[go: up one dir, main page]

WO2004061090A1 - Composition permettant l'activation de cellules tueuses naturelles et procede de production de cette composition, composition contenant des cellules tueuses naturelles activees par la composition precedente et procede de production de ladite composition - Google Patents

Composition permettant l'activation de cellules tueuses naturelles et procede de production de cette composition, composition contenant des cellules tueuses naturelles activees par la composition precedente et procede de production de ladite composition Download PDF

Info

Publication number
WO2004061090A1
WO2004061090A1 PCT/JP2004/000003 JP2004000003W WO2004061090A1 WO 2004061090 A1 WO2004061090 A1 WO 2004061090A1 JP 2004000003 W JP2004000003 W JP 2004000003W WO 2004061090 A1 WO2004061090 A1 WO 2004061090A1
Authority
WO
WIPO (PCT)
Prior art keywords
fraction
organic solvent
barley
insoluble
natural killer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2004/000003
Other languages
English (en)
Japanese (ja)
Inventor
Toshiro Omori
Yasufumi Umemoto
Yoshifumi Furuta
Hideki Hokazono
Mihoko Furutera
Hideki_____________________________________- Ohba
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Institute of Advanced Industrial Science and Technology AIST
Barley Fermentation Technologies Inc
Sanwa Shurui Co Ltd
Original Assignee
National Institute of Advanced Industrial Science and Technology AIST
Barley Fermentation Technologies Inc
Sanwa Shurui Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National Institute of Advanced Industrial Science and Technology AIST, Barley Fermentation Technologies Inc, Sanwa Shurui Co Ltd filed Critical National Institute of Advanced Industrial Science and Technology AIST
Priority to JP2005507954A priority Critical patent/JPWO2004061090A1/ja
Publication of WO2004061090A1 publication Critical patent/WO2004061090A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • A61K36/8998—Hordeum (barley)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars

