WO2007013609A1 - Fucoidan-derived oligosaccharide - Google Patents

Fucoidan-derived oligosaccharide Download PDF

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Publication number
WO2007013609A1
WO2007013609A1 PCT/JP2006/315020 JP2006315020W WO2007013609A1 WO 2007013609 A1 WO2007013609 A1 WO 2007013609A1 JP 2006315020 W JP2006315020 W JP 2006315020W WO 2007013609 A1 WO2007013609 A1 WO 2007013609A1
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Prior art keywords
fucoidan
oligosaccharide
molecular weight
oligosaccharides
formula
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PCT/JP2006/315020
Other languages
French (fr)
Japanese (ja)
Inventor
Yuji Nonaka
Takeshi Yasumoto
Hideo Naoki
Toshihide Kusumoto
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Suntory Limited
Tropical Technology Center Ltd.
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Publication of WO2007013609A1 publication Critical patent/WO2007013609A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C07H13/06—Fatty acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
    • C07H7/02—Acyclic radicals
    • C07H7/033—Uronic acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a novel compound which can be used for foods and drinks, health foods, functional foods, pharmaceuticals, cosmetics and the like for the purpose of enhancing immunity and regulating immune function, and a composition comprising the same
  • a novel compound which can be used for foods and drinks, health foods, functional foods, pharmaceuticals, cosmetics and the like for the purpose of enhancing immunity and regulating immune function, and a composition comprising the same
  • Fucoidan is a sulfite polysaccharide contained in algae, which has anti-blood coagulation, fat serum clarification (removing cholesterol and peracid lipids in blood), anti-tumor, and cancer metastasis It has been reported that it has various activities such as anti-AIDS virus infection. Among them, the action of normalizing the immune function of the living body is attracting attention, and it is considered useful as a material for foods and drinks and pharmaceuticals having the function of regulating the immune function.
  • fucoidan is known to vary depending on the algae from which it is derived and its growth environment.
  • One reason for this is that it varies depending on the compositional algae such as fucose, galactose, xylose, and glucuronic acid, which are constituents of fucoidan, and their growth environment.
  • the fact that the positions of ester bonds and dalcoside bonds on these constituent sugars can be varied contributes to the diversity of fucoidan structures. Therefore, many fucoidan structures have been identified! ⁇ ⁇ .
  • fucoidan is a sulfated polysaccharide having a very large molecular weight, and therefore, there are problems relating to absorbability, antigenicity, homogeneity, anticoagulant activity and the like when used as it is as a food or drink.
  • Patent Document 1 discloses a method for acid hydrolysis of fucoidan, and describes that the obtained low-molecular fucoidan has a molecular weight distribution of 5 ⁇ 10 3 or less.
  • Patent Document 2 describes a method of hydrolyzing fucoidan to obtain an oligosaccharide without adding an acid or external force. A method of hydrolyzing fucoidan with an enzyme as described in Patent Document 3 has also been reported! Speak.
  • Patent Document 4 reports that an oligosaccharide was produced by acid hydrolysis of fucoidan obtained from algae such as mozuku, and the structures of several low-molecular fucoidan-derived oligosaccharides were identified. ing.
  • Patent Documents 5 and 6 disclose the structures of oligosaccharides obtained by enzymatic hydrolysis of fucoidan.
  • Patent Document 1 JP-A-7-215990
  • Patent Document 2 JP 2002-226496
  • Patent Document 3 JP 2000-236889
  • Patent Document 4 JP 2000-351790 A
  • Patent Document 5 JP 2003-199596
  • Patent Document 6 JP 2001-226408
  • Non-Patent Document 1 Carbohydrate research 4, 189-195 (1967)
  • Non-Patent Document 2 Carbohydrate research 37, 75-79 (1974)
  • Non-Patent Document 3 Carbohydrate research 41, 308-312 (1975)
  • Patent Documents 1 to 3 the structure of the substances obtained by the methods described in Patent Documents 1 to 3 is not specified. Therefore, when using these oligosaccharides with unknown structures in food, there is a problem that quality control is not easy.
  • the oligosaccharide described in Patent Document 4 has a clear function when used in foods and the like. It is hard to say that it is not safe. Further, the oligosaccharide described in Patent Document 5 has a problem that the molecular weight is large, and the production method described in Patent Document 6 has a problem that the operation is complicated.
  • an object of the present invention is to provide a novel fucoidan-derived oligosaccharide having a specified structure.
  • Another object of the present invention is to provide a fucoidan-derived oligosaccharide that contributes to normalizing immune functions of a living body with high safety.
  • the normalization of immune function as used herein refers to some of the functions that improve the gut microbiota and indirectly improve immune function, such as xylooligosaccharides known as prebiotics. As indicated by fucoidan, it directly activates immunocompetent cells.
  • a further object of the present invention is to provide a fucoidan-derived oligosaccharide that can be accurately added in an effective amount to foods and drinks, pharmaceutical compositions, and the like.
  • a further object of the present invention is to provide the oligosaccharide without requiring complicated steps.
  • the present inventors have completed the present invention by producing novel oligosaccharides such as Fucoidanka and confirming their immunostimulatory activity.
  • these oligosaccharides are also referred to as fucoidan oligosaccharides.
  • the present invention relates to
  • a pharmaceutical composition comprising at least one selected from compounds represented by formulas (I) to (XI);
  • the present invention relates to a cosmetic containing at least one selected from compounds represented by formulas (I) to (XI).
  • the novel fucoidan oligosaccharide of the present invention has an immune function regulating action.
  • the fucoidan oligosaccharide of the present invention has a mild action because it has a separated food material strength, and is extremely safe. Therefore, the oligosaccharide of the present invention is very useful, and its application range is applicable not only to health foods but also to pharmaceuticals and cosmetics.
  • a food / beverage product a pharmaceutical composition, or a cosmetic product that contributes to normalizing the immune function of a living body can be provided.
  • the fucoidan oligosaccharide of the present invention can be provided by a less complicated method.
  • FIG. 1 is an HPLC chart showing the sugar composition analysis of fucoidan obtained by hot water extraction from offshore mozuku riki.
  • FIG. 2 is a diagram showing an MS spectrum of a fucoidan oligosaccharide having a molecular weight of 340 represented by formula (I).
  • FIG. 3 is a diagram showing an MS spectrum of a fucoidan oligosaccharide having a molecular weight of 486 represented by formula (II).
  • FIG. 4 is a diagram showing a 1 H-NMR ⁇ vector of a labeled oligosaccharide corresponding to a compound of formula (I).
  • FIG. 5 is a diagram showing a 13 C-NMR ⁇ vector of the corresponding labeled I spoon oligosaccharide compound of the formula (I).
  • FIG. 6 is a diagram showing a 1 H-NMR spectrum of a labeled oligosaccharide corresponding to a compound of formula (II).
  • FIG. 7 is a diagram showing a 13 C-NMR spectrum of a labeled oligosaccharide corresponding to a compound of formula (IV).
  • FIG. 8 is a diagram showing a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 539 corresponding to the compound of formula (III).
  • FIG. 9 is a diagram showing a 13 C-NMR spectrum of a labeled oligosaccharide having a molecular weight of 539 corresponding to the compound of formula (III).
  • FIG. 10 is a diagram showing a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 715 corresponding to the compound of formula (V).
  • FIG. 11 is a diagram showing a 13 C-NMR spectrum of a labeled oligosaccharide having a molecular weight of 715 corresponding to the compound of formula (V).
  • FIG. 12 shows a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 861 corresponding to the compound of formula (VI).
  • FIG. 13 is a diagram showing a 13 C-NMR spectrum of a labeled oligosaccharide having a molecular weight of 861 corresponding to the compound of formula (VI).
  • FIG. 14 is a diagram showing a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 903 corresponding to the compound of formula (VII).
  • FIG. 15 is a view showing a “C-petal” of a labeled oligosaccharide having a molecular weight of 903 corresponding to the compound of the formula (VII).
  • FIG. 16 shows a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 957 corresponding to the compound of formula (VIII).
  • FIG. 17 shows a 13 C-NMR spectrum of a labeled oligosaccharide having a molecular weight of 957 corresponding to the compound of formula (VIII).
  • FIG. 18 is a diagram showing a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 999 corresponding to the compound of formula (IX).
  • FIG. 19 shows a 13 C-NMR spectrum of a labeled oligosaccharide having a molecular weight of 999 corresponding to the compound of formula (IX).
  • FIG. 20 is a diagram showing a 1 H-NMR ⁇ vector of a fucoidan oligosaccharide having a molecular weight of 754 represented by the formula (VII).
  • FIG. 21 is a view showing a TOF-MS spectrum of a fucoidan oligosaccharide having a molecular weight of 754 represented by the formula (VII).
  • FIG. 22 is a diagram showing an MSZMS spectrum after regeneration of a fucoidan oligosaccharide having a molecular weight of 754 represented by formula (VII).
  • FIG. 23 is a diagram showing a FAB-MS spectrum of a fucoidan oligosaccharide having a molecular weight of 420 represented by the formula (IV).
  • FIG. 24 is a diagram showing an MSZMS spectrum of a fucoidan oligosaccharide having a molecular weight of 420 represented by the formula (IV).
  • FIG. 25 is a view showing a FAB-MS spectrum of a fucoidan oligosaccharide having a molecular weight of 858 represented by formula (X) and a molecular weight of 900 represented by formula (XI).
  • FIG. 26 is a diagram showing an MSZMS spectrum of a fucoidan oligosaccharide having a molecular weight of 858 represented by formula (X).
  • FIG. 27 is a diagram showing an MSZMS spectrum of a fucoidan oligosaccharide having a molecular weight of 900 represented by the formula (XI).
  • FIG. 28 is a diagram showing an ESI-MS chart that has been hydrolyzed offshore mozuku and then fluorescently labeled with ABEE.
  • FIG. 29 is a graph showing the effect of inducing IFN- ⁇ on spleen cells of fucoidan oligosaccharide.
  • FIG. 30 is a diagram showing IFN- ⁇ -inducing effects of multiple fucoidan oligosaccharides on spleen cells.
  • FIG. 31 shows IL-12-inducing effects of fucoidan oligosaccharides on rod cells.
  • FIG. 32 is a diagram showing an IL-10-inducing effect of fucoidan oligosaccharides on rod cells.
  • FIG. 33 is a diagram showing the maturation effect of rod cells by fucoidan oligosaccharides.
  • FIG. 34 is a graph showing the CTL activity-inducing effect of fucoidan oligosaccharide.
  • Fucoidan is a generic name for algae-derived sulfated polysaccharides and contains galactose, glucuronic acid, xylose, etc. in addition to the main constituent sugar, fucose.
  • the type and amount of constituent sugars vary depending on the algae from which fucoidan is derived and its growth environment.
  • the fucoidan used as a raw material for the fucoidan oligosaccharide of the present invention may have any structure, and any algal power may be obtained.
  • Examples of algae include the order of Sphacelariales, Chordariales, Scytosiphonales, Dictyosipnonaies, Mutomoles, Sporochnales, Dictyotales), Kombu (Laminariales), Hibamata
  • mozuku Contains brown alga Phaeophyceae seaweed containing (Fucales).
  • mozuku more preferably fucoidan derived from offshore mozuku is used.
  • algae for example, offshore mozuku
  • algae is filled with 5 to 10 times the amount of distilled water, and it is 0 to 5 hours at 50 to 100 ° C, preferably 0.5 to 2 hours at 80 to 100 ° C. More preferably, about 1 hour at 90-100 ° C Extract between.
  • the algae extract thus obtained can be cooled, suction filtered, desalted and dried to obtain a fucoidan fraction that is easily dissolved in water.
  • the fucoidan fraction thus obtained may be used in the next step without further purification, or may be used after further purification.
  • Fucoidan is preferably extracted from algal power as described above, but may be used in a state of being contained in algae.
  • the compound according to the present invention can be obtained by subjecting fucoidan or algae containing it to the subsequent hydrolysis step.
  • a fucoidan oligosaccharide mixture is obtained by hydrolyzing fucoidan by a method using an acid or an enzyme.
  • the following acid hydrolysis conditions are used.
  • the fraction or fucoidan containing fucoidan obtained as described above by algal power is decomposed using an acid, preferably hydrochloric acid. More specifically, 0.1 to 5.
  • an acid preferably hydrochloric acid. More specifically, 0.1 to 5.
  • the hydrolysis is carried out in an aqueous solvent at 50 to 90 ° C. for 0.1 to 3 hours, preferably 0.25 to 2.5 hours, more preferably 0.5 to 2 hours.
  • the obtained reaction product is neutralized with a base such as about 1N NaOH, desalted by an appropriate means such as electrodialysis or gel filtration, and dried (for example, freeze-dried) to obtain a fucoidan oligosaccharide mixture. This comes out.
  • a base such as about 1N NaOH
  • the mixture force obtained in this way can be used alone or in combination with methods such as chromatography, recrystallization, dialysis, and alcohol precipitation.
  • the oligosaccharide is purified according to the following procedure.
  • an oligosaccharide mixture obtained by hydrolysis of fucoidan is subjected to chromatography using anion exchange resin, and is passed through without adsorption and contains an oligosaccharide containing no sulfate group.
  • the fraction neutral / acidic sugar fraction
  • the fraction is separated from the fraction containing sulfated oligosaccharides (sulfated sugar fraction) that are eluted by the acidic eluate.
  • each component of sulfated oligosaccharides (III) to (XI) can be isolated by subjecting the sulfated sucrose fraction to chromatography.
  • Each oligosaccharide may be appropriately labeled or derivatized for easier purification and structural analysis.
  • oligosaccharides can be fluorescently labeled with a reagent such as 4-amino benzoate (ABEE), which facilitates oligosaccharide detection.
  • ABEE 4-amino benzoate
  • Pure oligosaccharides can be obtained by separating each labeled oligosaccharide and then removing the labeled portion.
