JPH0892303A - New saccharide, its production and immunopotentiator - Google Patents

Abstract

PURPOSE: To obtain an acidic saccharide, derived from a red algal mucilaginous polysaccharide and expected to have antitumor activities due to excellent immunopotentiating activities and the capability of inducing a tumor necrosis factor(TNF) possessed thereby. CONSTITUTION: This new acidic saccharide contains constituent monosaccharides of A and B. A is L-galactose-6-sulfate or 3,6-anhydro-L-galactose and B is D- galactose or 6-O-methyl-D-galactose. Both A and B are alternately arranged and the bonding mode of the A→B is α 1→+3 and the bonding mode of B→A is β1→4. The monosaccharide constituting the nonreducing terminal is the L- galactose-6-sulfate. The saccharide contains >=2 monosaccharides of L- galactose-6-sulfate as the constituent monosaccharide in one molecule. The number of the constituent monosaccharides is an even number of 4-500. This method for producing the acidic saccharide is provided and the immunopotentiator contains the acidic saccharide as an active ingredient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel acidic sugar obtainable from red algae, a method for producing the same, and an immunostimulant containing the same as an active ingredient.

[0002]

2. Description of the Related Art Seaweeds have long been eaten in Japan.
It has also been used as a folk medicine. In recent years, research and development focusing on its physiological activity has been conducted. For example, JP-A-63-316732 and JP-A-5-271306 disclose antiviral agents derived from red algae, and JP-A 64-66126 discloses antitumor agents derived from red algae. Has been done. Furthermore, JP-A-3-284626 and JP-A-5-13
Japanese Patent Publication No. 9988 discloses an immunostimulant containing a substance extracted with an aqueous solvent from seaweeds of the genus Amanori and the genus Ogonori, both of which are red algae. It is known that substances extracted from seaweeds belonging to the genus Ogonori and the genus Anoly are mainly composed of polymeric acidic polysaccharides having agarose as a basic skeleton. Since these acidic polysaccharides are highly viscous and sometimes form a strong gel, it is extremely difficult to mix them with food or feed or to formulate themselves into a dosage form suitable for oral or parenteral administration. . Moreover, handling in the manufacturing process is difficult,
There was a big problem that the operation became complicated.

As an enzyme that hydrolyzes such a mucilage acidic polysaccharide, Pseudomonus atlantica ( Pseudomonus)
atlantica) , Cytophaga
sp.), Vibrio sp. AP-2 ( Vibrio sp. AP-2) and the like produce β-agarase having the ability to hydrolyze β-1,4 bond in the viscous acidic polysaccharide. (Biochem., 105, 317-321 (1967), Chin.J.Ocean)
ol.Limnol., 8 (2) 135-149 (1990), Eur.J.Biochem., 13
3, 673-684 (1983), Eur. J. Biochem., 187, 461-465 (199
0) etc.). In addition, agar-containing substances using agarase (β- or α-agarase) (progenitor algae such as hornwort, agar and the like, intermediate products in the agar production process, agar, agarose, etc.)
A method for producing an oligosaccharide from the above is described in Japanese Patent Application Laid-Open No. 2-65788. However, regarding the oligosaccharide to be produced, the oligosaccharide having only two, four, six or more sugars should be present in the reaction solution. Is only confirmed by high performance liquid chromatography. Further, a method for producing an oligosaccharide by causing a polysaccharide-degrading enzyme produced by a microorganism belonging to the genus Vibrio to act on seaweed belonging to the genus Red seaweed Alanori is described in JP-A-4-271791, but it is produced. For oligosaccharides it is only stated that the gel filtration fraction range is 100-1800 Daltons.

Porphyran-type polysaccharides are known as polysaccharides obtained from seaweeds of the genus Rhodophyta of the genus Amanori. Porphyra belonging to the genus Malva Chishima Chrono Li (Porphyra um
porphyran obtained from B. bilicalis) is D-galactose or 6-O- which has an α1 → 3 bond at the non-reducing end side.
It has a structure in which methyl-D-galactose and 3,6-anhydro-L-galactose or L-galactose-6-sulfate having β1 → 4 bond at the non-reducing end are alternately bonded (integrated polysaccharide science (bottom)). , Kodansha Scientific, p. 306 (1973)). By hydrolyzing porphyran with β-agarase, 6-O-sulfato-α-L
-Galactopyranosyl- (1 → 3) -β-D-galactopyranosyl- (1 → 4) -3,6-anhydro-α-L
-Galactopyranosyl- (1 → 3) -D-galactopyranose (according to the notation described below, the formula

[0005]

[Chemical 2]

(Represented by) and oligosaccharides (Eu
rJ Biochem. 133, 673-684 (1983)). In the true Rhodophyta, the main component of the viscous polysaccharides that make up the intercellular spaces is agar. Agar is obtained from red algae belonging to the genus Astragalus, Scutellariae, Scutellariae, Scutellariae, etc., and has an agarose skeleton containing many neutral agarose and agarose skeletons containing pyruvic acid and sulfate ester. It is a mixture of three polysaccharides. The amount and characteristics of the components of agar vary greatly depending on the growth stage of red algae and the type of red algae. Agarose is a neutral polysaccharide, and D-galactose or 6-O-methyl-D-galactose having α1 → 3 bond at the non-reducing terminal side and 3,6-anhydro-L having β1 → 4 bond at the non-reducing terminal side. -Has a structure in which galactose is linked alternately. The sulfate ester-containing polysaccharide has a structure in which the 3,6-anhydro-L-galactose residue of agarose is partially replaced by a galactose-6-sulfate residue (see Comprehensive Polysaccharide Science (above), 298). -Page 304).

[0007]

DISCLOSURE OF THE INVENTION The present inventors extracted β-agarase into an extract obtained by extracting mucilage polysaccharide (mucilage acidic polysaccharide) from seaweed belonging to Rhodophyta with an aqueous solvent and subjecting it to solid-liquid separation. It was found that the low-viscosity acidic sugar obtained by the action of the above has an immunostimulant action (Japanese Patent Application No. 5-67479, filed March 3, 1993). On the other hand, the present inventors
SP O-148 (FERM P-13767) "catalyses the reaction of hydrolyzing red algae mucilage polysaccharides to the endo type and producing oligosaccharides mainly having a degree of polymerization of 4" Novel red algae mucilage polysaccharides decomposition It was found that an enzyme is produced (Japanese Patent Application No. 6-57469).

The present inventors have found that a culture solution of the above strain further contains an enzyme that hydrolyzes a β1 → 4 galactoside bond, which is different from the above enzyme. The present inventors hydrolyze the above-mentioned viscous polysaccharide with these enzymes to obtain a low-viscosity acidic sugar with greatly improved immunostimulatory action, and by fractionating this low-viscosity acidic sugar, immunity It was found that an acidic sugar having a significantly improved activation activity can be obtained. The present invention has been completed based on these findings, and an object thereof is to provide a novel substance excellent in immunostimulatory activity, a method for producing the same, and a novel immunostimulant containing the same as an active ingredient. .