Definitions

  • the present invention relates to a composition having an action of activating natural killer cells and a method for producing the same, and a composition containing natural killer cells activated using the composition and a method for producing the same.
  • the present invention obtains a liquid component by solid-liquid separation of a barley shochu distillation residue (hereinafter abbreviated as “barley shochu distillation residue”) by-produced in the production of shochu using barley as a raw material,
  • barley shochu distillation residue a barley shochu distillation residue
  • the fraction that has not been adsorbed to the synthetic adsorbent which has been collected by exhausting the fraction or subjecting the liquid to an adsorption separation treatment using a synthetic adsorbent, is subjected to ion exchange treatment using an ion exchange resin.
  • the fraction that is not adsorbed on the ion exchange resin is fractionated, and the fraction that is not adsorbed on the ion exchange resin is subjected to a concentration treatment by ultrafiltration using an ultrafiltration membrane.
  • the present invention also includes a composition containing a natural killer cell and a natural killer cell activated by using a composition comprising a natural killer cell and a fraction insoluble in the organic solvent, and a method for producing the same.
  • Natural killer cells are found in the lymphoid tissues of animals (humans, mice, hamsters, rats, chickens, pigs, etc.). These sputum cells are morphologically somewhat large lymphocytes (LGLs) that contain azul granules in the cytoplasm, occupying a few percent of lymphocytes, and without immune memory. It is thought that it plays a part in the defense mechanism of the body against tumor development because it can destroy non-specific tumor cells (mainly hematopoietic tumors).
  • LGLs lymphocytes
  • sputum cells play an important role in non-antigen-specific innate immunity that acts at the very beginning of the immune response. Even though it does not have a sputum cell receptor, it can accurately identify self and non-self and can cause necrosis of virus-infected cells and tumor cells without prior antigen sensitization. Are known.
  • Patent Document 1 describes a filamentous fungus of the genus Aspergillus or Basidiomycetus as a plant tissue raw material of rice bran, bran, rice straw or bagasse. Describes a method for producing hemicellulose having an immunostimulatory action, characterized by being assimilated in the culture medium.
  • the hemicellulose having an immunostimulatory effect obtained through the production method is described as having main components xylose and arabinose and having an average molecular weight of 550,000 or 600,000.
  • the bran, rice straw, or bagasse is the residue of barley shochu that is a by-product of the distillation of the whitened grains that have undergone whitening to remove the hulls of barley grains, followed by distillation after alcohol fermentation. It is completely different from liquid.
  • water-soluble polysaccharides are obtained by preparing water-soluble polysaccharides of plant tissues such as gramineous plants, especially rice bran.
  • An immunity enhancing substance obtained by preparing an extracellular enzyme of a filamentous fungus to obtain an enzyme complex, mixing the water-soluble polysaccharide and the enzyme complex, and reacting at a predetermined pH for a predetermined time, It is described that it has an effect of activating NK cells. Further, it is described that the substance for enhancing immunity has an average molecular weight of 600,000 or 650,000, and main components are xylose and arabinose.
  • the rice bran in Patent Document 2 is a by-product generated when whitening the threshed brown rice, and after subjecting the whitened grains that have been whitened to remove the hulls of barley grains to alcohol fermentation, distillation is performed. It is completely different from the barley shochu distillation residue, which is a distillation residue produced as a by-product during the production of shochu.
  • Patent Document 3 an aqueous extract of sake lees produced as a by-product when rice is subjected to alcoholic fermentation to produce sake is activated by NK cell activation. It is described to have
  • the sake lees in Patent Document 3 is a solid content which is a residue after separating the liquid obtained by subjecting rice to alcoholic fermentation and then squeezing it as a sake product. Therefore, the sake lees in Patent Document 3 is a distillation residue produced as a by-product in producing shochu by subjecting the refined grains that have undergone whitening to remove the hulls of barley grains to alcohol fermentation and then distilling them. It is completely different from a barley shochu distillation residue.
  • Patent Document 4 describes an immunostimulation comprising a water extract of rice bran and comprising an extract having a molecular weight of about 30000 to 40000. A composition for use is described. Patent Document 4 describes that the composition has mitogenic activity and excellent NK cell activation action. Patent The rice bran in Ref. 4 is a by-product generated when whitening the threshed brown rice, and the whitened grains that have undergone whitening to remove the hulls of barley grains are subjected to alcoholic fermentation followed by distillation to produce shochu. It is completely different from the barley shochu distillation residue, which is a by-product distillation residue.
  • Patent Document 5 discloses a partial decomposition of a hemicell mouthpiece obtained from corn hull, which is commercially available under the trade name “Celuse”. An antitumor composition containing the product as an active ingredient is described. According to Patent Document 5, the partial degradation product of hemicellulose has an average molecular weight of 20,000 to 200,000, and NK cell activity is significantly increased in colon26 cancer-bearing mice administered with the partial degradation product. Furthermore, it is described that the ability to produce cytokines (IL-2 and INF-a) is also significantly increased.
  • cytokines IL-2 and INF-a
  • Non-Patent Document 1 a partially decomposed product of hemicellulose obtained from corn hull marketed under the trade name “Cel Ace” described in Patent Document 5 contains arabinose and It is mainly composed of arabinoxylan consisting of xylose and has a composition of 40.32% xylose, 27.76% arabinose, 11.86% uronic acid, 5.91% galactose and 2.30% glucose. It is described that there is.
  • the corn hull which is the source of the “partial degradation product of hemicellulose obtained from corn hull” described in Patent Document 5
  • There is a barley shochu distillation residue that is a distillation residue by-produced when producing white spirits by subjecting the whitened grains that have undergone whitening to remove the hulls of barley grains to alcohol fermentation and then distilling them.
  • Patent Document 6 The barley shochu distillation residue (i.e., obtained from the distillation residue produced as a by-product in producing shochu by subjecting the whitened grains that had undergone whitening to remove the hull of barley grains to alcohol fermentation and then performing distillation) A composition having a fatty liver inhibitory action different from the activating action on M cells is described, that is, Patent Document 6 obtains a liquid component by solid-liquid separation of the barley shochu distillation residue. Then, Al force is added to the obtained liquid to fractionate the Al force soluble fraction, and the fraction obtained is neutralized with an acid to obtain a neutral soluble fraction.
  • Patent Document 6 A composition having a fatty liver inhibitory action obtained by adding ethanol to the neutral soluble fraction is described in Patent Document 6.
  • Patent Document 6 describes the composition described above. , which contains a molecular weight of 3000 or less as a main component, and the composition contains hemicellulose It is described that the constituent element is xylose, and therefore, in any of the above-mentioned known documents, whether or not the barley shochu distillation residual liquid contains a component having an activating action on M cells. It is clear that there is no suggestion about this.
  • the present invention further provides a barley shochu distillation residue (hereinafter referred to simply as “barley shochu distillation residue”) produced as a by-product when producing barley shochu.
  • barley shochu distillation residue hereinafter referred to simply as “barley shochu distillation residue”
  • An object of the present invention is to provide a composition having a significant activating effect on natural killer cells (hereinafter abbreviated as “NK cells”) collected from a barley shochu distillation residue and a method for producing the same. There is.
  • NK cells natural killer cells
  • Another object of the present invention is to provide a composition containing NK cells activated using the composition having a significant activating effect on NK cells, and a method for producing the same.
  • the composition having a significant activating effect on NK cells of the present invention includes the following two embodiments.
  • the composition having a significant activating effect on NK cells of the first aspect is obtained by solid-liquid separation of a barley shochu distillation residual liquid to obtain a liquid component, and the liquid component is subjected to an ion exchange treatment using an ion exchange resin.
  • the fraction that is not adsorbed on the ion-exchange resin is fractionated, and the fraction that is not adsorbed on the ion-exchange resin that has been collected is subjected to a concentration treatment by ultrafiltration using an ultrafiltration membrane.
  • a concentrated solution and a fraction insoluble in the organic solvent fractionated by adding an organic solvent to the concentrated solution, and the fraction insoluble in the organic solvent substantially contains uronic acid. It contains polysaccharides consisting of xylose, arabinose, and glucose.
  • the molecular weight distribution is 5% for 100,000 or more, 18% for 30,000 to 100,000, 23% for 10,000 to 30,000, 000 to 10,000 is 31%, 1,000 to 3,000 is 11%, 500 to 1,000 is 3%, and 500 or less is 9%. Has an activating effect.
  • the fraction insoluble in the organic solvent is about 84.1% by weight of polysaccharide, about 54.4% by weight of total arabinose and xylose derived from polysaccharide, about 4.2% by weight of crude protein, organic acid Has a component composition consisting of about 0.3% by weight and about 1.2% by weight of free sugars.
  • the composition having a significant activating effect on NK cells according to the second aspect is obtained by subjecting the barley shochu distillation residue to solid-liquid separation to obtain a liquid component, which is then subjected to an adsorption separation process using a synthetic adsorbent. And fractionating a non-adsorbed fraction on the synthetic adsorbent, and subjecting the non-adsorbed fraction to the fractionated synthetic adsorbent to an ion exchange treatment using an ion exchange resin. A non-adsorbed fraction is fractionated, and the non-adsorbed fraction is subjected to a concentration treatment by ultrafiltration using an ultrafiltration membrane to obtain a concentrated solution.
  • the fraction insoluble in the organic solvent fractionated by adding an organic solvent to the organic solvent is substantially free of uronic acid and consists of xylose, arabinose and glucose.
  • uronic acid consists of xylose, arabinose and glucose.
  • composition having an activating effect on NK cells according to the first and second aspects of the present invention is very remarkable for NK cells, comparable to IL-2 which is a known NK cell activator. It has an activating effect and can be used very suitably for the treatment of cancer including leukemia as a pharmaceutical.
  • composition containing the activated NK cell of the present invention includes the following two embodiments.
  • the composition containing activated NK cells according to the first aspect contains activated NK cells using the composition having a significant activation action on the NK cells according to the first aspect. It is a cell-containing composition.
  • the composition containing activated NK cells according to the second aspect contains NK cells activated using the composition having a significant activating effect on the M cells according to the second aspect. It is a cell-containing composition.
  • the activated NK cell-containing composition containing activated NK cells according to the first and second aspects of the present invention can be used very suitably for the treatment of cancer including leukemia as a pharmaceutical. .
