TWI237026B - Fucose sulfuric acid-containing polysaccharide, the preparing method thereof and the anti-cancer agent and preventing agent for inducement of cancer - 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

1237026 (1) Description of the invention: [Technical field to which the invention belongs] The present invention relates to a cell self-deactivation 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 art] In recent years, the death of cellular tissues has focused on the so-called cell death (apoptosis, also known as dephagy; self-violence or cell death). 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, the main cause of any external or internal factors is the activation of the gene that causes the self-destruction of the planned cells, and the use of this gene will biosynthesize the protein that plans the dead gene, and the cell will be generated by the generated planned dead protein. Decomposed by itself, and died. 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 compound of 7 1237026. [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. A second invention of the present invention relates to a method for inducing cell self-destruction, which is characterized by using a fucose-containing sulfated polysaccharide and / or a degradation product thereof as an active ingredient. The present invention; the third invention relates to a carcinostatic agent, which contains the fucose-containing sulfated polysaccharide and / or a degradation product thereof according to 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. (2) Fucoidan produced by Flavobacterium sp. SA-0082 (CCRC 910069) decomposes enzymes to reduce molecular weight, and generates at least the following formula (I), (II ), (III) One or more compounds selected from the compounds shown in (III). 8 1237026 CH2 Oil

OH

o = s = o I OH

OH 9 1237026

10

L 1237026 The sixth invention of the present invention relates to a fucose-containing sulfated polysaccharide having the following physical and chemical properties. (1) Constitutive sugar: It does not substantially contain sugar acid. (2) The low molecular weight of fucoidan-degrading enzyme produced by Beibei 0082 (CCRC 9 1 0069) cannot be reduced. The seventh invention of the present invention relates to a method for preparing fucose-containing sulfated polysaccharides according to the fifth invention of the present invention, which is characterized by including the treatment of fucose-containing sulfated polysaccharide mixtures in the presence of salts with a chemical agent having an acidic polysaccharide aggregation ability And remove the sedimentation process. The eighth invention of the present invention is 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 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 a fucose-containing sulfuric acid polysaccharide according to the sixth invention of the present invention, which is characterized by including a fucose-containing sulfuric acid polysaccharide mixture to decompose the fucose-containing sulfuric acid A polysaccharide-decomposing enzyme, or a process for treating and collecting a target polysaccharide with a microorganism having the degrading enzyme. The eleventh invention of the present invention is a method for preparing fucose-containing bran 1237026 sulfated polysaccharide according to the sixth invention of the present invention, which is characterized by including a fucose-containing sulfated polysaccharide mixture in the presence of salt to have an acidic polysaccharide aggregation ability. The process of medicament precipitation of 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 relates to a method for preparing a fucose-containing sulfated polysaccharide mixture, which is characterized in that the fucose-containing sulfuric acid used in the seventh, eighth, tenth, tenth, or twelfth invention of the present invention is extracted from seaweed. In the case of a polysaccharide mixture, acetate ions and calcium ions 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 fucose-containing sulfated polysaccharide having the following physical and chemical properties' and reduces the molecular weight of the stone bath sugar-containing polyacetate. (a) Structural sugar: It does not substantially contain uronic acid. (1) The fucoidan-degrading enzyme produced by Flavobacterium sp. SA · 0082 (CCRC 910069) cannot be reduced in molecular weight. It has no effect on fucose-containing sulfate polysaccharide having the following physical and chemical properties. (c) Structural sugar: contains uronic acid. 12 1237026 (d) The fucoidan-degrading enzyme produced by Flavobacterium sp. SA-0082 (CCRC 9 1 0069) is reduced in molecular weight, and is produced by at least the following formula (I), (II ), (III) One or more compounds selected from the compounds shown in (III).

CH2 OH

OH

〇

13 1237026

/ OH 0 (111 14 1237026 (11) Optimum pH: The optimum pH of this enzyme is around 7 to 8. (iii) Optimum temperature: The optimum temperature of this enzyme is around 30-35 ° C. The first of the present invention The 6th invention is composed of an enzyme containing a calcium source and the terminal fucose-containing sulfated polysaccharide-decomposing enzyme of the 15th invention of the invention. The 17th invention of the invention is the terminal rock-containing enzyme of the 15th invention of the invention A method for producing a fucose sulfate polysaccharide degrading enzyme, characterized by culturing a Symbiotic genus cell having the ability to produce a terminal fucose sulfate polysaccharide decomposing enzyme according to the 15th invention of the present invention, and collecting the enzyme from a culture thereof The eighteenth invention of the present invention relates to 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 sulfated polysaccharides. The inventors have successfully obtained various purified fucose-containing sulfated polysaccharides and their decomposition products, and then reviewed their biological activity, and found that these substances cause cancer cells to induce cell self-destruction And It shows a strong carcinogenic effect. It was also found that fucose-containing sulfated polysaccharide and / or its decomposed product exhibited strong carcinogenic inhibitory effect. Moreover, the fucose-containing sulfated polysaccharide decomposing enzyme useful for preparing the decomposed product of the present invention was successfully prepared. The present invention has been completed. [Embodiment] The present invention will be described in detail below. 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 Gaogu can be used. GAGOME kelp, kelp-derived, wakame-derived, and other 15 1237026 all brown-algae-derived materials can also be used. It is also known that the sea cucumber body wall also contains fucose-containing sulfated polysaccharides Therefore, fucose-containing sulfated polysaccharides from sea cucumbers are also used in the present invention. Decomposed products of fucose-containing sulfated polysaccharides can also be used in the present invention. Decomposition methods of fucose-containing sulfated polysaccharides include acid treatment. Chemical decomposition method, etc., physical decomposition method such as ultrasonic treatment, or enzyme decomposition method, etc. The inventors have discovered that various fucoid When sulfated polysaccharides and / or their degradation products are added to the culture medium of cancer cells, the cells self-destruct within 1 to several days after the addition. It was also confirmed that they do not show toxicity to normal cells. In the present invention, the so-called Fucose sulfate polysaccharide is a polysaccharide containing fucose sulfate in the molecule, and it is not particularly limited. For example, it is contained in brown algae plants, sea cucumbers, etc. [supervised by Toshiro Soda, edited by Fujikami Ekami, Kyoritsu Publishing Co., Ltd., Showa 3 Published on February 15, 2010, Polysaccharide Chemistry, Page 3 119, Page 321]. 尙 Fucose-containing sulfated polysaccharides from brown algae plants are commonly known as fucoidan, fucoid Polysaccharides, 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 SIGMA company can be divided into 13 molecular species ( Carbohydrate research, Vol. 255, pp. 213 ~ 224 (1994)], which includes fucose as one of the main components, and a constituent sugar containing several% of uronic acid, which mostly contains one of fucose and mannose. Molecular species. Various cases of enhanced macrophage activity, inhibition of cancer metastasis, and anticoagulation in its biological activity have been reported. However, since there are molecular types in the fucose-containing sulfur acid polysaccharide, it is necessary to investigate the activity itself. Molecular species 16 1237026, 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. In the following description, those who do not substantially contain uronic acid will be referred to as fucose-containing sulfated polysaccharide-F, and those who contain gluconic 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 the After purification, but the soluble barium salt is not easy to dispose in waste liquid, etc., and the pH will change if calcium chloride is mixed with seaweed. Is necessary. At the time of pH adjustment, since the seaweed powder is agglomerated with viscosity, the subsequent extraction efficiency is lowered, and the filtration by separating and recovering the liquid becomes difficult. That is, 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 described above, due to its difficulty in grading 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, 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 difficult to remove from fucose-containing sulfated polysaccharides, which generally causes enzyme reaction inhibition and resin pollution. Furthermore, according to the present invention ', there is provided a substantially purified fucose-containing sulfated polysaccharide-F, a simple extraction method thereof, and a method for preparing fucose-containing sulfated polysaccharide_F. According to the present invention, a fucose-containing sulfated polysaccharide F, which is generally difficult to remove and has a highly reactive coloring substance that inhibits enzyme reaction and resin pollution, is provided. The fucose-containing sulfated polysaccharides used in the present invention may be fucose-containing sulfated polysaccharides such as brown algae plants, 18 1237026 sea cucumbers, and the like, for example, dried to be 'pulverized for use' or derived from fucoids 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 rock bath sulphuric acid-containing polysaccharide extraction solution can be performed by a known method without any particular limitation. 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 pi nna tifi da can be used to prepare fucose-containing sulfated polysaccharides. Sea cucumbers containing fucose-containing sulfated polysaccharides are, for example, sea cucumbers described in Japanese Patent Application Laid-Open No. 4-9 1 02 7. For example, sea cucumbers (S t 丨 chopus japonicus) and fake black sea cucumbers (Holothuria leucospilota) can be used. According to the method described in the publication, a fucose-containing sulfated polysaccharide can be prepared. The fucose-containing sulfated polysaccharide has a sulfate group in the molecule, and this group forms a reaction salt with various bases. These fucose-containing sulfated polysaccharides and their decomposition products are stable in the state of 19 1237026 salt, and are usually isolated in the form of salts such as sodium and / or potassium. The salts of these substances are treated with cation exchange resins such as Dowex 50W to free fucose-containing sulfated polysaccharides, and may lead to free decomposition products thereof. Moreover, it is also possible to perform a well-known and conventional salt exchange as needed, and to exchange various salts as desired. Salts of fucose-containing sulfated polysaccharides and their decomposed products may be pharmaceutically acceptable salts. Examples include alkali metal salts such as potassium, sodium, alkaline earth metal salts such as calcium, magnesium, barium, and salts of organic bases such as pyridine. And ammonium salts. The fucose-containing sulfated polysaccharide-containing brown algae plants and sea cucumbers are dried and then pulverized to prepare a fucose-containing sulfated polysaccharide powder. The fucose-containing sulfated polysaccharide powder was subjected to hot water extraction. Dilute acid extraction to prepare fucose-containing sulfated polysaccharide extract. The extraction temperature and time of the fucose-containing sulfated polysaccharide content can be selected according to the purpose in the range of 0 ~ 200 ° C and 1 ~ 360 minutes, usually 10 ~ 150 ° C and 5 ~ 240 minutes, preferably It is preferable to select a range of 50 to 130 ° C and 10 to 180 minutes. In order to improve the extraction rate of fucose-containing sulfated polysaccharides, there are methods to classify fucose-containing sulfated polysaccharides such as calcium chloride and barium acetate, and acidic polysaccharide agglutinants such as cetylpyridinium chloride. Fractionation method of fucose-containing sulfated polysaccharides, grading method of fucose-containing sulfated polysaccharides using an acidic polysaccharide aggregating agent in the presence of salts, gel filtration, ion exchange chromatography, etc., and can be combined as needed to perform refined. The method for decomposing fucose-containing sulfated polysaccharides can be a method using fucose-containing sulfated polysaccharides to decompose enzymes, a method of acid decomposition, a method of ultrasonic treatment 20 1237026, and the like. Announcement method, purification of decomposed matter can be performed by the method described above. 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, and for example, those from the loquat leaf tip, those from the high fruit beauty kelp, and those from the true kelp may be used. , From wakame, others from all brown algae. For the production of fucose-containing sulfated polysaccharides, first, an aqueous solution of brown algae was used to obtain an extract. Also, the seaweed for extraction may be raw seaweed. Before obtaining the extract, if brown algae is dried, it is made into a dry powder, washed with 60 to 100% ethanol and acetone, and immersed in formaldehyde, In aqueous solutions such as acetaldehyde, glutaraldehyde, and ammonia, it is advantageous because it can greatly reduce the mixing of coloring substances into fucose-containing sulfated polysaccharides. When extracting fucose-containing sulfated polysaccharides from brown algae or brown algae's ethanol washing residues, the use of soluble barium acetate, barium chloride, or calcium chloride is advantageous because it can inhibit the mixing 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, the initial extraction efficiency of 0.2 M or more will reduce the extraction efficiency. Therefore, first extract with water and then add calcium acetate to remove the generated alginic acid. Precipitation is sufficient. However, in the case where the fucose-containing sulfated polysaccharide and alginic acid are to be simultaneously extracted, 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 / 3-of uronic acid under an alkaline condition, 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 under weakly alkaline 100 ° C and heating for 1 hour, 'If a molecular sieve membrane with a pore size of 300 is used, the fucose-containing sulfated polysaccharide-U with a molecular weight distribution of about 1,000 to 10,000 can be modulated. 2. Fucose-containing sulfated polysaccharide-F 'and the fucose-containing sulfate of the present invention which can be adjusted to any molecular weight and molecular weight distribution according to the use conditions are 22 1237026 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, cetylpyridinium chloride can be added in the presence of salts such as edible salt at a concentration of 0.2 to 0.6 M. And so on 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 0.6M, and the cetylpyridinium chloride in the precipitate is washed away with a 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 present inventors have discovered that in the presence of one or more salts of 0.6 to 3M, the rock-containing sugar polyacetate-F of the present invention and the fucose-containing sulfate polysaccharide-U of the present invention are acidic polysaccharides. The coagulant showed a completely different behavior. 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 kinds of salts are added to an aqueous solution containing a fucose-sulfate polysaccharide mixture so that the total concentration thereof becomes 0.6 to 2 M. The added salts are not particularly limited, such as sodium chloride and calcium chloride. 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 1.5 M, if acidic polysaccharide agglutinating agents such as cetylpyridinium chloride are added so as not to produce 23 1237026 Shenyuan again, the fucose-containing sulfate polysaccharide_ F forms a precipitate, so if the precipitate is removed, a fucose-containing sulfate 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 salt-saturated 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 can also be dissolved and then subjected to ultrafiltration. 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 that 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 1237026, it is expected to be less than 0.5. Μ. In addition, the method used in this method as a source of divalent cations is particularly excellent in the effects of calcium salts and barium salts, but it is not particularly limited. Magnesium sulfate, bell 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, for example, to a solution containing a fucose-containing sulfated polysaccharide, Ce 1 1 u 1 〇fine, GCL-2 0 0 (manufactured by Biochemical Industry Co., Ltd.), and Sephacryl S-500, Sephadex G-200, and Sepharose CL are added. -2B (also manufactured by PHARMAICA) and other polysaccharide resins, or DEAE-Cellulofine A-800 (manufactured by Biochemical Industry Co., Ltd.), DEAE-Sepharose FF, DEAE-Sephadex A-50, QAE_Sephadex A-50, DEAE-Sephacel (Also manufactured by PHARMA CIA), TSK-Gel DEAE-Toyopearl 6 50, TSK-Gel DEAEToyopearl 5 50 (TOS〇), Amberlite anion exchange resin (sold by ORGAN0), Kitopearl Anion-exchange resins (manufactured by Fuji Textile Co., Ltd.) and other substances with anion-exchange groups can be removed after stirring, or a solution containing fucose-containing sulfated polysaccharide can be passed through the column filled with it to easily remove this highly reactive Coloring substance. 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 25 1237026. 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%, and were mixed and dissolved in various concentrations of chlorine by mixing in various proportions. 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. To make 2% of the fucose-containing sulfated polysaccharide-U and fucose-containing sulfated polysaccharide-F of the present invention dissolved in water completely precipitate in 1.25% cetylpyridinium chloride, 3.2 times the capacity is required. Therefore, with respect to 125 microliters each of 2% fucose-containing sulfated polysaccharide-U and fucose-containing sulfated polysaccharide-F dissolved in 2% of each concentration of sodium chloride, cetyl wax dissolved in each concentration of sodium chloride After adding 4 0 0 26 1237026 microliter of pyridine solution, stir thoroughly, leave it for 30 minutes, centrifuge and separate the sugar content of the supernatant liquid according to the benzene sulfuric acid method [Analytical Chemistry, Volume 28, page 3 50 (1 95 6)] Measurement can calculate the precipitation formation rate of each fucose-containing sulfate polysaccharide at each sodium chloride concentration. The molecular weight of the fucose-containing sulfated polysaccharide-U of the present invention, when calculated by the gel filtration method using Sephacry 1 S-500, shows a molecular weight distribution centered on about 190,000 (see 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: 0.2 M sodium chloride and 10 mM sodium phosphate buffered solution (pH 6 · 0) Flow rate: 1.5 ml / min Sample concentration: 0.2 5% sample liquid volume: 20 ml molecular weight reference material: Shodex STANDARD P-82 (manufactured by Showa Denko) 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). Next, the obtained dried sample of fucose-containing sulfated polysaccharide-U was dissolved in 1 equivalent of hydrochloric acid at a concentration of 0.5%, and treated at 110 ° C for 2 hours. 27 1237026 The constituent monosaccharide was hydrolyzed . Next, the reducing ends of the monosaccharides obtained by hydrolysis with GlycoTAG reagent kit (also manufactured by Takara Shuzo Co., Ltd.) were subjected to Pit D amidation- (2amination (PA)) and investigated by hPLC. The ratio of constituent sugars. Alas, the conditions of HPLC are as follows. Apparatus ... Model L-6200 (manufactured by Hitachi)

