WO2023083226A1 - 一种α-红景天苷及其制备方法与应用 - Google Patents

一种α-红景天苷及其制备方法与应用 Download PDF

Info

Publication number
WO2023083226A1
WO2023083226A1 PCT/CN2022/130955 CN2022130955W WO2023083226A1 WO 2023083226 A1 WO2023083226 A1 WO 2023083226A1 CN 2022130955 W CN2022130955 W CN 2022130955W WO 2023083226 A1 WO2023083226 A1 WO 2023083226A1
Authority
WO
WIPO (PCT)
Prior art keywords
salidroside
preparation
free radicals
glycosyltransferase
enzyme
Prior art date
Application number
PCT/CN2022/130955
Other languages
English (en)
French (fr)
Inventor
广濑芳彦
上田诚
方诩
颜秉强
殷文成
刘大明
张爽
Original Assignee
山东恒鲁生物科技有限公司
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 山东恒鲁生物科技有限公司 filed Critical 山东恒鲁生物科技有限公司
Priority to EP22892020.3A priority Critical patent/EP4349843A4/en
Publication of WO2023083226A1 publication Critical patent/WO2023083226A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • 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.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • 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
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • 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
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/06—Arthrobacter

Definitions

  • the invention is required to be submitted to the China Patent Office on November 10, 2021, the application number is 202111328730.0, the invention name is "a kind of ⁇ -salidroside and its preparation method and application” and the Chinese patent is submitted on November 2, 2022
  • the priority of the Chinese patent application with application number 202211359878.5 and the title of the invention is "a kind of ⁇ -salidroside and its preparation method and application", the entire content of which is incorporated in the present invention by reference.
  • the invention relates to ⁇ -salidroside and its preparation method and application, in particular to a method for synthesizing ⁇ -salidroside by using glycosyltransferase, which belongs to the field of biotechnology.
  • Rhodiola is a precious traditional Chinese medicine.
  • the 2020 edition of "Chinese Pharmacopoeia” includes the dried roots and rhizomes of Rhodiola rosea (scientific name: Rhodiolacrenulata (Hook.f.et Thoms.) H.Ohba), produced in China, etc. It grows in areas with an altitude of about 4050 meters to 5400 meters.
  • Rhodiola tablets, Xinnaoxin capsules and other medicines prepared from Rhodiola rosea have the effects of promoting blood circulation and removing blood stasis, dredging channels and relieving pain.
  • the functions of health foods such as Rhodiola rosea capsules are to enhance immunity and relieve physical fatigue.
  • Salidroside is the effective active ingredient of the traditional Chinese medicine Rhodiola rosea. According to reports, salidroside has the functions of protecting cardiovascular and cerebrovascular, anti-fatigue, anti-depression, anti-aging, anti-hypoxia, anti-radiation, anti-tumor, immune regulation, whitening Physiological activities such as freckle removal.
  • Salidroside (plant salidroside) in the natural state is ⁇ -salidroside, and its glycosidic bond is ⁇ -type. Its chemical formula is as follows:
  • the glycosyltransferase in this patent document is derived from the plant Rhodiola sachalinensis
  • the glycosyltransferase gene ugt73b6 of the glycosyltransferase gene ugt73b6 was randomly mutated by error-PCR to obtain the glycosyltransferase mutant gene ugt73b6 MK that catalyzes the synthesis of gastrodin or salidroside, specifically the methionine at position 264 of the glycosyltransferase ugt73b6 Mutation to lysine;
  • Chinese patent document CN108220264A application number 201611200315.6 discloses a glycosyltransferase and its application in the biosynthesis of ⁇ -salidroside.
  • the glycosyltransferase in this patent document is derived from pseudo The UDP-glycosyltransferase of Arabidopsis thaliana (Genbank GI: 66774038); Chinese patent document CN106543243 (application number 201610979792.0) discloses a salidroside derivative and its preparation method.
  • the sugar used in the patent document is a cyclodextrin glucosyltransferase derived from Bacillus subtilis ATCC6699;
  • Chinese patent document CN106543244A (application number 201610979793.5) discloses the use of galactosidase to prepare ⁇ -galactosyl salidroside and its derivatives
  • the galactosidase used in this patent document is a transglycosyl ⁇ -galactosidase derived from Enterobacter cloacae;
  • Chinese patent document CN109321615A (application number 201811363071.2) discloses a starch degrading enzyme with high transglycosylation activity The application of Bacillus in the non-aqueous phase biosynthesis of salidroside.
  • the glucose glycosyltransferase in this patent document is derived from Leuconostoc enterococci; Butyl- ⁇ -D-glucoside carries out the method for synthesizing salidroside by transglycoside reaction, and the ⁇ -glucosidase used in this patent document is almond ⁇ -glucosidase, Agrobacterium (Agrobacterium) ⁇ -glucosidase Any one of , white-rot fungus (Phanerochaetechrysosporium) ⁇ -glucosidase, and marine Thermotoga maritima (Thermotoga maritima) ⁇ -glucosidase.
  • the above salidrosides prepared by artificial means are all ⁇ -salidrosides.
  • Purpose of the invention to provide an excellent form of salidroside, or to provide a salidroside preparation process suitable for industrial production.
  • the applicant conducted research on salidroside, and unexpectedly found that the synthesized glycoside was different from the existing ⁇ -salidroside, which was identified as ⁇ -salidroside and ⁇ -salidroside Isomers of sedroside.
  • the invention also provides a preparation method and application of ⁇ -salidroside.
  • the present invention provides ⁇ -salidroside, the structural formula of which is formula I, which has an ⁇ -glucosidic bond on the alcoholic hydroxyl group of tyrosol.
  • the ⁇ -salidroside has a molecular weight of 300.23 through LC-MS analysis.
  • the ⁇ -salidroside of the present invention is an isomer of the natural ⁇ -salidroside, and its chemical name is: 2-(4-hydroxyphenyl)ethyl- ⁇ -D-glucoside.
  • the ⁇ -salidroside of the present invention is superior to ⁇ -salidroside (natural salidroside) in anti-oxidation and free radical scavenging abilities.
  • the present invention further provides the application of the ⁇ -salidroside in the preparation of products with the function of scavenging free radicals.
  • the free radicals include DPPH free radicals and hydroxyl free radicals.
  • the application of the ⁇ -salidroside in the preparation of a product with the function of scavenging free radicals further preferably, the product is a cosmetic with the function of scavenging free radicals.
  • the application of the ⁇ -salidroside in the preparation of a product with the function of scavenging free radicals further preferably, the product is an anti-fatigue health product.
  • the present invention further provides a composition containing ⁇ -salidroside.
  • the composition has the function of scavenging free radicals, and the free radicals include DPPH free radicals and hydroxyl free radicals.
  • the cosmetic composition containing ⁇ -salidroside is selected from water, oil, emulsion, gel, paste, or others. It can be produced according to the existing technical specifications of cosmetics, and there is no special requirement.
  • the health product composition containing ⁇ -salidroside can be in the form of capsules, tablets, pastes, liquids, or others. It can be produced according to the existing technical specifications of health care products, and there is no special requirement.
  • the present invention further provides a preparation method of the ⁇ -salidroside, which is prepared by dissolving tyrosol and a glycosyl donor in a buffer solution, adding glycosyltransferase, and carrying out a glycosylation reaction in a reaction system.
  • the glycosyltransferase has strict specificity and chiral catalytic ability, and the glycosyltransferase used in the present invention can specifically generate ⁇ -salidroside, which is the same as that recorded in the prior art
  • the glycosyltransferases that generate ⁇ -salidroside are different.
  • the glycosyl donor is one or more of maltose, maltotriose, glucose, fructose, starch, water-soluble starch, and dextrin.
  • the concentration of tyrosol is 5-400 mg/mL, more preferably 5-100 mg/mL; the concentration of the glycosyl donor is 0.5-30 times the concentration of tyrosol , more preferably 2 to 10 times.
  • the buffer solution is a phosphate buffer solution, acetate buffer solution or Good's buffers buffer solution, the concentration of the buffer solution is 0.01M-0.5M, and the pH of the buffer solution is 5-10.
  • the glycosyltransferase is a commercially available glycosyltransferase enzyme preparation or a crude glycosyltransferase enzyme solution after cultured and purified microorganisms.
  • the commercially available glycosyltransferase enzyme preparations are derived from Amano Enzyme Co., Ltd. and Novozymes, including L-glycosyltransferase, amylase, aromatase, cellulase, cycloglucose Enzymes that can produce or transfer ⁇ -glycosides such as glycantransferase and glycosidyltransferase.
  • the microorganisms include Arthrobacter sp., Aspergillus sp., Paenibacillus sp., Geobacillus sp., Thermoanaerobacter sp. ), Aerribacillus, Trichoderma (Trichodermasp.), Bacillus (Bacillussp.) or Penicilliumsp. Microorganisms of the genus Arthrobacter sp.
  • the microorganisms are Aspergillus niger and Arthrobacter sp.M-238.
  • the cleavage auxiliary solvent dimethoxysulfoxide (DMSO) or dimethylacetamide (DMA) that does not affect the glycosylation reaction can also be added to the reaction system, or the cleavage auxiliary solvent that does not affect the enzymatic reaction Surfactant Tween-20 or Span.
  • DMSO dimethoxysulfoxide
  • DMA dimethylacetamide
  • the temperature of the glycosylation reaction is 15-50° C., more preferably 30-50° C.; the time is 1-100 hours, more preferably 10-50 hours.
  • the preparation of the ⁇ -salidroside can also be carried out in an organic solvent, as long as the organic solvent used in the reaction is the type and concentration range that does not affect the reaction, it specifically includes methanol, DMSO, 2-propane One solvent or a mixed solvent of two or more of alcohol and ethanol; and the above buffer solution is used to adjust the pH of the reaction system to 5-10.
  • the organic solvent used in the reaction is the type and concentration range that does not affect the reaction, it specifically includes methanol, DMSO, 2-propane One solvent or a mixed solvent of two or more of alcohol and ethanol; and the above buffer solution is used to adjust the pH of the reaction system to 5-10.
  • the present invention further provides a method for scavenging free radicals, the method comprising administering an effective dose of ⁇ -salidroside or a composition comprising ⁇ -salidroside to a subject.
  • the free radicals include DPPH free radicals and hydroxyl free radicals.
  • the subject refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, most preferably a human.
  • the effective dose refers to the amount of active compound or composition or medicament including ⁇ -salidroside, which can cause the tissue system, animal or human being pursued by researchers, veterinarians, doctors or other medical personnel.
  • Biological or medical responses which include scavenging or partial scavenging of free radicals.
  • the present invention provides a new type of salidroside, that is, ⁇ -salidroside, which has an ⁇ -glucosidic bond on the alcoholic hydroxyl group of tyrosol.
  • ⁇ -salidroside which has an ⁇ -glucosidic bond on the alcoholic hydroxyl group of tyrosol.
  • the activity of ⁇ -salidroside is better than that of ⁇ -salidroside, which can be added to cosmetics or anti-fatigue health products as new functional ingredients; ⁇ -rhodiola
  • the development of new functions of glycosides is under further study.
  • the present invention also provides a preparation method for ⁇ -salidroside.
  • the conversion rate of tyrosol into ⁇ -salidroside is above 2%;
  • the conversion rate of tyrosol into ⁇ -salidroside is 20%, which greatly improves the conversion rate of glycosylation reaction and is suitable for industrial production.
  • the preparation method of ⁇ -salidroside in the present invention uses cheap starch as a glycosyl donor, tyrosol as a substrate, and under the action of a glycosyltransferase to synthesize ⁇ -salidroside, which is suitable for industrial production , and low cost.
  • Fig. 1 After separating and purifying the obtained enzyme-catalyzed reaction solution of embodiment 1, obtain the TLC spectrogram of sample; Among the figure, from left to right are respectively TLC plate 1, TLC plate 2; TLC plate 1 is from left to right Right are lane 1 and lane 2 respectively.
  • Fig. 4 After separating and purifying the enzyme-catalyzed reaction solution obtained in embodiment 1, obtain the HPLC spectrogram of the sample.
  • the ⁇ -salidroside of the present invention is an isomer of the natural ⁇ -salidroside, the chemical name is: 2-(4-hydroxyphenyl) ethyl- ⁇ -D-glucoside, and the structural formula is as follows :
