Method for separating and purifying dihydromyricetin from raspberry tea

Abstract

The invention relates to the technical field of medicine raw material manufacturing, and discloses a method for separating and purifying dihydromyricetin from raspberry tea, and the method comprises the following steps: S1, crushing, sieving and drying dried raspberry tea to obtain raspberry tea powder; s2, adding a compound enzyme preparation into the berry tea powder for enzymolysis to obtain enzymatic hydrolysate; s3, performing a ceramic membrane extraction process on the enzymatic hydrolysate, and collecting membrane permeate; s4, enriching the membrane permeate through a dihydromyricetin molecular imprinting chromatographic column; s5, performing rotary evaporation, crystallization, centrifugal separation and vacuum drying to obtain dihydromyricetin. The product has the advantages of high purity, high yield, low impurity residue, no need of complex equipment and mild and controllable operation parameters, meets the raw material quality requirements of high-end application scenarios, and has significant practical value and popularization prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical raw material manufacturing technology, specifically a method for separating and purifying dihydromyricetin from berry tea. Background Technology

[0002] Dihydromyricetin is a natural flavonoid compound mainly found in wild tea. It possesses various biological activities, including antioxidant, anti-inflammatory, hepatoprotective, hypoglycemic, and antibacterial properties, and has broad application prospects in the food, pharmaceutical, and health product industries. It is an important raw material for food and pharmaceuticals. With increasing market demand, the technology for the efficient separation and purification of dihydromyricetin from wild tea has attracted widespread attention. Currently, various process routes have been developed for the separation and purification of dihydromyricetin, mainly including solvent extraction, traditional column chromatography, gel filtration, and high-speed countercurrent chromatography. These methods generally suffer from insufficient dissolution of the target component, low extraction rates, and high levels of impurities such as polysaccharides, proteins, and organic acids, making subsequent purification difficult. Column chromatography lacks specific recognition capabilities and has limited effectiveness in separating structurally similar flavonoid impurities. High-speed countercurrent chromatography requires extremely high equipment precision, making it unsuitable for large-scale industrial production. Therefore, developing a dihydromyricetin separation and purification method that boasts high extraction rates, good purification effects, simple processes, environmental friendliness, and industrial applicability is of significant practical importance. Summary of the Invention

[0003] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for separating and purifying dihydromyricetin from berry tea.

[0004] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: A method for isolating and purifying dihydromyricetin from berry tea includes the following steps: S1. Grind the dried berry tea into powder, sieve and dry to obtain berry tea powder; S2. Add deionized water to the berry tea powder, adjust the pH, add a compound enzyme preparation for enzymatic hydrolysis, then heat to inactivate, and filter to obtain the enzymatic hydrolysate. S3. A ceramic membrane with a molecular weight cutoff of 600~1000 Da is laid at the bottom of the extraction chamber. The extraction process is carried out, and the primary membrane permeate and the secondary membrane permeate are collected and combined. S4. The combined membrane permeate was pumped into the dihydromyricetin molecularly imprinted column using a high-pressure pump. After loading, the column was flushed with rinsing solution. After flushing, the column was flushed with eluent. The column was monitored using a UV detector. The eluent was collected when the absorbance was ≥0.05 AU and collected when the absorbance was ≤0.05 AU. S5. Transfer the eluent to a rotary evaporator and concentrate it to 1 / 3 to 1 / 4 of its original volume. Then transfer it to a crystallization apparatus for cooling and crystallization, centrifugation, and vacuum drying to obtain dihydromyricetin.

[0005] Furthermore, in step S2, the compound enzyme preparation consists of cellulase, pectinase, and xylanase in a mass ratio of 2~3:1~2:1, and the amount of compound enzyme preparation added is 0.3~0.5% of the mass of the berry tea powder.

