α-Salidroside and its preparation method and application

Abstract

The present invention relates to α-salidroside and its preparation method and application. The present invention provides a new type of salidroside, namely α-salidroside. Compared with the known β-salidroside, α-salidroside has better activity in scavenging DPPH free radicals and hydroxyl free radicals than β-salidroside, and can be added to cosmetics or anti-fatigue health products as a new functional ingredient. The preparation method of α-salidroside of the present invention uses inexpensive starch as a glycosyl donor and tyrosol as a substrate. α-salidroside is synthesized under the action of a glycosyltransferase. The glycosyltransferase has strict specificity and chiral catalytic ability, and can specifically produce α-type salidroside.

Description

Technical Field

[0001] The present invention relates to α-salidroside and a preparation method and application thereof, in particular to a method for synthesizing α-salidroside by utilizing glycosyltransferase, and belongs to the field of biotechnology. Background Art

[0002] Rhodiola rosea is a precious traditional Chinese medicine. The 2020 edition of the Chinese Pharmacopoeia includes the dried roots and rhizomes of Rhodiola rosea (scientific name: Rhodiolacrenulata (Hook.f.et Thoms.) H.Ohba) of the Crassulaceae family. It is produced in Tibet and other places in China and grows in areas with an altitude of about 4050 to 5400 meters. Medicines such as Rhodiola rosea tablets and Xinnaoxin capsules prepared from Rhodiola rosea have the effects of promoting blood circulation and removing blood stasis, dredge meridians and relieving pain. The function of health foods such as Rhodiola rosea capsules is to enhance immunity and relieve physical fatigue. Salidroside is the effective active ingredient of the traditional Chinese medicine Rhodiola rosea. It is reported that salidroside has physiological activities such as protecting cardiovascular and cerebrovascular vessels, anti-fatigue, anti-depression, anti-aging, anti-hypoxia, anti-radiation, anti-tumor, immune regulation, whitening and freckle removal. Salidroside in its natural state (plant salidroside) is β-salidroside, and its glycosidic bond is β-type. Its chemical formula is as follows:

[0003] .

[0004] Due to the limited resources of the plant Rhodiola rosea, some scholars have conducted research on the preparation of salidroside. The production methods of salidroside include plant extraction, enzymatic method, fermentation method, chemical synthesis method, etc. Chinese patent document CN104774815A (application number 201510160496.3) discloses a glycosyltransferase that can catalyze the synthesis of gastrodin or salidroside. The glycosyltransferase in this patent document is derived from the glycosyltransferase gene ugt73b6 of the plant Rhodiola sachalinensis, which was randomly mutated by error-PCR to obtain a glycosyltransferase mutant gene ugt73b6 that catalyzes the synthesis of gastrodin or salidroside. MK Specifically, the methionine at position 264 of the glycosyltransferase UGT73B6 is mutated to lysine; Chinese patent document CN108220264A (application number 201611200315.6) discloses a glycosyltransferase and its application in the biosynthesis of β-salidroside. The glycosyltransferase in the patent document is derived from Arabidopsis thaliana ( Arabidopsis thaliana) UDP-glycosyltransferase (Genbank GI: 66774038); Chinese patent document CN106543243 (application number 201610979792.0) discloses a salidroside derivative and a preparation method thereof, and the glycosyltransferase 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 β-galactose-type salidroside and its derivatives, and the galactosidase used in the patent document is a galactosidase derived from Enterobacter cloacae ( Enterobacter cloacae )'s transglycosyl β-galactosidase; Chinese patent document CN109321615A (application number 201811363071.2) discloses an application of Bacillus amyloliquefaciens with high glycosyl transfer activity in the biosynthesis of salidroside in a non-aqueous phase, in which the wet cells of Bacillus amyloliquefaciens FJ18 are used to catalyze the glycosylation reaction; Chinese patent document CN102174619A (application number 201110005088.2) discloses a method for synthesizing salidroside or analogs catalyzed by glucose glycosyltransferase, and the glucose glycosyltransferase in the patent document is derived from Leuconostoc mesenteroides; Chinese patent document CN107937457A (application number 201711141952.5) ​​discloses a method for synthesizing salidroside by enzyme-catalyzed transglycosidation reaction of n-butyl-β-D-glucoside, and the β-glucosidase used in the patent document is almond β-glucosidase, Agrobacterium ( Agrobacterium )β-glucosidase, white rot fungi ( Phanerochaete chrysosporium )β-glucosidase, Thermotoga maritima ( Thermotoga maritima ) Any one of β-glucosidase. The above salidrosides prepared by artificial means are all β-salidroside. Summary of the Invention

[0005] Purpose of the invention: To provide an excellent form of salidroside, or to provide a salidroside preparation process suitable for industrial production.

[0006] Driven by a need for product iteration, the applicant conducted research on salidroside and unexpectedly discovered that the synthesized glycoside differed from the existing β-salidroside. It was identified as α-salidroside, an isomer of β-salidroside. Furthermore, the present invention provides a preparation method and application of α-salidroside.

[0007] There is no report on the α-salidroside isomer of β-salidroside in the prior art, and there is no research on the synthesis method and biological activity of α-salidroside.

[0008] In one aspect, the present invention provides an α-salidroside having a structural formula as shown in Formula I, which has an α-glucosidoside bond on the alcoholic hydroxyl group of tyrosol.

[0009] .

[0010] Preferably according to the present invention, the molecular weight of the α-salidroside is 300.23 as analyzed by LC-MS.

[0011] Preferably, according to the present invention, the H-NMR spectrum of the α-salidroside is: 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).