Column: PERPACK type A | 4.6mmXl50nim: manufactured by Takara Shuzo Co., Ltd.) Eluent: 700mM boric acid buffer (pH9.〇): acetonitrile = 9 ······ Fluorescence detector F-1 1 50 (manufactured by Hitachi, Ltd.) at an excitation wavelength of 310 nm and a fluorescence wavelength of 380 nm. Detection flow rate: 0.3 ml / min column temperature · 6 5 ° C Second: According to Analytical Biochemistry, Volume 4, The amount of uronic acid is determined on page 330 (1 96 2). Secondly, the sulfuric acid content was quantified as described in Biochemical Jouorna Vol. 84, page 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: mannose: galactose: uronic acid: sulfate group has a molar ratio of about 10 ·· 7: 4: 5: 20 ° Second, the fucose-containing sulfate polysaccharide-U calcium salt The IR spectrum was measured with a Fourier transform infrared spectrophotometer JIR-DIAMOND 20 (manufactured by Japan Electronics Co., Ltd.) to obtain the spectrum shown in FIG. 3. Alas, Figure 3 28 1237026 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_U 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), the spectrum shown in FIG. 4 was obtained. In FIG. 4, the vertical axis represents the signal intensity, and the horizontal axis represents the chemical shift (PPη0.). The chemical shift in 1H-NMR is the chemical shift of HOD, which is regarded as 4.65 ppm. 1H-NMR (D20 ) δ 5.27 (H at 1st mannose), 5.07 (Hat at fucose 1), 4.49 (H at fucose 3), 4.37 (glucuron 1), 4.04 (fucose 4th place), 3.82 (Fourth place H), 3.54 (Glucuronate 3 place H), 3.28 (Glucuronide place 2), 1.09 (Fucose place 5) (CH3, H) The specific optical rotation of the lyophilized product of fucose-containing sulfated polysaccharide-U of the present invention is 53.6 degrees when measured by a high-sensitivity polarimeter SEPA-300 (manufactured by Horiba). The inventors, etc. The structure of the fucose-containing sulfated polysaccharide-U of the present invention was determined as described below. The fucose-containing sulfated polysaccharide-U is decomposed and decomposed by a decomposing enzyme having the ability to decompose the fucose-containing sulfated polysaccharide-U. Refining: The refined fucose-containing sulfated polysaccharide _U is refined by the following terminal fucoidan-decomposing enzyme.

That is, 16 ml of a 1% fucose-containing sulfate polysaccharide_U solution, 12 ml of 50 mM 29 1237026 phosphate buffer solution (PH8.0) and 4 ml of sodium chloride 4 ml and 3 2 mU / m 1 of the end 8 ml of fucoidan-decomposing enzyme solution was mixed and reacted at 25 ° C for 4 8 hours. It was confirmed that the absorbance at 2 3 Q nm increased as the reaction progressed, and it was determined that the fucose-containing sulfated polysaccharide-U was decomposed by this enzyme. This reaction solution was desalted with MICR0ACILIZER-G3 (manufactured by Asahi Kasei Co., Ltd.), and three dissolving fractions (a), (b), and (c) 〇 尙, which were separated by DEAE-Sepha ros e FF, were obtained. Algae is prepared by the following method based on the glycolytic enzyme. 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 fungus is Brevibacterium wide 0 _ 8 ~ 1 · 0 // m length 1. 0 ~ 1.2 μm (2) spores (3) Gram-staining-negative b. Physiological properties 30 1237026 Since March 29, it has been deposited with FERM P-14872, and has been registered as FERM BP- 5 40 2 (On the transfer application date of international deposit: February 15th, 2008), it was deposited in the strain preservation and research center of the Food Industry Development Research Institute of your country on December 24, 1996 as CCRC Deposit No. 910069. 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 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-0082 strain is cultured in an appropriate culture medium, the bacterial cells are collected, and the cells are broken by conventional cell destruction methods, such as ultrasonic treatment, to obtain cell-free cells. Extract. 33 1237026 Secondly, a sample of purified enzyme can be obtained from the extract 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 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. 20 liters of culture medium consisting of 0.25% glucose, 1.0% protein gland, 0.05% yeast extract, and 0.01% artificial seawater (manufactured by Germalin Laboratory) pH 7.5 of an antifoaming agent (made by Shin-Etsu Chemical Co., Ltd. 070). Place in a 30 liter capacity fermentation tank and sterilize at 120 ° C for 20 minutes. After cooling, inoculate 600 ml of the above-mentioned culture solution and incubate at 24 ° C for 24 hours, with a ventilation volume of 10 liters per minute and a stirring speed of 125 revolutions per minute. After the end of the culture, the culture solution was centrifuged to obtain bacterial cells. The bacterial cells were suspended in a 20 mM acetic acid-phosphate buffer solution (PH7 · 5) containing 200 mM sodium chloride, and the cells were crushed by ultrasound and centrifuged to obtain a bacterial cell extract. When measuring the activity of this terminal fucoidan-degrading enzyme 34 1237026 in this bacterial cell extract, an activity of 5 mU was detected in 1 ml of 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. (PH7.5.5) was fully dialyzed. After the precipitate produced by dialysis was removed by centrifugation, it was adsorbed on a DEAE-Sepharose FF column equilibrated with 20 mM acetic acid · phosphate buffer (ρΗ7.5) containing 50 mM common salt, and the adsorbate was placed in the same buffer. After sufficient washing, dissolve it with a sodium chloride gradient of 50 mM to 600 mM and collect the active fraction. Secondly, add 4M table salt to this active dissolve, so as to adsorb to a phenyl-Sepharose CL-4B column equilibrated with a 20mM phosphate buffer solution (pH 8. 0) containing 4M table salt in advance and After the adsorbate was sufficiently washed with the same buffer solution, it was dissolved 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 was calculated from Sephacryl S-300 retention time to be approximately 460,000. This active fraction was then dialyzed against a 10 mM phosphate buffer (ρΗ7) containing 25 μM of salt. This enzyme solution was adsorbed on a Mono Q HR 5/5 column equilibrated with a 10 mM phosphate buffer (P7) containing 2 50 mM common salt, and the adsorbate was sufficiently washed with the same buffer, and then 250 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 1 1237026

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. 50 microliters of a 2.5% fucoidan solution from high fruit kelp, 10 microliters of this enzyme, and 60 microliters of a 83 mM phosphate buffer PH7.5 containing 66 7 mM sodium chloride were mixed and made After reacting at 37 ° C. for 3 hours, 105 μl 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 only for dissolving this enzyme in place of this enzyme and make the person react under the same conditions, and use only water instead of 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 mannose and uronic acid of 1 V mo i 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 uronic acid generated during the dissociation reaction as 5.5. Alas, the enzyme activity is 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 1237026 2 · 1 05: The amount of sample liquid for measuring absorbance ( Ml) 120: Liquid volume (μl) of enzyme reaction solution 5 · 5 ·· Molar molecular absorption coefficient (/ mM) of unsaturated uronic acid at 2 3 0ηπ 105: Liquid volume of reaction liquid used for dilution (micro L) 0.01: the amount of enzyme solution (ml) 180: the reaction time (minutes) The protein was determined by measuring the absorbance of the enzyme solution at 280 nm. At this time, the grade photometry of the 1 mg / ml protein solution was calculated as 1.0. Alas, the fucoidan from the high fruit kelp was prepared as follows. The dried high fruit kelp was crushed with a free-pulverizer M-2 (manufactured by Nara Machinery Co., Ltd.), and treated in 70% of 85% methanol at 70 ° C for 2 hours, filtered, and the residue was left at 1 A 0-fold amount of methanol was treated at 70 ° C for 2 hours, and then filtered. A 20-fold amount of water was added to the residue, and the solution was treated at 1000 ° C for 3 hours and filtered to obtain an extract. After the salt concentration of the extract was the same as that of a sodium chloride solution of 40 μm, cetylpyridinium chloride was added so as not to cause precipitation again, and centrifuged. This precipitate was thoroughly washed with ethanol, and desalted and low-molecules were removed with an ultrafilter (a filter membrane with a molecular weight of 100,000 (made by AM ICON)) capable of completely removing cetylpyridinium chloride, and the precipitate generated at this time was It is removed by centrifugation. The supernatant liquid is freeze-dried to obtain a refined high-fruit kelp fucoidan. The structure of the enzyme reaction product is analyzed by the above-mentioned terminal fucoidan-decomposing enzyme, and the fucoid sulfate-containing polysaccharide The α 1-4 bond between D-mannose and D-glucuronic acid present in -U is released from 37 1237026. An enzyme that is sexually decomposed. If the fucose-containing sulfated polysaccharide-U shell 11 obtained is acted on, Sugar collecting structure having the following formulae (I), (II), and (III).

OH

OH 11 38 1237026 ch2〇h

39 1237026 Details are described below. The three dissolved fractions (a), (b), and (c) separated and purified by the above-mentioned DEAE-Sepharose FF only partially used GlycoTAG and GlycoTAG reagent kits to give reducing ends to pyridyl- (2) -amine By basification (palation), each PA-sugar (PA-a), (PA-b), and (PA-c) can be obtained. (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 2.5 mm) (manufactured by Showa Denko Corporation) Eluent: 0.2 M sodium chloride: dimethylimine 2 9: 1 Detection: Fluorescence detector F-1 1 50 (made by Hitachi) at an excitation wavelength, 320 nm, and a fluorescence wavelength of 400 mm. Detection flow rate: 1 ml / min. Column temperature: 50 ° C. (2) HPLC analysis device using a reverse phase column: L-6200 ( (Hitachi Manufacturing Co., Ltd.) Column: L-column (4.6 X 2 50mm) [(Financial) Chemical Inspection Association] Eluent: 50 mM acetic acid-triethylamine (ρΗ5 · 5)

Detection: Fluorescence detector F-1 150 (made by Hitachi) at an excitation wavelength of 3 20 nm and a fluorescence wavelength of 400 mm. Flow rate: 1 ml / min. Column temperature: 40 ° C 40 1237026. Figures 7 and 7 show the HPLC dissolution patterns of each pyridyl- (2) -aminated sugar compound (PA-a), (PA-b), and (PA-c). Indicates the relative fluorescence intensity, and the horizontal axis indicates the residence time (minutes). The physical properties of the compound represented by the following formula (I), formula (I I), and formula (I 丨 ί) 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 (1)), the mass-mass spectrum of (c) is shown in 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. 15 are shown in FIG. 14, and the vertical axis represents the signal intensity and the horizontal axis represents Chemical shift 値 (PP m). That is, the chemical shift 値 in 1H-NHR is expressed in terms of the chemical shift of H0D 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_H2〇_h +]-, 1 75 [Unsaturated D-glucuronic acid-H +]-, 225 [L-Fucose-containing sulfuric acid · H2O-H +]-, 243 [L-Fucose sulfate-H +]-, 319 [Unsaturated D-Bu Combination of gluconic acid and D-mannose-H2〇_h +]-, 405 [M-unsaturated D-glucuronic acid-H +]-, 4 8 3 [MS〇3, H +]-41 1237026 1H -NMR (D2 〇) (55 · 78 (1H, d, J = 3.7Hz, 4 "-Η), 5.26 (1H, d, J = 1.2Hz, 1-H), 5.12 ( 1H, d, J = 4 · OHz, 1 '-Π), 5. 03 (1H, d, J = 6. 1Hz, 4 · 4 7 (1 H, d — d, J = 3. 4, 1 0 4 H z, 3 '-H), 4. 21 (1H, br — s, 2-H), 4 · 12 (1 H, m, 5' — H), 4 · 10 (1H, d-d , J = 3 · 7, 5 ·

8 H z, 3 " -H), 4. 03 (1H, d, J = 3.4 Hz, 4 '-Π), 3.86 (1H, m, 3-H), 3.83 (1H, d — d, J = 4. 0, 1.0. 4 H z, 2 '— H), 3 · 7 2 (1 H, m, 4-H), 3 · 7 2 (1 Π, m, 5-Π ) f, 3.70 (2H, m, H2 of 5-CH2), 3〆6 5 (1 H, dd, J = 5. 8, 6. · 1Hz, 2 "-Η), 1. 08 (3H, d, J = 6 · 7Hz, H3 of a C H3) Sugar composition L-Fucose: Unsaturated D-Glucuronic acid: D-Mannose = 1: 1 = 1 (1 molecule each) Sulfate 1 molecule (3-position of L-fucose) 尙, the number assigned to the peak in 1H-NMR is as shown in the following formula (IV).