  • the preparation method of ⁇ -salidroside in the present invention uses tyrosol as a substrate to realize glycosylation under the action of glycosyltransferases derived from microorganisms or commercialized glycosyltransferases.
  • Glycosyltransferases use cheap sugar sources, such as starch, dextrin, and maltose, for glycosylation, and their glycosides exist in the form of ⁇ -linkages.
  • Enzyme preparations include L-glycosyltransferase, amylase, aromatase enzymes, cellulases, cycloglucanases, and glycosyltransferases, etc.
  • the method of using the glycosyltransferase enzyme preparation includes adding the enzyme preparation into the reaction system in the form of powder or liquid, or fixing the enzyme preparation on the resin to make an immobilized enzyme preparation and adding it to the reaction system, and the immobilized enzyme preparation can be used repeatedly.
  • Glycosyltransferase enzyme preparations can use commercially available commercial enzyme preparations, or produce the required enzyme liquid through microbial culture, and then use fresh enzyme liquid for glycosylation modification.
  • the microbial source of glycosyltransferase can be Arthrobacter sp., Aspergillus sp., Paenibacillus sp., Geobacillus sp., Thermoanaerobacter sp. .), Aerribacillus, Trichoderma sp., Bacillus sp.
  • glycosyltransferase is isolated and purified from microbial culture As obtained, compositions comprising glycosyltransferases, purified glycosyltransferases, compositions containing ⁇ -glycosyltransferases, purified ⁇ -glycosyltransferases, or immobilized ⁇ -sugars bound to a carrier base transferase.
  • Preparation of enzyme solution the method of isolating and purifying glycosyltransferase from microbial culture is to use ultrasonic waves or glass beads to crush microbial cells, and then perform enzyme purification on the crushed bacteria or supernatant, including sulfate precipitation , ion exchange column chromatography, chelate affinity chromatography or gel filtration column chromatography, etc., and a combination of the above methods.
  • Enzyme immobilization There are no restrictions on the carrier for immobilizing microorganisms and the above-mentioned glycosyltransferases, such as diatomaceous earth, gypsum soil, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics, ceramic powder and other inorganic carriers , polyvinyl alcohol, polypropylene, acrylamide, carrageenan (carrageenan), chitosan, ion exchange resin, hydrophobic adsorption resin, chelating resin, synthetic adsorption resin and other organic polymers.
  • glycosyltransferases such as diatomaceous earth, gypsum soil, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics, ceramic powder and other inorganic carriers , polyvinyl alcohol, polypropylene, acrylamide, carrageenan (carrageenan), chito
  • Methods for immobilizing microorganisms, enzymes, etc. on carriers including adsorption methods, ionic bonding methods, covalent bonding methods, and biochemical specific binding methods.
  • the immobilized glycosyltransferase can be reused in a batch system or a continuous system to produce glycoside compounds.
  • the raw materials for glycosylation generally include high-molecular-weight sugar derivatives such as starch, water-soluble starch, and dextrin, and low-molecular-weight sugar derivatives such as maltose, maltotriose, glucose, and fructose.
  • high-molecular-weight sugar derivatives such as starch, water-soluble starch, and dextrin
  • low-molecular-weight sugar derivatives such as maltose, maltotriose, glucose, and fructose.
  • the preparation method of ⁇ -salidroside in the present invention is to add glycosyltransferase or microbial culture into the mixture of tyrosol and glycosyl donor, and carry out glycosyltransferase reaction.
  • a cleavage auxiliary solvent that does not affect the enzymatic reaction can be added to the enzyme reaction solution, such as organic solvents such as dimethoxysulfoxide (DMSO) or dimethylacetamide (DMA), or a surfactant that does not affect the enzymatic reaction, Examples include reagents such as Tween-20 and Span.
  • the amount of glycosyltransferases and reaction conditions have a great impact on production efficiency, so it is very important to choose the appropriate reaction conditions such as enzyme amount and reaction time .
  • the amount of enzyme added in the reaction should be controlled within 1 to 100 hours to complete the reaction.
  • the pH of the reaction system is preferably 5-10, and the reaction temperature is 15-50°C.
  • the concentration of tyrosol in the enzyme reaction liquid can usually be 5-400 mg/mL, and the preferred concentration is 20-300 mg/mL; , a concentration range of 0.5 to 30 times is preferred.
  • the reaction between tyrosol and glycosyl donor can be carried out in the presence of a solvent.
  • the solvent used in the reaction can be used as long as it does not affect the type and concentration range of the reaction. Specifically, it can include methanol, DMSO, 2-propanol, ethanol, etc. reagent. These solvents may be used alone or in combination of two or more.
  • the glycosylation reaction in the present invention can be carried out by contacting a culture of a microorganism in the presence of a glycosyl donor.
  • Contacting the culture of microorganisms refers to adding tyrosol and glycosyl donors to the system containing the culture of microorganisms to react, for example, allowing the solution of tyrosol and glycosyl donors to flow down and contact the immobilized microorganisms to complete the tyrosol Glycosylation.
  • microorganisms may or may not be present in the medium.
  • the "culture of microorganisms” includes dried cells of microorganisms and disrupted products thereof, free glycosyltransferases and immobilized glycosyltransferases.
  • the medium contains glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, galactose, starch, dextrin, molasses, sorbitol, glycerol and other sugars, these sugars can be used as sugar base donor.
  • the enzyme used in the present invention can be inactivated by heating or changing the pH value, thereby stopping the enzyme-catalyzed reaction.
  • the substrate tyrosol and glycosylated compounds have different solubility in water, they can usually be separated by extraction operation using the equilibrium distribution relationship of the liquid (organic phase)-liquid (water phase) interface.
  • Tyrosol derivative glycosylated compounds can be dissolved in a more hydrophilic solvent, and this hydrophilic solution can be further separated and purified by ion exchange column chromatography, gel filtration chromatography or hydrophobic chromatography, which can be easily easily obtain high-purity glycoside refined products.
  • the mixture of the above glycosides, the hydrolyzate of the mixture, the enzyme inactivation product of the mixture, the purified product of the mixture, and its dry powder products can be used in food, beverage and cosmetics containing glycosides, and can also be used as food, special medical food, health care products or pharmaceutical ingredients, used in food and beverages, cosmetics, special medical food, health care products or pharmaceuticals.
  • tyrosol As the substrate of enzyme-catalyzed reaction, tyrosol has benzene ring in its molecular structure, so it has ultraviolet absorption, which can be detected by ultraviolet detector at 254nm wavelength. Thin-layer chromatography is a simple and convenient method for the analysis of enzyme reaction solution, and the separation conditions are studied.
  • High performance liquid chromatography Hitachi HPLC 5440 chromatograph, column: Unison UK-C18 (150 ⁇ 4.6mm, 3 ⁇ m), detector: photodiode array (DAD 280nm), detection wavelength: 280nm, injection volume: 10 ⁇ L, Flow rate: 0.5mL/min, column temperature: 30°C, mobile phase: gradient elution of acetonitrile: 0.1% formic acid aqueous solution, the elution gradient (v/v) is shown in Table 1.
  • Embodiment 6 Glycosylation experiment of glycosyltransferase (Glucose trasferase-L)
  • the enzyme-catalyzed reaction solution obtained in Example 1 was separated and purified, and analyzed by TLC, HPLC, LC-MS and NMR.
  • hydrophobic polymer resin HP-20, macroporous adsorption resin, Solarbio brand
  • ODS silica gel resin
  • the HPLC analyzer is a Hitachi HPLC 5440 chromatograph
  • the chromatographic column is a C18 column (Unison UK-C18, 150mm ⁇ 4.6mm, 3 ⁇ m)
  • the column temperature is 30°C
  • the mobile phase is acetonitrile: 0.1% aqueous formic acid (v/v) gradient
  • a photodiode array detector with a detection wavelength of UV 280nm.
  • HPLC analysis was performed on the product after the enzyme-catalyzed reaction solution obtained in Example 1 was separated and purified in this example, and the HPLC spectrum is shown in Figure 4 for details.
  • the HPLC retention time of salidroside standard substance (molecular formula: C 14 H 20 O 7 , molecular weight: 300.23) is 9.72min, and through LC-MS analysis, the measured value of its molecular ion peak [M-1] is 299.11, determined It is the [MH]+ peak of tyrosol glycoside (molecular weight 300), see Figure 6 for details.
  • Salidroside standard substance ⁇ -salidroside
  • Example 1 The product of Example 1 is ⁇ -glucose glycosylated.
  • the two are the same point, from the LC-MS analysis, the two molecular weights are the same, from the HPLC analysis, the two are two substances, combined with the ⁇ bond feature in the NMR spectrum (different from the natural ⁇ bond ), confirm that the substance is ⁇ -salidroside, which is an isomer of ⁇ -salidroside (natural salidroside), and its structural formula is as shown in formula I:
  • the transformation rate that tyrosol is converted into ⁇ -salidroside is respectively 2% and 3%; - Salidroside, but in very low amounts.
  • Inoculate Arthrobacter sp.M-238 (NITE preservation number AP-02396) cultivated at 25°C for 2 to 3 days on the above agar medium into the liquid medium described in step 2, 2 inoculation loops, 25°C , shake at 160rpm for 42 hours; after cultivation, remove the bacterial cells by centrifugation, salt out the supernatant with ammonium sulfate, desalt, use anion exchange resin to fractionate the glycosyltransferase to obtain a crude enzyme solution.
  • ⁇ -salidroside was confirmed by TLC, HPLC and LC-MS analysis; the conversion rate of tyrosol into ⁇ -salidroside was about 20% by HPLC analysis.
  • the present invention utilizes glycosyltransferase, especially glycosyltransferase derived from Arthrobacter sp., to carry out glycosylation modification on tyrosol derivatives.
  • glycosyltransferase especially glycosyltransferase derived from Arthrobacter sp.
  • Glycosylated glycosides not only have the original properties of tyrosol derivatives, but also further enhance their physiological functions, thereby increasing the development value, and can be used as raw materials for cosmetics, health products, and medicines.
  • Salvianolic acids are the donors of phenolic hydroxyl groups and have the structural basis of antioxidant activity.
  • DPPH 1,1-diphenyl-2-trinitrophenylhydrazine, alias alias 1,1-diphenyl-2-pichydrazyl, (free radical)] free radical scavenging reaction was used to study ⁇ - Scavenging efficiency of salidroside and ⁇ -salidroside on DPPH free radicals.
  • ⁇ -salidroside solution prepared as a 500 mg/L aqueous solution according to conventional methods.
  • the DPPH free radical scavenging rate was calculated according to the following formula, and the results are recorded in Table 3.
  • Hydroxy free radical scavenging activity is an important index to measure the antioxidant capacity of substances.
  • the analysis and testing methods for hydroxyl radicals mainly include high performance liquid chromatography, chemiluminescence, fluorescence analysis, spectrophotometry and so on.
  • the most commonly used method for determining the scavenging ability of hydroxyl radicals is to measure the decrease in the absorbance of the sample to inhibit the chromogenic substance o-phenanthroline by spectrophotometry, thereby reflecting the ability of the sample to scavenge hydroxyl radicals.
  • the measurement principle is: H 2 O 2 /Fe 2+ generates hydroxyl radicals through the Fenton reaction, and oxidizes Fe 2+ in the o-phenanthroline-Fe 2+ aqueous solution to Fe 3+ , resulting in a decrease in the absorbance at 536nm, and a decrease in the absorbance of the sample at 536nm
  • the degree of inhibition of the rate reflects the ability of the sample to scavenge hydroxyl radicals.
  • the degree of inhibition of the absorbance decrease rate reflects the ability of the sample to scavenge hydroxyl radicals.
  • the production of hydroxyl radicals can be indirectly determined by measuring the change in the absorbance value of the indicator.
  • FeSO 4 solution Prepare 1.5mM and 5mM FeSO 4 aqueous solutions according to conventional methods.
  • Phenanthroline 0.75 mM and 3 mM aqueous solutions were prepared respectively according to conventional methods.
  • ⁇ -salidroside solution Prepare 1 mg/mL and 4 mg/mL aqueous solutions respectively according to conventional methods.
  • ⁇ -salidroside solution 1 mg/mL and 4 mg/mL aqueous solutions were prepared respectively according to conventional methods.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Birds (AREA)
  • Toxicology (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gerontology & Geriatric Medicine (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