[0006] Further, in step S3, the extraction process involves uniformly pumping the enzymatic hydrolysate at 85-90°C into the extraction chamber at a pressure of 10-20 MPa. After the enzymatic hydrolysate extraction is completed, the primary membrane permeate is collected, and the filter residue is retained. The filter residue is then extracted again with deionized water at 85-90°C at a pressure of 10-20 MPa for 20-40 minutes. The secondary membrane permeate is then collected, and the two membrane permeates are combined.

[0007] Furthermore, in step S4, the dihydromyricetin molecularly imprinted chromatographic column is filled with dihydromyricetin molecularly imprinted polymer microspheres.

[0008] Furthermore, the preparation method of dihydromyricetin molecularly imprinted polymer microspheres includes the following steps: A1. Take 1-3 parts of dihydromyricetin and add it to a three-necked flask. Add 40-60 parts of anhydrous ethanol and stir to dissolve. Add 4-8 parts of methacrylic acid and continue stirring for 10-30 min. Add 20-30 parts of ethylene glycol dimethacrylate and stir evenly. Add 0.5-0.8 parts of azobisisobutyronitrile and ultrasonically disperse for 10-15 min at a power of 200-300 W and a frequency of 20-40 kHz to obtain an oil-phase prepolymer solution. A2. Take 100-200 parts of deionized water, add it to the reaction vessel, add 4-6 parts of polyvinyl alcohol, heat to 60-70℃, stir until completely dissolved, cool to 30-40℃, add 0.02-0.03 parts of sodium dodecyl sulfate, stir for 10-15 min to form an aqueous dispersion. A3. Under a nitrogen atmosphere, slowly add the oil phase prepolymer to the aqueous phase dispersion at a dropping rate of 5-10 mL / min. During the dropping process, maintain a stirring speed of 600-800 r / min, raise the temperature to 50-60℃, react for 3-4 h, then raise the temperature to 70-75℃ and keep the temperature constant for 6-8 h. A4. After the reaction is complete, stop stirring and allow it to cool naturally to room temperature. Filter the reaction solution through a 100-120 mesh stainless steel sieve, wash it repeatedly with deionized water 1-3 times, collect the precipitate, add a mixture of ethanol and 1 mol / L hydrochloric acid at a volume ratio of 3-4:1 (3-5 times the volume of the precipitate), reflux and heat with stirring for 2-4 hours, wash with deionized water until neutral, and dry in a 60-80℃ forced-air drying oven for 8-12 hours to obtain dihydromyricetin molecularly imprinted polymer microspheres.

[0009] Furthermore, in step S4, the loading parameters are: loading flow rate of 1~2 BV / h, loading volume of 15~20 L / column, and loading time of 1.5~2 h.

[0010] Furthermore, in step S4, the rinsing solution is deionized water, and the pH is adjusted to 3-3.5 in advance with 8-10% citric acid solution. The rinsing parameters are: rinsing flow rate of 4-6 BV / h, rinsing volume of 3-5 BV, and rinsing time of 1-2 h.

[0011] Further, in step S4, the eluent is an aqueous solution of ethanol with a volume fraction of 40-45%, and the pH is adjusted to 3-3.5 with a citric acid solution with a mass fraction of 8-10%. The elution parameters are: elution flow rate of 1-2 BV / h, elution volume of 2-3 BV, and elution time of 1-2 h.

[0012] Further, in step S5, the parameters for the rotary evaporator are: temperature 45~50℃, vacuum degree -0.08~-0.09 MPa, and rotation speed 200~300 r / min; the parameters for the crystallization equipment are: stirring speed 150~200 r / min, stirring at 45~50℃ for 10~15 min, cooling to 20~25℃, holding for 1~2 h, cooling to 0~5℃, and holding for 6~8 h; the centrifugation parameters are: centrifugation at 8000~10000 r / min for 10~15 min; and the vacuum drying parameters are: temperature 50~60℃, vacuum degree -0.085~-0.09 MPa, and drying time 6~8 h.

