacryl S-300 7.24 19,600 2,710 19.4 Mono Q 0.824 15,000 18,200 14.9 The activity of this enzyme was measured as follows. Lu ίο Phosphorus instead of the acidity of the original sugar content 2.5% of the fucoidan solution from high fruit kelp 50 microliters, with microliters of the enzyme, and 60 microliters of 83mM acid buffer containing 667mM sodium chloride PH7. 5 was mixed and allowed to react at 37 ° C for 3 hours. 105 microliters of the reaction solution was mixed with 2 ml of water and stirred, and its absorbance (AT) at 230 nm was measured. Prepare the above-mentioned buffer solution for dissolving this enzyme instead of the enzyme and make the reaction under the same conditions, and use only water instead of the fucoidan solution and perform the reaction as a control group, and measure the absorbance in the same way | (AB1 and AB2 ). One unit of enzyme is the amount of enzyme that cleaves the glycosidic bond between 1 // mo 1 mannose and uronic acid in 1 minute in the above reaction system. The quantification of the cut bond is calculated by taking the absorption coefficient of the millimolar molecule of the unsaturated sugar aldehyde generated during the dissociation reaction as 5.5. Alas, the properties of enzymes are calculated by the following formula. (AT-AB1 -AB2) X 2. 1 0 5 X 1 20/5. 5 X 1 0 5 X 0. 0 1 X 1 80 = U / ml 36 200400195 2. 1 0 5: The amount of sample liquid for measuring absorbance (Ml) 120: Liquid volume of enzyme reaction solution (microliter) 5 · 5: Molar molecular absorption coefficient (/ mM) of unsaturated uronic acid at 230 nm 1 0 5: Liquid volume of reaction liquid used for dilution (Microliter) 〇1: The amount of the enzyme solution (ml) 180: The reaction time (minutes) The protein was quantified by measuring the absorbance of the enzyme solution at 280 nm. At this time, the grade photometric value of the 1 mg / ml protein solution is calculated as 1.0. The fucoidan from the high fruit beauty kelp, which is 尙 ', is prepared as follows. The dried high fruit kelp was crushed with a free-pulverizer M-2 (manufactured by Nara Machinery Co., Ltd.), and then treated at 70 ° C for 2 hours in a 10-fold amount of 85% methanol, and then filtered. The residue was 70% in 10 times the amount of methanol. (: 2 hours treatment, filtration. Add 20 times the amount of water to the residue, treat at 1000 ° C for 3 hours and filter to obtain an extract. The salt concentration of the extract is adjusted to 400 mM sodium chloride solution. After the same, cetylpyridinium chloride was added so as not to cause precipitation again, and centrifuged. The precipitate was thoroughly washed with ethanol, and an ultrafilter (filtration) capable of completely removing cetylpyridinyl chloride was filtered. The membrane has a molecular weight of 100,000 (manufactured by AM ICON) for desalination and removal of low molecular weight, and the precipitate generated at this time is removed by centrifugation. The supernatant liquid can be freeze-dried to obtain refined commercial kelp kelp flock Analysis of the structure of the enzyme reaction product The above-mentioned terminal fucoidan-decomposing enzymes contain the α 1-4 bond between D-mannose and D-glucuronic acid present in the fucoid sulfate-polysaccharide-U. If the enzyme decomposed by 37 200400195 is decomposed, and the fucose-containing sulfated polysaccharide-u is allowed to act, an oligosaccharide having a structure of the following formulae (I), (II), and (III) can be produced.

OH

OH 11 38 200400195

39 200400195 Below, detailed description. The three dissolving fractions (a), (b), and (c) separated and purified by the above-mentioned DEAE-Sepharose FF only used a part of G1 yc 〇TAG and G 1 yc 〇TAG respectively. Pyridyl- (2) -amidation (palation) can obtain each PA-sugar (PA-a), (PA-b), and (PA-c). (PA-a), (PA-b), and (pA-c) were analyzed by HPLC. Alas, the conditions of HPLC are as follows. (1) HPLC analysis using molecular weight fractionation device: L-6200 (manufactured by Hitachi)

Column ·· SHODEX SB- 803 (4.6 x 250mm) (manufactured by Showa Denko Corporation) Eluent: 0.2 M sodium chloride: dimethylimine = 9: 1 Detection: Fluorescence detector F-11 5 0 (Manufactured by Hitachi, Ltd.) Detected at an excitation wavelength of 320 nm and a fluorescence wavelength of 400 mm. Flow rate: 1 ml / min. Column temperature: 50 ° C. (2) HPLC analysis device using a reverse phase column: L-6200 Type (manufactured by Hitachi, Ltd.) Column: L-column (4 · 6 X 2 50mm) [(Finance) Chemical Inspection Association] Eluent · 50mM acetic acid · triethylamine (PΗ5 · 5)

Detection: Fluorescence detector F-1 150 (made by Hitachi) at an excitation wavelength of 320 nm and a fluorescence wavelength of 400 mm Flow rate: 1 ml / min Column temperature: 40 ° C 40 200400195 at Figures 5, 6, and 7 show the HPLC dissolution profiles of each pyridyl- (2) -aminated sugar compound (PA-a), (PA-b), and (PA-c). In the figure, the vertical axis represents the relative fluorescence intensity, and the horizontal axis represents the residence time (minutes). The physical properties of the compounds represented by the following formulas (I), (I I), and (I I I) are (a), (b), and (c). Mass spectra of (a), (b) and (c) of (10) are shown in Fig. 8, and (a) and (12) are shown in Fig. 11 The mass-mass spectrum of (c) is shown in (b) and FIG. 13, and the vertical axis represents the relative intensity (%) and the horizontal axis represents m / z 値 in each figure. Furthermore, the 1H-NHR spectra of (a), (b), and (c) of FIG. 16 are shown in FIG. 14, and the vertical axis represents the signal intensity and the horizontal axis represents Chemical shift 値 (ppm). The chemical shift 値 in 1H-NHR is expressed as the chemical shift of HOD as 4.65 ppm. (a) Physical molecular weight 564 MS m / z 563 [M-H +]-MS / MS m / z 97 [HS04]-, 157 [Unsaturated D-glucuronic acid-H20-H +]-, 175 [No Saturated D-glucuronic acid-H +]-, 22 5 [L-Fucose-containing sulfuric acid-[] 20-H +]-, 243 [L-Fucose sulfate-H +]-, 319 [Unsaturated D-glucose Combination of uronic acid and D-mannose-H20-H +]-, 405 [M-unsaturated D-glucuronic acid-H +]-, 4 8 3 [MS〇3-H +]-200400195 1H-NMR (D2 〇) (55 · 78 (1H, d, J = 3.7Hz, 4 "-Η), 5.26UH, d, J = 1.2Hz, 1-H), 5.12 (1 H, d , J = 4.OHz, 1 f-Π), 5.0 · 3 (1 H, d, J = 6. 1Hz, i " — li), 4.47 (f Π, d-d, J = 3. 4, 10 · 4Hz, 3 '-H), 4. 21 (1H, br — s, 2-H), 4. 12 (1H, m, 5' —H), 4. 10 (1H, dd, J = 3 · 7, 5 ·

8Hz, 3 " -H), 4.03 (1H, d, J = 3.4Hz, -1 ", 3.86 (1H, m, 3-Η), 3.83 (1H, d-d, J = 4. 0, 1.0. 4 H z, 2 '—H), 3.72 (1H, m, 4 —11), 3.72 (1H, m, 5-H) f, 3.70 (2H M, 5-H2 of CH2), Q. 6 5 (1 H, dd, J = 5. 8, 6. 1Hz, 2 ~ —H), 1 · 08 (3H, d, J = 6 · 7 H z, 5 '-C H3 of H3) sugar composition L-fucose: unsaturated D-glucuronic acid: D-mannose = 1: 1: 1 (1 molecule each) sulfate 1 molecule (L-rock The third position of fucose) 尙, the number assigned to the peak in 1H-NMR is as shown in the following formula (IV).

I rr 〇

IV η rr ο 42 200400195 (b) Physical and molecular weight 724 MS m / z 723 [M-H +]-, 361 [M-2H +] 2-, MS / MSm / z97 [HS04]-, 175 [Unsaturated D- Polyuronic acid-H +]-, 243 [L-fucoic acid-H +]-, 321 [M-S03-2H +] 2_, 405 [M-unsaturated D-glucuronic acid-2S〇3-H + ]-, 417 [ML Fucose-2S03-H +]-° 1H-NMR (D20)

δ 5. 6 6 (1 H, d, J = 3.4 Hz, 4 "-H), 5.27 (1H, d,

J = 7 · 3 H zt 1 " -H), 5. · 2 2 (1 H, d, J = 1. 8 II z, '一 H), 5. · 21 (1H, d, J = 3 · 7Πζ, 1 '-Π), 4.50 (1 H, d, J = 3.1 Hz · 4'-H), 4.32 (1 H, q, J = 6.7 M z, 5 #), 4.27 ( 1H, dd, J = 3 · 7, 10 · 4 H z, 2 '-H), 4. 2 1 (1H, dd, J = 3 · 4, 6 · 7 Hz, 3 "-Π), 4. 18 (1 H, d-d, J = 1. 8, 11 · OHz, 5-CH of H), 4 · 15 (1H, br-s,

2-H), 4 · 10 (1 H, d-d, J = 5. 8, 1 1. 〇 H z > 5 ~ C, 3. Θ 9 (1 H, dd, J = 3.1, 10 · 4 H z, 3 / -H) > 3. 9 0

(1H, m, 5-H), 3.82 (1H, m, 3-H), 3. 82 (1H, m, 4-H) 3. 54 (1H, br-t, J = 7 · 3 11z ， 2 "-H), 1. 11 (3H, d, J = 6.7 H z, 5′-CH3 Π3) sugar composition L-fucose: unsaturated D-glucuronic acid: D- Mannose = 1: 1: 1 (1 molecule each) 3 sulfates (2 and 4 positions of L-fucose and 6-position of D-mannose) 尙, at 1 Η-NMR assigned to the spike The number is as follows (v). 43 200400195

V) (c) Physical molecular weight 1 1 28 MS m / z 1 127 [M-H +]-MS / MS m / z 97 [HS04]-, 175 [Unsaturated D-glucuronic acid-H +]-, 225 [L-fucose-containing sulfuric acid-H20-H +]-, 24 3 [L-fucose-containing sulfuric acid-H +]-, 371 [M-unsaturated D-glucuronic acid-L-fucose-S03- 2H2 +] 2_, 40 5 [One of the sulfated L-fucose and D-mannose-H +]-, 721 [MD-mannose-L-fucose-so3-h2o-h +] 44 200400195 1H- NMR (D2 〇) 55 · 69 (1H, d, J = 3.7Hz, ⑷ " 一 Η), 5.34 (1H, s, (1) -Η), 5.16 (1Η, s, BuΗ ), 5. · 10 (1Η, d, J = 4.0 Hz, ⑴, 5.50 (" 丨, ", J = 3.7 Hz, 1 '-丨 1), 4.93 (1H, J = 6 · 4Hz, (l) " —11), 4 · 50 (1Π,

d-d, J = 3 · 4, 10 · 7 Hz, 3 '-Η), 4 · 47 (1H, d-d, J

= 3, 4, 10. 4Hz, (3) ^ -H), 4.39 (1H; d, J = 7. 9H z, 1 "-Η), 4.33 (1Η, br-s, (2 )-H), 4 · 14 (1H, meter m, 2-Η), 4 · 1 2 (1 H, m, (3) "-Η), 4 · 1 2 (1 H, m, 5 ~ H), 4.12 (1H, m, (5) '-H), 4.04 (lH, m, t-Η), 4.03 (1Η, m, (4)'-H) , 3.85 (lH'm, 2 H), 3.85 (1H, m, ⑵'-H), 3.82 (1H, m, 3 -H), 3. 8 2 (1H, m, (3) -H), 3.73 (lH, m, 4-H), 3.73 UH, m, 5-H), 3.73 (1H, m, H-H), 3.70 (2H, m, 5-C H2) H2), 3 · 7 0 (2 H, m, (5)-c H2), 3 · 6 7 (1 H, m, 5 "-H), 3. 62 UH, m, 4 ~ _H), 3 · 62 (1H, m, (2) ~ H), 3. · 62UH, m, (5)-Η), 3 · δΐ (1H, t, J = 8. 9Hz, 3 "-H), 3 · 28 (1H, t, J = 7.9 Hz, 2 "-# Η), 1 · 〇9 (3H, d, J = 6.7 Hz, (5K—H3 of CH3), 1. · 07 (1H, d, J = 6.7Hz, 5,-H3 of CH3) Sugar composition L-Fucose: Unsaturated D-Glucuronic acid: D-Pulonic acid: D-mannose = 2: 1: 1: 2 (2 molecules each for L-fucose and D-mannose and one molecule each for unsaturated D-glucuronic acid and D-glucuronic acid) 2 molecules (each L - 3 of the rock bit fucose) yet have, it belongs to the 1H-NMR peaks are numbered following formula (VI) 45 200400195.

CHzOH

(VI)

46 200400195 If the obtained fucose-containing sulfated polysaccharide-u causes the above-mentioned fucoidan-decomposing enzyme to decompose, the 23 Onm absorbance caused by the dissociation reaction increases as the reaction proceeds. The uronic acid group becomes all of the main reaction products, so it can be said that in the obtained fucose-containing sulfated polysaccharide-U molecule, there is a sugar chain in which hexuronic acid and mannose are interactively combined. Since the constituent sugars of the obtained fucose-containing sulfated polysaccharide-U are mostly fucose, fucose-containing sulfated polysaccharide-U is more easily decomposed into acids than ordinary polysaccharides. On the other hand, hexuronic acid and The mannose bond is a relatively strong acid. The present inventors and others, in order to clarify the existence of hexuronic acid and mannose in the fucose-containing sulfated polysaccharide mixture molecules of the high fruit kelp, are the types of hexuronic acid in the sugar chain that are interactively combined. Volume 125, pages 283 to 290 (1 984). First, the fucose-containing sulfuric acid polysaccharide mixture is dissolved in 0.3M oxalic acid, and the molecular weight fraction is collected for 3 hours at 100 ° C. The molecular weight is collected to 3 000 The above-mentioned dissolved fractions were collected by an anion exchange resin. This material was acid-hydrolyzed with 4N hydrochloric acid after lyophilization, adjusted to pH 8, and then PA-treated, and analyzed for uronic acid by HPLC. The conditions for HPLC were as follows. Device: L-6200 (manufactured by Hitachi) Column: PERPACK type N (4.6_X 2 50mm) (manufactured by Takara Shuzo Co., Ltd.) Eluent: 200mM acetic acid-triethylamine buffer (PH7 · 3) ·· acetonitrile = 25: 75 Detection: Fluorescence detector F-1 150 (made by Hitachi) at an excitation wavelength of 320 nm and a fluorescence wavelength of 400 mm Flow rate: 0.8 ml / min 47 200400195

Column temperature: 40 ° C 尙, the standard substance of PA-hexuronic acid glucuronic acid is manufactured by SIGMA, galacturonic acid is manufactured by Wako Pure Chemical Industries, and iduronic acid is manufactured by SI GMA 4- Methylumbellyl-a-L-iduronic acid hydrolyzed, mannoic acid I and gluconic acid are according to Acta Chemica Scandinavica, Vol. 15, No. 1 3 9 7 ~ 1 3 9 8 The method described in page (1961) is obtained by hydrolyzing alginic acid manufactured by Wako Pure Chemical Industries, Inc. and separating it with an anion exchange resin to obtain PA. As a result, it was determined that the hexuronic acid contained in the sugar chain of the fucose-containing sulfated polysaccharide mixture was only glucuronic acid. Furthermore, when the glucuronic acid in the sugar chain hydrolysate is separated from D-mannose via an anion exchange resin, and its specific optical rotation is measured after freeze-drying, it can be determined as D-glucuronic acid of dextran glucuronic acid. . In addition, in the case of a fucose-containing sulfated polysaccharide mixture derived from high fruit beauty treated with the aforementioned terminal fucoidan-decomposing enzyme in advance, D-glucoaldehyde was not detected even when acid-hydrolyzed with oxalic acid in the same manner as described above. Acid and D-mannose are cross-linked polymers. From this, it can be judged that the structure of the fucose-containing sulfated polysaccharide having the above-mentioned terminal fucoidan-decomposing enzyme separated and cut off has a structure in which D-glucuronic acid and D-mannose are interactively combined. Furthermore, in order to investigate the disposition of each binding position of D-glucuronic acid and D-mannose and the anomer of the sugar 22 · binding, the polymer obtained by the decomposition of oxalic acid was subjected to NMR analysis. The NMR measurement results of the polymer are shown below. However, the chemical shift in ih-NMR is the chemical shift of the methyl group of triethylamine, which is determined as 丨. 3, and in 48 200400195 "The chemical shift of the methyl group of triethylamine in C-NMR" It is expressed as 9 · 32ppm. 1H ^ NMR (D2 0)

55 · 25 (1H, br * -s, 1-Η), 4.32 (1H, d, J 2 7. 6H r —H), 4. · 〇〇 (HI, bru-11), 3. 71 ( 1H 'm · Η), 3 · 6 9 UH, m, 5-CH of 丨 丨), 3 · 6 8 (1 Π, m, 3-之), 3 · 63 (1Η, m, 5-CH of Η), 3.63 (1Η, m, 4 ~ Η), 3 57 (1Η,-H), 3.54 (lH, m, 3 Η), 3.53 (lH, m, 5-Η), 3. · 25 (1Η, t, J = 8.5 Hz, 2 ~ Η) 13C-NMR (D2 Ο) 5175 · 3 (5, -C of COO H), 102.5 (1 ~ 〇), 99 · 6 (

5-0, 78 · 5 (2-C), 77 · 9 (4, -C), 77 · 0 (3, -C), 76 · 7 (5 '-C), 73.9 (5-C ), 73.7 (2 '-C), 70.6 (3-C), 67.4 (4-C), 61.0 (5-CH2 〇Η of C) 尙, the number assigned to the spike is Such as the following formula (v II): Φ

Due to the stereo configuration of the 1-position of D-bucuronic acid, the fixed binding number is 7.6 Hz, so it is determined to be Lu-D-glucuronic acid. 49 200400195 In addition, due to the steric configuration of the 1-position mannose, the chemical shift 値 was 5.25 ppm, so it was determined to be a -D -mannose. The method of combining sugars was performed using the HMBC method of 1Η detection of heterogeneous nuclear detection. For the assignment of 1H-NMR, the DQF-COSY method and the Η0ΗΑΗΑ method were used, and for the assignment of 13C-NMR, the HSQC method was used. From the HMBC spectrum, it is confirmed that there are staggered peaks between 1-H and 4'-H and 1-C, 1-H and 2-C, and 2-H and 1'-C, respectively. From this, it can be seen that D-gluconic acid is bonded to the 2-position of mannose with a hydrazone-bond, and D-mannose is bonded to the 4-position of D-glucuronic acid with an X bond. If the above results are taken into consideration, (a) it can be judged that (a) has a structure in which unsaturated D-glucuronic acid is bound to L-fucose having a sulfate group in D-mannose as a reducing terminal residue. (B) Structure of unsaturated D_glucuronic acid in D-mannose as a reducing terminal residue bound to a sulfate group, and L-fucose bonded to two sulfate groups, ( c) It has D-glucuronic acid in D-mannose as a reducing terminal residue, binds to L-rock bath sugar with sulfate group, and binds D- in its D-glucose M acid. Mannose has a structure in which D-mannose has unsaturated D-glucuronic acid and is bound to L-fucose having a sulfate bond. Above, the obtained fucose-containing sulfated polysaccharide-U has a structure in which D-glucuronic acid and D-mannose are interactively combined, and has at least one D-mannose having an L-fucose bond. structure. In addition, it has a partial structure represented by the following general formula (VIII) (however, at least one of the 50,200,100,195 alcoholic hydroxide groups is sulfated, and η is an integer of 1 or more)

(V1D)

According to the present invention, there is provided a purified fucose-containing sulfated polysaccharide-U separated from the fucose-containing sulfated polysaccharide-F of the present invention. The fucose-containing sulfated polysaccharide-U of the present invention is a low-molecular-weight fucoidan-containing enzyme that contains uronic acid as a constituent sugar and is produced by fucoidan sp. SA- 00 82 (CCRC 9 1 0069). And generate at least one compound selected from the compounds represented by the formulae (I), (II), and (III). Its molecular weight, molecular weight distribution, and sugar composition are not limited if it is the fucose-containing sulfated polysaccharide-U of the present invention, and any molecular weight and molecular weight distribution of fucose-containing sulfated polysaccharide-U can be adjusted, and a sugar composition can be provided. Fucose-containing sulfated polysaccharide with well-defined physicochemical properties. The fucose-containing sulfated polysaccharide-U of the present invention does not have the strong anticoagulant activity of the fucose-containing sulfated polysaccharide_F, and therefore can provide fucose-containing sulfuric acid that does not substantially exhibit anticoagulation 51 200400195 Polysaccharide. The fucose-containing sulfated polysaccharide of the present invention-u can be used as a carcinogen, an anti-metastatic agent, a carcinogenic agent, and the like as a fucose-containing sulfated polysaccharide, and can also be used as an anti-fucose-containing sulfated polysaccharide antibody Of the antigen. Furthermore, fucose-containing sulfated polysaccharide _υ can be used to produce oligosaccharides having the structures of the formulae (I), (I I), (111) and the like, and it is also useful in the production of these novel compounds. Next, the fucose-containing sulfated polysaccharide_F of the present invention and a method for producing the same are described. When using the method of the present invention to manufacture the fucose-containing sulfated polysaccharide-F of the present invention, 'if the decomposing enzyme capable of decomposing fucose-containing sulfated polysaccharide_u is not allowed to act on the fucose-containing sulfated polysaccharide mixture, and After the enzyme reaction is completed, the low-molecular-weight fucose-containing sulfated polysaccharide-u may be removed by ultrafiltration or the like. The above-mentioned degrading enzyme may be any enzyme as long as it is capable of selectively decomposing fucose-containing sulfated polysaccharide-U. Specific examples thereof include Flavobacterium sp. SA-0082 strain (CCRC) 910069) produced the above-mentioned terminal fucoidan-decomposing enzyme. When the enzyme works, the substrate concentration, temperature, and pH can be set under the favorable enzyme reaction. The substrate concentration is usually from about 0.1 to 10%, the temperature is from about 20 to 40 ° C, and the pH is From 6 to 9 is expected. Further, the fucose-containing sulfated polysaccharide mixture is added to the culture medium, and microorganisms having the ability to decompose the fucose-containing sulfated polysaccharide-U to produce a degrading enzyme are cultured in the medium, and may be purified from the cultured medium. The microorganism used may be any microorganism as long as it can produce a degrading enzyme capable of decomposing fucose-containing sulfated polysaccharide-U. Specific examples include the Flavobacterium sp. SA-0082 strain described in 52 200400195 CCRC 9 1 00 6) or