本发明涉及一种α-红景天苷及其制备方法与应用。本发明提供了一种新型的红景天苷，即α-红景天苷，与已知的β-红景天苷相比，在清除DPPH自由基和羟基自由基方面，α-红景天苷的活性优于β-红景天苷，可作为新的功能性成分添加到化妆品或抗疲劳保健品中。本发明所述α-红景天苷的制备方法，以廉价的淀粉等为糖基供体、酪醇为底物，在糖基转移酶作用下，合成α-红景天苷，所述糖基转移酶具有严格的专一性和手性催化能力，可以专一性的生成α-红景天苷。

Description

一种α-红景天苷及其制备方法与应用
本发明要求于2021年11月10日提交中国专利局、申请号为202111328730.0、发明名称为“一种α-红景天苷及其制备方法与应用”以及于2022年11月2日提交中国专利局、申请号为202211359878.5、发明名称为“一种α-红景天苷及其制备方法与应用”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明涉及一种α-红景天苷及其制备方法与应用，特别涉及一种利用糖基转移酶合成α-红景天苷的方法，属于生物技术领域。
背景技术
红景天是一味珍贵中药，2020版《中国药典》收录为景天科大花红景天的干燥根和根茎(学名：Rhodiolacrenulata(Hook.f.et Thoms.)H.Ohba)，产于中国西藏等地，生长于海拔4050米至5400米左右的地区。由红景天制备的红景天片、心脑欣胶囊等药品具有活血化瘀，通脉止痛的功效，红景天胶囊等保健食品的功能为增强免疫力、缓解体力疲劳。红景天苷是中药红景天的有效活性成分，据报道，红景天苷具有保护心脑血管、抗疲劳、抗抑郁、抗衰老、抗缺氧、抗辐射、抗肿瘤、免疫调节、美白祛斑等生理活性。天然状态下的红景天苷(植物红景天苷)为β-红景天苷，其糖苷键为β型。其化学式如下：
Figure PCTCN2022130955-appb-000001
β-红景天苷的H-NMR谱：1H-NMR(CD 3OD)，δppm:7.06(2H，d,J＝8.2Hz,H-2和H-6),6.70(2H,d,J＝8.2Hz,H-3和H-5),2.82(2H,t,J＝7.2Hz,H-α),3.70和4.04(d，分别表示β-CH2上的一个氢)，4.28(1H,d,糖端基质子)，3.68和3.84(d,分别表示6'-CH2上的两个氢)，3.1～3.4(4H,m,为H-2’、H-3'、H-4'和H-5'的四个氢，其中δ3.24ppm为H-2'，δ3.27ppm为H-4'，δ3.25～3.44ppm为H-3'和H-5')。
β-红景天苷的C-NMR谱：13C-NMR(CD 3OD)，δpppm:36.37(α-C),72.09(β-C)，62.75(C-6'),78.10(C-5'),71.64(C-4'),75.11(C-2'),77.96(C-3'),104.37(C-1'),78.10(C-5'),71.64(C-4'),75.11(C-2'),77.96(C-3'),104.37(C-1'),130.93(C-1)，156.81(C-4)，116.11(C-3和C-5)，130.93(C-2和C-6)。
由于植物红景天资源有限，一些学者对红景天苷的制备开展了研究。红景天苷的生产方法包括植物提取法、酶法、发酵法、化学合成法等。中国专利文献CN104774815A(申请号201510160496.3)公开了一种能够催化天麻素或红景天苷合成的糖基转移酶，该专利文献中的糖基转移酶是来源于植物库页红景天(Rhodiolasachalinensis)的糖基转移酶基因ugt73b6经error-PCR随机突变后得到催化天麻素或红景天苷合成的糖基转移酶突变基因 ugt73b6 MK，具体的是糖基转移酶ugt73b6第264位的甲硫氨酸突变为赖氨酸；中国专利文献CN108220264A(申请号201611200315.6)公开了一种糖基转移酶及在生物合成β-红景天苷中的应用，该专利文献中的糖基转移酶是来源于拟南芥(Arabidopsis thaliana)的UDP-糖基转移酶(Genbank GI：66774038)；中国专利文献CN106543243(申请号201610979792.0)公开了一种红景天苷衍生物及其制备方法，该专利文献使用的糖基转移酶是来源于枯草芽孢杆菌ATCC6699的环糊精葡萄糖基转移酶；中国专利文献CN106543244A(申请号201610979793.5)公开了使用半乳糖苷酶制备β-半乳糖型红景天苷及其衍生物，该专利文献使用的半乳糖苷酶是来源于阴沟肠杆菌(Enterobacter cloacae)的转糖基β-半乳糖苷酶；中国专利文献CN109321615A(申请号201811363071.2)公开了一种高糖基转移活性解淀粉芽孢杆菌在非水相中生物合成红景天苷的应用，该专利文献中是以解淀粉芽孢杆菌FJ18的湿菌体催化糖基化反应；中国专利文献CN102174619A(申请号201110005088.2)公开了一种葡萄糖糖基转移酶催化合成红景天苷或类似物的方法，该专利文献的葡萄糖糖基转移酶来源于肠膜明串珠菌；中国专利文献CN107937457A(申请号201711141952.5)公开了一种酶催化正丁基-β-D-葡萄糖苷进行转糖苷反应合成红景天苷的方法，该专利文献中使用的β-葡萄糖苷酶为杏仁β-葡萄糖苷酶、农杆菌(Agrobacterium)β-葡萄糖苷酶、白腐真菌(Phanerochaetechrysosporium)β-葡萄糖苷酶、海栖热袍菌(Thermotoga maritima)β-葡萄糖苷酶中的任一种。以上通过人工手段制备的红景天苷均为β-红景天苷。
发明内容
发明目的：提供红景天苷的优异存在形式，或提供适合工业化生产的红景天苷制备工艺。
处于对产品迭代的需求，申请人对红景天苷进行了研究，意外发现合成的糖苷与现有的β-红景天苷存在差异，经鉴定为α-红景天苷，为β-红景天苷的同分异构体。另外，本发明还提供了α-红景天苷的制备方法及应用。
现有技术尚没有关于β-红景天苷同分异构体α-红景天苷的报道，更不会有对于α-红景天苷的合成方法和生物活性方面的研究。
技术方案：
一方面，本发明提供了一种α-红景天苷，其结构式如式I，其在酪醇的醇羟基上具有α-葡萄糖糖苷键。
Figure PCTCN2022130955-appb-000002
根据本发明优选的，所述α-红景天苷经LC-MS分析，其分子量为300.23。
根据本发明优选的，所述α-红景天苷的H-NMR谱：1H NMR(600MHz,DMSO):δ2.74(t,2H),3.05-3.06(m,1H),3.19(m,1H),3.31(m,2H),3.40-3.50(m,3H),3.71-3.72(m,1H),4.42-4.35(m,1H),4.59-4.60(m,1H),4.68(d,J＝3.6Hz,1H:β-H),4.74(m,1H),4.73-4.84(m,1H),6.67(d,J＝7.8Hz,2H:H-2,H-6),7.04(d,J＝7.8Hz,2H:H-3,H-5),9.1(s,1H:OH)。
所述α-红景天苷的C-NMR谱：13C NMR(151MHz,DMSO):δ35.2,61.4,68.8,70.7,72.4,73.3,73.7,99.0,115.5,129.3,130.2,156.0。
本发明所述α-红景天苷为天然β-红景天苷的同分异构体，化学名称为：2-(4-羟基苯基)乙基-α-D-葡萄糖苷。
本发明所述α-红景天苷，抗氧化和清除自由基能力均优于β-红景天苷(天然红景天苷)。
本发明进一步提供了所述α-红景天苷在制备具有清除自由基功能的产品中的应用。
所述α-红景天苷在制备具有清除自由基功能的产品中的应用，优选的，所述自由基包括DPPH自由基和羟基自由基。
所述α-红景天苷在制备具有清除自由基功能的产品中的应用，进一步优选的，所述产品为具有清除自由基功能的化妆品。
所述α-红景天苷在制备具有清除自由基功能的产品中的应用，进一步优选的，所述产品为抗疲劳保健品。
本发明进一步提供了一种组合物，含有α-红景天苷。所述组合物具有清除自由基的功能，所述自由基包括DPPH自由基和羟基自由基。
含有α-红景天苷的化妆品组合物，其品类选自水剂类、油剂类、乳剂类、凝胶类产品、膏状产品，或其他。可以按现有化妆品技术规范生产，没有特别的要求。
含有α-红景天苷的保健品组合物，可以是胶囊状、片状、膏状、水剂，或其他。可以按现有保健品技术规范生产，没有特别的要求。
本发明进一步提供了所述α-红景天苷的制备方法，是将酪醇和糖基供体溶解于缓冲溶液中，再加入糖基转移酶，在反应体系中进行糖基化反应制备得到。
本发明中，所述糖基转移酶具有严格的专一性和手性催化能力，本发明中使用的糖基转移酶可以专一性的生成α-红景天苷，与现有技术中记载的生成β-红景天苷的糖基转移酶不同。
根据本发明优选的，所述糖基供体为麦芽糖、麦芽三糖、葡萄糖、果糖、淀粉、水溶性淀粉、糊精中的一种或几种。
根据本发明优选的，所述反应体系中，酪醇的浓度为5～400mg/mL，更优选的浓度为5～100mg/mL；所述糖基供体浓度为酪醇浓度的0.5～30倍，更优选为2～10倍。
根据本发明优选的，所述缓冲溶液为磷酸盐缓冲溶液、醋酸盐缓冲溶液或Good's buffers缓冲溶液，缓冲溶液的浓度为0.01M～0.5M，缓冲溶液的pH为5～10。
根据本发明优选的，所述糖基转移酶为市售商品化糖基转移酶酶制剂或微生物经培养纯化后的糖基转移酶粗酶液。
