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WO2021131900A1 - Glycoside de prénylflavonoïde, son procédé de production, et procédé pour améliorer la solubilité dans l'eau de prénylflavonoïde - Google Patents

Glycoside de prénylflavonoïde, son procédé de production, et procédé pour améliorer la solubilité dans l'eau de prénylflavonoïde Download PDF

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Publication number
WO2021131900A1
WO2021131900A1 PCT/JP2020/046738 JP2020046738W WO2021131900A1 WO 2021131900 A1 WO2021131900 A1 WO 2021131900A1 JP 2020046738 W JP2020046738 W JP 2020046738W WO 2021131900 A1 WO2021131900 A1 WO 2021131900A1
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prenylflavonoid
general formula
hexose
amino acid
acid sequence
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Japanese (ja)
Inventor
美佐 落合
栄一郎 小埜
祐子 福井
慶造 藤井
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Suntory Holdings Ltd
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Suntory Holdings Ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B70/00—Preservation of non-alcoholic beverages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/116—Heterocyclic compounds
    • A23K20/121—Heterocyclic compounds containing oxygen or sulfur as hetero atom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans
    • C07H17/07—Benzo[b]pyran-4-ones
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic

Definitions

  • the present invention relates to a prenylflavonoid glycoside in which two or more hexoses are bound to prenylflavonoid, and a method for producing the same.
  • the present invention also relates to a method for producing a prenylflavonoid monoglycoside in which one hexose is bound to prenylflavonoid.
  • the present invention also relates to a method for improving the water solubility of prenylflavonoids.
  • the present invention further relates to methods for reducing the bitterness of prenylflavonoids, methods for improving stability, and the like.
  • Prenylflavonoids are known to have various useful biological activities such as antitumor activity and improvement of metabolic syndrome (Non-Patent Documents 1 to 3).
  • prenylflavonoids such as isoxanthohumol have extremely low polarity and are hardly soluble in water
  • the amount of prenylflavonoid added may be limited, for example, when blended in a beverage.
  • the present inventors have added hexose to the 7-position of isoxanthohumol and 8-prenylnaringenin by a bacterium belonging to the genus Rhizopus, which is a filamentous fungus. I found that I could do it.
  • the present inventors have found a plant-derived UDP-gluconosyltransferase (UGT) that adds hexose to the 7-position of isoxanthohumol.
  • UDP-gluconosyltransferase UDP-gluconosyltransferase
  • the present invention is not limited to this, but the present invention describes the following prenylflavonoid glycosides, a method for producing the same, a method for improving the water solubility of prenylflavonoid, a method for reducing the bitterness of prenylflavonoid, and stability of prenylflavonoid. Regarding methods for improving sex.
  • a prenylflavonoid glycoside represented by the following general formula (1).
  • a method for producing a prenylflavonoid glycoside which comprises a step of producing a prenylflavonoid monoglycoside (A2) and a step of adding 1 to 9 hexoses to the hexose residue of the prenylflavonoid monoglycoside (B).
  • (P1) Protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (p2) In the amino acid sequence shown in SEQ ID NO: 1, 1 to 9 amino acids consist of deleted, substituted, inserted and / or added amino acid sequences, and , A protein having an activity of adding a hex sauce to the 7-position of the prenyl flavonoid represented by the general formula (2) (p3), which comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1.
  • a method for producing a prenyl flavonoid monoglycoside which comprises a step (A2) of producing a prenyl flavonoid monoglycoside in which one hex source is bound to the 7-position of the prenyl flavonoid from the above prenyl flavonoid and UDP-hexose.
  • (P1) Protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (p2) In the amino acid sequence shown in SEQ ID NO: 1, 1 to 9 amino acids consist of an amino acid sequence deleted, substituted, inserted and / or added, and , A protein having the activity of adding hexose to the 7-position of the prenyl flavonoid represented by the general formula (2) (p3), which comprises an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1.
