Piceatannol glucoside with alpha-glucosidase inhibitory activity as well as preparation method and application of piceatannol glucoside

Abstract

The invention discloses piceatannol glucoside with alpha-glucosidase inhibitory activity as well as a preparation method and application of the piceatannol glucoside. The piceatannol glucoside is prepared from a glycosyl donor and piceatannol through a glycosylation reaction, and the structural formula of the piceatannol glucoside is # imgabs0 #. The piceatannol glucoside is obtained by adding piceatannol and an enzyme catalyst into a sugar solution, carrying out an oscillating reaction and carrying out separation and purification. The water solubility, pH stability, temperature stability and light stability of the synthesized piceatannol glucoside are obviously improved compared with those of an original compound, and the piceatannol glucoside also has antioxidant activity and alpha-glucosidase inhibitory activity. The application of piceatannol in the fields of food, medicine and the like is favorably promoted. The method has the advantages of simplicity in operation, mild conditions, environment friendliness, high selectivity and the like.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly relates to a piceatannol glucoside having α-glucosidase inhibitory activity, a preparation method thereof, and an application thereof. Background Art

[0002] Piceatannol is a stilbene compound, mainly derived from natural plants such as passion fruit, blueberry, and grape. A large number of studies have shown that piceatannol has various bioactive functions such as hypoglycemic, anti-obesity, antioxidant, and anti-cancer effects. However, the solubility of piceatannol in water at 25°C is only about 0.5 mg / mL, and it is easily degraded under high temperature and alkaline conditions. The poor water solubility and stability of piceatannol result in low bioavailability, which limits its application in the fields of functional foods and pharmaceuticals.

[0003] At present, glycosylation modification has become an effective means to improve the water solubility of polyphenolic compounds such as piceatannol. At present, the common glycosylation modification pathways mainly include chemical methods and biological methods. Although the chemical method has a certain degree of feasibility, this method usually requires a series of complex processes such as protection, activation, coupling, and deprotection, and has disadvantages such as poor selectivity, many by-products, and large solvent consumption. At the same time, it is also easy to cause environmental pollution problems. In contrast, biocatalysis shows significant advantages in the glycosylation modification process of polyphenolic compounds. The reaction process of biocatalysis is more streamlined, with less steric hindrance, high regioselectivity, and relatively few by-products. Moreover, the subsequent treatment steps of the enzymatic reaction are simple, the production cost can be effectively controlled, the reaction conditions are mild, and it is more environmentally friendly.

[0004] In the process of enzymatic glycosylation modification, glycosyltransferases and glycosidases are mainly used as catalysts. In the synthesis reaction catalyzed by glycosyltransferases, it is often necessary to use expensive activated nucleoside phosphosugars as glycosyl donors, which have low practical value. Glycosidases, although they have rich sources and broad substrate specificity, usually have the problem of low conversion rate. Cyclodextrin glucosyltransferase, as a glycosyltransferase, can catalyze four types of reactions: cyclization, coupling, disproportionation, and hydrolysis. In particular, its coupling and disproportionation activities can be applied to the glycosylation modification of polyphenolic compounds.

[0005] Le et al. (Highly regioselective hydroxylation of polydatin, a resveratrol glucoside, for one-step synthesis of astringin, a piceatannol glucoside, by P450BM3) used the cytochrome P450 enzyme CYP102A1 mutant to perform regioselective hydroxylation at the C-3′ position of polydatin, achieving highly regioselective one-step synthesis of piceatannol 3-O-β-D-glucoside. Although the synthesized piceatannol 3-O-β-D-glucoside showed good stability under weakly acidic conditions (pH 6), its retention rates decreased to 70% and 7% respectively after 8 hours of incubation under neutral (pH 7) and alkaline (pH 8) conditions. Li et al. (Scalable Total Synthesis of Piceatannol-3′-O-β-D-glucopyranoside and the 4′-Methoxy Congener Thereof: An Early Stage Glycosylation Strategy) constructed the glucosyl backbone through Fischer glycosylation, combined with regioselective iodination of phenolic hydroxyl groups under acidic conditions and Mizoroki-Heck coupling reaction, and prepared piceatannol-3'-O-β-D-glucopyranoside and its methoxy derivatives in a nine-step route. However, the Mizoroki-Heck reaction led to a side reaction of deacetylation of phenolic ester groups under alkaline conditions, which needed to be repaired by in-situ addition of acetic anhydride through post-treatment, increasing the operation complexity; at the same time, the regioselective iodination relied on a strongly acidic system (a mixed solvent of trifluoroacetic acid / dichloromethane), which put forward high requirements for the corrosion resistance of equipment and reaction control; in addition, multiple steps involved expensive reagents (such as the protecting group reagent TBSCl and the palladium catalyst for the Heck reaction) and low-temperature conditions (-78 °C), significantly increasing the large-scale production cost. Both of the above methods achieved glycosylation of piceatannol, but the glycosidic bonds were all in the β configuration, and no further studies were carried out on the solubility, stability, and biological activity of the piceatannol glycosylation products.