进一步优选的，所述市售商品化糖基转移酶酶制剂来源于天野酶制剂株式会社和诺维信制剂公司，包括L-糖基转移酶、淀粉酶、芳香酶、纤维素酶、环葡聚糖转移酶和糖苷转移酶等能产生或转移α-糖苷的酶。
进一步优选的，所述微生物包括节杆菌属(Arthrobactersp.)、曲霉属(Aspergillussp.)、类芽孢杆菌属(Paenibacillussp.)、地芽胞杆菌属(Geobacillussp.)、栖热厌氧杆菌属(Thermoanaerobactersp.)、Aerribacillus 菌属、木霉菌属(Trichodermasp.)、芽孢杆菌属(Bacillussp.)或青霉菌属(Penicilliumsp.)的微生物；更进一步优选的，所述微生物为曲霉属(Aspergillussp.)和节杆菌属(Arthrobactersp.)的微生物。
更进一步优选的，所述微生物为Aspergillus niger和Arthrobacter sp.M-238。
根据本发明优选的，所述反应体系中还可以加入不影响糖基化反应的裂解辅助溶剂二甲氧基亚砜(DMSO)或二甲基乙酰胺(DMA)，或不影响酶促反应的表面活性剂吐温(Tween-20)或司班(Span)。
根据本发明优选的，所述糖基化反应的温度为15～50℃，更优选为30～50℃；时间为1～100小时，更优选为10-50小时。
本发明中，所述α-红景天苷的制备也可以在有机溶剂中进行，反应中使用的有机溶剂只要是不影响反应的种类和浓度范围即可，具体包括甲醇、DMSO、2-丙醇、乙醇中的一种溶剂或两种以上的混合溶剂；并采用上述缓冲溶液调节反应体系pH为5～10。
本发明进一步提供一种清除自由基的方法，所述方法包括对受试者施用有效剂量的α-红景天苷或包含α-红景天苷的组合物。所述自由基包括DPPH自由基和羟基自由基。
所述受试者是指已经是治疗、观察或实验的对象的动物，优选指哺乳动物，最优选指人。
所述有效剂量是指包括α-红景天苷在内的活性化合物或组合物或药剂的量，该量可引起研究者、兽医、医生或其他医疗人员所追求的组织系统、动物或人的生物学或医学响应，这包括清除或部分清除自由基。
有益效果：
本发明提供了一种新型的红景天苷，即α-红景天苷，是在酪醇的醇羟基上具有α-葡萄糖糖苷键，与已知的β-红景天苷相比，在清除DPPH自由基和羟基自由基方面，α-红景天苷的活性优于β-红景天苷，可作为新的功能性成分添加入化妆品中或抗疲劳保健品中；α-红景天苷的新功能开发在进一步研究中。
本发明还提供了α-红景天苷的制备方法，当糖基转移酶来源于曲霉属(Aspergillus sp.)时，酪醇转化为α-红景天苷的转化率为2％以上；当糖基转移酶来源于节杆菌属(Arthrobacter sp.)时，酪醇转化为α-红景天苷的转化率为20％，极大的提高了糖基化反应的转化率，适合工业化生产。
本发明所述α-红景天苷的制备方法，以廉价的淀粉等为糖基供体、酪醇为底物，在糖基转移酶作用下，合成α-红景天苷，适合工业化生产，且成本低廉。
附图说明
图1.实施例10中，分离纯化实施例1所得酶催化反应溶液后，获得样品的TLC谱图；图中，从左至右分别为TLC板1、TLC板2；TLC板1从左到右分别为泳道1、泳道2。
图2.β-红景天苷标准品的HPLC图谱。
图3.酪醇标准品的HPLC图谱。
图4.实施例10中，分离纯化实施例1所得酶催化反应溶液后，获得样品的HPLC谱图。
图5.实施例10中，分离纯化实施例1所得酶催化反应溶液后，获得样品的HPLC谱图中rt＝10.16min物质的LC-MS谱图。
图6.β-红景天苷标准品的LC-MS图谱。
图7.实施例10中，分离纯化实施例1所得酶催化反应溶液后，获得样品的HPLC谱图中rt＝10.16min物质的H-NMR谱图。
图8.实施例10中，分离纯化实施例1所得酶催化反应溶液后，获得样品的HPLC谱图中rt＝10.16min物质的C-NMR谱图。
具体实施方式
下面结合具体实施例对本发明作进一步说明，所举实施例不作为对本发明的限定，本发明所保护范围不限于此。
本发明所述α-红景天苷为天然β-红景天苷的同分异构体，化学名称为：2-(4-羟基苯基)乙基-α-D-葡萄糖苷，结构式如下：
Figure PCTCN2022130955-appb-000003
本发明所述α-红景天苷的制备方法，以酪醇为底物，在微生物来源的糖基转移酶或商品化的糖基转移酶的作用下实现糖基化。
糖基转移酶利用廉价的糖源，如淀粉、糊精和麦芽糖等进行糖基化，其糖苷以α-键的形式存在。
具有实现形成α糖苷键的糖基化作用的市售商品化糖基转移酶酶制剂，来源于天野酶制剂株式会社以及诺维信公司，酶制剂包括L-糖基转移酶、淀粉酶、芳香酶、纤维素酶、环葡聚糖转移酶和糖苷转移酶等。
糖基转移酶酶制剂的使用方法包括以粉末或液体的形式加入反应体系中，也可将酶制剂固定在树脂上制成固定化酶制剂加入到反应体系中，固定化酶制剂可以重复使用。
糖基转移酶酶制剂可使用市售商品化酶制剂，也可通过微生物培养自行生产需要的酶液，然后利用新鲜酶液进行糖基化修饰。
糖基转移酶的微生物来源可以是节杆菌属(Arthrobactersp.)、曲霉属(Aspergillussp.)、类芽孢杆菌属(Paenibacillussp.)、地芽胞杆菌属(Geobacillussp.)、栖热厌氧杆菌属(Thermoanaerobactersp.)、Aerribacillus菌属、木霉菌属(Trichodermasp.)、芽孢杆菌属(Bacillussp.)或青霉菌属(Penicilliumsp.)等，不受特别限制，糖基转移酶从微生物培养物中经分离和纯化得到即可，包括糖基转移酶的组合物、纯化的糖基转移酶、其中含α-糖基转移酶的组合物、纯化的α-糖基转移酶或与载体结合的固定化α-糖基转移酶。
制备酶液：从微生物培养物中分离和纯化糖基转移酶的方法是使用超声波或玻璃珠粉碎微生物细胞，然后将破碎后的菌体或者上清液进行酶纯化处理，处理方法包括硫酸盐沉淀、离子交换柱层析、螯合亲和层析或凝胶过滤柱层析等，以及上述几种方法的组合。
酶固定化：作为固定微生物和上述糖基转移酶的载体不受限制，例如硅藻土、石膏土、高岭石、硅胶、分子筛、多孔玻璃、活性炭、碳酸钙、陶瓷、陶瓷粉等无机载体，聚乙烯醇、聚丙烯、丙烯酰胺、角叉菜胶(卡拉胶)、壳聚糖、离子交换树脂、疏水吸附树脂、螯合树脂、合成吸附树脂等有机聚合物等。
将微生物、酶等固定在载体上的方法，包括吸附法、离子键合法、共价键合法和生化特异性结合法等。固定化糖基转移酶可以在分批系统或连续系统中重复使用来生产糖苷化合物。
选择糖基供体：糖基化的原料，一般有淀粉、水溶性淀粉、糊精等高分子量的糖衍生物，以及麦芽糖、麦芽三糖、葡萄糖、果糖等低分子量的糖衍生物。
糖苷的制备：本发明所述α-红景天苷的制备方法，是将糖基转移酶或微生物培养物加入酪醇和糖基供体的混合物中，进行糖基转移酶反应。酶反应溶液中可加入不影响酶促反应的裂解辅助溶剂，例如二甲氧基亚砜(DMSO)或二甲基乙酰胺(DMA)等有机溶剂，或不影响酶促反应的表面活性剂，例如Tween-20和Span等试剂。
在使用上述糖基转移酶制备α-红景天苷化合物时，糖基转移酶的用量和反应条件对生产效率有很大的影响，因此选择合适的酶量和反应时间等反应条件至关重要。从经济学的角度出发，反应中的添加酶量应控制在1～100小时左右反应完成。此外，为了让糖基转移酶充分催化底物，优选反应体系pH为5～10，反应温度为15～50℃。当使用缓冲溶液调节pH时，可以使用磷酸盐缓冲溶液、醋酸盐缓冲溶液或者Good's buffers缓冲溶液等，以0.01M～0.5M浓度为宜。
本发明所述α-红景天苷制备方法，酶反应液中酪醇的浓度通常可以为5～400mg/mL，优选浓度为20～300mg/mL；糖基供体浓度相对于底物酪醇，优选0.5～30倍的浓度范围。
酪醇和糖基供体的反应可以在溶剂存在的情况下进行，反应中使用的溶剂只要是不影响反应的种类和浓度范围即可，具体可包括甲醇、DMSO、2-丙醇、乙醇等常用试剂。这些溶剂可以单独使用，也可以同时使用两种或两种以上。
本发明中的糖基化反应可以在糖基供体存在下，通过接触微生物的培养物来进行。“接触微生物的培养物”是指将酪醇和糖基供体加入含有微生物的培养物的体系中进行反应，例如，让酪醇和糖基供体的溶液流下并接触固定化微生物，完成酪醇的糖基化。在这种情况下，培养基中可以存在或不存在微生物。如上所述，“微生物的培养物”包括微生物的干燥细胞及其破碎产物，游离糖基转移酶以及固定化糖基转移酶。当培养基中含有葡萄糖、果糖、蔗糖、甘露糖、麦芽糖、甘露糖醇、木糖、半乳糖、淀粉、糊精、糖蜜、山梨糖醇、甘油等糖类时，这些糖类均可作为糖基供体。
通过加热或改变pH值等方法可以导致本发明使用的酶失活，从而停止酶催化反应。
由于底物酪醇和糖基化化合物在水中的溶解度不同，通常可以通过萃取操作利用液(有机相)-液(水相)界面的平衡分配关系进行分离。酪醇衍生物糖基化化合物能溶解在亲水性更高的溶剂中，这种亲水性溶液通过离子交换柱层析、凝胶过滤层析或疏水层析等方法进一步分离纯化，可以很容易地得到高纯度的糖苷精制产品。
上述糖苷的混合物、混合物的水解产物、混合物的酶灭活产物、混合物的纯化产物，其干燥的粉末产品可用 于含糖苷的食品、饮料和化妆品中，也可作为食品、特医食品、保健品或药品的成分，用于食品和饮料、化妆品、特医食品、保健品或药品。
TLC分析条件：
作为酶催化反应底物的酪醇，其分子结构含有苯环，因此具有紫外吸收，可用紫外检测器在254nm波长处进行检测。薄层色谱法作为一种简单方便的酶反应溶液分析方法，对分离条件进行了研究。