  • amino acid sequence shown in SEQ ID NO: 2 of the protein (q2) consisting of the amino acid sequence shown in SEQ ID NO: 2 of the protein (q1) having the activity of adding hexose to the 7-position of the prenyl flavonoid represented by the general formula (2).
  • a protein consisting of an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, inserted and / or added, and having an activity of adding a hex source to the 7-position of the prenyl flavonoid represented by the general formula (2) ( q3) A protein consisting of an amino acid sequence having 90% or more identity with respect to the amino acid sequence shown in SEQ ID NO: 2 and having an activity of adding a hex source to the 7-position of the prenyl flavonoid represented by the general formula (2).
  • [12] A method for reducing the bitterness of the prenylflavonoid by adding a sugar chain composed of 2 to 10 hexoses to the 7-position of the prenylflavonoid represented by the general formula (2).
  • [13] A method for improving the stability of the prenylflavonoid by adding a sugar chain composed of 2 to 10 hexoses to the 7-position of the prenylflavonoid represented by the general formula (2).
  • [14] The method according to any one of the above [7] to [13], wherein the hexose is glucose.
  • FIG. 3 is a diagram showing the results of NMR measurement of reaction products of UDP-glucose and isoxanthohumol by a soybean-derived UGT (Gm_UGT1) expressed in Escherichia coli.
  • FIG. 4A shows the results of HPLC analysis of the culture supernatant of Rhizopus oryzae (R. oryzae) IAM6049 cultured with the addition of isoxanthohumol.
  • FIG. 4B shows R. cerevisiae cultured without the addition of isoxanthohumol.
  • the HPLC analysis result of the culture supernatant (analytical sample for comparison) of oryzae IAM6049 is shown.
  • FIG. 5A shows the analysis results of isoxanthohumol (IX) and isoxanthohumol monoglucoside (IXG) in the culture supernatant obtained by adding isoxanthohumol to a bacterium belonging to the genus Risopas. ..
  • FIG. 5B shows the analysis results of isoxanthohumol (IX) and isoxanthohumol monoglucoside (IXG) in the cells when isoxanthohumol was added to the bacteria belonging to the genus Risopas and cultured. Is shown.
  • 8-prenylnaringenin (8-PN) was added and R.
  • FIG. 7A is an HPLC analysis result of a reaction solution (reaction time 0 hour) in which CGTase and cluster dextrin were added to isoxanthohumol monoglucoside (IXG).
  • FIG. 7B shows the results of HPLC analysis of the reaction solution obtained by adding CGTase and cluster dextrin to IXG and reacting for 2 hours (both detected: 280 nm).
  • R represents a methyl group or a hydrogen atom
  • X represents a hexose residue
  • n represents an integer of 2 to 10.
  • the 2 to 10 hexose residues are the same. It may or may not be different.
  • R represents a methyl group in the general formula (1).
  • the prenylflavonoid glycoside when R is a methyl group, is an isoxanthohumol glycoside.
  • R is a hydrogen atom in the general formula (1)
  • the prenylflavonoid glycoside is an 8-prenylnaringenin glycoside.
  • the prenylflavonoid glycoside of the present invention is preferably an isoxanthohumol glycoside.
  • the hexose in the present invention does not contain glucuronic acid.
  • the hexose is preferably D-form.
  • D-glucose and D-galactose are preferable, and D-glucose is more preferable.
  • the prenylflavonoid glycoside of the present invention exhibits excellent water solubility and has less bitterness than aglycones such as isoxanthohumol. Moreover, the prenylflavonoid glycoside of the present invention is excellent in stability. Therefore, the prenylflavonoid glycoside of the present invention is excellent in beverage suitability.
  • the composition of the present invention is preferably a liquid composition, more preferably a beverage.
  • the form of the beverage is not particularly limited, and can be a packaged beverage.
  • the container of the packaged beverage is not particularly limited, and a container of any form and material may be used.