Fucoidanobacter marinus SI-0098 strain (FERM BP-5403). If the nutrient source added to the culture medium is available for use, and the decomposition enzyme can be produced, the carbon source can be, for example, fucoidan, seaweed powder, alginic acid, fucose, glucose, mannitol, Glycerin, sucrose, maltose, lactose, starch, etc., and the nitrogen source is suitably yeast extract, protein gland, casein amino acid, corn pulp, meat extract, defatted soybean, ammonium sulfate, ammonium chloride, and the like. Other inorganic substances such as sodium salts, phosphates, potassium salts, magnesium salts, zinc salts, and metal salts may be added. In addition, of course, the culture conditions are based on the strain used, the composition of the culture medium, etc., and the decomposition activity of fucose-containing sulfate U-U is set to the maximum. Generally, if the culture temperature is 15 to 30 ° C, the pH of the culture medium is set. It is 5-9, and it can be cultured under aeration and agitation for 5 to 72 hours. After the end of the culture, the components other than the fucose-containing sulfated polysaccharide-U and the fucose-containing sulfated polysaccharide-F in the medium may be removed by ultrafiltration. That is, the above-mentioned Fucoidanobacter marinus SI-0098 strain is a strain newly obtained from the seawater of Aomori Prefecture and retrieved by the present inventors and the like, and its mycological properties are as follows. 1. Fucoidanobacter marinus SI- 0098 a. Type properties (1) The bacterium is Brevibacterium (brevibacterium) wide 0.5 to 0. 7 // m length 0.5 to 0.7 // m 53 200400195 (2) the presence or absence of spores None (3) Gram-staining-negative b. Physiological properties (1) Growth can be performed at a temperature range above 37 ° C. The appropriate growth temperature is 15 ~ 28 ° C. (2) Attitude to oxygen aerobic (3) Catalase positive (4) Oxidase negative (5) Urease negative (6) Hydrolyzed starch negative Gelatin negative Casein negative t Lutein positive (7) Nitric acid Negative reduction of salt (8) D-bend, negative generation of indole (9) Positive generation of hydrogen sulfide (10) milk coagulation negative (1 1) sodium requirement positive (1 2) salt requirement

Growth-negative in 0% salt medium Growth-negative in 1% salt medium Growth-negative in seawater medium 54 200400195 (1 3) quinone menaquinone 7 (14) GC content of DNA in bacteria 61% ( 15) Cell wall diamine pimelic acid negative (16) Ethyl alcohol test negative (17) Positive presence of hydroxy fatty acids (18) 0 F test 0 (19) The hue of the colony does not generate characteristically Colony pigment (2 0) motility with (2 1) slip away without (22) flagella extremely single hair This strain is classified into group 4 according to the basic classification described in Bergey's Manual of Determinative Bacteriology, Volume 9 (1994) (Gram-negative aerobic bacteria and cocci). However, this strain differs from the bacteria belonging to group 4 in that it contains menaquinone 7 in the electron transfer chain and has a GC content of 61%. Basically Gram-negative bacteria have ubiquinone in the electron transfer chain, while Gram-positive bacteria have menaquinone. As Gram-negative bacteria, Xanthomonas and Cellulobacterium (Cyt. Ophaga) exceptionally have menaquinone 'in the electron transfer chain, but the GC content of bacteria belonging to these genera, and the soil bacteria Cytophaga arvensicola Waiting 43 ~ 46%, the marine bacteria Cytophaga di ff luens' Cytophaga fermentans, Cytophaga marina and Cytophaga uliginosa are 42%, which is completely different from the properties of this strain. Furthermore, when the 16S r DNA sequence identity of this strain was compared with that of the identified strain ', the same identity was found in Verrucomicrobiura spinosum, which is the most homogeneous 55 200400195. The identity was 76 · 6%. Since it is generally known that when the 16S r DNA sequence has an identity of 90% or less, the genera of the two bacteria are different, the inventors have determined that this strain is a new genus bacteria that does not belong to the existing genera, and accordingly The strain was named Fucoidanobacter marinus SI-0098. The inventors also found that the fucose-containing sulfated polysaccharide-F and fucose-containing sulfated polysaccharide-U of the present invention are acid agglutinating agents in the presence of one or more salts of 0.6 to 3 M as described above Showing completely different behavior. For example, using the method of the present invention, the fucose-containing sulfated polysaccharide-F of the present invention can be separated from an aqueous solution of the fucose-containing sulfated polysaccharide mixture. First, one or two or more kinds of salts are added to the aqueous solution containing the fucose sulfate polysaccharide mixture and the total concentration is 0.6 to 3M. The added salts may be, for example, sodium chloride, calcium chloride, and the like, and are not particularly limited. After adjusting the salt concentration in this way, if an acidic polysaccharide agglutinating agent such as cetylpyridinium chloride is added so as not to cause precipitation again, and the precipitate is collected, the fucose-containing sulfated polysaccharide-F of the present invention can be obtained. However, if the above-mentioned salt concentration exceeds 2M, since the fucose-containing sulfated polysaccharide-F of the present invention is difficult to precipitate through cetylpyridinium chloride, it needs attention. For the purpose of separating the fucose-containing sulfated polysaccharide-F and the fucose-containing sulfated polysaccharide of the present invention, the purpose can usually be achieved with a salt concentration of about 1.5 M (refer to the description in FIG. 1). Secondly, if necessary, after washing the precipitate, the cetylpyridine chloride in the precipitate is washed with a salt-saturated alcohol, and the fucose-containing sulfuric acid 56 200400195 polysaccharide-F of the present invention is obtained. In order to remove the pigment from the fucose-containing sulfated polysaccharide-F of the present invention thus obtained, ultrafiltration may be performed after the precipitate is dissolved. Dry samples can also be obtained by freeze-drying after desalting. In addition, preservatives can be added to the project. The inventors also found that fucose-containing sulfated polysaccharides containing divalent cations coexisted when the fucose-containing sulfated polysaccharides were refined with an anion exchange resin as described above. The separation of sulfated polysaccharides becomes better. That is, when the fucose-containing sulfuric acid polysaccharide-F of the present invention is produced by the method of the present invention, it is preferable to firstly add 1 mM or more of the medicine containing divalent cations to the fucose-containing sulfuric acid polysaccharide mixture. Next, the anion exchange resin is equilibrated with a liquid containing divalent cations of preferably 1 mM or more, and the fucose-containing sulfated polysaccharide mixture is adsorbed. After thoroughly washing the anion exchange resin with a balanced solution, the fucose-containing sulfuric acid polysaccharide-F is dissolved out, for example, with a sodium chloride gradient. When using this method, the concentration of the divalent cation added should be 1 mM or more. In addition, the method used in this method as a source of divalent cations is particularly effective in the case of calcium salts and barium salts, but is not particularly limited. Magnesium sulfate, manganese chloride, and the like can also be used. The fucose-containing sulfated polysaccharide-F of the present invention can be obtained, for example, as described in Example 8. The physical and chemical properties of the fucose-containing sulfated polysaccharide are shown below, but the fucose-containing sulfated polysaccharide-F of the present invention is not limited to this example. The molecular weight distribution of the obtained fucose-containing sulfated polysaccharide-F of the present invention, when calculated by a gel filtration method using Sephacryl S-500, showed a molecular weight distribution of about 190,000 as the center (see FIG. 17). A. In Figure 17, the vertical axis 57 200400195 is used to determine the sugar content in the sample according to the phenol-sulfuric acid method. The absorbance is expressed as 48 n m. The horizontal axis is the number of the dissociated fraction. The conditions of "尙" gel filtration are shown below. Column size: 3 · 08 X 1 62 · 5 cm Solvent: 10 mM sodium phosphate buffer (PH6.0) containing 0.2 M sodium chloride and 10% ethanol Flow rate: 1 · 5 ml / min Sample concentration: 0 25% sample liquid volume: 20 ml molecular weight standard material: Shodex STANDARD P-82 (manufactured by Showa Denko Corporation) Next, the obtained fucose sulfated polysaccharide-F-containing component of the present invention was analyzed. First, the amount of fucose was quantified as described in Journal of Biological Chemistry, Vol. 175, p. 595 (1948). Next, the obtained dried sample of fucose-containing sulfated polysaccharide-F was dissolved in 1 equivalent of hydrochloric acid at a concentration of 0.5%, and treated at 110 ° C for 2 hours to hydrolyze the constituent monosaccharides. Next, the reducing end of the monosaccharide obtained by hydrolysis using Glyco TAG and Glyco TAG reagent kit (also manufactured by Takara Shuzo Co., Ltd.) was pyridyl- (2) · amination (PA), and the constituent sugars were investigated by HPLC. The ratio. A. The conditions of HPLC are as follows. Device: L-6200 (manufactured by Hitachi) Column: PERPACK type A (4.6mmX150mm: made by Takara Shuzo Co., Ltd.) Eluent: boric acid buffer (pH9 · 0); acetonitrile = 9 · · 1 200400195 Detection: detection by fluorescence Fl 150 (manufactured by Hitachi, Ltd.) at an excitation wavelength of 3 10 nm and a fluorescence wavelength of 38 nm. Flow rate: 0.3¾ liters / minute

Column temperature: 65 ° C Second: Quantify the amount of uronic acid as described in Analytical Biochemistry, Volume 4, page 330 (1962). Secondly, the sulfuric acid content was quantified as described in Biochemical Jouorna Vol. 84, p. 106 (1962). As a result, the fucose-containing sulfated polysaccharide-F has fucose, galactose, and its molar ratio is about 10: 1. It is essentially free of uronic acid and other neutral sugars. The molar ratio of fucose to sulfate was about 1: 2. 16 ml of a 1% fucoidan-F solution, 12 ml of 50 mM phosphate buffer (pH 8.0), 4 ml of sodium chloride 4 M, and 32 μm / Γη1 of the foregoing were from Xanthomonas sp. SA -0082 (CCRC 9 1 0069), 8 ml of fucoidan-decomposing enzyme solution was mixed and allowed to react at 25 ° C for 48 hours. No decomposition product was formed by the reaction, and it was also confirmed that there was no low molecular weight. Next, the IR spectrum of the fucose-containing sulfated polysaccharide-F calcium salt was measured by a Fourier transform infrared spectrophotometer IR-DIAMOND 20 (manufactured by Nippon Denshi) when the spectrum shown in the first δ chart was obtained. A. In Fig. 18, the vertical axis represents the transmittance (%), and the horizontal axis represents the wave number (cuT1). Next, the NMR spectrum of the fucose-containing sulfated polysaccharide-F calcium salt of the present invention was measured with a 500 MHz nuclear magnetic resonance apparatus. NM-α 500 type nuclear magnetic resonance apparatus (manufactured by Japan Electronics Corporation). spectrum. 59 200400195 In Figure 19, the vertical axis represents the signal strength and the horizontal axis represents the chemical shift ppm (ppm). The chemical shift 値 in 1H-NMR is represented by the chemical shift H of HOD as 4.65 ppm. 1H-NMR (D20) 5.30 (H at fucose position 1), 1.19 (CH32 at fucose position 5) Second, the specific optical rotation of the lyophilized product containing fucose sulfate polysaccharide-F is increased at a high speed The high-sensitivity polarimeter SEPA-3 00 (manufactured by Horiba) was measured at -135 degrees. According to the present invention, there is provided a purified fucose-containing sulfated polysaccharide-F separated from the fucose-containing sulfated polysaccharide-U of the present invention. The fucose-containing sulfated polysaccharide-F of the present invention is a fucoidan-decomposing enzyme that does not substantially contain uronic acid as a constituent sugar and is not produced by Xanthomonas sp. SA-0082 (CCRC 9 1 0069). And low molecular weight. Its molecular weight, molecular weight distribution, and sugar composition are not limited if it is the fucose-containing sulfated polysaccharide-F of the present invention, and any molecular weight and molecular weight distribution of the fucose-containing sulfated polysaccharide-F can be adjusted, and a sugar composition can be provided Fucose-containing sulfated polysaccharide-F with clear physical and chemical properties, such as reducing ends, and extremely high sulfate degree. The fucose-containing sulfated polysaccharide-F of the present invention has strong anticoagulant activity because it is separated from the fucose-containing sulfated polysaccharide-U, which has substantially no anticoagulant activity. F and / or its decomposed product can be used as an anticoagulant in a purified fucose-containing sulfuric acid form, and can also be used as an antigen of an anti-fucose-containing sulfated polysaccharide antibody. When the fucose-containing sulfated polysaccharide of the present invention is added to cancer cell culture 60 200400195 in a nutrient solution at a concentration of 1 microgram / ml or more ', the cancer cells cause the cells to self-destruct from one day to several days after the addition. That is, the fucose-containing sulfuric acid-containing polysaccharide of the present invention and its decomposed product have a strong cell-self-inducing effect.尙 'These substances do not induce cell self-destruction to normal cells and are not toxic. In particular, fucose-containing sulfated polysaccharides derived from edible brown algae plants and sea cucumbers and their decomposed products are highly safe. In formulating the cell autoinactivation inducer of the present invention, fucose-containing sulfated polysaccharide and / or its decomposed product may be used as an active ingredient, and it may be combined with a known pharmaceutical carrier. Generally, the fucose-containing sulfated polysaccharide and / or its decomposed product of the present invention is mixed with a pharmaceutically acceptable liquid or solid carrier, and a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, and an excipient are added as needed. , Binding agents, disintegrating agents, lubricants, etc., and solid preparations such as lozenges, granules, powders, powders, capsules, and other liquids, suspensions, emulsions and other liquids. In addition, it can be made into a liquid dried product by adding an appropriate carrier before use. The cell self-inactivation inducer of the present invention may be administered orally, or any parenteral agent such as an injection or an instillation agent may be administered. The pharmaceutical carrier can be selected according to the above-mentioned administration form and dosage form. In the case of oral preparations, for example, starch, lactose, white sugar, mannitol, carboxymethyl cellulose, corn starch, and inorganic salts can be used. In the preparation of an oral agent, a binding agent, a disintegrating agent, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a coloring agent, a fragrance, and the like may be further added. Specific examples are listed below. 61 200400195 < Binders> Starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, Polyvinylpyrrolidone, polyethylene glycol. < Disintegrating agent > Source powder, hydroxypropyl starch, sodium carboxymethylcellulose, carboxymethylcellulose, quaternary methylcellulose, and low-substituted hydroxypropylcellulose. < Surfactant > Sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80. < Lubricants > Talc, osmium, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol. Lu < Fluidity promoter > Light anhydrous silicic acid, dry aluminum hydroxide glue, synthetic aluminum silicate, magnesium silicate. In addition, liquid preparations for oral use can be used as suspensions, emulsions, syrups, and elixirs. Flavors, odorants, and colorants can also be added to these formulations. On the other hand, in the case of a non-oral preparation, the active ingredient of the present invention, the fucose-containing sulfated polysaccharide and / or its decomposition product, can be dissolved or suspended in distilled water for injection, physiological saline, and glucose aqueous solution as a diluent. For injection, use vegetable oil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, etc., and if necessary, add fungicides, stabilizers, isotonicity agents, analgesics and so on. The cell self-killing inducer of the present invention can be administered by an appropriate administration route depending on the formulation type. The administration method is also not particularly limited, and can be administered internally, externally, and by injection. The injection can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, and the like, and the external preparation may also include a raccoon agent and the like. 62 200400195 The dosage of the cell self-extinguishing inducer of the present invention can be appropriately set according to the type of preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient to which it is applied. The effective ingredient is contained in an amount of 1 to 1000 mg, preferably 10 to 200 mg per adult per day. Of course, the amount to be administered varies depending on various conditions. Therefore, a smaller amount than the above amount may be sufficient, or it may be necessary to exceed the range. The fucose-containing sulfated polysaccharide-U, fucose-containing sulfated polysaccharide-F, and / or its decomposed product of the present invention, which has a carcinogenic effect, can be manufactured by preparing it with a known pharmaceutical carrier. Cancer agent. The manufacturing of a carcinostatic agent can be performed according to the above method. Generally, the fucose-containing sulfated polysaccharide and / or its decomposed product of the present invention is mixed with a pharmaceutically acceptable liquid or solid carrier, and a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, and an excipient are added as needed. , Binding agents, disintegrating agents, lubricants, etc., and made into solids such as tablets, granules, powders, powders, capsules, and other liquids, suspensions, emulsions and other liquids. In addition, it can be made into a dry product in a liquid state by adding an appropriate carrier before use. The carcinostatic agent may be administered orally, or any parenteral agent such as an injection or an infusion solution may be administered. The pharmaceutical carrier can be selected according to the above-mentioned administration form and dosage form, and it can be used as the above-mentioned cell self-extinguishing inducer. The carcinostatic agent can be administered by an appropriate administration route depending on the type of preparation. The method of administration is also not particularly limited, and can be administered internally, externally, and by injection. Injectables can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, and the like, and external use 63 200400195 can also include raccoon and the like. The dosage of carcinogens can be appropriately set according to the type of preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient to which it is applied. Although it is not fixed, it is generally the active ingredient contained in the preparation The amount is 1 to 1000 mg per adult per day, preferably 10 to 200 mg. Of course, the dosage varies depending on various conditions, so there may be a smaller amount than the above-mentioned dosage, which is sufficient, or it may be necessary to exceed the range. The medicament of the present invention can be administered orally in this regard, and can also be added to any ravioli food for daily ingestion. It is appealed that fucose-containing sulfated polysaccharides and / or decomposed products thereof having carcinogenicity-inhibiting effects be used as an active ingredient and formulated with a well-known pharmaceutical carrier to produce a carcinogenic agent. The carcinogenic agent can be manufactured according to the above method. Generally, the fucose-containing sulfated polysaccharide and / or its decomposed product are mixed with a pharmaceutically acceptable liquid or solid carrier, and if necessary, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binding agent , Disintegrating agents, lubricants, etc., and made into solids such as lozenges, granules, powders, powders, capsules, and other liquids such as liquids, suspensions, and emulsions. In addition, it can be used as a dry product in liquid form by adding an appropriate carrier before use. The carcinogenicity preventive agent may be administered orally, or parenteral preparations such as injections and drip preparations. The pharmaceutical carrier can be selected according to the above-mentioned administration form and dosage form, and the above-mentioned cell self-inactivation inducer can be used as a standard. The cancer-preventing agent can be administered by an appropriate administration route depending on the type of preparation. 64 200400195 There are no particular restrictions on the method of administration, and it can be used internally, externally, and by injection. Injectables can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, and the like, and external preparations can also include raccoon and the like. The dosage of the cancer-preventing agent can be appropriately set according to the type of preparation, the method of administration, the purpose of use, and the age, weight and symptoms of the patient to which it is applied. Although it is not certain, it is generally effective in the preparation. The amount of the ingredients is 1 to 1000 mg, preferably 10 to 200 mg per adult per day. Of course, the amount to be administered varies depending on various conditions, so there may be cases where the amount is smaller than the above-mentioned amount and is sufficient, or it may be necessary to exceed the range. The medicament of the present invention can be administered orally in this way, and can also be added to any glutinous food for daily intake. The decomposed product of the fucose-containing sulfated polysaccharide of the present invention is derived from natural substances, and is not considered to be toxic even if administered to rats orally. The medicament of the present invention is expected to be used as a therapeutic agent for reducing or increasing immune function, or cancer diseases, viral diseases, and the like. It can be used as a carcinogen to maintain health. In addition, the method for inducing cell self-deactivation of the present invention can be used for research on biological defense mechanism, immune function or relationship with cancer, viral diseases, etc., and development of inhibitors for inducing cell self-destruction. In particular, if the fucose-containing sulfated polysaccharide of the present invention is prepared by edible brown algae plants and edible sea cucumber, the decomposed product thereof has a long history as a food, so the fucose-containing sulfated polysaccharide prepared by it, The decomposed substance is extremely safe when administered orally. Secondly, 'Fucose-containing sulfated polysaccharide is a sulfated polysaccharide with a very large molecular weight, 65 200400195. Therefore, in order to improve the antigenicity, uniformity, and anticoagulant activity, etc., it is necessary to improve the antigenicity, uniformity, and anticoagulant activity. The fucose sulfate polysaccharide is decomposed to a certain degree. According to the present invention, in order to provide an enzyme that selectively decomposes fucose sulfate polysaccharide F, and a low molecule of fucose sulfate polysaccharide F obtained by the action of the enzyme Compound. The strain used in the present invention may be any strain as long as it is a bacterium belonging to the genus Alteromonas and has the terminal-type fucose-containing sulfuric acid polysaccharide-decomposing enzyme-producing ability of the present invention. Specific examples of the strain having the terminal-type fucose-containing sulfated polysaccharide-decomposing enzyme-producing ability include, for example, S. sp. SN-1 009 strain. If the terminal type fucose-containing sulfated polysaccharide-decomposing enzyme from this strain is caused to act on the fucose-containing sulfated polysaccharide, the low-molecular-weight compound of the fucose-containing sulfated polysaccharide-F of the present invention can be obtained. This strain is a strain newly retrieved from the seawater of Aomori Prefecture by the present inventors and others, and its mycological properties are as follows. a. Morphological properties (1) The bacterium is a bacillus about 1 // m wide and about 2 β m (2) the presence or absence of spores (3) negative Gram staining b. physiological properties (1) temperature range for growth 66 200400195 Suitable growth temperature is 15 ~ 30 ° C. Cannot grow at 4 ° C or 4 (TC. (2) attitude to oxygen aerobic (3) catalase positive (4) oxidase positive (5) lipase positive (6) glucose-positive mannose Negative sucrose positive lactose negative cellobiose positive melibiose negative mannitol positive glycerol positive methanol negative DL-malate negative succinate negative fumaric acid negative citrate negative salicylate negative (7) hydrolyzed starch negative gelatin negative 67 200400195 (8) Negative reduction of nitrate (9) Negative denitrification reaction (10) Positive decomposition of alginic acid (11) /?-Hydroxybutyric acid negative (1 2) Accumulation of polyhydroxybutyric acid negative ( 1 3) Sodium requirement is positive (1 4) Salt is required to grow negative in ο% common salt medium