CHZ OH

42 1237026 _ (b) Physical molecular weight 724 MS m / z 723 [M-H +]-, 361 [M-2H +] 2-, MS / MSm / z97 [HS04, 175 [Unsaturated D-glucuronic acid -H +]-, 243 [L-fucoic acid-H +]-, 321 [MS〇3-2H +] 2_, 405 [M-unsaturated D-glucuronic acid-2S〇3-H +]-, 417 [ML Fucose-2S03-H +]-. 1H-NMR (D20) 55 · 6 6 (1 Η, d, J = 3.4 Hz, 4 "-Η) > 5. 2 7 (1 H, d, J = 7 · 3 Η ζ, 1 " --Η), 5.22 (111, d, J = l · 8Ηζ, '-Η), 5 · 2 1 (1 Η, d, J = 3 · 7 Η ζ, 1, one Π), 4 · 5 Ο (1 Η, d, J = 3. 1 Η ζ, 4 '1Η), 4.32 (1 Η, q, J = 6 · 7Ηζ, 5, -Η), 4. 27 (1Η, d- d, J = 3. 7, 10. 4 Η ζ, 2,-Η) Ν 4. 2 1 (1Η, dd, J = 3 · 4, 6. 7 II ζ, 3 " -Η), 4.1 8 (1 Η, d -d, J = 1.8, 1 i. 〇 Η ζ, 5-CH: tH), 4 · 1 5 (1 Η, br-s, 2-Η), 4 · 10 ( 1 Η, d-d, J = 5. 8, 1 1 · Ο Η ζ ν 5-C Η Η)

, 3. θ 9 (1 Η, d-d, J = 3, 1.1, 0.4 Η ζ, 3 '-Π), 3.90 (lH, pi, 5 — H), 3.82 (lH, m, 3 — H), 3.82 (lH, m, 4 aΗ) 3.54 (1Η, br-t, J = 7 · 3 H ζ, 2 ^-Η) > 1 · 11 (3 Η, d, J = 6. 7 Η ζ, 5'-CH3 of Η3) Sugar composition L-Fucose: Unsaturated D-Glucuronic acid: D-Mannose bis 1: 1: 1 (1 molecule each) 3 sulfates (The 2nd and 4th positions of L-fucose and the 6th position of D-mannose) 尙, the number assigned to the peak in 1 Η-NMR is shown by the following formula (V). 43 1237026 〇

(c) Physical molecular weight 1 1 2 8 MS m / z 1127 [M-H +]-MS / MSm / z97 [HS04]-, 175 [Unsaturated D-glucose fucoic acid-H20-H +]- , 24 3 [L-fucose sulfate-saturated D-glucuronofucose-S03-2H2 +] 2_, one of the couplers of fucose and D-mannose-H +]-, 721 [MD Sugar-S03-H2 〇-H +] Answer acid 1 +]-, 225 [1 ^ -H +]-, 371 [M- 不 4 0 5 [sulfated L-rock-mannose-L-fucoid 44 1237026 1H-NMR (〇2 O) (55 · 69 (1Η, d, J = 3.7Hz, (4) " _Η), 5.34 (1 s, (1)-H), 5.16 (1H, s, 1-H) 5.10 (1H, d, 4.0 Hz, (1) '--), 5.E30 (" i, d, J = 3 · 7 11 z ^ 1), 4.93 (1H, d, J = 6 · 4 Hz, (1) "-H), 4 · 50 (1 d-d, J = 3 · 4, 10 · 7 Hz, 3,-H), 4 · 47 (1H, d- d = 3.4, 10.4Hz, (3) '-H), 4.39 (1H, d, J = 7 · z, 1 " -H), 4.33 (1H, br-s, (2 )-H), 4 · 14 (1 m, 2-H), 4 · 1 2 (1 H, m, (3),-H), 4 · 1 2 (1 H, m, -H), 4 1 2 (1 H, m, (5) '-H), 4.0 04 (1H, m, 4 H) > 4. 0 3 (1 H, m, (4) ^-H), 3. 8 5 (1 H, m, 2 ^ -3. 8 5 (1 H, m, (2),-H), 3.82 (1H, m, 3 -H), 3 2 (1H, m, (3)-H), 3.73UH, m, 4-H), 3.73 (m, 5 — H), 3.73 (1H, m, (4) — H ), 3 · 70 (2H, m, H2 of CH2), 3 · 7 〇 (2H, (5)-C 1 忉 of H2), 3 · 6 7 H, m, 5G-H), 3.6 2 (1 H, m, 4 "-Π), 3 · 6 2 (1 H, (2) " -H), 3. 62 (iH, m, (5)-H), 3. 51 (1H , T, J = 8.9Hz, 3 "-H), 3.28UH, t, J = 7. 9 H z, 2 H), 1 · 0 9 (3 H, d, J = 6. 7H z, (5) '-H3 of C H3), 07 (1H, d, J = 6.7 Hz, 5, H3 of CH3) Sugar composition L-fucose: unsaturated D-glucuronic acid: D-glucose Uronic acid: D-mannose = 2: 1: 1: 2 (L-fucose and D-mannose and unsaturated D-glucuronic acid and D-glucuronic acid each molecule 1 sulfate 2 molecules (three positions of each L-fucose) 尙, the number assigned to the peak in 1 Η-NMR is as follows (VI) Η, J = -11 Π,, J 9 Η Η,, 5 # Η), .8 1 Η, 5-(1 m, i · 2 minutes 45 1237026 CH2〇h

Oil VI) 46 1237026 If the fucose-containing sulfated polysaccharide-U obtained causes the aforementioned terminal fucoidan to decompose the enzyme, the absorbance at 230 nm caused by the dissociation reaction increases as the reaction proceeds. Unsaturated hexuronic acid groups become 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 interacts with mannose. 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 have made reference to Carbohydrate Research for the purpose of clarifying that the hexuronic acid and mannose present in the molecule of the fucose-containing sulfated polysaccharide mixture of the high fruit kelp are interactively combined in the sugar chain. Volume 125, pages 283 ~ 290 (1 9 84). First, the fucose-containing sulfated polysaccharide mixture is first dissolved in 0.3 M oxalic acid and treated at 100 ° C for 3 hours. The molecular weight is classified and the molecular weight is collected as More than 3,000 dissolved fractions were collected by 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 of 尙 HPLC are as follows. Device: L-6200 (manufactured by Hitachi) Column: PERPACK type N (4.6mmX250_) (manufactured by Takara Shuzo Co., Ltd.) Eluent: 200 mM acetic acid-triethylamine buffer (PH7.3) · • acetonitrile = 25: 75 Detection : Detection with a fluorescence detector F-1 1 50 (made by Hitachi) at an excitation wavelength of 3 20 nm and a fluorescence wavelength of 400 mm. Flow rate: 0.8 ml / min 47 1237026

Column temperature: 4 (TC 尙, standard material of PA-hexuronic acid glucuronic acid is manufactured by s I GMA, galacturonic acid is manufactured by Wako Pure Chemical Industries, and iduronic acid is manufactured by SIG M A. The 4-methylumbellyl-a-L-idurose acid acid hydrolyzed, mannose acid I and gluconic acid are according to Acta Chemica Scandinavica, Vol. 15, No. 1 3 9 7 ~ 1 The method described on page 398 (1 961) 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, the fucose-containing sulfated polysaccharide mixture can be determined. The hexuronic acid contained in the sugar chain is only glucuronic acid. Moreover, the glucuronic acid in the above-mentioned sugar chain hydrolysate is separated from D-mannose through an anion exchange resin and freeze-dried to determine its specific optical rotation. At this time, it can be judged as D-glucuronic acid of dextran glucuronic acid. Also, for those who treat the fucose-containing sulfated polysaccharide mixture from high fruit beauty with the above-mentioned terminal fucoidan-decomposing enzyme in advance, Acid hydrolysis was performed with oxalic acid in the same manner as above, and D-glucuronic acid and D_mannose were not detected. A polymer that interacts with each other. From this, it can be determined that the skeleton structure of the fucose-containing sulfated polysaccharide having the above-mentioned terminal fucoidan-decomposing enzyme is separated and separated to have D_glucuronic acid and D-mannose It is a structure of interactive binding. Furthermore, in order to investigate the binding positions of D-glucuronic acid and D-mannose and the configuration of sugar 22. Anomer (c) binding, the polymer obtained through the decomposition of oxalic acid was used. NMR analysis. The NMR measurement results of the polymer are shown below. However, the chemical shift in 1H-NMR is the chemical shift with methyl group of triethylamine, which is set to 1. 13 and 48 1237026 " C The chemical shift of the methyl group of triethylamine in -NMR is set to 9.32 ppm without ° and Table 1H-NMR (D2 0) < 55 · 25 (1H, br-s, 1-Η), 4.32 (1 H, d, J-7. Z, i,-Η), 4. · 〇〇 (1H, br-s, 2-1 ", 3.71 (111, 5,-Η), 3.69 ( 1Η, m, 5-CH Η), 3 · (58 (1Π, m, Η), 3.63 (1Η, m, 5-CH Η), 3.63 (lH, m, 4 3 · 57 (1Η, m, 4 -Η), 3.54UH, m, 3 '-Η), 3. · 1Η, m, 5-Η), 3.25UH, t, J = 8.5 · 5Ηζ, 2' -Η) nc — NMR (D2 〇) 5175 · 3 (5 '-COO ΗC), 10 2.5 (1'-C), 99 lC), 78.5 (2-C), 77 · 9 (4 '-C), 77.0 (3'), 7 6. 7 (5 '-C), 73.9 (5-C), 73.7 (2 / -〇 > 6 (3 — C), 67.4 (4 -C) > 6 1. 0 (5 — C of CH2 OH) 6 Η m · 3 — Η), 3 (6 (C 7 0. 尙), the number assigned to the spike is as follows (V 丨 丨):

Due to the stereo configuration of the 1-position of D-glucuronic acid, the fixed binding number is 7.6 Hz, so it is determined as / S-D-glucuronic acid. 49 1237026 In addition, due to the steric configuration of the 1-position mannose, the chemical shift 値 is 5.25 ppm, so it is 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 can be confirmed that there are staggered spikes between 1-H and VC, between 4'-H and 1-C, between 1 '-Η and 2-C, and between 2-' and 1 '-C. . It can be known from this that D-glucuronic acid is bound to the 2-position of D_mannose by a / 3-bond, and D-mannose is bonded to the 4-position of D-mannuronic acid by an X bond. If the above results are considered in combination, it can be determined that (a) is a structure in which unsaturated D-glucuronic acid is bound to L-fucose having a sulfate group bound to 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-fucose with sulfate group, and binds D- to its D-glucuronic 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 (V I I I) (however, at least one of the alcoholic hydroxide groups of 50 1237026 is sulfated, and η is an integer of 1 or more)

C-OH CH2 OH

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 a fucoidan 3 ·· SA- 00 8 2 (CCRC 910069) 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 prepared, 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, so it can provide substantially no anticoagulant 51 /; I23702 «1 ~ blood activity Contains fucose sulfate polysaccharide. The fucose-containing sulfated polysaccharide-ϋ of the present invention can be used as a carcinogen, an anti-metastatic agent, a carcinogenic preventive agent, and the like, and can also be used as an anti-fucose-containing sulfated polysaccharide antibody. Of the antigen. Furthermore, fucose-containing sulfated polysaccharide-U can be used to produce oligosaccharides having the structures of the formulae (I), (I I), (I I I) and the like, and they are 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 the fucose-containing sulfated polysaccharide-F of the present invention is produced by using the method of the present invention, if a decomposing enzyme having the ability to decompose fucose-containing sulfated polysaccharide-U is allowed to act on the fucose-containing sulfated polysaccharide mixture, After the 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 an enzyme capable of selectively decomposing fucose-containing sulfated polysaccharide-U. Specific examples thereof include, for example, Xanthobacter (? 1 & ¥ (^ 3 (^ 6 ] ^ 11111) 8? .3 six-0082 strain ((^ 1 ^ 9 1 0069) produced by the above-mentioned terminal fucoidan-decomposing enzyme.

When the enzyme works, the substrate concentration, temperature, and pH can be set under 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 decompose enzymes are cultured in the medium, and the culture medium 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 Flavobacterium sp. SA- 0082 described in 52 1237026 ( CCRC 9 1 0069) or Fucoidanobacter mar inns 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, the culture conditions are, of course, to set the decomposition activity of fucose-containing sulfated polysaccharide-U to the maximum depending on the strain used and the composition of the culture medium. Generally, if the culture temperature is 15 to 30 ° C, the pH of the culture medium is 5 ~ 9, culture under aeration for 5 ~ 7 for 2 hours. After the end of the culture, the low molecular weight fucose-containing sulfated polysaccharide-U and the medium other than the fucose-containing sulfated polysaccharide-p1 in the culture 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- 009 8 a. Type properties (1) The bacterium is Brevibacterium (Bacteroides) wide 0.5 to 0.7 // IT1 length 0.5 to 0 · 7 μm 53 1237026 (2) 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 aesculin positive (7) nitric acid Negative reduction of salt (8) Negative generation of indole (9) Positive generation of hydrogen sulfide (1 0) Negative coagulation of milk (1 1) Positive requirement for sodium (1 2) Salt required for 0% salt medium Growth negative in 1% common salt medium Growth negative in seawater medium 54 1237026 (13) Xing • Methylnaphthalene Xing 7 (14) GC content of DNA in cells 61% (1 5) Diamine pimelic acid negative (16) Ethanol 醯 test negative (17) Positive presence of hydroxy fatty acids (18) OF-test 0 (1 9) The hue of the colonies does not generate characteristic colony pigments (2 0) movement Sexuality (2 1) Slip-out Sexuality (22) Flagella extremely monopilous This strain is classified into group 4 according to the basic classification described in Bergey's Manual of Determinative Bacteriology, Volume 9 (1 994) (Gram negative aura Bacilli 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 Cy to phag a have the exception of menaquinone in the electron transfer chain, but the GC content of bacteria belonging to these genera is found in the soil bacteria Cytophaga 43 to 46% of arvensicola and so on, 42% of marine bacteria Cytophaga diffluens, Cytophaga fermentans, Cytophaga marina and Cytophaga uliginosa, 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 identity of the identified strain Verrucomicrobium spinosum with the highest identity of 55 1237026 was 76.6%. Since it is generally known that the genus of the two bacteria is different when the identity of the 16Sr DNA sequence is less than 90%, the inventors have determined that this strain is a new genus bacterium that does not belong to the existing genus, and accordingly named this strain For 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. 6〜3M。 First add one or two or more kinds of salts to the aqueous solution containing the fucose sulfate polysaccharide mixture and make the total concentration of 0.6 ~ 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 sulfuric acid polysaccharide-F of the present invention is difficult to form a precipitate through cetylpyridinium chloride, it needs attention. For the purpose of separating the fucose-containing sulfated polysaccharide-F and the fucose-containing sulfated polysaccharide-U 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 to obtain the fucose-containing sulfuric acid 56 1237026 polysaccharide-F of the present invention. 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 lmM 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, according to the description of 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 using the gel filtration method of Sepephry 1 S-500, shows a molecular weight distribution centered at about 19,000 (refer to FIG. 17) . That is, in Figure 17, the vertical axis 57 1237026 is the sugar content in the sample measured by the phenylhydrazone-sulfuric acid method. The absorbance is expressed at 480 nm. The horizontal axis is the dissociation number. Alas, the conditions for gel filtration are shown below. Column size: 3.08X162.5 cm Solvent: Contains 0 · 2M sodium chloride and 10 mM sodium phosphate buffer solution (PH6 · 0) containing 10% ethanol Flow rate: 1.5 ml / min Sample concentration: 0.2 5% Sample volume : 20 halo; molecular weight reference material: Shodex STANDARD P-82 (manufactured by Showa Denko Corporation) Next, the components of the fucose-containing sulfated polysaccharide-F of the present invention were 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 monosaccharide. 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 (4.6mmXl50mm: manufactured by Takara Shuzo Co., Ltd.) Eluent: 700mM boric acid buffer (PH9 · 0); acetonitrile = 9: 1 58 1237026 Detection Fluorescence detector F-11 50 (manufactured by Hitachi, Ltd.) at an excitation wavelength of 310 nm and a fluorescence wavelength of 38 nm. Flow rate: 0.3 ml / min