[0012] The C-NMR spectrum of the α-salidroside: 13C NMR (151 MHz, DMSO): δ 35.2, 61.4, 68.8, 70.7, 72.4, 73.3, 73.7, 99.0, 115.5, 129.3, 130.2, 156.0.

[0013] The α-salidroside described in the present invention is an isomer of natural β-salidroside, and its chemical name is: 2-(4-hydroxyphenyl)ethyl-α-D-glucoside.

[0014] The α-salidroside of the present invention has better antioxidant and free radical scavenging abilities than β-salidroside (natural salidroside).

[0015] The present invention further provides the use of the α-salidroside in preparing a product with the function of scavenging free radicals.

[0016] The use of the α-salidroside in the preparation of a product having the function of scavenging free radicals, preferably, the free radicals include DPPH free radicals and hydroxyl free radicals.

[0017] The use of the α-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.

[0018] The use of the α-salidroside in the preparation of a product with the function of scavenging free radicals is further preferably an anti-fatigue health product.

[0019] The present invention further provides a composition containing α-salidroside. The composition has the function of scavenging free radicals, including DPPH free radicals and hydroxyl free radicals.

[0020] Cosmetic compositions containing α-salidroside are selected from aqueous solutions, oils, emulsions, gels, pastes, or other formulations. They can be produced according to existing cosmetic technical specifications, with no special requirements.

[0021] The health care product composition containing α-salidroside can be in the form of capsules, tablets, pastes, liquids, or other forms. It can be produced according to existing health care product technical specifications without special requirements.

[0022] The present invention further provides a method for preparing the α-salidroside, which comprises dissolving tyrosol and a glycosyl donor in a buffer solution, adding a glycosyltransferase, and performing a glycosylation reaction in a reaction system to prepare the α-salidroside.

[0023] In the present invention, the glycosyltransferase has strict specificity and chiral catalytic ability. The glycosyltransferase used in the present invention can specifically produce α-type salidroside, which is different from the glycosyltransferase recorded in the prior art for producing β-type salidroside.

[0024] Preferably according to the present invention, the glycosyl donor is one or more of maltose, maltotriose, glucose, fructose, starch, water-soluble starch, and dextrin.

[0025] Preferably according to the present invention, in the reaction system, 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 2-10 times.

[0026] Preferably, according to the present invention, the buffer solution is a phosphate buffer solution, an acetate buffer solution or a Good's buffer solution, the concentration of the buffer solution is 0.01M to 0.5M, and the pH of the buffer solution is 5 to 10.

[0027] According to the present invention, preferably, the glycosyltransferase is a commercial glycosyltransferase enzyme preparation or a crude glycosyltransferase enzyme solution obtained by culturing and purifying microorganisms.

[0028] Further preferably, the commercially available glycosyltransferase enzyme preparations are derived from Amano Enzyme Co., Ltd. and Novozymes Pharmaceuticals Co., Ltd., and include enzymes such as L-glycosyltransferase, amylase, aromatase, cellulase, cycloglucanotransferase and glycosidase that can produce or transfer α-glycosides.

[0029] Further preferably, the microorganisms include Arthrobacter ( Arthrobacter sp.), Aspergillus ( Aspergillus sp.), Paenibacillus spp. Paenibacillus sp.), Geobacillus sp. Geobacillus sp.), Thermoanaerobacterium spp. Thermoanaerobacter sp.)、 Aerribacillus Trichoderma Trichoderma sp.), Bacillus ( Bacillus sp.) or Penicillium ( Penicillium sp.); further preferably, the microorganism is Aspergillus ( Aspergillus sp.) and Arthrobacter sp. Arthrobacter sp.) microorganisms.

[0030] More preferably, the microorganism is Aspergillus niger and Arthrobacter sp. M-238.

[0031] Preferably, according to the present invention, the reaction system may further include dimethoxysulfoxide (DMSO) or dimethylacetamide (DMA), a cleavage auxiliary solvent that does not affect the glycosylation reaction, or Tween-20 or Span, a surfactant that does not affect the enzymatic reaction.

[0032] Preferably, according to the present invention, the temperature of the glycosylation reaction is 15-50° C., more preferably 30-50° C.; and the time is 1-100 hours, more preferably 10-50 hours.

[0033] In the present invention, the preparation of α-salidroside can also be carried out in an organic solvent. The organic solvent used in the reaction can be any type and concentration range that does not affect the reaction, specifically including one solvent or a mixed solvent of two or more of methanol, DMSO, 2-propanol, and ethanol; and the above-mentioned buffer solution is used to adjust the pH of the reaction system to 5-10.

[0034] Beneficial effects:

[0035] The present invention provides a novel salidroside, namely α-salidroside, which has an α-glucosidic bond on the alcoholic hydroxyl group of tyrosol. Compared with the known β-salidroside, α-salidroside has better activity in scavenging DPPH free radicals and hydroxyl free radicals than β-salidroside. α-salidroside can be added to cosmetics or anti-fatigue health products as a new functional ingredient. The development of new functions of α-salidroside is under further research.

[0036] The present invention also provides a method for preparing α-salidroside, wherein the glycosyltransferase is derived from Aspergillus ( Aspergillus sp.), the conversion rate of tyrosol to α-salidroside was more than 2%; when the glycosyltransferase was derived from Arthrobacter ( Arthrobacter sp.), the conversion rate of tyrosol to α-salidroside was 20%, which greatly improved the conversion rate of the glycosylation reaction and was suitable for industrial production.