Growth negative in 1% common salt medium Growth positive in seawater medium (15) quinone ubiquinone 8 (16) GC content of DNA in bacteria 36% (17) OF-test 〇 (18) color of colonies Does not produce characteristic colony pigments (19), luminescence negative (20), sports positive

(21) The flagellated monochaete strain was identified as Bergey's Manual of Systematic Bacteriology, Volume 1, pages 343 to 352, and Bergey's Manual of Determinative Bacteriology, volume 9, pages 75, 132 to 133 (1 994) The genus Zymomonas is described in the bacterium. However, the physiological characteristics of this bacterium are not consistent with any of the recorded species, and the GC content is also low. Thus, this strain was named as Symbiotic sp. SN- 丨 09. 68 200400195 尙, the above-mentioned strains are represented by the genus Symbiotic genus sp. SN-1009 at the Institute of Biotechnology and Industrial Technology, Industrial Technology Research Institute, Ministry of International Trade and Industry [East 1-Chome, Tsukuba, Tanjo Prefecture, Japan, No. 3 (Postal No. 30 5)] was deposited with FERM P-15436 from February 13, 2008, and in the aforementioned Institute of Industrial Technology, Industrial Technology Institute, Ministry of International Trade and Industry, FERM BP-5747 (Transferred from International Depository Application date: November 15, 2008) Deposited, and deposited in the strain conservation and research center of the Food Industry Development and Research Institute of your country, on December 24, 1996 with CCRC No. 910070. If the nutrient source added to the culture medium of this strain can be used by the strain of tadpoles and can produce a terminal type fucose-containing sulfate polysaccharide decomposition enzyme, the carbon source can be, for example, fucose-containing sulfate polysaccharide and seaweed powder. , Alginic acid, fucose, glucose, mannitol, glycerol, sucrose, maltose, etc. The nitrogen source is yeast extract, protein gland, casein amino acid, corn pulp, meat extract, defatted soybean, ammonium sulfate, Ammonium chloride and the like are suitable. Others such as sodium, phosphate, potassium, magnesium, zinc, and metal salts can also be added. This strain grows very well in seawater or artificial seawater containing the above nutrition sources. When cultivating the terminal type fucose-containing sulfated polysaccharide-decomposing enzyme of the present invention, the production amount varies depending on the culture conditions, but the culture temperature is generally 15. (: ~ 3 (TC, the pH of the culture medium is preferably 6-9). Under the aeration and agitation culture of 5 ~ 7 2 hours 69 200400195, the production amount of the terminal fucose-containing sulfate polysaccharide decomposing enzyme of the present invention reaches the highest. Cultivation conditions of course set the production amount of the terminal fucose-containing sulfated polysaccharide degrading enzyme of the present invention to the maximum depending on the strain used, the composition of the culture medium, and the like. Existing, and also in the culture solution. The above-mentioned Symbiotic species sp. SN-1 009, if cultured in an appropriate culture medium, and the bacterial cells are collected by the usual means of cell destruction, such as ultrasonography. Cell-free extracts can be obtained by breaking up the cells with sonication, etc. Second, from this extract, purified enzyme samples can be obtained by the usual purification methods. For example, 'salt precipitation, ion exchange column chromatography, hydrophobic bond columns Purification by chromatography, gel filtration, etc. can obtain a purified terminal fucose-containing sulfated polysaccharide-degrading enzyme of the present invention that does not contain other fucoidan-decomposing enzymes. Since the enzyme is also present in a large amount in the culture solution of the nutrient-removing bacterial cells, it can be purified by the same refining methods as the enzymes in the bacterial cells. The chemical and The physical and chemical properties are as follows: (1) Action: It acts on the fucose-containing sulfated polysaccharide having the following physical and chemical properties, that is, 70 200400195 rock-containing sugar-sulfur polysaccharide-F, so that the rock-containing sugar-sulfur polysaccharide-F is Reduced molecular weight. (A) Constituent sugars .. • Does not substantially contain uronic acid. (B) Fucoids are not produced by Flavobacterium sp. SA-0082 (CCRC 9 1 0069). The glycan-decomposing enzyme is low-molecular. It does not act on fucose-containing sulfated polysaccharides having the following physical and chemical properties, ie, fucose-containing sulfated polysaccharide-U. Β (c) constitutes a sugar: it contains uronic acid. (D) via yellowing Fucoidan produced by Flavobacterium sp. SA-0082 (CCRC 9 1 0069) decomposes the enzyme and degrades it, and generates at least the following formulas (I), (II), and (III). One or more compounds. 71 200400195

CL 2 Η

ο Q OMnsno Η ο

72 200400195

OH (in

73 200400195 (ii) Optimum pH: The optimum pH of this enzyme is around 7-8 (Figure 20). That is, Fig. 20 is a graph showing the relationship between the pH and relative activity of the enzyme, with the vertical axis representing relative activity (%) and the horizontal axis representing pH. The solid line is a curve using PA-derived fucose-containing sulfated polysaccharide-F (PA-FF) for the reducing end, and the dotted line is the fucose-containing sulfuric acid described in (V)-(2) below. Polysaccharide-F was used as the curve of the substrate. (iii) Optimum temperature: The optimum temperature of this enzyme is around 30 ~ 35 ° C (Figure 21). That is, Fig. 21 is a graph showing the relationship between the temperature and relative activity of the enzyme, with the vertical axis representing relative activity (%) and the horizontal axis representing temperature (° C). The solid line is the curve at the time of using PA-based fucose-containing sulfated polysaccharide-F (PA-FF) for the reducing end, and the dotted line is the fucoid containing the following (V)-(2) Sugar sulfate polysaccharide-F was used as the curve of the mass. (iv) Molecular weight: The molecular weight of this enzyme is about 100,000 when calculated by the gel filtration method using Sephacryl S-200 (manufactured by PHARMACIA). (v) Method for measuring enzyme activity: The terminal type fucose-containing sulfated polysaccharide-decomposing enzyme activity of the present invention was measured as follows. First, the fucose-containing sulfated polysaccharide-F and PA-FF, which are the terminal type fucose-containing sulfated polysaccharide decomposing enzymes of the present invention, are prepared by the following processes (1) to (3). (1) Preparation of a mixture of fucose-containing sulfated polysaccharides from high fruit kelp. 2 kg of dried high fruit kelp was crushed with a free crusher M-2 (manufactured by Nara Machine 74 200400195 Machinery Co., Ltd.) and crushed at 4.5 times The amount of 80% ethanol was treated at 80 ° C for 2 hours, and then filtered. The process of extracting and filtering the residue with the above 80% ethanol was repeated three times to obtain 1,870 g of ethanol washing residue. Add 36 liters of water to the residue, treat at 100 ° C for 2 hours, and obtain an extract by filtration. After the extraction solution had the same salt concentration as the 400 mM sodium chloride solution, 5% cetylpyridinium chloride was added so as not to cause precipitation again, and centrifuged. This precipitate was repeatedly washed with 80% ethanol. After completely removing the cetylpyridinium chloride, it was dissolved in 3 liters of 2M sodium chloride. The insoluble matter was removed by centrifugation and suspended in 2M sodium chloride. Balanced 100ml DEAE- Φ

Cel 1 ιΐ 〇 fine A- 800, filtered after stirring, and removed the resin. This filtrate was placed in 100 ml of DEAE-Cel lulof ine A-800 particles equilibrated with 2M sodium chloride, and the dissolved fractions were subjected to desalting and low molecular removal by an ultrafilter (the molecular weight of the filter membrane was 100,000). The precipitate produced at this time was removed by centrifugation. By freeze-drying the supernatant liquid, 82.2 g of refined fucose-containing fucose sulfate polysaccharide mixture can be obtained. (2) Preparation of fucose-containing sulfated polysaccharide-F | 6 g of the fucose-containing sulfated polysaccharide mixture derived from the high fruit kelp was placed in 600 ml of 20 mM sodium acetate (ρΗ6.0 · 0) containing 0.2M calcium chloride After dissolution, it was placed in a 3 600 ml DEAE-Sepharose FF column equilibrated with 20 mM sodium acetate (pH 6.0) containing 0.2M calcium chloride, and 20 mM sodium acetate (pH 6.0 containing 0.2M calcium chloride) was used. ) After fully washing the column, dissolve it with a sodium chloride gradient of 0 ~ 2M. Collect fucose-containing sulfuric acid containing more than 0.75M, and dissolve 75 200400195 sugar-F dissociated fractions, and concentrate and desalinate them with an ultrafilter equipped with an ultrafiltration membrane with a molecular weight of 1 million, and freeze-dry them. 3.3 g of a lyophilized sample containing fucose sulfate polysaccharide-F was obtained. (3) Preparation of PA-FF Dissolve 12 mg of the above-mentioned freeze-dried sample containing fucose sulfated polysaccharide-F in 480 microliters of water, and aliquot 36 parts in 12 microliters each. The Glyco TAG and Glyco TAG reagent kits were PA-reduced at the reducing end to obtain PA-FF. The obtained PA-FF was dissolved in 15 ml of a 10 mM ammonium acetate solution containing 10% methanol, and subjected to gel filtration on a Cel lulofine GCL-300⑩ column (40 X 90 Qmm), and the polymer fractions were collected. The obtained polymer fraction was fully dialyzed and desalted with a dialysis membrane with a pore size of 3,500, and then concentrated to 5 ml by an evaporator to obtain the terminal PA-containing fucose-containing sulfate polysaccharide-F decomposition enzyme of the present invention. FF. In addition, the PA-FF obtained in this way was quantitatively compared with the commercially available pyridyl_ (2) -aminated fucose (manufactured by Takara Shuzo Co., Ltd.) with fluorescence intensity (excitation wavelength 320 nm, fluorescence wavelength 400 nm). About 40nmol. _ When measuring the activity of the terminal fucose-containing sulfated polysaccharide-degrading enzyme of the present invention using the fucose-containing sulfated polysaccharide-F obtained from the above (1) and (2) processes, the following procedure is performed. Namely, 12 microliters of 2.5% fucose-containing sulfated polysaccharide-F solution, 6 microliters of a 1M calcium chloride solution, 12 microliters of a 1M sodium chloride solution, and 72 microliters 50 mM buffer (pH 7.5) containing acetic acid and imidazole and THs-hydrochloric acid was mixed with 18 microliters of the terminal fucose-containing sulfated polysaccharide-F decomposing enzyme of the present invention 76 200400195 and reacted at 30 ° C. After 3 hours, the reaction solution was treated with 100t. After centrifugation, 100 microliters were analyzed by HPLC to determine the degree of low molecularization. Prepare a buffer solution to dissolve the terminal fucose-containing sulfated polysaccharide-degrading enzyme of the present invention in place of the terminal fucose-containing sulfated polysaccharide-degrading enzyme of the present invention and make the person react under the same conditions, and use water instead of fucose-containing sulfate The responders of the polysaccharide-F solution were used as a control group, and analyzed by HPLC in the same manner. One unit of enzyme is the amount of enzyme that cleaves Ivmol's fucose-containing sulfated polysaccharide-F fucose bond in 1 minute in the above reaction system. The amount of the fucosyl bond to be cleaved is calculated by the following formula. {(12X2.5) / (100XMF)} X {(MF / M) -1} X {0.12 / (180X0.01} = U / ml (12 × 2 · 5) / 100: fucoids added to the reaction system Sugar sulfate polysaccharide_F (mg) MF: average molecular weight of the fucose-containing sulfated polysaccharide-F (M): average molecular weight of the reaction product (MF / M) -1: 1 molecule of fucose-containing sulfated polysaccharide-17 Number of enzyme cuts 180: reaction time (minutes) 0.01: amount of enzyme solution (ml) 0 · 1 2: total amount of reaction solution (ml) 尙, HPLC conditions are as follows: 77 200400195 Device: L-6200 type (Made by Hitachi, Ltd.) Column: OHpak KB-804 (8mmX300mm) (manufactured by Showa Denko Corporation) Eluent ·· 25 μM imidazole buffer solution (PH8) containing 5 mM sodium azide, 25 mM sodium chloride, and 50 mM sodium chloride Detection: Parallax refractive index detector (Shode XRI-7 1. manufactured by Showa Denko Corporation)

Flow rate: 1 ml / min. Column temperature: 2 5 ° C. To determine the average molecular weight of the reaction product, a commercially available polytriose (STANDARD P-82, manufactured by Showa Denko Corporation) was analyzed by the same HPLC as above. The analysis was performed under the conditions, and the relationship between the molecular weight of polytribuose and the residence time of OHpak KB-804 was expressed as a curve, and it was used as a standard curve for measuring the molecular weight of the above enzyme reaction product. When the activity of the terminal fucose-containing sulfated polysaccharide enzyme of the present invention is measured using PA-FF obtained from the above (1) to (3) processes, the following procedure is performed. That is, 2 μl of a PA-FF solution of 8 pmol / // 1 and 5 μl of a 1 M calcium chloride solution, 10 μl of a 1 M sodium chloride solution, and 23 μl of water, and 50 μl 50 mM of a buffer solution containing acetic acid, imidazole and Ti: is-hydrochloric acid (pH 8 · 2) was mixed with 10 microliters of the terminal fucose-containing sulfate polysaccharide degrading enzyme of the present invention, and reacted at 30 ° C for 3 hours. After hours, the reaction solution was treated at 100 ° C. for 10 minutes, and after centrifugation, 80 microliters thereof were analyzed by HPLC to determine the degree of low molecularization. A buffer solution for dissolving the terminal fucose-containing sulfated polysaccharide-degrading enzyme of the present invention 78 200400195 is prepared to replace the terminal fucose-containing sulfated polysaccharide-decomposing enzyme of the present invention and to react under the same conditions. Respondents who used water instead of the p A-FF solution as a control group were also analyzed by HPLC in the same manner. One unit of enzyme is the amount of enzyme that cleaves 1 Amol of fucose-containing sulfated polysaccharide containing fucosyl bond in 1 minute in the above reaction system. The amount of the fucosyl bond to be cleaved is calculated by the following formula. 16Xl0-6 (MF / M) -l} X {1 / (180x0.01)} = U / ml 16 X 10 · 6 PA-FF (/ z mol) added to the strain MF: PA- The average molecular weight of FF M: the average molecular weight of the reaction product (MF / M) -1: 1 molecule of the number of fucose-containing sulfated polysaccharide-F cut by enzymes 180: reaction time (minutes) 0.01: the amount of enzyme solution ( Ml) 尙, HPLC conditions are as follows. Device: L-62 00 type (manufactured by Hitachi) Column: OHpak SB-803 (8mmX 300mm) (manufactured by Showa Denko Corporation) Eluent: 200mM chlorination containing 5mM sodium azide and 10% dimethyl methylene maple Sodium solution detection: Fluorescence detector F-1150 (manufactured by Hitachi, Ltd.) was used to detect at an excitation wavelength of 320 nm and a fluorescence wavelength of 400 nm.

Flow rate: 1 ml / min Column temperature: 50 ° C 79 200400195 In order to determine the average molecular weight of the reaction product, commercially available polytriglyceride (STANDARD P-82, manufactured by Showa Denko Corporation) was used with Glyco TAG and Glyco TAG The reagent kit was PA-reduced at the reducing end to obtain PA-polyglucose of various molecular weights. The PA-polyglucose with various molecular weights obtained was analyzed under the same HPLC analysis conditions as described above, and the relationship between the molecular weight of polytriglucose and the retention time of OHpak SB-80 3 was plotted as a curve to determine the enzyme reaction product. Standard curve of molecular weight. Protein was quantified by measuring the 28 Onm absorbance of the enzyme solution. At this time, the absorbance of the 1 mg / ml protein solution was calculated as K 0. The inventors of the present invention determined the action mechanism of the terminal fucose-containing sulfate polysaccharide-decomposing enzyme of the present invention as described below. (1) The fucose-containing sulfated polysaccharide-F from the terminal type fucose-containing sulfated polysaccharide-decomposing enzyme and the decomposition of the decomposed product make refined fucose-containing sulfated polysaccharide-F from high fruit kelp according to the present invention The terminal type fucose-containing sulfated polysaccharide decomposes enzymes and regulates the decomposition products. First, production of a fucose-containing sulfate-decomposing enzyme is performed. That is, sp. SN-1 009 (CCRC 9 1 0070) was inoculated to artificial seawater (manufactured by Germaline Laboratory) containing 0.25% of glucose, 1.0% of protein glands, and 0.05% of yeast extract. ΡΗ8 · 2 600 ml of the culture medium composed and sterilized (12 (TC, 20 minutes) in a 2 liter Erlenmeyer flask 'and cultured at 25 ° C for 26 hours to prepare a seed culture medium. It will contain glucose 0.25%, protein gland 1.0% , Enzyme extract 0.02%, the aforementioned fucose-containing sulfated polysaccharide 0.2% from high fruit kelp, and defoamer (made by Shin-Etsu Chemical Industry 80 200400195 KM70) 0.01% artificial seawater (made by Germalin Laboratory) pH 8.0 20 liters of the culture medium is put into a 30 liter capacity fermentation tank and sterilized at 120 ° C for 20 minutes. After cooling, 'inoculate 600 ml with the above seed and incubate at 24 ° C for 24 hours, every minute Cultivate under the conditions of 10 liters of aeration and a stirring speed of 125 rpm. After the end of the culture, the culture solution is centrifuged to obtain bacterial cells and culture supernatant. The obtained culture supernatant is classified into a molecular weight of 1 Wanzhi ultrafilter is 85% saturated after being concentrated Salting out with ammonium sulfate, the resulting precipitate was collected by centrifugation, and fully dialyzed against 20 mM Tris-hydrochloric acid buffer (pH 8.2) containing artificial seawater at a concentration of 1/10, to obtain 600 ml of crude enzyme. 40 ml of the crude enzyme obtained from the treatment, 44 ml of artificial seawater, 510 mg of fucose-containing sulfated polysaccharide-F and 36 ml of water were mixed, the pH was adjusted to 8, and the reaction was performed at 25 ° C for 48 hours. After that, gel filtration was performed with Cellulofine GCL-300, and it was divided into 4 parts. From the order of large molecular weight, it was determined as F-Fd-1 (molecular weight more than 2 5000), F-Fd-2 (molecular weight 25000 ~ more than 12000). ), F-Fd-3 (molecular weight 12000 to more than 6500), and F-Fd-4 (molecular weight 6500 or less). The four fractions were desalted and freeze-dried, and 170 mg, 270 mg, and 300 mg of dried products were obtained. And 340 mg. The enzyme-decomposed product containing fucose sulfated polysaccharide-F, that is, the low-molecular-weight filtered product through a Ce 1 lulofi ne GCL- 3 00 gel, is shown in FIG. 22. The vertical axis is shown in FIG. 22. Indicates the absorbance at 48 Onm (the amount of coloration according to the phenol-sulfuric acid method), and the horizontal axis indicates dissolution Part number, 1 dissolving fraction is 10 ml. The column volume is 1075 81 2004 00 195 ml, and the eluent is a 0.2 M ammonium acetate solution containing 10% methanol. In the figure 22, the white circle is marked as fucose-containing sulfuric acid The gel filtration results of the polysaccharide-F enzyme degraded product, the black triangle mark indicates the gel filtration results of the fucose-containing sulfated polysaccharide-F before the enzyme decomposition. From the results of the above-mentioned Cellulofine GCL-300, it can be determined that the molecular weight distribution of the reaction product of the fucose-containing sulfated polysaccharide decomposition enzyme of the present invention is about 1,000 to 30,000. (2) Analysis of the composition of reducing terminal sugars and neutral sugars of the enzyme reaction products. Glyco TAG and F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4 were used as part of the above. The Glyco TAG reagent kit PA-reduced the terminal ends, and each of the PA-sugars (PA-F-Fd-1), (PA-F-Fd-2), (PA-F-Fd-3) ) And (PA-F-Fd-4) were treated with 4 equivalents of hydrochloric acid at 100 ° C for 3 hours to hydrolyze, and the reducing terminal sugar was investigated by HPLC. A. The conditions of HPLC are as follows. Device · L-6200 (manufactured by Hitachi) Column: Per Pack Type A (4.6mmX150mm) (manufactured by Takara Shuzo Co., Ltd.) Eluent: 700 mM boric acid buffer (ρΗ9): acetonitrile = 9: 1 Detection: The fluorescence detector F-1 150 (manufactured by Hitachi, Ltd.) is detected at an excitation wavelength of 310 nm and a camping light wavelength of 380 nm. Flow rate · 0.3 ml / min