  • the fucoidan oligosaccharide thus obtained can be used for, for example, foods and drinks, pharmaceuticals, cosmetics and the like to impart an immune function-modulating action thereto.
  • the function of regulating immune function in the present specification refers to an action of enhancing a lowered immune response in a living body and suppressing Z or an enhanced immune response. Therefore, the action includes immunostimulatory action and immunosuppressive action.
  • the function of regulating immune function in the present specification can be measured by a method known in the art. It can be measured using, for example, an effect of inducing a site force-in having an immune function regulating effect as an index. Site force ins used for this purpose include interferon- ⁇ , interleukin-10, 12 and the like.
  • the immune function-modulating action can be measured using maturation of rod cells involved in the immune response or the activity of cytotoxic rod cells (CTL) as an index.
  • CTL cytotoxic rod cells
  • the immune function regulator of the present invention can be used for health promotion, tumors, metastasis of cancer, viral diseases (for example, kaze, AIDS, viral hepatitis), allergic diseases (for example, pollen) It is also known to be effective for diseases or conditions such as infectious diseases, allergic rhinitis, atopy, asthma), autoimmune diseases (eg rheumatoid arthritis), inflammatory diseases, diabetes.
  • viral diseases for example, kaze, AIDS, viral hepatitis
  • allergic diseases for example, pollen
  • diseases or conditions such as infectious diseases, allergic rhinitis, atopy, asthma), autoimmune diseases (eg rheumatoid arthritis), inflammatory diseases, diabetes.
  • the food additive and food or drink containing the same and having an immune function modulating action, and the immune function prepared by adding the same It is preferable to implement as a health food having a moderating effect. They may be mixed with various components such as known sweeteners, acidulants, vitamins, etc. to make products that meet the user's taste.
  • Foods and drinks include, for example, tablets, capsules, soft drinks, tea drinks, drinks, dairy products such as yogurt and lactic acid bacteria drinks, seasonings, processed foods, desserts, confectionery (e.g. gum, candy, jelly) etc. It is possible to provide in the form.
  • the foods and drinks of the present invention also include functional foods (including foods for specified health use and conditionally specified health foods) with a label indicating that they have an immune function regulating effect on containers and instructions.
  • the labeling place includes, but is not limited to, a container or instructions attached to it.
  • Containers include but are not limited to bottles, cans, plastic bottles, plastic bottles, paper packs, and the like.
  • the display method is not limited to force including printing, engraving, sealing, and the like.
  • the food and drink may be pet food or animal feed that is used as pet food.
  • the fucoidan oligosaccharide of the present invention can be used, for example, as an immune function regulator, immunostimulator, antiallergic agent, and immunosuppressant. Accordingly, in one embodiment, the present invention is a pharmaceutical composition having an immune function modulating action, an immunostimulating action, an antiallergic action, and a Z or immunosuppressive action, comprising fucoidan oligosaccharide.
  • the pharmaceutical composition is generally used in the pharmaceutical formulation technical field such as diluents, carriers, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspension agents, coating agents, etc. as the main agent. It can be formulated using known adjuvants.
  • dosage forms include tablets, capsules, granules, powders, solutions, syrups, suppositories, creams, ointments, emulsions, tablets, injections, and the like, and are particularly limited. It is not a thing.
  • Examples of the route of administration of this drug include oral administration, rectal administration, enteral administration, etc., but are not particularly limited.
  • Cosmetics to which the oligosaccharide of the present invention is added or blended are, for example, facial, skin and hair creams, lotions, gels, mousses, shampoos, rinses and the like. [0064] Combination of other ingredients
  • the fucoidan oligosaccharide of the present invention may be used by itself in foods and drinks, pharmaceutical compositions, and cosmetics, but it is a food material having other immune function regulating action or immunostimulatory action or It is also suitable to use together with a substance.
  • Such food materials or substances include microorganisms such as lactic acid bacteria (Lactococcus, Lactobacilli), yeasts, fungi; mushrooms such as shitake, maitake, ganoderma, agaritas; Xinucleotide (CpG-ODN), Polyl: Nucleic acid-derived substances such as C; Lipopolysaccharide (LPS) and j8-glucan; Seaweed; Chinese medicine; Cancer vaccine (eg, MM46 cancer antigen); Concananoline A (ConA) BRM (Biological Response Modifier) such as Krestin (registered trademark), OK-432 (Pisibanil, registered trademark) and Lentinan (registered trademark); Known immune activators such as fucoidan;
  • the fucoidan oligosaccharides of the present invention in combination with lactic acid bacteria, synergistically enhance their immune function regulating action. Therefore, the present invention also includes foods and drinks, pharmaceutical compositions, and cosmetics to which fucoidan oligosaccharide is added in combination with or containing lactic acid bacteria. These foods and drinks, pharmaceutical compositions, and cosmetics are characterized by having a synergistically enhanced immune function regulating action as compared with the case where fucoidan oligosaccharide or lactic acid bacteria are used alone. Lactic acid bacteria used for this purpose include Lactobacillus sp. 3) Lactobacillus plantarum or 7 is Lactobacillus pentosus.
  • the preferred ratio of fucoidan oligosaccharide used to lactic acid bacteria is 1: 1 to L0000: 1 by weight, more preferably 250: 1 to 1000: 1.
  • the present invention also includes a method of synergistically enhancing the immune function regulating action by using a combination of fucoidan oligosaccharide and lactic acid bacteria.
  • the food / beverage products and compositions of the present invention contain general ingredients such as carriers, diluents, excipients or additives according to specific embodiments. Can be blended.
  • the carrier, diluent or excipient is not particularly limited as long as it does not interfere with the physiological activity of fucoidan oligosaccharide.
  • Sugars such as liquid sugar, ethanol, Alcohols such as propylene glycol and glycerin, sorbitol, mannitol, erythritol, latathitol, xylitol, maltitol, reduced palatinose, reduced starch and other sugar alcohols, solvents such as triacetin, gum arabic, carrageenan, xanthan gum , Polysaccharides such as guar gum, dielan gum, pectin, or water.
  • additives include auxiliaries such as chelating agents, fragrances, spice extracts, and preservatives. Carriers, additives and the like can be added as long as the effects of the present invention are not impaired.
  • the blending amount of the oligosaccharide in the food / beverage product, the pharmaceutical composition and the cosmetic product is appropriately selected depending on the relationship with the other combination components to be selected, and is not particularly limited.
  • fucoidan oligosaccharides are added to beverages or foods in a pharmaceutical composition in an amount of 0. Olg to: LOgZ days, preferably 0.05 g to lgZ days for an individual weight of 60 kg. Particularly preferred is 0.05 g to 0.5 gZ days.
  • 0.01 to 20% by weight, preferably 0.05 to 15% by weight is used.
  • the oligosaccharide of the present invention can be used alone in an extracted purified product or a synthetic product in foods, beverages, pharmaceutical compositions, and cosmetics, but is a mixture containing one or more of the oligosaccharides of the present invention. It can also be added to food and drink in the form of
  • NMR analysis was performed using an ECA-600 type nuclear magnetic resonance apparatus (JEOL Ltd.). Heavy water (D O) as the measurement solvent
  • the resulting aqueous solution was neutralized with 2NNaOH and fluorescently labeled with ABEE to prepare a monosaccharide analysis sample.
  • the oligosaccharide mixture was obtained by elution with 2NHC1 with 280 mg of the fraction containing oligosaccharides without sulfate groups (neutral'acid sugar fraction) obtained by elution with water.
  • the fraction containing oligosaccharides rich in sulfate groups (sulfated sugar fraction) was separated into 425 mg.
  • the sulfated sugar fraction was desalted by subjecting it to gel filtration (Biogel P-6 (Bio-Rad)).
  • gel filtration Biogel P-6 (Bio-Rad)
  • ABEE ethyl 4-aminoaminobenzoate
  • NaBH CN sodium cyanoborohydride
  • Acetic acid 410 1 was added and stirred at 65 ° C for 4 hours.
  • the resulting product is dried under vacuum, partitioned between water and black mouth form, and the aqueous layer is separated on a reverse phase column (support: Lichroprep RP—8 (25—40 m) (Merck), ⁇ 10 X 220 mm; Solvent conditions: 5% CH CNZO. 1% TFA (100ml)
  • ABEE was bound on each oligosaccharide, and a regenerated oligosaccharide product was obtained. That is, to each of these separated labeled oligosaccharides lOmg (100 1), 10 1 each of hydrogen peroxide and acetic acid was added, and the mixture was allowed to stand overnight and then dried. Among the regenerated oligosaccharides obtained in this way, those obtained from a compound with a molecular weight of 903 were analyzed using 1 H-NMR (Fig.
  • C57BLZ6 mice (8 weeks old, male, Nippon Charles River Co., Ltd.) force Spleen cells were isolated, prepared to 5 ⁇ 10 6 cells / ml, and cultured in 24-well plates.
  • the fucoidan oligosaccharides of compounds (1) and (II) were added to each well so as to be 100 g / ml.
  • fucose, sulfated fucose (MW164 and 244, respectively) and xylooligosaccharide (XOS) were used in equal amounts.
  • lactic acid bacteria Lactobacillus pentosus: FERM A BP-10028 (dead bacteria) (dead bacteria) were added to each well in an amount of 0.1 gZml. I went. With regard to compound (I), when it was used in combination with lactic acid bacteria, a remarkably strong IFN- ⁇ induction ability was obtained. This effect can be achieved by using a combination of both compounds (I) and lactic acid bacteria that are so strong that they cannot be predicted from the results obtained by using the lactic acid bacteria alone (second left force and third right force in Fig. 29). Supports synergistic effects. Such an action is also ineffective in xylo-oligosaccharides widely used as prebiotics, which have no such activity in the constituent components fucose and sulfated fucose.
  • the fucoidan oligosaccharides of compound (I) and compound (II) have a function of activating splenocytes and can regulate the immune function of the living body.
  • BALBZc mice (8 weeks old, male, Japan SLC, Inc.) the femoral force marrow cells were separated, so as to l X 10 6 cells / ml, 10% FBS, 20ng / ml GM- CSF (Peprotec), 20ng It was suspended in RPMI 1640 medium containing / ml IL-3 (Peprotec) and cultured in a 24-well plate. On the 3rd and 5th days from the start of the culture, the medium was changed to obtain adherent immature rod-like cells. Compounds (1), (11), (111), (V), (VI), and (VII) were each added to the obtained rod-shaped cells at a concentration of 50 gZml (pretreatment).
  • lactic acid bacteria Lactobacillus pentosus: FERM ABP-10028 (dead bacteria) (dead bacteria) were added to each well in an amount of 0.1 ⁇ g Zml, and the same operation as described above was performed.
  • Figs. 31 and 32 The collected culture supernatant was measured for IL-12 and IL-10 using an ELISA kit. The results are shown in Figs. 31 and 32, respectively. Compounds (11), (111), and (VI) alone showed IL-12 induction ability. Compound (I) induced IL-12 synergistically when used in combination with lactic acid bacteria (FIG. 31). Compounds (11), (III) and (V) induced IL-10 when used in combination with lactic acid bacteria (Fig. 32). For these reasons, the fucoidan oligosaccharides found in the present invention have a function of regulating the immune system of the living body positively or negatively (activate reduced immunity or suppress excessive immune responses). I was divided. Xylooligosaccharides did not show this effect.
  • the collected cells were counted and prepared to 1 X 10 5 cells Zml, and then 1 ml of cell suspension was treated with mitomycin C (50 gZml) and reacted at 37 ° C for 30 minutes. After completion of the reaction, mitomycin C was removed by washing with PBS, and 1 ml of C57BL / 6 mouse spleen cells (5 ⁇ 10 6 cells / ml) prepared by a conventional method was added and mixed and cultured for 4 days. After culturing the cells Targeted P-815 cells (SOOOcellsZlOO / zl) with the C57BLZ6 mouse alloantigen and killed by reacting for 4 hours at an E: T ratio of 40: 1 and 80: 1.

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Abstract

Fucoidan is a sulfated polysaccharide having an extremely large molecular weight. In the development of pharmaceuticals and health foods using fucoidan, there are problems with respect to absorption property, antigenicity, homogeneity, anti-coagulant activity and the like. Then, provided is a low molecular weight compound derived from a low molecular weight fucoidan having a specified structure and function, which can overcome the above-mentioned problems. Novel fucoidan oligosaccharides (I) to (XI) as disclosed in the description can be found by the hydrolysis of fucoidan with an acid. It is found that these oligosaccharides have various immune function-modulating effects including IFN-Ϝ-inducing capability, IL-10 and 12-inducing capability, dendritic cell-maturating effect and CTL activity-inducing effect.

Description

明 細 書  Specification
フコィダン由来オリゴ糖  Fucoidan-derived oligosaccharides
技術分野  Technical field
[0001] 本発明は免疫力の増強、免疫機能の調節などを目的とした飲食品、健康食品、機 能性食品、医薬品、化粧品等に利用可能な新規ィ匕合物およびそれを含む組成物に 関する。  [0001] The present invention relates to a novel compound which can be used for foods and drinks, health foods, functional foods, pharmaceuticals, cosmetics and the like for the purpose of enhancing immunity and regulating immune function, and a composition comprising the same Related to
背景技術  Background art
[0002] フコィダンは藻類に含まれる硫酸ィヒ多糖であり、抗血液凝固作用、脂血清澄作用（ 血液中のコレステロールや過酸ィ匕脂質を除去する作用）、抗腫瘍作用、癌転移抑制 作用、抗エイズウイルス感染作用等の様々な活性を有することが報告されている。中 でも、生体の免疫機能を正常化する作用が注目されており、免疫機能調節作用を有 する飲食品や医薬品の素材として有用であると考えられている。  [0002] Fucoidan is a sulfite polysaccharide contained in algae, which has anti-blood coagulation, fat serum clarification (removing cholesterol and peracid lipids in blood), anti-tumor, and cancer metastasis It has been reported that it has various activities such as anti-AIDS virus infection. Among them, the action of normalizing the immune function of the living body is attracting attention, and it is considered useful as a material for foods and drinks and pharmaceuticals having the function of regulating the immune function.