[0037] The preparation method of α-salidroside of the present invention uses cheap starch or the like as a glycosyl donor and tyrosol as a substrate to synthesize α-salidroside under the action of glycosyltransferase, is suitable for industrial production, and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 In Example 10, after the enzyme-catalyzed reaction solution obtained in Example 1 was separated and purified, a TLC spectrum of the sample was obtained; in the figure, from left to right are TLC plate 1 and TLC plate 2; from left to right on TLC plate 1 are lanes 1 and lanes 2.

[0039] Figure 2 HPLC spectrum of β-salidroside standard.

[0040] Figure 3 . HPLC spectrum of tyrosol standard.

[0041] Figure 4 In Example 10, after the enzyme-catalyzed reaction solution obtained in Example 1 was separated and purified, an HPLC spectrum of the sample was obtained.

[0042] Figure 5 In Example 10, after the enzyme-catalyzed reaction solution obtained in Example 1 was separated and purified, an LC-MS spectrum of the substance at rt=10.16 min in the HPLC spectrum of the sample was obtained.

[0043] Figure 6 LC-MS spectrum of β-salidroside standard.

[0044] Figure 7 In Example 10, after the enzyme-catalyzed reaction solution obtained in Example 1 was separated and purified, an H-NMR spectrum of the substance at rt=10.16 min in the HPLC spectrum of the sample was obtained.

[0045] Figure ⑧ In Example 10, after the enzyme-catalyzed reaction solution obtained in Example 1 was separated and purified, a C-NMR spectrum of the substance at rt=10.16 min in the HPLC spectrum of the sample was obtained.

[0046] Figure 9 .H-NMR spectrum of β-salidroside standard. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to specific embodiments. The embodiments are not intended to limit the present invention, and the protection scope of the present invention is not limited thereto.

[0048] The α-salidroside described in the present invention is an isomer of natural β-salidroside, and its chemical name is: 2-(4-hydroxyphenyl)ethyl-α-D-glucoside, and its structural formula is as follows:

[0049] .

[0050] The preparation method of α-salidroside of the present invention uses tyrosol as a substrate and realizes glycosylation under the action of glycosyltransferase derived from microorganisms or commercial glycosyltransferase.

[0051] Glycosyltransferases utilize cheap sugar sources such as starch, dextrin, and maltose for glycosylation, and their glycosides exist in the form of α-bonds.

[0052] Commercially available glycosyltransferase enzyme preparations with the ability to achieve glycosylation to form α-glycosidic bonds are sourced from Amano Enzyme Co., Ltd. and Novozymes. The enzyme preparations include L-glycosyltransferase, amylase, aromatase, cellulase, cycloglucanotransferase, and glycosidic transferase.

[0053] The method of using the glycosyltransferase enzyme preparation includes adding it to the reaction system in the form of powder or liquid, or fixing the enzyme preparation on a resin to make an immobilized enzyme preparation and adding it to the reaction system. The immobilized enzyme preparation can be reused.

[0054] The glycosyltransferase enzyme preparation can use a commercial enzyme preparation, or the required enzyme solution can be produced by microbial culture, and then the fresh enzyme solution is used for glycosylation modification.

[0055] The microbial source of the glycosyltransferase may be Arthrobacter ( Arthrobacter sp.), Aspergillus ( Aspergillus sp.), Paenibacillus spp. Paenibacillus sp.), Geobacillus sp. Geobacillus sp.), Thermoanaerobacterium spp. Thermoanaerobacter sp.)、 AerribacillusTrichoderma Trichoderma sp.), Bacillus ( Bacillus sp.) or Penicillium ( Penicillium sp.), etc., are not particularly limited, and the glycosyltransferase can be obtained by separation and purification from a microbial culture, including a composition of glycosyltransferases, a purified glycosyltransferase, a composition containing α-glycosyltransferase, a purified α-glycosyltransferase, or an immobilized α-glycosyltransferase bound to a carrier.

[0056] Preparation of enzyme solution: The method for isolating and purifying glycosyltransferase from microbial culture is to use ultrasound or glass beads to crush the microbial cells, and then subject the crushed cells or supernatant to enzyme purification treatment. Treatment methods include sulfate precipitation, ion exchange column chromatography, chelate affinity chromatography or gel filtration column chromatography, etc., as well as combinations of the above methods.

[0057] Enzyme immobilization: There is no limitation on the carriers for immobilizing microorganisms and the above-mentioned glycosyltransferases, for example, inorganic carriers such as diatomaceous earth, gypsum, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics, ceramic powder, and organic polymers such as polyvinyl alcohol, polypropylene, acrylamide, carrageenan (carrageenan), chitosan, ion exchange resins, hydrophobic adsorption resins, chelating resins, and synthetic adsorption resins.

[0058] Methods for immobilizing microorganisms, enzymes, etc. on carriers include adsorption, ionic bonding, covalent bonding, and biochemical specific binding. Immobilized glycosyltransferases can be reused in batch or continuous systems to produce glycoside compounds.

[0059] Selection of glycosyl donors: The raw materials for glycosylation generally include high molecular weight sugar derivatives such as starch, water-soluble starch, and dextrin, as well as low molecular weight sugar derivatives such as maltose, maltotriose, glucose, and fructose.

[0060] Preparation of Glycosides: The method for preparing α-salidroside described herein comprises adding a glycosyltransferase or a microbial culture to a mixture of tyrosol and a glycosyl donor to carry out a glycosyltransferase reaction. A cleavage-aiding solvent that does not affect the enzymatic reaction, such as an organic solvent such as dimethoxysulfoxide (DMSO) or dimethylacetamide (DMA), or a surfactant that does not affect the enzymatic reaction, such as Tween-20 or Span, may be added to the enzyme reaction solution.