购买商业化的高纯度酪醇和β-红景天苷(天然红景天苷)作为标准品用于产品的分析或鉴定(下文分别称为：酪醇标准品，β-红景天苷标准品)，使用市售的Merck公司的TLC板(Merck Kieselgel 60 F254)，氯仿:甲醇:乙酸＝5:1:1(体积比)的溶液作为展开液，酪醇标准品：Rf＝0.9，红景天苷标准品：Rf＝0.4。
HPLC分析条件：
高效液相色谱仪：日立HPLC 5440色谱仪，色谱柱：Unison UK-C18(150×4.6mm，3μm)，检测器：光电二极管阵列(DAD 280nm)，检测波长：280nm，进样量：10μL，流速：0.5mL/min，柱温：30℃，流动相：乙腈：0.1％甲酸水溶液梯度洗脱，洗脱梯度(v/v)见表1。
表1梯度洗脱表
检测时间min 乙腈 0.1％甲酸水溶液(v/v)
0-10 10％-20％ 90％-80％
10-15 20％-21％ 80％-79％
15-20 21％-95％ 79％-5％
20-35 95％-10％ 5％-90％
LC-MS分析条件：
色谱柱：Unison UK-C18(150x4.6mm，3μm)，检测器：光电二极管阵列(DAD 280nm)，检测波长：280nm，进样量：10μL，流速：0.5mL/min，柱温：30℃，流动相同表1检测时间为10-15min的流动相；H-ESI模式，分子量扫描范围50～800。
实施例1.L-糖基转移酶糖基化实验
取酪醇(分子量138.16)10mg、可溶性淀粉20mg和麦芽糖20mg，加入0.9mL0.2M醋酸钠缓冲液(pH5.6)，搅拌均匀，再加入120μL酶液，酶液中的L-糖基转移酶(TG-L，天野酶制剂株式会社)来源于Aspergillus niger，50℃搅拌反应，过夜，得酶催化反应溶液。
实施例2.纤维素酶A糖基化实验
取酪醇(分子量138.16)10mg、可溶性淀粉20mg和麦芽糖20mg，加入0.9mL0.2M醋酸钠缓冲液(pH5.6)，搅拌均匀，再加入120μL酶液，酶液中的纤维素酶A(cellulase A，天野酶制剂株式会社)来源于Aspergillus niger，50℃搅拌反应，过夜，得酶催化反应溶液。
实施例3.环葡聚糖转移酶(CGTase)糖基化实验
取酪醇(分子量138.16)10mg、可溶性淀粉60mg，加入0.8mL0.2M醋酸钠缓冲液(pH5.6)，搅拌均匀，再加入120μL酶液，酶液中环葡聚糖转移酶(CGTase(Contizyme)，天野酶制剂株式会社)来源于Paenibacillusmacerans， 50℃搅拌反应，过夜，得酶催化反应溶液。
实施例4.环葡聚糖转移酶(CGT-SL)糖基化实验
取酪醇(分子量138.16)10mg、可溶性淀粉60mg，加入0.8mL0.2M醋酸钠缓冲液(pH5.6)，搅拌均匀，再加入120μL酶液，酶液中环葡聚糖转移酶(CGT-SL，天野酶制剂株式会社)来源于Geobacillus sp.，50℃搅拌反应，过夜，得酶催化反应溶液。
实施例5.环葡聚糖转移酶(Toruzyme)糖基化实验
取酪醇(分子量138.16)10mg、可溶性淀粉60mg，加入0.8mL0.2M醋酸钠缓冲液(pH5.6)，搅拌均匀，再加入120μL酶液，酶液中环葡聚糖转移酶(Toruzyme，诺维信公司)来源于Thermoanaerobacter sp.，50℃搅拌反应，过夜，得酶催化反应溶液。
实施例6.糖苷转移酶(Glucose trasferase-L)糖基化实验
取酪醇(分子量138.16)10mg、可溶性淀粉20mg和麦芽糖20mg，加入0.9mL0.2M醋酸钠缓冲液(pH5.6)，搅拌均匀，再加入120μL酶液，酶液中的糖苷转移酶(Glucose trasferase-L，天野酶制剂株式会社)来源于Aerribacillussp.，50℃搅拌反应，过夜，得酶催化反应溶液。
实施例7.纤维素酶T糖基化实验
取酪醇(分子量138.16)10mg、可溶性淀粉20mg和麦芽糖20mg，加入0.9mL0.2M醋酸钠(pH5.6)缓冲液，搅拌均匀，再加入120μL酶液，酶液中的纤维素酶(cellulase T，天野酶制剂株式会社)来源于Trichoderma viride，50℃搅拌反应，过夜，得酶催化反应溶液。
实施例8.β-淀粉酶糖基化实验
取酪醇(分子量138.16)10mg、可溶性淀粉20mg和麦芽糖20mg，加入0.9mL0.2M醋酸钠缓冲液(pH5.6)，搅拌均匀，再加入120μL酶液，酶液中的淀粉酶(β-amylase F，天野酶制剂株式会社)来源于Bacillus flexus，50℃搅拌反应，过夜，得酶催化反应溶液。
实施例9.芳香酶糖基化实验
将酪醇(分子量138.16)10mg、可溶性淀粉20mg和麦芽糖20mg，加入0.9mL0.2M醋酸钠(pH5.6)缓冲液，搅拌均匀，再加入120μL酶液，酶液中的芳香酶(Aromase，天野酶制剂株式会社)来源于Penicillium multicolor，50℃搅拌反应，过夜，得酶催化反应溶液。
实施例10：α-红景天苷的制备、纯化及结构分析
分离纯化实施例1所得酶催化反应溶液，并进行TLC、HPLC、LC-MS、NMR分析。
(1)分离纯化
使用疏水性高分子树脂(HP-20，大孔吸附树脂，Solarbio品牌)以5％乙醇溶液对反应液进行分离纯化，再用硅胶树脂(ODS)以40％乙醇溶液进行分离纯化，得到高纯度酶催化反应溶液。
(2)TLC分析
使用Merck公司生产的TLC板(Merck Kieselgel 60 F254)，以氯仿:甲醇:乙酸＝5:1:1的溶液为展开溶剂，酪醇 (标准品，泳道1)和红景天苷(β-红景天苷标准品，泳道2)展开在TLC板1，实施例1所得酶催化反应溶液展开在TLC板2，进行TLC分析。TLC板干燥后，通过UV灯确认TLC板上的斑点。因为TLC板1中酪醇标准品Rf值0.9，β-红景天苷标准品Rf值0.4，从而确认TLC板2中Rf值0.4附近的斑点，推测酶催化反应溶液中合成了β-红景天苷的类似物。详见图1。
(3)HPLC分析
HPLC分析仪为日立HPLC 5440色谱仪，色谱柱为C18柱(Unison UK-C18，150mm×4.6mm，3μm)，柱温为30℃，流动相为乙腈:0.1％甲酸水溶液(v/v)梯度洗脱，使用光电二极管阵列检测器，检测波长为UV 280nm。
依照上述色谱条件，对β-红景天苷标准品进行HPLC分析，其HPLC图谱详见图2。
依照上述色谱条件，对酪醇标准品进行HPLC分析，其HPLC图谱详见图3。
依照上述色谱条件，对本实施例分离纯化实施例1所得酶催化反应溶液后的产品进行HPLC分析，HPLC图谱详见图4。
图2、图3、图4HPLC图谱显示，β-红景天苷标准品出峰时间rt＝9.72min，酪醇标准品出峰时间rt＝12.78min，实施例1所得酶催化反应溶液有两个峰，出峰时间分别是rt＝10.16min和rt＝12.83min，其中rt＝12.83min处的峰是酪醇，rt＝10.16min的峰与β-红景天苷标准品出峰时间有明显的差异。
(4)LC-MS分析
将rt＝10.16min的物质进行LC-MS分析，得到分子离子峰[M-1]的测量值为299.11，结果详见图5。
红景天苷标准品(分子式：C 14H 20O 7，分子量：300.23)的HPLC保留时间为9.72min，经LC-MS分析，其分子离子峰[M-1]的测量值为299.11，确定为酪醇糖苷(分子量300)的[M-H]+峰，详见图6。这表明保留时间为10.16min处的物质属于糖苷，分子量为300.23。根据光电二极管阵列检测器显示的紫外光谱图和LC-MS图谱，HPLC图谱中保留时间rt＝10.16min的新峰被认为是酪醇糖苷。因为具有相同分子量的单糖苷，初步分析该物质是葡萄糖与酪醇结合形成的糖苷。
(5)H-NMR和C-NMR谱图分析
将rt＝10.16min的物质进行NMR分析，H-NMR谱图见图7，C-NMR谱图见图8。
实施例1产品的H-NMR谱：1H NMR(600MHz,DMSO):δ2.74(t,2H),3.05-3.06(m,1H),3.19(m,1H),3.31(m,2H),3.40-3.50(m,3H),3.71-3.72(m,1H),4.42-4.35(m,1H),4.59-4.60(m,1H),4.68(d,J＝3.6Hz,1H:β-H),4.74(m,1H),4.73-4.84(m,1H),6.67(d,J＝7.8Hz,2H:H-2,H-6),7.04(d,J＝7.8Hz,2H:H-3,H-5),9.1(s,1H:OH)；
实施例1产品的C-NMR谱：13C NMR(151MHz,DMSO):δ35.2,61.4,68.8,70.7,72.4,73.3,73.7,99.0,115.5,129.3,130.2,156.0。
依据实施例1产品的H-NMR谱和C-NMR谱，结合红景天苷标准品(β-红景天苷)的核磁数据，实施例1产品的H-NMR谱在4.68ppm处，耦合常数为3.6Hz,(d,J＝3.6Hz,1H:β-H)，这是“α糖苷键的特征”。红景天苷标准品(β-红景天苷)在4.29ppm处有两个峰，耦合常数为8.0Hz，这是β-红景天苷的特征，二者明显不同，结合文献资料(Bassanini I,J Krejzová,Panzeri W,et al.A Sustainable One‐Pot,Two‐Enzyme Synthesis of Naturally  Occurring Arylalkyl Glucosides[J].Chemsuschem,2017,10,2040-2045.doi:10.1002/cssc.201700136)，确定实施例1产品是α-葡萄糖糖基化。从TLC图谱分析，二者是同一个点，从LC-MS分析，二者分子量相同，从HPLC分析，二者是两种物质，再结合NMR图谱中的α键特征(不同于天然的β键)，证实该物质为α-红景天苷，是β-红景天苷(天然红景天苷)的同分异构体，其结构式如式I所示：