[0003] 一方、フコィダンの構造は、由来となる藻類やその生育環境などにより異なることが 知られている。その理由の一つは、フコィダンの構成成分であるフコース、ガラクトー ス、キシロース、グルクロン酸等の組成力 藻類やその生育環境によって変動するた めである。また、それら構成糖上のエステル結合およびダルコシド結合の位置が変動 し得ることも、フコィダン構造の多様性に寄与している。そのため、未だに多くのフコィ ダンの構造が特定されて！ヽな ヽ。  [0003] On the other hand, the structure of fucoidan is known to vary depending on the algae from which it is derived and its growth environment. One reason for this is that it varies depending on the compositional algae such as fucose, galactose, xylose, and glucuronic acid, which are constituents of fucoidan, and their growth environment. In addition, the fact that the positions of ester bonds and dalcoside bonds on these constituent sugars can be varied contributes to the diversity of fucoidan structures. Therefore, many fucoidan structures have been identified!ヽ ヽ.
[0004] これらの理由により、フコィダンを利用して飲食品や医薬品などを開発する場合に は、それらに適切なフコィダンを選定するのに多大な時間を要することが多力つた。 また、消費者にとっても、どのフコィダンを選べばよいのか明確ではな力つた。さらに 、フコィダンは分子量が極めて大きい硫酸ィ匕多糖であり、このためそのまま飲食品や 医薬品として用いる際には、吸収性、抗原性、均一性、抗凝血活性等に関する問題 がある。  [0004] For these reasons, when developing foods and beverages, pharmaceuticals, etc. using fucoidan, it often takes a lot of time to select an appropriate fucoidan. It was also clear for consumers which fucoidan to choose. Furthermore, fucoidan is a sulfated polysaccharide having a very large molecular weight, and therefore, there are problems relating to absorbability, antigenicity, homogeneity, anticoagulant activity and the like when used as it is as a food or drink.
[0005] これらの問題を解決するために、特定された構造と機能を有する低分子のフコイダ ン由来オリゴ糖が望まれる。  [0005] In order to solve these problems, a low-molecular fucoidan-derived oligosaccharide having a specified structure and function is desired.
[0006] これまでに、化学合成によるフコース含有オリゴ糖が報告されているが（非特許文献 1、 2および 3)、これらは有機合成反応により調製されているため食品などに使用す るのは好ましくなかった。 [0006] So far, fucose-containing oligosaccharides by chemical synthesis have been reported (non-patent literature). 1, 2 and 3), which are prepared by organic synthesis reactions, are not preferred for use in foods.
[0007] 一方、フコィダンを加水分解してフコィダンを低分子化する方法も報告されている。 [0007] On the other hand, a method for hydrolyzing fucoidan to lower the molecular weight of fucoidan has also been reported.
例えば、特許文献 1には、フコィダンを酸加水分解する方法が開示されており、得ら れた低分子フコィダンは、 5 X 103以下の分子量分布を有していたことが記載されて いる。また、特許文献 2には、酸を外部力も添加することなくフコィダンを加水分解し てオリゴ糖を得る方法が記載されている。また、特許文献 3のように酵素によりフコィ ダンを加水分解する方法も報告されて！ヽる。 For example, Patent Document 1 discloses a method for acid hydrolysis of fucoidan, and describes that the obtained low-molecular fucoidan has a molecular weight distribution of 5 × 10 3 or less. Patent Document 2 describes a method of hydrolyzing fucoidan to obtain an oligosaccharide without adding an acid or external force. A method of hydrolyzing fucoidan with an enzyme as described in Patent Document 3 has also been reported! Speak.
[0008] また、フコィダンの加水分解により得られ、構造が決定されたオリゴ糖カ^、くつか報 告されている。例えば、特許文献 4においては、モズク等の藻類カゝら得たフコィダンを 酸加水分解してオリゴ糖を製造したことが報告されており、数種の低分子フコィダン 由来オリゴ糖の構造が特定されている。また、特許文献 5および 6においては、フコィ ダンを酵素的に加水分解して得たオリゴ糖の構造が開示されている。 [0008] Further, some oligosaccharides obtained by hydrolysis of fucoidan and whose structure has been determined have been reported. For example, Patent Document 4 reports that an oligosaccharide was produced by acid hydrolysis of fucoidan obtained from algae such as mozuku, and the structures of several low-molecular fucoidan-derived oligosaccharides were identified. ing. Patent Documents 5 and 6 disclose the structures of oligosaccharides obtained by enzymatic hydrolysis of fucoidan.
特許文献 1 :特開平 7— 215990  Patent Document 1: JP-A-7-215990
特許文献 2：特開 2002— 226496  Patent Document 2: JP 2002-226496
特許文献 3：特開 2000 - 236889  Patent Document 3: JP 2000-236889
特許文献 4：特開 2000 - 351790  Patent Document 4: JP 2000-351790 A
特許文献 5 :特開 2003— 199596  Patent Document 5: JP 2003-199596
特許文献 6：特開 2001— 226408  Patent Document 6: JP 2001-226408
非特許文献 1 : Carbohydrate research 4, 189-195 (1967)  Non-Patent Document 1: Carbohydrate research 4, 189-195 (1967)
非特許文献 2 : Carbohydrate research 37, 75-79 (1974)  Non-Patent Document 2: Carbohydrate research 37, 75-79 (1974)
非特許文献 3 : Carbohydrate research 41, 308-312 (1975)  Non-Patent Document 3: Carbohydrate research 41, 308-312 (1975)
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0009] し力しながら、特許文献 1〜3に記載の方法で得られた物質は構造が特定されてい ない。したがって、これら構造不明のオリゴ糖を食品に使用する際には、品質の管理 が容易ではな ヽと 、う問題がある。 However, the structure of the substances obtained by the methods described in Patent Documents 1 to 3 is not specified. Therefore, when using these oligosaccharides with unknown structures in food, there is a problem that quality control is not easy.
[0010] また、特許文献 4に記載のオリゴ糖は、食品などに利用した場合の機能が明らかに されておらず安全性が高いとは言い難い。さらに、特許文献 5に記載のオリゴ糖には 分子量が大きいという問題が、特許文献 6に記載の製法にはその操作が煩雑である という問題もある。 [0010] Furthermore, the oligosaccharide described in Patent Document 4 has a clear function when used in foods and the like. It is hard to say that it is not safe. Further, the oligosaccharide described in Patent Document 5 has a problem that the molecular weight is large, and the production method described in Patent Document 6 has a problem that the operation is complicated.
[0011] そこで、様々な用途に用いることができる素材として、上記のように、特定された構 造を有し、簡便かつ正確に品質管理できるフコィダン由来オリゴ糖を開発することが 望まれている。また、飲食品や医薬品における用途を考慮すると、低分子で扱いや すくまた簡便に製造できるものであること、さらには安全性が高いものであることが必 要とされる。  [0011] Therefore, as a material that can be used in various applications, it is desired to develop fucoidan-derived oligosaccharides having the specified structure as described above and capable of quality control simply and accurately. . In addition, considering applications in foods and beverages and pharmaceuticals, it is necessary that they are easy to handle with small molecules and can be easily manufactured, and that they are highly safe.
そこで、本発明の目的は、構造が特定された新規なフコィダン由来オリゴ糖を提供 することである。  Accordingly, an object of the present invention is to provide a novel fucoidan-derived oligosaccharide having a specified structure.
[0012] また、本発明の別の目的は、安全性が高ぐ生体の免疫機能を正常化するのに寄 与するフコィダン由来オリゴ糖を提供することである。なお、ここでいう免疫機能の正 常化とは、例えばプレバイオテイクスとして知られているキシロオリゴ糖のように腸内細 菌叢を改善して間接的に免疫機能を整えるものではなぐいくつかのフコィダンで示 されているように直接的に免疫担当細胞を活性ィ匕するものを指す。  [0012] Another object of the present invention is to provide a fucoidan-derived oligosaccharide that contributes to normalizing immune functions of a living body with high safety. The normalization of immune function as used herein refers to some of the functions that improve the gut microbiota and indirectly improve immune function, such as xylooligosaccharides known as prebiotics. As indicated by fucoidan, it directly activates immunocompetent cells.
[0013] 本発明のさらなる目的は、効果量を的確に飲食品、医薬組成物等に添加すること ができるフコィダン由来オリゴ糖を提供することである。  [0013] A further object of the present invention is to provide a fucoidan-derived oligosaccharide that can be accurately added in an effective amount to foods and drinks, pharmaceutical compositions, and the like.
[0014] 本発明のさらなる目的は、煩雑な工程を必要とせずに当該オリゴ糖を提供すること でもある。  [0014] A further object of the present invention is to provide the oligosaccharide without requiring complicated steps.
課題を解決するための手段  Means for solving the problem
[0015] 本発明者は、フコィダンカゝら新規オリゴ糖を製造し、それらの免疫賦活活性を確認 することにより、本発明の完成に至った。本発明においては、これらオリゴ糖をフコィ ダンオリゴ糖とも称する。 [0015] The present inventors have completed the present invention by producing novel oligosaccharides such as Fucoidanka and confirming their immunostimulatory activity. In the present invention, these oligosaccharides are also referred to as fucoidan oligosaccharides.
[0016] すなわち本発明は、 That is, the present invention relates to
(1)下記構造式 (1)、 (Π)、（111)、（IV)、 (V)ゝ (VI)、（VII)、（VIII)、（IX)、（x)、または (1) The following structural formula (1), (Π), (111), (IV), (V) ゝ (VI), (VII), (VIII), (IX), (x), or
(XI)で表されるフコィダンオリゴ糖； A fucoidan oligosaccharide represented by (XI);
[0017] [化 1]
Figure imgf000005_0001
[0017] [Chemical 1]
Figure imgf000005_0001
[0018] (分子量 340) [0019] [化 2]  [0018] (Molecular weight 340) [0019] [Chemical 2]
Figure imgf000005_0002
Figure imgf000005_0002
[0020] (分子量 486) [0021] [化 3]  [0020] (Molecular weight 486) [0021] [Chemical 3]
Figure imgf000005_0003
Figure imgf000005_0003
[0022] (分子量 390) [0023] [化 4] [0022] (Molecular weight 390) [0023] [Chemical 4]
Figure imgf000005_0004
Figure imgf000005_0004
[0024] (分子量 420) [0024] (Molecular weight 420)
[0025] [化 5] [0025] [Chemical 5]
Figure imgf000006_0001
Figure imgf000006_0001
[0026] (分子量 566) [0027] [化 6] [0026] (Molecular weight 566) [0027] [Chemical 6]
Figure imgf000006_0002
Figure imgf000006_0002
[0028] (分子量 712) [0029] [化 7] [0028] (Molecular weight 712) [0029] [Chemical 7]
Figure imgf000006_0003
Figure imgf000006_0003
[0030] (分子量 754) [0031] [化 8] [0030] (Molecular weight 754) [0031] [Chemical 8]
Figure imgf000007_0001
Figure imgf000007_0001
[0032] (分子量 808) [0033] [化 9] [0032] (Molecular weight 808) [0033] [Chemical 9]
Figure imgf000007_0002
Figure imgf000007_0002
[0034] (分子量 850) [0035] [化 10] [0034] (Molecular weight 850) [0035] [Chemical 10]
Figure imgf000007_0003
[0036] (分子量 858)
Figure imgf000007_0003
[0036] (Molecular weight 858)
[0037] [化 11] [0037] [Chemical 11]
Figure imgf000008_0001
Figure imgf000008_0001
[0038] (分子量 900) [0038] (Molecular weight 900)
(2)式 (I)〜 (XI)で表される化合物力 選択される少なくとも一種を含む免疫機能調 節剤；  (2) Compound function represented by the formulas (I) to (XI): an immune function modulator comprising at least one selected from;
(3)式 (I)〜 (XI)で表される化合物力 選択される少なくとも一種を添加した飲食品； (3) Compound power represented by formulas (I) to (XI)
(4)式 (I)〜 (XI)で表される化合物から選択される少なくとも一種を含有し、免疫機能 調節作用を有する旨の表示を付した飲食品； (4) A food or drink containing at least one selected from the compounds represented by formulas (I) to (XI) and labeled as having an immune function-modulating effect;
(5)式 (I)〜 (XI)で表される化合物から選択される少なくとも一種を含む医薬組成物； および  (5) a pharmaceutical composition comprising at least one selected from compounds represented by formulas (I) to (XI); and
(6)式 (I)〜 (XI)で表される化合物から選択される少なくとも一種を含む化粧品に関 する。  (6) The present invention relates to a cosmetic containing at least one selected from compounds represented by formulas (I) to (XI).
発明の効果  The invention's effect
[0039] 本発明の新規フコィダンオリゴ糖は、免疫機能調節作用を有する。また、本発明の フコィダンオリゴ糖は、食品素材力も分離されたものであるため穏ゃ力な作用を有し、 極めて安全性が高い。従って、本発明のオリゴ糖は非常に有用であり、その応用範 囲は健康食品のみならず、医薬品およびィ匕粧品にも適用可能である。  [0039] The novel fucoidan oligosaccharide of the present invention has an immune function regulating action. In addition, the fucoidan oligosaccharide of the present invention has a mild action because it has a separated food material strength, and is extremely safe. Therefore, the oligosaccharide of the present invention is very useful, and its application range is applicable not only to health foods but also to pharmaceuticals and cosmetics.
[0040] 即ち、本発明のオリゴ糖を添加することにより、生体の免疫機能を正常化するのに 寄与する飲食品、医薬組成物、または化粧品を提供することができる。 That is, by adding the oligosaccharide of the present invention, a food / beverage product, a pharmaceutical composition, or a cosmetic product that contributes to normalizing the immune function of a living body can be provided.
[0041] また、本発明により、煩雑でない方法で本発明のフコィダンオリゴ糖を提供すること ができる。 [0041] Further, according to the present invention, the fucoidan oligosaccharide of the present invention can be provided by a less complicated method.