[0009]

In the present invention, the constituent monosaccharides are A and B, A is L-galactose-6-sulfate or 3,6-anhydro-L-galactose, and B is D-galactose or 6-. O-methyl-D-galactose, A and B are arranged alternately, the binding mode of A → B is α1 → 3, the binding mode of B → A is β1 → 4, and the non-reducing end is The monosaccharide constituting L-galactose-6 is
-Sulfuric acid, L-galactose-6- as the constituent monosaccharide
Containing 2 or more of sulfuric acid in one molecule, the number of constituent monosaccharides is 4-50
An acidic sugar that is an even number of 0; a mucilage polysaccharide contained in seaweed belonging to red algae and extracted with an aqueous solvent is an enzyme source containing at least one of the following red algae slime polysaccharide degrading enzymes I and II. The number of constituent monosaccharides in the low-viscosity acidic sugar obtained by hydrolyzing or hydrolyzing with an enzyme source containing the following red algal mucopolysaccharide degrading enzymes III and at least one of I and II is 50.
The above-mentioned acidic compound, characterized in that a fraction greater than 0 is removed and an acidic sugar mixture in which the number of sulfate groups in each molecule is 2 or more is fractionated or the acidic sugar mixture is further appropriately separated. Method for producing sugar: Red alga mucilage polysaccharide degrading enzyme I: 3,6-anhydro-L-galactose, D-galactose, 6-O-methyl-D-
When galactose and L-galactose-6-sulfate are represented by A-L-Gal, D-Gal, Me-D-Gal and L-GalS, respectively, A-L- in the viscous polysaccharide.
Gal-D-Gal residue or AL-Gal-Me-
An enzyme having specificity for hydrolyzing a β1 → 4 bond on the reducing end side of a D-Gal residue Red alga mucilage polysaccharide degrading enzyme II: A-L-G in the mucilage polysaccharide
an al-D-Gal-AL-Gal-D-Gal residue,
AL-Gal-D-Gal-AL-Gal-Me-
D-Gal residue, A-L-Gal-Me-D-Gal-
AL-Gal-D-Gal residue or AL-Gal
-Me-D-Gal-AL-Gal-Me-D-Ga
Endo-type enzyme having specificity to hydrolyze β1 → 4 bond on the reducing end side of l residue

Red alga mucilage polysaccharide degrading enzyme III: L-GalS-D-Gal residue or L-GalS in the mucilage polysaccharide
The β1- → 4 bond on the reducing end side of the -Me-D-Gal residue has an AL-Gal-D-Gal residue or an AL-Gal-Me-D-Gal residue on the reducing end side. Exists,
Furthermore, the L-GalS-D-Gal residue or A-L-
A- on the non-reducing end side of the GalS-Me-D-Gal residue
L-Gal-D-Gal residue, A-L-Gal-Me-
An endo-type enzyme having specificity of hydrolyzing only when a D-Gal residue, an L-GalS-D-Gal residue or an L-GalS-Me-D-Gal residue is present; An immunostimulant containing one as an active ingredient (hereinafter sometimes referred to as immunostimulant 1); and a viscous polysaccharide contained in seaweed belonging to red algae and extracted with an aqueous solvent, Hydrolyzed with an enzyme source containing at least one of the polysaccharide degrading enzymes I and II, or hydrolyzed with an enzyme source containing the above red alga slime polysaccharide degrading enzyme III and at least one of I and II. The low-viscosity acidic sugars have a molecular weight of 100,0 measured by the GPC method.
An immunostimulant which is obtained by removing a fraction larger than 00, and which contains an acidic sugar mixture in which the number of sulfate groups in the molecule is 2 or more as an active ingredient (hereinafter referred to as immunostimulant 2). In some cases). Hereinafter, the red algal mucilage polysaccharide degrading enzymes I, II, and III may be collectively referred to simply as red algal mucilage polysaccharide degrading enzymes. Before describing the present invention in detail, the notation of sugar units used in the present invention will be described. In the present invention, the formula

[0011]

[Chemical 3]

6-O-sulfato-α-L-galactopyranosyl- (1 → 3) -D-galactopyranose or 6-O represented by the formula (wherein R is a hydrogen atom or a methyl group) -Sulfato-α-L-galactopyranosyl- (1 → 3) -6-O-methyl-D-galactopyranose

[0013]

[Chemical 4]

It is abbreviated as As well as the formula

[0015]

[Chemical 5]

3,6-anhydro-α-L-galactopyranosyl-represented by the formula (wherein R has the same meaning as above)
(1 → 3) -D-galactopyranose or 3,6-
Anhydro-α-L-galactopyranosyl- (1 → 3)
-6-O-methyl-D-galactopyranose

[0017]

[Chemical 6]

It is abbreviated as Also, the formula

[0019]

[Chemical 7]

Β-D-galactopyranosyl- (1 → 4) -6-O- represented by the formula (wherein R has the same meaning as above)
Sulfato-L-galactopyranose or 6-O-
Methyl-β-D-galactopyranosyl- (1 → 4) -6
-O-sulfato-L-galactopyranose

[0021]

Embedded image

It is abbreviated as As well as the formula

[0023]

[Chemical 9]

Β-D-galactopyranosyl- (1 → 4) -3,6-represented by the formula (wherein R has the same meaning as above)
Anhydro-L-galactopyranose or 6-O-
Methyl-β-D-galactopyranosyl- (1 → 4)-
3,6-anhydro-L-galactopyranose

[0025]

[Chemical 10]

It is abbreviated as According to this abbreviation, the formula

[0027]

[Chemical 11]

6-O-sulfato-α-L-galactopyranosyl- (1 → 3) -β-D-galactopyranosyl (1 in which R is as defined above) is represented. → 4)
-3,6-anhydro-α-L-galactopyranosyl-
(1 → 3) -D-galactopyranose, 6-O-sulfato-α-L-galactopyranosyl- (1 → 3) -β-
D-galactopyranosyl (1 → 4) -3,6-anhydro-α-L-galactopyranosyl- (1 → 3) -6-O
-Methyl-D-galactopyranose, 6-O-sulfato-α-L-galactopyranosyl- (1 → 3) -6-O
-Methyl-β-D-galactopyranosyl (1 → 4)-
3,6-anhydro-α-L-galactopyranosyl-
(1 → 3) -D-galactopyranose or 6-O-
Sulfato-α-L-galactopyranosyl- (1 → 3)
-6-O-methyl-β-D-galactopyranosyl (1 →
4) -3,6-anhydro-α-galactopyranosyl-
(1 → 3) -6-O-methyl-D-galactopyranose is

[0029]

[Chemical 12]

It is abbreviated as As can be seen from the above abbreviations, D-galactose or 6-O-methyl-D- at the reducing end of 6-O-sulfato-L-galactopyranose or 3,6-anhydro-L-galactose.
When the OH group of galactose is bonded (glycoside bond), α1 → 3 bond is always formed, and conversely, 6 is added to the reducing terminal of D-galactose or 6-O-methyl-D-galactose.
When the OH group of -O-sulfato-L-galactopyranose or 3,6-anhydro-L-galactose is bonded (glycosidic bond), β1 → 4 bond is always present.

As mentioned above, the acidic sugar of the present invention is (1)
The constituent monosaccharides are A and B, and A is L-galactose-6-
Sulfuric acid (= 6-sulfato-L-galactopyranose) or 3,6-anhydro-L-galactose (= 3.
6-anhydro-L-galactopyranose) and B is D-
Galactose (= D-galactopyranose) or 6
-O-methyl-D-galactose (= 6-O-methyl-
D-galactopyranose), A and B are alternately arranged, the binding mode of A → B is α1 → 3, and B → A.
Has a binding mode of β1 → 4, the monosaccharide constituting the non-reducing end is L-galactose-6-sulfate, and contains two or more L-galactose-6-sulfates as a constituent monosaccharide in one molecule, It is an acidic sugar with an even number of constituent monosaccharides of 4 to 500. Among the acidic sugars of (1) above, the preferable one is the acidic sugar of (1) above, wherein the number of (2) constituent monosaccharides is an even number of 4 to 200, and more preferable is the number of (3) constituent monosaccharides. Is 4 ~
It is the acidic sugar of (1) above, which is an even number of 14. Further, preferred among the acidic sugars of the present invention are (4) each monosaccharide unit having 1, 2, 3, 4, ... N- from the non-reducing end toward the reducing end.
Assuming that the numbers 1 and n are attached, the constituent monosaccharides of n-1 are 3, 6
-Anhydro-L-galactose, 3,5 ... n
-3 constituent monosaccharide is L-galactose-6-sulfate or 3,6-anhydro-L-galactose, and the number of constituent monosaccharides is an even number of 6 or more, or n-1 constituent monosaccharide Is L-galactose-6-sulfate, and 3, 5, ...
The constituent unit of n-3 is also L-galactose-6-sulfate, and the number of constituent monosaccharides is an even number of 4 or more (1),
It is the acidic sugar of (2) or (3). Further, more preferable among the acidic sugars of the present invention is the acidic sugar of (1) above, wherein the sequence of the constituent monosaccharide (5) is represented by the following formula:

[0032]

[Chemical 13]

Next, the process for producing the acidic sugar of the present invention is carried out by the following process 1.
And 2 Method 1 for producing acidic sugar In the acidic sugars of the present invention of the above (1) to (5), the n-1st constituent monosaccharide, that is, the second constituent monosaccharide from the reducing end is 3,6-
An acidic sugar, which is anhydro-L-galactose, is contained in seaweed belonging to red algae, and the viscous polysaccharide (mucilage acidic polysaccharide) extracted with an aqueous solvent is used as a red algae slime polysaccharide degrading enzyme I defined above. Or II, or a mixture of these or a substance containing them (culture filtrate, etc.), which has a low-viscosity acidic sugar obtained by hydrolysis and has a molecular weight of more than 100,000, preferably more than 45,000, or 3,000 to 100,000 or Fractions greater than any value up to 45,000, such as greater than 7,500, or the number of constituent monosaccharides greater than 500, preferably
Fractions of molecular weight greater than 200, or greater than any value from 14 to 500 or 200, for example greater than 36, are removed by solvent precipitation, salting out, ultrafiltration or gel filtration etc. A fraction having a relatively low molecular weight is subjected to a method for separating substances by difference in ionicity, for example, anion exchange chromatography, electrophoresis, electroosmosis treatment, or the like,
Or ultrafiltration using an ultrafiltration membrane having a smaller molecular weight cut off or gel filtration using gel particles suitable for fractionation of a polysaccharide having a smaller molecular weight, and the number of sulfate groups in each molecule is 2 or more. It can be obtained by fractionating the acidic sugar mixture. In addition to the above, the molecular weight referred to in the present specification is pullulan (Shodex standard P-82), neoagarobiose, neoagarotetraose and neoagarohexaose as standard, and the column Asahipak G
SM-700 (7.6 x 500 mm) + Asahipak GS-220 HQ (7.6 x 300 mm)
(Manufactured by Asahi Kasei Co., Ltd.), the molecular weight is determined by the GPC method using 50 mM NaCl as an eluent.

This production method 1 is also a production method of acidic sugar which is an active ingredient of the immunostimulants 1 and 2. In the present invention, an acidic polysaccharide having agarose as a basic skeleton or seaweed belonging to the red algae containing porphyran as one of the main components is used as a raw material. As seaweeds that can be preferably used, seaweeds belonging to the genus Ogonori, for example, Ogonori, Tsurushimo, Shiramo, Ogonori, Mizogonori, Kabanori, seaweeds belonging to the genus Amanori, such as Marubaa Nori, Tsukusia Manori, Onia Manori, Kosuno Nori, Samurais nori, Upu Nori, Urinosa, Mention may be made of Tishima macronori, Ono nori, and Firitasa. In addition to this, Ushikenori and Funorinoushige of the genus Ushikenori can also be used in the present invention, and some seaweeds belonging to the genera of Genus Rhizophora and Funori are also usable in the present invention. Among the seaweeds belonging to these red algae, more preferred are seaweeds belonging to the genus Ogonori or the genus Amanori.

These seaweeds can be obtained from the beginning by extraction with an aqueous solution to obtain the active ingredient, but first, in order to remove foreign substances and to impart flexibility to the tissue to facilitate the subsequent treatment, Washing with water or warm water (for example, warm water of 80 ° C or lower) for a short time, or treating seaweed with an organic solvent that can dissolve oils, oligosaccharides, and oil-soluble dyes, etc. Alternatively, they may be combined as needed, and this is preferable. As such an organic solvent, for example, lower alkanol such as methanol, ethanol, n-propanol and isopropanol, lower alkanone such as acetone and the like can be used. Also,
It may be a mixture of these solvents and a small amount of water (up to about 80:20 V / V). Preferred are methanol, ethanol and a mixed solvent of these and water. The amount of such an organic solvent (including a mixed solvent with water) is not particularly limited, but 0.5 to 10 L is suitable for 100 g of seaweed (dry matter standard). The temperature time for extraction with an organic solvent is not particularly limited, but may be room temperature or higher, for example, 30 ° C to 5 minutes or more at the boiling point of the organic solvent, for example 15
Minutes to 1 hour is appropriate.

Next, the organic solvent is removed by decanting, filtering, centrifuging, etc., and the resulting residue is subjected to extraction with an aqueous solvent. As the aqueous solvent, water, an acidic aqueous solution or a basic aqueous solution is used, but it may further contain a small amount of a hydrophilic organic solvent such as 10% by volume or less. It is an acid and a base which give “acidic” and “basic”, respectively, but a commonly used buffer may be used. The acid is not particularly limited, but inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, acetic acid,
Organic acids such as propionic acid and citric acid can be used. The base is not particularly limited, but ammonia, hydroxide of alkali or alkaline earth metal, carbonate or bicarbonate such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, bicarbonate. Usually potassium or the like is used. The base may be a hydrophilic organic base such as pyridine. As the buffering agent, a conventional buffering agent such as Tris-based, phosphoric acid-based, citric acid-based, boric acid-based can be used.

The pH of the aqueous solvent is not particularly limited, but usually 1 to
13 and especially 2 to 12 are suitable. The amount of aqueous solvent used is not particularly limited, but is usually 1 per 100 g of raw seaweed (dry matter basis).
About 10L is suitable. The temperature time for extraction with an aqueous solvent is not particularly limited, but it is usually 4 minutes to the boiling temperature of the system, usually room temperature to the boiling temperature of the system, and usually 5 minutes or more, for example, 30 minutes to 20 hours is suitable. Note that two or more extractions with water, an acidic aqueous solution, and a basic aqueous solution may be combined in series (for example, a water extraction residue is extracted with an acidic aqueous solution).

The extract is separated from the residue by a usual solid-liquid separation means (decantation, filtration, centrifugation, etc.) to obtain a viscous polysaccharide-containing aqueous solution. Usually, the enzyme treatment is carried out at this stage, but when the extraction is carried out using an acidic aqueous solution or a basic aqueous solution, the pH is adjusted to a suitable pH for the enzyme treatment and then the enzyme treatment is carried out, or preferably, the extract is once subjected to medium extraction. The mixture was neutralized (the base and acid used for neutralization may be the base and acid used for the basic aqueous solution and the acidic aqueous solution, respectively), and then desalted (dissolved in water after precipitation of the organic solvent, dialyzed, ultrafiltered, etc.). It is later subjected to enzyme treatment. If you want to know the quantitative relationship, add ethanol or ethanol and salt (sodium chloride etc.) to the solution suitable for enzyme treatment obtained above,
The resulting precipitate may be recovered, freeze-dried and then subjected to enzyme treatment.

An example of the red alga mucilage polysaccharide degrading enzymes I and II which can be used in the present production method 1 is β-agarase. The definition of β-agarase is “D in agarose.
An enzyme that hydrolyzes the β1 → 4 galactoside bond between galactose and 3,6-anhydro-L-galactose residues. Examples of β-agarase that can be used in the present invention include β-agarase derived from Pseudomonas atlantica, Cytoferga spp, and Vibrio spp AP-2 as exemplified above.
Of these, β-agarase preparations originating from Pseudomonas microorganisms are commercially available (Sigma Co .; a mixture of red algal mucopolysaccharide degrading enzymes I and II).