[0006] Therefore, it is of great significance to develop a low-cost, low-pollution and efficient method for preparing piceatannol glucoside with high water solubility, high stability, strong antioxidant activity and hypoglycemic activity. In view of the current research status, the present invention proposes a green and efficient synthetic strategy for piceatannol glucoside. This strategy uses an enzyme catalyst and a green reaction solvent to realize the greening of the whole process of glycosylation reaction. In the present invention, piceatannol and an enzyme catalyst are added to a sugar solution and subjected to an oscillating reaction. The sugar solution can not only dissolve piceatannol well, but also provide a glycosyl donor for the glycosylation reaction, thus successfully realizing the efficient and green biosynthesis of piceatannol glucoside, and further exploring the physicochemical properties and biological activities of this glycosylation product, including water solubility, pH stability, temperature stability, light stability, antioxidant activity and hypoglycemic activity. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a method for biocatalytic synthesis of piceatannol glucoside, which has the characteristics of high reaction efficiency, mild conditions, strong specificity and less environmental pollution. At the same time, the present invention provides a novel piceatannol derivative that can effectively inhibit the activity of α-glucosidase and has strong antioxidant activity.

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] A piceatannol glucoside is obtained by reacting piceatannol and an enzyme catalyst in a sugar solution, and its structural formula is as follows:

[0010]

[0011] The present invention provides a method for preparing piceatannol glucoside, comprising the following steps:

[0012] Piceatannol and an enzyme catalyst are added to a sugar solution, and after mixing evenly, a mixed solution is obtained. The glycosylation reaction is carried out under an oscillating state, and then piceatannol glucoside is obtained by separation and purification.

[0013] The obtained piceatannol glucoside is piceatannol-3′-O-α-D-glucoside.

[0014] The obtained piceatannol glucoside has improved water solubility, pH stability, temperature stability and light stability compared with piceatannol; the IC 50 for the DPPH radical scavenging rate is < 6.5 μg / mL; the IC 50 for the ABTS radical scavenging rate is < 4.0 μg / mL; the IC 50 for the α-glucosidase inhibitory activity is < 15 μg / mL.

[0015] Preferably, the solute in the sugar solution is at least one of soluble starch, maltose, α-cyclodextrin, β-cyclodextrin, and maltodextrin.

[0016] Preferably, the mass ratio of the solute in the sugar solution to piceatannol is 25:6 to 400:6.

[0017] More preferably, the concentration of the sugar solution is 25 mg / mL to 400 mg / mL.

[0018] Preferably, the enzyme catalyst is cyclodextrin glucosyltransferase Toruzyme 3.0L.

[0019] More preferably, the concentration of cyclodextrin glucosyltransferase in the reaction solution is 0.1% to 2.5% (v / v).

[0020] Preferably, the temperature of the glycosylation reaction is 30 to 60 °C, the time is 0.25 to 24 h, the pH of the reaction solution is 4 to 9, and the oscillation speed is 100 to 180 rpm.

[0021] More preferably, the reaction temperature is 50 °C, the reaction time is 8 h, the pH of the reaction solution is 6.0, and the oscillation speed is 150 rpm.

[0022] After treating piceatannol with the enzyme catalyst as described above, piceatannol glucoside is generated, and the substrate conversion rate reaches more than 70%. The water solubility of piceatannol-3′-O-α-D-glucoside is measured to be 28.63 mg / mL.

[0023] Application of the piceatannol glucoside obtained by the above method in the preparation of an antioxidant.

[0024] Application of the piceatannol glucoside obtained by the above method in the preparation of an α-glucosidase inhibitor.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] (1) The present invention provides a method for biocatalytic synthesis of piceatannol glucoside. The prepared piceatannol glucoside has better water solubility, pH stability, temperature stability, and light stability than the original compound, and the substrate conversion rate can reach more than 70%.