図面の簡単な説明 [図 1]沖縛モズク力ゝら熱水抽出により得られるフコィダンの糖組成分析を示す HPLC チャートである。 Brief Description of Drawings FIG. 1 is an HPLC chart showing the sugar composition analysis of fucoidan obtained by hot water extraction from offshore mozuku riki.
[図 2]式 (I)で表される分子量 340のフコィダンオリゴ糖の MSスペクトルを示す図であ る。  FIG. 2 is a diagram showing an MS spectrum of a fucoidan oligosaccharide having a molecular weight of 340 represented by formula (I).
[図 3]式 (II)で表される分子量 486のフコィダンオリゴ糖の MSスペクトルを示す図で ある。  FIG. 3 is a diagram showing an MS spectrum of a fucoidan oligosaccharide having a molecular weight of 486 represented by formula (II).
[図 4]式 (I)の化合物に対応する標識ィ匕オリゴ糖の1 H— NMR ^ベクトルを示す図で ある。 FIG. 4 is a diagram showing a 1 H-NMR ^ vector of a labeled oligosaccharide corresponding to a compound of formula (I).
[図 5]式 (I)の化合物に対応する標識ィ匕オリゴ糖の13 C— NMR ^ベクトルを示す図で ある。 5 is a diagram showing a 13 C-NMR ^ vector of the corresponding labeled I spoon oligosaccharide compound of the formula (I).
[図 6]式 (II)の化合物に対応する標識ィ匕オリゴ糖の1 H— NMRスペクトルを示す図で ある。 FIG. 6 is a diagram showing a 1 H-NMR spectrum of a labeled oligosaccharide corresponding to a compound of formula (II).
[図 7]式 (Π)の化合物に対応する標識ィ匕オリゴ糖の13 C— NMRスペクトルを示す図で ある。 FIG. 7 is a diagram showing a 13 C-NMR spectrum of a labeled oligosaccharide corresponding to a compound of formula (IV).
[図 8]式 (III)の化合物に対応する分子量 539の標識ィ匕オリゴ糖の1 H— NMR^ぺクト ルを示す図である。 FIG. 8 is a diagram showing a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 539 corresponding to the compound of formula (III).
[図 9]式 (III)の化合物に対応する分子量 539の標識ィ匕オリゴ糖の13 C—NMRスぺタト ルを示す図である。 FIG. 9 is a diagram showing a 13 C-NMR spectrum of a labeled oligosaccharide having a molecular weight of 539 corresponding to the compound of formula (III).
[図 10]式 (V)の化合物に対応する分子量 715の標識ィ匕オリゴ糖の1 H— NMRスぺク トルを示す図である。 FIG. 10 is a diagram showing a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 715 corresponding to the compound of formula (V).
[図 11]式 (V)の化合物に対応する分子量 715の標識ィ匕オリゴ糖の13 C— NMR^ぺク トルを示す図である。 FIG. 11 is a diagram showing a 13 C-NMR spectrum of a labeled oligosaccharide having a molecular weight of 715 corresponding to the compound of formula (V).
[図 12]式 (VI)の化合物に対応する分子量 861の標識ィ匕オリゴ糖の1 H - NMR^ぺク トルを示す図である。 FIG. 12 shows a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 861 corresponding to the compound of formula (VI).
[図 13]式 (VI)の化合物に対応する分子量 861の標識ィ匕オリゴ糖の13 C - NMRスぺク トルを示す図である。 FIG. 13 is a diagram showing a 13 C-NMR spectrum of a labeled oligosaccharide having a molecular weight of 861 corresponding to the compound of formula (VI).
[図 14]式 (VII)の化合物に対応する分子量 903の標識ィ匕オリゴ糖の1 H— NMRスぺ タトルを示す図である。 [図 15]式 (VII)の化合物に対応する分子量 903の標識ィ匕オリゴ糖の" C - ぺ タトルを示す図である。 FIG. 14 is a diagram showing a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 903 corresponding to the compound of formula (VII). FIG. 15 is a view showing a “C-petal” of a labeled oligosaccharide having a molecular weight of 903 corresponding to the compound of the formula (VII).
[図 16]式 (VIII)の化合物に対応する分子量 957の標識ィ匕オリゴ糖の1 H— NMR^ぺ タトルを示す図である。 FIG. 16 shows a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 957 corresponding to the compound of formula (VIII).
[図 17]式 (VIII)の化合物に対応する分子量 957の標識ィ匕オリゴ糖の13 C— NMRスぺ タトルを示す図である。 FIG. 17 shows a 13 C-NMR spectrum of a labeled oligosaccharide having a molecular weight of 957 corresponding to the compound of formula (VIII).
[図 18]式 (IX)の化合物に対応する分子量 999の標識ィ匕オリゴ糖の1 H— NMRスぺク トルを示す図である。 FIG. 18 is a diagram showing a 1 H-NMR spectrum of a labeled oligosaccharide having a molecular weight of 999 corresponding to the compound of formula (IX).
[図 19]式 (IX)の化合物に対応する分子量 999の標識ィ匕オリゴ糖の13 C—NMR^ぺク トルを示す図である。 FIG. 19 shows a 13 C-NMR spectrum of a labeled oligosaccharide having a molecular weight of 999 corresponding to the compound of formula (IX).
[図 20]式 (VII)で表される分子量 754のフコィダンオリゴ糖の1 H— NMR ^ベクトルを 示す図である。 FIG. 20 is a diagram showing a 1 H-NMR ^ vector of a fucoidan oligosaccharide having a molecular weight of 754 represented by the formula (VII).
[図 21]式 (VII)で表される分子量 754のフコィダンオリゴ糖の TOF- MSスペクトルを 示す図である。  FIG. 21 is a view showing a TOF-MS spectrum of a fucoidan oligosaccharide having a molecular weight of 754 represented by the formula (VII).
[図 22]式 (VII)で表される分子量 754のフコィダンオリゴ糖を再生した後の MSZMS スペクトルを示す図である。  FIG. 22 is a diagram showing an MSZMS spectrum after regeneration of a fucoidan oligosaccharide having a molecular weight of 754 represented by formula (VII).
[図 23]式（IV)で表される分子量 420のフコィダンオリゴ糖の FAB- MSスペクトルを示 す図である。  FIG. 23 is a diagram showing a FAB-MS spectrum of a fucoidan oligosaccharide having a molecular weight of 420 represented by the formula (IV).
[図 24]式（IV)で表される分子量 420のフコィダンオリゴ糖の MSZMSスペクトルを示 す図である。  FIG. 24 is a diagram showing an MSZMS spectrum of a fucoidan oligosaccharide having a molecular weight of 420 represented by the formula (IV).
[図 25]式 (X)で表される分子量 858および式 (XI)で表される分子量 900のフコィダン オリゴ糖の FAB- MSスペクトルを示す図である。  FIG. 25 is a view showing a FAB-MS spectrum of a fucoidan oligosaccharide having a molecular weight of 858 represented by formula (X) and a molecular weight of 900 represented by formula (XI).
[図 26]式（X)で表される分子量 858のフコィダンオリゴ糖の MSZMSスペクトルを示 す図である。  FIG. 26 is a diagram showing an MSZMS spectrum of a fucoidan oligosaccharide having a molecular weight of 858 represented by formula (X).
[図 27]式 (XI)で表される分子量 900のフコィダンオリゴ糖の MSZMSスペクトルを示 す図である。  FIG. 27 is a diagram showing an MSZMS spectrum of a fucoidan oligosaccharide having a molecular weight of 900 represented by the formula (XI).
[図 28]沖縛モズクを加水分解後、 ABEEで蛍光標識ィ匕した ESI— MSのチャートを示 す図である。 [図 29]フコィダンオリゴ糖の脾細胞に対する IFN— γ誘導効果を示す図である。 FIG. 28 is a diagram showing an ESI-MS chart that has been hydrolyzed offshore mozuku and then fluorescently labeled with ABEE. FIG. 29 is a graph showing the effect of inducing IFN-γ on spleen cells of fucoidan oligosaccharide.
[図 30]複数のフコィダンオリゴ糖の脾細胞に対する IFN— γ誘導効果を示す図であ る。  FIG. 30 is a diagram showing IFN-γ-inducing effects of multiple fucoidan oligosaccharides on spleen cells.
[図 31]フコィダンオリゴ糖の榭状細胞に対する IL— 12誘導効果を示す図である。  FIG. 31 shows IL-12-inducing effects of fucoidan oligosaccharides on rod cells.
[図 32]フコィダンオリゴ糖の榭状細胞に対する IL— 10誘導効果を示す図である。  FIG. 32 is a diagram showing an IL-10-inducing effect of fucoidan oligosaccharides on rod cells.
[図 33]フコィダンオリゴ糖による榭状細胞の成熟化効果を示す図である。  FIG. 33 is a diagram showing the maturation effect of rod cells by fucoidan oligosaccharides.
[図 34]フコィダンオリゴ糖の CTL活性誘導効果を示す図である。  FIG. 34 is a graph showing the CTL activity-inducing effect of fucoidan oligosaccharide.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0043] フコィダン [0043] Fucoidan
フコィダンとは藻類由来の硫酸ィ匕多糖の総称であり、主な構成糖であるフコースに カロえて、ガラクトース、グルクロン酸、キシロース等を含有する。構成糖の種類や量は 、フコィダンの由来となる藻類やその生育環境により異なる。  Fucoidan is a generic name for algae-derived sulfated polysaccharides and contains galactose, glucuronic acid, xylose, etc. in addition to the main constituent sugar, fucose. The type and amount of constituent sugars vary depending on the algae from which fucoidan is derived and its growth environment.
[0044] 本発明のフコィダンオリゴ糖のための原料として用いられるフコィダンは、どのような 構造のものでもよぐまたいずれの藻類力も取得してもよい。藻類の例には、クロガシ ラ目 (Sphacelariales)、ナガマツモ目 (Chordariales)、カャモノリ目 (Scytosiphonales)、 ウイ =Τヨウモ目 (Dictyosipnonaies)、ムテモ目 (し utlenalesノ、ケャリモ目 (Sporochnales )、アミジグサ目（Dictyotales)、コンブ目（Laminariales)、ヒバマタ目  [0044] The fucoidan used as a raw material for the fucoidan oligosaccharide of the present invention may have any structure, and any algal power may be obtained. Examples of algae include the order of Sphacelariales, Chordariales, Scytosiphonales, Dictyosipnonaies, Mutomoles, Sporochnales, Dictyotales), Kombu (Laminariales), Hibamata
(Fucales)を含む褐藻綱 Phaeophyceaeの海藻が含まれる。好ましくはモズク、より好ま しくは沖縛モズク由来のフコィダンを用 、る。  Contains brown alga Phaeophyceae seaweed containing (Fucales). Preferably, mozuku, more preferably fucoidan derived from offshore mozuku is used.
[0045] フコィダンの抽出方法  [0045] Method for extracting fucoidan
フコィダンを藻類力 抽出する方法は種々検討されており、広く知られている（例え ば、特開平 10— 245334に記載されているような水を用いる方法、特開平 10— 195 106に記載されて ヽるような酸を用いる方法、特開 2002— 262788に記載されて ヽ るようなアルカリ水性溶媒を用いる方法等である)。本発明オリゴ糖の原料として用い るフコィダンはこれら公知の方法により取得することができる。本発明においては、例 えば、以下の方法により取得したものを用いる。  Various methods for extracting fucoidan from algae force have been studied and widely known (for example, a method using water as described in JP-A-10-245334, and JP-A-10-195106). For example, a method using an acid as described above, and a method using an alkaline aqueous solvent as described in JP-A-2002-262788). Fucoidan used as a raw material for the oligosaccharide of the present invention can be obtained by these known methods. In the present invention, for example, the one obtained by the following method is used.
[0046] 即ち、藻類（例えば沖縛モズク）に蒸留水 5〜10倍量をカ卩え、 50〜100°Cで 0〜5 時間、好ましくは 80〜100°Cで 0. 5〜2時間、さらに好ましくは 90〜100°Cで約 1時 間抽出する。このようにして得られた藻類抽出物を冷却、吸引濾過、脱塩および乾燥 することにより、容易に水に溶解するフコィダン画分を得ることができる。このようにし て得られたフコィダン画分は、さらに精製することなく次の工程に用いてもよいし、更 に精製してカゝら用いてもよい。 [0046] That is, algae (for example, offshore mozuku) is filled with 5 to 10 times the amount of distilled water, and it is 0 to 5 hours at 50 to 100 ° C, preferably 0.5 to 2 hours at 80 to 100 ° C. More preferably, about 1 hour at 90-100 ° C Extract between. The algae extract thus obtained can be cooled, suction filtered, desalted and dried to obtain a fucoidan fraction that is easily dissolved in water. The fucoidan fraction thus obtained may be used in the next step without further purification, or may be used after further purification.
[0047] フコィダンは、好ましくは上記のように藻類力も抽出したものを用いるが、藻類に含 まれた状態で用いてもょ ヽ。フコィダンまたはそれを含む藻類を次の加水分解工程 に付すことによって本発明による化合物が得られる。  [0047] Fucoidan is preferably extracted from algal power as described above, but may be used in a state of being contained in algae. The compound according to the present invention can be obtained by subjecting fucoidan or algae containing it to the subsequent hydrolysis step.
[0048] フコィダンオリゴ糖混合物の製造方法  [0048] Method for producing fucoidan oligosaccharide mixture
本発明のフコィダンオリゴ糖を製造するには、先ず、特許文献 1〜3に記載されてい るように、フコィダンを酸や酵素を用いる方法により加水分解することにより、フコイダ ンオリゴ糖の混合物を得る。好ましくは、以下の様な酸加水分解条件を用いる。  In order to produce the fucoidan oligosaccharide of the present invention, as described in Patent Documents 1 to 3, first, a fucoidan oligosaccharide mixture is obtained by hydrolyzing fucoidan by a method using an acid or an enzyme. Preferably, the following acid hydrolysis conditions are used.