Column temperature: 65 ° C Secondly, the amount of uronic acid was quantified as described in Analytical Biochemistry, Volume 4, page 330 (1962). Second, the sulfuric acid content was quantified as described in Biochemical Jouornal, Volume 84, Page 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, 50 ml of phosphoric acid buffer, 12 ml of washing solution (ρΗ8 · 0), 4 ml of sodium chloride 4 ml, and 32 μm / m of the aforementioned from Xanthomonas sp. 8 ml of a fucoidan-decomposing enzyme solution of SA-0082 (CCRC 910069) 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 with a Fourier transform infrared spectrophotometer IR-DIAMOND 20 (manufactured by Nippon Denshi) when the spectrum shown in FIG. 18 was obtained. A. In Fig. 18, the vertical axis indicates the transmittance (%), and the horizontal axis indicates the wave number (cm · 1). 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 JNM-α 5 00 type nuclear magnetic resonance apparatus (manufactured by Japan Electronics Co., Ltd.), as shown in Figure 19 Of the spectrum. 59 1237026 In Figure 19, the vertical axis represents the signal strength and the horizontal axis represents the chemical shift 値 (ppm).尙, the chemical shift Η in 1Η-NMR is represented by the chemical shift H of HOD as 4.65 ppm. 5.30 (H f in fucose position), 1.19 (H in fucose 5 position CH3) Second, the specific optical rotation of the freeze-dried product containing fucose sulfate polysaccharide-F is determined by high speed and high sensitivity polarimeter SEPA -3 00 (manufactured by Horiba) is -135 degrees during measurement. According to the present invention, there is provided a purified fucose-containing sulfated polysaccharide-F which is separated from the fucose-containing sulfated polysaccharide-ϋ of the present invention. The fucose-containing sulfate poly preparation-F of the present invention is a fucoidan that does not substantially contain uronic acid as a constituent sugar and does not undergo decomposition by fucoidan sp. SA-0082 (CCRC 9 1 0069). Enzymes 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. The fucose-containing sulfated polysaccharide of the present invention, if the decomposition product is added to the nourishing liquid of cancer cells 60 1237026 at a concentration of 1 microgram / ml or more, the cancer cells will cause the cells to self-destruct from a few 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. Alas, 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 1237026 < 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 > Starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose. < Surfactant > Sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80. < Lubricants > Talc, waxes, hydrogenated vegetable oils, sucrose fatty acid esters, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol. < 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 parenteral preparations, the fucose-containing sulfated polysaccharide and / or its decomposed product, which is an effective ingredient of the present invention, can be dissolved or suspended in distilled water for injection, physiological saline, and glucose aqueous solution as a diluent. , Vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, etc., and if necessary, can be prepared by adding a fungicide, stabilizer, isotonicity agent, analgesic agent and the like. 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 1237026 The administration amount 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 of parenteral agents such as injections and drip preparations 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. Injections can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, and the like, and external use 63 1237026 can also include raccoon and the like. The dosage of carcinogens may vary depending on the system and the age and body of the patient to which it is applied, but it is generally 1 to 1000 mg per day in the preparation, and it is preferably changed under various conditions, so there are comparative situations. Or if it is necessary, it can be taken orally and daily intake. If the carcinogenic inhibitory substance is used as an active ingredient, it should be combined with a carcinogenic agent. Carcinogenic agent OK. Generally, a liquid or solid carrier containing more fucose and sulfuric acid is mixed with an emulsifier, a buffer, a stabilizer, a slip agent, etc., and used as a lozenge, a granule solid agent, a usual liquid agent, and suspended in use. Before the addition of an appropriate carcinogenic agent, an oral agent can be administered, and any of the oral agents can be administered. The pharmaceutical carrier can be administrated by the above-mentioned cell auto-inactivation inducer as a quasi-carcinogenic preventive agent, and can be appropriately set according to the formulation type _ dosage form, administration method, purpose of use, and symptoms, although it contains active ingredients The amount is ~ 200 mg per adult. It is a matter of course that the amount to be administered is a sufficient range in accordance with the above-mentioned amount that is still small. The medicament of the present invention can be added to any glutinous food, and can be prepared by preparing fucose-containing sulfated polysaccharide and / or its known pharmaceutical carrier. The sugar can be prepared according to the above method and / or Decomposed products and pharmaceuticals are acceptable, and if necessary, solvents, dispersants, excipients, binding agents, disintegrating agents, emollients, powders, powders, capsules and other floating agents, emulsions and other liquids are added. It can also be used as a liquid and dried product. It can be selected as a non-preservative form and dosage form for injections, drips, etc., and can be used if necessary. Invest in an appropriate route. 64 1237026 The method of administration is not particularly limited, 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 a case where the amount is smaller than the above-mentioned amount and it 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 '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, the fucose-containing sulfated polysaccharide is a sulfated polysaccharide with a very large molecular weight, 65 1237026. Therefore, in order to improve the antigenicity, uniformity, and anticoagulant activity, etc., than to use it as a pharmaceutical product, it is necessary to make fucose-containing Sugar sulfate polysaccharide is decomposed to a certain degree. According to the present invention, in order to provide an enzyme that selectively decomposes fucose-containing sulfate polysaccharide-F, and a low-molecular-weight compound of fucose-containing sulfate polysaccharide-F obtained by the action of the enzyme . 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, a strain of Symbiotic sp. Sp. SN-109. 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 bacterium with a width of about 1 // m and a length of about 2 A m (2) the presence or absence of spores (3) negative Gram staining b. physiological properties (1) temperature range for growth 66 1237026 Suitable growth temperature is 15 ~ 3 (TC. Cannot grow at or below 40t. (2) Attitude to oxygen Aerobic (3) Catalytic enzyme positive (4) Oxidase positive (5) Lipase positive (6 ) Information glucose positive mannose negative sucrose positive lactose negative cellobiose positive melibiose negative mannitol positive glycerol positive methanol negative DL-malate negative succinate negative fumarate negative citrate negative salicylate negative (7) Hydrolyzed starch-negative gelatin-negative 67 1237026 (8) Nitrate reduction-negative (9) Negative denitrification reaction (1 0) Alginic acid decomposition-positive (1 1) / 3 -hydroxybutyric acid negative (12) Polyhydroxybutyric acid accumulation negative (1 3) Sodium positive requirement (1 4) Salts required to grow negative in ο% common salt medium Negative growth positive in 1% common salt medium Growth positive in seawater medium ( 1 5) quinone series GC content of DNA in ubiquinone (16) bacteria 36% (17) OF-test 0 (18) hues (19) luminous (20) motility (21) flagella does not produce characteristic colony pigments Negative positive monopilus strains were identified as Bergey's Manual of Systematic Bacteriology, Volume 1 'Pages 343 to 352, and Bergey's Manual of Determinative Bacteriology, Volume 9, Page 75, Pages 13 2 to 133 (1 The symbiotic bacterium described in 994) is a bacterium. However, the physiological characteristics of this bacterium are not consistent with any of the species described, and the GC content is also low. Therefore, this strain was named as a symbiotic bacterium. Genus sp. SN-1009. 68 1237026 菌株, the above strains are represented by Symbiotic sp. SN-1 009, in the Institute of Biotechnology and Industrial Technology, Industrial Technology Institute, Ministry of International Trade and Industry Shidong 1chome 1fan 3 (postal code 3 0 5)] was deposited with FERM P-15436 from February 13th, 2008, and was registered at the Institute of Biotechnology, Industrial Technology, Industrial Technology Institute, Ministry of International Trade and Industry China-Israel FERM BP-5747 (Application for transfer in International Deposit: Heisei (November 15, 8), and was deposited in the strain preservation 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 used strain, and can produce terminal fucose-containing sulfate polysaccharide decomposing enzyme, the carbon source can be, for example, fucose-containing sulfate polysaccharide, 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, chlorine Ammonium chloride and the like are suitable. Others, such as sodium salt, phosphate, potassium salt, magnesium salt, zinc salt, etc., and metal salts may be added. In addition, this strain grows very well in seawater or artificial seawater containing the above-mentioned nutrient sources. When the terminal type fucose-containing sulfate-polysaccharide-decomposing enzyme-producing bacteria of the present invention is cultured, the production amount varies depending on the culture conditions, but generally the culture temperature is 15 t: ~ 30 ° C, and the pH of the medium is 6 It is better to be ~ 9. Under aeration and agitation culture for 5 to 7 2 hours 69 1237026, the production amount of the terminal type fucose-containing sulfate polysaccharide decomposing enzyme of the present invention reaches the maximum. The culture conditions of course depend on the strain used, the composition of the culture medium, etc., and set the production amount of the terminal fucose-containing sulfated polysaccharide-degrading enzyme of the present invention to be the most terminal type of fucose-containing sulfated polysaccharide-degrading enzyme of the present invention that exists in the cell Also present in the mash on the culture. If the above-mentioned Symbiotic genus sp. SN-1 009 is cultured in an appropriate medium, and the bacterial cells are collected, the bacterial cells are broken by the usual cell destruction means, such as ultrasonic treatment, etc. Cell extract. Secondly, a sample of the purified enzyme can be obtained from the extract by the usual purification methods. For example, purification by salting out, ion exchange column chromatography, hydrophobic bond column chromatography, gel filtration, etc. can obtain a purified fucose-containing terminal type of the present invention that does not contain other fucoidan-decomposing enzymes. Sulfated polysaccharides decompose enzymes. In addition, since the present enzyme is also abundantly present in the mash on the culture solution in which the bacterial cells are removed from the culture solution, it can be purified by the same purification method as the enzyme in the bacterial cells. The chemical and physical and chemical properties of the terminal fucose-containing sulfated polysaccharide-decomposing enzyme of the present invention are as follows. (i) Action: The action of fucose-containing sulfated polysaccharide having the following physical and chemical properties, that is, 70Ϊ237026 fucose-containing sulfated polysaccharide-F ', makes the fucose-containing sulfated polysaccharide-F low-molecular. (a) Constituting sugars ... It does not substantially contain uronic acid. (b) The fucoidan-decomposing enzyme produced by Flavobacterium sp. SA_ 0082 (CCRC 9 1 0069) is not substantially reduced in molecular weight. Does not act on fucose-containing sulfated polysaccharides that have the following physical and chemical properties, that is, fucose-containing sulfated polysaccharide-U (c) Constituting sugar: contains uronic acid. (d) The fucoidan produced by Flavobac ter ium sp. SA-0082 (CCRC 910069) is degraded and reduced to a low molecular weight, and at least the following formulae (I), (II) (III) Select at least one compound. 71 1237026

CH2 OH

OH t 0

CH2 0 — S-OH

〇 A

0 = 5 = 0

OH

OH 72 1237026

OH 〇 II Ho— s— o II 〇

OH

73 1237026 (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 shows that the PA-containing rock bath sulphuric acid polysaccharide-f (pa _ f p1) is used as the receiving curve for the reducing end, and the dotted line is the fucose-containing sugar as described in (V)-(2) below. Sulfated polysaccharide_F was used as the curve of mass. (i i i) Optimum temperature: The optimum temperature of this enzyme is around 30 ~ 35 (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 (t). The solid line shows the curve at the time of mass reduction using PA-modified fucose-containing sulfated polysaccharide-ρ (PA-FF), and the dotted line is the fucoid containing the following (V)-(2) The sulphuric acid polysaccharide_F is used as the curve of the mass. (iv) Molecular weight: The molecular weight of this enzyme is about 100,000 when calculated by a gel filtration method using sepharcryl S-2 00 (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, as the terminal type fucose-containing sulfated polysaccharide decomposing enzyme of the present invention, the fucose-containing sulfated polysaccharide F and PA-FF 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 1237026 Machinery Co., Ltd.) and crushed at 4.5 times. The amount of 80% ethanol in 8 (TC, 2 hours after treatment, filtration. The residue was extracted with the above 80% ethanol, the filtration process was repeated 3 times to obtain 1 870 grams of ethanol washing residue. 36 liters of water was added to the residue After being treated at 100 ° C for 2 hours and filtered, an extract can be obtained. After the salt concentration of the extract is the same as that of a 400 mM sodium chloride solution, 5% cetylpyridinium chloride is used so as not to cause precipitation again. Add and centrifuge.

This precipitate was repeatedly washed with 80% ethanol, and after completely removing 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. Make 100 ml of DEAE-Cellulofine A-800, filter after stirring, and remove the resin. This filtrate was placed in 100 ml of DEAE-Cellulofine A-800 particles equilibrated with 2M sodium chloride, and the fractions passed therethrough were subjected to desalting and low-molecular removal using an ultrafilter (the molecular weight of the filter membrane was 100,000). The resulting precipitate 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 dissolved in 600 ml of 20 mM sodium acetate (pH 6.0) containing 0.2 M calcium chloride. After that, it was placed in a 3600 ml DEAE-Sepharose FF column equilibrated with 20 mM sodium acetate (pH 6.0) containing 0.2 M calcium chloride, and 20 mM sodium acetate (pH 6.0) containing 0.2 M calcium chloride. After the column was thoroughly washed, it was dissolved with a sodium chloride gradient of 0 to 2M. Collect dissolving fucose-containing sulfuric acid with more than 127.55M sodium and more than 75 1237026 sugar-F dissolving fractions, collect and desalinate them with an ultrafilter equipped with an ultrafiltration membrane with a molecular weight of 100,000, and freeze-dry them. 3.3 g of a freeze-dried sample containing fucose sulfate polysaccharide F was obtained. (3) Preparation of PA-FF After dissolving 12 mg of the above-mentioned freeze-dried sample containing fucose sulfated polysaccharide-F in 480 microliters of water, and aliquot 36 microliters in 12 microliters each, The lyophilized Glyco TAG and Glyco TAG reagent kits were used to PA-reducing the reducing end to obtain PA-FF. The obtained PA-FF was dissolved in 15 ml of a 10 mM solution of 10 mM methanol in 10 mM ammonium acetate, and subjected to gel filtration on a Cellulofine GCL-300 column (40 X 900 mm), 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. The PA-FF obtained in this way was quantitatively compared with the commercially available pyridyl- (2) -amidated fucose (manufactured by Takara Shuzo Co., Ltd.) with fluorescence intensity (excitation wavelength 320 nm, fluorescence wavelength 400 nm). About 40nmol. To determine 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. That is, 12 microliters of a 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 containing acetic acid, imidazole, and Ti · is-hydrochloric acid (ρΗ7.5), mixed with 18 microliters of the terminal fucose-containing sulfated polysaccharide-F decomposing enzyme of the present invention, and mixed with 76 1237026. After 3 hours of reaction at ° C, the reaction solution was treated at 100 ° C. 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 mol of fucose-containing sulfated polysaccharide-F fucosyl bond in the above reaction system at 1 minute. The amount of the fucosyl bond to be cleaved is calculated by the following formula. {(1 2 X 2.5. 5) / (1 00 X MF)} X {(MF / M)-1} X {0. 12 / (180X0.01) 2 U / ml (12X2.5) / 100: Fucose-containing sulfated polysaccharide-F (mg) added in the reaction system MF: average molecular weight of the fucose-containing sulfated polysaccharide-F (M): average molecular weight of the reaction product (MF / M)-1: 1 molecule Number of Fucose Sulfate Polysaccharide-F Cut by Enzymes 1 8 0: Reaction time (minutes) 0.01: Amount of enzyme solution (ml) 0. 1 2 ·· Total reaction solution (ml) 尙, HPLC conditions are See the following: 77 1237026 Device: L-6200 (manufactured by Hitachi) Column: OHpak KB- 804 (8mmX 3 00mm) (manufactured by Showa Denko Corporation) Eluent: 5mM sodium azide, 25mM calcium chloride, and 50 mM sodium chloride in 25 mM imidazole buffer (pH 8) 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 polytriglucose (STANDARD P-82, manufactured by Showa Denko Corporation) was analyzed under the same conditions as the HPLC analysis described above. The relationship between the molecular weight of polytriglucose and the residence time of OHp ak KB-8 04 is expressed as a curve, which is used as a standard curve for determining 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 pL of 8-pmol / μ1 PA-FF solution, and 5 pL of 1M calcium chloride solution, 10 pL of 1 M sodium chloride solution, and 23 pL of water, and 50 pL 5 mM of a buffer solution (pH 8.2) containing acetic acid and imidazole and Tris-hydrochloric acid 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 1237026 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 PA-FF solution as a control group were analyzed by HPLC in the same manner. One unit of enzyme is the amount of enzyme that cleaves the fucosyl bond of 1 # m0 1 containing fucose sulfate polysaccharide in 1 minute in the above reaction system. The amount of the fucosyl bond to be cleaved is calculated by the following formula. 16X10-6 (MF / M) -1} X {1 / (180X0.01)} = U / ml 16 X 10 · 6 PA-FF (// mol) added to the system MF: PA-FF Average molecular weight M: average molecular weight of reaction product (MF / M) -1: 1 molecule of fucose-containing sulfated polysaccharide_f number cut by enzyme 180: reaction time (minutes) 0 · 0 1: enzyme solution Amount (ml) 尙, HPLC conditions are as follows. Device: L-6200 (manufactured by Hitachi) Column: OHpak SB-803 (8mmX300mm) (manufactured by Showa Denko Corporation) Eluent: 5mM sodium azide and 10% dimethylsulfonate 200 mM Sodium chloride solution detection: Fluorescence detector F-1 150 (manufactured by Hitachi) was used to detect at an excitation wavelength of 3 20nm and a fluorescence wavelength of 400nm.