[0061] When using the above-mentioned glycosyltransferase to prepare α-salidroside compounds, the amount of glycosyltransferase used and the reaction conditions have a great influence on the production efficiency. Therefore, it is crucial to select the appropriate reaction conditions such as the amount of enzyme and reaction time. From an economic point of view, the amount of enzyme added in the reaction should be controlled to complete the reaction in about 1 to 100 hours. In addition, in order for the glycosyltransferase to fully catalyze the substrate, the pH of the reaction system is preferably 5 to 10 and the reaction temperature is 15 to 50°C. When using a buffer solution to adjust the pH, phosphate buffer solution, acetate buffer solution or Good's buffer solution can be used, with a concentration of 0.01M to 0.5M being appropriate.

[0062] In the method for preparing α-salidroside of the present invention, the concentration of tyrosol in the enzyme reaction solution can generally be 5 to 400 mg / mL, preferably 20 to 300 mg / mL; the concentration of the glycosyl donor is preferably in the range of 0.5 to 30 times the concentration of the substrate tyrosol.

[0063] The reaction between tyrosol and the glycosyl donor can be carried out in the presence of a solvent. The solvent used in the reaction can be of any type and concentration range as long as it does not affect the reaction. Specific examples include commonly used reagents such as methanol, DMSO, 2-propanol, and ethanol. These solvents can be used alone or in combination of two or more.

[0064] The glycosylation reaction in the present invention can be carried out by contacting a culture of microorganisms in the presence of a glycosyl donor. "Contacting a culture of microorganisms" means adding tyrosol and a glycosyl donor to a system containing a culture of microorganisms to carry out the reaction. For example, a solution of tyrosol and a glycosyl donor is allowed to flow down and contact an immobilized microorganism to complete the glycosylation of tyrosol. In this case, the microorganism may or may not be present in the culture medium. As mentioned above, "culture of microorganisms" includes dried microbial cells and their fragments, free glycosyltransferases, and immobilized glycosyltransferases. When the culture medium contains sugars such as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, galactose, starch, dextrin, molasses, sorbitol, and glycerol, these sugars can serve as glycosyl donors.

[0065] The enzyme used in the present invention can be inactivated by heating or changing the pH value, thereby stopping the enzyme catalytic reaction.

[0066] Due to the different solubilities of the substrate tyrosol and glycosylated compounds in water, they can typically be separated by partitioning them into an oil phase and an aqueous phase via extraction. Tyrosol-derived glycosylated compounds can be dissolved in a more hydrophilic solvent. This hydrophilic solution can then be further separated and purified using methods such as ion exchange column chromatography, gel filtration chromatography, or hydrophobic chromatography, easily yielding a highly purified glycoside product.

[0067] The above-mentioned glycoside mixture, hydrolysis product of the mixture, enzyme-inactivated product of the mixture, purified product of the mixture, and dry powder products thereof can be used in glycoside-containing foods, beverages, and cosmetics, and can also be used as ingredients of foods, special medical foods, health products, or medicines, and used in foods and beverages, cosmetics, special medical foods, health products, or medicines.

[0068] TLC analysis conditions:

[0069] Tyrosol, the substrate for the enzyme-catalyzed reaction, has a benzene ring in its molecular structure, resulting in UV absorption, which can be detected using a UV detector at a wavelength of 254 nm. Thin-layer chromatography, a simple and convenient method for analyzing enzyme reaction solutions, was used to investigate the separation conditions.

[0070] Commercially available high-purity tyrosol and β-salidroside (natural salidroside) were purchased as standards for product analysis or identification (hereinafter referred to as: tyrosol standard, β-salidroside standard). Commercially available Merck TLC plates (Merck Kieselgel 60 F254) were used, and a solution of chloroform: methanol: acetic acid = 5:1:1 (volume ratio) was used as the developing solution. Tyrosol standard: Rf = 0.9, salidroside standard: Rf = 0.4.

[0071] HPLC analysis conditions:

[0072] High-performance liquid chromatography: Hitachi HPLC 5440 chromatograph, chromatographic column: Unison UK-C18 (150×4.6 mm, 3 μm), detector: photodiode array (DAD 280 nm), detection wavelength: 280 nm, injection volume: 10 μL, flow rate: 0.5 mL / min, column temperature: 30°C, mobile phase: acetonitrile: 0.1% formic acid aqueous solution gradient elution, elution gradient (v / v) is shown in Table 1.

[0073] Table 1 Gradient elution table

[0074] .

[0075] LC-MS analysis conditions:

[0076] Chromatographic column: Unison UK-C18 (150x4.6mm, 3μm), detector: photodiode array (DAD 280nm), detection wavelength: 280nm, injection volume: 10μL, flow rate: 0.5mL / min, column temperature: 30℃, mobile phase with the same detection time of 10-15min as shown in Table 1; H-ESI mode, molecular weight scan range: 50-800.

[0077] Example 1. L-glycosyltransferase glycosylation experiment

[0078] Take 10 mg of tyrosol (molecular weight 138.16), 20 mg of soluble starch and 20 mg of maltose, add 0.9 mL of 0.2 M sodium acetate buffer (pH 5.6), stir evenly, and then add 120 μL of enzyme solution. The L-glycosyltransferase (TG-L, Amano Enzyme Preparation Co., Ltd.) in the enzyme solution is derived from Aspergillus niger , react with stirring at 50°C overnight to obtain an enzyme-catalyzed reaction solution.