  • the medium contains nitrogen sources (for example, various peptones (potato peptone, etc.), various extracts, ammonium sulfate, urea, etc.) and inorganic substances (dipotassium hydrogen phosphate, potassium phosphate, magnesium sulfate, zinc sulfate, iron, etc.), if necessary. , Manganese, molybdenum, sodium chloride, potassium chloride, magnesium chloride, etc.) may be added.
  • a potato dextrose medium (PD medium) or the like can be used as the medium.
  • immobilized cells in which bacteria belonging to the genus Rhizopus are immobilized on a carrier can also be used.
  • the method for preparing the immobilized cells is not particularly limited, and a known method can be adopted.
  • spores of a bacterium belonging to the genus Rhizopus are added to an aqueous solution of sodium alginate, stirred and suspended, and then added dropwise to a calcium chloride solution to form beads in which the spores are immobilized. You can get a gel.
  • Immobilized cells can be obtained by culturing this bead-shaped gel in the medium and culture conditions used for culturing bacteria belonging to the genus Rhizopus.
  • step (A1) by carrying out the above culture, a prenylflavonoid monoglycoside in which one hexose is bound to the 7-position of the prenylflavonoid represented by the general formula (2) is produced and accumulated in the medium. To do.
  • the step (C) for separating the bacterial cells and the culture supernatant containing the prenylflavonoid monoglycoside may be performed. It does not have to be.
  • the method for separating the bacterial cells and the culture supernatant is not particularly limited, and a method such as centrifugation or filtration can be adopted.
  • the step (B) can also be carried out using the medium containing the prenylflavonoid monoglycoside and the bacterium belonging to the genus Lysopas as it is.
  • amino acid sequence shown in SEQ ID NO: 2 of the protein (q2) consisting of the amino acid sequence shown in SEQ ID NO: 2 of the protein (q1) having the activity of adding hexose to the 7-position of the prenyl flavonoid represented by the general formula (2).
  • the amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of isoflavone 7-O-glucose transferase, which is a UDP-glycosyltransferase derived from soybean (Glycine max).
  • the amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of flavonoid UDP-glycosyltransferase (VvGT6), which is a UDP-glycosyltransferase derived from grape (Vitis vinifera). It has not been previously reported that these UDP-glycosyltransferases have the activity of adding hexose to the 7-position of isoxanthohumol or 8-prenylnaringenin.
  • the number of amino acids deleted, substituted, inserted and / or added in the proteins (p2) and (q2) is preferably 1 to 8, 1 to 7, or 1 to 6, and more preferably 1.
  • the number is ⁇ 5, more preferably 1 to 4, particularly preferably 1 to 3, particularly preferably 1 or 2, and most preferably 1.
  • step (A2) preferably, a protein consisting of the amino acid sequence shown in (p1) SEQ ID NO: 1 and / or a protein consisting of the amino acid sequence shown in (q1) SEQ ID NO: 2 is used.
  • step (A2) it is also preferable to use a protein consisting of the amino acid sequence shown in (p1) SEQ ID NO: 1 or a protein consisting of the amino acid sequence shown in (q1) SEQ ID NO: 2.
  • examples of the glucose source include glucose, cyclodextrin, dextrin (cluster dextrin and the like), starch and the like.
  • the amount of the hexose source added to the reaction solution is preferably 10 to 1000 times the molar ratio of prenylflavonoid monoglycoside in the reaction solution, for example.
  • the amount of hexose source in the reaction solution at the start of the reaction is preferably 10 to 1000 times the molar ratio of prenylflavonoid monoglycoside.
  • the prenylflavonoid monoglycoside produced in the step (A2) may be purified from the above reaction solution by a known method and subjected to the step (B), but the step (A2) and the step (A2) and the step (A2) without purification may be performed. (B) may be performed continuously.