As the red algal mucilage polysaccharide degrading enzyme which can be used in the present production method 1, the red algal mucilage polysaccharide degrading enzyme (red algal mucilage degrading enzyme described in the above-mentioned Japanese Patent Application No. 6-57469) can be used. Polysaccharide-degrading enzyme II). In addition to the properties defined above, this enzyme has the following properties: 1) Action Catalyses the reaction that hydrolyzes red algal slime polysaccharides into endo type and produces oligosaccharides mainly having a degree of polymerization of 4; 2) Substrate specificity Acts on galactan-based polysaccharides having a β1 → 4 galactoside bond and oligosaccharides of neo-agarohexaose or higher,
It does not act on carrageenan, neoagarotetraose, neoagarobiose, and lactose; 3) Optimal pH and pH stability The optimal pH when using the hot water extract of red seaweed mucilage, Ogonori. 4.5-5.5, stable at pH 5.5-8.0 under conditions of 28 ° C., 16 hours and no substrate;

4) Optimum temperature and thermostability The optimum temperature is 55 when the hot water extract of Scutellaria japonica is used as a substrate.
C., pH 7.5, 10 minutes, in the absence of substrate, stable at 50.degree. C., about 72.5% activity remains at 95.degree. C .; 5) Deactivation condition pH 6.0, About 10% of the activity remains under the condition that the substrate is absent at 120 ° C. for 10 minutes, and is completely inactivated at 120 ° C. for 60 minutes; 6) Molecular weight 41000 (SDS-PAGE method); 7) Isoelectricity Points 4.7 to 5.6 (isoelectric focusing); 8) Effect of metal salts, etc. pH 6.0, 28 ° C., 1 mM under salt concentration of metal salts, etc., 1
After 6 hours of treatment, Ag ⁺ , N-bromosuccinimide 5% or less, Cu ²⁺ , Fe ³⁺ 15 to 50% Fe ²⁺ , Hg
²⁺ shows a residual activity of 70 to 80%, and Na ⁺ , Mg2 ⁺ ,
Ca ²⁺ , Mn ²⁺ , Co ²⁺ , Ni ²⁺ , Zn ²⁺ , Ba ²⁺ , P
It is stable with b ²⁺ and Al ³⁺ . Reference Example 2 shows an example of the production of this red alga mucilage polysaccharide degrading enzyme.

As the enzyme source containing at least one of the red algal mucilage polysaccharide degrading enzymes I and II, each enzyme alone or enzyme I can be used.
And a mixture of the same, a crude enzyme containing enzyme I and / or II and another enzyme that does not adversely affect the acquisition of the target substance, a culture solution or a culture filtrate of a production bacterium containing enzyme I and / or II, Alternatively, a suspension of bacteria producing the enzymes I and / or II and the like can be mentioned. The red-algae mucilage polysaccharide degrading enzyme I referred to in the present invention of the aqueous solvent-extracted mucilage acidic polysaccharide and
The enzyme treatment with an enzyme source containing at least one of II is 0.01 to 10,000 of the enzyme (as a whole) per 1 g of polysaccharide.
u, more preferably 0.1-1000u, pH 4-9, temperature 1
0.5 to 72 hours at 5 to 95 ° C, more preferably pH 4 to
6. The reaction can be carried out at a temperature of 30 to 70 ° C. for 4 to 48 hours. Here, the enzyme 1u means pH 6.0,
It means the amount of an enzyme that produces a reducing sugar corresponding to 1 μmol of galactose per minute when a mucilage acidic polysaccharide derived from sorghum is used as a substrate under the condition of a temperature of 37 ° C.
As the reaction medium, the above-mentioned aqueous solvent extract after pH adjustment, desalting, etc., if necessary, or water, a buffer, or the like can be used. The reaction can be allowed to proceed in a stationary state, stirred, or adjusted while the pH is constantly adjusted to a value at which the enzyme reaction can be most efficiently performed. After the enzymatic reaction, the enzyme is usually inactivated by heating or the like.

By the above enzyme treatment, the viscous polysaccharide has its β1.
→ 4 bonds are usually partially hydrolyzed to acidic sugars of low viscosity (acidic polysaccharides + acid oligosaccharides). The molecular weight of the acidic sugar is usually about 300 to about 1,400,000. The enzyme treatment is performed on the aqueous solvent extract in the above, but it can also be performed at the same time as the aqueous solvent extraction, that is, by performing extraction using a red algal mucopolysaccharide-degrading enzyme-containing aqueous solvent. . In this case, the enzyme reaction conditions may be the same as above, but the extraction conditions are restricted by the enzyme reaction conditions.

From the enzyme-treated solution, the molecular weight is greater than 100,000, preferably greater than 45,000, or greater than any value from 3,000 to 100,000 or 45,000, eg, a polysaccharide fraction greater than 7,500, or the number of constituent monosaccharides is greater than 500. Polysaccharide fractions of high molecular weight, preferably greater than 200, or greater than any value between 14 and 500 or 200, such as greater than 36, are subjected to solvent precipitation, salting out, ultrafiltration or gel filtration, etc. Remove. Solvent precipitation or salting-out is carried out by setting a solid fraction having a relatively high molecular weight as a precipitation fraction and a solid fraction having a relatively low molecular weight as a dissolved fraction.

Solvent precipitation and salting out can be carried out by a conventional method. As a solvent for solvent precipitation, hydrophilic organic solvents such as lower alkanols such as ethanol, methanol, n-propanol and isopropanol and lower alkanones such as acetone, especially Ethanol is used as a salt in salting out, and includes salts containing polyvalent anions such as phosphate ion and sulfate ion, for example, ammonium sulfate, sodium sulfate, potassium phosphate, etc., and sodium chloride, potassium chloride, barium carbonate, cetyl pyrichloride. It is possible to use dium and the like, especially ammonium sulphate. The amount of the solvent or salt for precipitating the polysaccharide of the molecular weight fraction as described above to the enzyme-treated solution is determined by gradually increasing the ratio of the solvent to the enzyme-treated solution or the degree of saturation of the salt in steps of, for example, 5 to 15%. Easy to find. For example, in the case of solvent precipitation with ethanol, ethanol may be added so as to be 50 to 90% by volume with respect to the enzyme solution. During solvent precipitation or salting out, the temperature and pH may not be adjusted, but the temperature may be adjusted to about 0 to 20 ° C. and the pH to about 4 to 9, for example. Removal of the precipitate by solvent precipitation or salting out can be carried out by centrifugation, filtration or the like.

In the ultrafiltration, ultrafiltration is carried out using an ultrafiltration membrane having a molecular weight cutoff suitable for removing polysaccharides having the above molecular weight or more. As the ultrafiltration membrane, a commercially available one can be used, and as an appropriate one, for example, an ultra filter Q0
100, P0200 (Toyo Filter Paper), Diaflow Membrane YM5, YM10, PM10, YM30, XM50 (Amicon), Type PLGC, Type PLCC, Type PLT
K (Millipore) or the like can be used. Further, a reverse osmosis membrane, an ion exchange membrane, a dialysis membrane, etc. can be used depending on the case. At the time of gel filtration, gel filtration is performed using gel particles suitable for fractionation of the above-mentioned molecular weight polysaccharides. As the gel particles, commercially available ones can be used, and as suitable ones, for example, Superdex 75 pg, Sepha
crylS-200; Sephadex G-25, G-5
0, G-75 (Pharmacia); TSKgel Toyopearl HW-40, HW-50 (Tosoh); Cellulofine GC
L-90, 300 (Seikagaku Corporation) or the like can be used. The eluent is not particularly limited, but for example, a 0.05M NaCl aqueous solution or the like can be used. Other conditions in ultrafiltration and gel filtration may be the same as in the case of fractionation of polysaccharides in ordinary ultrafiltration and gel filtration, and are not particularly limited.