[0079] Example 2. Cellulase A glycosylation experiment

[0080] Take 10 mg of tyrosol (molecular weight 138.16), 20 mg of soluble starch and 20 mg of maltose, add 0.9 mL of 0.2 M sodium acetate buffer (pH 5.6), stir evenly, and then add 120 μL of enzyme solution. The cellulase A in the enzyme solution (cellulase A, Amano Enzyme Preparation Co., Ltd.) is derived from Aspergillus niger , react with stirring at 50°C overnight to obtain an enzyme-catalyzed reaction solution.

[0081] Example 3. Cycloglucanotransferase (CGTase) glycosylation experiment

[0082] Take 10 mg of tyrosol (molecular weight 138.16) and 60 mg of soluble starch, add 0.8 mL of 0.2 M sodium acetate buffer (pH 5.6), stir evenly, and then add 120 μL of enzyme solution. The enzyme solution contains cycloglucan transferase (CGTase (Contizyme), Amano Enzyme Co., Ltd.) Paenibacillus macerans , react with stirring at 50°C overnight to obtain an enzyme-catalyzed reaction solution.

[0083] Example 4. Cycloglucanotransferase (CGT-SL) glycosylation experiment

[0084] Take 10 mg of tyrosol (molecular weight 138.16) and 60 mg of soluble starch, add 0.8 mL of 0.2 M sodium acetate buffer (pH 5.6), stir evenly, and then add 120 μL of enzyme solution. The enzyme solution contains cycloglucan transferase (CGT-SL, Amano Enzyme Co., Ltd.) Geobacillus sp. , react with stirring at 50°C overnight to obtain an enzyme-catalyzed reaction solution.

[0085] Example 5. Cycloglucanotransferase (Toruzyme) glycosylation experiment

[0086] Take 10 mg of tyrosol (molecular weight 138.16) and 60 mg of soluble starch, add 0.8 mL of 0.2 M sodium acetate buffer (pH 5.6), stir evenly, and then add 120 μL of enzyme solution. The enzyme solution contains cycloglucan transferase (Toruzyme, Novozymes) fromThermoanaerobacter sp. , react with stirring at 50°C overnight to obtain an enzyme-catalyzed reaction solution.

[0087] Example 6. Glycosylation experiment with glycosyltransferase-L

[0088] Take 10 mg of tyrosol (molecular weight 138.16), 20 mg of soluble starch and 20 mg of maltose, add 0.9 mL of 0.2 M sodium acetate buffer (pH 5.6), stir evenly, and then add 120 μL of enzyme solution. The glycosyltransferase in the enzyme solution (Glucosetrasferase-L, Amano Enzyme Preparation Co., Ltd.) is derived from Aerribacillus sp., react with stirring at 50°C overnight to obtain an enzyme-catalyzed reaction solution.

[0089] Example 7. Cellulase T glycosylation experiment

[0090] Take 10 mg of tyrosol (molecular weight 138.16), 20 mg of soluble starch and 20 mg of maltose, add 0.9 mL of 0.2 M sodium acetate (pH 5.6) buffer, stir evenly, and then add 120 μL of enzyme solution. The cellulase in the enzyme solution (cellulase T, Amano Enzyme Preparation Co., Ltd.) is derived from Trichoderma viride , react with stirring at 50°C overnight to obtain an enzyme-catalyzed reaction solution.

[0091] Example 8. β-amylase glycosylation experiment

[0092] Take 10 mg of tyrosol (molecular weight 138.16), 20 mg of soluble starch and 20 mg of maltose, add 0.9 mL of 0.2 M sodium acetate buffer (pH 5.6), stir evenly, and then add 120 μL of enzyme solution. The amylase in the enzyme solution (β-amylase F, Amano Enzyme Co., Ltd.) is derived from Bacillus flexus , react with stirring at 50°C overnight to obtain an enzyme-catalyzed reaction solution.

[0093] Example 9. Aromatase glycosylation experiment

[0094] 10 mg of tyrosol (molecular weight 138.16), 20 mg of soluble starch and 20 mg of maltose were added to 0.9 mL of 0.2 M sodium acetate (pH 5.6) buffer and stirred evenly. Then 120 μL of enzyme solution was added. The aromatase (Aromase, Amano Enzyme Co., Ltd.) in the enzyme solution was derived from Penicillium multicolor , react with stirring at 50°C overnight to obtain an enzyme-catalyzed reaction solution.

[0095] Example 10: Preparation, purification and structural analysis of α-salidroside

[0096] The enzyme-catalyzed reaction solution obtained in Example 1 was separated and purified, and analyzed by TLC, HPLC, LC-MS, and NMR.

[0097] (1) Separation and purification

[0098] The reaction solution was separated and purified using a hydrophobic polymer resin (HP-20, macroporous adsorption resin, Solarbio brand) with a 5% ethanol solution, and then separated and purified using a silica gel resin (ODS) with a 40% ethanol solution to obtain a high-purity enzyme-catalyzed reaction solution.

[0099] (2) TLC analysis

[0100] Using a TLC plate (Merck Kieselgel 60 F254) manufactured by Merck, a solution of chloroform:methanol:acetic acid = 5:1:1 was used as the developing solvent. Tyrosol (standard, lane 1) and salidroside (β-salidroside standard, lane 2) were developed on TLC plate 1, and the enzyme-catalyzed reaction solution obtained in Example 1 was developed on TLC plate 2 for TLC analysis. After the TLC plate was dried, the spots on the TLC plate were confirmed by UV light. Because the Rf value of the tyrosol standard on TLC plate 1 was 0.9 and the Rf value of the β-salidroside standard was 0.4, the spot near the Rf value of 0.4 on TLC plate 2 was confirmed, and it was speculated that an analog of β-salidroside was synthesized in the enzyme-catalyzed reaction solution. See for details. Figure 1 .