  • the present invention has been completed based on the facts obtained by the present inventors through experiments. The experiment conducted by the present invention will be described below.
  • the present inventors obtained a liquid component by solid-liquid separation of a barley shochu distillation residue produced as a by-product in the production of shochu using barley as a raw material, and an organic solvent (preferably ethanol) was added to the liquid component.
  • an organic solvent preferably ethanol
  • the collected fraction insoluble in the organic solvent contains polysaccharide as one of its main components
  • the polysaccharide contained in the fraction insoluble in the organic solvent contains NK cells.
  • components other than polysaccharides contained in the fraction insoluble in the organic solvent that is, amino acid, peptide, protein, organic acid, or barley-derived
  • various ion exchange resins are prepared, and the liquid components obtained by solid-liquid separation of the barley shochu distillation residual liquid are ion exchange treatments using these ion exchange resins individually.
  • a fraction insoluble in the organic solvent collected by fractionation is obtained, and whether each of the obtained fractions insoluble in the organic solvent has an NK cell activation effect or not, has an NK cell activation effect.
  • the following experiment was conducted to clarify the degree of the NK cell activation effect. Barley shochu was produced for the purpose of the following Experiment 1 to Experiment 14. Barley (70% refined) was used as a raw material.
  • Steamed barley was produced by absorbing 40% by weight of barley, steaming for 40 minutes, and allowing to cool to 40 ° C.
  • the first preparation add 3.6 ml of water and lkg (wet weight) of cultured cells of shochu yeast as yeast to the barley koji (3 tons as barley) produced by the method described above to obtain primary mash.
  • the obtained primary moromi was subjected to 5 days of fermentation (1st stage fermentation).
  • 11.4 kiloliters of water and 7% ton of steamed barley (7 tons of barley) produced by the method described above were added to the primary mash that had been subjected to the first stage fermentation. (Stage fermentation).
  • the fermentation temperature was 25 ° C for both the first and second charge.
  • the second mash after the second stage fermentation was subjected to simple distillation by a conventional method to obtain 10 kiloliters of barley shochu and 15 kiloliters of barley shochu distillation residue.
  • the obtained barley shochu distillation residue was Used for Experiment 1 to Experiment 14.
  • a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue is centrifuged under the conditions of 8000 rpm and lOmin to obtain a liquid component, the Brix of the obtained liquid component is adjusted to 10, and the liquid component adjusted to BrixlO is adjusted to 1 L. Ethanol is added so that the concentration becomes 75% by volume, and the mixture is centrifuged at 8000 rpm and lOmin to collect a fraction insoluble in an organic solvent (ethanol). The obtained fraction is lyophilized. 11.8 g of a freeze-dried product was obtained.
  • a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue is centrifuged at 8000 rpm and lOmin to obtain a liquid component. After adjusting the liquid component to BrixlO, 1 ml of the liquid component adjusted to BrixlO is stored in a volume of 500 ml. The fraction not adsorbed on the ion exchange resin was fractionated through a column packed with Amberlite IRC76 (weakly acidic cation exchange resin) manufactured by Organo Co., Ltd., and the obtained fraction was converted to Brix60.
  • Amberlite IRC76 weakly acidic cation exchange resin
  • a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm and lOmin to obtain a liquid component. After adjusting the obtained liquid component to Brixl O, 1 ml of the liquid component adjusted to BrixlO was added to 500 ml. Through a column packed with Amberlite IRA67 (weakly acidic anion exchange resin) manufactured by Organo Co., Ltd., fractions not adsorbed on the ion exchange resin were collected, and the obtained fractions were combined with Brix60.
  • Amberlite IRA67 weakly acidic anion exchange resin
  • a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue is centrifuged under the conditions of 8000 rpm and lOmin to obtain a liquid component. After the liquid component thus obtained is adjusted to BrixlO, 1 L of the liquid component adjusted to BrixlO is added to a 500 ml organopolyester. Pass through a column packed with Amberlite 200CT (Strongly Acidic Cation Exchange Resin), and collect fractions not adsorbed on the ion exchange resin, and adjust the resulting fractions to Brix60.
  • Amberlite 200CT Shortly Acidic Cation Exchange Resin
  • NK cell activity ie, activation effect on NK cells was performed in the same manner as described in the Examples below. ) was measured.
  • the present inventors obtain a liquid component by solid-liquid separation of the barley shochu distillation residual liquid by-produced in the production of shochu using barley as a raw material, and add an organic solvent (preferably ethanol) to the liquid component.
  • an organic solvent preferably ethanol
  • the polysaccharides contained in the fraction insoluble in the organic solvent activate M cells. Further studies were conducted through experiments to clarify whether or not they are involved in the process. That is, the following experiments were conducted for the purpose of removing amino acids, peptides, proteins, organic acids, polyphenols derived from barley, etc., which are components other than polysaccharides contained in the fraction insoluble in the organic solvent. .
  • the liquid content obtained by solid-liquid separation of the barley shochu distillation residue was synthesized for the purpose of removing polyphenol derived from barley contained in the liquid content of the barley shochu distillation residue.
  • a fraction that is not adsorbed to the synthetic adsorbent is separated by an adsorption separation process using an adsorbent, and then the amino acids and peptides contained in the fraction that is not adsorbed to the synthesized adsorbent.
  • the ion-exchange resin is obtained by subjecting the non-adsorbed fraction to the synthetic adsorbent to ion exchange treatment using various ion-exchange resins.
  • Barley shochu distillation residue obtained in “Manufacture of barley shochu and barley shochu distillation residue” above From the above, a fraction insoluble in an organic solvent was collected by the method shown below. That is, the barley shochu distillation residue is centrifuged under the conditions of 8000 rpm and lOmin to obtain a liquid component, the Brix of the obtained liquid component is adjusted to 10, and the liquid component adjusted to BrixlO is adjusted to 1 L. Ethanol is added so that the concentration is 75% by volume, and the mixture is centrifuged at 8000 rpm and lOmin to collect a fraction insoluble in the organic solvent (ethanol), and the resulting fraction is freeze-dried. As a result, 11.8 g of a freeze-dried product was obtained.
  • a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm and lOmin to obtain a liquid component, and the resulting liquid component was packed with a synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation. The fraction that was not adsorbed to the synthetic adsorbent consisting of a flow-through solution exhibiting non-adsorbability with respect to the synthetic adsorbent of the column was fractionated.
  • the obtained fraction was adjusted to BrixlO, and 1 L of the fraction adjusted to BrixlO was passed through a column packed with 500 ml of Amberlite IRC76 (weakly acidic cation exchange resin) manufactured by Organo Corporation. The fraction not adsorbed on the ion exchange resin is collected, and the resulting fraction is adjusted to Brix60. Ethanol is added to a final concentration of 75% by volume, followed by centrifugal separation under the conditions of 8000 rpm and lOmin. A fraction insoluble in an organic solvent (ethanol) was collected, and the obtained fraction was freeze-dried to obtain 5.3 g of a lyophilized product.
  • Amberlite IRC76 weakly acidic cation exchange resin
  • a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm and lOmin to obtain a liquid component, and the resulting liquid component was placed in a column packed with Synthetic Adsorbent Amperai® XAD-16 manufactured by Organo Corporation. The fraction adsorbed on the synthetic adsorbent consisting of a flow-through liquid exhibiting non-adsorbability to the synthetic adsorbent of the column was fractionated by subjecting it to an adhesion separation treatment.
  • the obtained fraction Adjust to BrixlO, and pass 1 L of this adjusted fraction to BrixlO through a column packed with 500 ml of Amberlite IRA67 (weakly acidic anion exchange resin) manufactured by Organo Corp. After the fraction was adjusted to Brix60, ethanol was added to a final concentration of 75% by volume, and the mixture was centrifuged at 8000 rpm and lOmin to obtain the organic solvent (ethanol). The insoluble fraction was collected, and the obtained fraction was freeze-dried to obtain 5.9 g of a freeze-dried product.
  • Amberlite IRA67 weakly acidic anion exchange resin
  • the barley shochu distillation residue obtained in the above-mentioned “Production of barley shochu and barley shochu distillation residue” fractions insoluble in an organic solvent were collected by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 kg, lOmin to obtain a liquid component, and the obtained liquid component was applied to a column packed with a synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation. Through the adsorption separation process, fractions that were not adsorbed to the synthetic adsorbent consisting of a flow-through solution exhibiting non-adsorbability to the synthetic adsorbent of the column were fractionated.
  • the obtained fraction was adjusted to BrixlO, and 1 L of the fraction adjusted to BrixlO was passed through a column filled with 500 ml of Amberlite IR120B (strongly acidic cation exchange resin) manufactured by Organo Corporation. Fraction that is not adsorbed on the ion exchange resin is collected, and the resulting fraction is adjusted to Brix 60. Ethanol is added to a final concentration of 75% by volume, followed by centrifugation at 8000 rpm and lOmin. A fraction insoluble in an organic solvent (ethanol) was collected, and the obtained fraction was freeze-dried to obtain 5.6 g of a freeze-dried product.
  • Amberlite IR120B strongly acidic cation exchange resin
  • a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm and lOmin to obtain a liquid component, and the resulting liquid component was applied to a column filled with synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation. Through the adsorption separation process, fractions that were not adsorbed to the synthetic adsorbent consisting of a flow-through solution exhibiting non-adsorbability to the synthetic adsorbent of the column were fractionated.
  • the obtained fraction The fraction 1 L adjusted to BrixlO was passed through a column filled with 500 ml of Amberlite 200CT (strongly acidic cation exchange resin) manufactured by Organo Corp., and the fraction not adsorbed to the ion exchange resin. After fractionation, the fraction obtained was adjusted to Brix 60, ethanol was added to a final concentration of 75% by volume, and the mixture was centrifuged at 8000 rpm and lOmin, and insoluble in the organic solvent (ethanol). The fraction was collected, and the obtained fraction was freeze-dried to obtain 4.1 g of a freeze-dried product.
  • Amberlite 200CT strongly acidic cation exchange resin
  • a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged under the conditions of 8000 n> m and lOmin to obtain a liquid, and the resulting liquid was filled with a synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation. By passing through a column and subjecting to an adsorption separation treatment, a fraction that was not adsorbed to the synthetic adsorbent consisting of a flow-through liquid that showed non-adsorbability to the synthetic adsorbent of the column was fractionated.
  • the obtained fraction was adjusted to BrixlO, and 1 L of the fraction adjusted to BrixlO was passed through a column filled with 500 ml of Amberlite IRA402BL (strongest basic anion exchange resin) manufactured by Organo Corp. Fraction that is not adsorbed on the exchange resin, the resulting fraction is adjusted to Brix60, ethanol is added to a final concentration of 75% by volume, and the organic material is centrifuged at 8000 rpm and lOmin. The fraction insoluble in the solvent (ethanol) was collected, and the obtained fraction was freeze-dried to obtain 2.3 g of a freeze-dried product.
  • Amberlite IRA402BL strongest basic anion exchange resin