Flow rate: 1 ml / min. Column temperature: 50 ° C 79 1237026 In order to determine the average molecular weight of the reaction product, commercially available polytriglucose (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 of 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 residence time of 0Hp ak SB-803 was plotted as a curve to measure the enzyme reaction. Standard curve of product molecular weight. Protein was quantified by measuring the absorbance at 280 nm of the enzyme solution. At this time, the absorbance of the 1 mg / ml protein solution was calculated as 1.0. 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 the high fruit kelp. 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. In other words, sp. SN-1 009 (CCRC 9 1 0070) was inoculated into artificial seawater (made by Germaline Laboratory) containing 0.25% glucose, 1.0% protein gland, and 0.05% yeast extract. 600 ml of the medium consisting of pH 8.2 and sterilized (120 ° C, 20 minutes) in a 2 liter Erlenmeyer flask, and cultured at 25 ° C for 26 hours to make a seed culture solution. Contains 0.25% glucose, 1.0% protein gland, 0.02% enzyme extract, 0.2% fucose-containing sulfated polysaccharide from high fruit kelp, and an antifoaming agent (manufactured by Shin-Etsu Chemical Co., Ltd. 80 1237026) KM70) 0.01% artificial seawater (manufactured by Germalin Laboratory) pH 8.0. 20 liters of culture medium was placed in a 30 liter capacity fermentation tank and sterilized at 120 ° C for 20 minutes. After cooling, inoculate 600 ml of the above-mentioned seed and incubate at 24 ° C for 24 hours, with a ventilation volume of 10 liters per minute and a stirring speed of 125 revolutions per minute. After the end of the culture, the culture solution is centrifuged to obtain bacterial cells and culture supernatant. The resulting culture supernatant was concentrated in an ultrafiltration filter with a molecular weight of 10,000, and then salted out with 85% saturated ammonium sulfate. The resulting precipitate was collected by centrifugation, and 20 mM Tris containing 1/10 concentration of artificial seawater was collected. -Fully dialyzed with hydrochloric acid buffer (pH 8.2) to obtain 600 ml of crude enzyme. 40 ml of the crude enzyme obtained in this way, 44 ml of artificial seawater, 510 mg of fucose-containing sulfated polysaccharide-F and 36 ml of water were mixed, and the pH was adjusted to 8 at 25 ° C After 48 hours of reaction, gel filtration was performed with Cellulofine GCL-300, and it was divided into 4 parts. The order of the molecular weight was F-Fd-1 (molecular weight of more than 25,000) and F-Fd-2 (molecular weight of 25000). To 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 to obtain 170 mg, 270 mg, 300 mg, and 340 mg of dried products. Fig. 22 shows the results of the enzyme degradation product containing fucose sulfated polysaccharide-F, that is, low molecular weight filtration through Cel 1 ulof ine GCL-3 00 gel filtration. In Fig. 22, the vertical axis represents the absorbance at 480 nm (the amount of color produced by the phenol-sulfuric acid method), the horizontal axis represents the number of the dissociated fraction, and the 1 dissociated fraction is 10 ml. The column volume was 107 5 81 1237026 ml, and the eluent was a 0.2M ammonium acetate solution containing 10% methanol. In Figure 22, the white circle marks indicate the gel filtration results of the fucose-containing sulfated polysaccharide-F-containing enzyme degradation products, and the black triangle marks indicate the fucose-containing sulfated polysaccharide-F-containing gel filtration results 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 decomposing enzyme of the present invention is about 10,000 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 reducing end, and each of the PA-sugars (PA-F-Fd-1), (PA-F-Fd-2), (PA-F-Fd-3) ) And (PA-F-Fd-4) were hydrolyzed with 4 equivalents of hydrochloric acid and 100 ° C for 3 hours, 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 (4.6_xi50mm) (manufactured by Takara Shuzo Co., Ltd.) Eluent: 700mM boric acid buffer (pH9): acetonitrile = 9: 1 Detection: Fluorescence F-1 150 (made by Hitachi) is detected at an excitation wavelength of 310 nm and a fluorescent wavelength of 380 nm. Flow rate: 0.3 ml / min

Column temperature: 6 5 ° C The results of (PA-F-Fd-1), (PA-F-Fd-2), (PA-F-Fd-3), and (PA-F-Fd-4) The reducing terminal sugar is fucose. 82 Ϊ237026 The neutral sugar composition of F-Fd-1, F-Fd-2, F-Fd-3 and F-Fd-4 &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 in accordance with the instructions using an F-set 'lactose / galactose (manufactured by Bailing Kazanouchi Co., Ltd.), F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4, which were hydrolyzed with 4 equivalents of hydrochloric acid at 1000 ° C for 2 hours for 2 hours, were used as the reaction system. Determination. Furthermore, in order to quantify the other L-fucose, which constitutes sugar, according to the method described in Clinical Chemistry, Vol. 36, pp. 474-476 (1 990), another 4 equivalents of hydrochloric acid was added at 100 ° C. 2, F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4 hydrolyzed for 2 hours after neutralization, and measured by this reaction system. From the above results, the ratio of L-fucose to D-galactose is about 100: 44, 100: 27 for 卩 ^ 1, F-Fd-2, F-Fd-3, and F-Fd-4, respectively. , 100: 5 and 100: 1. The results summarized above can be used to judge that the terminal type fucose-containing sulfated polysaccharide decomposing enzyme of the present invention acts on the fucose-containing sulfated polysaccharide-F and hydrolyzes the fucose 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. Thallium, 83 1237026 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 polysaccharide sulfate_U is acted by the fucose-containing sulfate polysaccharide decomposing enzyme of the present invention. That is, '2.5 microliters of fucose-containing sulfate polysaccharide solution u 12 microliters, and 6 microliters of 1M calcium chloride solution and microliters of 1M sodium chloride solution, and 72 microliters of 50mM imidazole buffer (PH 7.5), mixed with 18 microliters of the terminal fucose-containing sulfated polysaccharide-degrading enzyme (1.6mU / ml) of the present invention, and reacted at 30 ° C for 3 hours, and then the reaction solution was subjected to 100%. After treatment at 10 ° C for 10 minutes, after centrifugation, 100 microliters were analyzed 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, Ltd.) Column: OHpak KB-804 (8mmX300mm) (manufactured by Showa Denko) Eluent: 25mM imidazole containing 5mM sodium azide, 25mM chloride, and 50mM sodium chloride Buffer (pH 8) Detection: Parallax refractive index detector (Shodex RI-71, manufactured by Showa Denko Corporation) Flow rate: 1 ml / min

Column temperature: 2 5 ° C As a result, the fucose-containing sulfated polysaccharide-degrading enzyme of the present invention is completely free of 84 1237026 method to lower the fucose-containing sulfated polysaccharide-U. 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 dissolving solution may be selected according to the purpose, such as water and a buffer solution. The pH of the dissolving solution is usually neutral, and the enzyme reaction is usually carried out near 30 ° C. By adjusting the amount of enzyme, reaction time, etc., the molecular weight of low molecular compounds can be adjusted. 85 1237026 Secondly, the molecular weight fractionation of the low-molecular compounds can prepare a more uniform molecular weight distribution of the fucose-containing sulfated polysaccharide-F low-molecular 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, can be subjected to refining operations such as ion-exchange resin treatment and activated carbon treatment, and desalted, aseptically processed if necessary, and freeze-drying can also be used to prepare the low-molecular-weight compounds of the present invention. 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 the dried fruit kelp, 2 kg of the dried product was crushed by a free crusher (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, 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 40 liters of water, treated at 95 ° C for 2 hours, and filtered. The residue was washed with hot water to obtain 36 liters of fucose-containing sulfated polysaccharide extract of high fruit kelp. 4.8 liters of the obtained extract was freeze-dried to obtain 15.4 g of a fucose-containing sulfated polysaccharide sample. Next, 0.4 M common salt was added to the residual extract, and then 5% cetylpyridine 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 0.4 M saline, centrifuged, and washed. This washing operation was repeated three times, and 1 liter of 4M food 86 1237026 brine was added to the precipitate. After being stirred well, ethanol was added to 80%. After stirring, the precipitate was obtained by centrifugation. The operation of suspending this precipitate in 80% ethanol and centrifugation was repeated until the absorbance at 260 nm in the supernatant solution became zero. This Shen Dian was dissolved in 3 litres 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 sulfate polysaccharide mixture weighed 76 grams. Example 2 After the real kelp was sufficiently dried, 2 kg of the dried material 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 hour. 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 40 liters of water, treated at 95 ° C for 2 hours, and filtered. The residue was washed with hot water to obtain 36 liters of fucose-sulfuric acid-containing polysaccharide extract of true kelp. 0.4M common salt was added to the obtained extract, and 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, centrifuged, and washed. This washing operation was repeated three times, and 1 liter of 4M common salt was added to the precipitate. 87 1237026 will be added until the precipitate regenerates, and the precipitate is collected by centrifugation. The precipitate 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. 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-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 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, 0.2 M sodium chloride, 0.2 M calcium chloride, 0 Among 2M magnesium chloride, 4 500ml DEAE-Sepharose FF columns were prepared, and 2 of them were sodium chloride of 0.2M, 1 was calcium chloride of 0.2M, 1 was 0.2 M 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. The fucose-sulfur-containing 1237026 acid polysaccharide mixture dissolved in water, sodium chloride, bromide, and magnesium chloride was respectively placed in DEAE-Sephar ose which was equilibrated with water, sodium chloride, calcium chloride, and magnesium chloride, respectively. In the FF column, each 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 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. Φ Example 6 After fully drying Komaki kelp, 2 kg of it was crushed with a free crusher (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, filter paper was used to filter 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 95 ° C 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 supernatant solution was ultrafiltered to a concentration of 1 liter, and 4 liters of ethanol was added, and the resulting precipitate was collected by centrifugation and centrifuged. After adding 100 ml of 4M saline to the precipitate and stirring well, ethanol was added to 80%. After stirring, the precipitate was obtained by centrifugation. The 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. The resin was removed 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 weight of the freeze-dried fucose-containing sulfated polysaccharide-U was 15 g. The fucose-containing sulfated polysaccharide-U of the present invention does not contain a coloring substance adsorbed to a polysaccharide resin. The fucose-containing sulfated polysaccharide-U reacts with the aforementioned terminal fucoidan-decomposing enzyme to generate oligosaccharides represented by the above formulae (I), (Π), and (I I I). Example 7 After fully drying Gaogomei kelp, 2 kg of it was crushed with a free pulverizer (Nara | Jiki Seisakusho), and the obtained dry powder was suspended in 9 liters of 80% ethanol, and at 80 ° C for 2 hours. After treatment, filter paper was used to obtain radon slag. 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 0.2 M 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 1237026 calcium solution to obtain 36 liters of fucose-containing sulfate polysaccharide extract of high fruit kelp. In the obtained filtrate, a 5% cospirochloride π ratio of 0 was set so as not to cause precipitation again, and the precipitate was collected by centrifugation. The precipitate was suspended in 3 liters of 0.4 M 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 pellet was suspended in 80% ethanol and the operation of centrifugation was repeated.

Until the absorbance at 260 nm in the mash is 0. This precipitate was dissolved in 3 liters of 2M saline, and the insoluble matter was removed by centrifugation. 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-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 0.2 M 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, with a sodium chloride gradient of 0 ~ 4M. Its dissolution. Collect the dissolving fractions with sodium chloride concentration of 0.5 ~ 0.8 Μ in the dissolving fraction and desalting after desalting with dialysis, and then freeze-drying to obtain fucose containing fucosose which is substantially separated from fucose-containing sulfated polysaccharide_F. Polysaccharide sulfate-U 2.1 g. The 'sodium chloride concentration within the above-mentioned dissolution fraction was collected from 0.9 to 1.5 M, and the solution was freeze-dried after desalting with dialysis to obtain fucose containing substantially separated from fucose-containing sulfated polysaccharide-U. Polysaccharide sulfate-F 4.7 g. Example 8 Manufacture of fucose-containing sulfated polysaccharide-F. Weigh 1.2 g of the fucose-containing sulfated polysaccharide mixture obtained in Example 7 and the final concentration was 0.2% in a 1.5 M sodium chloride solution. Dissolve and add 1.5% sodium cetylpyridinium chloride in 1.5M sodium chloride solution so that it will not cause precipitation again. The resulting precipitate was collected by centrifugation, and the precipitate was suspended in 500 ml of 1.5M saline, 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 26 Onm absorbance in the supernatant solution became 0. 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 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 weight of freeze-dried fucose-containing sulfated polysaccharide-93 1237026 F was 710 grams. The fucose-containing sulfated polysaccharide-F does not contain a coloring substance adsorbed to a polysaccharide resin. Further, the fucose-containing sulfated polysaccharide-F is a 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 An enzymatic refining 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- 00 82 (CCRC 910069) breaks down 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. The fucose-containing sulfated polysaccharide-F of the present invention was determined to be fucosyl-containing sulfated polysaccharide-F of the present invention that does not contain uronic acid and contains fucose as a main component of sugar. Example 10 0 Cultivation purification 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 gland and yeast 4 g of the extract was placed in a 30 liter fermentation tank and sterilized. The aforementioned Flavobacterium sp. SA-0082 strain (CCRC 9 1 0069) was planted and cultured at 25 ° C for 24 hours. After centrifuging the culture solution to remove the bacterial cells, ultrafiltration is performed by an ultrafiltration device equipped with a hollow wire with a removal volume of 94 1237026 and a volume of less than 100,000. After the low-molecular substances are completely removed, the insoluble substances are removed by centrifugation and filtration. And freeze-dried. The weight of the freeze-dried fucose-containing sulfated polysaccharide-F was 36 grams. The fucose-containing sulfated polysaccharide-F is a coloring substance that does not contain a polysaccharide resin. This fucose-containing sulfated polysaccharide-F was also judged to be 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 1 1

The freeze-dried product of the fucose-containing sulfated polysaccharide mixture described in Example 7 was weighed into 4 parts of 1 g each, and dissolved in water, 0.2M sodium chloride, 0.2M calcium chloride, and 0.2M respectively. Among the magnesium chlorides, 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, respectively. Be balanced. One of the columns equilibrated with 0.2M 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 1237026 Example 1 2

7 g 'of the fucose-containing sulfated polysaccharide mixture described in Example 1 was weighed and dissolved in 800 ml of 0.2 M 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 washed with 8 liter 0.2M calcium chloride solution. It was dissolved with a sodium chloride gradient of 0 to 4M. 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 desalted after freeze-drying, and 1.4 g and 4.8 g of dried products were obtained, respectively. Example 1 3 In the fucose-containing sulfated polysaccharide mixture obtained in Example 1, the terminal fucoidan produced by Xanthomonas sp. SA-〇82 (CCRC 9 1 0069) was produced. # 角 | _ A vegetative effect produces an oligosaccharide having the following structure.