A liquid containing a relatively low molecular weight sugar fraction (oligosaccharide + polysaccharide) obtained by removing a relatively high molecular weight polysaccharide fraction having a specific molecular weight or more by the various methods described above, and then the substance is ionic Or a neutral sugar and / or an acidic sugar having a sulfate group number of 1 in the molecule, which is subjected to ultrafiltration or gel filtration, to obtain an acid having a sulfate group number of 2 or more in the molecule. A sugar mixture is obtained. As a method of separating substances by difference in ionicity, for example, anion exchange chromatography using an anion exchange resin, electrophoresis, or electroosmosis membrane treatment can be used, and the above-mentioned relatively low molecular weight sugar fractions can be used. By adding the containing liquid thereto, the neutral sugar fraction and the acidic sugar fraction in the dissolved solid content are fractionated, and the acidic sugar fraction is fractionated. From these acidic sugar fraction fractions, a fraction consisting of an acidic sugar mixture in which the number of sulfate groups in each molecule is 2 or more is obtained. Such fractions can also be obtained by normal phase partition chromatography using a carrier having a hydrophilic group bonded thereto, such as a silica gel system, by utilizing the difference in the adsorptivity of substances.

Anion exchange chromatography, electrophoresis, electro-osmosis membrane treatment, etc., and normal phase partition chromatography are used for neutral sugar fractions and acidic sugar mixtures in which the number of sulfate groups in each molecule is 1 from the above-mentioned precipitate-removed liquid. Can be removed to give acidic sugars each having 2 or more sulfate groups in one molecule, which can be carried out by a conventional method. For example, in the case of anion exchange chromatography, the ion exchange resin used may be a strong anion type or a weak anion type, but a strong anion type is preferable. Specifically, Diaion HPA-75 (manufactured by Mitsubishi Kasei), Q Sepharose, QAE Sephadex (Pharmacia), TSKgel Super Q-Toyopearl, DEAE
-Toyopearl 650 (Tosoh), DEAE-Cellulofine (Seikagaku Corporation), etc. can be used.

The above-mentioned high molecular sugar fraction removal liquid is usually placed on an ion exchange column and usually eluted by a stepwise elution method using a water → salt aqueous solution or a concentration gradient elution method. As a result, a neutral sugar mixture, a mixture of acidic sugars having one sulfate group in one molecule, a mixture of acidic sugars having two sulfate groups in one molecule, 1
A mixture of acidic sugars having 3 sulfate groups in the molecule is eluted in this order. The salt of the aqueous salt solution is a strong acid-strong base salt,
For example, sodium chloride, potassium chloride or the like can be used. The final salt concentration varies depending on the ionicity of the target fraction, but generally 0.05 to 2M is suitable. Collect an appropriate amount of eluate.

It is also possible to use ultrafiltration or gel filtration to obtain an acidic sugar mixture in which the number of sulfate groups in the molecule is 2 or more, respectively, from a liquid containing a sugar fraction having a relatively low molecular weight. The reason is that the number of sulfate groups is 1 due to the specificity of the enzyme.
There is a clear difference in the molecular weight between the acidic sugars and the two or more acidic sugars, and the neutral sugar fraction is enzymatically decomposed to have a sulfate group number of 2
This is because there is a clear difference in molecular weight between the above acidic sugars. This ultrafiltration and gel filtration can be carried out in the same manner as described above except that the molecular weight range intended for fractionation is different from the above. That is, the upper limit of the molecular weight of neutral sugars or acidic sugars having one sulfate group is approximately 1,000, and acidic sugars having two or more sulfate groups have a larger molecular weight, and therefore, it is necessary to fractionate polysaccharides having a molecular weight of 1,000 or more. A suitable ultrafiltration membrane and gel particles may be used. As a specific ultrafiltration membrane, for example, Diaflow membrane YM5 (Amicon), type PLC
Gel particles such as C (Millipore), for example, Superdex 75p
g, Sephadex G-15, Sephadex G-25
(Pharmacia), TSKgel Toyopearl HW-40 (Tosoh), etc. can be used. The identification of the obtained target substance-containing liquid can be performed, for example, by the GPC method.

Method 2 for producing acidic sugar In the acidic sugars of the present invention of the above (1) to (5), the constituent monosaccharide at the n-1th position, that is, at the second position from the reducing end, is L-galactose-6-sulfate. Acidic sugars are contained in seaweeds belonging to red algae, and are composed of mucilage polysaccharides (mucoacidic polysaccharides) extracted with an aqueous solvent, which are III of the red algae mucopolysaccharide degrading enzyme and at least one of I and II. A low-viscosity acidic sugar obtained by hydrolysis with an enzyme source containing a molecular weight of more than 100,000, preferably more than 45,000, or 3,000 to 100,0
Greater than 00 or any value up to 45,000, for example 7,500
A larger fraction, or a fraction with a molecular weight such that the number of constituent monosaccharides is greater than 500, preferably greater than 200, or greater than any value from 14 to 500 or 200, such as greater than 36. Methods such as anion exchange chromatography, electrophoresis, and electrophoresis, in which the relatively low molecular weight fraction obtained after removal by solvent precipitation, salting out, ultrafiltration or gel filtration, etc. 1 molecule after being subjected to permeation membrane treatment or the like, or subjected to ultrafiltration using an ultrafiltration membrane having a smaller molecular weight cutoff or gel filtration using gel particles suitable for fractionation of a polysaccharide having a smaller molecular weight It can be obtained by fractionating an acidic sugar mixture having a sulfate group number of 2 or more.

Red alga mucilage polysaccharide degrading enzymes III, I and II
As an enzyme source containing at least one of red algal mucus polysaccharide degrading enzyme III, an enzyme mixture containing at least one of I and II, these and other enzymes that do not adversely affect the acquisition of the target substance, etc. Crude enzymes contained, Enzyme III and Enzyme I
And at least one of II and II, or a culture broth or a culture filtrate of the producing strain, or enzyme III and enzyme I and / or
Alternatively, a suspension of a bacterium that produces at least one of II and the like can be mentioned. Pseudomonas sp. O-148 (FERM) described in the specification of Japanese Patent Application No. 6-57469 mentioned above.
It was found by the present inventors that all of the red alga mucilage polysaccharide degrading enzymes I, II and III are present in the culture filtrate of P-13767). Isolation of the red alga mucilage polysaccharide degrading enzyme III (or I or II) from the culture broth can be performed by a conventional method utilizing the properties of the enzyme. The ratio of the enzyme solution to the acidic polysaccharide to be enzyme-treated is such that the total amount of the enzyme at the start of the enzyme treatment is 0.01 to 10,000 u / g, preferably 0.1 to 1000 u / g.
g, and the ratio of III and at least one of I and II used is such that the enzyme activity of the latter is 1 to 10,000, preferably 10 to 50, relative to 1 of the enzyme activity of the former. 1u is synonymous with 1u in the red alga mucilage polysaccharide degrading enzymes I and / or II.

When the method for separating substances by the difference in ionicity is ion exchange chromatography, a mixture of neutral sugars, a mixture of acidic sugars having one sulfate group in one molecule,
An acidic sugar mixture in which the number of sulfate groups in each molecule is 2,
An acidic sugar mixture in which the number of sulfate groups in each molecule is 3,
It will be eluted in the order of ……, so just collect the necessary fractions. The salt concentration in this case is such that when the acidic sugar mixture is an acidic sugar mixture in which the number of sulfate groups in each molecule is 2, respectively.
About 0.2 to 0.5 M is suitable, and those having 3 or more sulfate groups can be fractionated by appropriately increasing the salt concentration with reference to the case of 2. Other operations in the production method 2 may be the same as those in the production method 1, including an operation before the enzyme treatment.