  • a fraction insoluble in an organic solvent was fractionated by the following method. That is, the barley shochu distillation residue was centrifuged at 8000 rpm and lOmin to obtain a liquid component, and the resulting liquid component was applied to a column filled with synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation. Through the adsorption separation process, fractions that were not adsorbed to the synthetic adsorbent consisting of a flow-through solution exhibiting non-adsorbability to the synthetic adsorbent of the column were fractionated.
  • the obtained fraction The fraction 1 L adjusted to Br ixlO was adjusted to 350 ml of Amberlite IRC76 (weakly acidic cation exchange resin) by Organo Co., Ltd. and 150 ml of Amberlite IRA67 (organo Co., Ltd.) A weakly basic anion exchange resin) is passed through a column packed with a mixed bed ion exchange resin, and a fraction not adsorbed on the mixed bed ion exchange resin is collected. After adjustment to Brix60, ethanol was added to a final concentration of 75 volumes, and the mixture was centrifuged under the conditions of 8000 rpiii, lOmin, and the fraction insoluble in the organic solvent (ethanol) was collected. By subjecting the portion to freeze-drying, 1.9 g of a freeze-dried product was obtained.
  • the NK cell activity (ie, the activation action on M cells) was performed in the same manner as described in the Examples below. ) was measured.
  • the polysaccharide content and the total content of arabinose and xylose derived from the polysaccharide were measured by the following methods. That is, the fraction insoluble in each organic solvent obtained in Experiment 1 to Experiment 14 (lyophilized product) was dissolved by adding 0.5 ml of ion-exchanged water to 0.05 g, and 200 l of concentrated hydrochloric acid was added thereto to obtain 95 ° C. C.
  • the M cell activity exhibited by NK cells cultured separately by adding each of the fractions (lyophilized product) insoluble in the organic solvent obtained in Experiment 1 to Experiment 14 is the fraction insoluble in the organic solvent. It was revealed that the content was increased in proportion to the total content of arabinose and xylose derived from the polysaccharide contained in the minute. The correlation coefficient between the NK cell activity and the total content of arabinose and xylose derived from polysaccharide was found to be 0 ⁇ 961.
  • Shodex standard P-82 (molecular weight 1300 to 1660000) and maltotriose (molecular weight 504) manufactured by Showa Denko Co., Ltd. were separately dissolved in O. lmol / L sodium nitrate solution. A standard solution of 0.05 W / V agricultural power was obtained, and the standard solution was injected into a high performance liquid chromatograph to prepare a calibration curve. Next, prepare 0.02 g of the fraction insoluble in each organic solvent (lyophilized product) obtained in Experiment 1 to Experiment 10, and add 10 ml of O.
  • the barley shochu distillation residue obtained in the above-mentioned “Manufacture of barley shochu and barley shochu distillation residue” was centrifuged at 8000 rpm and lOmin to obtain a liquid component, and the liquid obtained After adjusting the volume to Brixl O, 1 L of the liquid volume adjusted to Brixl O was passed through a column packed with 500 ml of Amberlite 200CT (strongly acidic cation exchange resin) manufactured by Organo Corporation. Fraction that is not adsorbed on the exchange resin is obtained, and the obtained fraction is subjected to concentration treatment with an ultrafiltration membrane UFP-30-E-4MA (fraction molecular weight 30,000) manufactured by A / G Technology.
  • Amberlite 200CT strongly acidic cation exchange resin
  • the total content of polysaccharide-derived arabinose and xylose was measured by the method described in “Measurement of Polysaccharide Content” above. As a result, it was found that the total content of polysaccharide-derived arabinose and xylose increased to about 54.4 wt.
  • the molecular weight distribution of the fraction insoluble in the organic solvent was 100,000 to 5%, 30,000 to 100,000 are 18%, 10,000 to 30,000 are 23%, 3,000 to 10,000 are 31%, 1,000 From 3,000 to 11%, 500 to 1,000 was found to be 3%, and 500 or less was found to be 9%. From the results of the chromatogram obtained, it was revealed that the chromatogram of the fraction insoluble in the organic solvent had the highest peak in the molecular weight range of 3,000 to 10,000.
  • the NK cell activity of NK cells cultured by adding a fraction insoluble in the organic solvent was measured by the method described in the Examples below.
  • the NK cell activation action of the fraction insoluble in the organic solvent was extremely strong, and was comparable to the positive control IL-2.
  • the NK cell activation action of the fraction insoluble in the organic solvent fractionated from the liquid fraction of the barley shochu distillation residue is contained in the fraction insoluble in the organic solvent, arabinose and xylose.
  • a composition containing a significant amount of such polysaccharides mainly composed of arabinose and xylose is derived from a polysaccharide mainly composed of arabinose.
  • the liquid is subjected to solid-liquid separation to obtain a liquid component, which is subjected to an ion exchange treatment using an ion exchange resin to fractionate a fraction not adsorbed on the ion exchange resin, and the resulting ion exchange is obtained.
  • a fraction that is not adsorbed to the resin is subjected to a concentration treatment by ultrafiltration using an ultrafiltration membrane to obtain a concentrate, and an organic solvent is added to the concentrate to obtain a fraction insoluble in the organic solvent. It was found that it can be sorted as .
  • the fraction insoluble in the organic solvent contains about 84.1% by weight of polysaccharide, about 4.2% by weight of crude protein, and about 0.3% by weight of organic acid. About 1.2% by weight The fraction was found to contain polysaccharides as the main component.
  • the barley shochu distillation residue obtained in “Manufacture of barley shochu and barley shochu distillation residue” above was used.
  • the liquid is centrifuged under the conditions of 8000 rpm and lOmin to obtain a liquid component, and the obtained liquid component is passed through a column filled with a synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation for adsorption separation treatment.
  • a synthetic adsorbent Amberlite XAD-16 manufactured by Organo Corporation for adsorption separation treatment.
  • Obtained fraction was adjusted to Brixl O, and then 1 L of the fraction adjusted to BrixlO was passed through a column filled with 500 ml of Amberlite 200CT (strongly acidic cation exchange resin) manufactured by Organo Corporation.
  • Fractionated non-adsorbed fraction is subjected to concentration treatment with ultrafiltration membrane UFP-30-E-4MA (fractional molecular weight 30,000) manufactured by A / G Technology.
  • the resulting concentrate is adjusted to Brix20, ethanol is added to a final concentration of 75% by volume, and the mixture is centrifuged at 8000 n) m and lOmin, and the organic solvent (ethanol) is obtained.
  • Fraction-insoluble fraction was collected, and the obtained fraction was freeze-dried to obtain 1.3 g of a freeze-dried product.
  • the total content of polysaccharide-derived arabinose and xylose was measured by the method described in “Measurement of Polysaccharide Content” above. As a result, it was found that the total content of arabinose and xylose derived from polysaccharides increased to about 58.6% by weight.
  • the molecular weight distribution of the fraction (lyophilized product) insoluble in the obtained organic solvent was measured by the method described in “Measurement of molecular weight distribution” above, the fraction insoluble in the organic solvent was obtained.
  • the molecular weight distribution is 11% for 100,000 or more, 29% for 30,000 to 100,000, 24% for 10,000 to 30,000, 21% for 3,000 to 10,000, 1,000 to 3 , 000 was found to be 6%, 500 to 1,000 was 2%, and 500 or less was 7%. From the results of the obtained chromatogram, it was revealed that the chromatogram of the fraction insoluble in the organic solvent had the highest peak in the molecular weight range of 30,000 to 100,000. Therefore, the NK cell activity of NK cells cultured by adding a fraction insoluble in the organic solvent was measured by the method described in Examples below. As a result, the NK cell activation action of the fraction insoluble in the organic solvent was extremely remarkable, and was comparable to that of IL-2 as a positive control.
  • the NK cell activation action of the fraction insoluble in the organic solvent fractionated from the liquid content of the barley shochu distillation residue by the above-described method is the arabinose contained in the fraction insoluble in the organic solvent
  • the main composition of arabinose and xylose in this way A composition containing a significant amount of polysaccharide as an element is obtained by solid-liquid separation of the barley shochu distillation residue produced as a by-product in the production of shochu using barley as a raw material, and this liquid is used as a synthetic adsorbent.
  • a fraction that is non-adsorbed on the synthetic adsorbent is obtained by subjecting it to an adsorption separation treatment, and the obtained fraction is subjected to an ion exchange treatment using an ion exchange resin to obtain a fraction that is not adsorbed on the ion exchange resin.
  • an ion exchange treatment using an ion exchange resin to obtain a fraction that is not adsorbed on the ion exchange resin.
  • the fraction insoluble in the organic solvent is about 78.5% by weight of polysaccharide, about 3.3% by weight of crude protein, about 0.2% by weight of organic acid, and free. It was found that the saccharide contained about 1.5% by weight, and the fraction contained polysaccharide as a main component.
  • the hemicellulos described in Patent Document 1 is similar to the composition having the action of activating NK cells of the present invention in that the main constituents are xylose and arabinose.
  • the hemicellulose has an average molecular weight of 550,000 or 600,000, it is clearly different from the composition having an action of activating NK cells of the present invention.
  • the immunity enhancing substance described in Patent Document 2 is similar to the composition having an action of activating M cells of the present invention in that the main components are xylose and arabinose. Since the force-enhancing substance has an average molecular weight of 600,000 or 650,000, it is clearly different from the composition having the action of activating NK cells of the present invention.
  • Patent Document 3 describes that an aqueous extract of sake lees produced as a by-product in sake production produced using rice as a raw material only through fermentation has an NK cell activation effect. However, Patent Document 3 does not describe the component composition of the aqueous extract at all, and does not specifically describe the component involved in the NK cell activation action.
  • Patent Document 4 describes an aqueous extract of rice bran from a fraction having a molecular weight of about 30,000 to 40,000. It is described that the composition for immunostimulation comprising the extract as described above has mitogenic activity and excellent NK cell activation action. However, Patent Document 4 does not describe the component composition of the immunostimulatory composition at all, and does not specifically describe the component involved in the immunostimulatory action. Patent Document 5 discloses that hemicellulose obtained by alkali extraction of the residue obtained by removing starch and protein from corn hull, which is marketed under the name of “Cel Ace”, is treated with xylanase.
  • Non-Patent Document 1 includes “Celace” (partially decomposed hemicellulose obtained from corn hulls) described in Patent Document 5 containing xylose, arabinose, uronic acid, galactose, and glucose. It is described that it is. Therefore, Patent Document 5 and Non-Patent Document 1 relate to the same “partially decomposed product of hemicellulose obtained from corn hulls”, that is, commercially available “Cell Ace”.
  • the composition having an action of activating NK cells of the present invention produces barley koji and steamed barley using barley as a raw material, and the starch contained in the obtained barley koji and steamed barley is added to the barley koji. Saccharified, and then subjected to alcoholic fermentation with yeast to obtain shochu-ripened mash, and the resulting shochu-ripened mash is subjected to distillation to produce shochu, that is, a by-product distillation residue, that is, barley shochu mash distillation residue It is obtained from the liquid. That is, the source for obtaining the composition having the effect of activating NK cells of the present invention is barley shochu distillation residue.