[0101] (3) HPLC analysis

[0102] The HPLC analyzer was a Hitachi HPLC 5440 chromatograph, the chromatographic column was a C18 column (Unison UK-C18, 150 mm × 4.6 mm, 3 μm), the column temperature was 30°C, the mobile phase was acetonitrile:0.1% formic acid aqueous solution (v / v) gradient elution, and a photodiode array detector was used with a UV detection wavelength of 280 nm.

[0103] According to the above chromatographic conditions, β-salidroside standard was analyzed by HPLC. The HPLC spectrum is shown in [[ID= .

[0104] According to the above chromatographic conditions, tyrosol standard was subjected to HPLC analysis. The HPLC spectrum is shown in ​ .

[0105] According to the above chromatographic conditions, the product after separation and purification of the enzyme catalytic reaction solution obtained in Example 1 was subjected to HPLC analysis. The HPLC spectrum is shown in ​ .

[0106] ​ 、 ​ 、​ The HPLC spectrum showed that the peak time of the β-salidroside standard was rt=9.72 min, and the peak time of the tyrosol standard was rt=12.78 min. The enzyme-catalyzed reaction solution obtained in Example 1 had two peaks, with peak times of rt=10.16 min and rt=12.83 min, respectively. The peak at rt=12.83 min was tyrosol, and the peak at rt=10.16 min had a significant difference from the peak time of the β-salidroside standard.

[0107] (4) LC-MS analysis

[0108] The material with rt = 10.16min was subjected to LC-MS analysis, and the measured value of the molecular ion peak [M-1] was 299.11. ​ .

[0109] Salidroside standard (molecular formula: C 14 H 20 O7, molecular weight: 300.23) had an HPLC retention time of 9.72 min. LC-MS analysis showed that the molecular ion peak [M-1] had a measured value of 299.11, which was identified as the [MH]+ peak of tyrosol glycoside (molecular weight 300). ​ This indicates that the substance at retention time 10.16 min is a glycoside with a molecular weight of 300.23. Based on the UV spectrum displayed by the photodiode array detector and the LC-MS spectrum, the new peak at retention time rt = 10.16 min in the HPLC spectrum is believed to be a tyrosol glycoside. Because monoglycosides with the same molecular weight exist, preliminary analysis suggests that this substance is a glycoside formed by the combination of glucose and tyrosol.

[0110] (5) H-NMR and C-NMR spectrum analysis

[0111] The substance with rt=10.16 min was subjected to NMR analysis, and the H-NMR spectrum is shown in ​ , C-NMR spectrum see ​ .

[0112] H-NMR spectrum of the product of Example 1: 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) .

[0113] C-NMR spectrum of the product of Example 1: 13C NMR (151 MHz, DMSO): δ 35.2, 61.4, 68.8, 70.7, 72.4, 73.3, 73.7, 99.0, 115.5, 129.3, 130.2, 156.0.

[0114] According to the H-NMR spectrum and C-NMR spectrum of the product in Example 1, combined with the H-NMR spectrum of the salidroside standard (see ​ ), the H-NMR spectrum of the product of Example 1 has a coupling constant of 3.6 Hz at 4.68 ppm (d, J = 3.6 Hz, 1H: β-H), which is a "characteristic of α-glycosidic bond". The standard salidroside (β-salidroside) has two peaks at 4.29 ppm with a coupling constant of 8.0 Hz, which is a characteristic of β-salidroside. The two are significantly different. Combined with the literature (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), it was determined that the product of Example 1 was α-glucose glycosylated. TLC analysis showed that the two substances were at the same point. LC-MS analysis showed that the two substances had the same molecular weight. HPLC analysis showed that the two substances were two different substances. Combined with the α-bond characteristics in the NMR spectrum (different from the natural β-bond), it was confirmed that the substance was α-salidroside, an isomer of β-salidroside (natural salidroside). Its structural formula is as follows:

[0115] .

[0116] (6) The enzyme-catalyzed reaction solutions obtained in Examples 2 to 9 were separated and purified, and analyzed by TLC, HPLC, LC-MS, and NMR. The results were consistent with those in Example 1.

[0117] HPLC detection showed that the conversion rates of tyrosol to α-salidroside in Examples 1 and 2 were 2% and 3%, respectively. α-salidroside was detected in the enzyme-catalyzed reaction solutions obtained in Examples 3 to 9, but the content was very low.

[0118] Example 11. Glycosylation using microbial cultures

[0119] Step 1. Preparation of agar medium

[0120] Maltose (Nippon Shokuhin Kako) 5g, yeast extract (Becton Dickinson) 100mg, ammonium sulfate (Wako Pure Chemical Industries, Japan) 2g, potassium dihydrogen phosphate (Kanto Chemical Co., Inc., Japan) 1g, dipotassium hydrogen phosphate (Wako Pure Chemical Industries, Japan) 1g, magnesium sulfate heptahydrate (Kanto Chemical Co., Inc., Japan) 0.2g, ferric sulfate heptahydrate (Kanto Chemical Co., Inc., Japan) 0.01g, and 15g agar BA-10 (Ina Food Industry, Japan) Co., Ltd.), transfer the above reagents to a 2-L beaker, add 1 L of distilled water to dissolve, adjust the pH to 7.0 with NaOH, sterilize the solution in an autoclave at 121°C for 20 min, and then dispense into culture dishes on a clean bench, 15 mL / dish; gently open the culture dish lid, let it stand for 20 minutes to cool, and use it as an agar medium.