In any of the above production methods 1 and 2,
Purification operations such as deproteinization and desalting can be performed before starting the process, at any stage of the process, or after the end of the process. Deproteinization can be performed by means such as precipitating a protein or polypeptide with trichloroacetic acid or the like. Desalination is suitably carried out by dialysis, ultrafiltration (ultrafiltration for desalination), reverse osmosis membrane treatment or ion exchange treatment (ion exchange treatment for desalination).

The acidic sugar mixture-containing liquid obtained by any one of the above-mentioned production methods 1 and 2 or further subsequent treatment is usually dried by a method appropriately selected from spray drying, freeze drying, hot air drying and the like to give an acidic sugar mixture. obtain. The acidic sugar mixture obtained by the above production methods (1) and (2) is the above (1).
As described later, having the structure as shown in (5) to (5), known knowledge about the structure of red algal mucilage polysaccharide, enzyme specificity, component analysis value, thin layer chromatography (T
It is estimated from the degree of polymerization of the molecule obtained by LC). These acidic sugar mixtures can be isolated into individual acidic sugars by repurification, for example by ion exchange column chromatography.

The acidic sugar (mixture) of the present invention has immunostimulatory activity. The immunostimulatory activity has, for example, the ability to induce tumor necrosis factor produced by immunocompetent cells, in addition to the macrophage activating effect. This latter activity suggests that these acidic sugars (mixtures) have anti-tumor activity via activation of the immune system. Therefore, the immunostimulant of the present invention described below includes use as an antitumor agent. The acidic sugar mixture obtained through any one of the above production methods 1 and 2 or the subsequent purification operation, or the solution containing the individual acidic sugars can be directly used as an immunostimulant, but is usually spray-dried or freeze-dried. It is dried by a method appropriately selected from hot air drying and the like, and is used as an immunostimulant as it is or in the form of a mixture with various pharmaceutically acceptable carriers.

In the case of oral administration, the tablets, granules, powders, capsules and the like applied to the composition include binders, lubricants, excipients, etc. which are commonly used for formulation in their compositions. It contains additives such as disintegrants and wetting agents. When used as an oral liquid preparation, an internal solution, a shaking mixture,
It may be in the form of a suspension, emulsion, syrup, or in the form of a dry product which is redissolved before use. Further, such a liquid preparation may contain any of commonly used additives and preservatives. For injection, the composition usually contains additives such as stabilizers, buffers, preservatives, and isotonicity agents, and is usually provided in the form of unit-dose ampoule or multi-dose container. In addition,
The composition may be in the form of an aqueous solution, suspension, solution, emulsion in an oily or aqueous vehicle, while the active ingredient is in a suitable vehicle, eg sterile pyrogen-free water, before use. It may be powder to be redissolved.

The immunostimulant of the present invention is orally or parenterally administered to humans or animals, for example, to people with weakened immunity, particularly those with weakened immune function due to aging or disease. Oral administration includes sublingual administration. Parenteral administration includes injections such as subcutaneous, intramuscular, intravenous and infusion. The amount of the solid of the active ingredient in the immunostimulant of the present invention can be variously changed, but usually 5 to 100% (w / w), particularly 10 to 60% (w / w).
Is appropriate. Since the dose of the immunostimulant of the present invention is affected by humans or animals, age, individual difference, medical condition, etc., it may be sometimes administered in an amount outside the following range, but it is generally intended for humans. In this case, the oral dose is 0.5 to 1,00 per kg of adult daily body weight as the solid amount of the active ingredient.
The dose is 0 mg, preferably 1 to 300 mg, and is administered once or in 2 or 3 divided doses.

The acute toxicity of the active ingredient of the present immunostimulant (specifically, the dried product obtained in each Example) is LD _50.
(ICR mouse, oral administration)> 3 g / kg. In addition, since the present immunostimulant active ingredient has an advantage that it does not adversely affect the living body even if it is ingested in a large amount, as it is, or by adding various nutrients or the like, or by including it in a food or drink, it has an immunostimulatory action. It may be consumed as a functional food or a health food having a function. That is, for example, by adding nutrients such as various vitamins and minerals, for example, liquid foods such as nutritional drinks, soy milk, soups and solid foods in various shapes, and further as powders as they are or added to various foods for use. You can also The content and intake of the active ingredient in the present immunostimulant as such functional food and health food may be the same as the content and dose in the above-mentioned pharmaceutical preparation, respectively.

[0060]

【Example】

Reference Example 1 Water Extraction of Mucilage Acidic Polysaccharide 20 g of Protophora algae was washed with a sufficient amount of water, 500 ml of distilled water was added, and the mixture was treated at 40 ° C. for 30 minutes and washed with warm water. After the washing solution was completely removed, 500 mL of ethanol was added and the mixture was treated at 45 ° C for 15 minutes and washed in the same manner. Next, 2 L of distilled water was added, and extraction was performed in a boiling water bath for 3 hours while appropriately stirring. The obtained extracts were combined and suction-filtered with a Nutsche to completely remove solid components. To the obtained filtrate, 4-fold amount of ethanol was added, the precipitate was collected, and freeze-dried to obtain 2.6 g of a dried product (viscous acidic polysaccharide).

Reference Example 2 Preparation of red alga mucilage polysaccharide degrading enzyme Pseudomonas sp. O-148 (FERM P-1376)
7) to agar 0.1 g / dL, polypeptone 0.1 g / dL, K ₂ HPO
_{4 0.1g / dL, NaCl 0.1g /} dL and MgSO ₄ · 7
It was cultured in a pre-culture medium of pH 7.0 consisting of 0.05 g of H ₂ O / dL. Then, the obtained pre-culture liquid 300
mL, Ogonori powder 1.5 g / dL, polypeptone 0.1 g / dL,
Yeast extract _{0.1g / dL, MgSO 4 · 7H} 2 O O.25g / d
L, NaCl 0.1 g / dL, CaCl ₂ 0.01 g / dL, Adecanol LG126 (Asahi Denka Kogyo Co., Ltd.) as an antifoaming agent, 30 L jar fermenter containing 15 L of pH 6.0 medium having a composition of 0.03 g / dL The cells were inoculated into (bacteria culture medium) and cultured at a temperature of 28 ° C. for 3 days. The culture was performed at an aeration rate of 0.5 VVM (superficial velocity) and a stirring rate of 200 rpm.

After the culture, the bacterial cells are removed by filtration,
12.7 L of culture filtrate was obtained. The enzyme activity of this culture filtrate was 0.5 u / mL. In addition, the culture filtrate was concentrated about 15 times with an ultrafiltration concentrator: Sartokonmini (Sartorius, Germany), and 860 mL of a concentrated solution (activity:
7.7 u / mL), and the concentrated solution was further adsorbed on a 500 mL column of agarose gel: Sepharose CL-6B (Pharmacia, Sweden) to obtain 750 m.
L / hr flow rate, 20 mM sodium acetate buffer, pH
After washing at 6.0 (4 ° C), additional 1250 mL / h
at a flow rate of r in 20 mM Tris-HCl buffer,
Elution was performed at pH 8.0 (35 ° C.) to obtain the active category. The obtained section is designated as a gel filtration resin Toyopearl HW
Adsorbed on a column of 65S (Tosoh Corp.) 500mL, and 4M NaCl at a flow rate of 1,000mL / hr.
/ 20 mM sodium acetate buffer (pH 6.0), and then washed with 2M NaCl / at a flow rate of 1500 mL / hr.
Eluted with a 20 mM sodium acetate buffer (pH 6.0) to give a purified enzyme activity category (46.7 u / m) that is almost pure.
260 mL of g protein) was obtained. This activity yield is about 2
It was 7.6%.

Reference Example 3 Hydrolysis of mucilage acidic polysaccharide by red alga mucilage polysaccharide degrading enzyme 1.5 g of the dried product (mucilage acidic polysaccharide) obtained in Reference Example 1 was dissolved in 150 mL of distilled water, and Reference Example 2 3 u of the red alga mucilage polysaccharide degrading enzyme obtained in step 3 was hydrolyzed by allowing it to act at 37 ° C. for 48 hours to obtain a low-viscosity acidic sugar-containing aqueous solution, which was freeze-dried to obtain a dried product (sample A 1.48 g). .