Figure PCTCN2022130955-appb-000004
(6)分别分离纯化实施例2～9所得酶催化反应溶液，并进行TLC、HPLC、LC-MS、NMR分析，结果与实施例1一致。
通过HPLC的检测，实施例1和实施例2中，酪醇转化为α-红景天苷的转化率分别为2％和3％；实施例3～9所得酶催化反应溶液，可以检出α-红景天苷，但是含量很低。
实施例11.使用微生物培养物进行糖基化
步骤1.琼脂培养基的制备
麦芽糖(日本食品化工Nippon ShokuhinKako)5g、酵母提取物(Becton Dickinson)100mg、硫酸铵(日本和光Wako Pure Chemical Industries)2g、磷酸二氢钾(日本关东化学Kanto Chemical Co.,Inc.)1g、磷酸氢二钾(日本和光Wako Pure Chemical Industries)1g、七水硫酸镁(日本关东化学Kanto Chemical Co.,Inc.)0.2g、七水硫酸铁(日本关东化学Kanto Chemical Co.,Inc.)0.01g以及15g琼脂BA-10(伊那食品工业Ina Food Industry Co.,Ltd.)，将以上试剂用转移到2L烧杯中加入1L蒸馏水溶解，用NaOH调节至pH7.0，将该溶液在高压釜中121℃下灭菌20min，然后在洁净工作台上分装到培养皿中，15mL/皿；轻轻打开培养皿盖，静置20min冷却，用作琼脂培养基。
步骤2.液体培养基的制备
麦芽糖(日本食品化工Nippon ShokuhinKako)50g、多价蛋白胨(日本和光Wako Pure Chemical Industries)2g，酵母提取物(Becton Dickinson)100mg、硫酸铵(日本和光Wako Pure Chemical Industries)2g、磷酸二氢钾(日本关东化学Kanto Chemical Co.,Inc.)1g、磷酸氢二钾(日本和光Wako Pure Chemical Industries)1g，将以上试剂加入2L烧杯中，加入1L蒸馏水，溶解，用NaOH调pH至7.0；取50mL溶液放入200mL锥形瓶中，在高压釜中121℃灭菌20min，冷却，用作液体培养基。
步骤3.粗酶溶液的制备
将在上述琼脂培养基上，25℃温度下培养了2～3天的Arthrobacter sp.M-238(NITE保藏号AP-02396)接种到步骤2所述液体培养基中，2接种环，25℃，160rpm振荡42小时；培养后，通过离心除去菌体，用硫酸铵盐析上清液，脱盐，使用阴离子交换树脂分级分离糖基转移酶，得粗酶溶液。
步骤4.糖基化反应
将10mg酪醇(分子量138.16)和20mg麦芽糖溶解在1mL0.1M磷酸盐缓冲液(pH7.0)中，加入20μL上述所得粗酶溶液，30℃搅拌过夜，得酶催化反应溶液。
通过TLC、HPLC和LC-MS分析，确认α-红景天苷的生成；经HPLC分析，酪醇转化为α-红景天苷的转化率约为20％。
以上实验发现，所述糖基转移酶来源于节杆菌属(Arthrobacter sp.)时，酪醇转化为α-红景天苷的转化率为20％，和以上商品酶制剂相比，酪醇转化为α-红景天苷的转化率大幅度提高。
工业用途的可行性：
本发明利用糖基转移酶，特别是来源于节杆菌属(Arthrobacter sp.)的糖基转移酶，可将酪醇衍生物进行糖基化修饰。糖基化的糖苷类物质不仅具有酪醇衍生物原有的性质，并且进一步增强其生理学功能，从而提高了开发价值，可作为化妆品、保健品、药品的原料。
如下实施例为自由基清除实验，所用物料与试剂如见表2。
表2.物料与试剂
Figure PCTCN2022130955-appb-000005
实验例12.DPPH自由基的清除实验
自由基具有直接或间接地发挥强氧化作用，广泛地参与机体的生理与病理过程。机体自由基过量时，能通过氧化作用损伤机体。丹酚酸类化合物是酚羟基的供体，具有抗氧化活性的结构基础。本实验采用DPPH【1,1-二苯基-2-三硝基苯肼，别名别名1,1-二苯基-2-苦肼基，(自由基)】自由基清除反应,研究α-红景天苷、β-红景天苷对DPPH自由基的清除效率。
1.溶液制备：
(1)α-红景天苷溶液，按常规方法制备为500mg/L水溶液。
(2)β-红景天苷溶液，按常规方法制备为500mg/L水溶液。
(3)DPPH乙醇溶液，按常规方法制备为0.04mg/ml的无水乙醇溶液。
2.DPPH自由基清除实验：
(1)分别取α-红景天苷溶液、β-红景天苷水溶液各0.9mL，分别加入DPPH无水乙醇溶液0.9mL，混合均 匀，置于暗处静置于，分别于0.5h、1h、2h、4h取样，于517nm波长处测定吸光度，计算三个平行的平均值，记为Ai；
(2)分别取α-红景天苷溶液、β-红景天苷水溶液各0.9mL，分别加入无水乙醇0.9mL，混合均匀，置于暗处静置1h，分别于0.5h、1h、2h、4h取样，于517nm波长处测定吸光度，计算三个平行的平均值，记为Aj；
(3)取蒸馏水0.9mL，加入DPPH乙醇溶液0.9mL，混合均匀，于517nm波长处测定吸光度，计算三个平行的平均值，记为A4；
(4)0.9mL无水乙醇，加蒸馏水0.9mL，混合均匀，作为空白。
依据如下公式计算DPPH自由基清除率，结果记录于表3。
Figure PCTCN2022130955-appb-000006
表3.DPPH自由基清除率
Figure PCTCN2022130955-appb-000007
表3数据说明：α-红景天苷清除DPPH自由基的能力高于β-红景天苷，特别是反应时间在1小时以上的情况下。
实验例13.羟基自由基清除能力
羟基自由基清除能力(Hydroxy free radical scavenging activity)是一种衡量物质抗氧化能力的重要指标。目前有关羟基自由基的分析测试方法主要有高效液相色谱法、化学发光法、荧光分析法、分光光度法等。其中测定羟基自由基清除能力最常用的是通过分光光度法测定样本抑制显色物邻二氮菲的吸光度的下降，从而反映样品清除羟基自由基的能力。测定原理为：H 2O 2/Fe 2+通过Fenton反应产生羟基自由基，将邻二氮菲-Fe 2+水溶液中Fe 2+氧化为Fe 3+，导致536nm吸光度下降，样品对536nm吸光度下降速率的抑制程度，反映了样品清除羟基自由基的能力。
吸光度下降速率的抑制程度，反映了样品清除羟基自由基的能力。
通过测定指示剂吸光值的变化可间接测定生产的羟基自由基。
1.溶液制备：
(1)FeSO 4溶液：按常规方法分别制备1.5mM、5mMFeSO 4水溶液。
(2)PBS缓冲液：
1)配制0.2mol/L的磷酸二氢钠和0.2mol/L的磷酸氢二钠水溶液；
2)取磷酸二氢钠水溶液19ml，磷酸氢二钠水溶液81ml，混匀，调整pH为7.4(pH计测量，可用0.1mol/L氢氧化钠或0.1mol/L磷酸调整pH值)。
(3)邻二氮菲：按常规方法分别制备0.75mM、3mM水溶液。
(4)过氧化氢溶液：按常规方法制备0.1％(v/v)水溶液。
(5)α-红景天苷溶液：按常规方法分别制备1mg/mL、4mg/mL水溶液。
(6)β-红景天苷溶液：按常规方法分别制备1mg/mL、4mg/mL水溶液。
2.羟基自由基清除实验：
方案1：使用5mMFeSO 4水溶液、0.1％双氧水、1mg/mLα-红景天苷水溶液或1mg/mLβ-红景天苷水溶液、3mM邻二氮菲水溶液，按如下操作步骤进行反应。
方案2：使用1.5mMFeSO 4水溶液、0.1％双氧水、4mg/mLα-红景天苷水溶液或4mg/mLβ-红景天苷水溶液、0.75mM邻二氮菲水溶液，按如下操作步骤进行反应。
具体操作步骤(1)分别取α-红景天苷溶液、β-红景天苷溶液40μL，分别加入FeSO 4溶液40μL、PBS缓冲液(pH7.4)60μL、邻二氮菲溶液40μL，混合均匀，再加入H 2O 2溶液40μL，混匀，37℃避光反应30min，以蒸馏水作空白于536nm波长处测定吸光度，计算三个平行的平均值，记为A i；
(2)取蒸馏水80μL，加入FeSO 4溶液40μL、PBS缓冲液(pH7.4)60μL、邻二氮菲溶液40μL，混合均匀，混合均匀，37℃避光反应30min，以蒸馏水作空白于536nm波长处测定吸光度，计算三个平行的平均值，记为A j；
(3)取蒸馏水40μL，加入FeSO 4溶液40μL、PBS缓冲液(pH7.4)60μL、邻二氮菲溶液40μL，混合均匀，再加入H 2O 2溶液40μL，混匀，37℃避光反应30min，以蒸馏水作空白于536nm波长处测定吸光度，计算三个平行的平均值，记为A 0；
Figure PCTCN2022130955-appb-000008
羟基自由基清除结果记录于表4。
表4.羟基自由基清除率
Figure PCTCN2022130955-appb-000009
表4两种检测方法的数据说明：α-红景天苷清除羟基自由基的能力远高于β-红景天苷。
虽然出于清楚理解的目的，已经通过说明以及实例的方式相当详细描述了本发明，本领域普通技术人员将清楚的是，可以实施任何等效方面或修饰。因此，该说明和实例不应当解释为限制本发明的范围。