[0121] Step 2. Preparation of liquid culture medium

[0122] 50 g of maltose (Nippon Shokuhin Kako), 2 g of polyvalent peptone (Wako Pure Chemical Industries, Japan), 100 mg of yeast extract (Becton Dickinson), 2 g of ammonium sulfate (Wako Pure Chemical Industries, Japan), 1 g of potassium dihydrogen phosphate (Kanto Chemical Co., Inc., Japan), and 1 g of dipotassium hydrogen phosphate (Wako Pure Chemical Industries, Japan) were added to a 2 L beaker and dissolved in 1 L of distilled water. The pH was adjusted to 7.0 with NaOH. 50 mL of the solution was placed in a 200 mL conical flask and sterilized in an autoclave at 121°C for 20 min. The solution was cooled and used as a liquid culture medium.

[0123] Step 3. Preparation of crude enzyme solution

[0124] The above agar medium was cultured at 25 ° C for 2 to 3 days. ​ sp. M-238 (NITE Accession No. AP-02396) was inoculated into the liquid culture medium described in step 2 using two loops of inoculation and shaken at 160 rpm at 25°C for 42 hours. After incubation, the cells were removed by centrifugation, the supernatant was salted out with ammonium sulfate, desalted, and the glycosyltransferases were fractionated using anion exchange resin to obtain a crude enzyme solution.

[0125] Step 4. Glycosylation reaction

[0126] Dissolve 10 mg of tyrosol (molecular weight 138.16) and 20 mg of maltose in 1 mL of 0.1 M phosphate buffer (pH 7.0), add 20 μL of the crude enzyme solution obtained above, and stir at 30°C overnight to obtain an enzyme-catalyzed reaction solution.

[0127] The formation of α-salidroside was confirmed by TLC, HPLC and LC-MS analysis; HPLC analysis showed that the conversion rate of tyrosol to α-salidroside was approximately 20%.

[0128] The above experiments found that the glycosyltransferase is derived from Arthrobacter ( ​ sp.), the conversion rate of tyrosol to α-salidroside was 20%. Compared with the above commercial enzyme preparations, the conversion rate of tyrosol to α-salidroside was greatly improved.

[0129] Feasibility of industrial use:

[0130] The present invention utilizes glycosyltransferases, particularly those derived from the genus Arthrobacter ( ​Sp.) glycosyltransferases can glycosylate tyrosol derivatives. Glycosylated glycosides not only retain the inherent properties of tyrosol derivatives but also further enhance their physiological functions, thereby increasing their development value and enabling them to be used as raw materials for cosmetics, health products, and pharmaceuticals.

[0131] The following examples are free radical scavenging experiments, and the materials and reagents used are shown in Table 2.

[0132] Table 2. Materials and reagents

[0133] .

[0134] Experimental Example 12. DPPH free radical scavenging experiment

[0135] Free radicals exert strong oxidative effects, both directly and indirectly, and are widely involved in physiological and pathological processes. Excessive free radicals can damage the body through oxidative damage. Salvianolic acid compounds are phenolic hydroxyl group donors, providing the structural basis for their antioxidant activity. This study used the DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) radical scavenging reaction to investigate the scavenging efficiency of α-salidroside and β-salidroside against DPPH radicals.

[0136] 1. Solution Preparation:

[0137] (1) α-Salidroside solution was prepared as a 500 mg / L aqueous solution according to conventional methods.

[0138] (2) β-Salidroside solution was prepared as a 500 mg / L aqueous solution according to conventional methods.

[0139] (3) DPPH ethanol solution: Prepare 0.04 mg / ml anhydrous ethanol solution according to conventional methods.

[0140] 2. DPPH free radical scavenging experiment:

[0141] (1) Take 0.9 mL of α-salidroside solution and 0.9 mL of β-salidroside aqueous solution, add 0.9 mL of DPPH anhydrous ethanol solution, mix well, and place in a dark place for 2 h. Samples were taken at 0.5 h, 1 h, 2 h, and 4 h, and the absorbance was measured at a wavelength of 517 nm. The average value of the three replicates was calculated and recorded as Ai.

[0142] (2) Take 0.9 mL of α-salidroside solution and 0.9 mL of β-salidroside solution, add 0.9 mL of anhydrous ethanol, mix well, and place in the dark for 1 h. Samples are taken at 0.5 h, 1 h, 2 h, and 4 h, and the absorbance is measured at a wavelength of 517 nm. The average value of the three parallel samples is calculated and recorded as Aj;

[0143] (3) Take 0.9 mL of distilled water, add 0.9 mL of DPPH ethanol solution, mix well, measure the absorbance at a wavelength of 517 nm, and calculate the average of three parallel measurements, which is recorded as A4;

[0144] (4) Add 0.9 mL of anhydrous ethanol to 0.9 mL of distilled water and mix well. This will serve as a blank.

[0145] The DPPH free radical scavenging rate was calculated according to the following formula and the results are recorded in Table 3.

[0146] .

[0147] Table 3. DPPH free radical scavenging rate

[0148] .

[0149] The data in Table 3 show that α-salidroside has a higher ability to scavenge DPPH free radicals than β-salidroside, especially when the reaction time is more than 1 hour.

[0150] Experimental Example 13. Hydroxyl radical scavenging ability

[0151] Hydroxy free radical scavenging activity is an important indicator for measuring the antioxidant capacity of a substance. Currently, the main analytical testing methods for hydroxyl radicals include high-performance liquid chromatography, chemiluminescence, fluorescence analysis, and spectrophotometry. The most commonly used method for determining hydroxyl radical scavenging activity is spectrophotometry, where the sample inhibits the decrease in the absorbance of the color-developing substance o-phenanthroline, thereby reflecting the sample's ability to scavenge hydroxyl radicals. The determination principle is: H2O2 / Fe 2+ The hydroxyl radicals are generated by Fenton reaction and the o-phenanthroline-Fe 2+ Fe in aqueous solution 2+ Oxidized to Fe 3+ , resulting in a decrease in the absorbance at 536 nm. The degree to which the sample inhibits the rate of decrease in absorbance at 536 nm reflects the ability of the sample to scavenge hydroxyl radicals.