[0013] (iii) Beneficial technical effects This invention provides a method for separating and purifying dihydromyricetin from wild tea. Through a combination of enzymatic hydrolysis, high-pressure ceramic membrane extraction, molecularly imprinted chromatography, and cooling crystallization, the method achieves efficient purification and high-quality preparation of the target component. The method utilizes the degradation effect of the complex enzyme preparation on the cell wall of wild tea, disrupting structural barriers such as cellulose and pectin, promoting the full release of dihydromyricetin, and reducing the dissolution of large molecular weight impurities. The high-pressure ceramic membrane, with its specific molecular weight cutoff, further retains large molecular weight impurities that have not been enzymatically hydrolyzed, providing a pure raw material system for subsequent separation. The polymer microspheres in the molecularly imprinted chromatography column specifically recognize and bind to dihydromyricetin through pre-constructed specific imprinted sites, achieving efficient enrichment of the target component through targeted elution. The cooling crystallization process, based on the solubility characteristics of dihydromyricetin, promotes its directional precipitation through gradient cooling, ensuring a regular crystal form in the product.

[0014] The process of this invention does not require complex equipment, the operating parameters are mild and controllable, the solvent can be recycled and reused, and it takes into account both production efficiency and environmental protection requirements. The resulting product has low impurity residue, and the heavy metal and solvent residues meet relevant standards. It can meet the raw material quality requirements of high-end application scenarios and has significant practical value and promotion prospects. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] Unless otherwise specified, all components of the dihydromyricetin molecularly imprinted polymer microsphere formulation of this invention are commercially available. All parts used in this invention are parts by weight; The dried berry tea was purchased from Zhangjiajie Nuokang Ecological Tea Industry Co., Ltd. Cellulase activity ≥5000 U / g, pectinase activity ≥3000 U / g, xylanase activity ≥10000 U / g, purchased from Novozymes (China) Investment Co., Ltd. The ceramic membrane material is a titanium dioxide / silicon dioxide composite material, with an outer diameter of φ10~41 mm and a length of 100~1200 mm; Molecularly imprinted chromatographic column: The column body is made of 316L stainless steel, with an inner diameter of 80~100 mm and a column length of 500~1000 mm. It is packed with dihydromyricetin molecularly imprinted polymer microspheres with a particle size of 80~120 μm. UV detector: Model: Elite UV3100, wavelength range 190~700 nm, purchased from Elite (Dalian) Analytical Instruments Co., Ltd.; Example

[0017] A method for isolating and purifying dihydromyricetin from berry tea includes the following steps: S1. Grind the dried berry tea into 40 mesh, sieve it, and then dry it in a 60℃ forced-air drying oven for 2 hours to obtain berry tea powder. S2. Add 15 times the mass of deionized water to the berry tea powder, adjust the pH to 4.5 with 8% citric acid solution, add the compound enzyme preparation, and enzymatically hydrolyze at 50℃ for 2 h. Then raise the temperature to 90℃ and keep it at 90℃ for 15 min to inactivate the enzyme. Filter to obtain the enzymatic hydrolysate. S3. A ceramic membrane with a molecular weight cutoff of 600 Da is laid at the bottom of the extraction chamber for extraction. The primary membrane permeate and the secondary membrane permeate are collected and combined. S4. The combined membrane permeate was pumped into the dihydromyricetin molecularly imprinted column using a high-pressure pump. After sample loading, rinsing solution was introduced into the column. After rinsing, eluent was introduced into the column. The column was monitored using a UV detector with a detection wavelength of 290 nm, a flow cell path length of 10 mm, a time constant of 1.0 s, and a sampling rate of 10 pt / s. Eluent collection was started when the absorbance was ≥0.05 AU and stopped when the absorbance was ≤0.05 AU. S5. Transfer the eluent to a rotary evaporator and concentrate it to 1 / 3 of its original volume. Then transfer it to a crystallization apparatus for cooling and crystallization, centrifugation, and vacuum drying to obtain dihydromyricetin.

[0018] In step S2, the compound enzyme preparation consists of cellulase, pectinase, and xylanase in a mass ratio of 2:1:1, and the amount of compound enzyme preparation added is 0.3% of the mass of the berry tea powder.