[0027] (2) The present invention uses a green solvent sugar solution as the reaction system for biocatalytic reaction. Compared with chemical methods and other organic solvent systems, the conditions of the present invention are simple, the cost is low, and it is environmentally friendly.

[0028] (3) The present invention uses cyclodextrin glucosyltransferase as a catalyst for the glycosylation reaction of piceatannol. Under certain enzyme-catalyzed conditions, this enzyme can efficiently catalyze the glycosylation reaction of phenolic glycoside compounds. The reaction steps are simple, with high regioselectivity and fewer by-products. In addition, this enzyme has a wide source, mild reaction conditions and is environmentally friendly.

[0029] (4) Cyclodextrin glucosyltransferase is expensive, and previous methods are difficult to be applied industrially. The method of the present invention overcomes the defect of high cost, realizes the efficient catalysis of glycosylation reaction with a very small amount of enzyme. Even when the enzyme dosage is significantly reduced, it can still efficiently promote the reaction to achieve the glycosylation of piceatannol. It not only ensures that the reaction product has good water solubility, but also maintains excellent α-glucosidase inhibitory activity, providing a more economical, efficient and environmentally friendly solution for industrial production and application in related fields.

[0030] (5) The piceatannol glucoside of the present invention has a stronger ABTS radical scavenging rate than the commonly used antioxidant VC, and the inhibition of the activity of α-glucosidase is significantly better than that of the original compound piceatannol. Description of the Drawings

[0031] Figure 1 It is the liquid chromatography diagram of the substrate detection in the reaction system at 24 hours of the glycosylation reaction described in Example 1.

[0032] Figure 2 It is the mass spectrum of piceatannol-3′-O-α-D-glucoside.

[0033] Figure 3 It is the nuclear magnetic resonance carbon spectrum of piceatannol.

[0034] Figures 4 to 7 They are respectively the nuclear magnetic resonance carbon spectrum, nuclear magnetic resonance hydrogen spectrum, HSQC spectrum, and NOESY spectrum of piceatannol glucoside.

[0035] Figure 8 It is the retention rate of piceatannol and piceatannol glucoside at different pH values.

[0036] Figure 9 It is the retention rate of piceatannol and piceatannol glucoside at different temperatures.

[0037] Figure 10 It is the retention rate of piceatannol and piceatannol glucoside under different ultraviolet irradiation times.

[0038] Figure 11 It is the inhibitory activity of acarbose (a), piceatannol (b) and piceatannol glucoside (c) on α-glucosidase.

[0039] Figure 12The ability of VC (a), piceatannol (b), and piceatannol glucoside (c) to scavenge DPPH free radicals.

[0040] Figure 13 The ability of VC (a), piceatannol (b), and piceatannol glucoside (c) to scavenge ABTS free radicals. Detailed implementation mode

[0041] The following further illustrates the specific implementation of the present invention in conjunction with embodiments, but the implementation and protection of the present invention are not limited thereto. It should be noted that for the processes not specifically described in detail below, those skilled in the art can implement or understand them with reference to the prior art. For those not specified in the embodiments, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0042] In the embodiments of the present invention, the cyclodextrin glucosyltransferase used is the commercial Toruzyme 3.0L (from Thermoanaerobacter.sp), purchased from Novozymes.

[0043] The detection method used in the following examples is the high performance liquid chromatography (HPLC) determination method. The chromatographic column used in the HPLC determination method is an Agilent Zorbax SB-C18 column (4.6 mm × 250 mm, 5 μm); the mobile phase is 30% (v / v) acetonitrile-aqueous solution (containing 0.1% (v / v) formic acid); the elution method is isocratic elution; the flow rate is 0.9 mL / min, the detection wavelength is 308 nm; the injection volume is 20 μL; the column temperature is 30 °C.

[0044] Among them, the calculation method of the substrate conversion rate of the glycosylation reaction of piceatannol is shown in Formula 1-1.

[0045] Conversion rate (%) = [(S0 – S t ) / S0] × 100 (1-1)

[0046] In the formula, S0 and S t respectively represent the peak areas of piceatannol at the start of the reaction and after reaction time t.