  • the reaction solution containing the prenylflavonoid monoglycoside obtained in the step (A2) and the UGT protein is added with the enzyme having glycosyltransferase activity used in the step (B) and, if necessary, a hexose source in the step (B). Can also be done.
  • the present invention also includes the following methods for producing prenylflavonoid monoglycosides.
  • a method for producing a prenylflavonoid monoglycoside which comprises a step (A1) of producing a prenylflavonoid monoglycoside.
  • Examples of the extraction method include an extraction method using an ethanol solvent, which is used as a method for preparing a hop extract used for beer brewing.
  • the hop extract is commercially available, and a commercially available hop extract can also be used.
  • the heating of the hop extract for producing isoxanthohumol is preferably carried out at 80 to 140 ° C. (more preferably 85 to 100 ° C.) for 15 minutes to 5 hours (more preferably 20 minutes to 3 hours).
  • Purification of the hop extract for preparing isoxanthohumol is carried out by known methods. Examples of the purification method include the use of HPLC, an adsorption column, and the like, and a method such as precipitation utilizing a change in solubility.
  • Flavonoid 3-position Glucuronosyltransferase VvGT6 (Reference 3: Ono E et al (2010) Plant Cell 22 (8): 2856-2871) was examined for its reactivity to IX.
  • the amino acid sequence of VvGT6 (ACCESSION AB499075), which is a grape-derived UGT, is shown in SEQ ID NO: 2.
  • the nucleotide sequence of the DNA encoding Gm_UGT1 is shown in SEQ ID NO: 3
  • the nucleotide sequence of the DNA encoding VvGT6 is shown in SEQ ID NO: 4.
  • the transformant (recombinant Escherichia coli) was cultured in LB medium (50 mL), and the protein was expressed using Overnight Expression TM Autumn System 1 (manufactured by Novagen). Recombinant protein was extracted from the collected Escherichia coli using BugBuster (registered trademark) Protein Extension Reagent (manufactured by Merck Millipore), and the target UG was obtained using a HisTrap TM High Performance (manufactured by GE Healthcare) column. did.
  • the peak fraction of the product (IX glucoside) was purified by eluting stepwise using a Sep-Pak column.
  • the peak fraction of the reaction product was loaded onto 35 cc of Sep-pakC18 (manufactured by Waters) and stepwise with 3 CV (column volume) washed with water, 3 CV of 5% acetonitrile aqueous solution, 3 CV of 40% acetonitrile aqueous solution, and 3 CV of 80% acetonitrile aqueous solution. Eluted into.
  • Table 1 and FIG. 3 show the results of NMR measurement of the reaction products of UDP-glucose and isoxanthohumol (IX) by recombinant UGT (Gm_UGT1).
  • Table 1 shows the chemical shifts of 1 H and 13 C.
  • FIG. 3 shows the results of HMBC (Heteroncular Multiple Bond Correlation) and NOE (Nuclear Overhauser Effect), which are the keys to determine the sugar binding position to IX.
  • HMBC Heteroncular Multiple Bond Correlation
  • NOE Nuclear Overhauser Effect
  • the culture supernatant and acetonitrile were mixed 1: 1 and filtered through a GL chromatodisk 4P 0.45 ⁇ m (manufactured by GL Sciences Co., Ltd.) to prepare an LC analysis sample of the culture supernatant.
  • 2 mL of ethyl acetate was added to the recovered cells, and the mixture was vigorously stirred and then centrifuged to recover the solvent layer. After removing the solvent with a centrifugal concentrator, the residue was dissolved in 200 ⁇ L of a 50% aqueous acetonitrile solution and filtered through a GL chromatodisk 4P 0.45 ⁇ m to prepare an LC analysis sample of the cells.
  • LC analysis sample was analyzed by HPLC under the following LC analysis condition (B).
  • FIG. 4A R. IX was added and cultured.
  • the results of HPLC analysis of the culture supernatant of oryzae IAM6049 are shown (detection: 280 nm).