96 1237026

97 1237026

/ OH ο (in 98 1237026 The inventors and others artificially performed the following enzyme reaction to obtain the above oligosaccharide. That is, 2.5% of the fucose-containing sulfated polysaccharide mixture solution of Example 1 in 80 ml, and 50 mM 60 ml of phosphate buffer solution (pH 7.5) and 20 ml of 4 M sodium chloride and 40 ml of a terminal fucoidan-decomposing enzyme solution of 32 mU / ml were mixed and allowed to react at 251 for 48 hours. The column of Cellulof ine GCL- 3 0 0 (manufactured by Biochemical Industry Co., Ltd.) was classified by molecular weight, and fractions with molecular weight below 2000 were collected. This fraction was desalted with MICR0ACILIZERG3, and 3 fractions were separated with DEAE-SepharoseFF, and then After desalting, it was freeze-dried. In this way, 250 mg, 310 mg, and 52 mg of each of the oligosaccharides of the above formulae (1), (11), and (III) were obtained. Example 1 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 gel filtered with Cel lu lo fine GCL-25 to classify the molecular weight (molecular weight of 5000 or more, 5000 to over 3000, 3000 to super 2000, 2000 to super! 〇〇〇, 1 000 to over 500, 500 or less), in the order of the larger molecular weight, named GFd-01i-1, GFd-01i-2, GFd-01i-3, GFd-01i-4, GFd-oli-5, and. ? 0-〇1 i-6. After desalting these 6 parts, freeze-drying can obtain 2.3 g, 1.7 g, 0.88 g, 1.8 g, 1.4 g, and 0.72 g of dried products, respectively. Example 1 5 99 1237026 Weigh 60 g of the fucose-containing sulfated polysaccharide mixture obtained in Example 1 and dissolve it in 20 liters of artificial seawater. Add 200 g of protein glands and 4 g of yeast extract, and place in 30 liters of After being sterilized in a fermentation tank, the aforementioned Flavobacterium sp. SA-0082 strain (CCRC 9 1 00 6 9) was planted and cultured at 25 ° C for 24 hours. The culture solution was centrifuged to remove bacterial cells, and then ultra-filtered with an ultrafiltration device having a hollow fiber with a molecular weight of less than 100,000. After the low-molecular substances were completely removed, 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 grams. Example 16 6 kg of real sea cucumber was disassembled, the internal organs were removed, and the body wall was collected. 500 ml of acetone was added per 200 grams of wet body wall weight, treated with a homogenizer, and filtered, and the residue was washed with acetone until there were no more coloring matter. The residue was evacuated and dried to obtain 140 g of a dried product. To this dried product was added 2.8 liters of 0.4M saline, and after processing at 100 ° C for 1 hour, it was filtered and the residue was thoroughly washed with 0.4M saline to obtain 3.7 liters of extract. In this extract, 5% cetylpyridinium chloride was added so as not to cause precipitation again, and the resulting precipitate was collected by centrifugation. This precipitate was suspended in 0.4M saline, and then centrifuged again, and 1 liter of 4M saline 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 precipitation.

For this precipitation, the process of filtering and suspending 80% ethanol was repeated until the absorbance at 260 nm of the supernatant solution became zero. The obtained precipitate was suspended in 2 liters of 2M 1237026 saline, and insoluble matter was removed by centrifugation. The supernatant liquid was ultra-filtered with an ultrafiltration device with a molecular weight of 30,000 membranes, and after completely desalting, freeze-drying could obtain 3.7 g of fucose-containing sulfated polysaccharide. Example 17 After fully drying the loquat leaf tip (F ucus Vesicu 10 sus), 2 kg of the dried product was crushed with a free grinder (manufactured by Nara Machinery Co., Ltd.), and the obtained dry powder was suspended in 80% of 9 liters. In 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 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. 0.5M sodium chloride was added to the filtrate, 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 0.4M saline, centrifuged, and washed.

This washing operation was repeated three times, and 250 grams of sodium chloride was added to Shen Dian, and suspended in 3 liters 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 solution 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. 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 filament excluding molecular weight of less than 100,000. The coloring substance and sodium chloride were completely removed. After 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 wakame (Undaria pinnatifida) was sufficiently dried, 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 ethanol and treated at 75 ° C 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. After 684 g of this residue was suspended in 9 liters of 0.2 M calcium acetate, it was treated at 95 ° C. for 1 hour and left to stand for 24 hours to obtain the supernatant liquid. 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 performed while adding 2 mM sodium chloride. After the calcium acetate was completely removed, it was freeze-dried to obtain 3.2 g of freeze-dried product. 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 with a free crusher M-2 (manufactured by Nara Machinery Co., Ltd.), and was crushed at 4.5 Double the amount at 80 ° C in 80% ethanol for 2 hours 102 1237026 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. 36 liters of water was added to the residue and treated at 100 ° C for 2 hours, and filtered to obtain an extract. After the salt concentration of the extract was the same as that of a 40 mM sodium chloride solution, 5% cetylpyridinium chloride was added so as not to cause any further shock, and the mixture was separated by centrifugation. This precipitate was repeatedly washed with 80% ethanol, and the cetylpyridinium chloride was completely removed, and then dissolved in 3 liters of 2M sodium chloride. The insoluble matter was removed by centrifugation, and suspended in 2M chloride. One hundred milliliters of DEAE-Cel lulofine Α-800, sodium balance, was stirred, filtered, and the resin was removed. The 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). The resulting precipitate was removed by centrifugation. By freeze-drying this solution, 82.2 g of refined high-fruit kelp-containing fucose-containing sulfate polysaccharide mixture can be obtained. (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 in medium, it was placed in a 3000 ml DEAE-Sepharose FF column equilibrated with 20 mM sodium acetate (pH 6.0) containing 0.2 M calcium chloride, and 20 mM sodium acetate (pH 6 containing 0.2 M sodium chloride) was used. 0) After the column is sufficiently washed, it is dissolved out with a sodium chloride gradient of 0 to 2M. Collect the fucose-containing sulfated polysaccharide-F dissolving fractions with a concentration of sodium chloride of more than 0.75M, concentrate and desalinize it with an ultrafilter equipped with an ultrafiltration membrane with a molecular weight of 100,000, and freeze-dry it. 3.3 grams of a freeze-dried sample of Fucose 1237026 Sulfate-F. (3) Modulation of a terminal type fucose-containing sulfated polysaccharide-decomposing enzyme will produce Flavobacterium sp. SN-1 009 (CCRC 9 1 0070), which will contain glucose 0.25%, protein gland 1.0%, yeast Extract: 0. 05% artificial seawater (manufactured by Germalin Laboratory), pH 8.2, culture medium consisting of 600 ml, dispensed and sterilized (120 ° C, 20 minutes) in a 2 liter Erlenmeyer flask, and inoculated at 25 ° C Cultivate for 25 hours to make a seed culture solution. 18 liters of a medium consisting of 200 g of protein glands, 4 g of yeast extract, and an antifoaming agent (made by Shin-Etsu Chemical Industry Co., Ltd. 0 7 0) pH 8 · 0 in artificial seawater was placed in a 30 liter capacity fermentation tank Medium and sterilize at 120 ° C for 20 minutes. After cooling, 20 grams of an additional 2 liters of artificial seawater sterilized at 120 ° C for 15 minutes was prepared using fucose-containing sulfated polysaccharide-F from the high fruit kelp prepared by the method of Example 8 and the above-mentioned culture solution. Inoculate 600 ml and incubate at 24 ° C for 20 hours, aeration volume of 10 liters per minute, and stirring speed of 250 rpm. After the end of the culture, the culture solution is centrifuged to obtain bacterial cells and culture supernatant. The activity of the fucose-containing sulfated polysaccharide degrading enzyme of the present invention in the culture supernatant was 5 mU / m 1 of culture medium when the fucose-containing sulfated polysaccharide-F was used as the substrate. After the obtained culture supernatant was concentrated by an ultrafiltration filter with a molecular weight of 10,000, the resulting precipitate was removed by centrifugation, salted out with 85% saturated ammonium sulfate, and the resulting precipitate was collected by centrifugation. / 10 Concentrated artificial seawater (Germalin 5) in 20mM Tris-hydrochloric acid buffer (ρΗ8 · 2) 104 1237026 Fully dialyzed to obtain 400 ml of crude enzyme. The obtained crude enzyme solution was adsorbed to DEAE-Cellulofine A- 800 (produced in 20 mM Tris-hydrochloric acid buffer solution (PH8.2) containing 5 μM sodium azide and 1/10 concentration artificial seawater (Germalin S) in advance. (Manufactured by Chemical Industry Co., Ltd.), and the adsorbate was sufficiently washed with the same buffer solution, and then dissolved in a solution containing 100 mM, 200 mM, 300 mM, 400 mM, and 600 mM sodium chloride in the same buffer solution, and Collect active dissolution fractions.

The activity of the obtained partially purified enzyme was 1,200 mU (10.2 U) when measured using fucose-containing sulfated polysaccharide-F as a substrate. Alas, it was confirmed that no other fucose-containing sulfate polysaccharide-decomposing enzyme was mixed. One part of the obtained partially purified enzyme was coagulated with Sephacryl S-200 equilibrated with 10 mM Tris-HCl buffer (PH8.0) containing artificial seawater (Germalin S) at a concentration of 1/10 and 5 mM sodium azide in advance. It was filtered through a gel and the molecular weight was calculated to be 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 5 OmM acetic acid, imidazole, and Tris-hydrochloric acid. In the reaction solution, sodium chloride was dissolved in a final concentration of 400 mM. The calcium chloride concentration in the reaction solution was changed to 0 to 100 mM, and the enzyme activity was measured to obtain the results shown in FIG. 23. A. In Figure 23, the vertical axis represents relative activity (%), and the horizontal axis represents the calcium concentration () 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 1237026 (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 buffers and holding at 5 ° C for 20 hours. 1 · 20 m Μ Tris-hydrochloric acid buffer (p Η 8. 2) 2. 20 mM Tris-hydrochloric acid buffer (ρΗ8 · 2) containing 5 mM sodium azide 3 · 5 m mM sodium azide and 50 m Μ sodium chloride 20 m Μ T ris-hydrochloric acid buffer (ρ Η 8 · 2)

4. 20mM Tris-hydrochloric acid buffer solution containing 5mM sodium azide and 500mM sodium chloride (ρΗ8 · 2) 5. 20mM Tris-hydrochloride buffer solution containing 5mM sodium azide, 50mM sodium chloride and 10mM bromide Solution (ρ Η 8 · 2) 6. 20mM Tris-hydrochloric acid buffer solution (ρΗ8 · 2) containing 5mM sodium azide and 1/10 concentration artificial seawater (Germalin S)

From the above results, the fucose-containing sulfate polysaccharide degrading enzyme of the present invention dialyzed with 1, 2, and 3 buffers was inactive, and the fucose-containing sulfate polysaccharide degrading enzyme of the present invention was dialyzed with 4, 5, and 6 buffers Stay active. From this, it can be judged that this enzyme is stabilized in the presence of 500 mM sodium chloride or 10 mM calcium ion. (6) Weigh the fucose-containing sulfated polysaccharide F 5 g 'prepared in the above example, and mix 471 ml of 50 mM imidazole buffer solution (pH 8), 12.5 ml of 4 M sodium chloride, and 6.5 ml of 4M calcium chloride and part of the refined product containing fucose sulfate polysaccharide decomposing enzyme of the present invention obtained in Examples 19-(3) 10 ml (only 6mU), and the reaction was performed at 25t for 120 minutes to obtain Fucose sulfate 106 10637026 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 with Cellulofine 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 2 0

Bone marrow leukemia cells HL-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 filtering treatment solution containing fucose sulfate polysaccharide physiological saline solution (5 mg / ml) obtained in Examples 1, 12, 2, and 15 was added to the suspension, respectively. [Filtered with cellulose acetate membrane (KON IC Co., Ltd.) having a pore size of 0.20 // m (the following filtration treatment is performed under this condition)], and cultured in the presence of 371, 5% carbon dioxide for 40 hours. The cultured cells were separated from the supernatant by centrifugation. The obtained cells were suspended in 50 mM Tr 1 s-hydrochloric acid buffer solution (pH 7 · 8) 20 # 1 containing 1 OmM ethylenediamine tetraacetate and 0.5% lauryl sarcosinate, and added 1 / 10 mg / ml ribonuclease A (manufactured by SIGMA) at 50 ° C, 107 1237026

After 30 minutes of treatment, 1 // 1 of 10 mg / ml proteinase K was added and treated at 5 ° 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, the DNA ladder characteristic of cell self-extinguishment was confirmed when the DN Α state in the gel was confirmed using a super-illumination device. Furthermore, for confirmation, use known as 10 μg / ml solution of radiobactin D that induces cell self-killing test reagent to replace the above-mentioned fucose-containing sulfated polysaccharide. When the same operation is performed, the same DNA as in the case of fucose-containing sulfated polysaccharide can be confirmed after 20 hours of culture As a result, it can be known that HL-60 cells induced fucose-containing sulfated polysaccharides obtained in Examples 1, 12, and 15 to induce cell self-destruction.

Using HL-60 (ATCC CCL-2 40), each of the fucose-containing sulfated polysaccharide solutions obtained in Examples 1, 12, and 15 [dissolved at 5 mg / ml in 30 mMHEPES buffer solution (pH 7.2 containing 120 mM sodium chloride) ), And the cell self-killing induction effect at 121 ° C and autoclaving for 20 minutes] was measured as described above, and the same results were obtained. Example 2 1 Bone marrow leukemia cells HL-60 before culture 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 / 9 ml. To this suspension 9 ml, 1 ml of the fucose-containing sulfated polysaccharide physiological saline solution (5 mg / ml) obtained in Example 15 was filtered and treated 108 I237〇26 1 ml of Examples 1, 2, 1 2 ' And a filtered solution of the fucose-containing sulfated polysaccharide physiological saline solution (5 mg / ml) obtained in 16 and cultured at 37 ° C in the presence of 5% carbon dioxide for 60 hours. The cultured cells were separated from the supernatant by centrifugation. The obtained cells were suspended in 50 mM Tris · HCl buffer (pH 7 · 8) 20 // 1 containing 10 mM ethylenediamine tetraacetate and 0. 5% sodium lauryl sarcosinate, and added 1 / 1/10 mg / ml of RNA 18. A (manufactured by SIG M A), after 30 ° C treatment at 50 ° C, add 1 # 1 of 10 mg / ml of proteinase K, at 5 ( TC treatment for 1 hour. Using the treated cells as samples, electrophoresis was performed using a 2% agarose gel at a constant voltage of 100 V. This gel was immersed in ethidium bromide solution for 30 minutes, and the coagulation was confirmed using an ultra-lighting device. In the DNA state in the gel, the DNA ladder characteristic of cell self-destruction is confirmed. Furthermore, for confirmation, a 10 mg / ml solution of radiobactin D known as a test agent for inducing cell self-destruction was used instead of the above-mentioned fucoid When the same procedure was performed for sugar sulphuric acid polysaccharides, the same DNA ladder as in the case of fucose-containing sulfuric acid polysaccharides could be confirmed after 20 hours of culture. From this result, the content of the sugars obtained in Examples 1, 2, 12, 2, and 16 could be determined. Fucose Sulfate Polysaccharide induces self-destruction of MOLT-3 cells. Obtained from Examples 1, 2, 12 and 16 Fucose-containing sulfated polysaccharide solution [dissolved in PBS at 5 mg / ml (8 g of sodium chloride, 0.2 g of potassium chloride, 2.9 g of disodium hydrogen phosphate 12 hydrate, and 0.2 g of potassium dihydrogen dihydrochloride (Dissolved in 1 liter of water), and at 121 ° C. The autoclavable induction effect of autoclaved sterilization for 2 () minutes) was determined as described above to obtain the same results. Example 24 111 1237026 Example 15 The fucose-containing sulfated polysaccharide and dextran sulfuric acid show substantially the same curves as those of the fucose-containing sulfated polysaccharide obtained in Example 15.