  • the acquisition source of “cell ace”, a partial degradation product of hemicellulose described in Patent Document 5 is corn hull, that is, hull of corn kernel that is removed when corn kernel is refined.
  • both sources are completely different.
  • the composition having the action of activating NK cells of the present invention is obtained by a production method completely different from the method for producing a partially decomposed product of hemicellulose described in Patent Document 5.
  • the NK cells of the present invention The composition having an activating effect contains arabinose, xylose and glucose, but does not substantially contain uronic acid.
  • the partially decomposed product of hemicellulose described in Patent Document 5 contains a relatively large amount of uronic acid. In this respect both
  • the composition having the effect of activating the M cells of the present invention is clearly distinguished from the partially decomposed product of hemicellulose (trade name Cellace) obtained from corn hull described in Patent Document 5. Obviously it is different.
  • the average molecular weight of the partially degraded hemicellulose is preferably 20,000 to 200,000, more preferably 20,000 to 100,000, and 20,000 to 40,000.
  • the experimental data that led to the determination of the average molecular weight is not described at all, and a specific example of obtaining a partially decomposed product of hemicellulose having such an average molecular weight is as follows. It is not described at all.
  • Patent Document 5 a partially decomposed product of hemicellulose is marketed under the trade name “Cel Ace” (manufactured by Nippon Shokuhin Kako Co., Ltd.).
  • Cellace was obtained, and the molecular weight distribution of Cellace was measured by the method described in“ Measurement of molecular weight distribution ”described above.
  • the molecular weight distribution of Cell Ace is 1% for 1 million or more, 9% for 300,000 to 1 million, 30% for 100,000 to 300,000, 30% for 30,000 to 100,000, and 10,000 to 30,000.
  • the molecular weight distribution of the composition having the effect of activating the M cells of the present invention is only 5% or 11% of 100,000 or more, and has a molecular weight of 3,000 to 100,000.
  • Components with molecular weight distribution in the range account for 72% or 74% of the total.
  • the highest peaks observed in the chromatogram are molecular weights of 3,000 to 10,000, respectively. Is present in the molecular weight range of 30,000 to 100,000.
  • the molecular weight distribution of the composition having the action of stimulating NK cells of the present invention is “cell suspension”, that is, a composition comprising a partial degradation product of hemicellulose obtained from corn hulls. It is completely different from the molecular weight distribution. Therefore, it is clear that the composition having the effect of activating NK cells of the present invention is different from the partial degradation product of hemicellulose described in Patent Document 5.
  • Patent Document 6 describes a composition having an action to suppress fat dryness from a barley shochu distillation residue.
  • the source of the composition having an action to suppress fatty liver is barley shochu distillate as in the case of the composition having an action to activate NK cells of the present invention. It is different from the action to become.
  • the main component of the composition described in Patent Document 6 has a molecular weight of 3000 or less, and the micelle mouth contained in the composition contains xylose as a main component. It is a distinct distinction from a composition that has the effect of activating cells.
  • the composition having the effect of activating the M cells of the present invention is produced as follows. That is, Step A to obtain a liquid component by solid-liquid separation of the barley shochu distillation residue that is a by-product in the production of distilled liquor using barley, and the obtained liquid component is subjected to an ion exchange treatment using an ion exchange resin. Step B 1 for obtaining a non-adsorbed fraction on the ion exchange resin, and subjecting the obtained non-adsorbed fraction to the ion exchange resin to a concentration treatment by ultrafiltration using an ultrafiltration membrane. It is produced by sequentially performing Step C for obtaining a concentrated solution and Step D for fractionating a fraction insoluble in the organic solvent by adding an organic solvent to the obtained concentrated solution.
  • the composition having an action of activating NK cells of the present invention is produced as follows. That is, Step A to obtain a liquid component by solid-liquid separation of a barley shochu distillation residue, which is a by-product in the production of distilled liquor using barley, and the obtained liquid component using a synthetic adsorbent. Step E to obtain a fraction that is not adsorbed to the synthetic adsorbent by subjecting it to an adsorption separation treatment, and the fraction that is not adsorbed to the synthetic adsorbent thus obtained is subjected to an ion exchange treatment using an ion exchange resin.
  • Step B 2 for obtaining a non-adsorbed fraction on the ion exchange resin, and subjecting the obtained non-adsorbed fraction to the ion exchange resin to a concentration treatment by ultrafiltration using an ultrafiltration membrane.
  • the step C is obtained by sequentially performing the step C for obtaining a concentrated liquid and the step D for fractionating a fraction insoluble in the organic solvent by adding an organic solvent to the obtained concentrated liquid.
  • the barley shochu distillation residue used in the present invention typically comprises barley koji and steamed barley using barley or polished barley as a raw material, and the starch contained in the barley koji and steamed barley obtained is When saccharifying with barley koji, subjecting them to alcoholic fermentation with yeast to obtain shochu-ripened mash, and distilling the obtained shochu-ripened mash with a single distillation apparatus such as vacuum distillation or atmospheric distillation Means a by-product as a distillation residue, that is, a distillation residue of barley shochu.
  • the barley koji used for the production of barley shochu may be produced under the koji-making conditions used in normal barley shochu production.
  • the Aspergillus kawachi i generally used in the production of barley shochu is preferable.
  • Aspergi lus genus strains such as Aspergillus awamori used in awamori manufacture and Aspergi lus oryzae used in sake production and the like can also be used.
  • various yeasts for brewing shochu can be used as the yeast used in the production of barley shochu.
  • the step A for obtaining a liquid component by solid-liquid separation of the barley shochu distillation residue obtained in the distillation step in the production of barley shochu is made from raw barley or barley koji derived from the barley shochu distillation residue.
  • the purpose is to remove SS components such as water-insoluble fermentation residues.
  • the solid-liquid separation in step A can be performed by screw press or -Preliminary separation is performed by a solid-liquid separation method using the Ler press method or using a filter-press type solid-liquid separator, and then using a centrifuge, a diatomaceous earth filter, a ceramic filter, or a filter-press
  • the solid-liquid separation process that can be performed according to the present invention is performed.
  • the step E of obtaining a non-adsorbed fraction on the synthetic adsorbent by subjecting the liquid content of the barley shochu distillation residue obtained in the step A to an adsorption separation process using a synthetic adsorbent is a barley shochu distillation
  • the purpose is to remove components such as polyphenol contained in the liquid content of the residual liquid.
  • an aromatic, aromatic modified, or methacrylic synthetic adsorbent can be used.
  • Preferable specific examples of the synthetic adsorbent used in Step E include Amberlay XAD-4, Amberlay XAD-16, Amberlite XAD-1180 and Amperlite XAD-2000 manufactured by Organo Corporation.
  • Aromatic (or styrene) synthetic adsorbents such as Sepabeads SP850 and Diaion HP20 manufactured by Mitsubishi Chemical Co., Ltd. Amperlite XAD-7 manufactured by Organo Co., Ltd. and Diamond manufactured by Mitsubishi Chemical Co., Ltd. Examples include methacrylic (or acrylic) synthetic adsorbents such as Ion HP2MG. In addition to these, aromatic modified synthetic adsorbents such as Sepapies SP207 manufactured by Mitsubishi Chemical Corporation may be used in some cases.
  • step B2 in which the fraction adsorbed on the synthetic adsorbent obtained in E is subjected to an ion exchange treatment using an ion exchange resin to obtain a fraction not adsorbed on the ion exchange resin, the liquid fraction or Components other than the above-mentioned polysaccharides contained in the non-adsorbed fraction of the synthetic adsorbent, that is, amino acids, peptides, proteins, organic acids, or barley-derived polyphenols are removed using an ion exchange resin.
  • the ion exchange resin used in Step B1 and Step B2 includes a cation exchange resin, an anion exchange resin, or a mixed bed ion in which both are mixed.
  • Exchange resins can be used.
  • a cation exchange resin either a strong acid cation exchange resin or a weak acid cation exchange resin can be used.
  • an anion exchange resin a strong base anion exchange resin and a weak base are used. Any sex anion exchange resin can be used.
  • a mixed bed ion exchange resin the above-described cation exchange resin and anion exchange resin can be freely combined and used at a predetermined ratio.
  • ion exchange resins include strong acid cation exchange resins such as Amberlite 200CT and Amberlite IR120B manufactured by Organo Corp., weak acid cation exchange resins such as Amberly MRC76, and amperite. Strongest basic anion exchange resin such as IRA402BL, weak basic anion exchange measurement such as Amberlite IRA67, or both the weak acid cation exchange resin and the weak basic anion exchange resin at a predetermined ratio A mixed bed type ion exchange resin obtained by mixing can be used.
  • strong acid cation exchange resins such as Amberlite 200CT and Amberlite IR120B manufactured by Organo Corp.
  • weak acid cation exchange resins such as Amberly MRC76
  • Strongest basic anion exchange resin such as IRA402BL, weak basic anion exchange measurement such as Amberlite IRA67, or both the weak acid cation exchange resin and the weak basic anion exchange resin at a predetermined ratio
  • a mixed bed type ion exchange resin obtained by mixing can be used.
  • the weak acid cation exchange resin Amberlite IRC76 and the above-mentioned weak It is particularly preferable to use a mixed bed type ion exchange resin obtained by mixing both basic anion exchange resin ampere lye IRA67 at a predetermined ratio, or Amberlite 200CT, a strongly acidic cation exchange resin. preferable.
  • the purpose is to concentrate a polysaccharide mainly composed of arabinose and xylose, which are components involved in NK cell activation, using an ultrafiltration membrane.
  • the ultrafiltration membrane used in Step C any membrane material and membrane module type can be used, and the molecular weight cut-off is preferably 3000 or more, particularly preferably 10,000 to 50,000. Can be used.
  • the concentration rate of ultrafiltration to preferably 2 times or more, particularly preferably 5 times or more, a polysaccharide composed of arabinose and xylose.
  • a composition having an effect of activating the M cells of the present invention containing a significant amount of can be obtained.
  • step D in which an organic solvent is added to the concentrate obtained in step C to fractionate a fraction insoluble in the organic solvent, an appropriate organic solvent is added until a predetermined final concentration is reached.
  • an organic solvent is optimal as the organic solvent, but the organic solvent is not limited to this.
  • the final concentration of the organic solvent affects the production efficiency of the components, and the optimum final concentration of the organic solvent is preferably 5 volume% or more, more preferably 30 to 75 volume%.
  • the composition having the action of activating NK cells of the present invention obtained as described above is obviously of course administered directly as it is, as is apparent from the results of test examples in the examples described later.
  • the desired NK cell activation effect can also be obtained by dispersing the composition in a suitable carrier such as physiological saline and administering it to a patient by intravenous injection.