[0049] 即ち、上記のようにして藻類力 得られたフコィダンを含む画分またはフコィダンを 、酸、好ましくは塩酸を用い分解する。より具体的には、 0. 1〜5. ON、好ましくは 0. 5〜4.0N、さらに好ましくは 0.5〜3.0Nの HC1を含む、 25〜100°C、好ましくは 30〜 95°C、さらに好ましくは 50〜90°Cの水性溶媒中で、 0.1〜3時間、好ましくは 0.25〜 2.5時間、さらに好ましくは 0.5〜2時間加水分解を行なう。得られた反応物を塩基、 例えば約 1Nの NaOHで中和した後、電気透析もしくはゲル濾過等の適切な手段で 脱塩し、乾燥 (例えば、凍結乾燥)することにより、フコィダンオリゴ糖混合物を得るこ とがでさる。  That is, the fraction or fucoidan containing fucoidan obtained as described above by algal power is decomposed using an acid, preferably hydrochloric acid. More specifically, 0.1 to 5. ON, preferably 0.5 to 4.0 N, more preferably 0.5 to 3.0 N containing HC1, 25 to 100 ° C, preferably 30 to 95 ° C, Preferably, the hydrolysis is carried out in an aqueous solvent at 50 to 90 ° C. for 0.1 to 3 hours, preferably 0.25 to 2.5 hours, more preferably 0.5 to 2 hours. The obtained reaction product is neutralized with a base such as about 1N NaOH, desalted by an appropriate means such as electrodialysis or gel filtration, and dried (for example, freeze-dried) to obtain a fucoidan oligosaccharide mixture. This comes out.
[0050] オリゴ糖の精製  [0050] Purification of oligosaccharide
このようにして得られる混合物力もフコィダンオリゴ糖をさらに精製するためには、ク 口マトグラフィー、再結晶、透析、アルコール沈殿等の方法を単独で、または組み合 わせて用いることができる。例えば、以下の操作に従ってオリゴ糖を精製する。  In order to further purify the fucoidan oligosaccharide, the mixture force obtained in this way can be used alone or in combination with methods such as chromatography, recrystallization, dialysis, and alcohol precipitation. For example, the oligosaccharide is purified according to the following procedure.
[0051] 先ず、フコィダンの加水分解により得られたオリゴ糖混合物を陰イオン交換榭脂を 用いたクロマトグラフィーに付して、吸着されずに通過する、硫酸基を含まないオリゴ 糖を含有する画分（中性 ·酸性糖画分)と、酸性溶出液により溶出する、硫酸基を多く 含むオリゴ糖を含有する画分 (硫酸化糖画分)とを分離する。  [0051] First, an oligosaccharide mixture obtained by hydrolysis of fucoidan is subjected to chromatography using anion exchange resin, and is passed through without adsorption and contains an oligosaccharide containing no sulfate group. The fraction (neutral / acidic sugar fraction) is separated from the fraction containing sulfated oligosaccharides (sulfated sugar fraction) that are eluted by the acidic eluate.
[0052] 中性 ·酸性糖画分をさらにゲル濾過に付すことにより、式 (I)で表される二糖と式 (II) で表される三糖を得ることができる。 [0052] By subjecting the neutral and acidic sugar fraction to gel filtration, the disaccharide represented by the formula (I) and the formula (II) Can be obtained.
[0053] 一方、硫酸ィ匕糖画分をクロマトグラフィーに付すことにより、硫酸ィ匕オリゴ糖 (III) 〜 (XI)の各成分を単離することができる。  [0053] On the other hand, each component of sulfated oligosaccharides (III) to (XI) can be isolated by subjecting the sulfated sucrose fraction to chromatography.
[0054] 各オリゴ糖は、精製、構造解析をより容易にするために適宜標識化または誘導体化 してもよい。例えば、オリゴ糖は、 4—ァミノ安息香酸ェチル (ABEE)のような試薬で 蛍光標識ィ匕することができ、これによりオリゴ糖の検出が容易となる。標識化された各 オリゴ糖を分離した後にその標識ィ匕部分を除去することにより、純粋なオリゴ糖を取 得することができる。  [0054] Each oligosaccharide may be appropriately labeled or derivatized for easier purification and structural analysis. For example, oligosaccharides can be fluorescently labeled with a reagent such as 4-amino benzoate (ABEE), which facilitates oligosaccharide detection. Pure oligosaccharides can be obtained by separating each labeled oligosaccharide and then removing the labeled portion.
[0055] こうして得られるフコィダンオリゴ糖は、例えば飲食品、医薬品、化粧品等に用いて 、これらに免疫機能調節作用を付与することができる。  [0055] The fucoidan oligosaccharide thus obtained can be used for, for example, foods and drinks, pharmaceuticals, cosmetics and the like to impart an immune function-modulating action thereto.
[0056] 騰 翻  [0056] Rise
本明細書における免疫機能調節作用とは、生体において低下している免疫反応を 高める、および Zまたは亢進している免疫反応を抑制する作用のことをいう。従って、 その作用には、免疫賦活作用および免疫抑制作用が含まれる。  The function of regulating immune function in the present specification refers to an action of enhancing a lowered immune response in a living body and suppressing Z or an enhanced immune response. Therefore, the action includes immunostimulatory action and immunosuppressive action.
[0057] 本明細書における免疫機能調節作用は、当該技術分野で知られて 、る方法で測 定することができる。それは、例えば、免疫機能調節作用を有するサイト力インを誘導 する作用を指標として測定することができる。この目的に用いられるサイト力インには 、インターフェロン— γ、インターロイキン— 10、 12等が含まれる。また、免疫機能調 節作用は、免疫応答に関与する榭状細胞の成熟化や、細胞障害性 Τ細胞 (CTL)の 活性を指標として測定することもできる。  [0057] The function of regulating immune function in the present specification can be measured by a method known in the art. It can be measured using, for example, an effect of inducing a site force-in having an immune function regulating effect as an index. Site force ins used for this purpose include interferon-γ, interleukin-10, 12 and the like. In addition, the immune function-modulating action can be measured using maturation of rod cells involved in the immune response or the activity of cytotoxic rod cells (CTL) as an index.
[0058] 本発明の免疫機能調節剤は健康増進のために用いることができるが、腫瘍、癌の 転移、ウィルス性疾患 (例えば、カゼ、エイズ、ウィルス性肝炎）、アレルギー性疾患（ 例えば、花粉症、アレルギー性鼻炎、アトピー、喘息）、自己免疫疾患 (例えば、リウ マチ様関節炎）、炎症性疾患、糖尿病のような疾患または状態に有効であることも知 られている。  [0058] Although the immune function regulator of the present invention can be used for health promotion, tumors, metastasis of cancer, viral diseases (for example, kaze, AIDS, viral hepatitis), allergic diseases (for example, pollen) It is also known to be effective for diseases or conditions such as infectious diseases, allergic rhinitis, atopy, asthma), autoimmune diseases (eg rheumatoid arthritis), inflammatory diseases, diabetes.
[0059] フコィダンオリゴ糖を含む食品添加物および飲食品、並びにそれを添カロした飲食品  [0059] Food additive and food / drink containing fucoidan oligosaccharide, and food / drink containing the food
本発明のフコィダンオリゴ糖を飲食品に用いる場合には、それを含み免疫機能調 節作用を有する食品添加物および飲食品として、並びにそれを添加した免疫機能調 節作用を有する健康食品として実施することが好適である。それらは、公知の甘味料 、酸味料、ビタミン等の各種成分と混合してユーザーの嗜好に合う製品とすればよい 。飲食品は、例えば、錠剤、カプセル剤、清涼飲料、茶飲料、ドリンク剤、ヨーグルト や乳酸菌飲料等の乳製品、調味料、加工食品、デザート類、菓子 (例えば、ガム、キ ヤンディ、ゼリー)等の形態で提供することが可能である。本発明の飲食品には、免疫 機能調節作用を有する旨の表示を容器や説明書に付した機能性食品 (特定保健用 食品や条件付き特定保健用食品が含まれる)も含まれる。表示場所は容器またはそ れに添付した指示書などが挙げられるが、これらに限られない。容器には、瓶、缶、 ペットボトル、プラスチックボトル、紙パック等が含まれる力 それらに限定されない。ま た、表示の方法には、印刷、刻印、シール等が含まれる力 それらに限定されない。 また、飲食品は、ペットの餌としてカ卩ェしたペットフード等や動物飼料等でもよい。 When the fucoidan oligosaccharide of the present invention is used in a food or drink, the food additive and food or drink containing the same and having an immune function modulating action, and the immune function prepared by adding the same It is preferable to implement as a health food having a moderating effect. They may be mixed with various components such as known sweeteners, acidulants, vitamins, etc. to make products that meet the user's taste. Foods and drinks include, for example, tablets, capsules, soft drinks, tea drinks, drinks, dairy products such as yogurt and lactic acid bacteria drinks, seasonings, processed foods, desserts, confectionery (e.g. gum, candy, jelly) etc. It is possible to provide in the form. The foods and drinks of the present invention also include functional foods (including foods for specified health use and conditionally specified health foods) with a label indicating that they have an immune function regulating effect on containers and instructions. The labeling place includes, but is not limited to, a container or instructions attached to it. Containers include but are not limited to bottles, cans, plastic bottles, plastic bottles, paper packs, and the like. In addition, the display method is not limited to force including printing, engraving, sealing, and the like. In addition, the food and drink may be pet food or animal feed that is used as pet food.
[0060] フコィダンオリゴ糖 含す 靠鉬成物  [0060] Fucoidan oligosaccharide-containing composition
本発明のフコィダンオリゴ糖は、例えば、免疫機能調節剤、免疫賦活剤、抗アレル ギー剤および免疫抑制剤として用いることができる。したがって、 1つの態様において 、本発明はフコィダンオリゴ糖を含む、免疫機能調節作用、免疫賦活作用、抗アレル ギー作用および Zまたは免疫抑制作用を有する医薬組成物である。  The fucoidan oligosaccharide of the present invention can be used, for example, as an immune function regulator, immunostimulator, antiallergic agent, and immunosuppressant. Accordingly, in one embodiment, the present invention is a pharmaceutical composition having an immune function modulating action, an immunostimulating action, an antiallergic action, and a Z or immunosuppressive action, comprising fucoidan oligosaccharide.
[0061] 医薬組成物は、主薬に希釈剤、担体、結合剤、崩壊剤、滑沢剤、矯味矯臭剤、溶 解補助剤、懸濁剤、コーティング剤等の医薬の製剤技術分野において通常使用する 公知の補助剤を用いて製剤化することができる。剤型としては、錠剤、カプセル剤、 顆粒剤、散剤、液剤、シロップ剤、座剤、クリーム剤、軟膏剤、エマルシヨン、ノ、ップ剤 、注射剤等を挙げることができ、特に限定されるものではない。本医薬品の投与経路 としては、例えば、経口投与、直腸投与、経腸投与等を挙げることができるが、特に 限定されるものではない。  [0061] The pharmaceutical composition is generally used in the pharmaceutical formulation technical field such as diluents, carriers, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspension agents, coating agents, etc. as the main agent. It can be formulated using known adjuvants. Examples of dosage forms include tablets, capsules, granules, powders, solutions, syrups, suppositories, creams, ointments, emulsions, tablets, injections, and the like, and are particularly limited. It is not a thing. Examples of the route of administration of this drug include oral administration, rectal administration, enteral administration, etc., but are not particularly limited.
[0062] フコィダンオリゴ糖を含む化粧品  [0062] Cosmetics containing fucoidan oligosaccharide
本発明のフコィダンオリゴ糖を用いることにより、免疫機能調節作用を有する化粧品 を製造することができる。  By using the fucoidan oligosaccharide of the present invention, a cosmetic product having an immune function regulating action can be produced.