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 are 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 sulfuric acid (molecular weight 500,000 ' The self-killing induction effect of each solution (made by Wako Pure Chemicals Co., Ltd.) (dissolved in 0.5 mg / ml in PBS, at 1 21 ° C, autoclaved for 20 minutes) was determined in accordance with the above, and obtained Same result. Example 2 5 PRMI 1640 medium (manufactured by GIBC0) treated with bovine fetal blood pupa (JRH BIOSCIENCE) for 30 minutes at 10% at 56 ° C for 37 minutes (: myeloid leukemia cells HL-60 before subculture) (ATCC CCL-240) was suspended in ASF 104 medium (manufactured by Ajinomoto Co., Ltd.) at 5x104 cells / 9 ml. This suspension was prepared in 4 portions of 9 ml ', and 10 0 # 1 physiological saline was added to each suspension', And fucose-containing sulfated polysaccharide samples obtained in Example 1, F-Fd-1 to F-Fd-4, and three kinds of fucose-containing 113 1237026 oligosaccharide sulfate aqueous physiological salt solution (10 Mg / ml), and cultured at 37 ° C in the presence of 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, a sample containing fucose-containing sulfated polysaccharide was added , F-Fd-1 ~ F-Fd-4, and three kinds of HL-60 cells containing fucose sulfate oligosaccharides exhibited cell self-destructive characteristics such as cell shrinkage and cell fragmentation. Furthermore, physiological saline solution was added. The number of HL-60 cells increased by about 4 times, but the fucose-containing sulfuric acid was added. Sugar samples, F-Fd-1 ~ F-Fd-4, and three kinds of fucose-containing oligosaccharide HL-60 cells did not increase the number of cells at all to less than 1/10. Samples of fucose sulfate polysaccharide, F-Fd-1 ~ F-Fd-4, and three kinds of fucose sulfate oligosaccharide-containing cell self-knock-inducing effects inhibited the proliferation of HL-60 cells. Furthermore, for confirmation, we used known as induction A 10 μg / ml solution of radiobactin D of the cell self-killing test agent was used in place of the fucose-containing oligosaccharide sulfate. When the same operation was performed, the same situation as the fucose-containing oligosaccharide sulfate was observed after 20 hours of culture. Cell shrinkage and cell fragmentation. From this result, it can be seen 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 content obtained in Example 13. Fucose sulfate oligosaccharide induces cell self-destruction. Fucose sulfate-containing polysaccharide samples obtained in Example 1, F-Fd-1 to F-Fd-4, and three kinds of fucose sulfate-containing oligosaccharides obtained in Example 13 Each solution of sugar [dissolved in 10 mg / ml in 30 mM HEPES buffer (pH 7) containing 120 mM sodium chloride, and sterilized by autoclaving at 121 ° C ] Cells by 20 minutes of self-extinguishing action induced by the above-described hair 1141237026 measurement subject, obtaining the same results. Example 26

Lung cancer cells A-549 (ATCC CCL-185), SV40 transformed lung cells WI-38VA13 (ATCC CCL-75.1), and liver cancer cells Hep G2 (ATCC HB-8065) were suspended at 104 cells / ml each. PRMI 1 640 medium containing bovine fetal blood pupa (JRH BIOSCIENCE) treated with i0% 56 ° C for 30 minutes. 1.8 ml of this suspension was dispensed, and 200 / z 1 of physiological saline solution and fucose-containing sulfated polysaccharide obtained in Examples 1 and 15 were added to each suspension and each cancer cell, and Example 1 The obtained 6 kinds of fucose sulfate oligosaccharide-containing physiological salt solution (1 mg / ml) were filtered and treated 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 sulfuric acid polysaccharides obtained in Examples 1 and 15 and the six types of fucose-containing sulfuric acid oligosaccharides obtained in Example 14 were added to the liver cancer cells A-549 and SV with a molecular weight of 3 or more and a molecular weight of 200. 40 transformed lung cells WI-3 8VA13, and hepatocellular carcinoma 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 various cancer cells with molecular weights above 2,000 and 3 divisions is reduced, and it can be determined that the proliferation of various cancer cells can be inhibited through the self-inducing induction of cells containing fucose sulfate polysaccharides and oligosaccharides. Fucose-containing sulfated polysaccharides obtained in Examples 1 and 15 and 6 fucose-containing sulfated oligosaccharides in each of the four PBS solutions (1 mg / ml) obtained in Example 1 4 115 1237026 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 2 7 Colon cancer cells HCT 1 16 (ATCC CCL-247) and gastric cancer cells AGS (ATCC CRL- 1 7 3 9) were each suspended in 10 cells / 1.8 ml at 10% 56 ° C. McCroy's 5a medium (manufactured by GIBCO) and HanTsF12 medium (manufactured by GIBCO) for 30 minutes of JRB BIOSCIENCE. 1.8 ml of this suspension was dispensed, and 200/1 physiological saline was added to each suspension and each cancer cell, and the fucose-containing sulfated polysaccharides obtained in Examples 1, 12, 2, and 15 were added. And F-Fd-1 ~ F-Fd-4 4 kinds of fucose sulfate oligosaccharide-containing physiological saline solution (10 mg / ml) filtered and treated at 37 ° C in the presence of 5% carbon dioxide 48 hours. The cultured cells were observed under a microscope to investigate the degree of proliferation and cell type. As a result, fucose-containing sulfated polysaccharides obtained in Examples 1, 12, and 15 and four kinds of fucose-containing oligosaccharide-containing colon cancer cells HCT 1 16 and gastric cancer cells F-Fd-1 to F-Fd-4 were added. AGS all exhibit 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 oligosaccharides 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 1237026 to F-Fd-4 obtained in Examples 1, 12, and 15 were each PBS solution containing fucose-sulfate oligosaccharides (10 mg / ml) ) The cell self-destructive induction effect of the autoclaved sterilized product at 1 2 1 ° C for 20 minutes is the same as above, and the same result is obtained. Example 2 8

Human colon cancer cells HCT 1 16 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. In McCoy's 5a The culture medium was suspended at 5 × 103 cells / ml, and 1.8 ml was dispensed into each well on a 24-well plate manufactured by FALCON. To each suspension, 0.2 ml of a fucose-containing sulfated polysaccharide sample obtained in Example 1 dissolved in PBS at 10 mg / ml, a fucose-containing sulfated polysaccharide mixture, and fucose-containing sugar obtained in Example 12 were added. Sulfate-Sulfur-F, Fucose-Containing Sulfate-u, F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4, the fucose-containing sulfated polysaccharide obtained in Example 15 Each solution and 5 mg / ml heparin (manufactured by Wako Pure Chemical Industries, Ltd.) and dextran sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in PBS were autoclaved at 1 2 ° C for 20 minutes. The treated material was cultured in the presence of 37 t: 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 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 Example 1, 1 370 1237026 fucose-containing sulfated polysaccharide mixture obtained in Example 1, and the sample obtained in Example 12 in HCT 1 16 cell culture broth. Fucose-containing sulfated polysaccharide-U and fucose-containing sulfated polysaccharide-F, F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4, the rock-containing material obtained in Example 15 The relationship between the culture time when the fucose sulfate polysaccharide is 1 mg / ml, and the heparin and dextran sulfate are added at 0.5 mg / ml and the number of progenitor cells in the culture medium. The horizontal axis indicates the culture time (days). The vertical axis indicates the number of viable cells in the culture medium (XI 04 cells / 2 ml). In Figure 29, the type of fucose-containing sulfated polysaccharide or oligosaccharide added to the culture medium is: 0 is marked as no addition (control ), • Labeled as the fucose-containing sulfated polysaccharide sample obtained in Example 1 'Labeled as the fucose-containing sulfated polysaccharide sample 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 significantly increased, but the fucose-containing sulfated polysaccharide sample obtained in Example 1, the fucose-containing sulfated polysaccharide mixture, and the fucose-containing sulfuric acid obtained in Example 12 were added. Polysaccharide-F, fucose-containing sulfated polysaccharide-U, F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4, fucose-containing sulfated polysaccharide obtained in Example 15 ' Heparin and HCT 1 16 cells with dextran sulfate have almost no increase or decrease in the number of cells. Furthermore, the fucoid bran-containing sulfuric acid multi-fermentation sample obtained in Example 1, the fucose-containing sulfated polysaccharide mixture, and the examples were added. The obtained fucose-containing sulfated polysaccharide-F, fucose-containing sulfated polysaccharide-U, F-Fd-1, F-Fd · 2, F-Fd_3, and 118 1237026 F-Fd-4, Example 1 The obtained HCT 1 16 cells containing fucose sulfated polysaccharide, heparin, and aposulfuric acid all exhibited cell self-destructive characteristics 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, and The cell self-killing-inducing effect of each filtration 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 2 9 ^ 0 (^ '8 5 & culture medium (013 (made by 0)) 37 ° C in bovine fetal blood pupa (JRH 6103 (: 化 化)) treated with 10% 56t for 30 minutes Human colon cancer cell line HCT 1 16 cultured under suspension was suspended in McCoy's 5a medium at 5 X 103 cells / ml, and each well was dispensed in each well on a 24-well plate manufactured by FALCON Corporation. 1 · 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 1 2 1 ° C. The autoclaved sterilized product was cultured in 20 minutes at 37 ° C and 5% carbon dioxide. 尙 'Only the same amount of PBS was used as a control group, and 1237026 was cultured in the same way. The number of viable cells 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, 1990) (pages 26 to 28). Measured by the method of pan-blue staining.

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 to the HCT 116 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 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 as no addition (control), 2 is marked as 2 mg / ml, 3 mg / ml is marked, and the black triangle is 5 Mg / ml. As a result, the number of cells of HCT 116 cells added with PBS increased significantly, 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 autoinduction-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 1237026 results. Example 30 0 Human gastric cancer cell line AGS was cultured in 37 t in a HanTs F12 medium (manufactured by GIBCO) containing bovine fetal blood pupa (jRH BIOSCIENCE) containing 10% 56t for 30 minutes, and was cultured in Ham's F12 medium. 5 X 103 cells / ml were suspended, and 1.8 ml was dispensed in each well on a 24-well plate manufactured by FALCON. 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 1 21 ° C, The treated material was autoclaved 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 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 31, the amount of the fucose-containing sulfated polysaccharide sample in the culture medium is: 0 is marked without addition (control), φ is marked as 2 mg / ml, _ is marked as 3 mg / ml, and the black triangle is marked 5 mg / ml. 121 1237026 As a result, the number of cells of AGS cells added with PBS increased significantly, 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. Φ Dissolve 20 mg / ml, 30 mg / ml, and 50 mg / ml of the fucose-containing sulfated polysaccharide sample solution obtained in Example 1 to dissolve the cell auto-inactivation effect in PBS. Same result. ~ Example 3 1

Human colon cancer cells SW 480 (ATCC CCL- 228) cultured at 37 ° (:) in L-15 medium (manufactured by GIBCO) containing bovine fetal blood pupa (JRH BIOSCIENCE) treated at 10% 56 ° C for 30 minutes. ), Suspended in L-15 medium at 5 X 103 cells / ml, and dispensed 1.8 ml in each well on a 24-well plate manufactured by FALCON. 0.2 ml of each suspension was added to dissolve The fucose-containing sulfated polysaccharide sample solution obtained in Example 1 at 10 mg / ml, 30 mg / ml, and 50 mg / ml in PBS was autoclaved at 1 21 ° C for 20 minutes, and then at 37 ° C, cultured in the presence of 5% carbon dioxide. 尙 Only the same amount of PBS as the control group was cultured in the same way as 122 1237026. The number of time-lapsed cells after the start of culture was based on "Tissue Culture Technology" (2nd Edition) ) (Asakura Publishing, Japan Tissue Culture Association, 1990) measurement (page 26 ~ 28) measurement. That is, the method using the trypan blue staining on the blood cell calculation board. The results are shown in Figure 32. That is, Fig. 32 shows the fucose-containing solution obtained in Example 1 in SW 480 cell culture solution. The relationship between the culture time and the number of viable cells in the culture medium when the sulfated polysaccharide samples were added at various concentrations. The horizontal axis represents the culture time (time), and the vertical axis represents the number of viable cells in the culture solution (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), spring is marked as 1 mg / ml, 3 mg / ml is marked, and black triangle is marked. It was 5 mg / ml. As a result, the number of SW 480 cells with PBS was significantly increased, but the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added to SW 480 cells with a final concentration of 3 mg / ml or more. The number of cells decreased and the cell proliferation was significantly inhibited by adding 1 mg / ml. In addition, the SW 480 cells added with the fucose-containing sulfated polysaccharide sample obtained in Example 1 all exhibited cell self-destructive characteristics such as cell shrinkage and cell fragmentation. It can be determined that the fucose-containing sulfated polysaccharide sample obtained in Example 1 has a cell self-extinguishing induction effect on SW 480 cells at a concentration of at least 1 mg / ml, and can inhibit cell proliferation. The 123-1237026 fucose-containing sulfated polysaccharide sample solution obtained in Example 1 containing 10 mg / ml, 30 mg / ml, and 50 mg / ml was dissolved in BS. The same result was obtained. Example 32 2 DEME medium (Da Nihon Pharmaceutical Co., Ltd.) containing bovine fetal blood pupa (j rh BI0SCIENCE) and NEAA (manufactured by Dainippon Pharmaceutical Co., Ltd.) treated at 10% 5 6 ° C for 30 minutes. Human colon cancer cell WiDr (ATCC CCL-218) cultured at 37 ° C at 37 ° C, suspended in the above medium at 5 X 103 cells / ml, and placed in each well on a 24-well plate manufactured by FALCON Dispense 1.8 ml. To each suspension, 0.2 ml of a fucose-containing sulfated polysaccharide mixture obtained in Example 1 dissolved in PBS at 10 mg / ml, and fucose-containing sulfated polysaccharide obtained in Example 12, F, F-Fd were added. -3 and F-Fd-4, and each solution containing fucose sulfate polysaccharide obtained in Example 15 was autoclaved at 12 1 ° C for 20 minutes, and at 37 ° C, 5% carbon dioxide Culture in the presence. Alas, only those who added the same amount of PBS as a control group were cultured in the same manner. The number of germline cells 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, 1 990) (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 1237026 culture time (time) The vertical axis indicates the number of viable cells in the culture medium (x 1 ο 4/2 ml). In Figure 33, the type of fucose-containing sulfated polysaccharide in the culture medium is: ◦ is marked as no addition (control), _ is marked as fucose-containing sulfated polysaccharide-F obtained in Example 12, 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 polysaccharides-F, F-Fd-3, and F-Fd-4 obtained in Example 12 and the fucose-containing sulfated polysaccharide-containing W i D r cells obtained in Example 15 all showed cell shrinkage and cells. Fragmentation and other cell self-destructive features. 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. 10 mg / ml of the fucose-containing sulfated polysaccharide mixture obtained in Example 1 and the fucose-containing sulfated polysaccharide-F, F-Fd-3, and F-Fd-4 obtained in Example 12 were dissolved in PBS, and The cell self-killing-inducing effect of the fucose-containing sulfated polysaccharide solution-containing filtering solution obtained in Example 15 was determined in accordance with the above-mentioned measurement "125 1237026, and the same results were obtained. Example 3 3

In bovine fetal blood pupa (JRH) containing 10% 56 ° C for 30 minutes

Human colon cancer cells (WiDrUTCC CCL-218) cultured at 37 ° C in NEMEM (manufactured by Dainippon Pharmaceutical Co., Ltd.) in NEAA (manufactured by Dainippon Pharmaceutical Co., Ltd.), suspended at 5X103 cells / ml in the above medium 1.8 ml was dispensed into each orifice on a 24-well plate made by FALCON. To each suspension, 0.2 ml of a 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 added at 1 21 ° C. The product was autoclaved and sterilized at 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 "Technology of Tissue Culture" (Second Edition) (Asakura Publishing, Japan Tissue Culture Society, 1990) (pages 26-28). That is, it was measured by trypan blue staining on a blood cell calculation board.