  • the present invention also provides a composition containing NK cells activated with the composition having an action of activating NK cells (hereinafter referred to as an activated NK cell-containing composition).
  • the activated NK cell-containing composition collects blood from an affected person such as leukemia, separates the obtained blood into lymphocytes and serum composed of immune cells such as NK cells, and the lymphocytes are separated into the NK cells.
  • the activated NK cell-containing composition obtained in this way is washed with a drip physiology Can be suspended in saline and administered via intravenous infusion.
  • Such activated NK cell-containing composition has the effect of necrotizing K562 cells. Therefore, the activated NK cell-containing composition can be used as a therapeutic agent for (myeloid) leukemia.
  • UFP-30-E-4MA fractional molecular weight 30,000
  • the resulting concentrated solution is adjusted to Brix20, and the final concentration is 75 volumes. Ethanol was added so that the concentration of the lyophilized product was 10%. Centrifugation was performed at 8000 rpm and lOmin, and a fraction insoluble in the organic solvent (ethanol) was collected. 2. 5g was obtained. The lyophilized product was powdered to obtain a grayish white, tasteless and odorless composition.
  • the barley shochu distillation residue obtained in “Manufacture of barley shochu and barley shochu distillation residue” was centrifuged at 8000 rpm and lOmin to obtain a liquid content of the barley shochu distillation residue.
  • a column filled with a synthetic adsorbent amperite XAD-16 manufactured by Co., Ltd. By passing through a column filled with a synthetic adsorbent amperite XAD-16 manufactured by Co., Ltd. and subjecting it to an adsorption separation treatment, the above-mentioned flow-through liquid that exhibits non-adsorbability to the synthetic adsorbent of the column is used. The fraction not adsorbed on the synthetic adsorbent was collected.
  • the fraction that was not adsorbed to the resulting synthetic adsorbent was adjusted to Br ixlO, and 1 L of the fraction adjusted to Br ixlO was added to 500 ml of Amberlite 200CT (strongly acidic cation exchange resin) manufactured by Organo Corporation.
  • a fraction that is not adsorbed to the ion exchange resin is obtained, and the obtained fraction is filtered with an ultrafiltration membrane UFP-30-E-4MA manufactured by A / G Technology (fractionated molecular weight 3
  • the resulting concentrate is adjusted to Brix20, ethanol is added to a final concentration of 75% by volume, and the mixture is centrifuged at 8000 rpm and lOmin.
  • a fraction insoluble in the organic solvent (ethanol) was collected, and the obtained fraction was freeze-dried to obtain 1.3 g of a lyophilized product.
  • the freeze-dried product was powdered to obtain an off-white, tasteless and odorless composition.
  • Example 2 In order to clarify whether the compositions obtained in Example 2 and Comparative Example 1 have an activating effect on NK cells, the following Test Example 1 was performed.
  • heparin-added peripheral blood was collected from healthy humans, diluted twice with physiological saline, and the diluted blood was overlaid on Lymphosepar I (manufactured by Immune Biology Laboratories) at room temperature. After centrifugation at 1500 ⁇ for 30 minutes, the plasma was removed using a pipette to obtain a fraction of peripheral blood mononuclear cells ( ⁇ C). Next, the PBMC fraction obtained to obtain a S t emSep NK cell fraction Ri by the subjecting (manufactured by Veritas), the NK cell fraction obtained 5% C0 2 atmosphere at 37 ° C for Culture was performed to obtain a NK cell culture solution.
  • the obtained M cell culture solution was centrifuged at 1000 rpm for 5 minutes to collect NK cells.
  • the number of NK cells collected was adjusted to IX cells / ml using RPMI-1640 medium supplemented with 10% FCS to obtain an NK cell suspension.
  • K562 cells chronic myeloid leukemia cells obtained by subculture are centrifuged to separate the K562 cells, which are then washed with PBS (Phosphat e-bufered saline).
  • PBS Phosphat e-bufered saline
  • NK cell culture solution thus obtained was centrifuged at 1000 rpm for 5 minutes to collect NK cells.
  • the number of NK cells collected was adjusted to IX cells / ml using RPMI-1640 medium supplemented with 10% FCS to obtain effect cells.
  • the target cells adjusted to the optimum concentration were dispensed 100 1 into a 96-well V-bottom plate, and the effector cells were mixed separately. Plate mixing said target cell and said effector cells are, 800 rpm, centrifuged for 2 minutes, 5% C0 2 atmosphere, after 4 hours at 37 ° C, each culture 100 1 were taken, respectively, Measure the K562 cell viability using the following formula, using the CeilTiter-Glo Luminescent Cel 1 Vi ab i 1 i Assay Assay kit (Promega), and determine the NK cells of each sample. It was used as an index of activation ability.
  • K562 cell viability ⁇ (Luminescence of [NK cells + K562 cells treated with each sample]) 1 (Luminescence of NK cells only) ⁇ / (Luminescence of K562 cells treated with PBS) X 100
  • Table 1 shows the measurement results of NK cell activity against K562 cells. The following facts were found from the results shown in Table 1. That is, NK cells obtained by adding a predetermined amount of the composition obtained in Comparative Example 1 and subjecting it to culture are more than NK cells obtained by adding the predetermined amount of IL-2 and subjecting it to culture. Slightly low NK cell activity was shown. On the other hand, NK cells obtained by adding a predetermined amount of the composition obtained in Example 1 and Example 2 and subjecting them to culture are obtained by adding the predetermined amount of IL-2 and subjecting them to culture. It was revealed that the cells have extremely remarkable NK cell activation ability comparable to that of M cells.
  • NK cells By the way, in afflicted patients such as malignant tumors, the activity of NK cells is reduced.
  • the cytotoxic activity against tumor cells caused by the NK cells is also extremely low. Therefore, the following tests were conducted for the purpose of more practically evaluating the NK cell activation by the fraction insoluble in the organic solvent and the resulting cytotoxic activity against tumor cells.
  • heparin-added peripheral blood was collected from healthy humans, diluted twice with physiological saline, and the diluted blood was overlaid on Lymphosepar I (manufactured by Immune Biology Laboratories) at room temperature. After centrifugation at 1500 rpm for 30 minutes, plasma was removed using a pipette to obtain a fraction of peripheral blood mononuclear cells (IC). Then, the resulting: PBMC fraction to obtain a StemSep NK cell fraction Ri by the subjecting (manufactured by Veritas), and the NK cell fraction obtained 5% C0 2 atmosphere, the culture at 37 ° C for The NK cell culture solution was obtained.
  • Lymphosepar I manufactured by Immune Biology Laboratories
  • the obtained NK cell culture solution was centrifuged at 1000 rpm for 5 minutes to collect NK cells.
  • the number of cells of each collected NK cell was adjusted to IX 10 6 cells / ml using RPMI-1640 medium supplemented with 10% FCS to obtain an NK cell suspension.
  • K562 cells chronic myeloid leukemia cells obtained by subculture are centrifuged to separate the K562 cells and washed twice with PBS (Phosphate-buf fered saline). Subsequently, the cells were further washed with RPMI-1640 medium supplemented with CS, to obtain target cells consisting of K562 cells.
  • the NK cell suspension was dispensed into a 96-well V-bottom plate at a rate of 1001, and the target cells adjusted to the optimum concentration were added at a rate of 100 U.
  • the cells were cultured for 4 hours at 37 ° C. in a 5% CO 2 atmosphere.
  • 100 L of the target cells adjusted to the optimal concentration was added to another M cell suspension 100 1, and then the RPMI-1640 medium with 10% FCS was added to 22 ⁇ 1. The same treatment was applied to the material to which the slag was added.
  • the target cell 100 ⁇ ⁇ adjusted to the optimal concentration was added to another ⁇ cell suspension 100 1, and then the RPMI-1640 medium supplemented with 10% FCS was used.
  • IL-2 water prepared to a concentration of lmg / ml The same treatment was applied to the solution to which 22 1 was added.
  • 100 1 of each culture was collected, and the amount of ATP was measured using CelTiter-Glo Luminescent Cell Viability Assay Kit (Promega), and the survival rate of K562 cells was calculated using the following formula.
  • K562 cell viability (%) ⁇ ([Luminescence of [NK cells + K562 cells treated in each sample])]
  • Table 2 shows the measurement results of the cytotoxic activity when the lyophilized product obtained in Comparative Example 1 was added to the mixture of M cells and K562 cells.
  • the following facts were found from the results shown in Table 2. That is, when the lyophilized product obtained in Comparative Example 1 was added to the mixture of NK cells and K562 cells in the predetermined amount, it was clearer than the case where the predetermined amount of IL-2 was added to the mixture. Showed low cytotoxic activity. On the other hand, when the predetermined amount of the lyophilized product obtained in Example 1 and Example 2 was added to the mixture of NK cells and K562 cells, it was comparable to the case where the predetermined amount of IL-2 was added to the mixture. It was found to exhibit extremely strong cytotoxic activity.
  • Example 1 and Example 2 when the lyophilized product of Example 1 and Example 2 is added to a mixture of NK cells and K562 cells, it not only significantly activates NK cells but also suppresses the proliferation of K562 cells. From the above, it was revealed that there is an excellent leukemia treatment effect.
  • Example 2 In order to clarify the in vivo NK cell activation action of the freeze-dried fraction insoluble in the organic solvent obtained in Example 1 and Example 2, the following test was performed. That is, C57BL / 6 mice (3 weeks old, 10 mice per group) were fed with a diet (control group) that did not contain the composition having the effect of activating NK cells of the present invention, and the organic obtained in Example 1 Diet containing 0.5% and 1.0% of lyophilized fraction insoluble in solvent (test group 1), and lyophilized fraction insoluble in organic solvent obtained in Example 2 of 0. 5% and 1.0% diet (Test group 2) was ingested, and after 5 weeks, the spleens were removed from both groups of mice, the number of cells was counted and NK fine Cell activity was measured as follows.
  • spleen cells were suspended in C-RMI medium, and cell-labeled with fluorescein isothiocyanate (FITC) -conjugated anti-mouse IL-2 / 3 receptor antibody and phycoerythrin 0PE) -conjugated anti-mouse CD3 antibody,
  • FITC fluorescein isothiocyanate
  • the NK cell fraction was measured by flow cytometry, and the CD3-IL-2 ⁇ + fraction was identified as M cells.
  • NK cell activity (%) was calculated from the following formula using a scintillation counter.
  • NK cell activity (%) [(experimental release-spontaneous release) / (maximum release-spontaneous release)] X 100
  • Table 3 shows the results of measurement of the number of spleen cells, NK cell fraction, and spleen cell-derived NK cell activity.
  • test group 1 ingesting a diet containing 0.5% and 1.0% of the composition having the effect of activating NK cells obtained in Example 1, and the organic solvent obtained in Example 2 above.
  • the spleen cell-derived NK cell activity in test group 2 that received diets containing 0.5% and 1.0% lyophilized fractions insoluble in erythrocyte significantly increased compared to the control group, respectively. It has been found.
  • the number of spleen cells and NK cell fraction there was no significant difference between the three groups consisting of test group 1, test group 2 and control group. From these results, it became clear that the composition having an action of activating NK cells of the present invention activates NK cells even in vivo.
  • the M cells of the present invention which are mainly composed of polysaccharides consisting of alapinose and xylose, obtained from the barley shochu distillation residue, are activated. It has been found that a composition having an action has an excellent activation effect on NK cells.
  • composition having the effect of activating NK cells of the present invention has a very remarkable NK cell activation effect comparable to IL-2, which is a known NK cell activator, so leukemia is repelled. It is extremely suitable for the treatment of cancer.
  • NK cell activity (3 ⁇ 4) 2.70 ⁇ 0.25 18.8 ⁇ 2.97 21.6 ⁇ 5.18 20.2 ⁇ 4.31 22.8 ⁇ 3.75 fl Number of base cells (x10 a ) 1.17 ⁇ 0.31 1.18 ⁇ 0.16 1.08 + 0.61 1.22 ⁇ 0.38 1.09 ⁇ 0.45 NK cell fraction ( .81 ⁇ 0.14 62 ⁇ 0.71 4.43 ⁇ 0.29 4,29 ⁇ 0.87 4.58 ⁇ 1.27