[0047] Example 1

[0048] A method for catalytically synthesizing piceatannol glucoside using cyclodextrin glucosyltransferase Toruzyme 3.0L includes the following steps:

[0049] Using piceatannol as the reaction substrate, with a reaction system volume of 2 mL, 12 mg of piceatannol and cyclodextrin glucanotransferase enzyme solutions at concentrations of 0.1%, 1.5%, and 2.5% (v / v) were respectively added to 1.8 mL of a 150 mg / mL α-cyclodextrin solution. After mixing evenly, the reaction was carried out with shaking in a constant temperature shaking incubator (50 °C, 150 r / min, pH = 6.0). After 24 h of reaction, 50 μL of the sample was taken, centrifuged at 8000 r / min for 5 min, and 20 μL of the supernatant was diluted 50 times with methanol and then detected and analyzed by high performance liquid chromatography (HPLC).

[0050] After 24 h of reaction, the substrate conversion rates of the enzyme solutions at 0.1%, 1.5%, and 2.5% (v / v) for the glycosylation reaction of piceatannol were 71.67%, 75.83%, and 79.04% respectively. The HPLC analysis results of the products are as Figure 1 shown, and a new product peak was generated at 3.463 min. By comparing with the piceatannol standard, it was known that the retention time of the substrate peak was 5.734 min, and it was speculated that the newly generated peak was the peak of the glycosylation derivative of piceatannol.

[0051] The products catalyzed by cyclodextrin glucanotransferase as the catalyst were identified by high resolution mass spectrometry (HRMS). The mass spectra are respectively as Figure 2 shown. The glycosylation reaction product of piceatannol showed an [M+Na] + ion peak at m / z 429.1164, and its molecular formula was C 20 H 22 O9, and the molecular weight was 162 higher than that of the substrate piceatannol (C 14 H 12 O4), that is, the product differed from piceatannol by one glucose residue, and it was speculated that the glycosylation reaction of piceatannol produced piceatannol monoglucosylated derivative.

[0052] The structure of the biocatalytically obtained piceatannol glucoside was identified using nuclear magnetic resonance carbon spectrum ( 13 C-NMR), nuclear magnetic resonance hydrogen spectrum ( 1 H-NMR), HSQC spectrum, and NOESY spectrum. The nuclear magnetic resonance spectra of piceatannol and its glucoside are as Figures 3 to 7 shown.

[0053] By comparing the signal peaks in the 13 C-NMR spectra of piceatannol and piceatannol glucoside, it was found that piceatannol glucoside had 6 more C atoms than piceatannol. According to the HSQC spectrum of this compound, the anomeric carbon (C-1”) and hydrogen signal (H-1”) of the sugar ring could be quickly found to be δ C 100.3 and δ H5.25 (d, J = 3.6 Hz, 1H). The coupling constant of the anomeric proton is relatively small, confirming that the glycosidic bond configuration is α. In the NOESY spectrum, a correlation signal was observed between H-2' (7.45 ppm) and H-1'' (5.25 ppm), so it was inferred that the sugar ring was located at the 3'-position carbon. In summary, the structure of the piceatannol glycosylation derivative was identified as piceatannol-3′-O-α-D-glucoside.

[0054] Separation and purification method of piceatannol-3′-O-α-D-glucoside: The reaction solution was heated in a boiling water bath for 10 min to denature and precipitate the enzyme protein, and then centrifuged at 10000 r / min for 15 min and filtered to remove the precipitate. A n-butanol-aqueous solution was prepared according to a volume ratio of 1:1, shaken vigorously and allowed to stand for phase separation, and the upper water-saturated n-butanol phase was collected for later use. An equal volume of water-saturated n-butanol was added to the enzyme-removed reaction solution, shaken and extracted with a separating funnel and then allowed to stand for phase separation, and the upper organic phase was taken for rotary evaporation to remove the solvent at 60 °C. The obtained residue was redissolved with an acetonitrile aqueous solution to obtain a crude product solution. The above crude product solution was filtered through a 0.22 μm filter membrane, and then separated and purified using a semi-preparative chromatographic column Zorbax SB-C18 (9.4 mm × 250 mm, 5 μm). Mobile phase: 20% (v / v) acetonitrile-aqueous solution (containing 0.1% (v / v) formic acid) gradient elution; flow rate: 2.5 mL / min; column temperature: 25 °C; injection volume: 100 μL. The separated product was freeze-dried for later use.