[0063] 本発明オリゴ糖が添加または配合される化粧品は、例えば、顔用、皮膚用、頭髪用 のクリーム、ローション、ゲル、ムース、シャンプー、リンス等である。 [0064] 他の成分 の併用 [0063] Cosmetics to which the oligosaccharide of the present invention is added or blended are, for example, facial, skin and hair creams, lotions, gels, mousses, shampoos, rinses and the like. [0064] Combination of other ingredients
本発明のフコィダンオリゴ糖は、飲食品、医薬組成物、およびィ匕粧品において、そ れ自体単独で使用してもよ!、が、他の免疫機能調節作用または免疫賦活作用を有 する食品素材または物質と併用することも好適である。そのような食品素材または物 質としては、乳酸菌 (乳酸球菌、乳酸桿菌）、酵母、真菌類のような微生物;シィタケ、 マイタケ、霊芝、ァガリタスなどのキノコ類; CpGモチーフを含む免疫原性オリゴデォ キシヌクレオチド（CpG - ODN)、 Polyl： Cのような核酸由来物質；リポ多糖 (LPS) 及び j8—グルカン;海藻類;漢方薬;癌ワクチン (例えば、 MM46癌抗原）；コンカナ ノ リン A(ConA)；クレスチン (登録商標）、 OK— 432 (ピシバニール、登録商標）、レ ンチナン (登録商標）のような BRM (生物学的応答修飾剤）；フコィダン等の公知の免 疫賦活剤が挙げられる。  The fucoidan oligosaccharide of the present invention may be used by itself in foods and drinks, pharmaceutical compositions, and cosmetics, but it is a food material having other immune function regulating action or immunostimulatory action or It is also suitable to use together with a substance. Such food materials or substances include microorganisms such as lactic acid bacteria (Lactococcus, Lactobacilli), yeasts, fungi; mushrooms such as shitake, maitake, ganoderma, agaritas; Xinucleotide (CpG-ODN), Polyl: Nucleic acid-derived substances such as C; Lipopolysaccharide (LPS) and j8-glucan; Seaweed; Chinese medicine; Cancer vaccine (eg, MM46 cancer antigen); Concananoline A (ConA) BRM (Biological Response Modifier) such as Krestin (registered trademark), OK-432 (Pisibanil, registered trademark) and Lentinan (registered trademark); Known immune activators such as fucoidan;
[0065] フコィダンオリゴ糖 乳酸菌の組み合わせ  [0065] Fucoidan oligosaccharide combination of lactic acid bacteria
本発明のフコィダンオリゴ糖は、特に、乳酸菌と組み合わせることにより、それらの 免疫機能調節作用が相乗的に増強される。したがって、本発明には、フコィダンオリ ゴ糖を乳酸菌と組み合わせて添加した、またはそれらを含む、飲食品、医薬組成物、 および化粧品も含まれる。これら飲食品、医薬組成物、およびィ匕粧品は、フコィダン オリゴ糖または乳酸菌を単独で用いた場合と比較して相乗的に増強された免疫機能 調節作用を有することを特徴とする。この目的に用いられる乳酸菌には、 Lactobacillu s属カ S含まれる。好 3;しくは、孚 L酸菌は Lactobacillus plantarumま 7こは Lactobacillus pe ntosusである。用いられるフコィダンオリゴ糖の乳酸菌に対する好ましい比率は、重 量で 1： 1〜： L0000 : 1、より好ましくは 250 : 1〜1000： 1である。また、本発明には、フ コィダンオリゴ糖と乳酸菌とを組み合わせて用いることによりそれらの免疫機能調節 作用を相乗的に増強する方法も含まれる。  The fucoidan oligosaccharides of the present invention, in combination with lactic acid bacteria, synergistically enhance their immune function regulating action. Therefore, the present invention also includes foods and drinks, pharmaceutical compositions, and cosmetics to which fucoidan oligosaccharide is added in combination with or containing lactic acid bacteria. These foods and drinks, pharmaceutical compositions, and cosmetics are characterized by having a synergistically enhanced immune function regulating action as compared with the case where fucoidan oligosaccharide or lactic acid bacteria are used alone. Lactic acid bacteria used for this purpose include Lactobacillus sp. 3) Lactobacillus plantarum or 7 is Lactobacillus pentosus. The preferred ratio of fucoidan oligosaccharide used to lactic acid bacteria is 1: 1 to L0000: 1 by weight, more preferably 250: 1 to 1000: 1. The present invention also includes a method of synergistically enhancing the immune function regulating action by using a combination of fucoidan oligosaccharide and lactic acid bacteria.
[0066] 本発明の飲食品、組成物には、これら活性成分以外に、具体的な態様に応じて、 一般的な成分、例えば、担体、希釈剤、賦形剤または添加剤等の成分を配合するこ とができる。ここで担体、希釈剤または賦形剤としては、フコィダンオリゴ糖の生理活 性を妨げないものであれば特に制限されず、例えばシュクロース、グルコース、果糖 、マルトース、トレハロース、乳糖、澱粉、水飴、異性化液糖などの糖類、エタノール、 プロピレングリコール、グリセリン等のアルコール類、ソルビトール、マン-トール、エリ スリトール、ラタチトール、キシリトール、マルチトール、還元パラチノース、還元澱粉 分解物等の糖アルコール類、トリァセチン等の溶剤、アラビアガム、カラギナン、キサ ンタンガム、グァーガム、ジエランガム、ぺクチン等の多糖類、または水を挙げることが できる。また添加剤としては、キレート剤等の助剤、香料、香辛料抽出物、防腐剤など を挙げることができる。担体、添加剤等を本発明の効果を損なわない限り配合するこ とがでさる。 [0066] In addition to these active ingredients, the food / beverage products and compositions of the present invention contain general ingredients such as carriers, diluents, excipients or additives according to specific embodiments. Can be blended. Here, the carrier, diluent or excipient is not particularly limited as long as it does not interfere with the physiological activity of fucoidan oligosaccharide. Sugars such as liquid sugar, ethanol, Alcohols such as propylene glycol and glycerin, sorbitol, mannitol, erythritol, latathitol, xylitol, maltitol, reduced palatinose, reduced starch and other sugar alcohols, solvents such as triacetin, gum arabic, carrageenan, xanthan gum , Polysaccharides such as guar gum, dielan gum, pectin, or water. Examples of additives include auxiliaries such as chelating agents, fragrances, spice extracts, and preservatives. Carriers, additives and the like can be added as long as the effects of the present invention are not impaired.
[0067] 飲食品、医薬組成物およびィ匕粧品におけるオリゴ糖の配合量は、選択する他の配 合成分との関係等により適宜選択されるものであり、特に限定されるものではない。し 力しながら、通常、フコィダンオリゴ糖は、飲料または食品中、医薬組成物中に添カロ する場合、個体の体重 60kgに対して 0. Olg〜： LOgZ日、好ましくは 0. 05g〜lgZ 日、特に好ましくは 0. 05g〜0.5gZ日である。化粧品中には、 0. 01〜20重量％、 好ましくは 0. 05〜15重量％用いられる。  [0067] The blending amount of the oligosaccharide in the food / beverage product, the pharmaceutical composition and the cosmetic product is appropriately selected depending on the relationship with the other combination components to be selected, and is not particularly limited. However, normally, fucoidan oligosaccharides are added to beverages or foods in a pharmaceutical composition in an amount of 0. Olg to: LOgZ days, preferably 0.05 g to lgZ days for an individual weight of 60 kg. Particularly preferred is 0.05 g to 0.5 gZ days. In cosmetics, 0.01 to 20% by weight, preferably 0.05 to 15% by weight is used.
[0068] 本発明のオリゴ糖は、抽出精製品や合成製品を単独で飲食品、医薬組成物、およ びィ匕粧品に用いることもできるが、本発明オリゴ糖の 1つ以上を含む混合物の形態で 飲食品等に添加することもできる。  [0068] The oligosaccharide of the present invention can be used alone in an extracted purified product or a synthetic product in foods, beverages, pharmaceutical compositions, and cosmetics, but is a mixture containing one or more of the oligosaccharides of the present invention. It can also be added to food and drink in the form of
[0069] なお本発明は上述した各実施形態に限定されるものではなぐ請求項に示した範 囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を 適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。  Note that the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims, and the technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.
[実施例]  [Example]
以下、実施例に基づいて本発明を具体的に説明するが、本発明の範囲はこれらの 実施例に限定されな ヽことは言うまでもな ヽ。  EXAMPLES Hereinafter, the present invention will be specifically described based on examples, but it goes without saying that the scope of the present invention is not limited to these examples.
[0070] 以下の実施例においては、特に明記しない限り、 ECA— 600型核磁気共鳴装置（ 日本電子株式会社)を用いて NMR分析を行なった。測定溶媒として重水 (D O)を In the following examples, unless otherwise specified, NMR analysis was performed using an ECA-600 type nuclear magnetic resonance apparatus (JEOL Ltd.). Heavy water (D O) as the measurement solvent
2 用いた。構成糖の結合様式は、 2D-NMRを用いて行なった。  2 Used. The conjugation pattern of the constituent sugars was performed using 2D-NMR.
実施例 1  Example 1
[0071] a)フコィダン画分の調製 [0071] a ) Preparation of fucoidan fraction
沖縛モズク藻体 100gに蒸留水を 1000mlカ卩え、 100°Cで 1時間抽出した。得られ た抽出物を冷却後、吸引濾過、電気透析 (脱塩)をして凍結乾燥し、フコィダン画分 を 2g得た。このフコィダンを 2NH SOを含む 100°Cの水溶液で 1時間加水分解し、 1000 ml of distilled water was added to 100 g of offshore mozuku alga and extracted at 100 ° C for 1 hour. Obtained The extract was cooled, suction filtered, electrodialyzed (desalted), and lyophilized to obtain 2 g of fucoidan fraction. This fucoidan is hydrolyzed with 100 ° C aqueous solution containing 2NH SO for 1 hour,
2 4  twenty four
得られた水溶液を 2NNaOHを用いて中和し、 ABEEで蛍光標識ィ匕することで単糖 分析サンプルを調製した。その構成糖の組成は SFuc (硫酸ィ匕フコース）： GlcA:Fu c :Xyl=49. 3 :4. 9 : 12. 1： 1であることを確認した（図 1)。 The resulting aqueous solution was neutralized with 2NNaOH and fluorescently labeled with ABEE to prepare a monosaccharide analysis sample. The composition of the constituent sugar was confirmed to be SFuc (sulfuric acid sulfate): GlcA: Fuc: Xyl = 49.3: 4.9: 12: 1: 1 (FIG. 1).
カラム： Cosmosil C18 AR— Π (4. 6mm X 250mm) Column: Cosmosil C18 AR—Π (4.6 mm x 250 mm)
移動相： 10%ァセトニトリル含有 0. 2Mホウ酸カリウム緩衝液 Mobile phase: 10% acetonitrile containing 0.2M potassium borate buffer
流速： 1. OmlZ分 Flow rate: 1. OmlZ min
温度： 45°C Temperature: 45 ° C
検出：蛍光検出器 (株式会社島津製作所)、 Ex: 305nm、 Em: 360nm Detection: Fluorescence detector (Shimadzu Corporation), Ex : 305nm, Em: 360nm
b)フコィダンの加 7k分解およびオリゴ糖の分離  b) Fucoidan 7k degradation and oligosaccharide separation
得られたフコィダン画分 lgに 2NHC1を 100ml加えて 50〜100°Cで 1時間酸カロ水 分解を行ない、次いで INNaOHで中和した。得られた反応液をゲル濾過 (バイオゲ ル P— 6 (Bio— Rad) )に付して脱塩し、凍結乾燥を行なうことによりフコィダンオリゴ 糖混合物を 895mg得た。得られたフコィダンオリゴ糖混合物は、蟻酸で活性化した 陰イオン交換榭脂 (東ソ一株式会社)を用いるクロマトグラフィーに付した。結果として 、オリゴ糖混合物は、水で溶出することで得られた硫酸基を含まないオリゴ糖を含有 する画分（中性'酸性糖画分） 280mgと、 2NHC1で溶出することで得られた硫酸基 を多く含むオリゴ糖を含有する画分 (硫酸化糖画分) 425mgに分離された。  100 ml of 2NHC1 was added to the obtained fucoidan fraction lg, acid-hydrolysis was carried out at 50-100 ° C. for 1 hour, and then neutralized with INNaOH. The obtained reaction solution was subjected to gel filtration (Biogel P-6 (Bio-Rad)) for desalting and lyophilization to obtain 895 mg of a fucoidan oligosaccharide mixture. The obtained fucoidan oligosaccharide mixture was subjected to chromatography using an anion exchange resin activated by formic acid (Tosohichi Corporation). As a result, the oligosaccharide mixture was obtained by elution with 2NHC1 with 280 mg of the fraction containing oligosaccharides without sulfate groups (neutral'acid sugar fraction) obtained by elution with water. The fraction containing oligosaccharides rich in sulfate groups (sulfated sugar fraction) was separated into 425 mg.
c)化合物（I)および (II)の単離  c) Isolation of compounds (I) and (II)
b)で得られた中性 ·酸性糖画分 lOOmgをゲル濾過（バイオゲル P— 4 (Bio— Rad) 、溶出溶媒: 0. 2Mホウ酸カリウム (K B O )水溶液）に付すことにより、当該画分から  Apply the lOOmg neutral / acid sugar fraction obtained in b) to gel filtration (Biogel P-4 (Bio-Rad), elution solvent: 0.2M potassium borate (KBO) aqueous solution).
2 4 7  2 4 7
分子量 340の二糖と分子量 486の三糖が分離された (化合物 I、 11)。これらの分子量 は、 FAB— MSにより求めた (図 2、 3 ;化合物（I) [M— H]— : 339. 2、化合物（II) [M -H]":485. 0)。これらの化合物 5mgに、水 lml、 ABEE (4—ァミノ安息香酸ェチ ル） 1.6g、 NaBH CN (水素化シァノほう素ナトリウム） 350mg、メタノール 3.5ml、酢 A disaccharide with a molecular weight of 340 and a trisaccharide with a molecular weight of 486 were separated (compounds I and 11). Their molecular weights were determined by FAB-MS (Figures 2 and 3; Compound (I) [M—H] —: 339.2, Compound (II) [M−H] ”: 485. 0). Compound 5mg, water lml, ABEE (ethyl 4-aminobenzoate) 1.6g, NaBH CN (sodium cyanoborohydride) 350mg, methanol 3.5ml, vinegar
3  Three
酸 410 1を加え、 65°C、 4時間撹拌した。反応液をクロ口ホルムと水で分配すること により蛍光標識ィ匕された上記二糖および三糖を約 7〜9mg取得した。これら標識ィ匕 オリゴ糖について測定した1 H— NMRおよび13 C— NMRのチャートを図 4〜7に示し 、それらを解析した結果を表 1、 2に示した。これらの結果より、得られた分子量 340の 二糖は式（I)で表される —D— GlcA—(l→2)—L—Fucであり、分子量 486の三 糖は式（Π)で表される at - D - GlcA- ( 1→2) a—L— Fuc—（1→3)—L— Fuc であることが分力つた。 Acid 410 1 was added and stirred at 65 ° C. for 4 hours. By partitioning the reaction solution with black mouth form and water, about 7 to 9 mg of the above-mentioned disaccharide and trisaccharide labeled with fluorescence were obtained. These signs The 1 H-NMR and 13 C-NMR charts measured for the oligosaccharides are shown in FIGS. 4 to 7, and the analysis results are shown in Tables 1 and 2. From these results, the obtained disaccharide having a molecular weight of 340 is represented by the formula (I) —D-GlcA— (l → 2) —L-Fuc, and the trisaccharide having a molecular weight of 486 is represented by the formula (Π). It was expressed that at-D-GlcA- (1 → 2) a—L—Fuc— (1 → 3) —L—Fuc.