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Communicable Diseases (AREA)
  • Medical Informatics (AREA)
  • Mycology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne une composition permettant l'activation de cellules tueuses naturelles comprenant une fraction qui ne contient sensiblement pas d'acide uronique, qui contient des polysaccharides comprenant du xylose, de l'arabinose et du glucose et qui est insoluble dans un solvant acide organique. Il est possible d'obtenir cette fraction en soumettant une vinasse de shochu d'orge (eau-de-vie distillée) à une séparation solide-liquide de manière à obtenir un liquide, en soumettant ce liquide à un traitement d'échange ionique au moyen d'une résine échangeuse d'ions de manière à obtenir une fraction (a) non adsorbée par la résine échangeuse d'ions, ou en soumettant le liquide décrit ci-dessus à un traitement de séparation par adsorption à l'aide d'un adsorbant synthétique de manière à obtenir une fraction (b) non adsorbée par l'adsorbant synthétique, puis en soumettant cette fraction (b) à un traitement d'échange ionique au moyen d'une résine échangeuse d'ions afin de produire la fraction décrite ci-dessus (a), en concentrant la fraction ainsi obtenue (a) par ultrafiltration à l'aide d'une membrane d'ultrafiltration de manière à obtenir un concentré liquide, et en ajoutant le solvant organique décrit ci-dessus à ce concentré liquide. L'invention se rapporte également à une composition contenant des cellules tueuses naturelles qui ont été inactivées au moyen de la composition décrite ci-dessus.
PCT/JP2004/000003 2002-12-27 2004-01-05 Composition permettant l'activation de cellules tueuses naturelles et procede de production de cette composition, composition contenant des cellules tueuses naturelles activees par la composition precedente et procede de production de ladite composition Ceased WO2004061090A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005507954A JPWO2004061090A1 (ja) 2002-12-27 2004-01-05 ナチュラルキラー細胞を賦活化する作用を有する組成物及びその製造方法、及び前記組成物を使用して賦活化したナチュラルキラー細胞を含有する組成物及びその製造方法