Claims (9)

  1. 一种α-红景天苷，其结构式如式I，是在酪醇的醇羟基上具有α-葡萄糖糖苷键，
    Figure PCTCN2022130955-appb-100001
  2. 权利要求书1所述的α-红景天苷在制备具有清除自由基功能的产品中的应用；
    优选的，所述自由基包括DPPH自由基和羟基自由基；
    优选的，所述产品为具有清除自由基功能的化妆品；
    优选的，所述产品为抗疲劳保健品。
  3. 一种组合物，其特征在于，含有α-红景天苷；
    优选的，所述组合物具有清除自由基的功能；
    优选的，所述自由基包括DPPH自由基和羟基自由基；
    优选的，所述组合物为化妆品组合物，所述化妆品组合物的品类选自水剂类、油剂类、乳剂类、凝胶类产品、和膏状产品；
    优选的，所述组合物为保健品组合物，所述保健品组合物的剂型选自胶囊状、片状、膏状和水剂。
  4. 权利要求1所述的α-红景天苷的制备方法，其特征在于，包括：将酪醇和糖基供体溶解于缓冲溶液中，再加入糖基转移酶，在反应体系中进行糖基化反应制备得到。
  5. 如权利要求4所述的制备方法，其特征在于，所述糖基供体选自麦芽糖、麦芽三糖、葡萄糖、果糖、淀粉、水溶性淀粉和糊精中的一种或几种。
  6. 如权利要求4所述的制备方法，其特征在于，所述反应体系中，酪醇的浓度为5～400mg/mL，优选为5～100mg/mL；糖基供体的浓度为酪醇浓度的0.5～30倍，优选为2～10倍；
    优选的，所述缓冲溶液为磷酸盐缓冲溶液、醋酸盐缓冲溶液或Good's buffers缓冲溶液，缓冲溶液的浓度为0.01M～0.5M，缓冲溶液的pH为5～10。
  7. 如权利要求4所述的制备方法，其特征在于，所述糖基转移酶为市售商品化糖基转移酶酶制剂或微生物经培养纯化后的糖基转移酶粗酶液。
  8. 如权利要求7所述的制备方法，其特征在于，所述市售商品化糖基转移酶酶制剂选自L-糖基转移酶、淀粉酶、芳香酶、纤维素酶、环葡聚糖转移酶和糖苷转移酶中的一种或多种；
    优选的，所述微生物选自以下属中的至少一种：节杆菌属(Arthrobactersp.)、曲霉属(Aspergillussp.)、类芽孢杆菌属(Paenibacillussp.)、地芽胞杆菌属(Geobacillussp.)、栖热厌氧杆菌属(Thermoanaerobactersp.)、 Aerribacillus菌属、木霉菌属(Trichoderma sp.)、芽孢杆菌属(Bacillus sp.)和青霉菌属(Penicillium sp.)；
    优选的，所述微生物选自曲霉属(Aspergillussp.)和节杆菌属(Arthrobactersp.)；
    优选的，所述微生物为Aspergillus niger或Arthrobacter sp.M-238。
  9. 如权利要求4至8中任一项所述的制备方法，其特征在于，所述反应体系中还可以加入不影响糖基化反应的裂解辅助溶剂二甲氧基亚砜或二甲基乙酰胺，或不影响酶促反应的表面活性剂Tween-20或Span；
    优选的，所述糖基化反应的温度为15～50℃，更优选为30～50℃；所述糖基化反应的时间为1～100小时，更有选为10-50小时。
PCT/CN2022/130955 2021-11-10 2022-11-09 一种α-红景天苷及其制备方法与应用 WO2023083226A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP22892020.3A EP4349843A4 (en) 2021-11-10 2022-11-09 ALPHA-SALIDROSIDE, ITS PREPARATION METHOD AND ITS USE

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202111328730 2021-11-10
CN202111328730.0 2021-11-10
CN202211359878.5A CN115819479A (zh) 2021-11-10 2022-11-02 一种α-红景天苷及其制备方法与应用
CN202211359878.5 2022-11-02