Column temperature · 6 5 ° C The results are (PA-F-Fd-1), (PA-F-Fd-2), (PA-F-Fd-3), and (PA-F-Fd-4) The reducing terminal sugar is fucose. 82 200400195 The neutral sugar composition of F-Fd-1, F-Fd-2, F-Fd-3 and F-Fd-4 was measured by the following method.尙 After fucose-containing sulfated polysaccharide-F used in the substrate was hydrolyzed by sulfuric acid, Glyco TAG and Glyco TAG reagent kits were used to PA-reducing the reducing end of the sugar, and the enzyme reaction product was reduced in the same analysis as above. When analyzed under HPLC at the end of sex, only fucose and galactose were detected, and because their stereo coordination was L and D, respectively, only L-fucose and D-galactose were also determined for the product. That is, in order to investigate the content of D-galactose, which constitutes one of the sugars, a reaction system capable of measuring only D-galactose was constructed according to the instruction manual using an F-set, lactose / galactose (manufactured by Bailing Kamanouchi Co., Ltd.), After neutralizing 4 equivalents of hydrochloric acid at 1000 ° C for 2 hours, F-Fd · 1, F-Fd-2, F-Fd-3, and F-Fd-4 were neutralized to form a reaction system. Determination. In addition, in order to quantify the other L-fucose, which constitutes sugar, according to the method described in Clinical Chemistry, Vol. 36, pp. 474-476 (1990), 4 equivalents of hydrochloric acid was further hydrolyzed at 100 ° C for 2 hours. After F-Fd · 1, F-Fd-2, F-Fd-3, and F-Fd-4 were neutralized for one hour, the reaction system was used to measure the neutralization. From the above results, what is the ratio of L-fucose to D-galactose? -? ^ 1, F-Fd-2, F-Fd-3, and F-Fd-4 are about 100: 44, 100: 27, 100: 5 and 100: 1. If the above results are summarized, it can be judged that the terminal fucose-containing sulfated polysaccharide decomposing enzyme of the present invention acts on fucose-containing sulfated polysaccharide-F and hydrolyzes the fucose-based bond to generate a molecular weight of about 1,000 to 30,000. Left and right low molecular compounds, and this low molecular compound has a high galactose content in the larger molecular weight. Alas, 83 200400195 The reducing ends of the low molecular compounds are all L-fucose. Secondly, in order to investigate the specificity of the quality of this enzyme, fucose-containing sulfated polysaccharide-U was acted with the fucose-containing sulfated polysaccharide-decomposing enzyme of the present invention. That is, a 2.5% solution of fucose-containing sulfated polysaccharide-U was used. 12 μl with 6 μl of 1 M calcium chloride solution and 12 μl of 1 M sodium chloride solution, and 72 μl of 50 mM imidazole buffer (ρ 缓冲 7.5), and 18 μl of The terminal type fucose-containing sulfated polysaccharide decomposing enzyme (1.6mU / ml) was mixed and reacted at 30 ° C for 3 hours, and then the reaction solution was treated at 100 ° C for 10 minutes. The analysis was performed by HPLC to determine the degree of low molecularization. The control group was prepared, and a buffer solution for dissolving the terminal fucose-containing sulfated polysaccharide-degrading enzyme of the present invention was used instead of the terminal fucose-containing sulfated polysaccharide-decomposing enzyme of the present invention, and those who responded under the same conditions were similarly treated with Analysis was performed by HPLC. A. The conditions of HPLC are as follows. Device: L-6200 (manufactured by Hitachi) Column: OHpak KB- 804 (8mmX 300mm) (manufactured by Showa Denko) Eluent: 25mM containing 5mM sodium azide, 25mM calcium chloride, and 50mM sodium chloride Imidazole buffer solution (PH8) detection: parallax refractive index detector (Shode XRI-7) 1, manufactured by Showa Denko Corporation

Flow rate: 1 ml / min. Column temperature: 25 ° C. As a result, the fucose-containing sulfate polysaccharide-decomposing enzyme of the present invention is completely free of 84 200400195 method to reduce the fucose-containing sulfate polysaccharide-u to a low molecular weight. As described above, the present invention relates to an enzyme composition containing the fucose-containing sulfated polysaccharide decomposing enzyme of the present invention and a calcium source. Examples of usable calcium sources in the solid composition include calcium chloride, calcium carbonate, calcium salts of calcium acetate, calcium oxide, calcium hydroxide, and hydrates thereof. In addition, in a liquid composition in which water, ethanol and the like are dissolved, suspended, and emulsified, the calcium source may be a monomer as described above, or may be in an ionized state by dissolution. These calcium sources are effective because they can activate or stabilize the enzyme. Therefore, the above-mentioned enzyme composition may also contain commonly used additives depending on the application, as long as the above-mentioned calcium source does not hinder the action of the calcium source. By causing the fucose-containing sulfated polysaccharide-decomposing enzyme of the present invention to act on the fucose-containing sulfated polysaccharide-F content, a low-molecular-weight compound containing fucose-containing sulfated polysaccharide-F can be prepared. The fucose-containing sulfated polysaccharide-F content may be, for example, a fucose-containing sulfated polysaccharide-F refined product, or may be the aforementioned fucose-containing sulfated polysaccharide mixture, and may also be an aqueous solvent extract of brown algae seaweed. . The dissolution of the fucose-containing sulfated polysaccharide-F content can be performed by a common method, and although the concentration of the fucose-containing sulfated polysaccharide in the dissolving liquid may be the highest dissolved concentration, it is generally considered to be operable. It is better to choose enzyme. The fucose-containing sulfated polysaccharide · F solution can be selected according to the purpose, such as water or a buffer solution. The pH of the dissolving solution is usually neutral, and the enzyme reaction is usually carried out around 30 ° C. By adjusting the amount of enzyme, reaction time, etc., the molecular weight of low molecular compounds can be adjusted. 85 200400195 Secondly, molecular weight fractionation of low-molecular-weight compounds' can prepare a more uniform molecular weight distribution of fucose-containing sulfated polysaccharide-F low-molecular-weight compounds. The molecular weight classification can be performed by a method generally used, for example, a gel filtration method and a molecular weight classification membrane can be used. Low-molecular-weight compounds, if necessary, may be subjected to refining operations such as ion exchange resin treatment, activated carbon treatment, etc., and desalting treatment, aseptic processing may be performed as needed, and freeze-drying of the low-molecular-weight compounds of the present invention may be prepared. Product. Examples Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited by these descriptions. Alas,% in the examples means% by weight. Example 1 After fully drying Komami kelp, 2 kg of the dried product was crushed by a free grinder (manufactured by Nara Machinery Co., Ltd.), and the obtained dried powder was suspended in 9 liters of 80% ethanol, and treated at 8 (TC 2 hours. After treatment, filter the filter residue to obtain the residue. This residue is washed with the above ethanol. Repeated 3 times of filtration to obtain the ethanol washing residue. This residue is suspended in 40 liters of water, treated at 95 t for 2 hours, and filtered. The residue was washed with hot water to obtain an extract of fucose-containing sulfated polysaccharide of high fruit kelp 36 liters. The obtained extract was freeze-dried to obtain a fucose-containing sulfated polysaccharide sample 1 5 4 g. Secondly, 0.4 M common salt was added to the remaining extract, and 5% cetylpyridinium chloride was added so that it would not cause precipitation again, and the precipitate was collected by centrifugation. This precipitate was collected in 3 liters. Suspend in 0 · 4M saline and centrifuge and wash. Repeat this washing operation three times and add 1 liter of 4M saline 86 200400195 to the precipitate. After stirring well, add ethanol to 80%. After stirring, Centrifuge to obtain the precipitate. The suspension of the precipitate in 80% ethanol and centrifugation was repeated until the absorbance at 260 nm in the supernatant solution became 0. This precipitate was dissolved in 3 liters of 2M saline and the insoluble matter was removed by centrifugation, and 2M saline was added. Equilibrated 100 ml of DEAE-Cellulofine A-800 (manufactured by Biochemical Industry Co., Ltd.), after stirring, the resin added was filtered off. The filtrate was placed in a 2M saline-equipped DEAE-Cellulofine A-800 column, non-adsorbed Part of it is ultra-filtered by an ultrafiltration device with a hollow fiber with a molecular weight of less than 100,000. After completely removing the coloring matter and salt, the insoluble matter is removed by centrifugation and filtration, and freeze-dried. The weight of the sugar sulfate polysaccharide mixture was 76 g. Example 2 After the real kelp was sufficiently dried, 2 kg of the dried product was crushed by a free grinder (manufactured by Nara Machinery Co., Ltd.), and the obtained dried powder was suspended in 9 liters of 80% ethanol And treated at 80 ° C for 2 hours. After the treatment, the residue was filtered through filter paper. The residue was washed with the above ethanol. The filtration was repeated 3 times to obtain the ethanol residue. The residue was suspended in 40 liters of water, and then treated at 95 ° C for 2 hours, and filtered. The residue was washed with hot water to obtain 36 liters of fucose-containing sulfate polysaccharide extract of true kelp. 0.4M common salt was added to the extract, and then 5% cetylpyridinium chloride was added so as not to cause precipitation again, and the precipitate was collected by centrifugation. The precipitate was suspended in 3 liters of 0.3M saline Centrifuge and wash. After repeating this washing operation 3 times, add 1 liter of 4M common salt 87 200400195 water to the precipitate, add ethanol to 80% after stirring well, and obtain the precipitate by centrifugation after stirring. This pellet was suspended in 80% ethanol and centrifuged again until the 260 nm absorbance in the supernatant solution became zero. This precipitate was dissolved in 3 liters of 2M saline, and insoluble matter was removed by centrifugation. Then, 100 ml of DEAE-Cellulofine A-800 (manufactured by Biochemical Industry Co., Ltd.) equilibrated with 2M saline was added. After stirring, the resin was added to Remove by filtration. The filtrate was placed in a DEAE-Cellulofine A-800 column equilibrated with 2M saline, and the non-adsorbed part was ultra-filtered with an ultrafiltration device with a hollow fiber excluding molecular weight of less than 100,000. After the coloring matter and salt were completely removed, The insoluble matter was removed by centrifugation and filtration, and freeze-dried. The freeze-dried fucose-containing sulfated polysaccharide mixture weighed 52 grams. Example 3 Extraction of fucose-containing sulfated polysaccharide mixture from real kelp After the real kelp was sufficiently dried, 2 kg of the dried product was crushed by a free grinder (manufactured by Nara Machinery Co., Ltd.), and the obtained dried powder was suspended in 9 liters of 80 % Ethanol and treated at 80 ° C for 2 hours. After the treatment, the residue was filtered with filter paper. This residue was washed with the ethanol described above. Filtration and repeating the operation 3 times can obtain ethanol washing residue. This residue was suspended in 36 liters of a 0.2M calcium acetate solution, and then treated at 95 ° C for 2 hours and filtered. The residue was washed with 4 liters of a 0.2M calcium acetate solution to obtain 36 liters of fucose-containing sulfate polysaccharide extract of true kelp. Example 4 Preparation of a mixture of fucose-containing sulfated polysaccharides from true kelp In the filtrate obtained in Example ·, 5% cetylpyridinium chloride was added to the mixture until it was re-precipitated by 88 200400195 and centrifuged. Collect the precipitate. This pellet was suspended in 3 liters of 0.3M saline, centrifuged, and washed. This washing operation was repeated three times, and 1 liter of 4M saline was added to the precipitate. After good stirring, ethanol was added to 80%. After stirring, the precipitate was obtained by centrifugation. This precipitate was suspended in 80% ethanol and the operation of centrifugation was repeated until the absorbance at 260 nm in the supernatant solution became 0. This precipitate was dissolved in 3 liters of 2M saline, and insoluble matter was removed by centrifugation. Then, 100 ml of DEAE-Cellulofine A-800 (manufactured by Biochemical Industry Co., Ltd.) equilibrated with 2M saline was added. Remove by filtration. The filtrate was placed in a 2M saline-equipped DEAE-Cel lulofine A- 800 column, and the non-adsorbed part was ultra-filtered with an ultrafiltration device with a hollow fiber excluding molecular weight of less than 100,000. The coloring matter and salt were completely removed. After removal, the insoluble matter was removed by centrifugation and filtration, and freeze-dried. The freeze-dried fucose-containing sulfated polysaccharide mixture weighed 52 grams. The fucose-containing sulfated polysaccharide mixture does not contain a coloring substance adsorbed to a polysaccharide resin. Example 5 The lyophilized product containing the fucose-sulfate polysaccharide mixture described in Example 4 was weighed into 4 parts of 1 g each, and dissolved in water, 0.2M sodium chloride, 0.2M calcium chloride, 0.2 M magnesium chloride, secondly, prepare 4 500 ml DEAE-SepharoseFF columns, and in them 2 with 0.2M sodium chloride, 1 with 0.2M calcium chloride, 1 with 0.2M Magnesium chloride was equilibrated separately. One of the columns equilibrated with 0.2M sodium chloride was washed with 10 times the amount of water in the column. Fucose-containing sulfur 89 200400195 dissolved in water, sodium chloride, calcium chloride, and magnesium chloride was separately placed in a DEAE-Sepharose FF column equilibrated with water, sodium chloride, calcium chloride, and magnesium chloride, respectively. In each case, the solution was thoroughly washed with the solution used for equilibration, and secondly, it was dissolved out with a sodium chloride gradient of 0 to 4M. As a result, the total amount of the fucose-containing sulfated polysaccharide mixture was adsorbed only in the system using calcium chloride and magnesium chloride. Only 0.4 g of fucose-containing sulfuric acid polysaccharide was adsorbed in a column of water and saline solution equilibrium. Further, in any of the columns, the fucose-containing sulfated polysaccharide-F and the fucose-containing sulfated polysaccharide-U of the present invention were substantially separated. Example 6 After fully drying Komaki kelp, 2 kg of it was crushed with a free pulverizer (manufactured by Nara Machinery Co., Ltd.), and the resulting dried powder was suspended in 9 liters of 80% ethanol and treated at 80 ° C. 2 hour. After treatment, filter paper was used to obtain the residue. This residue was washed with the aforementioned ethanol. Filtration was repeated 3 times to obtain ethanol washing residue. This residue was suspended in 36 liters of a 0.2 M calcium acetate solution, treated at 9 51 for 2 hours, and filtered. The residue was washed with 4 liters of 0.2M calcium acetate solution to obtain 36 liters of fucose-containing polysaccharide extract of high fruit kelp. This filtrate was concentrated to 2 liters with an ultrafilter equipped with an ultrafiltration membrane with a molecular weight of 100,000. Secondly, a common salt with a final concentration of 1.5 M was added and 5% cetylpyridinium chloride was not regenerated. Add until precipitation. The resulting precipitate was removed by centrifugation. The resulting mash solution was ultra-filtered to a concentration of 1 liter, and 4 liters of ethanol was added, and the resulting precipitate was collected by centrifugation and 90 200400195. After adding 100 ml of 4M saline to the precipitate and stirring well, 80% of ethanol was added, and the precipitate was obtained by centrifugation after stirring. This precipitate was suspended in 80% ethanol and the operation of centrifugation was repeated until the 26 Onm absorbance in the supernatant solution was 0. This precipitate was dissolved in 2 liters of 2M saline, and insoluble matter was removed by centrifugation. Then, 50 ml of DEAE-Cellulofine A-800 (manufactured by Biochemical Industry Co., Ltd.) equilibrated with 2M saline was added. After stirring, add The resin was removed by filtration. The filtrate was placed in a 2M saline-equipped DEAE-Cellulofine A-800 column, and the non-adsorbed part was ultra-filtered with an ultrafiltration device with a hollow fiber excluding molecular weight of less than 100,000. After the coloring matter and salt were completely removed, The insoluble matter was removed by centrifugation and filtration, and freeze-dried. The weight of the freeze-dried fucose-containing sulfated polysaccharide-U was 15 grams. The fucose-containing sulfated polysaccharide-U of the present invention does not contain a coloring substance adsorbed to a polysaccharide resin. In addition, the fucose-containing sulfated polysaccharide-U reacts with the aforementioned terminal fucoidan-decomposing enzyme to generate oligosaccharides represented by the formulae (I), (II), and (; [; [丨) . Example 7 After the dried commercial kelp was sufficiently dried, 2 kg of it was crushed with a free pulverizer (manufactured by Nara Machinery Co., Ltd.), and the obtained dried powder was suspended in 9 liters of 80% ethanol 'and treated at 80 ° C. 2 hours. After treatment, the residue was filtered with filter paper. This residue was washed with the aforementioned ethanol. Filter and repeat the operation 3 times to obtain ethanol washing residue. This residue was suspended in 36 liters of a 0.2M calcium acetate solution, and then treated at 95 ° C for 2 hours and filtered. The residue was washed with 4 liters of 0.2M acetic acid 91 200400195 calcium solution to obtain 36 liters of fucose-containing sulfated polysaccharide extract of high fruit kelp. To the obtained filtrate, 5% cetylpyridinium chloride was added so as not to cause precipitation again, and the precipitate was collected by centrifugation. The precipitate was suspended in 3 liters of saline, centrifuged, and washed. This washing operation was repeated three times, and 1 liter of 4M saline was added to the precipitate. After good stirring, ethanol was added to 80%. After stirring, the precipitate was obtained by centrifugation. This precipitate was suspended in 80% ethanol and the operation of centrifugation was repeated until the 26 Onm absorbance in the supernatant solution became 0. This precipitate was dissolved in 3 liters of 2M saline and insoluble matter was removed by centrifugation. Then, 100 ml of DEAE-Cel liilo fine A-800 (manufactured by Biochemical Industry Co., Ltd.) equilibrated with 2M saline was added. After stirring, add The resin was removed by filtration. The filtrate was placed in a 2M saline-equipped DEAE-Cellulofine A-800 column, and the non-adsorbed part was ultra-filtered by an ultrafiltration device with a hollow fiber excluding molecular weight of less than 100,000, and the coloring matter and salt were completely removed. The insoluble matter was removed by centrifugation and filtration, and freeze-dried. The weight of the freeze-dried fucose-containing sulfated polysaccharide mixture was 90 g. The fucose-containing sulfated polysaccharide mixture does not contain a coloring substance adsorbed to a polysaccharide resin. 7 g of this lyophilized mixture containing the fucose sulfate polysaccharide was weighed and dissolved in 0.2M potassium chloride. Next, a 4000 ml DEAE-Sepharose FF column was equilibrated with 0.2M calcium chloride. The fucose-containing sulfated polysaccharide mixture dissolved in 0.2M calcium chloride was placed in a DEAE-Sepharose FF column, washed thoroughly with 0.2M calcium chloride, and secondly, it was subjected to a sodium chloride gradient of 0 to 4M. Dissolve. Collect the dissolving fraction with sodium chloride concentration of 0.0 5 ~ 0.8M in the dissolving fraction and obtain the fucose-containing sulfated polysaccharide separated from the fucose-containing sulfuric acid _F by freeze-drying after desalting with dialysis. -U 2. 丨 gram. In addition, the dissolving fraction having a sodium chloride concentration within the above-mentioned dissolution fraction of 0.9 to 1.5 M was collected and freeze-dried after being desalted by dialysis to obtain fucose containing substantially separated from rock-containing bath sugar sulfate polysaccharide-U. Polysaccharide sulfate_F 4.7 g. Example 8 Manufacture of fucose-containing sulfated polysaccharide-F. Weigh 1.2 f 'of the fucose-containing sulfated polysaccharide mixture obtained in Example 7 in a 1.5 M sodium chloride solution to a final concentration of 0.2. % Dissolved 'and a 1.5 M sodium cetylpyridinium solution in 1.5 M sodium chloride was added so as not to cause precipitation again. The resulting precipitate was collected by centrifugation, and the precipitate was suspended in 500 ml of 1.5 M saline and centrifuged 'and washed. This washing operation was repeated three times, and 1 liter of 4M saline was added to the precipitate. After being stirred well, ethanol was added to 80%. After stirring, the precipitate was obtained by centrifugation. This precipitate was suspended in 80% ethanol and the operation of centrifugation was repeated until the absorbance at 260 nm in the supernatant solution became zero. This precipitate was dissolved in 500 ml of 2M saline, and insoluble matter was removed by centrifugation. Then, 1 ml of DEAE-Cel lulofine A-800 (manufactured by Biochemical Industry Co., Ltd.) equilibrated with 2M saline was added, and the resin was added after stirring. Removed by filtration. The filtrate was placed in a 2M saline-balanced DEAE-Cel lulofine A- 800 column, and the non-adsorbed part was ultra-filtered by an ultrafiltration device with a hollow fiber with a molecular weight of less than 100,000. The coloring substance and salt were completely removed. After removal, the insoluble matter was removed by centrifugation and filtration, and freeze-dried. The weight of freeze-dried fucose-containing sulfated polysaccharide-93 200400195 F was 710 grams. The fucose-containing sulfated polysaccharide_F does not contain a coloring substance adsorbed to a polysaccharide resin. And this fucose-containing sulfated polysaccharide-F is the fucose-containing sulfated polysaccharide-F of the present invention which does not contain uronic acid and contains fucose as a main component of sugar. Example 9 Enzymatic purification method of fucose-containing sulfated polysaccharide-F. Weigh 10 g of the fucose-containing sulfated polysaccharide mixture obtained in Example 7 and dissolve it in 500 ml of artificial seawater. The terminal type fucoidan of the genus sp, SA-0082 (CCRC 910069) decomposes the enzyme and reacts at 25 ° C for 50 hours. The reaction solution was subjected to ultrafiltration by preparing an ultrafiltration device excluding hollow filaments having a molecular weight of less than 100,000, and after completely removing the low-molecular substances, the insoluble substances were removed by centrifugation and filtration, and freeze-dried. The weight of the freeze-dried fucose-containing sulfated polysaccharide-F was 6 g. The fucose-containing sulfated polysaccharide-F is a coloring substance that does not contain a polysaccharide resin. It was also determined that the fucose-containing sulfated polysaccharide_F is fucosyl-containing sulfated polysaccharide-F of the present invention that does not contain uronic acid and uses fucose as a main component of sugar. Example 10 Culture-refining method of fucose-containing sulfated polysaccharide-F 60 g of the fucose-containing sulfated polysaccharide mixture obtained in Example 7 was weighed and dissolved in 20 liters of artificial seawater. 200 g of protein glands were added with yeast. 4 g of the extract was placed in a 30 liter fermentation tank and sterilized, and the aforementioned Flavobacterium sp. SA-008 2 strain (CCRC 9 1 0069) was planted and cultured at 25 ° C for 24 hours. After centrifuging the culture solution to remove bacterial cells, ultrafiltration was performed by an ultrafiltration device equipped with a hollow wire of 94 200400195 sub-M 100,000 or less. After removing the low molecular weight stomach, the centrifugal separation and centrifugation were performed. Filter to remove insoluble materials, and #? 东 干 丨 喿 ° Freeze-dried fucose-containing sulfated polysaccharide-F is 3 6 g ° 51 # Fucose-containing sulfated polysaccharide-F is free from adsorption to polysaccharide resin & amp With 1 f biomass. The fucose-containing sulfated polysaccharide-F was determined to be the fucose-containing sulfated polysaccharide-F of the present invention which does not contain uronic acid as the main component of sugar. Example 1 1 3 quart # 5S The freeze-dried product of the fucose-containing sulfated polysaccharide mixture described in Example 7 was weighed into 4 parts each of 1 g, and dissolved in water, 0.2M sodium chloride, 0.2M chlorine Among calcium chloride and 0.2M magnesium chloride, four 500 ml DEAE-SepharoseFF columns were prepared, and two of them were 0.2M sodium chloride, one was 0.2M calcium chloride, and one was 0.2M magnesium chloride was equilibrated separately. One of the columns equilibrated with 0.2 M sodium chloride was washed with 10 times the amount of water in the column. The fucose-containing sulfated polysaccharide mixtures dissolved in water, sodium chloride, calcium chloride, and magnesium chloride were respectively placed in DEAE-Sepharose FF columns equilibrated with water, sodium chloride, calcium chloride, and magnesium chloride, respectively. Wash thoroughly with the solution used for equilibration, and then dissolve it with a sodium chloride gradient of 0 to 4M. As a result, the total amount of the fucose-containing sulfated polysaccharide mixture placed in the column was adsorbed only in the system using calcium chloride and magnesium chloride. Only 0.4 g of fucose-containing sulfated polysaccharide was adsorbed in a column equilibrated with water and saline. Further, in any of the columns, the fucose-containing sulfated polysaccharide-F and the fucose-containing sulfated polysaccharide-U of the present invention were substantially separated. 95 200400195 Example 1 2 Weigh 7 g 'of the fucose-containing sulfated polysaccharide mixture described in Example 1 and dissolve it in 800 ml of 0.2M calcium chloride. Next, a 4 liter DEAE_Sepharose FF column was equilibrated with 0.2M calcium chloride, and the entire amount of the fucose-containing sulfated polysaccharide solution was placed in the column, and after washing with 8 liter 0.2M chlorinated chlorin solution, It was dissolved in a gradient of 0 to 4 M sodium chloride. Detected fractions of uronic acid (sodium chloride concentration of about 0.9M or less: containing fucose sulfate polysaccharide-U), and no detected fractions of uronic acid (sodium chloride concentration of about 1) · Near 2M: fucose-containing sulfated polysaccharide-F) was desalinated and freeze-dried 'to obtain 1.4 g and 4.8 g of dried products, respectively. Example 1 3 In the fucose-containing sulfated polysaccharide mixture obtained in Example 1, the terminal fucoidan-producing enzyme produced by Xanthomonas sp. SA-0082 (CCRC 9 1 0069) was used to produce Oligosaccharide of the following structure. 96 200400195