[0055] Example 2

[0056] Water solubility determination of piceatannol glucoside

[0057] Weigh 3 mg of dry piceatannol and its glycosylation derivative ground into fine powder in centrifuge tubes, add 50 μL of ultrapure water, sonicate at room temperature for 1 h, and after sonication, place the centrifuge tubes in a constant temperature shaker at 25 °C and 100 r / min for shaking culture for 24 h. When the solid / liquid two-phase reaches equilibrium, centrifuge at a speed of 6000 r / min for 10 min in a small centrifuge. Take 20 μL of the supernatant, dilute it with chromatographically pure methanol and filter it through a 0.22 μm filter membrane, and perform HPLC detection and analysis, and then calculate the saturated solubility of each sample at 25 °C according to the corresponding standard curve.

[0058] The calculated saturated solubilities of piceatannol and piceatannol-3′-O-α-D-glucoside at 25 °C were 0.83 mg / mL and 28.63 mg / mL respectively, indicating that the water solubility of the synthesized piceatannol glucoside was significantly improved compared with piceatannol.

[0059] Example 3

[0060] pH stability evaluation of piceatannol glucoside

[0061] Prepare acetate buffer solutions with a concentration of 50 mM (pH 3.0, 4.0, 5.0) and phosphate buffer solutions with a concentration of 50 mM (pH 6.0, 7.0, 8.0, 9.0) to evaluate the pH stability of piceatannol glucoside. Mix 500 μL of a sample solution with a concentration of 1 mg / mL with 1500 μL of buffer solutions with different pH values. Place the mixture in a constant temperature shaking incubator at 25 °C and 120 r / min, shake it, take samples after 24 h, dilute with methanol, and perform HPLC analysis to determine the retention amounts of piceatannol and its glycosylated derivatives and calculate the retention rate.

[0062] The results are as Figure 8 shown. AST and AST-G1 in the figure represent piceatannol and piceatannol-3′-O-α-D-glucoside respectively. The pH stability of piceatannol after glycosylation modification is significantly improved, and its stability is significantly better than that of piceatannol in the range of pH 6-8.

[0063] Example 4

[0064] Evaluation of the temperature stability of piceatannol glucoside

[0065] Mix 500 μL of a 1 mg / mL sample solution with 1500 μL of a phosphate buffer solution with pH = 6.0. Place the mixed solution in a water bath at different temperatures (40, 50, 60, 70, 80, 90 °C) for 1 h, quickly cool the reaction solution, take samples, and perform HPLC detection and analysis to determine the retention amounts of piceatannol and its glycosylated derivatives and calculate the retention rate.

[0066] The results are as Figure 9 shown. AST and AST-G1 in the figure represent piceatannol and piceatannol-3′-O-α-D-glucoside respectively. In the temperature range (40-90 °C) studied in this experiment, the retention amount of piceatannol modified by glucosylation is higher than that of the original compound, indicating that glycosylation modification improves the stability of the product of piceatannol in the range of 40-90 °C.

[0067] Example 5

[0068] Evaluation of the light stability of piceatannol glucoside

[0069] Mix 500 μL of a 1 mg / mL sample solution with 1500 μL of methanol solution. Place the mixed solution under ultraviolet light at 254 nm for irradiation for 0, 5, 10, 20, 40, 60, 90, and 120 minutes. Take samples and perform HPLC detection and analysis to determine the retention amounts of piceatannol and its glycosylated derivatives and calculate the retention rate.

[0070] The results are as Figure 10As shown, within 0 to 120 minutes of ultraviolet light irradiation, the retention of piceatannol glucoside was significantly higher than that of the original compound piceatannol, indicating that glycosylation modification improved the stability of piceatannol under ultraviolet light irradiation.

[0071] Example 6

[0072] Evaluation of the inhibitory activity of piceatannol glucoside against α-glucosidase

[0073] Mix 20 μL of piceatannol and its glycosylated derivatives respectively with 20 μL of α-glucosidase at 1 U / mL. Use the same volume of acarbose as the positive control. Incubate at 37 °C for 15 min. Then add 20 μL of 2.5 mM p-nitrophenyl-α-glucoside to the above solution. Incubate at 37 °C, 80 r / min for 30 min. Then keep the reaction mixture in an ice-water bath for 5 min. Finally, terminate the reaction with 80 μL of 0.2 mM Na2CO3. Measure the absorbance at 405 nm. The calculation method of the inhibition rate is shown in formula 2-1:

[0074] α-glucosidase inhibition rate (%) = [(A1 - A2) - (A3 - A4)] / (A1 - A2) × 100 (2-1)

[0075] In the formula: A1 is the absorbance value of the blank group at 405 nm

[0076] A2 is the absorbance value of the blank control group at 405 nm

[0077] A3 is the absorbance value of the sample group at 405 nm

[0078] A4 is the absorbance value of the sample control group at 405 nm

[0079] Among them, the blank group is α-glucosidase + water; the blank control group is PBS + water; the sample group is α-glucosidase + the sample to be tested; the sample control group is PBS + the sample to be tested; the sample to be tested is piceatannol, piceatannol monoglucoside or acarbose.

[0080] The results are as Figure 11 shown. Both piceatannol and its monoglucoside showed inhibitory activity against α-glucosidase. The IC 50 values were 26.31 μg / mL and 11.18 μg / mL respectively. The inhibitory activity of piceatannol monoglucoside was about 2.4 times higher than that of its aglycone. Although the inhibitory strengths of both were lower than that of the positive control acarbose (IC 50= 0.015 μg / mL), but piceatannol compounds act through a non-competitive inhibition mechanism, and this property may reduce the risk of gastrointestinal side effects caused by substrate accumulation, indicating that piceatannol derivatives have potential advantages over traditional competitive inhibitors in the treatment of diabetes.

[0081] Example 7

[0082] Scavenging effect of piceatannol glucoside on DPPH free radicals

[0083] Prepare piceatannol solutions with concentrations of 1, 2, 3, 5, 10, 20, 40, 60, 80, 100, 200, 400 μg / mL and piceatannol glucoside solutions with concentrations of 1, 2.5, 5, 10, 15, 20, 30, 80, 100, 200 μg / mL. The positive control is replaced with the same volume of ascorbic acid (VC). Take 100 μL of DPPH solution with a concentration of 0.065 mg / mL, mix it with 100 μL of the sample solution to be tested and 100 μL of absolute ethanol respectively. After rapid shaking and mixing, react in the dark at room temperature for 30 min. Use an enzyme-linked immunosorbent assay reader to measure the absorbance at 515 nm. According to the following formula 2-2, calculate the DPPH free radical scavenging ability of piceatannol and its glycosylated derivatives, and then calculate the IC of the corresponding sample according to the fitting equation 50 .

[0084] DPPH scavenging rate (%) = (1 - (A1 - A2) / A0) × 100 (2-2)

[0085] In the formula: A0 is the absorbance value of the blank group at 515 nm

[0086] A1 is the absorbance value of the sample group at 515 nm

[0087] A2 is the absorbance value of the sample control group at 515 nm

[0088] Among them, the blank group is DPPH + water; the sample group is DPPH + the sample to be tested; the sample control group is absolute ethanol + the sample to be tested, where the sample to be tested is piceatannol, piceatannol monoglucoside or VC.

[0089] The results are as Figure 12 shown. Piceatannol (IC 50 = 5.10 μg / mL) shows strong antioxidant ability comparable to that of the positive control VC (IC 50 = 5.09 μg / mL), and piceatannol monoglucoside (IC 50The DPPH scavenging ability of piceatannol glucoside (6.40 μg / mL) is weaker than that of its aglycone, piceatannol. Generally, the antioxidant ability of glycosylated products is weaker than that of the original compounds. Even attaching glycosides at certain sites can lead to the direct loss of antioxidant activity. The enzymatic glycosylation reaction improved in the present invention has a specific selectivity and does not shield the key groups responsible for antioxidant activity. After glycosylation modification of piceatannol, although its antioxidant activity decreases, due to the significant increase in water solubility, it can be efficiently absorbed in the gastrointestinal tract and maintain a high concentration. Hepatic-enteral circulation and enzymatic deglycosylation finally convert it into piceatannol with stronger activity, thereby exerting antioxidant, anti-proliferation and other effects. This "prodrug strategy" improves the bioavailability and efficacy of piceatannol by optimizing the balance between solubility and activity.