[0152] The degree of inhibition of the absorbance decrease rate reflects the ability of the sample to scavenge hydroxyl radicals.

[0153] The produced hydroxyl radicals can be indirectly determined by measuring the change in the absorbance value of the indicator.

[0154] 1. Solution Preparation:

[0155] (1) FeSO4 solution: Prepare 1.5 mM and 5 mM FeSO4 aqueous solutions respectively according to conventional methods.

[0156] (2) PBS buffer:

[0157] 1) Prepare 0.2 mol / L sodium dihydrogen phosphate and 0.2 mol / L sodium hydrogen phosphate aqueous solutions;

[0158] 2) Take 19 ml of sodium dihydrogen phosphate aqueous solution and 81 ml of disodium hydrogen phosphate aqueous solution, mix well, and adjust the pH to 7.4 (measure with a pH meter; 0.1 mol / L sodium hydroxide or 0.1 mol / L phosphoric acid can be used to adjust the pH).

[0159] (3) 1,2-Diazophenanthroline: Prepare 0.75 mM and 3 mM aqueous solutions respectively according to conventional methods.

[0160] (4) Hydrogen peroxide solution: Prepare a 0.1% (v / v) aqueous solution according to conventional methods.

[0161] (5) α-Salidroside solution: Prepare 1 mg / mL and 4 mg / mL aqueous solutions respectively according to conventional methods.

[0162] (6) Preparation of β-salidroside solution: 1 mg / mL and 4 mg / mL aqueous solutions were prepared according to conventional methods.

[0163] 2. Hydroxyl radical scavenging experiment:

[0164] Scheme 1: Use 5 mMFeSO4 aqueous solution, 0.1% hydrogen peroxide, 1 mg / mL α-salidroside aqueous solution or 1 mg / mL β-salidroside aqueous solution, and 3 mM phenanthroline aqueous solution to carry out the reaction according to the following steps.

[0165] Scheme 2: Use 1.5 mM FeSO4 aqueous solution, 0.1% hydrogen peroxide, 4 mg / mL α-salidroside aqueous solution or 4 mg / mL β-salidroside aqueous solution, and 0.75 mM phenanthroline aqueous solution to carry out the reaction according to the following steps.

[0166] Specific operation steps (1) Take 40 μL of α-salidroside solution and 40 μL of β-salidroside solution, add 40 μL of FeSO4 solution, 60 μL of PBS buffer (pH 7.4), and 40 μL of o-phenanthroline solution respectively, mix well, then add 40 μL of H2O2 solution, mix well, react at 37℃ in the dark for 30 minutes, measure the absorbance at a wavelength of 536 nm with distilled water as blank, calculate the average value of three parallels, and record it as A i ;

[0167] (2) Take 80 μL of distilled water, add 40 μL of FeSO4 solution, 60 μL of PBS buffer (pH 7.4), and 40 μL of o-phenanthroline solution, mix well, and react at 37°C in the dark for 30 minutes. Measure the absorbance at a wavelength of 536 nm using distilled water as a blank, and calculate the average value of three replicates, which is recorded as A. j ;

[0168] (3) Take 40 μL of distilled water, add 40 μL of FeSO4 solution, 60 μL of PBS buffer (pH 7.4), and 40 μL of o-phenanthroline solution, mix well, then add 40 μL of H2O2 solution, mix well, and incubate at 37°C in the dark for 30 min. Measure the absorbance at 536 nm using distilled water as a blank, and calculate the average of three replicates, which is recorded as A0.

[0169] .

[0170] The hydroxyl radical scavenging results are recorded in Table 4.

[0171] Table 4. Hydroxyl radical scavenging rate

[0172] .

[0173] The data of the two detection methods in Table 4 show that the ability of α-salidroside to scavenge hydroxyl radicals is much higher than that of β-salidroside.

[0174] Although the present invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that any equivalent aspects or modifications may be implemented. Therefore, the description and examples should not be construed as limiting the scope of the invention.

Claims

1. A method for preparing α-salidroside, characterized in that: The method 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 microbial source of the glycosyltransferase is selected from the genus Arthrobacter (Arthrobacter sp.).

2. The preparation method according to claim 1, wherein The glycosyl donor is one or more of maltose, maltotriose, glucose, fructose, starch, water-soluble starch, and dextrin.

3. The preparation method according to claim 1, wherein In the reaction system, the concentration of tyrosol is 5-400 mg / mL.

4. The preparation method according to claim 1, wherein In the reaction system, the concentration of tyrosol is 5-100 mg / mL.

5. The preparation method according to claim 1, wherein In the reaction system, the concentration of the glycosyl donor is 0.5 to 30 times the concentration of tyrosol.

6. The preparation method according to claim 1, wherein In the reaction system, the concentration of the glycosyl donor is 2 to 10 times the concentration of tyrosol; 7. The preparation method according to claim 1, wherein The buffer solution is a phosphate buffer solution, an acetate buffer solution or a Good's buffer solution. The concentration of the buffer solution is 0.01M to 0.5M, and the pH of the buffer solution is 5 to 10.

8. The preparation method according to claim 1, wherein The microbial source of the glycosyltransferase enzyme is selected from Arthrobacter sp. M-238.

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