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP2002379476 2002-12-27
JP2002379486 2002-12-27
JP2002-379486 2002-12-27
JP2002-379476 2002-12-27
JP2003-429972 2003-12-25
JP2003429972A JP2005104957A (ja) 2002-12-27 2003-12-25 ナチュラルキラー細胞を賦活化する作用を有する組成物及びその製造方法、及び賦活化したナチュラルキラー細胞含有組成物及びその製造方法

Publications (1)

Publication Number Publication Date
WO2004061090A1 true WO2004061090A1 (fr) 2004-07-22

Family

ID=32718766

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2004/000003 Ceased WO2004061090A1 (fr) 2002-12-27 2004-01-05 Composition permettant l'activation de cellules tueuses naturelles et procede de production de cette composition, composition contenant des cellules tueuses naturelles activees par la composition precedente et procede de production de ladite composition

Country Status (2)

Country Link
JP (2) JP2005104957A (fr)
WO (1) WO2004061090A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006115826A (ja) * 2004-10-25 2006-05-11 Yasunobu Kobayashi ナチュラルキラー細胞の増殖促進方法およびそれに用いる培養組成物
WO2007034958A1 (fr) * 2005-09-26 2007-03-29 Sanwa Shurui Co., Ltd. Composition anti-angiogénique comprenant un composant dérivé d'un grain comme ingrédient actif
IT201900006858A1 (it) * 2019-05-15 2020-11-15 Abresearch Srl Preparazione di polisaccaridi insolubili ottenuti da colture cellulari vegetali in sospensione per il trattamento di infezioni da Clostridium difficile

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0923895A (ja) * 1995-07-12 1997-01-28 Daiwa Yakuhin Kk 免疫力増強物質及びその製造方法
JPH10146166A (ja) * 1996-11-18 1998-06-02 Nippon Shuzo Kumiai Chiyuuoukai 生理活性組成物
JP2001145472A (ja) * 1999-09-07 2001-05-29 Sanwa Shiyurui Kk 大麦焼酎蒸留残液から分取した脂肪肝抑制作用を有する組成物及び該組成物の製造方法
JP2001161339A (ja) * 1999-12-03 2001-06-19 Asahi Kasei Corp 二次仕込み水の安定化方法
JP2002255843A (ja) * 2001-03-02 2002-09-11 Oriza Yuka Kk 免疫賦活用組成物
JP2003073294A (ja) * 2001-09-05 2003-03-12 Sanwa Shiyurui Kk 白血病細胞増殖阻害剤及びその製造方法
JP2003221342A (ja) * 2001-11-21 2003-08-05 Sanwa Shiyurui Kk 肝炎の発症抑制作用及び治癒作用を有する組成物及び該組成物の製造方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0923895A (ja) * 1995-07-12 1997-01-28 Daiwa Yakuhin Kk 免疫力増強物質及びその製造方法
JPH10146166A (ja) * 1996-11-18 1998-06-02 Nippon Shuzo Kumiai Chiyuuoukai 生理活性組成物
JP2001145472A (ja) * 1999-09-07 2001-05-29 Sanwa Shiyurui Kk 大麦焼酎蒸留残液から分取した脂肪肝抑制作用を有する組成物及び該組成物の製造方法
JP2001161339A (ja) * 1999-12-03 2001-06-19 Asahi Kasei Corp 二次仕込み水の安定化方法
JP2002255843A (ja) * 2001-03-02 2002-09-11 Oriza Yuka Kk 免疫賦活用組成物
JP2003073294A (ja) * 2001-09-05 2003-03-12 Sanwa Shiyurui Kk 白血病細胞増殖阻害剤及びその製造方法
JP2003221342A (ja) * 2001-11-21 2003-08-05 Sanwa Shiyurui Kk 肝炎の発症抑制作用及び治癒作用を有する組成物及び該組成物の製造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KUKIZAKI MASATO: "Shochu joryu haieki jikomi no yoru shcho seizo no closed-ka ni kansuru kenkyu", MIYAZAKI-KEN KOGYO SHIKENJO KENKYU HOKOKU, vol. 32, 1987, pages 51 - 54, XP002982751 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006115826A (ja) * 2004-10-25 2006-05-11 Yasunobu Kobayashi ナチュラルキラー細胞の増殖促進方法およびそれに用いる培養組成物
WO2007034958A1 (fr) * 2005-09-26 2007-03-29 Sanwa Shurui Co., Ltd. Composition anti-angiogénique comprenant un composant dérivé d'un grain comme ingrédient actif
IT201900006858A1 (it) * 2019-05-15 2020-11-15 Abresearch Srl Preparazione di polisaccaridi insolubili ottenuti da colture cellulari vegetali in sospensione per il trattamento di infezioni da Clostridium difficile
WO2020230080A1 (fr) * 2019-05-15 2020-11-19 Abresearch Srl Préparation de polysaccharides insolubles obtenue à partir de cultures de cellules végétales en suspension pour le traitement d'infections par clostridium difficile

Also Published As

Publication number Publication date
JPWO2004061090A1 (ja) 2006-09-28
JP2005104957A (ja) 2005-04-21

Similar Documents

Publication Publication Date Title
JP4826696B2 (ja) 血管新生阻害剤
KR100797016B1 (ko) 면역증강 활성을 나타내는 인삼다당체 고농축정제액과 그제조방법
WO2015052849A1 (fr) Nouveau dérivé ayant une activité de production de collagène, et son procédé de production
CN101249259A (zh) 一种高含量、高活性口服云芝糖肽及其制备方法和应用
TWI386485B (zh) 瓦茸菌株（Ｔｒａｍｅｔｅ Ｖｅｒｓｉｃｏｌｏｒ）及其萃取物，以及其用途
WO2004061090A1 (fr) Composition permettant l'activation de cellules tueuses naturelles et procede de production de cette composition, composition contenant des cellules tueuses naturelles activees par la composition precedente et procede de production de ladite composition
JP4451100B2 (ja) タモギタケ子実体組成物を有効成分として含有する免疫賦活剤、及び抗癌剤
CN117562893A (zh) Pd-l1抑制的表达和pd1增强的表达
CN118147260B (zh) 一种人参寡肽和稀有皂苷共提取的方法及提取产物的应用
CN114989258B (zh) 植物提取组合物在制备治疗便秘、减肥产品上的应用
JP2003342188A (ja) 抗ガン剤
EP1398036B1 (fr) Spores de ganoderma lucidum pour le traitement de lupus érythémateux disséminé (LED)
JPH11246427A (ja) 糖・脂質代謝活性化剤
KR100506950B1 (ko) 진세노사이드를 함유함을 특징으로 하는 면역증강효과에 대한 조성물
JP5756270B2 (ja) がんペプチドワクチン療法効果向上剤
JP2004217928A (ja) ナチュラルキラー細胞を賦活化する作用を有する組成物の製造方法、及び賦活化したナチュラルキラー細胞含有組成物の製造方法
CN109998055B (zh) 一种汉麻籽抗氧化多肽咀嚼片及其制备方法
WO2022158368A1 (fr) Composition de matière cosmétique inhibant le gène gls1, inhibiteur du gène gls1, composition de matière cosmétique pour l'élimination de cellules sénescentes et agent d'élimination de cellules sénescentes
JPH02134325A (ja) エイズ治療剤およびその製造方法
US20130022641A1 (en) Anti-Tumor Agent and Immunostimulating Agent
JP4524342B2 (ja) 大麦焼酎蒸留残液から分取した記憶学習能向上の薬理作用を呈する組成物
CN103450354A (zh) 一种黄芪糖蛋白及其制备方法和用途
WO2004004748A1 (fr) Extrait d'agaricus blazei murill pour la prevention de l'apparition du cancer ou de la metastase
US8034386B2 (en) Extract from Agaricus blazei Murill capable of suppressing breast cancer
JP7162542B2 (ja) 食品因子センシング関連遺伝子発現促進用食品組成物及び食品因子センシング関連遺伝子発現促進剤

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2005507954

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: GB0519678.7

Country of ref document: GB

122 Ep: pct application non-entry in european phase