[0073] [表 1] 表 1 ：：¾'光標識した化合物 （I) についての1 H— NMRおよび1 3 C NM R解析結果 [0073] [Table 1] Table 1 :: ¾ 'light-labeled Compound (I) 1 H- NMR and 1 3 C NM R analysis results for
Figure imgf000018_0001
Figure imgf000018_0001
[0074] [表 2] [0074] [Table 2]
表 2 ：蛍光標識した化合物 （I I) についての1 H NMRおよび1 3 C— NMR解析結果 Table 2: Results of 1 H NMR and 1 3 C-NMR analyzes of fluorescently labeled compound (II)
Figure imgf000019_0001
d)化合物（III)〜 (XI)の製造
Figure imgf000019_0001
d) Production of compounds (III) to (XI)
次に、硫酸化糖画分をゲル濾過 (バイオゲル P— 6 (Bio— Rad) )に付すことにより 脱塩した。得られた硫酸ィ匕糖画分 lOOmgに、水 lml、 ABEE (4—ァミノ安息香酸ェ チル） 1. 6g、 NaBH CN (水素化シァノほう素ナトリウム） 350mg、メタノール 3. 5ml  Next, the sulfated sugar fraction was desalted by subjecting it to gel filtration (Biogel P-6 (Bio-Rad)). To the obtained sulfate-sucrose fraction lOOmg, water lml, ABEE (ethyl 4-aminoaminobenzoate) 1.6 g, NaBH CN (sodium cyanoborohydride) 350 mg, methanol 3.5 ml
3  Three
、酢酸 410 1を加え、 65°C、 4時間撹拌した。得られた生成物を真空で乾燥させ、 水とクロ口ホルムに分配し、水層を逆相カラムで（担体： Lichroprep RP— 8 (25—4 0 m) (Merck)、 φ 10 X 220mm;溶媒条件： 5%CH CNZO. 1%TFA( 100ml)  Acetic acid 410 1 was added and stirred at 65 ° C for 4 hours. The resulting product is dried under vacuum, partitioned between water and black mouth form, and the aqueous layer is separated on a reverse phase column (support: Lichroprep RP—8 (25—40 m) (Merck), φ 10 X 220 mm; Solvent conditions: 5% CH CNZO. 1% TFA (100ml)
3  Three
、 8%CH CN/0. l%TFA(100ml)、 15%CH CN/0. l%TFA(100ml)、 20  8% CH CN / 0.1% TFA (100ml), 15% CH CN / 0.1% TFA (100ml), 20
3 3  3 3
%CH CN/0. l%TFA(100ml) )処理することにより蛍光標識ィ匕されたオリゴ糖の % CH CN / 0. L% TFA (100 ml)) treatment of the fluorescently labeled oligosaccharide
3 Three
混合物を得た。得られた蛍光標識化合物を HPLC (カラム： cosmosil 5C18— AR —Π、 10. O X 250mm;溶媒条件： 12. 5%CH CN/0. 1%TFA(5分）、 12. 5 A mixture was obtained. The resulting fluorescently labeled compound was analyzed by HPLC (column: cosmosil 5C18—AR —Π, 10. O X 250 mm; solvent condition: 12.5% CH CN / 0.1% TFA (5 min), 12.5
3  Three
- 27. 5%CH CN/0. 1%TFA(50分）；流速： 3mlZ分）でァセトニトリル： 0. 1%  -27. 5% CH CN / 0.1% TFA (50 min); flow rate: 3 ml Z min), acetonitrile: 0.1%
3  Three
TFA水溶液を 5〜30%の濃度勾配で溶出して、分子量力 39、 715、 861、 903、 9 57、 999であり硫酸基を有する 6つの標識ィ匕フコィダンオリゴ糖を混合物力 分離し た (分子量は、 ESI— MSにより決定した)。得られた標識ィ匕オリゴ糖について NMR スペクトルを測定し、その結果を解析した。標識ィ匕オリゴ糖の1 H— NMRおよび13 C— NMRのチャートを図 8 19に示し、それらを解析した結果を表 3 8に示す。これら 結果力ら、分子量 539 715 861 903 957 999のィ匕合物は、それぞれ、ィ匕合 物 (111)、 (V) (VI) （VII) （VIII) （IX)の標識体であることが明らかとなった。 Elution of a TFA aqueous solution with a concentration gradient of 5 to 30% was performed to separate six labeled 匕 fucoidan oligosaccharides having molecular weights of 39, 715, 861, 903, 957, and 999 and having sulfate groups. (Molecular weight was determined by ESI-MS). The obtained labeled oligosaccharide was measured for NMR spectrum and analyzed. The 1 H-NMR and 13 C-NMR charts of labeled oligosaccharides are shown in FIG. 8 19, and the analysis results are shown in Table 38. Based on these results, the compounds with molecular weights of 539 715 861 903 957 999 must be labeled with the compounds (111), (V) (VI) (VII) (VIII) (IX), respectively. Became clear.
[0076] 確認のため、各オリゴ糖上に結合して 、た ABEEを除去し、再生したオリゴ糖の純 品を得た。即ち、これら分離した各標識ィ匕オリゴ糖 lOmg (100 1)に、過酸化水素、 酢酸を各 10 1加えて一昼夜放置した後、乾固した。こうして得られた再生オリゴ糖の うち、分子量 903の化合物から得られたものを1 H— NMR (図 20)、 TOF— MS (装 ¾\ oyager D -STR (Applied Biosystems) Ion mode: negative ^ Mode of operation： reflector ^ Accelerating voltage： 20kV Matrix： 2,5-dihydroxybenzoic acid) (|¾|21) MSZMS (図 22)で解析したところ、その結果は、確かに式 (VII)の構造を示して いた。 [0076] For confirmation, ABEE was bound on each oligosaccharide, and a regenerated oligosaccharide product was obtained. That is, to each of these separated labeled oligosaccharides lOmg (100 1), 10 1 each of hydrogen peroxide and acetic acid was added, and the mixture was allowed to stand overnight and then dried. Among the regenerated oligosaccharides obtained in this way, those obtained from a compound with a molecular weight of 903 were analyzed using 1 H-NMR (Fig. 20), TOF- MS (equipment oyager D-STR (Applied Biosystems) Ion mode: negative ^ Mode of operation: reflector ^ Accelerating voltage: 20kV Matrix: 2,5-dihydroxybenzoic acid) (| ¾ | 21) When analyzed by MSZMS (Fig. 22), the result certainly showed the structure of formula (VII) .
[0077] また、上記方法で分離できなかった分子量 420 858 900の化合物（それぞれ (I V)、 （X)、 （XI) )に関しては、反応混合物を FAB— MSZMS (装置： HX110A/HX11 0A(JEOL)、 Ion mode : MS, MS/MS、negative)、 Xe atom beam : 5kV Ion source accele rating potential : 10kV Collision energy : 2keV Matrix : Glycerol)、および ESI— MS — MSで分析することによりその存在を確認した。これら未標識化オリゴ糖の分析結 果を図 23 27に示す。図 23に（IV)の FAB— MSチャート、図 24に MSZMSチヤ ートを示した。また図 25に（X)および（XI)の FAB— MSチャートを、図 26 27にそれ ぞれの MSZMSチャートを示した。  [0077] For compounds having a molecular weight of 420 858 900 that could not be separated by the above method (respectively (IV), (X), (XI)), the reaction mixture was FAB-MSZMS (apparatus: HX110A / HX11 0A (JEOL ), Ion mode: MS, MS / MS, negative), Xe atom beam: 5kV Ion source accele rating potential: 10kV Collision energy: 2keV Matrix: Glycerol), and ESI-MS-MS did. The analysis results of these unlabeled oligosaccharides are shown in FIG. Fig. 23 shows the (IV) FAB-MS chart, and Fig. 24 shows the MSZMS chart. Fig. 25 shows the FAB-MS chart of (X) and (XI), and Fig. 2627 shows the MSZMS chart of each.
[0078] これらの結果より、分子量 390の二糖は化学式（III)で表される α— L Fuc— [0078] From these results, a disaccharide having a molecular weight of 390 is represented by α-L Fuc- represented by the chemical formula (III).
4-O - SO (1→3) L Fuc、分子量 420の二糖は化学式（IV)で表される α 4-O-SO (1 → 3) L Fuc, a disaccharide with a molecular weight of 420 is represented by the formula (IV) α
3  Three
-D-GlcA- (1→2) L Fuc、分子量 566の三糖は化学式 (V)で表される α— L-Fuc -4-O- SO—― (1→3) - [ a— D— GlcA— (1→2) ]— L— Fuc、分子量  -D-GlcA- (1 → 2) L Fuc, molecular weight 566 trisaccharide is represented by the chemical formula (V) α— L-Fuc -4-O-SO—— (1 → 3)-[a— D — GlcA— (1 → 2)] — L—Fuc, molecular weight
3  Three
712の四糖は化学式 (VI)で表される a -L-Fuc-4-O - SO (1→3) a  712 tetrasaccharide is represented by chemical formula (VI) a -L-Fuc-4-O-SO (1 → 3) a
3  Three
-D-GlcA- (1→2) ] - a— L— Fuc— (l→3)— L— Fuc、そして分子量 754の 四糖は化学式 (VII)で表される α -L-Fuc -4-O - SO 1→3)— [ a D— GlcA- (1→2)]- a— L— Fuc— 4— O ァセチル— (1→3)— L— Fuc、分子量 8 08の 5糖は化学式 (VIII)で表される [ a—D— GlcA— (1→2) a—L— Fuc— (1 →3) ]-[a -D-GlcA- (1→2) ] a—L— Fuc—（1→3)—L— Fuc、分子量 8 50の 5糖は化学式（IX)で表される [ a— D— GlcA— (1→2) a L— Fuc— (1→ 3) ]— [ a -D-GlcA- (1→2) ]—4— 0 ァセチノレー a—L— Fuc—（1→3)—L -Fuc,分子量 858の 5糖は化学式（X)で表される a—L—Fuc—4— 0— SO—— ( -D-GlcA- (1 → 2)]-a— L— Fuc— (l → 3) — L— Fuc and a tetrasaccharide with a molecular weight of 754 are represented by the formula (VII) α -L-Fuc -4 -O-SO 1 → 3) — [a D— GlcA- (1 → 2)]-a— L— Fuc— 4— O acetyl — (1 → 3) — L— Fuc, molecular weight 8 08 pentasaccharide is represented by the chemical formula (VIII) [a—D— GlcA— (1 → 2) a—L— Fuc— (1 → 3)]-[a -D-GlcA- (1 → 2)] a—L—Fuc— (1 → 3) —L—Fuc, molecular weight 8 50 pentasaccharide is represented by chemical formula (IX) [a- D- GlcA- (1 → 2) a L- Fuc- (1 → 3)]-[a -D-GlcA- (1 → 2) ] —4— 0 Acetinolay a—L— Fuc— (1 → 3) —L-Fuc, molecular weight 858 pentasaccharide is represented by chemical formula (X) a—L—Fuc—4— 0—SO—— (
3 Three
1→3) a L— Fuc— (1→3)— [ a D— GlcA— (1→2) ] a L— Fuc— (1 →3) L Fuc、分子量 900の 5糖は化学式（XI)で表される a— L Fuc— 4 O -SO—― (1→3) - a—L— Fuc— (1→3) - [ a— D— GlcA— (l→2) ]— a— L1 → 3) a L— Fuc— (1 → 3) — [a D— GlcA— (1 → 2)] a L— Fuc— (1 → 3) L Fuc, a pentasaccharide with a molecular weight of 900, has the chemical formula (XI) A— L Fuc— 4 O -SO—— (1 → 3)-a—L— Fuc— (1 → 3)-[a— D— GlcA— (l → 2)] — a— L
3 Three
—Fuc— 4— O ァセチルー (1→3)—L—Fucであることが分かった。  —Fuc— 4—O acetylene (1 → 3) —L—Fuc.
[0079] [表 3] 表 3. ¾光標識した化 fr物 (III) についての1 H― NMKぉょび':iC—]\'MR解折¾果 [0079] [Table 3] Table 3. 1 H- NMK Oyobi about ¾ light labeled reduction fr compound (III) ': i C - ] \' MR Kaiori ¾ results
Figure imgf000021_0001
Figure imgf000021_0001
[0080] [表 4] 表 4. 蛍光標識した化^物 （V) についての 41一 NMRおよび13 C— NMR解析結栗 [0080] [Table 4] Table 4. 41-NMR and 13 C-NMR analysis of fluorescently labeled compounds (V)
Figure imgf000022_0001
5]
Figure imgf000022_0001
Five]
表 5. 蛍光標識した化合物 （VI) についての1 H— NMRおよび13 C— NMR解析結^ Table 5. Results of 1 H-NMR and 13 C-NMR analyzes of fluorescently labeled compound (VI)
Figure imgf000023_0001
6]
Figure imgf000023_0001
6]
表 6 . 蛍光標識した化 t物 （VI I) についての1 H— NM Rおよび 1 :i C— NMR解析お Table 6. 1 H—NMR and 1: i C—NMR analysis of fluorescently labeled compounds (VI I)
Figure imgf000024_0001
実施例 2
Figure imgf000024_0001
Example 2
ォキナヮモズク藻体 lOOgに 2NHC11000mlを入れ、 1時間、 50 100°Cで酸カロ 水分解を行った。得られた抽出物を冷却後、吸引濾過、電気透析 (脱塩)を行ない凍 結乾燥しフコィダン画分を 2g得た。得られたフコィダン画分を ABEEで蛍光標識ィ匕し たものを ESI— MS (4000Q TRAP LCZMSZMSシステム（Applied Biosyst ems) 条件 Polarity： Negative ion mode； Declustering Potential：― 50v; Collision energy:— lOeV; Temperature : 550°C)で分析した結果、図 28 で示されるチャートが得られ、式 (I)〜(XI)に示されるフコィダンオリゴ糖の存在が確 認できた。  11000 ml of 2NHC was put into lOOg of okina mozuku alga, and acid carolysis was carried out at 50 100 ° C for 1 hour. The obtained extract was cooled and then subjected to suction filtration and electrodialysis (desalting), followed by freezing and drying to obtain 2 g of a fucoidan fraction. ESI—MS (4000Q TRAP LCZMSZMS system (Applied Biosyst ems) conditions) Polarity: Negative ion mode; Declustering Potential: -50v; Collision energy: —lOeV; Temperature As a result of the analysis at 550 ° C, the chart shown in Fig. 28 was obtained, and the presence of the fucoidan oligosaccharide represented by the formulas (I) to (XI) was confirmed.