Example 1 Solvent precipitation of low viscosity acidic sugar and anion exchange resin chroma
Production of Acidic Sugar of the Present Invention by Fractionation by Topography 1/4 of the low-viscosity acidic sugar-containing aqueous solution obtained in Reference Example 3
After concentration, 4 times amount of ethanol was added to precipitate a hardly soluble polymer substance, which was removed by centrifugation (5000 rpm). The supernatant was freeze-dried to obtain a dried product. The lyophilized product was dissolved in a small amount of distilled water and placed on a column (2.3 φ × 20 cm) packed with strong anion exchange resin Diaion HPA-75 (Mitsubishi Kasei Co., Ltd.), water, 0.02 M sodium chloride and 0.50. Linear gradient of M sodium chloride (0 to 80 mL: H ₂ O, 80 to 480 mL: 0.02 M NaCl, 480 to 880 m
L: 0.02-0.50M NaCl) was performed at a flow rate of 40 mL / hr, and 8 mL of the eluate was collected. The sugar content of each fraction was colorimetrically determined by the phenol-sulfuric acid method (410 nm), and fractions No. 3 to 10, fractions No. 30 to 40 and fractions No. 75 to 85 were freeze-dried and dried. Sample B (neutral sugar) 0.840 g, Sample C
(Acid sugar) 0.170 g and Sample D (acid sugar of the present invention) 0.
115 g were obtained.

Reference Example 4 Pseudomonas SP O-148 (FERM P-1376)
Preparation of culture filtrate (enzyme solution) of 7) Pseudomonas sp. O-148 (FERM P-1376)
7) to agar 0.1 g / dL polypeptone 0.1 g / dL, K ₂ HPO ₄
0.1g / dL, NaCl 0.1g / dL and MgSO ₄ · 7
It was cultured in a pre-culture medium of pH 7.0 consisting of 0.05 g of H ₂ O / dL. Then, the obtained pre-culture liquid 300
mL, Ogonori powder 1.5g / dL, Polypeptone 0.1g / dL
, Yeast extract _{0.1g / dL, MgSO 4 · 7H} 2 O 0.25g
15 L of pH 6.0 medium having a composition of / dL, NaCl 0.1 g / dL, CaCl ₂ 0.01 g / dL and Adecanol LG126 (Asahi Denka Kogyo Co., Ltd.) 0.03 g / dL as an antifoaming agent
Inoculate into a 30L jar fermenter containing
The culture was carried out at a temperature of ° C for 3 days. Aeration rate is 0.5V
VM, stirring speed 200 rpm. After completion of the culture, bacterial cells were removed by filtration to obtain 12.7 L of culture filtrate.
The enzyme activity of this culture filtrate was 0.5 u / mL.

Example 2 Solvent precipitation of low viscosity acidic sugar and anion exchange resin chroma
Production of Acid Sugar of the Present Invention by Fractionation by Topography 1.5 g of the dried product (mucilage acidic polysaccharide) obtained in Reference Example 1 was dissolved in 150 mL of distilled water, and 2 u of the culture filtrate concentrate obtained in Reference Example 4 was dissolved. / G, and the mixture was allowed to act at 37 ° C for 48 hours for hydrolysis to obtain a low-viscosity acidic sugar-containing aqueous solution.
After concentrating this aqueous solution to 1/4, add 4 times the amount of ethanol to precipitate the hardly soluble polymer and centrifuge (5,000
rpm). The supernatant was freeze-dried to obtain a dried product. This lyophilized product was dissolved in a small amount of distilled water and loaded onto a column (2.3 φ × 20 cm) packed with a strong anion exchange resin, Diaion HPA-75, and water, 0.02M sodium chloride and 0.50M sodium chloride were added. Linear gradient (0 to 80 mL: H ₂ O, 80 to 880 mL: 0.02M N
aCl, 880 to 1680 mL: 0.02 to 0.50 M NaCl) was eluted at a flow rate of 40 mL / hr, and 8 mL of the eluate was collected. The content of each fraction was colorimetrically determined by the phenol-sulfuric acid method (410 nm), and five fractions each containing a separate component, neutral sugar fraction, acidic sugar fraction 1, acidic sugar fraction 2, acidic sugar Fraction 3 and acidic sugar fraction 4 were obtained in the order of elution, and the last eluted fraction 4 (fraction No. 120 to 130) of these 5 was freeze-dried to obtain a dried product (Sample E: acidic sugar of the present invention). Obtained 45 mg.

Example 3 Production of Acid Sugar by Fractionation of Low Viscosity Acid Sugar by Ultrafiltration 1.5 g of the dried product (mucoacidic polysaccharide) obtained in Reference Example 1 was dissolved in 150 mL of distilled water, and Pseudomonas sp. Β-Agarase (Sigma) derived 1,000 u was added and reacted at 30 ° C for 48 hours to hydrolyze, and after completion of the reaction, kept in a boiling water bath for 10 minutes to inactivate the enzyme, and a low viscous acidic sugar-containing aqueous solution. Got This aqueous solution was freeze-dried to obtain a dried product. This 0.5g
Was dissolved in 10 ml of distilled water and dispensed into two molcut L (LTK24, nominal molecular weight cutoff 30,000, manufactured by Millipore) to obtain a permeate. Further, the obtained permeated liquid was molcut L (LC
C24, nominal molecular weight cut-off of 5,000, manufactured by Millipore), and concentrated 5 times, and then 5 times more distilled water was added and concentrated in the same manner. The obtained concentrated liquid was freeze-dried to obtain 32 g of a dried product (Sample F: acidic sugar of the present invention). When the molecular weight of Sample F was measured, it was found to be distributed in the range of 4,000 to 45,000.

Example 4 Various Analytical Values and Estimation of Structure of Obtained Sample Various analyzes were performed on the dried products (Samples A, B, C, D and E) obtained in Reference Example 3 and Examples 1 and 2. The results are shown in Table 1. Among the analysis items, 3,6-anhydrogalactose is W. Yaph with neo-agarobiose as standard.
e et al. [Yaphe W. et al., Anal. Biochem. 13 , 14
3-148 (1965)] and calculated as weight% based on the total sugar value. The total sugar value here is the phenol-sulfuric acid method [Dubois
M. et al., Anal. Chem., 28 , 350-356 (1956)], and calculated as galactan, and the content of 3,6-anhydrogalactose was corrected and determined. Sulfate group content is IN HCl, 10
After 8 hours hydrolysis at 5 ° C., liberated sulfate ions were measured by ion chromatography system, in terms of sulfate (-SO ₃ H), it was calculated as% by weight relative to the total sugar level. The degree of polymerization was determined by thin layer chromatography after removing the sulfate group by alkali treatment.

[0069]

[Table 1]

From the known findings on the chemical structure of red algal mucilage polysaccharide and the results in Table 1, the basic skeleton and degree of polymerization of the molecule, 1
The number of sulfate ester groups bonded in the molecule becomes clear,
Further, the binding position of the sulfate ester group is determined by the substrate specificity of the enzyme used. As a result, samples B, C, D
And E are presumed to be mainly composed of sugars having the following structures.

[0071]

[Chemical 14]

Example 5 Measurement of immunostimulatory activity of samples A, B, C, D and F The macrophage activating action was examined as an index of immunostimulatory activity. Peritoneal cells of mice induced by proteose peptone were collected according to the conventional method "Functions and function measurement of macrophages", Nane Publishing, p. 72 (1985), and dispensed into a 96-well plate at 4 × 10 ⁵ / well. The cells were cultured for 1 hour to attach macrophages. After removing the floating cells, add the sample 10
The cells were cultured in RPMI-1640 medium containing g / dL fetal bovine serum, penicillin, and streptomycin for 72 hours, and the residual glucose and secreted nitrite ion concentration in the culture supernatant were measured and used as an index of macrophage activation. That is, the larger the glucose consumption amount and the nitrite ion production amount, the greater the macrophage activating effect. Laminarin derived from kelp, which is a known macrophage activator, was used as a control. The results are shown in Table 2.