The results are shown in Figs. That is, Fig. 34 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 Wi D r cell culture medium, and the number of proliferating 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 per 2 ml). In Figure 34, the amount of the fucose-containing sulfated polysaccharide sample in the culture medium is: 0 is marked without addition (control), # is marked as 1 mg / ml, 3 mg / ml is marked, and black triangle is 5 Mg / ml. 126 1237026 As a result, the number of HCT 1 16 cells with PBS significantly increased. However, the fucose-containing polysulfate sample obtained in Example 1 was added to Wi Dr cells at a final concentration of 3 mg / ml or more. The number of cells decreased, and the addition of 1 mg / liter also significantly inhibited cell proliferation. In addition, all the Wi Dr 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 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. Φ Dissolve 10 mg / mL, 30 mg / mL, and 50 mg / mL of the fucose-containing sulfated polysaccharide sample solution obtained in Example 1 in the PBS solution of the cell solution to induce the cell self-killing effect. Same result. '' Example 3 4

Human pre-myeloid leukemia cells HL-60 (ATCC CCL) cultured at 37 ° C in PRMI 1 640 medium (manufactured by GIBCO) containing bovine fetal blood pupa (JRHBIOSCIENCE) treated at 10% 5 61 for 30 minutes -2 40), suspended in ASF 104 medium (manufactured by Ajinomoto Co., Ltd.) at 5 x 104 cells / 900 ml, and dispensed 4.5 ml into each well on a 6-well plate manufactured by FALCON. To each suspension, 0.5 ml of the low molecular weight fucose-containing sulfated polysaccharide · F as described in Example 19 (6) was added and dissolved in 30 mM HEPES containing 120 mM sodium chloride at 10 mg / ml. The buffer was treated in a buffer solution (pH 7) and filtered through a filter and cultured at 37 ° C in the presence of 5% dioxide 127 1237026 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 was determined that the HL-60 cells were induced to self-destruct by the lyophilized product of the fucose-containing polysaccharide sulfate-F low-molecular-weight compound described above, and inhibited the cell proliferation rate. Example 3 5 Human premyeloid leukemia cells HL-60 were treated with PRMI 1 640 medium (manufactured by GIBC0) containing bovine fetal blood pupa (JRH BIOSCIENCE) containing 10% of 56 ° C for 30 minutes. X 104/900 ml suspension. Six parts of this suspension were prepared, and to each suspension, 100 μl of 30 mM HEPES buffer (pH 7) containing 120 mM sodium chloride and Example 19 which was dissolved in the same buffer at 10 mg / ml 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 it exists in 371, 5% carbon dioxide Incubate for 46 hours. The number of progenitor cells in the culture medium was measured 22 hours and 46 hours after the start of the culture. In addition, 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- 1237026

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, cells that were cultured in ASF 104 medium with fucose-containing sulfated polysaccharide-F, F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4 were: 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 cells with 1 ^ (1-1,? 彳 (1-2, F-Fd-3, and F-Fd-4) showed cell shrinkage. Cell fragmentation and cell fragmentation characteristics, and the cells are almost dead. The number of cells in the medium with only buffer solution increased by about 3 times, and the number of cells in the medium with fucose sulfate polysaccharide-F increased by about 2.5 times.

From the above results, it can be judged that fucose-containing sulfated polysaccharide-F has a strong cell-self-inducing effect on cancer cells in a bloodless agar medium, but F-Fd-1, F-Fd-2, F-Fd-3, And F-Fd-4 has a very strong induction effect on cell self-destruction regardless of whether it is in the blood-free maggot medium or in the blood maggot medium. In order to confirm, 5 × 104 human pre-myeloid leukemia cells HL-60 were prepared in PRMI 1640 medium (manufactured by GIBCO) containing bovine fetal blood pupa (JRHBIOSCIENCE) containing 10% of 56 ° C for 30 minutes. / 9 00 ml suspension. Prepare 2 parts of this suspension in 9 ml, and add 1 mmol 129 1237026 liters of 30 mM HEPES buffer (pH 7) containing 120 mM sodium chloride and F-Fd _ 4 dissolved in the same buffer at 10 mg / ml And cultured at 37 ° C in the presence of 5% carbon dioxide for 16 hours. The cultured cells were separated from the supernatant by centrifugation. The obtained cells were suspended in 20 μl of 50 mM Tr 1 s-hydrochloric acid buffer solution (pH 7.8) containing 10 mM glyoxalate and 0.5 mg sodium lauryl sarcosinate, and 10 mg / ml was added. 1 microliter of ribonuclease A (manufactured by SIGMA Co., Ltd.) was added at 50 ° C for 30 minutes, and then added

One microliter of 10 mg / ml proteinase K was processed at 50 ° C for 30 minutes. The treated cells were used as samples and electrophoresed at a voltage of 100V using a 2% agarose gel. 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. In addition, for confirmation, when the same operation was performed using a radiobacterin D 10 microgram / ml solution known as a test agent for inducing cell self-destruction, the same operation as that of F-Fd-4 described above was confirmed after 20 hours of incubation. The same DNA ladder.

That is, it can be determined that if the fucose-containing sulfated polysaccharide F is decomposed by the terminal type fucose-containing sulfated polysaccharide F-decomposing enzyme of the present invention, the self-killing-inducing effect on cancer cells becomes stronger. Example 36 A venous blood was taken from a 21-year-old female and a 32-year-old male normal person, and contained 100 mg of dextran, Caci2.2H 200.74 mg, MgCl2 19.92 pen gram 'KC1 40.26 mg, per liter, NaCl 7371 mg, Tris-hydrochloric acid 175.6.5 mg solution was diluted twice, and then placed in a centrifugal separation tube with twice the volume of diluted blood in a lymphosphere separation solution (sold by Daiichi 130 1237026). The overlapping layer was left to stand 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 RPM 1-1640 medium containing 10% bovine fetal blood pupa (56t, treated for 30 minutes) was added. In each case, 0.2 ml of 5 mg / ml of fucose-containing sulfated polysaccharide and its culture medium obtained in 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 (4X 106 cells / mouse) was injected subcutaneously into 8-week-old male BALB / C mice (approximately 20 g in weight) )abdomen. 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 grams, and the fucose sulfur-containing 131 1237026 acid polysaccharide-U administration group was 0.28 grams, which showed significance (relative to the control group P < 0. 01). The inhibition rate was 77.6%. Table 2 Rat (η) tumor weight (g) Mean soil SD inhibitory 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

Carcinogenic preventive effect of fucose-containing sulfated polysaccharides (1) Nine 19 Spragure-D aw 1 ey mice (male) at 6 weeks of age, 7.4 mg / kg of azomethane (NAKARI TESC) was administered subcutaneously on the back Company), and once a week thereafter, subcutaneously on the back until the 10th week. At the time of administration, azomethane oxide was dissolved in 0.1M phosphate buffer containing pH 6.5 containing 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 azo oxymethane administration, 70 ml of Gaogumei kelp hot water extract prepared in accordance with Example 1 as described in Example 1 will be administered orally in the form of water. 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. (4) Fourteen of the above 19 were not administered with fucose-containing sulfated polysaccharides, but tap water was given as water for the mash, and it was regarded as a control group. 132 1237026 As of 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. Significant carcinogenic inhibitory effect.对照 As of the 30th week, the control group had died of 3 animals, but the fucose-containing sulfate-polysaccharide mixture administration group had survived. The average weight of the control group at week 30 was 716 g, and the average weight of the fucose-containing sulfated polysaccharide mixture was 817 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 non-administered rats. The same. Next, four control groups were selected, and 40 ml of the above-mentioned hot fruit extract of hot fruit kelp (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 the chemical carcinogen, the preventive effect of the chemical carcinogen against weight gain, and the shrinkage of cancer tissue 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 50 ml of a glass vial for freeze-drying with 50 mg of fucose-containing sulfated polysaccharide, and then freeze-dried. Another 2 ml of physiological saline was added as a dissolving solution. Example 40 Injection An injection was prepared according to the following formulation. 133 1237026 Fucose-containing sulfated polysaccharide-U [Example 12] 40 mg of physiological saline solution Amount 2 ml per ampule 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 1 tablet Example 42 Injection

F-Fd-1 was 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 of fucose sulfate polysaccharide-F 40 mg of physiological saline solution 2 ml per 1 ampoule 134 1237026 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 polyvinylpyrrolidone 3 mg magnesium stearate 2 mg The effect of the invention is 100 mg per tablet. According to the present invention, it has the effect of inducing cell self-destruction on unwanted or pathogenic cells, and can induce cell self-destruction 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 The fungicide of the present invention as an active ingredient is very suitable for a carcinogen for digestive system cancer. And through its carcinogenic effect, it can also prevent carcinogenesis by chemical carcinogens. The medicament of the present invention uses edible materials such as edible brown algae plants and edible sea cucumbers as raw materials and 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 condiments containing fucose sulfated polysaccharides and / or their degradation products. In addition, according to the present invention, a simple method for inducing cell self-destruction 135 1237026 is provided. 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 which does not substantially contain fucose sulfated polysaccharide-F and removes highly reactive colored substances. Its decomposition product, and also provides its efficient production 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 1 does not show the rate of formation of the fucose-containing sulfated polysaccharides. Figure 2 does not show the molecular weight distribution of fucose-containing sulfated polysaccharide-U measured by gel filtration method using s p h a c r y 1 S-5 0 0. Figure 3 does not show the uiiR spectrum of fucose-containing sulfated polysaccharides. 136 1237026 The relationship between the culture time when the fucose-containing sulfated polysaccharide obtained was added at 1 mg / m1 and the number of viable cells in the culture solution. Figure 28 shows the fucose-containing sulfated polysaccharide obtained in Example 1 added to MOLT-3 cell culture solution, fucose-containing sulfated polysaccharide F obtained in Example 12, and those obtained in Examples 15 and 17 The relationship between the culture time when the fucose sulfate polysaccharide and dextran sulfate are contained and the number of viable cells in the culture medium. Figure 29 illustrates the addition of a fucose-containing sulfated polysaccharide sample obtained in Example 1, a fucose-containing sulfated polysaccharide mixture obtained in Example 1, and a fucose-containing sugar obtained in Example 12 to HCT 116 cell culture broth. Sulfate-containing 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 , And the relationship between the culture time of heparin and aposulfuric acid 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 1237026 sugar-F, F-Fd- 3 and F-Fd-4 and the relationship between the culture time when the fucose-containing sulfated polysaccharide obtained in Example 15 was added and the number of progenitor cells in the culture solution. Fig. 34 is a graph showing the relationship between the culture time when the fucose-containing sulfated polysaccharide sample obtained in Example 1 was added to the Wi Dr cell culture solution at various concentrations and the number of progenitor cells in the culture solution.

140

Claims (1)

I ----------------J2570261 1a, Patent application scope: No. 92120936 "Fucose-containing sulfated polysaccharide-F, its preparation method, carcinogen and carcinogen containing it "Prophylactics" patent case (Amended on 03.04.1994) 1. A fucose-containing sulfated polysaccharide-F, which is characterized by the following physical and chemical properties, and is a constituent sugar: fucose, galactose, essentially Contains no gluconic acid, and ⑵ does not substantially reduce the molecular weight of fucoidan decomposing enzymes produced by FuvobacteriunOsp. SA-OOSacCCRC 910069. ⑶ Via Alter omonas sp. SN-1009 (CCRC 910070) is a terminal type fucose-containing sulfate polysaccharide degrading enzyme and / or a culture supernatant of the strain and is low-molecular, and the optimum pH is: the optimum pH of this enzyme is 7 ~ 8, the most suitable Temperature: The optimal temperature for this enzyme is between 30 and 35. . In the presence of one or more salts of a total concentration of 0.6-3M, hydrazone produces a precipitate of fucose-containing sulfated polysaccharide-F salts by adding cetylpyridinium chloride. 2. A carcinogen, which is characterized by containing fucose-containing sulfated polysaccharide-F and / or a decomposition product thereof as described in item 1 of the scope of patent application. 3. A carcinogen preventive agent, which is characterized in that it contains fucose-containing sulfated polysaccharide-F and / or a degradation product thereof as described in item 1 of the scope of patent application. 4. A method for preparing fucose-containing sulfated polysaccharide-F as described in item 1 of the scope of patent application, characterized in that it comprises making a fucose-containing sulfated polysaccharide mixture to have the ability to decompose fucose-containing sulfated polysaccharide The fucoidan-degrading enzyme produced by Flavobacterium 1237026 (Flavobacterium) sp. SA-0082 (CCRC 910069), or the step of collecting the target polysaccharide by treating the microorganism with the degrading enzyme. 5. A method for preparing fucose-containing sulfated polysaccharide-F, such as the item 1 of the scope of the patent application, which comprises making the fucose-containing sulfated polysaccharide mixture in the presence of salts with a medicament order having the ability to aggregate acidic polysaccharides. The step of precipitation of the target polysaccharide. 6. A method for preparing fucose-containing sulfated polysaccharide-F as described in item 1 of the scope of patent application, characterized in that the fucose-containing sulfated polysaccharide mixture is treated with an anion exchange resin in the presence of a divalent cation mixture for collection purposes Polysaccharide step. 7. A method for preparing fucose-containing sulfated polysaccharide-F as described in item 1 of the scope of patent application, which includes the characteristics of coexisting colors when manufacturing fucose-containing sulfated polysaccharide-F as described in item 1 of the scope of patent application The step of removing the sexual substance using a polysaccharide substance or a substance having an anion exchange group. 8. A terminal fucose-containing type of fucose-containing sulfated polysaccharide-F activity produced by decomposing the first patent application scope, which is produced from S. spp. SN- 1 009 (CCRC9 1 0 0 70) The sulfated polysaccharide degrading enzyme is characterized by the following physical and chemical properties. (I) Action: acting on the fucose-containing sulfated polysaccharide-F as described in the first item of the patent application scope, so that the fucose-containing sulfated polysaccharide is reduced in molecular weight. But does not act on fucose-containing sulfated polysaccharide-U, which has the following physical and chemical properties: ⑴ constitutes a sugar: it contains uronic acid, and ⑵ is produced by Flavobacterium sp. SA-0082 (CCRC 1237026 910069) Fucoidan is reduced in molecular weight by decomposing enzymes, and generates at least one compound selected from compounds represented by the following formulae (I), (II), (III), OH 0 = S = 0 'I OH OH (11) 1237026 OH -4- 1237026 (Π) Optimum pH: The optimum pH of this enzyme is 7 ~ 8, (iii) Optimum temperature: The optimum temperature of this enzyme is 30-35 ° C. 9. An enzyme composition comprising a calcium source and a terminal fucose-containing sulfated polysaccharide-decomposing enzyme as described in item 8 of the patent application scope. j 〇. — A method for preparing a terminal fucose-containing sulfate polysaccharide degrading enzyme such as the item No. 8 in the scope of the patent application, which is characterized by culturing the production of a terminal fucose-containing sulfate polysaccharide degrading enzyme having the type 8 in the scope of the patent application. A capable Symbiotic bacterium of the genus, the enzyme is collected from its culture. } 丨. A low-molecular-weight compound containing fucose-containing sulfated polysaccharide, which is characterized in that the terminal type fucose-containing sulfated polysaccharide decomposing enzyme such as the scope of patent application item 8 and the fucose-containing It is obtained by the action of sulfated polysaccharides, and the molecular weight according to the gel overspray method is 1000 ~ 30,000.