[0090] Example 8

[0091] Scavenging effect of piceatannol glucoside on ABTS radicals

[0092] The experimental steps for determining the ABTS radical scavenging ability of piceatannol and its glycoside derivatives based on the Solarbio BC4775 kit are as follows: First, prepare piceatannol solutions with concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 60, 80, 100 μg / mL and piceatannol glucoside solutions with concentrations of 10, 20, 25, 35, 40, 50, 60, 80, 100, 120, 140, 160, 200 μg / mL. The positive control is replaced with the same volume of ascorbic acid (VC). Subsequently, prepare the ABTS working solution. Add 10 μL of the sample solution, 20 μL of reagent four working solution and 170 μL of ABTS working solution to a 96-well plate respectively. After mixing, let it stand in the dark for 6 min. Measure the absorbance at 405 nm using a microplate reader. According to the following formula 2-3, calculate the ABTS radical scavenging ability of piceatannol and its glycosylated derivatives, and then calculate the IC of the corresponding sample according to the fitting equation. 50 。

[0093] ABTS scavenging rate (%) = (1 - (A1 - A2) / A0) × 100 (2-3)

[0094] In the formula: A0 is the absorbance value of the blank group at 405 nm

[0095] A1 is the absorbance value of the sample group at 405 nm

[0096] A2 is the absorbance value of the sample control group at 405 nm

[0097] Among them, the blank group is ABTS + water; the sample group is ABTS + the sample to be tested; the sample control group is water + the sample to be tested, where the sample to be tested is piceatannol, piceatannol monoglucoside or VC.

[0098] As shown in Figure 13 , piceatannol exhibited the strongest antioxidant capacity (IC 50 = 1.63 μg / mL), and its activity was significantly better than that of the positive control VC (IC 50 = 4.42 μg / mL). This is because the polyphenolic hydroxyl structure can enhance the electron transfer efficiency. Although the introduction of sugar groups may reduce the contact efficiency between phenolic hydroxyl groups and free radicals through steric hindrance, resulting in a decrease in antioxidant activity, the scavenging ability of piceatannol monoglucoside (IC 50 = 3.57 μg / mL) against ABTS free radicals was still better than that of the positive control VC.

[0099] In summary, the present invention synthesized piceatannol glucoside based on the strategy of glycosylating piceatannol with a very small amount of cyclodextrin glycosyltransferase, which improved the water solubility, pH stability, temperature stability and light stability of piceatannol. At the same time, it also had antioxidant activity and α-glucosidase inhibitory activity, which was beneficial to promoting the application of piceatannol in the fields of food, medicine and so on.

[0100] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A piceatannol glucoside, characterized in that, It is obtained by the glycosylation reaction of piceatannol with a glycosyl donor, and its structural formula is as follows:

2. The piceatannol glucoside according to claim 1, wherein The piceatannol glucoside is piceatannol-3′-O-α-D-glucoside.

3. The piceatannol glucoside according to claim 1, wherein Its water solubility, pH stability, temperature stability and light stability are all improved compared with piceatannol; α-glucosidase inhibitory activity IC 50 <15 μg / mL; DPPH radical scavenging rate IC 50 <6.5 μg / mL; ABTS radical scavenging rate IC 50 <4.0 μg / mL.

4. The preparation method of piceatannol glucoside according to any one of claims 1-3, characterized in that, The reaction steps are as follows: Add piceatannol and an enzyme catalyst into a sugar solution, mix evenly to obtain a mixed solution, carry out a glycosylation reaction under an oscillating state, and separate and purify to obtain piceatannol glucoside.

5. The preparation method according to claim 4, wherein The mass ratio of the solute in the sugar solution to piceatannol is 25:6 to 400:6; the solute in the sugar solution is at least one of soluble starch, maltose, α-cyclodextrin, β-cyclodextrin, maltodextrin.

6. The preparation method according to claim 4, wherein, The concentration of the sugar solution is 25 mg / mL to 400 mg / mL.

7. The preparation method according to claim 4, characterized in that, The enzyme catalyst is cyclodextrin glucosyltransferase Toruzyme 3.0L; the concentration of the enzyme in the reaction solution is 0.1% to 2.5% (v / v).

8. The preparation method according to claim 4, characterized in that, The temperature of the glycosylation reaction is 30 to 60 °C, the time is 0.25 to 24 h, the pH of the reaction solution is 4 to 9, and the oscillation speed is 100 to 180 rpm.

9. Use of the piceatannol glucoside according to any one of claims 1-3 in the preparation of an antioxidant.

10. Use of the piceatannol glucoside according to any one of claims 1-3 in the preparation of an α-glucosidase inhibitor.

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