  • FIG. 4B shows the results of HPLC analysis of the culture supernatant (comparative analysis sample) of IAM6049 cultured without adding IX (detection: 280 nm).
  • the retention time of the product was the same in all the strains, and it was also consistent with the retention time of IXG in which one glucose was added to the 7-position of the previously identified IX.
  • the analysis results of the culture supernatants of all the investigated strains are shown in FIG.
  • FIGS. 5A and 5B are both peak areas per medium.
  • ⁇ is an isoxanthohumol glycoside (IXG) in which one glucose is glycosidic bonded to the 7-position of isoxanthohumol (in FIGS. 5A and 5B), and ⁇ is isoxanthohumol. It is a mall (IX).
  • IXG isoxanthohumol glycoside
  • Rhizopus filamentous fungi Rhizopus filamentous fungi (R. microsporus, R. oligosporus, R. chinensis, R. chinensis var. It was.
  • PP (0.2) G (0.2) indicates that the medium contains 0.2% potato peptone CP and 0.2% glucose.
  • R. 100 ⁇ L of a spore suspension of oryzae IAM6256 was added, and the mixture was shake-cultured at 25 ° C. for 2 days.
  • the cells and the culture supernatant were separated with Kiriyama filter paper (5A).
  • the pH of each culture supernatant was measured, treated according to the method described in saccharification of isoxanthohumol (1), and analyzed by HPLC. The results are shown in Table 4. IX in the medium was almost converted to IXG in 2 days of culturing, and there was no significant difference in the conversion rate when glucose was added at 0.2% or more.
  • Example 5 (Sample preparation of CGTase reaction) 100 mL of PD medium was placed in a 500 mL Sakaguchi flask, and 200 ⁇ L of 50 mg / mL isoxanthoflav (containing 90% or more of IX, manufactured by Hopstainer) ethanol solution was added. R. 100 ⁇ L of spore suspension of oryzae IAM6022, IAM6049, IAM6067, IAM6256 or NRRL395 was inoculated into each of the two Sakaguchi flasks and cultured at 25 ° C. for 3 days.
  • the reaction solution having a reaction time of 0 hours (at the start of the reaction) and the reaction solution after the reaction for 2 hours were analyzed by HPLC under the condition (A) of Example 1.
  • the reaction solution after the reaction for 2 hours is also referred to as a CGTase reaction solution below.
  • LC-MS analysis of the CGTase reaction solution was carried out under the following conditions, and the number of glucose transferred from the precise mass was determined.
  • LC equipment UHPLC manufactured by Shimadzu Corporation MS device: Waters Q-TOF Premier Column: COSMOCORE Packed Volume (oven temperature: 40 ° C)
  • Mobile phase A Water containing 0.1% formic acid
  • Mobile phase B Acetonitrile LC gradient conditions containing 0.1% formic acid are shown in Table 6 below.
  • FIG. 8C is an MS spectrum of IXG2 (corresponding to the peak to the left of IX
  • the water base is the result of adding IX, IXG or IXGs to water (black bar)
  • the malt extract base is the result of adding IX, IXG or IXGs to the malt extract (white bar).
  • FIGS. 12A, 12B and 12C show the residual rates of IX, IXG and IXGs stored at 45 ° C. in citrate buffer (pH 3)
  • FIG. 12A residual rate of IX
  • FIG. 12B residual rate of IXG
  • FIG. 12C Residual rate of IXGs
  • the vertical axis (residual rate) of the graphs of FIGS. 12A, 12B and 12C is the concentration of IX, XG or IXGs in the sample immediately after preparation (IX conversion) in the sample after storage when the concentration (IX conversion) is 100%. It is a relative value of the concentration (IX conversion) of IX, XG or IXGs.
  • Example 6 Preparation of immobilized cells and production of IXG and IXGs R.