97 200400195

98 200400195 The present inventors and others obtained the above oligosaccharide for the following enzyme reaction. That is, 2.5% of the fucose-containing sulfuric acid polysaccharide mixture solution of Example 80 80 ml, and 50 ml of phosphate buffer solution (ρΗ7.5) 60 ml and 4 M sodium chloride 20 ml and 32 mU / ml end The fucoidan-degrading enzyme solution was mixed in 40 ml, and allowed to react at 25 ° C for 48 hours. The reaction solution was passed through a column of Cell ul of ine GCL- 3 00 (manufactured by Biochemical Industry Co., Ltd.) to carry out molecular weight fractionation and collect fractions having a molecular weight of 2000 or less. After this salt was demineralized with MICROACILIZER G3, 3 particles were separated with DEAE-Sepharose FF, and after desalting again, it was freeze-dried. In this way, oligosaccharides of each of the above formulae (I), (I), (III) were obtained at 250 mg, 310 mg, and 52 mg each. Example 14 4 10 g of the fucose-containing sulfated polysaccharide mixture obtained in Example 1 was dissolved in 500 ml of 0. 2M citric acid, the pH was adjusted to 2.9, and then treated at 100 ° C for 3 hours. 150 ml of a 1M calcium acetate solution was added to this hydrolysate, and the resulting precipitate was removed by centrifugation and then subjected to gel filtration with Cell ulofine GCL-25 for molecular weight classification (molecular weight of 5000 or more, 50000 ~ 3 0 0 0, 3 0 0 0 ~ super 2 0 0 0, 2 0 0 0 ~ super 1 〇〇〇, i 〇〇〇 ～ super 5 00, 500 or less), in the order of the larger molecular weight, GFd -Oli-1, GFd-oli-2, GFd-01i-3, GFd-01i-4, GFd-oli-5, and 0? (1- 0 1 i-6. After desalting these 6 parts Freeze-drying can obtain dry products of 2.3 g, 1.7 g, 0.88 g, 1.8 g, 1.4 g, and 0.72 g. Example 1 5 99 200400195 Weigh the fucose-containing sulfated polysaccharide mixture obtained in Example 60. 0 G, and dissolved in 20 liters of artificial seawater, 200 g of protein glands and 4 g of yeast extract were added, and placed in a 30 liter fermentation tank and sterilized, and the aforementioned Xanthomonas sp. SA- 00 8 Two strains (CCRC 9 1 0069) were planted and cultured at 25t for 24 hours. After the culture solution was centrifuged to remove the bacterial cells, they were subjected to ultrafiltration with an ultrafiltration device that eliminated hollow silk with a molecular weight of less than 100,000. After filtration, the low-molecular substances were completely removed, and the insoluble substances were removed by centrifugation and filtration, and freeze-dried. The weight of the freeze-dried fucose-containing sulfated polysaccharide-F was 36 g. Example 1 6 5 kg of real sea cucumber Disintegrate, remove the internal organs, and collect the body wall. Add 500 ml of acetone per 200 grams of wet weight of the body wall, filter with a homogenizer, and wash the residue with acetone until no more coloring matter. 140 g of dried product was obtained. To this dried product was added 0.4 M of saline 2.8 liters, which was treated at 100 ° C for 1 hour, filtered, and the residue was thoroughly washed with 0.4 M of saline. To obtain an extract of 3.7 liters, 5% cetylpyridinium chloride was added to this extract until it did not cause precipitation again, and the resulting precipitate was collected by centrifugation. This precipitate was collected at 0.4 M After suspending in the saline solution, centrifugation was performed again, and 1 liter of 4M saline solution was added to the obtained precipitate. After processing with a homogenizer, 4 liters of ethanol was added while stirring, and after stirring for 1 hour, it was filtered to obtain a precipitate.