実施例 3 [0084] 免疫機能調節作用の測定 (1) Example 3 [0084] Measurement of immune function regulating action (1)
マウス！!縣田 に針する IFN— m im  mouse! ! IFN—m im
C57BLZ6マウス（8週齢、雄、 日本チャールズリバ一株式会社)力 脾細胞を分離 し、 5 X 106cells/mlになるように調製し、 24wellプレートで培養した。化合物（1)、 (I I)のフコィダンオリゴ糖を 100 g/mlになるように各 wellに添加した。比較対照とし てフコースと硫酸化フコース (それぞれ MW164、 244)、キシロオリゴ糖 (XOS)を等 量用いた。 24時間培養後、培養上清を回収し IFN— γの産生量を ELISAキット（Ο ptEIA、 BD Pharmingen)を用いて測定した（図 29)。化合物（II)に強 、IFN— y 誘導作用が見られた。 C57BLZ6 mice (8 weeks old, male, Nippon Charles River Co., Ltd.) force Spleen cells were isolated, prepared to 5 × 10 6 cells / ml, and cultured in 24-well plates. The fucoidan oligosaccharides of compounds (1) and (II) were added to each well so as to be 100 g / ml. As comparative controls, fucose, sulfated fucose (MW164 and 244, respectively) and xylooligosaccharide (XOS) were used in equal amounts. After culturing for 24 hours, the culture supernatant was collected, and the amount of IFN-γ produced was measured using an ELISA kit (ΟptEIA, BD Pharmingen) (FIG. 29). Compound (II) exhibited a strong IFN-y inducing action.
[0085] さらに、オリゴ糖を添加して 30分後に、乳酸菌（Lactobacillus pentosus： FERM A BP- 10028) (死菌）を 0. 1 gZmlずつ各 wellに添カ卩して上記と同様の操作も行つ た。化合物 (I)に関しては、それを乳酸菌と併用すると著しく強い IFN— γ誘導能が 得られた。この作用は、化合物 (I)と乳酸菌をそれぞれ単独で用いて得られた結果（ 図 29中、左力 2番目および右力 3番目）から予測できない程強ぐ両者を組み合 わせて用いることにより相乗的効果が生じることを裏付けて 、る。構成成分のフコース や硫酸ィ匕フコースにはこのような活性が無ぐプレバイオテイクスとして広く使われて いるキシロオリゴ糖にもこのような作用が無力つた。  [0085] Furthermore, 30 minutes after adding the oligosaccharide, lactic acid bacteria (Lactobacillus pentosus: FERM A BP-10028) (dead bacteria) were added to each well in an amount of 0.1 gZml. I went. With regard to compound (I), when it was used in combination with lactic acid bacteria, a remarkably strong IFN-γ induction ability was obtained. This effect can be achieved by using a combination of both compounds (I) and lactic acid bacteria that are so strong that they cannot be predicted from the results obtained by using the lactic acid bacteria alone (second left force and third right force in Fig. 29). Supports synergistic effects. Such an action is also ineffective in xylo-oligosaccharides widely used as prebiotics, which have no such activity in the constituent components fucose and sulfated fucose.
[0086] したがって、化合物（I)、化合物（II)のフコィダンオリゴ糖には脾細胞を活性ィ匕させ る機能があり、生体の免疫機能を調節できる。  Therefore, the fucoidan oligosaccharides of compound (I) and compound (II) have a function of activating splenocytes and can regulate the immune function of the living body.
[0087] また、同様に調製した脾細胞に、化合物 (I)〜 (VII)の化合物を任意に 3種類均等 に混合したものを 50 μ gZmlの濃度で添加し、 30分後に乳酸菌 (Lactobacillus pent osus ;FERM ABP- 10028) (死菌）を 0. 1 gZmlずつ各 wellに添カ卩した。 24時 間培養後、培養上清を回収し IFN— yの産生量を測定した（図 30)。図に示すとおり 、 (V)、 (VI)および (VII)の混合物、 (I)、 (V)および (VI)の混合物、および (I)、 (V)お よび (VII)の混合物を用いると、高い IFN— γ産生量が観測された。したがって、化 合物 (I)〜 (XI)が混在して ヽて ヽる場合でも乳酸菌と併用することで免疫機能調節 活性が上昇することがわ力つた。  [0087] In addition, to the spleen cells prepared in the same manner, an arbitrary mixture of three types of compounds (I) to (VII) was added at a concentration of 50 μgZml, and after 30 minutes, lactic acid bacteria (Lactobacillus pent osus; FERM ABP-10028) (dead bacteria) was added to each well in an amount of 0.1 gZml. After 24 hours of culture, the culture supernatant was collected and the amount of IFN-y produced was measured (FIG. 30). As shown in the figure, use a mixture of (V), (VI) and (VII), a mixture of (I), (V) and (VI), and a mixture of (I), (V) and (VII) And high IFN-γ production was observed. Therefore, even when the compounds (I) to (XI) were mixed together, the combined use with lactic acid bacteria increased the immune function regulating activity.
実施例 4 [0088] 免疫機能調節作用の測定 (2) Example 4 [0088] Measurement of immune function regulating action (2)
マウス ώ 針 纏 に する、 IL 10ぉよび 12 乍用、 ^田 )^ ィ乍) ¾およ び CTL活件増強作用  Mouse 針 Needle wrap, IL 10 ぉ and 12 乍, ^) ^ 乍 乍) ¾ and CTL activity enhancing action
BALBZcマウス (8週齢、雄、 日本 SLC株式会社)の大腿骨力 骨髄細胞を分離 し、 l X 106cells/mlになるように、 10%FBS、 20ng/ml GM— CSF (Peprotec )、 20ng/ml IL— 3 (Peprotec)を含む RPMI 1640培地中に懸濁し、 24wellプレ ートで培養した。培養開始 3、 5日目に培地交換を行ない、付着性の未成熟榭状細 胞を得た。得られた榭状細胞に、化合物 (1)、（11)、（111)、（V)、（VI)、および (VII)を それぞれ 50 gZmlの濃度で加えた (前処理)。 2日間培養後、培養上清と細胞を 回収した。また、前処理 30分後に、乳酸菌（Lactobacillus pentosus： FERM ABP- 10028) (死菌） )を 0. 1 μ gZmlずつ各 wellに添カ卩して上記と同様の操作を行なつ た。 BALBZc mice (8 weeks old, male, Japan SLC, Inc.) the femoral force marrow cells were separated, so as to l X 10 6 cells / ml, 10% FBS, 20ng / ml GM- CSF (Peprotec), 20ng It was suspended in RPMI 1640 medium containing / ml IL-3 (Peprotec) and cultured in a 24-well plate. On the 3rd and 5th days from the start of the culture, the medium was changed to obtain adherent immature rod-like cells. Compounds (1), (11), (111), (V), (VI), and (VII) were each added to the obtained rod-shaped cells at a concentration of 50 gZml (pretreatment). After culturing for 2 days, the culture supernatant and cells were collected. In addition, 30 minutes after the pretreatment, lactic acid bacteria (Lactobacillus pentosus: FERM ABP-10028) (dead bacteria)) were added to each well in an amount of 0.1 μg Zml, and the same operation as described above was performed.
[0089] 回収した培養上清は ELISAキットを用いて IL— 12と IL— 10を測定した。結果をそ れぞれ図 31、 32に示した。化合物（11)、（111)、および (VI)は、単独で IL— 12誘導能 を示した。化合物 (I)は、それを乳酸菌と併用することで相乗的に IL— 12を誘導した (図 31)。また、化合物 (11)、（III)および (V)は乳酸菌と併用することで IL— 10を誘導 した（図 32)。これらのことにより、本発明で見つかったフコィダンオリゴ糖には生体の 免疫系を正にも負にも調節 (低下した免疫を活性ィ匕し、または過剰な免疫反応を抑 制する）する機能があることが分力つた。キシロオリゴ糖には、このような作用は認めら れなかった。  [0089] The collected culture supernatant was measured for IL-12 and IL-10 using an ELISA kit. The results are shown in Figs. 31 and 32, respectively. Compounds (11), (111), and (VI) alone showed IL-12 induction ability. Compound (I) induced IL-12 synergistically when used in combination with lactic acid bacteria (FIG. 31). Compounds (11), (III) and (V) induced IL-10 when used in combination with lactic acid bacteria (Fig. 32). For these reasons, the fucoidan oligosaccharides found in the present invention have a function of regulating the immune system of the living body positively or negatively (activate reduced immunity or suppress excessive immune responses). I was divided. Xylooligosaccharides did not show this effect.
[0090] 回収した細胞の表面の成熟化マーカーである CD86の陽性率をフローサイトメトリ 一 (EPICS XL、ベックマンコールター株式会社製)を用いて解析した。その結果、 フコィダンオリゴ糖には単独で榭状細胞を成熟化させる作用があることが確認できた [0090] The positive rate of CD86, a maturation marker on the surface of the collected cells, was analyzed using flow cytometry (EPICS XL, manufactured by Beckman Coulter, Inc.). As a result, it was confirmed that fucoidan oligosaccharides have the effect of maturating rod cells alone.
(図 33)。 (Figure 33).
[0091] さらに、回収した細胞をカウントし、 1 X 105cellsZmlに調製後、 1mlの細胞懸濁液 中にマイトマイシン C (50 gZml)を処理し、 37°Cで 30分間反応させた。反応終了 後、 PBSの洗浄によりマイトマイシン Cを除去し、常法により調製した C57BL/6マウ スの脾細胞（5 X 106cells/ml)を lml添加し、 4日間混合培養した。培養後細胞を 回収し、 C57BLZ6マウスのァロ抗原を持つ P— 815細胞（SOOOcellsZlOO /z l)を ターゲットにし、 E :T比 40 : 1および 80 : 1の割合で 4時間反応させ、殺傷された P— 8 15の数をフローサイトメトリーでカウントすることにより CTL活性を求めた（図 34)。化 合物（11)、（VI)、（VII)は単独で CTL活性を高める作用があることを確認した。また、 化合物 (111)、（V)、（VI)、（VII)は乳酸菌と併用することで CTL活性が増強されること も分かった。 [0091] Further, the collected cells were counted and prepared to 1 X 10 5 cells Zml, and then 1 ml of cell suspension was treated with mitomycin C (50 gZml) and reacted at 37 ° C for 30 minutes. After completion of the reaction, mitomycin C was removed by washing with PBS, and 1 ml of C57BL / 6 mouse spleen cells (5 × 10 6 cells / ml) prepared by a conventional method was added and mixed and cultured for 4 days. After culturing the cells Targeted P-815 cells (SOOOcellsZlOO / zl) with the C57BLZ6 mouse alloantigen and killed by reacting for 4 hours at an E: T ratio of 40: 1 and 80: 1. P— 8 15 CTL activity was determined by counting the number of cells by flow cytometry (FIG. 34). It was confirmed that the compounds (11), (VI), and (VII) alone have the effect of increasing CTL activity. It was also found that compounds (111), (V), (VI), and (VII) enhance CTL activity when used in combination with lactic acid bacteria.

Claims

請求の範囲 下記構造式 (I)、（11)、（111)、（IV)、（V)、（VI)、（VII)、（VIII)、（IX)、 (X)、または (XI)で示される化合物。 The following structural formula (I), (11), (111), (IV), (V), (VI), (VII), (VIII), (IX), (X), or (XI) A compound represented by
[化 1]  [Chemical 1]
Figure imgf000028_0001
Figure imgf000028_0001
[化 2] [Chemical 2]
Figure imgf000028_0002
Figure imgf000028_0002
[化 3] [Chemical 3]
Figure imgf000028_0003
Figure imgf000028_0003
[化 4][Chemical 4]
Figure imgf000028_0004
Figure imgf000028_0004
[化 5] [Chemical 5]
Figure imgf000029_0001
Figure imgf000029_0001
[化 6]
Figure imgf000029_0002
Figure imgf000029_0003
Figure imgf000029_0004
[Chemical 6]
Figure imgf000029_0002
Figure imgf000029_0003
Figure imgf000029_0004
Figure imgf000030_0001
Figure imgf000030_0001
Figure imgf000030_0002
Figure imgf000030_0002
Figure imgf000030_0003
請求項 1記載の式 (I)〜 (XI)で表される化合物から選択される少なくとも一種を含む 免疫機能調節剤。
Figure imgf000030_0003
An immune function regulator comprising at least one compound selected from the compounds represented by formulas (I) to (XI) according to claim 1.
請求項 1記載の式 (I)〜 (XI)で表される化合物から選択される少なくとも一種を添加 した飲食品。 Add at least one selected from compounds represented by formulas (I) to (XI) according to claim 1. Food and drink.
[4] 請求項 1記載の式 (I)〜 (XI)で表される化合物から選択される少なくとも一種を含有 し、免疫機能調節作用を有する旨の表示を付した飲食品。  [4] A food or drink comprising at least one compound selected from the compounds represented by formulas (I) to (XI) according to claim 1 and labeled as having an immune function regulating action.
[5] 請求項 1記載の式 (I)〜 (XI)で表される化合物から選択される少なくとも一種を含む 医薬組成物。 [5] A pharmaceutical composition comprising at least one compound selected from the compounds represented by formulas (I) to (XI) according to claim 1.
[6] 請求項 1記載の式 (I)〜 (XI)で表される化合物から選択される少なくとも一種を含む 化粧品。  [6] A cosmetic comprising at least one selected from the compounds represented by formulas (I) to (XI) according to claim 1.
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