  • Spores of oryzae IAM6256 were added to 30 mL of a 2% sodium alginate solution to 1 ⁇ 10 5 cells / mL, and the suspension was added dropwise to 150 mL of an ice-cooled 2% calcium chloride solution. , Gelled into beads and left as it was at 4 ° C. for 2 hours. After removing the solution, the beads were washed with sterile water several times. 200 mL of the medium shown in Table 7 was added to the beads, and the beads were cultured at 24 ° C. with gentle stirring for 2 days to obtain immobilized cells.

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Abstract

Le but de la présente invention est de fournir un glycoside de prénylflavonoïde dans lequel deux hexoses ou plus ont été liés à de l'isoxanthohumol ou de la 8-prénylnaringénine, un procédé de production de celui-ci, un procédé de production d'un monoglycoside de prénylflavonoïde dans lequel un hexose a été lié à de l'isoxanthohumol ou de la 8-prénylnaringénine, un procédé pour améliorer la solubilité dans l'eau des prénylflavonoïdes, un procédé pour réduire l'amertume des prénylflavonoïdes, un procédé pour améliorer la stabilité des prénylflavonoïdes, et similaires. La présente invention concerne un glycoside de prénylflavonoïde représenté par la formule générale (1). (Dans la formule générale (1), R représente un groupe méthyle ou un atome d'hydrogène, X représente un résidu d'hexose, et n représente un nombre entier de 2 à 10. Les 2 à 10 résidus d'hexose peuvent être identiques ou différents.)
PCT/JP2020/046738 2019-12-27 2020-12-15 Glycoside de prénylflavonoïde, son procédé de production, et procédé pour améliorer la solubilité dans l'eau de prénylflavonoïde Ceased WO2021131900A1 (fr)

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WO2022131129A1 (fr) * 2020-12-18 2022-06-23 サントリーホールディングス株式会社 Xanthohumol glycoside, procédé de production de xanthohumol glycoside et procédé d'amélioration de la solubilité dans l'eau du xanthohumol
WO2023044365A1 (fr) * 2021-09-17 2023-03-23 Doublerainbow Biosciences Inc. Utilisation de cyclodextrine pour améliorer la solubilité de substrats et augmenter l'efficacité de réaction de glycosylation enzymatique

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DATABASE UniProtKB 11 December 2013 (2013-12-11), ANONYMOUS: "RecName: Full=Isoflavone 7-0-glucosyltransferase 1; AltName: Full=UDP-glucose:isoflavone 7-0- glucosyltransferase ; Flags: Precursor.", XP055837942, retrieved from UniProtKB Database accession no. A6BM07 *
HYUN JUNG KIM, IK-SOO LEE: "Microbial Metabolism of the Prenylated Chalcone Xanthohumol", JOURNAL OF NATURAL PRODUCTS, AMERICAN CHEMICAL SOCIETY, US, vol. 69, no. 10, 1 October 2006 (2006-10-01), US , pages 1522 - 1524, XP055369850, ISSN: 0163-3864, DOI: 10.1021/np060310g *
SHALASHVILI K G; SUTIASHVILI M G; ALANIYA M D; KAVTARADZE N S; SKHIRTLADZE A V: "Flavanonol glycosides from leaves of Phellodendron lavallei introduced in Georgia", CHEMISTRY OF NATURAL COMPOUNDS., CONSULTANTS BUREAU, NEW YORK, NY, US, 1 February 2018 (2018-02-01), US , pages 263 - 266, XP018531643, ISSN: 0009-3130 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022131129A1 (fr) * 2020-12-18 2022-06-23 サントリーホールディングス株式会社 Xanthohumol glycoside, procédé de production de xanthohumol glycoside et procédé d'amélioration de la solubilité dans l'eau du xanthohumol
WO2023044365A1 (fr) * 2021-09-17 2023-03-23 Doublerainbow Biosciences Inc. Utilisation de cyclodextrine pour améliorer la solubilité de substrats et augmenter l'efficacité de réaction de glycosylation enzymatique

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