For this precipitation, the process of filtering and suspending 80% ethanol was repeated until the absorbance at 260 nm of the supernatant liquid became 0. The obtained precipitate was suspended in 2 liters of 2M 200400195 saline, and insoluble matter was removed by centrifugation. The supernatant liquid is ultra-filtered with an ultrafiltration device with a molecular weight of 30,000 membranes, and after completely desalting, freeze-drying can obtain 3.  7 grams of fucose-containing sulfated polysaccharide. Example 17 After fully drying the loquat leaf tip (Fucus Vesicu 10sus), 2 kg of the dried material was crushed with a free crusher (manufactured by Nara Machinery Co., Ltd.), and the obtained dry powder was suspended in 9 liters of 80% ethanol Medium and treated at 80 ° C for 2 hours. After the treatment, the residue was filtered with filter paper. This residue was washed with the aforementioned ethanol. After filtering and repeating the operation 3 times, the ethanol washing residue was obtained. This residue was suspended in 40 liters of water, treated at 100 ° C for 2 hours, and filtered. Add 0 to the filtrate.  5M sodium chloride, 5% cetylpyridine chloride was added so that it would not cause precipitation again, and the precipitate was collected by centrifugation. This precipitate is 0 in 3 liters.  After being suspended in 4M saline, it was centrifuged and washed. After this washing operation was repeated three times, 250 g of sodium chloride was added to the precipitate, and the suspension was suspended in 3 liter of ethanol, and the precipitate was obtained by centrifugation. This precipitate was suspended in 80% ethanol and centrifuged repeatedly until the absorbance at 260 nm in the supernatant solution became zero. This precipitate was dissolved in 3 liters of 2M saline, and insoluble matter was removed by centrifugation. Then, 100 ml of DEAE-Cel lulofine A-800 (manufactured by Biochemical Industry Co., Ltd.) equilibrated with 2M saline was added. After stirring, the resin was added. Removed by filtration. The filtrate was placed in a 2M saline-equipped DEAE-Cel lulofine A · 800 column, and the non-adsorbed part was ultra-filtered with an ultrafiltration device with a hollow fiber excluding molecular weight of less than 100,000, and the coloring substance and sodium chloride were filtered. After complete removal, the soluble matter was removed by centrifugation and filtration, and freeze-dried. The weight of freeze-dried fucose-containing polysaccharide was 92 grams. Example 1 8 After the Wakame (Undaria pinnatifida) is sufficiently dried, 2 kg of it is crushed with a free pulverizer (manufactured by Nara Machinery Co., Ltd.), and the resulting dry powder is suspended in 9 liters of ethanol and treated at 75 t for 2 hours. . After treatment, the residue was filtered with filter paper. To this residue, 9 liters of 80% ethanol was added, stirred, and treated at 80 ° C for 1 hour, and then filtered with filter paper to obtain a residue. With regard to this residue, the above-mentioned operation of washing and filtering with 80% ethanol was repeated 3 times to obtain 1,908 g of ethanol washing residue. Add 684 grams of this residue to 9 liters of 0.  After suspended in 2M calcium acetate, it was treated at 95 ° C for 1 hour, and the supernatant was obtained after standing for 24 hours. To the precipitate from which the supernatant liquid was removed, 9 liters of 0.2 M calcium acetate was added and stirred. After standing for 1 hour, the supernatant liquid was obtained and combined with the above supernatant liquid. The supernatant liquid obtained after the treatment was filtered with filter paper, and then ultra-filtered with an ultrafiltration device having a molecular weight of 10,000 hollow filaments, and concentrated to 350 ml. The concentrated solution was separated by centrifugation. After removing the precipitate, ultrafiltration was added while adding 2mM sodium chloride. After the calcium acetate was completely removed, it was freeze-dried to obtain a freeze-dried product 3.  2 grams. The weight of the fucose-containing sulfated polysaccharide in the freeze-dried product was 3. 1 g. Example 1 9 (1) Preparation of a mixture of fucose-containing sulfated polysaccharides from high fruit kelp Kelp 2 kg of dried high fruit kelp was crushed by a free crusher M-2 (manufactured by Nara Machinery Co., Ltd.) Double the amount in 80% ethanol at 80 ° C for 2 hours. 102 200400195 After treatment 'filter. The residue was subjected to the above-mentioned 80% ethanol extraction and filtration process and repeated three times' to obtain 1,870 g of ethanol instead of washing the residue. Add 36 liters of water to the residue, treat at 100 ° C for 2 hours, and filter to obtain the extract. After the salt concentration of the extract was the same as that of the 40 OmM sodium chloride solution, 5% residual chloropyridyl chloride was added so as not to cause precipitation again, and the precipitate was centrifuged. The precipitate was repeated with 80% ethanol. After washing, completely remove cetylpyridinium chloride and dissolve it in 3 liters of 2M sodium chloride, remove insoluble matter by centrifugation, and suspend in 100 ml of DEAE equilibrated with 2M sodium chloride. -Cellulofine A-800, filtered after stirring, and removed the resin. This filtrate was placed in a 100 ml DEAE-Cellulofine A-800 column equilibrated with 2M sodium chloride, and the dissolved fraction was subjected to desalting and low-molecular removal using an ultrafilter (the molecular weight of the filter membrane was 100,000). At this time, The resulting precipitate was removed by centrifugation. Freeze-drying the supernatant liquid can obtain a refined high-fruit kelp fucose-containing sulfate polysaccharide mixture82.  2 grams. (2) Preparation of fucose-containing sulfated polysaccharide-F 6 g of the above-mentioned fucose-containing sulfated polysaccharide mixture from high fruit kelp was added to 600 ml of 20 mM sodium acetate (pH 6 · 0) containing 0.2 M calcium chloride. After dissolving, place in advance to contain 0. 211 Calcium Chloride ^^ sodium acetate (? 116. 0) Equilibrated 3000 ml DEAE-Sepharose FF column to contain 0. 2M sodium chloride, 20mM sodium acetate (PH6. 0) After washing the column sufficiently, it was dissolved with a sodium chloride gradient of 0 to 2M. Collect fucose-containing sulfated polysaccharide-F dissolved fractions with a concentration of sodium chloride of 0.75M or more, and concentrate and desalinize it with an ultrafilter equipped with an ultrafiltration membrane with a molecular weight of 100,000, and freeze-dry it. Sugar 103 200400195 Freeze-dried sample of sulfated polysaccharide-F 3.  3 grams. (3) Modulation of terminal fucose-containing sulfated polysaccharide-decomposing enzymes Flavobacterium sp. SN-1 00 9 (CCRC 9 1 0070) will be contained in glucose. 25%, protein gland 1. 0%, yeast extract 0. 05% artificial seawater (manufactured by Germalin Laboratory) ρΗ8 · 2, 600 ml of the medium was dispensed and sterilized (120 ° C, 20 minutes) in a 2 liter Erlenmeyer flask, and cultured at 25 ° C for 25 hours to prepare Seed culture fluid. 18 liters of culture medium consisting of 200 g of peptone, 4 g of yeast extract, and 4 ml of artificial seawater pH 8 · 0 (anti-foaming agent (070 manufactured by Shin-Etsu Chemical Industry Co., Ltd.) was placed in a 30-liter capacity fermentation tank Sterilize at 120 ° C for 20 minutes. After cooling, dissolve 20 grams of 2 liters of artificial seawater that was sterilized at 120 ° C for 15 minutes. Fucose sulfate polysaccharide-F and 600 ml of the above-mentioned culture solution were inoculated, and cultured at 24 ° C for 20 hours at a rate of 10 liters of ventilation per minute and a stirring speed of 250 revolutions per minute. After the culture was completed, the The culture solution can be centrifuged to obtain bacterial cells and culture supernatant. The activity of the fucose-containing sulfate polysaccharide decomposing enzyme in the culture supernatant is 5 mU / when the fucose-containing sulfate polysaccharide-F is used as the substrate. m 1 culture broth. The obtained culture supernatant was concentrated by an ultrafiltration filter with a molecular weight of 10,000, and the resulting precipitate was removed by centrifugation, salted out with 85% saturated ammonium sulfate, and the resulting precipitate was centrifuged. Collected, contains 1/1 〇 Concentrated artificial seawater (Germalin 5), 20 mM Tris-hydrochloric acid buffer (ρΗ8 · 2) 104 200400195 Fully dialyzed to obtain 400 ml of crude enzyme. The crude enzyme solution obtained was adsorbed to a solution containing 5 mM sodium azide and 1 in advance. 10mM artificial seawater (Germalin S) in 20mM Tris-HCl buffer (pH 8. 2) Equilibrated DEAE-Cellulofine A-800 (manufactured by Biochemical Industry Co., Ltd.) column, and thoroughly washed the adsorbate with the same buffer solution. 'The same buffer solution contains 100 mM, 200 mM, 300 mM, 400 mM, and 600 mM chlorine. The sodium sulphate solution was dissolved and the active fraction was collected. The activity of the obtained partially purified enzyme was determined as 1,200 mU (10.  2U). Alas, it was confirmed that no other fucose-containing sulfate polysaccharide-decomposing enzyme was mixed. A part of the obtained partially purified enzyme was passed through a 10 mM Tris · hydrochloric acid buffer solution (PH8. 1) containing artificial seawater (Germalin S) and 5 mM sodium azide in advance. 0) Equilibrated Sephacryl S-200 was subjected to gel filtration to calculate a molecular weight of about 100,000. (4) Using the partially purified enzyme and pa-FF obtained in the above examples as the enzyme source and substrate, respectively, to conduct a review of the calcium concentration affecting the activity of the enzyme. The buffer used in the enzyme reaction was a PH7 buffer containing 50 mM acetic acid, imidazole, and Tris-hydrochloric acid. The reaction solution contained sodium chloride at a final concentration of 400 mM. The calcium chloride concentration in the reaction solution was changed from 0 to 100 mM, and the enzyme activity was measured to obtain the results shown in FIG. 23. A. In Fig. 23, the vertical axis represents relative activity (%), and the horizontal axis represents calcium concentration (mM) in the reaction solution. As a result, it can be judged that the activity of this enzyme is significantly improved in the presence of calcium salt. 105 200400195 (5) The terminal fucose-containing sulfated polysaccharide-degrading enzyme of the present invention was measured for the remaining viability after dialysis of the following 6 kinds of buffer solutions and holding at 5 ° C for 20 hours. 1 · 20mM Tris-HCl buffer (pH 8. twenty two. 20mM Tris-hydrochloric acid buffer solution containing 5mM sodium azide (ρΗ8.2) 3. 20mM Tris-hydrochloric acid buffer solution containing 5mM sodium azide and 50mM sodium chloride (pH 8. 2) 4. 20 mM Tris-hydrochloric acid buffer solution containing 5 mM sodium azide and 500 mM sodium chloride (ρΗ 8. 2) # 5. 20 mM Ding containing 5 mM sodium azide, 50 mM sodium chloride and 10 mM calcium chloride Hydrochloric acid buffer solution ^) 6. 20mM Tris-hydrochloric acid buffer (pH 8. containing 5mM sodium azide and artificial seawater (Germalin S)) 2) The results above, the fucose-containing sulfate polysaccharide decomposing enzyme of the present invention dialyzed with 1, 2, and 3 buffers is inactive, and the fucose-containing sulfate polysaccharide of the present invention is dialyzed with 4, 5, and 6 buffers Decomposing enzymes remain active. | It can be judged that this enzyme is stabilized in the presence of 500mM sodium chloride or 1 OmM calcium ion. (6) Weigh 5 g of fucose-containing sulfated polysaccharide-F prepared in the above example, and mix 471 ml of 50 mM imidazole buffer solution (pH 8), 12.5 ml of 4M sodium chloride, and 6.25 ml of 4M calcium chloride and 10 ml (equivalent to 6mU) of a part of the refined product containing fucose sulfate polysaccharide decomposing enzyme of the present invention obtained in Example 19-(3), and reacted at 25 ° C for 120 minutes to obtain Fucose sulfate 106 200400195 Low molecular weight of sugar-F. The analysis results of IR and NMR of the obtained low-molecular compound are shown in Figs. 25 and 26, respectively. The results obtained when filtering through a Ce 1 1 ul of in GCL-300 gel are shown in FIG. 24. That is, this substance is distributed with a molecular weight of 1,000 to 30,000. The sulfuric acid content of this material was 46% of S04 (molecular weight 46). The neutral sugar composition is fucose: galactose = 100: 4. Alas, the vertical and horizontal axes in Figs. 24 to 26 are synonymous with Figs. 2 to 4 respectively. _ Example 20 PRMI 1 6 40 medium (manufactured by GIBC0) treated with bovine fetal blood pupa (JRH BIOSCIENCE) at 10% at 56 ° C for 30 minutes in 37 ° (: myeloid leukemia cells HL before subculture) -60 (ATCC CRL-1 964) was suspended in ASF 104 medium (manufactured by Ajinomoto Co., Ltd.) at 5 × 105 cells / 9 ml. This suspension was prepared in 4 parts of 9 ml, and 1 ml of the suspension was added to the example. Filtering treatment solution of fucose sulfate-containing polysaccharide physiological foods | saline solution (5 mg / ml) obtained from 1, 1, 2, and 15 [with a pore size of 0.  2 0 // m cellulose acetate membrane (manufactured by KONIC) filter (the following filtration treatment is performed under these conditions)], and cultured at 37 ° C in the presence of 5% carbon dioxide for 40 hours. The cultured cells were separated from the supernatant by centrifugation. The resulting cells contained 10 mM ethylenediamine tetraacetate and 0.1 mM.  5% Laurel tincture was suspended in 50 mM Ti * is s-hydrochloric acid buffer solution (pH7 · 8) 20 // 1, and 1 # 1 of 10 mg / ml ribonuclease A (manufactured by SIGMA Corporation) was added. ) After treatment at 50 ° C, 107 200400195 for 30 minutes, add 1 mg / ml of 10 mg / ml proteinase K and treat at 50 ° C for 1 hour. The treated cells were used as samples, and electrophoresis was performed using a 2% agarose gel at a constant voltage of 100V. After the gel was immersed in ethidium bromide solution for 30 minutes, and the DNA status in the gel was confirmed using a super-illumination device, the DNA ladder characteristic of cell self-destruction was confirmed. Furthermore, for the purpose of confirmation, when the same operation was performed using a solution of radiobactin D 10g / ml which is known as a test agent for inducing cell self-killing, instead of the fucose-containing sulfated polysaccharide described above, it was confirmed that the same as DNA ladder in the same situation with fucose sulfate polysaccharide. _ As a result, it can be known that HL-60 cells induced fucose-containing sulfated polysaccharides obtained in Examples 1, 12, 2, and 15 to induce cell self-destruction. HL-60 (ATCC CCL-2 40), each of the fucose-containing sulfated polysaccharide solutions obtained in Examples 1, 12, and 15 was dissolved at 5 mg / ml in 30 mMHEPES buffer solution containing 120 mM sodium chloride (ρΗ7 · 2 ), And the cell self-killing induction effect at 12TC and autoclaving for 20 minutes] was measured as above, and the same results were obtained. Example 2 ^ Bone marrow leukemia cells HL- were cultured at 37 ° C in PRMI 1 640 medium (manufactured by GIBC0) treated with bovine fetal blood pupa (jRH BIOSCIENCE) for 30 minutes at 10% at 56 ° C. 60 (ATCC CCL-240) was suspended in ASF 104 medium (manufactured by Ajinomoto Co.) at 5 x 105 cells / 9 ml. 9 ml of this suspension was added with 1 ml of a fucose-containing sulfated polysaccharide physiological saline solution (5 mg / ml) obtained in Example 15 and filtered and treated. 2004 2004 195 liquid was cultured at 37 ° C in the presence of 5% carbon dioxide 20 hours. The cultured cells were separated from the supernatant by centrifugation. The obtained cells were subjected to Gisham staining according to the "Handbook of Cell Self-Extermination Experiment" (Shuyunsha, Shinuma Yasu_Supervision, pp. 93 ~ 95, 1995). That is, the obtained cells were treated with Kanoya fixative solution (acetic acid : Methanol = 1: 3) fixed on the section glass, stained with Gissam pigment (Merck), and observed the fragmentation of the cell's unique nucleus with an optical microscope. Based on the results, it can be determined that HL-60 cells were treated by The fucose-containing sulfated polysaccharide obtained in Example 15 caused cell self-destruction. The fucose-containing sulfated polysaccharide solution obtained in Example 15 was dissolved at 30 mg MHEPES buffer (pH 7. 2), and the cell self-killing inducing effect in the 12th factory C, autoclaved sterilization treatment for 20 minutes] was determined as above, and the same results were obtained. Example 2 2 Myeloid leukemia cells HL-60 were cultured at 37 ° C in PRMI 1 640 medium (manufactured by GIBCO) treated with bovine fetal blood pupa (JRH BIOSCIENCE) for 30 minutes at 10% at 56 ° C. (ATCC CCL-240) was suspended in ASF 104 medium (manufactured by Ajinomoto Co., Ltd.) at 5 X 105 cells / 4.5 ml. Prepare 4 suspensions of this suspension.  5 ml and separately add 0 to this suspension.  5 ml containing the fucose-containing sulfated polysaccharide solution obtained in Examples 1, 15, and 18 [dissolved at 10 mg / ml in 30 mM HEPES buffer (ρΗ7.2) containing 120 mM sodium chloride, and 121 ° C, autoclaved for 20 minutes] and 120mM sodium chloride in 30mM HEPES buffer, and cultured at 37 ° C in the presence of 200400195 5% carbon dioxide, 24 hours and 40 hours after the start of culture The cell number was measured by the pan-blue staining method. The results are shown in Fig. 27. Fig. 27 is a graph showing the culture time and the number of viable cells in the culture medium when the fucose-containing sulfated polysaccharide obtained in Examples 1, 15, and 18 was added at 1 mg / ml in HL-cell culture medium. For the relationship diagram, the horizontal axis represents the culture time (time), and the vertical axis represents the number of viable cells in the culture solution (X105 cells / 5 ml). In Figure 27, the type of fucose-containing sulfated polysaccharide added to the culture medium is as follows: the mouth mark indicates no addition (control), the ten mark indicates Example 1, and the mark indicates Example 1 5. The X mark indicates Example 1 8 The obtained fucose-containing sulfated polysaccharide. In addition, the dead cells at this time exhibited a peculiar pattern of cell self-destruction such as cell shrinkage and fragmentation. That is, from these results, it can be determined that HL-60 cells induced cell self-destruction and significantly inhibited cell proliferation through the fucose-containing sulfated polysaccharides obtained in Examples 1, 15, and 18. Each of the fucose-containing sulfated polysaccharide solutions obtained in Examples 1, 15, and 18 was dissolved at 10 mg / ml in a 30 mM HEPES buffer solution (PH7.  2), and the cell self-killing-inducing effect of the filter processor] was determined as described above, and the same result was obtained. Example 2 3 Acute lymphoblastic leukemia cell MOLT-3 was treated with 9 ml of PRMI 1 640 medium (manufactured by GIBCO) treated with bovine fetal blood pupa (JRH BIOSCIENCE) for 30 minutes at 10% at 56 ° C. (ATCC CRL- 1 5 5 2) Suspended at 5 X 105 cells / 9 ml. 5 parts of 9 ml of this suspension, and 110 200400195 were respectively added with 1 ml of a filtering treatment solution containing fucose sulfate polysaccharide physiological saline solution (5 mg / ml) obtained in Examples 1, 2, 12, 2, and 16, and Incubate at 37 ° C in the presence of 5% carbon dioxide for 60 hours. The cultured cells were separated from the supernatant by centrifugation. The resulting cells were suspended in a solution containing 10 mM ethylenediamine tetraacetate and 0.1%.  5% sodium lauryl sarcosinate in 50 mM Tris-HCl buffer (pH 7.  8) 2 0 // 1 and add 1 μ 1 of 10 mg / ml RNA 18.  A (manufactured by SIGMA). After treating at 50 ° C for 30 minutes, proteinase K was added at a concentration of 10 mg / ml to 1 and treated at 50 ° C for 1 hour. The treated cells were used as samples, and electrophoresis was performed using a 2% agarose gel at a constant voltage of 100V. After the gel was immersed in ethidium bromide solution for 30 minutes, and the DNA state in the gel was confirmed using a super-illumination device, the DNA ladder characteristic of cell self-destruction was confirmed. Furthermore, for the purpose of confirmation, when the same operation was performed by using a 10 mg / ml solution of radiobactin D, which is known as a drug for inducing cell self-extinguishing, instead of the above-mentioned fucose-containing sulfated polysaccharide, the same operation was confirmed after 20 hours of culture. DNA ladder in the same situation with fucose sulfate polysaccharide. From this result, it was determined that the fucose-containing sulfated polysaccharides obtained in Examples 1, 2, 12, 2, and 16 induced MOLT-3 cells to self-destruct. The fucose-containing sulfated polysaccharide solution obtained in Examples 1, 2, 12, and 16 (dissolved in PBS at 5 mg / ml (8 g of sodium chloride,.  2 g of potassium chloride, 2.9 g of disodium hydrogen phosphate 12 hydrate, and 0.2 g of potassium dihydrogen phosphate were dissolved in 1 liter of water) at 121 ° C. The auto-killing-inducing effect of the autoclaved sterilization treatment for 20 minutes] was measured as described above, and the same results were obtained. Example 24 111 200400195 Human acute lymphoblasticity cultured at 37 ° C in PRMI 1 640 medium (manufactured by GIBC0) treated with bovine fetal blood pupa (JRH BIOSCIENCE) for 30 minutes at 10% at 56 ° C Leukemia cells MOLT-3 (ATCC CRL- 1 5 5 2) were suspended in RPMI 1 640 medium at 5 × 103 cells / ml, and dispensed into each well on a 24-well plate made by FALCON company. 1. 8 milliliters. For each suspension, add 0.2 ml of each.  5 mg / ml dissolved in PBS, filtered and sterilized, the fucose-containing sulfated polysaccharide mixture obtained in Example 1, the fucose-containing sulfated polysaccharide F obtained in Example 12, and the fucose-containing algae obtained in Examples 15 and 17. A solution of sugar sulfate polysaccharide and glucose sulfate (molecular weight 500,000, manufactured by Wako Pure Chemical Industries, Ltd.), and cultured in the presence of 3,71,5% carbon dioxide. Alas, only those who added the same amount of PBS as a control group were cultured in the same manner. The number of viable cells 2 days, 4 days, 6 days, and 8 days after the start of culture was measured by Trypan Blue staining. The results are shown in FIG. 28. That is, FIG. 28 shows the fucose-containing sulfated polysaccharide obtained in Example 1, the fucose-containing sulfated polysaccharide obtained in Example 12, and the rock-containing materials obtained in Examples 15 and 17 in MOLT-3 cell culture solution The relationship between the culture time and the number of progenitor cells in the culture medium when the fucose sulfate polysaccharide and dextran sulfate are added at 0.5 mg / ml. The horizontal axis represents the culture time (days), and the vertical axis represents the culture time. Number of viable cells (X 104 cells / 2 ml). In Figure 28, the types of sulfated polysaccharides added to the culture medium are as follows: ○ indicates no addition (control), · indicates the fucose-containing sulfated polysaccharide-F obtained in Example 12, and the mouth indicates the examples. The fucose-containing sulfated polysaccharide obtained in 1 is marked with △ as in Example 17 and the fucose-containing sulfated polysaccharide obtained in Example 7 is marked with 112. 200400195 Example 1 The fucose-containing sulfated polysaccharide and dextran sulfuric acid obtained in Example 1 are shown. A curve substantially the same as that of the fucose-containing sulfated polysaccharide obtained in Example 15 was obtained. In addition, the dead cells at this time exhibited a peculiar pattern of cell self-destruction such as cell shrinkage and fragmentation. That is, from these results, it can be determined that MOLT-3 cells were obtained through the fucose-containing sulfated polysaccharide obtained in Example 1, the fucose-containing sulfated polysaccharide F obtained in Example 12, and those obtained in Examples 15 and 17. Fucose-containing sulfated polysaccharides and dextran sulfates induced cell self-destruction and cell proliferation was significantly inhibited. · Fucose-containing sulfated polysaccharide obtained in Example 1, fucose-containing polysaccharide-F obtained in Example 12; fucose-containing sulfated polysaccharide obtained in Examples 15 and 17; and dextran sulfate (molecular weight) 500,000, manufactured by Wako Pure Chemical Industries, Ltd.)  After 5 mg / ml was dissolved in PBS, the cell self-killing induction effect at 1 2 1 ° C, autoclaved for 20 minutes was measured as above, and the same result was obtained. Example 2 5 Myeloid leukemia cells HL-60 were cultured at 37 ° C in PRMI 1 640 medium (manufactured by GIBC0) treated with bovine fetal blood pupa (JRH BIOSCIENCE) for 30 minutes at 10% at 56 ° C. (ATCC CCL-240) was suspended in ASF 104 medium (manufactured by Ajinomoto Co., Ltd.) at 5 x 104 cells / 9 ml. This suspension was prepared in 4 portions of 9 ml. To each suspension, 100 # 1 physiological saline solution and the fucose-containing sulfated polysaccharide sample obtained in Example 1, F-Fd-1 to F-Fd-4 were added. And three kinds of fucose-containing 113 200400195 filtered aqueous solutions of physiological saline solution (10 mg / ml) of oligosaccharide sulfate, and cultured at 37 ° C and 5% carbon dioxide for 40 minutes . The cultured cells were observed under a microscope to investigate the degree of proliferation and cell type. As a result, all the HL-60 cells containing fucose-containing sulfated polysaccharide samples, F-Fd-1 to F-Fd-4, and three kinds of fucose-containing oligosaccharides exhibited cell shrinkage and cell fragmentation. Self-extinguishing characteristics. In addition, the number of cells in HL-6 0 cells with physiological saline solution increased by about 4 times, but fucose-containing sulfated polysaccharide samples, F-Fd-1 to F-Fd-4, and three kinds of fucose-containing sulfuric acid were added. The number of oligosaccharide HL-60 cells did not increase to less than 1/10, and it can be judged that these fucose-containing sulfated polysaccharide samples, F-Fd-1 ~ F-Fd-4 and 3 kinds of rock containing Cell self-killing effect of fucose sulfate oligosaccharide inhibits the proliferation of HL-60 cells. Furthermore, in order to confirm, when the same operation was performed using a 10 μg / ml solution of radiobactin D, which is known as a drug for inducing cell self-destruction, when the same operation was performed instead of the fucose-containing oligosaccharide sulfate, as shown in FIG. In the case of fucose-containing oligosaccharide sulfate, cell shrinkage and cell fragmentation were the same. From this result, it can be known that HL-60 cells were obtained from the fucose-containing sulfated polysaccharide sample obtained in Example 1, F-Fd-1 to F-Fd-4, and the fucose-containing sulfated oligosaccharide obtained in Example 13. Induces cell self-destruction. Samples of fucose-containing sulfated polysaccharides obtained in Example 1, F-Fd-1 to F-Fd-4, and solutions of the three types of fucose-containing sulfated oligosaccharides [at 10 mg / ml] Cells self-inactivated when dissolved in 30 mM HEPES buffer (pH 7) containing 120 mM sodium chloride at 121 ° C and autoclaved for 20 minutes] 114 200400195 The effect was measured as described above and the same results were obtained. Example 26 Lung cancer cells A-549 (ATCC CCL-1 85) and SV40 transformed lung cells WI-38VA13 (ATCC CCL-75. 1) and liver cancer cells Hep G2 (ATCC HB-8065) at a rate of 104 cells / ml each suspended in PRMI 1 6 40 medium containing 10% 56 ° C bovine fetal blood cells (JRH BIOSCIENCE) for 30 minutes in. Dispense this suspension 1.  8 ml, and to each suspension, each cancer cell was added 200 physiological saline solution of 1 and fucose-containing sulfated polysaccharides obtained in Examples 1 and 15 and 6 kinds of fucose-containing algae obtained in Example 14 Filtration treatment solution of each physiological saline solution (1 mg / ml) of oligosaccharide sulphate and cultured at 37 ° C in the presence of 5% carbon dioxide for 6 days. The cultured cells were observed under a microscope to investigate the degree of proliferation and cell type. As a result, the fucose-containing sulfated polysaccharides obtained in Examples 1 and 15 and the six types of fucose-containing sulfated oligosaccharides obtained in Example 14 were added with 3 lysed liver cancer cells A-5 4 9. SV 40 transformed lung cells WI-3 8 VA13 and liver cancer cells Hep G2 all exhibited cell self-destructive features such as cell shrinkage and cell fragmentation. In addition, the number of cells of each cancer cell in which physiological saline was added significantly increased, but the fucose-containing sulfated polysaccharides obtained in Examples 1 and 15 and the six kinds of fucose-containing sulfated oligosaccharides obtained in Example 14 were added. The number of cancer cells with molecular weights ranging from 2000 to 3 is reduced, and it can be determined that the proliferation of various cancer cells can be inhibited by the self-inactivation of cells containing fucose sulfated polysaccharides and oligosaccharides. Fucose-containing sulfated polysaccharides obtained in Examples 1 and 15 and 6 types of fucose-containing sulfated oligosaccharides in PBS solutions (1 mg / ml) at 121 ° C for 20 minutes The auto-killing-inducing effect of the autoclaved sterilized product was measured as described above, and the same results were obtained. Example 27: Colon cancer cells HCT 116 (ATCC CCL-247) and gastric cancer cells AGS (ATCC CRL-1 7 3 9) were each divided into 104 cells per 1. 8 ml was suspended in McCoy ’s 5a medium (manufactured by GIBCO) and HanTs F12 medium (manufactured by GIBCO) containing bovine fetal blood pupa (JRH BI0SCIENCE) treated at 10% 56 ° C for 30 minutes. Dispense this suspension 1.  8 ml, and to each suspension, each cancer cell was added 200 // 1 of physiological saline, and the fucose-containing sulfated polysaccharides and F-Fd-1 to F obtained in Examples 1, 12, 2, and 15. -Fd-4 is a filtered treatment solution of 4 kinds of physiological saline solution (10 mg / ml) containing fucose sulfate oligosaccharide, and cultured at 37 ° C in the presence of 5% carbon dioxide for 48 hours. The cultured cells were observed under a microscope to investigate the degree of proliferation and cell type. As a result, the fucose-containing sulfated polysaccharides obtained in Examples 1, 12, and 15 and four kinds of fucose-containing oligosaccharides F-Fd-1 to F-Fd-4 colon cancer cells HCT 116 and gastric cancer were added. Cell AGS all exhibited cell self-destructive features such as cell shrinkage and cell fragmentation. In addition, the number of various cancer cells added with physiological saline significantly increased the number of cells, but four types of fucose-containing sulfated polysaccharides and F-Fd-1 to F-Fd-4 obtained in Examples 1, 12, and 15 were added. The number of various cancer cells containing fucose sulphate oligosaccharide is reduced. It can be determined that the proliferation of various cancer cells can be controlled by the self-killing induction effect of these fucose sulphate polysaccharides and oligosaccharides. Each of the fucose-containing sulfated polysaccharides and F-Fd-1 116 200400195 to F-Fd-4 obtained in Examples 1, 12, 2, and 15 each contained a fucose-containing sulfate oligosaccharide PBS solution (1. Mg / ml) of 1 2 1 ° C, 20 minutes autoclaving induced cell autoinactivation induced by the same effect as above, the same results were obtained. Example 2 8 Human colon cancer cells HCT cultured at 37 ° C in McCoy's 5a medium (manufactured by GIBCO) containing bovine fetal blood pupa (jrh BIOSCIENCE) treated at 10% 56 ° C for 30 minutes. 1 1 6 , Suspended in McCoy, s 5a medium at 5 × 103 cells / ml, and dispensed 1.8 ml in each well on a 24-well plate made by FALCON. For each suspension, add .0.  2 ml of a fucose-containing sulfated polysaccharide sample obtained in Example i, 10 mg / ml dissolved in PBS, a mixture of fucose-containing sulfated polysaccharides, fucose-containing sulfated polysaccharide-F obtained in Example 12, and fucose-containing Sugar sulfate polysaccharide_u, F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4, each solution of fucose-containing sulfate polysaccharide obtained in Example 15, and dissolved in PBS 5 mg / ml heparin (manufactured by Wako Pure Chemical Industries, Ltd.) and dextran sulfate (molecular weight 500,000, manufactured by Wako Pure Chemical Industries, Ltd.) were autoclaved at 121 ° C for 20 minutes, and 37 ° C, 5% Culture in the presence of carbon dioxide. Alas, only those who added the same amount of PBS as a control group were cultured in the same manner. The number of viable cells on the 1st, 2nd, 3rd, and 4th days after the start of the culture is based on the method described in "Technology of Tissue Culture" (2nd Edition) (Asakura Publishing, Japanese Society for Tissue Culture, 1900). (Pages 2 6 to 28). In other words, it was measured by trypan blue staining on a blood cell calculation board. The results obtained are shown in Figure 29. That is, Fig. 29 shows the fucose-containing sulfated polysaccharide sample obtained in Example 1 in η CT 1 16 cell culture solution, and the mixture of fucose-containing sulfated polysaccharide obtained in Example 1 and Example 1 2 The fucose-containing sulfated polysaccharide-U and fucose-containing sulfated polysaccharide_F, F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4 obtained in Example 15 Fucose-containing sulfated polysaccharide at 1 mg / ml, and heparin and dextran sulfate at 0.  The relationship between the culture time when 5 mg / ml was added and the number of living cells in the culture medium. The horizontal axis represents the culture time (days), and the vertical axis represents the number of living cells in the culture solution (XI 04 cells / 2 ml). In the figure 29, the types of fucose-containing sulfated polysaccharides or oligosaccharides added to the culture medium are: ◦ marked as no addition (control), • labeled as fucose-containing sulfated polysaccharide samples obtained in Example 1, and labeled as implemented The fucose-containing sulfated polysaccharide mixture obtained in Example 1. Example 12 The fucose-containing sulfated polysaccharide-U and fucose-containing sulfated polysaccharide-F'F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4, and The fucose-containing sulfated polysaccharide obtained in Example 15 is a curve substantially the same as that in the case of the fucose-containing sulfated polysaccharide mixture obtained in Example 1. As a result, the number of HCT 116 cells with PBS was significantly increased, but the fucose-containing sulfated polysaccharide sample obtained in Example 1, the fucose-containing sulfated polysaccharide mixture, and the fucose-containing mixture obtained in Example 12 were added. Sulfate polysaccharide-F, fucose-containing sulfate polysaccharide-U, F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4, the fucose-containing sulfate polysaccharide obtained in Example 15 The number of HCT 1 16 cells of heparin and dextran sulfate did not increase or decrease. In addition, the fucose-containing sulfated polysaccharide sample obtained in Example 1, the fucose-containing sulfated polysaccharide mixture, the fucose-containing sulfated polysaccharide-F obtained in Example 12, and the fucose-containing sulfated polysaccharide-U, F- were added. Fd-1, F-Fd-2, F-Fd-3, and 200400195 F-Fd-4 'HCT 1 1 6 cells containing fucose sulfated polysaccharide, heparin, and glucose sulfate obtained in Example 15 Cell self-destructive features such as cell shrinkage and cell fragmentation. That is, it can be judged that the proliferation of HCT 116 cells can be inhibited by the self-inactivation of cells containing these fucose sulfated polysaccharides and oligosaccharides, heparin, and dextran sulfate. The fucose-containing sulfated polysaccharide sample obtained in Example 1 was dissolved in PBS at 10 mg / ml, the fucose-containing sulfated polysaccharide mixture, the fucose-containing sulfated polysaccharide-F obtained in Example 12, and the fucose-containing Sulfated polysaccharide-U, F-Fd-1, ® F-Fd-2, F-Fd-3, and F-Fd-4, the filtered treatment solution of each solution containing fucose sulfated polysaccharide obtained in Example 15, The self-killing-inducing effect of each filtering treatment solution in which 5 mg / ml of heparin and dextran sulfuric acid were dissolved in PBS was measured as described above, and the same results were obtained. Example 29 Human colon cancer cells HCT 116 cultured at 37 ° C in McCoy's 5a medium (manufactured by GIBC0) containing bovine fetal blood pupa (JRH BIOSCIENCE) treated at 10% 56 ° C for 30 minutes. McCoy's 5a medium was suspended at 5 × 103 cells / ml, and dispensed into each well on a 24-well plate made by FALCON company.  8 ml. To each suspension, 0.2 ml of a fucose-containing sulfated polysaccharide sample solution obtained in Example 1 dissolved in PBS at 20 mg / ml, 30 mg / ml, and 50 mg / ml was added at 121 ° C for 20 minutes. Heat sterilize the treated material and incubate at 37 ° C in the presence of 5% carbon dioxide. Alas, only those with the same amount of PBS as the control group were cultured in the same way. The number of cells over time after the start of culture was measured according to the method described in "Technology of Tissue Culture" (Second Edition) (Asakura Publishing, Japanese Tissue Culture Society, 1900) (pages 26-28). That is, it was measured by trypan blue staining on a blood cell calculation board. The results are shown in Figure 30. That is, Fig. 30 shows the relationship between the culture time when the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added at various concentrations in the HCT 116 cell culture medium, and the number of viable cells in the culture medium. Indicates the culture time (time), and the vertical axis indicates the number of viable cells in the culture solution (X 104 cells / ml). In Figure 30, the amount of the fucose-containing sulfated polysaccharide sample in the culture medium is: 0 is marked without addition (control), 2 is marked as 2 mg / ml, 3 mg / ml is marked, and 5 is marked as 5 in the black triangle. Mg / ml. As a result, the number of cells of HCT 116 cells added with PBS significantly increased, but the number of cells of HCT 116 cells added with the fucose-containing sulfated polysaccharide sample obtained in Example 1 decreased. In addition, all the HCT 116 cells containing the fucose-containing sulfated polysaccharide sample obtained in Example 1 exhibited cell self-destructive characteristics such as cell shrinkage and cell fragmentation. That is, it can be judged that the fucose-containing sulfated polysaccharide sample obtained in Example 1 has a cell self-inducing effect on HCT 116 cells at a concentration of at least 2 mg / ml, and can inhibit cell proliferation. The cell self-killing induction of the fucose-containing sulfated polysaccharide sample solution obtained by dissolving 20 mg / ml, 30 mg / ml, and 50 mg / ml in PBS in PBS was measured as described above, and the same was obtained. 120 200400195. Example 30 0 Human gastric cancer cell line AGS cultured at 37 ° C in Ham's F12 medium (manufactured by GIBCO) containing 10% 56t of bovine fetal blood pupa (JRH BI0SCIENCE) for 30 minutes, and Ham's F1 2 The medium was suspended at 5 × 103 cells / ml, and dispensed into each well on a 24-well plate made by FALCON company.  8 ml. For each suspension, add 0.  2 ml of fucose-containing sulfated polysaccharide sample solution obtained in Example 1 of 20 mg / ml, 30 mg / ml, and 50 mg / ml dissolved in PBS was autoclaved at 1 2 ° C for 20 minutes, And cultured at 37 ° C in the presence of 5% carbon dioxide. Alas, only those who added the same amount of PBS as a control group were cultured in the same manner. The number of cells over time after the start of culture was measured according to the method described in "Technology of Tissue Culture" (Second Edition) (published by Asakura, edited by the Japan Society for Tissue Culture, 1 990) (pp. 26-28). That is, it was measured by trypan blue staining on a blood cell calculation board. The results are shown in Figure 31. That is, Fig. 31 shows the relationship between the culture time when the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added to the AGS cell culture solution at various concentrations and the number of proliferating cells in the culture solution, and the horizontal axis represents the culture time ( (Time), and the vertical axis represents the number of proliferating cells in the culture medium (x 104 cells per 2 ml). In Figure 31, the amount of the fucose-containing sulfated polysaccharide sample in the culture medium is: 0 is marked as no addition (control), 2 is marked as 2 mg / ml, 3 mg / ml is marked, and the black triangle is 5 Mg / ml. 121 200400195 As a result, the number of cells of AGS cells added with PBS significantly increased, but the number of cells of AGS cells added with the fucose-containing sulfated polysaccharide sample obtained in Example 1 at a final concentration of 3 mg / ml or less reduced the number of cells, and added 2 Mg / ml also significantly inhibited cell proliferation. In addition, all the AGS cells containing the fucose-containing sulfated polysaccharide sample obtained in Example 1 exhibited cell self-destructive characteristics such as cell shrinkage and cell fragmentation. That is, it can be judged that the fucose-containing sulfated polysaccharide sample obtained in Example 1 has a cell autoinduction-inducing effect on AGS cells at a concentration of at least 2 mg / ml, and can inhibit cell proliferation. The cell self-killing induction of the fucose-containing sulfated polysaccharide sample solution obtained by dissolving 20 mg / ml, 30 mg / ml, and 50 mg / ml in PBS in PBS was measured as described above, and the same was obtained. The result. Example 3 1 Human colon cancer cells SW 480 (ATCC) cultured at 37 ° C in L-15 medium (manufactured by GIBCO) containing bovine fetal blood pupa (JRH BIOSCIENCE) treated at 10% 56 ° C for 30 minutes. CCL-228), suspended in L-15 medium at 5 X 103 cells / ml, and dispensed into each well on a 24-well plate made by FALCON company. 1.  8 ml. For each suspension, add 0.  2 ml of the fucose-containing sulfated polysaccharide sample solution obtained in Example 1 dissolved in PBS at 10 mg / ml, 30 mg / ml, and 50 mg / ml was autoclaved at 121 ° C for 20 minutes, and then Incubate at 37 ° C in the presence of 5% carbon dioxide. Alas, only those with the same amount of PBS as the control group were cultured in the same way as 122 200400195. The number of cells over time after the start of the culture was measured according to the method described in "Technology of Tissue Culture" (2nd Edition) (Asakura Publishing Co., Ltd., 1990), pages 26-28. That is, it was measured by trypan blue staining on a blood cell calculation board. The results are shown in Figure 32. That is, Fig. 32 shows the relationship between the culture time when the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added at various concentrations in SW 480 cell culture medium and the number of progenitor cells in the culture medium, and the horizontal axis represents the culture time. (Time), the vertical axis represents the number of proliferating cells in the culture medium (X 104 cells / ml). In Figure 32, the amount of fucose-containing sulfated polysaccharide sample in the culture medium is: 0 is marked without addition (control), Lu is marked as 1 mg / ml, 3 mg / ml is marked, and black triangle is 5 Mg / ml. As a result, the number of SW 480 cells added with PBS significantly increased. However, the number of SW 480 cells added with a fucose-containing sulfated polysaccharide sample obtained in Example 1 at a final concentration of 3 mg / ml or more reduced the number of cells, and 1 Mg / ml also significantly inhibited cell proliferation. In addition, all SW 480 cells added with the fucose-containing sulfated polysaccharide sample obtained in Example 1 exhibited cell self-destructive characteristics such as cell shrinkage and cell fragmentation. That is, it can be determined that the fucose-containing sulfated polysaccharide sample obtained in Example 1 has a cell self-killing induction effect on SW 480 cells at a concentration of at least 1 mg / ml, and can inhibit cell proliferation. The fucose-containing sulfated polysaccharide sample solution obtained in Example 1 of 10 mg / ml, 30 mg / ml, and 50 mg / ml was dissolved in PBS. 123 200400195 The induction effect of cell self-extinguishing of the filtering treatment solution was as described above. Quasi-measurement and achieved the same results. Example 3 2 Cultured at 37 ° C in DEME medium (manufactured by Dainippon Pharmaceutical Co., Ltd.) containing NEV (manufactured by Dainippon Pharmaceutical Co., Ltd.) and NEAA (manufactured by Dainippon Pharmaceutical Co., Ltd.) which were treated with 10% 56 ° C for 30 minutes. Human colon cancer cell WiDr (ATCC CCL-218) was suspended at 5 X 103 cells / ml in the above culture medium, and dispensed in each well on a 24-well plate made by FALCON company. 1. 8 ml. For each suspension, add 0.  2 ml of 10 mg / ml sinus dissolved in PBS. The fucose-containing sulfated polysaccharide mixture obtained in Example 1 and the fucose-containing sulfated polysaccharide-F, F-Fd-3 and F-Fd- 4. Each solution of fucose-containing sulfated polysaccharide obtained in Example 15 was autoclaved at 12 1 ° C for 20 minutes, and cultured at 37 ° C in the presence of 5% carbon dioxide. Alas, only those who added the same amount of PBS as a control group were cultured in the same manner. The number of cells over time after the start of the culture was measured according to the method described in "Tissue Culture Techniques" (2nd Edition) (Asakura Publishing, Japan Tissue Culture Society, 1990) (pages 26 to 28). That is, it was measured by trypan blue staining on a blood cell calculation board. The results obtained are shown in Figure 33. That is, FIG. 33 shows the fucose-containing sulfated polysaccharide mixture obtained in Example 1 and the fucose-containing sulfated polysaccharide-F, F-Fd-3, and F-Fd-4 and the fucose-containing sulfated polysaccharide obtained in Example 15 at a concentration of 1 mg / ml and the relationship between the culture time and the number of progenitor cells in the culture medium, the horizontal axis represents 124 200400195 culture time (time) The vertical axis indicates the number of proliferating cells in the culture medium (χ 1 ο 4 cells / 2 ml). In FIG. 33, the type of fucose-containing sulfated polysaccharide in the culture medium is: 0 is marked with no addition (control), the fucose-containing sulfated polysaccharide F obtained in Example 12 is marked, and ♦ is marked as implemented Example 15 The fucose-containing sulfated polysaccharide obtained. F-Fd-3 and F-Fd-4 are substantially the same curves as those of the fucose-containing sulfated polysaccharide obtained in Example 15. As a result, the number of HCT 116 cells with PBS significantly increased, but the fucose-containing sulfated polysaccharide mixture obtained in Example 1 and the fucose-containing sulfated polysaccharide-F and F-Fd- 3 and F-Fd-4, and the fucose-containing sulfated polysaccharide-containing W i D r cells obtained in Example 1 were reduced in cell number. Furthermore, the fucose-containing sulfated polysaccharide mixture obtained in Example 1 was added to implement The fucose-containing sulfated polysaccharide-F, F-Fd-3, and F-Fd-4 obtained in Example 12 and the fucose-containing sulfated polysaccharide-containing Wi Dr cells obtained in Example 15 all showed cell shrinkage and cell fragmentation. Cell self-extinguishing characteristics. That is, the fucose-containing sulfated polysaccharide mixture obtained in Example 1, the fucose-containing sulfated polysaccharide-F, F-Fd-3, and F-Fd-4 obtained in Example 12, and Fucose sulfated polysaccharides have the effect of inducing cell self-destruction on WiDr cells and can inhibit cell proliferation. The fucose-containing sulfated polysaccharide mixture obtained in Example 1 and the fucose-containing sulfated polysaccharide obtained in Example 12 were dissolved in 10 mg / ml in PBS, and F-Fd-3, F-Fd-4, And the cell self-killing inducing effect of the fucose-containing sulfated polysaccharide solution-containing filtering solution obtained in Example 15 was determined as described above, and 125 200400195 obtained the same result. Example 3 3 In a DMEM medium (manufactured by Dainippon Pharmaceutical Co., Ltd.) containing NEAA (manufactured by Dainippon Pharmaceutical Co., Ltd.) containing 10% 5 61: of bovine fetal blood pupa (JRH BIOSCIENCE) treated for 30 minutes. Cultured human colon cancer cell WiDi: (ATCC CCL-218), suspended at 5 X 103 cells / ml in the above medium, and dispensed in each well on a 24-well plate made by FALCON company.  8 ml. For each suspension, add 0.  2 ml of a fucose-containing sulfated polysaccharide sample solution obtained in Example 1 dissolved at 10 mg / ml, 30 mg / ml, and 50 mg / ml in PBS was autoclaved at 1 2 ° C for 20 minutes. And cultured at 37 ° C in the presence of 5% carbon dioxide. Alas, only those who added the same amount of PBS as a control group were cultured in the same manner. The number of cells over time after the start of the culture was measured according to the method described in "Tissue Culture Techniques" (2nd Edition) (Asakura Publishing Co., Ltd., 1990), pages 26 to 28. g. Measured by trypan blue staining on a blood cell calculation board. The results are shown in Figure 34. That is, Fig. 34 shows the relationship between the culture time when the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added to the WiDr cell culture medium at various concentrations and the number of proliferating cells in the culture medium, and the horizontal axis represents the culture time ( Time), the vertical axis represents the number of proliferating cells in the culture medium (X 104 cells per 2 ml). In Figure 34, the amount of fucose-containing sulfated polysaccharide sample in the culture medium is: 0 is marked as no addition (control), 1 is marked as 1 mg / ml, 3 mg / ml is marked, and 5 is marked as 5 in the black triangle. Mg / ml. 126 200400195 As a result, the number of HCT 1 16 cells with PBS was significantly increased, but the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added to Wi Dr cells at a final concentration of 3 mg / ml or more. The number was reduced, and the addition of mg / ml also significantly inhibited cell proliferation. In addition, all the W i D r cells to which the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added showed cell shrinkage, cell fragmentation, and other cell self-killing characteristics. That is, it can be judged that the fucose-containing sulfated polysaccharide sample obtained in Example 1 has a cell self-destructive induction effect on HCT 116 cells at a concentration of less than 1 mg / ml, and can inhibit cell proliferation. The self-killing induction effect of the filter treatment solution of the fucose-containing sulfated polysaccharide sample solution obtained in Example 1 was dissolved in 10 mg / ml, 30 mg / ml, and 50 mg / ml in PBS. The result. Example 3 4 Human pre-myeloid leukemia cells HL-60 cultured at 37 ° C in PRMI 1 640 medium (manufactured by GIBCO) containing bovine fetal blood pupa (JRHBIOSCIENCE) treated at 10% 56 ° C for 30 minutes. (ATCC CCL- 2 40), suspended in ASF 104 medium (manufactured by Ajinomoto Co., Ltd.) at 5 X 104 cells / 900 ml, and dispensed into each well on a 6-well plate manufactured by Falcon Corporation 4.  5 ml. For each suspension, add 0.  5 ml of the low-molecular-weight compound containing fucose sulfated polysaccharide-F as described in Example 19 (6) was dissolved in 30 mM HEPES buffer (PH7) containing 120 mM sodium chloride at 10 mg / ml by freeze-drying, and The filter was filtered through a filter and cultured at 37 ° C in the presence of 5% dioxide 127 200400195 carbon. Alas, only those who added the same amount of the buffer as a control group were cultured in the same manner. The number of cells after 2 to 2 hours and 4 to 6 hours after the start of the culture was measured according to the method described in Tissue Culture Technology (2nd Edition) (Asakura Publishing, Japan Tissue Culture Society) (Pages 26 to 28). That is, it is measured by trypan blue staining on a blood cell calculator. As a result, it can be determined that the HL-60 cell is a lyophilized product of the fucosyl sulfate-containing polysaccharide-F low molecular compound described above, which induces cell self-destruction and suppresses the cell proliferation rate. Example 3 5 PRMI 1 640 medium (manufactured by GIBC0) of human premyeloid leukemia cells HL-60 was treated with bovine fetal blood cells (JRH BIOSCIENCE) containing 10% 56 ° C for 30 minutes. Take 5 x 104/900 ml suspension. Six parts of this suspension were prepared, and to each suspension, 1000 μl of 30 mM HEPES buffer (pH 7) containing 120 mM sodium chloride and 10 mg / ml of Example 19 dissolved in the same buffer were added. (2) The filtered treatment solution of fucose-containing sulfated polysaccharide-F, F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4, and the temperature was 37 ° C, Incubate for 46 hours in the presence of 5% carbon dioxide. The number of progenitor cells in the culture medium was measured 22 hours and 46 hours after the start of the culture. Furthermore, human pre-myeloid leukemia cells HL-60 were suspended in ASF 104 medium (manufactured by Ajinomoto Co., Ltd.) at 5 × 104 cells / 900 ml. Prepare 6 parts of this suspension, and add 100 μl of 30 mM HEPES buffer (pH 7) containing 120 mM sodium chloride to each suspension, and fucose-containing sulfate polysaccharide dissolved in the same buffer at 10 mg / ml -F, F-Fd-1, F-Fd-2, F- 128 200400195