Publications (1)

Publication Number Publication Date
WO2023083226A1 true WO2023083226A1 (zh) 2023-05-19

Family

ID=85526146

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/130955 WO2023083226A1 (zh) 2021-11-10 2022-11-09 一种α-红景天苷及其制备方法与应用

Country Status (3)

Country Link
EP (1) EP4349843A4 (zh)
CN (1) CN115819479A (zh)
WO (1) WO2023083226A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117126781A (zh) * 2023-09-01 2023-11-28 贵州省材料产业技术研究院 一种降解二甲基乙酰胺的原玻璃蝇节杆菌及应用

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101254198A (zh) * 2007-11-07 2008-09-03 中国医学科学院医药生物技术研究所 红景天苷延缓衰老的新用途
CN101347470A (zh) * 2008-08-01 2009-01-21 石任兵 女贞子总三萜提取物和总酚提取物及其同时制备方法
CN102174619A (zh) 2011-01-12 2011-09-07 郑州轻工业学院 葡萄糖糖基转移酶催化合成红景天苷或类似物的方法
CN104606066A (zh) * 2015-02-06 2015-05-13 深圳唯美度生物科技有限公司 一种含红景天苷的抗光损伤凝胶
CN104774815A (zh) 2015-04-07 2015-07-15 中国科学院天津工业生物技术研究所 催化天麻素或红景天苷合成的糖基转移酶及编码该酶的基因及应用
CN106031708A (zh) * 2015-03-17 2016-10-19 天津药物研究院有限公司 一种用于护肤除皱抗衰的药物组合物及其制备方法
CN106543244A (zh) 2016-11-08 2017-03-29 山东大学 半乳糖型红景天苷及其衍生物的制备方法
CN106543243A (zh) 2016-11-08 2017-03-29 山东大学 一种红景天苷衍生物及其制备方法
CN107937457A (zh) 2017-11-16 2018-04-20 江南大学 一种酶催化正丁基‑β‑D‑葡萄糖苷转糖苷制备红景天苷的方法
CN108220264A (zh) 2016-12-22 2018-06-29 中国科学院天津工业生物技术研究所 一种糖基转移酶在生物合成红景天苷中的应用
CN109321615A (zh) 2018-11-14 2019-02-12 南京中医药大学 一种高糖基转移活性解淀粉芽孢杆菌在非水相中生物合成红景天苷的应用
CN113897406A (zh) * 2021-09-29 2022-01-07 山东恒鲁生物科技有限公司 一种从红景天粉末中提取并纯化红景天苷的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112481336B (zh) * 2020-11-27 2022-12-13 上海交通大学 利用木质纤维素衍生物生物合成化合物的方法

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101254198A (zh) * 2007-11-07 2008-09-03 中国医学科学院医药生物技术研究所 红景天苷延缓衰老的新用途
CN101347470A (zh) * 2008-08-01 2009-01-21 石任兵 女贞子总三萜提取物和总酚提取物及其同时制备方法
CN102174619A (zh) 2011-01-12 2011-09-07 郑州轻工业学院 葡萄糖糖基转移酶催化合成红景天苷或类似物的方法
CN104606066A (zh) * 2015-02-06 2015-05-13 深圳唯美度生物科技有限公司 一种含红景天苷的抗光损伤凝胶
CN106031708A (zh) * 2015-03-17 2016-10-19 天津药物研究院有限公司 一种用于护肤除皱抗衰的药物组合物及其制备方法
CN104774815A (zh) 2015-04-07 2015-07-15 中国科学院天津工业生物技术研究所 催化天麻素或红景天苷合成的糖基转移酶及编码该酶的基因及应用
CN106543244A (zh) 2016-11-08 2017-03-29 山东大学 半乳糖型红景天苷及其衍生物的制备方法
CN106543243A (zh) 2016-11-08 2017-03-29 山东大学 一种红景天苷衍生物及其制备方法
CN108220264A (zh) 2016-12-22 2018-06-29 中国科学院天津工业生物技术研究所 一种糖基转移酶在生物合成红景天苷中的应用
CN107937457A (zh) 2017-11-16 2018-04-20 江南大学 一种酶催化正丁基‑β‑D‑葡萄糖苷转糖苷制备红景天苷的方法
CN109321615A (zh) 2018-11-14 2019-02-12 南京中医药大学 一种高糖基转移活性解淀粉芽孢杆菌在非水相中生物合成红景天苷的应用
CN113897406A (zh) * 2021-09-29 2022-01-07 山东恒鲁生物科技有限公司 一种从红景天粉末中提取并纯化红景天苷的方法

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. 66774038
"Pharmacopoeia of the People's Republic of China", 2020, article "Rhodiola crenulata"
BASSANINI IJ KREJZOVAPANZERI W ET AL.: "A Sustainable One-Pot, Two-Enzyme Synthesis of Naturally Occurring Arylalkyl Glucosides[J", CHEMSUSCHEM, vol. 10, 2017, pages 2040 - 2045
CAI ZHEN, SHI LI-FU, SHEN QIAN, XU YU-LIAN: "Effects of p-Hydroxyphenethyl-α-D-glucoside on Mouse Natural Killer Cells in Vitro", CHINESE JOURNAL OF PHARMACOLOGY AND TOXICOLOGY, vol. 12, no. 3, 31 August 1998 (1998-08-31), pages 207, XP009545637, ISSN: 1000-3002 *
See also references of EP4349843A4
SHI LIFU, WANG PENG, CHEN HAISHENG, DONG JIANPING: "STUDIES ON WATEER-SOLUBLE CONSTITUENTS FROM THE FRUITSO FLIGUSTRUM LUCIDUM AIT", ACTA PHARMACEUTICA SINICA, vol. 30, no. 12, 1 January 1995 (1995-01-01), pages 935 - 938, XP093066983 *
TRINCONE ANTONIO; PAGNOTTA EDUARDO; TRAMICE ANNABELLA: "Enzymatic routes for the production of mono- and di-glucosylated derivatives of hydroxytyrosol", BIORESOURCE TECHNOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 115, 1 January 1900 (1900-01-01), AMSTERDAM, NL , pages 79 - 83, XP028924307, ISSN: 0960-8524, DOI: 10.1016/j.biortech.2011.10.073 *
WANG QING, WEI XIONG, LIAO KAIJUN, LI HUI, MENG XIANGBAO, LI ZHONGJUN: "A convenient preparation of glycosyl sulfoxides and its application to the synthesis of Salidroside epimer", TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM , NL, vol. 57, no. 21, 1 May 2016 (2016-05-01), Amsterdam , NL , pages 2277 - 2279, XP093066969, ISSN: 0040-4039, DOI: 10.1016/j.tetlet.2016.04.045 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117126781A (zh) * 2023-09-01 2023-11-28 贵州省材料产业技术研究院 一种降解二甲基乙酰胺的原玻璃蝇节杆菌及应用
CN117126781B (zh) * 2023-09-01 2024-05-28 贵州省材料产业技术研究院 一种降解二甲基乙酰胺的原玻璃蝇节杆菌及应用

Also Published As

Publication number Publication date
EP4349843A4 (en) 2024-04-17
CN115819479A (zh) 2023-03-21
EP4349843A1 (en) 2024-04-10

Similar Documents

Publication Publication Date Title
US10683525B2 (en) Method for producing 2-O-glyceryl-alpha-D-glucopyranoside
Ribeiro Naringinases: occurrence, characteristics, and applications
US6140487A (en) DNA encoding kojibiose phosphorylase obtainable from thermoanaerobium, ' its preparation and uses
Han et al. Functions, applications and production of 2-O-D-glucopyranosyl-L-ascorbic acid
AU2005288314B2 (en) Isocyclomaltooligosaccharide, isocyclomaltooligosaccharide synthase, process for producing them and use thereof
US7951530B2 (en) Cyclic maltosylmaltose, cyclic maltosymaltose-forming enzyme, their preparation and uses
Kochikyan et al. Combined enzymatic modification of stevioside and rebaudioside A
Vic et al. Enzymatic glucosylation of hydrophobic alcohols in organic medium by the reverse hydrolysis reaction using almond‐β‐D‐glucosidase
WO2023083226A1 (zh) 一种α-红景天苷及其制备方法与应用
JPH0649715B2 (ja) 末端にイノシト−ル残基を結合したグルコオリゴ糖およびその製造方法
AU2002255280B2 (en) Process for producing isomaltose and use thereof
Seo et al. Highly selective biotransformation of arbutin to arbutin-α-glucoside using amylosucrase from Deinococcus geothermalis DSM 11300
JP2017123844A (ja) 配糖体の製造方法
JP5274700B2 (ja) 還元末端にアルドン酸残基を有しα１→６グルコシド結合またはβ１→６グルコシド結合を有するオリゴ糖の製造方法
JP6156947B2 (ja) 配糖体の製造方法
JP2019216633A (ja) 配糖体の製造方法
CN105039463B (zh) 一种区域选择性专一的转糖基β-N-乙酰氨基已糖苷酶的应用
JP3916682B2 (ja) 配糖体の製造方法
KURIKI et al. Production of isomalto/branched-oligosaccharide syrup by using immobilized neopullulanase and preliminary evaluation of the syrup as a food additive
EP3895711A1 (en) Cycloisomaltotetraose, cycloisomaltotetraose production enzyme, and production methods and uses thereof
WO2017078137A1 (ja) 微生物変換によるアシルグルクロン酸抱合体の製造方法及びその変換活性を有する微生物
JPH0685716B2 (ja) 固定化酵素
Hsieh et al. Production of ascorbic acid glucoside by alginate-entrapped mycelia of Aspergillus niger
JP2010159225A (ja) テアンデロース含有シロップ
JPH07165761A (ja) 新規生理活性物質ｓｆ２７７１物質及びその製造法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22892020

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2022892020

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2022892020

Country of ref document: EP

Effective date: 20240102