[0073]

[Table 2]

As is clear from Table 2, the glucose consumption of macrophages was obtained by treating the enzymatically treated product of acidic polysaccharides derived from Ogonori with ethanol precipitation and anion exchange resin, or by treating the enzymatically treated product with gel filtration. Both the amount and the nitrite producing capacity were significantly increased. This clearly shows that these treatments significantly enhance the immunostimulatory activity. Example 6 Measurement of Antitumor Immunostimulatory Activity of Samples A, C, D and F As an index of antitumor immunostimulatory activity, TNF in mouse blood
The (tumor necrosis factor) inducing ability was measured. Mouse (C3H /
HeN, 7 weeks old, male) was intravenously administered with a sample dissolved in filter-sterilized PBS (physiological phosphate buffer), and 3 hours later, OK-432 (Chugai Pharmaceutical “Picibanil” 1K) was further administered.
E / animal was administered as a trigger for TNF, and 2 hours after that, blood was collected and serum was separated. TNF concentration in serum is enzyme-immunoassay kit for mouse TNFα Factor-Test m TNF
It was measured using -α (Zenzyme). The results are shown in Fig. 1. As is clear from FIG. 1, Samples D and F are Samples A and C.
It showed a drastic TNF-inducing ability compared to.

Example 7 Injectable Solution Sample D 600 g Polyethylene ethylene hydrogenated castor oil 500 g Pharmacopoeial distilled water 10 liter An injectable solution is prepared by a conventional method using the above components, and each ampoule is filled with 5 mL. Example 8 Capsule Sample F 200 g Corn starch 150 g Talc 80 g Magnesium stearate 30 g The above ingredients are thoroughly mixed and passed through a 60 mesh wire mesh to adjust the particle size, and then filled into 1,000 gelatin capsules.

[0076]

INDUSTRIAL APPLICABILITY The acidic sugar obtained from red algal mucilage polysaccharide, which is an active ingredient of the immunostimulant of the present invention or has an excellent immunostimulatory activity.

[Brief description of drawings]

FIG. 1 is an acidic sugar TNF (tumor necrosis factor) according to the present invention.
Shows inducibility.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12P 19/04 Z 7432-4B // (C12P 19/04 C12R 1:89) (72) Inventor Yamamoto Ryo 19-10 Showa-cho, Anjo City, Aichi Prefecture, Japan Shin-Nippon Chemical Industry Co., Ltd. (72) Inventor Masao Ito 19-10 Showa-machi, Anjo City, Aichi Prefecture Nippon Steel Chemical Industry Co., Ltd. (72) Inventor, Kazu Nomura 19-10 Showa-machi, Anjo City, Aichi Prefecture Shin-Nippon Chemical Industry Co., Ltd.

Claims (9)

[Claims] 1. The constituent monosaccharides are A and B, and A is L-galactose-6-sulfate or 3,6-anhydro-L-.
In galactose, B is D-galactose or 6-O-
Methyl-D-galactose, A and B are alternately arranged, the binding mode of A → B is α1 → 3, and B → A
Has a binding mode of β1 → 4, the monosaccharide constituting the non-reducing end is L-galactose-6-sulfate, and contains two or more L-galactose-6-sulfates as a constituent monosaccharide in one molecule, An acidic sugar in which the number of constituent monosaccharides is an even number of 4 to 500. 2. The acidic sugar according to claim 1, wherein the number of constituent monosaccharides is an even number of 4 to 200. 3. The acidic sugar according to claim 1, wherein the number of constituent monosaccharides is an even number of 4 to 14. 4. Assuming that each monosaccharide unit is numbered 1, 2, 3, 4, ... N-1, n from the non-reducing end toward the reducing end, the constituent monosaccharides of n-1 are 3,6. -Anhydro-L-galactose, wherein the constituent monosaccharide of 3,5 ... n-3 is L-
Galactose-6-sulfuric acid or 3,6-anhydro-L
-Galactose, wherein the number of constituent monosaccharides is an even number of 6 or more, or the constituent monosaccharide of n-1 is L-galactose-6-sulfate, and the constituent monosaccharides of 3, 5, ... Sugar is also L-
Galactose-6-sulfate, wherein the number of constituent monosaccharides is an even number of 4 or more. 5. The acidic sugar according to claim 1, wherein the constituent monosaccharide sequence is represented by the following formula. [Chemical 1] 6. A viscous polysaccharide contained in a seaweed belonging to the red algae and extracted with an aqueous solvent is hydrolyzed with an enzyme source containing at least one of the following red algae viscous polysaccharide degrading enzymes I and II. Or the following red algae mucilage polysaccharide degrading enzymes III, I and II
Of the low-viscosity acidic sugar obtained by hydrolysis with an enzyme source containing at least one of the A certain acidic sugar mixture is fractionated, or the acidic sugar mixture is further appropriately separated.
Method for producing acidic sugar described: Red algal slime polysaccharide degrading enzyme I: 3,6-anhydro-L-galactose, D-galactose, 6-O-methyl-D-
When galactose and L-galactose-6-sulfate are represented by A-L-Gal, D-Gal, Me-D-Gal and L-GalS, respectively, A-L- in the viscous polysaccharide.
Gal-D-Gal residue or AL-Gal-Me-
An enzyme having specificity for hydrolyzing a β1 → 4 bond on the reducing end side of a D-Gal residue Red alga mucilage polysaccharide degrading enzyme II: A-L-G in the mucilage polysaccharide
an al-D-Gal-AL-Gal-D-Gal residue,
AL-Gal-D-Gal-AL-Gal-Me-
D-Gal residue, A-L-Gal-Me-D-Gal-
AL-Gal-D-Gal residue or AL-Gal
-Me-D-Gal-AL-Gal-Me-D-Ga
Endo-type enzyme having specificity for hydrolyzing β1 → 4 bond on the reducing end side of l residue Red algal mucin polysaccharide degrading enzyme III: L-Ga in the mucilage polysaccharide
1S-D-Gal residue or L-GalS-Me-D-
A β1 → 4 bond on the reducing end side of the Gal residue is attached to the reducing end side by an AL-Gal-D-Gal residue or A-L-G.
al-Me-D-Gal residue is present, and the LG
alS-D-Gal residue or AL-GalS-Me
-AL-Gal-D on the non-reducing end side of the -D-Gal residue
-Gal residue, AL-Gal-Me-D-Gal residue, L-GalS-D-Gal residue or L-GalS
An endo-type enzyme with the specificity of hydrolyzing only if a Me-D-Gal residue is present. 7. An immunostimulant containing at least one of the acidic sugars according to claim 1, 2, 3, 4 or 5 as an active ingredient. 8. An enzyme source containing at least one of the red algal slime polysaccharide degrading enzymes I and II according to claim 6, which is contained in seaweed belonging to red algae and is extracted with an aqueous solvent. Or by a GPC method in a low-viscosity acidic sugar obtained by hydrolysis with an enzyme source containing the red alga mucilage polysaccharide degrading enzyme III and at least one of I and II according to claim 6. An immunostimulant which is an acidic sugar mixture obtained by removing a fraction having a molecular weight of more than 100,000, wherein the acidic sugar mixture has two or more sulfate groups in the molecule as an active ingredient. 9. An acidic sugar mixture obtained by removing a fraction of the low-viscosity acidic sugar having a molecular weight of more than 45,000, wherein the number of sulfate groups in the molecule is 2 or more, respectively. The immunostimulant according to claim 8, which is