Fd-3 and F-Fd-4 were filtered and treated at 37 ° C and 5% carbon dioxide for 40 hours. The number of progenitor cells in the culture medium was measured 16 hours and 40 hours after the start of the culture. In addition, the cells cultured in the above two types were observed under a microscope, and the degree of proliferation and cell type were investigated. As a result, to the cells cultured in ASF 104 medium, cells containing fucose-containing sulfated polysaccharide-F, F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4 were added. All of them exhibited the characteristics of cell self-destruction such as cell shrinkage and cell fragmentation, and the number of viable cells was almost no increase or almost completely died. The number of cells was increased approximately three-fold in the buffer-only medium. On the other hand, in cells cultured in PRMI 1 640 medium, only added? The cells of 邛 (1-1,? 邛 (1-2, F · Fd-3, and F-Fd-4) all exhibit cell self-destructive characteristics such as cell shrinkage and cell fragmentation, and the cells are almost dead. Only the buffer is added The number of cells in the medium was increased by about 3 times, and the number of cells in the medium containing fucose sulfated polysaccharide-F was increased by about 2.5 times. From the above results, the fucose-containing sulfated polysaccharide- F has a strong cell-self-inducing effect on cancer cells in blood-free maggot culture medium, but F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4 are in the blood-free culture medium or in The blood maggot culture medium has a very strong induction effect of cell self-destruction. To confirm, human pre-myeloid leukemia cells HL-60 were treated with bovine fetal blood maggot containing 56% of 10% 56 ° C for 30 minutes (JRHBIOSCIENCE Corporation ) In PRMI 1 640 medium (manufactured by GIBC0) at 5 x 104 cells / 900 ml. Prepare 2 parts of this suspension 9 ml and add 1 milli 129 200400195 liters of 30 mM HEPES containing 120 mM sodium chloride Buffer (pH 7) and F-Fd-4 dissolved in the same buffer at 10 mg / ml And cultured at 37 ° C for 16 hours in the presence of 5% carbon dioxide. The cultured cells were separated from the supernatant by centrifugation. The obtained cells were suspended in 20 microliters containing 10 mM ethanedioate tetraacetate and 〇. . May lauryl sarcosinate 50 mM Tris-hydrochloric acid buffer (ρΗ7.8), and 10 mg / ml RNA 18. A (manufactured by SIGMA) was added 1 microliter, at 5 (TC, treatment After 30 minutes, 1 μl of 10 mg / ml protein 18. K was added and treated at 50 ° C. for 30 minutes. The treated cells were used as samples and performed at a voltage of 100 V using a 2% agarose gel. Electrophoresis. After the gel was immersed in ethidium bromide solution for 30 minutes, when the DNA state in the gel was confirmed using a super-illumination device, the DNA ladder characteristic of cell self-extinguishment was confirmed. Furthermore, for confirmation, a known When the same operation was performed instead of the above-mentioned F-Fd-4, a radiobiotin D 10 microgram / ml solution as a test agent for inducing cell self-killing was performed, and the same DNA ladder as in the case of F-Fd-4 was confirmed after 20 hours of culture. That is, it can be determined that if the terminal type fucose-containing sulfated polysaccharide _F decomposition enzyme of the present invention will be The fucose sulfate polysaccharide-F is decomposed, and the self-killing effect on cancer cells becomes stronger. Example 36 6 Venous blood was taken from 21-year-old females and 32-year-old normal males, and contained glucose per liter}. 〇mg, CaC12.2H20074mg, MgC12 19.92 mg, KC1 40.26 mg, NaCl 7371 mg, Tris-hydrochloric acid 1 7 5 6 · 5 mg, diluted twice, and then separated in the lymphosphere solution (Daily 130 200400195 (sold by this pharmaceutical company) The overlapping layer was placed in a centrifugal separation tube with twice the volume of diluted blood in advance, and centrifuged at 400 g at 18-20 ° C for 30 minutes. After centrifugation, the lymphosphere portion of the upper layer of the lymphosphere separation solution was collected. The normal lymphocytes obtained in this way were added to a 24-well plate every 1.9 X 105, and 1.8 ml of RPMI-1 containing 10% bovine fetal blood pupa (56 ° C, treated for 30 minutes) was added. In 640 medium, 0.2 ml of 5 mg / ml of fucose-containing sulfated polysaccharide and its decomposed medium obtained in each of the above examples were added and cultured at 37 ° C. The control group was added with physiological saline instead of the fucose-containing sulfated polysaccharide solution. After the start of the culture, the type change of the cells in each orifice and the number of progenitor cells were measured under a microscope. As a result, the pore openings in which various fucose-containing sulfated polysaccharides and their decomposed products were added were different from the control pore openings in cell type, and the number of regenerating cells was almost the same. The cells were almost dead. From this result, it can be determined that the concentration of fucose-containing sulfated polysaccharide and its decomposed product does not show toxicity to normal cells at a concentration that induces strong cell self-destruction of cancer cells. Example 37 7 Fucose-containing sulfated polysaccharide-U on carcinogenesis of solid cancer Murine solid cancer Meth A (4 X 106 cells / mouse) was injected subcutaneously into male BALB / C mice (body weight about 20) G) belly. Thereafter, fucose-containing sulfuric acid-U (100 mg / kg / day) described in Example 6 was continuously injected subcutaneously in the same place for 10 days. On the other hand, the control group was similarly subcutaneously injected with saline. The cancerous tissue formed in the abdomen of the rat 2 weeks later was excised and its weight was measured. The results are shown in Table 2. That is, the average cancer weight in the control group was 1.25 g, and the fucose sulfur-containing 131 200400195 acid polysaccharide-U administration group was 0.28 g, which showed significance (p < 0 relative to the control group). · 01) carcinogenesis. The inhibition rate was 77.6%. Table 2 Rat (η) tumor weight (g) Mean soil SD inhibition rate (%) Control group (8) 1.25 ± 0.10-Fucose-containing sulfated polysaccharide-U administration (8) 0.28 soil 0.07 77.6 Example 3 8 Including Carcinogenic Preventive Effect of Fucose Sulfate Polysaccharides (1) on Spragure-D aw 1 ey mice (male) at 6 weeks of age, 7.4 mg / kg of azomethane (NAKARI TESC) System), and then subcutaneously on the back until the 10th week.尙 When administered, azomethane oxide was dissolved in 0.1M phosphate buffer containing pH 6 · 5 of 0.9% sodium chloride, and the solution concentration was adjusted by 100 microliters each time. For 5 of the 19 above, at the same time as the initial azomethane administration, 70 ml of hot fruit extract of Gaogumei kelp prepared according to Example 1 will be used in the form of water. Vote until the 30th week. This hot water extract contains a mixture of 2 mg / kg fucose-containing sulfated polysaccharide, so it was orally administered daily with a fucose-containing sulfated polysaccharide mixture of 140 mg / kg. 14 To 14 of the above 19 animals, tap water was not administered as fucose-containing sulfated polysaccharide ', and it was regarded as a control group. From 200400195 to the 30th week, the number of ear nest cancers outside the control group was 14 out of 14, compared with 1 out of 5 in the fucose-containing sulfated polysaccharide mixture administration group, which was significant. Carcinogenic inhibitory effect.对照 Until the 30th week, the control group was dead, but the fucose-containing sulfated polysaccharide mixture administered to the group was all alive. At the 30th week, the average weight of the control group was 7 16 g, and the average weight of the fucose-containing sulfated polysaccharide mixture was 8 17 g. On the other hand, the average weight of non-administered rats (5 rats) was 788 g, and the weight of the fucose-containing sulfated polysaccharide mixture was increased to that of rats not administered The same. Next, four control groups were selected, and 40 ml of the above-mentioned hot fruit kelp hot water extract (containing fucose-sulfate polysaccharide mixture 80 mg) was orally administered daily in the 30th week. At 36 weeks, two of the four outer ear nest cancers regressed significantly, confirming the carcinostatic effect of fucose-containing sulfated polysaccharides. Through the oral administration of the fucose-containing sulfated polysaccharide as described above, the carcinogenic preventive effect of chemical carcinogens, the preventive effect of chemical carcinogens against weight gain, and the shrinkage of cancerous tissues were confirmed. Example 3 9 Injection The fucose-containing sulfated polysaccharide mixture prepared in Example 1 was dissolved in distilled water for injection to make a 5% solution. This solution was filled in 1 watt of a glass vial for freeze-drying, filled with 50 mg of fucose-containing sulfated polysaccharide, and lyophilized @ °. 2 ml of physiological saline was added as a dissolving solution. Example 40 Injection An injection was prepared according to the following formulation. 133 200400195 Fucose-containing sulfated polysaccharide-U [Example 12] 40 mg of physiological saline solution Amount 2 ml per ampoule Similarly, the fucose-containing sulfated polysaccharide-F described in Example 12 was used to prepare an injection. Example 41 Lozenges Lozenges were prepared according to the following formulation. Fucose-containing sulfated polysaccharide sample (Example 1) 10 mg corn starch 65 mg carboxymethyl cellulose 20 mg polyvinyl pyrrolidone 3 mg magnesium stearate 2 mg 100 mg per tablet Example 42 F-Fd_1 Dissolved in distilled water for injection to make a 5% solution. This solution was filled in 50 ml of a glass vial for freeze-drying with 50 mg of fucose-containing sulfated polysaccharide, and freeze-dried. Another 2 ml of physiological saline was added as a dissolving solution. Example 43 Injection An injection was prepared according to the following formulation. Example 19- (6) The lyophilized product containing the low-molecular weight fucose sulfate polysaccharide-F 40 mg physiological saline, an appropriate amount 2 ml per 1 ampoule 134 200400195 Example 44 Lozenges are prepared according to the following formula . Example 19- (6) The lyophilized product containing the low molecular weight fucose sulfate polysaccharide-F 10 mg corn starch 65 mg carboxymethyl cellulose 20 mg polyvinyl pyrrolidone 3 mg magnesium stearate 2 mg The effect of the invention per 100 mg per 1 mg according to the present invention provides a cell self-killing induction effect on unwanted or pathogenic cells, and can induce cell self-killing on diseased cells in abnormally proliferating cell diseases such as cancer and viral diseases. Effective medicine for the prevention and treatment of diseases. In particular, in the case of cancers of the digestive system such as colorectal cancer and gastric cancer, since the administration of the agent of the present invention can cause cancer cells to cause cell self-destruction, fucose-containing sulfated polysaccharides derived from natural foods and / or their degradation products are used. The medicament of the present invention as an active ingredient is very suitable for use as a carcinogen for digestive system cancer. And through its carcinogenic effect, carcinogenesis by chemical carcinogens can also be prevented. The medicament of the present invention uses edible substances such as edible brown algae plants and edible sea cucumbers as raw materials, can be supplied in large quantities at low cost, and is also excellent in terms of safety. It is also possible to maintain and enhance health by daily ingestion of foods or beverages containing fucose sulfate polysaccharide and / or its degradation products. In addition, according to the present invention, a simple method for inducing cell self-destruction is provided, and by using the method of the present invention, it is possible to investigate the mechanism of cell self-destruction, and to develop inhibitors of cell self-destruction induction. According to the present invention, there is provided the fucose-containing sulfated polysaccharide-U of the present invention, which is useful in the fields of sugar chain engineering, medicine, and the like, and contains substantially no fucose sulfated polysaccharide and removes highly reactive colored substances. , And also provides its efficiency method. Furthermore, according to the present invention, there is provided the fucose-containing sulfated polysaccharide of the present invention, which is useful in the fields of sugar chain engineering, medicine, etc., and which does not substantially contain fucose-containing sulfated polysaccharide-U and removes highly reactive colored substances. F and its decomposition products, and also provides its efficient production method. According to the present invention, a structural analysis and decomposition of fucose-containing sulfated polysaccharide-F is provided, which can be used to produce a fucose-containing sulfated polysaccharide-F terminal type useful in bioactivity retrieval. Fucose-containing sulfated polysaccharide-decomposing enzyme, its preparation method, and a low-molecular-weight compound of fucose-containing sulfated polysaccharide-F, which has a strong induction effect on the cell self-destruction of cancer cells. Furthermore, according to the present invention, the terminal fucose-containing sulfated polysaccharide-F degrading enzyme of the present invention, which has not been able to be produced stably, can be produced extremely stably in the presence of calcium ions. Furthermore, according to the present invention, the terminal fucose-containing sulfated polysaccharide-decomposing enzyme of the present invention can be activated with excellent efficiency in the presence of calcium ions. [Schematic description] Figure 1K shows the formation rate of Shen Dian containing fucose sulfate polysaccharide. Fig. 2 shows the molecular weight distribution of fucose-containing sulfated polysaccharide-U measured by a gel filtration method using Sephacryi S-500. Figure 3 shows the spectrum of fucose-containing sulfated polysaccharides. 136 200400195 Figure 4 shows the 1H-NMR spectrum of fucose-containing sulfated polysaccharide-U. Fig. 5 shows a dissolution pattern of a pyridyl- (2) -aminated sugar compound (PA-a) of the sugar compound (a) when separated by an L-column. Fig. 6 shows a dissolution pattern of the pyridyl- (2) -aminated sugar compound (PA-b) of the sugar compound (b) when separated on an L-column. Fig. 7 shows a dissolution pattern of a pyridyl- (2) -aminated sugar compound (PA-c) of a sugar compound (c) when separated by an L-column. Fig. 8 shows the results obtained by mass analysis (negative determination) of the sugar compound (a). Fig. 9 shows the results obtained by mass analysis (negative determination) of the sugar compound (b). Fig. 10 shows the results obtained by mass analysis (negative determination) of the sugar compound (c). Figure 11 shows the results obtained by mass-mass analysis (negative determination) of the sugar compound (a). Fig. 12 shows the results obtained by mass-mass analysis (negative determination) of the sugar compound (b). Fig. 13 shows the results obtained by mass-mass analysis (negative measurement) of the sugar compound (c). Fig. 14 shows the 1H-NMR spectrum of the sugar compound (a). Fig. 15 shows the 1H-NMR spectrum of the sugar compound (b). Fig. 16 shows the 1H-NMR spectrum of the sugar compound (c). FIG. 17 shows the molecular weight distribution of fucose-containing sulfated polysaccharide-F as measured by a gel filtration method using Sephacryl S-500. Figure 18 shows the IR spectrum of fucose-containing sulfated polysaccharide-F. Fig. 19 shows the 1H-HMR spectrum of fucose-containing sulfated polysaccharide-F. Fig. 20 is a graph showing the relationship between the pH and the relative activity of the terminal fucose-containing sulfated polysaccharide degrading enzyme obtained according to the present invention. Fig. 21 is a graph showing the relationship between the temperature and the relative activity of the terminal fucose-containing sulfated polysaccharide degrading enzyme obtained according to the present invention. Fig. 22 shows the molecular weight distributions of fucose-containing sulfated polysaccharide-F measured before and after decomposition by the terminal fucose-containing sulfated polysaccharide-degrading enzyme obtained by the present invention, as determined by the gel filtration method using Cellulofine GCL-300. Figure 23 does not show the relationship between the calcium ion concentration and the relative activity in the terminal fucose-containing sulfate-decomposing enzyme-containing reaction solution obtained according to the present invention. FIG. 24 shows the fucose-containing sulfated polysaccharide-F determined by the gel filtration method using Cellulofine GCL-300 in Example 19- (6), and the terminal fucose-containing sulfated polysaccharide obtained by the present invention is decomposed. The molecular weight distribution of a substance that an enzyme breaks down. Fig. 25 shows the IR spectrum of the fucose-containing sulfated polysaccharide-F, which is decomposed by the terminal fucose-containing sulfated polysaccharide-decomposing enzyme obtained by the present invention. Fig. 26 shows the 1H-NMR spectrum of fucose-containing sulfated polysaccharide-F, which was decomposed by the terminal fucose-containing sulfated polysaccharide-decomposing enzyme obtained by the present invention. Figure 2-7 shows the order of Examples 1 and 5 from the HL-60 cell culture medium! 8 138 200400195 The relationship between the culture time when fucose-containing sulfated polysaccharides were added at 1 mg / m1 and the number of viable cells in the culture solution. Figure 28 shows the addition of fucose-containing sulfated polysaccharide obtained in Example 1, to fucose-containing sulfated polysaccharide obtained in Example 12, to MOLT-3 cell culture solution, and those obtained in Examples 15 and 17. Relationship between the culture time when fucose sulfate polysaccharide and dextran sulfate are contained and the number of viable cells in the culture solution. Figure 29 illustrates the addition of the fucose-containing sulfated polysaccharide sample obtained in Example 1, the fucose-containing sulfated polysaccharide mixture obtained in Example 1, and the fucose-containing sulfuric acid obtained in Example 12 to HCT 116 cell culture solution. Polysaccharide-U and fucose-containing sulfated polysaccharide-F, F-Fd-1, F-Fd-2, F-Fd-3 and F-Fd-4, fucose-containing sulfated polysaccharide obtained in Example 15, The relationship between the culture time in the case of heparin and polysaccharide sulfate and the number of progenitor cells in the culture medium. Figure 30 shows the relationship between the culture time when the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added at various concentrations in the HCT 116 cell culture medium and the number of viable cells in the culture medium. Fig. 31 shows the relationship between the culture time when the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added to the AGS cell culture solution at various concentrations and the number of progenitor cells in the culture solution. Fig. 32 is a graph showing the relationship between the culture time when the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added at various concentrations in SW 480 cell culture solution and the number of progenitor cells in the culture solution. Figure 3 3 shows that the fucose-containing sulfated polysaccharide mixture obtained in Example 1 and the fucose-containing sulfated polysaccharide obtained in Example 12 are more concentrated in WiDr cell culture medium. 139 200400195 Sugar-F, F-Fd -3 and F-Fd-4 and the relationship between the culture time when the rock bath sugar sulfated polysaccharide obtained in Example 15 was added and the number of progenitor cells in the culture solution. Figure 34 shows the relationship between the culture time when the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added in various concentrations to the W r D cell culture medium and the number of viable cells in the culture medium.

140

Claims (1)

200400195 The scope of patent application: 1. A fucose-containing sulfated polysaccharide, which is characterized by the following physical and chemical properties: ⑴ constitutes a sugar: does not substantially contain uronic acid, and ⑵ does not substantially pass through the genus Xanthomonas (? 1 & ¥ (^ & (^ 61 * 丨 11111) 3? .3 8- 008 2 (CCRC 9 1 0069) produced a low-molecular-weight fucoidan-degrading enzyme, ⑶ via Alteromonas sp. SN-1 009 (CCRC 9 1 0070) terminal type fucose-containing sulfate polysaccharide degrading enzyme and / or culture supernatant of the strain to reduce the molecular weight, · ⑷ at a total concentration of 0.6-3M of 1 In the presence of two or more salts, precipitates are produced by adding cetylpyridinium chloride. 2. A cell self-inactivation inducer, which is characterized in that it contains fucose-containing sulfated polysaccharide as described in item 1 of the scope of the patent application. And / or its degradation product. 3 · As the cell self-extinguishing inducer of item 2 of the patent application, wherein the decomposition product is classified by molecular weight. 4 · As cell self-extinguishing inducer of the item 2 of patent application, wherein the decomposition product 0 In order to make the fucose-containing sulfated polysaccharide as in item 1 of the patent application scope The terminal type fucose-containing sulfated polysaccharide-decomposing enzyme produced by Alternaria sp. SN-109 (CCRC 9 1 0070) has the following physical and chemical properties and acts as a decomposed enzyme, (i) the action : Acting on fucose-containing sulfated polysaccharides such as in the scope of the first patent application, which makes the fucose-containing sulfated polysaccharides low in molecular weight, but does not act on fucose-containing sulfated polysaccharides according to the scope of the patent application '(1 ii) Optimum pH: The optimum pH of this enzyme is between 7 ~ 8, 141 200400195 (iii) Optimum temperature: The optimum temperature of this enzyme is between 30-35 t. 5 · If the cell self-extinguishing inducer of item 1 of the scope of patent application , Wherein the decomposition product is added with the culture supernatant of Alter onionas sp. SN-109 (CCRC 9 1 0 0 7 0) in the fucose-containing solution as described in the second item of the patent application scope Sulfated polysaccharides, the resulting decomposed products. 6 · Cell self-killing inducers such as those in the scope of patent application No. 4 or 5, where the decomposed products are classified by molecular weight. 7-A method for cell self-killing induction. Fucose-containing sulfated polysaccharides and / or components thereof If the substance is an active ingredient. 8 · If the method for inducing cell self-destruction in the scope of patent application item 7, wherein the decomposition product is classified by molecular weight. 9 · If in the method for the self-destruction induction of cell scope, the decomposition product is used for For example, the fucose-containing sulfated polysaccharide containing the first range of the patent application and the genus Zymomonas sp. SN-1 009 (CCRC 9 1 007 0) produced a terminal fucose-containing sulfated polysaccharide having the following physical and chemical properties Decomposition enzymes act, and the resulting decomposed product, (i): acts on the fucose-containing sulfated polysaccharide such as the first item in the scope of patent application, makes the fucose-containing sulfated polysaccharide low-molecular, but does not act on Physicochemical properties of fucose-containing sulfated polysaccharides, ⑴ Constituting sugar: Contains uronic acid, 岩 Fucoidan oligodegrading enzyme produced by Flavobacterium sp. SA-0082 (CCRC 9 1 006 9) While low-molecular, and generate 142 200400195 (II), (III) OH 11 OH 143 200400195 ch2oh • on 111 144 200400195 (ii) Optimum pH: The optimum pH of this enzyme is 7 ~ 8, (iii) Optimum temperature: The optimum temperature of this enzyme is 30-35 ° C. 10. The method for inducing cell self-killing according to item 7 of the scope of the patent application, wherein the decomposition product is a culture supernatant obtained by adding S. sp. SN-1 009 (CCRC 9 1 0070) in the scope of the patent application scope 1 The decomposed product of the fucose-containing sulfated polysaccharide. 1 1. The method for inducing cell self-destruction according to item 9 or 10 of the scope of patent application, wherein the decomposition product is a molecular weight fraction. 1 2 · The method for inducing cell self-killing according to item 7 of the scope of patent application, wherein the fucose-containing sulfated polysaccharide is a fucoid from any of GAGOME kelp, true kelp, coriander leaf fork, apical leaf, and true sea cucumber Sugar sulfate polysaccharide. 1 3. The method for inducing cell self-destruction according to item 7 of the scope of application, wherein the decomposition product is a decomposition product obtained by acid-decomposing the fucose-containing sulfated polysaccharide. 1 4. A carcinostatic agent characterized by containing a fucose-containing sulfated polysaccharide and / or a degradation product thereof according to any one of claims 1 to 6 of the scope of patent application. 1 ··· A carcinogen preventive agent, characterized in that it contains fucose-containing sulfated polysaccharide and / or a degradation product thereof according to any one of claims 1 to 6. 16 · The method for preparing fucose-containing sulfuric acid polysaccharide according to item 1 of the patent application scope, which comprises making the fucose-containing sulfuric acid polysaccharide mixture to decompose an enzyme having the ability to decompose fucose-containing sulfuric acid polysaccharide containing uronic acid. Or, the process of collecting the target polysaccharide with a microorganism having the degrading enzyme. 17. If the method for preparing rock-sweetened sulfuric acid polysaccharides according to item 1 of the scope of the patent application, it is desirable to include a fucose-containing sulfuric acid polysaccharide mixture in the presence of salts. 145 200400195 Engineering of polysaccharide precipitation. 18. The method for preparing fucose-containing sulfated polysaccharide according to item 丨 of the patent application scope, which comprises a process of collecting the target polysaccharide by treating the fucose-containing sulfated polysaccharide mixture in the presence of a divalent cation mixed with an anion exchange resin. . 19 · The method for preparing fucose-containing sulfated polysaccharides according to item 1 of the patent application scope, which includes the use of polysaccharides in coexisting colored substances when manufacturing fucose-containing sulfated polysaccharides as described in item 1 of the patent application. Removal of substances or substances with anion-exchange groups. ® 20. A terminal type fucose-containing sulfated polysaccharide degrading enzyme, which is characterized by the following physicochemical properties, (i) Action: It acts on the fucose-containing sulfated polysaccharide such as the first item in the scope of patent application, The fucose-containing sulfated polysaccharide is reduced in molecular weight, but does not act on the fucose-containing sulfated polysaccharide having the following physical and chemical properties. ⑴Constitutes sugar: Contains uronic acid. (CCRC ^ 9 1 0069) produced fuco-oligosaccharides that decompose enzymes and become low-molecular, and generate at least one selected from the compounds represented by the following formulae (I), (II), (I Π) Compound '146 200400195 οII • S — OH 〇H II 147 200400195 ρπ ch2oh -ο 111 148 200400195 (ii) Optimum pH: The optimum pH of this enzyme is 7 ~ 8 ′ (iii) Optimum temperature: The optimum temperature of this enzyme is 30-35 ° C. 2 1-An enzyme composition comprising a calcium source and a terminal fucose-containing sulfated polysaccharide-decomposing enzyme as described in item 20 of the patent application scope. 2 2 · —A method for preparing a terminal fucose-containing sulfate polysaccharide-decomposing enzyme such as the scope of application for patent No. 20, which is characterized by cultivating a terminal fucose-containing sulfate-polysaccharide decomposing enzyme as described in scope of the application for patent 20 A productivity-promoting bacteria of the genus Zymomonas is obtained from its culture. 23. —A low-molecular-weight compound containing fucose-containing sulfated polysaccharide, which is characterized in that the terminal fucose-containing sulfated polysaccharide decomposing enzyme such as the scope of patent application No. 20 and fucose-containing Acquired by the action of sulfated polysaccharides. 149