[0019] Step S3 extraction process involves uniformly pumping the 85°C enzymatic hydrolysate into the extraction chamber at a pressure of 10 MPa. After the enzymatic hydrolysate extraction is completed, the primary membrane permeate is collected, and the filter residue is retained. The filter residue is then further extracted with 85°C deionized water at a pressure of 10 MPa for 20 min. The secondary membrane permeate is then collected, and the two membrane permeates are combined.

[0020] In step S4, the dihydromyricetin molecularly imprinted chromatographic column is packed with dihydromyricetin molecularly imprinted polymer microspheres. The preparation method of the dihydromyricetin molecularly imprinted polymer microspheres includes the following steps: A1. Take 1 part of dihydromyricetin and add it to a three-necked flask. Add 40 parts of anhydrous ethanol and stir to dissolve. Add 4 parts of methacrylic acid and continue stirring for 10 min. Add 20 parts of ethylene glycol dimethacrylate and stir evenly. Add 0.5 parts of azobisisobutyronitrile and ultrasonically disperse for 10 min at a power of 200 W and a frequency of 20 kHz to obtain an oil-phase prepolymer solution. A2. Take 100 parts of deionized water, add it to the reaction vessel, add 4 parts of polyvinyl alcohol, heat to 60°C, stir until completely dissolved, cool to 30°C, add 0.02 parts of sodium dodecyl sulfate, stir for 10 min to form an aqueous dispersion. A3. Under a nitrogen atmosphere, the oil phase prepolymer is slowly added dropwise to the aqueous phase dispersion at a rate of 5 mL / min. During the addition process, the stirring speed is maintained at 600 r / min. The temperature is raised to 50℃ and reacted for 3 h. Then the temperature is raised to 70℃ and reacted at a constant temperature for 6 h. A4. After the reaction is complete, stop stirring and allow it to cool naturally to room temperature. Filter the reaction solution through a 100-mesh stainless steel sieve, wash it repeatedly with deionized water once, collect the precipitate, add a 3:1 volume ratio of ethanol-1 mol / L hydrochloric acid mixture (3 times the volume of the precipitate), reflux and heat with stirring for 2 h, wash with deionized water until neutral, and dry in a 60℃ forced-air drying oven for 8 h to obtain dihydromyricetin molecularly imprinted polymer microspheres.

[0021] In step S4, the loading parameters are: loading flow rate 1 BV / h, loading volume 15 L / column, and loading time 1.5 h.

[0022] In step S4, the rinsing solution is deionized water, and the pH is adjusted to 3 in advance with 8% citric acid solution. The rinsing parameters are: rinsing flow rate of 4 BV / h, rinsing volume of 3 BV, and rinsing time of 1 h.

[0023] In step S4, the eluent is a 40% (v / v) aqueous ethanol solution, and the pH is adjusted to 3 with an 8% (w / w) citric acid solution. The elution parameters are: elution flow rate of 1 BV / h, elution volume of 2 BV, and elution time of 1 h.

[0024] In step S5, the parameters for the rotary evaporator are: temperature 45℃, vacuum degree -0.08 MPa, and rotation speed 200 r / min; the parameters for the crystallization equipment are: stirring speed 150 r / min, stirring at 45℃ for 10 min, cooling to 20℃, holding for 1 h, cooling to 0℃, and holding for 6 h; the centrifugation parameters are: centrifugation at 8000 r / min for 10 min; and the vacuum drying parameters are: temperature 50℃, vacuum degree -0.085 MPa, and drying time 6 h. Example

[0025] A method for isolating and purifying dihydromyricetin from berry tea includes the following steps: S1. Grind the dried berry tea into 50 mesh, sieve it, and then dry it in a 65℃ forced-air drying oven for 2.5 h to obtain berry tea powder; S2. Add 18 times the mass of deionized water to the berry tea powder, adjust the pH to 5 with 9% citric acid solution, add the compound enzyme preparation, and hydrolyze at 52℃ for 2.5 h. Then raise the temperature to 92℃ and keep warm for 18 min to inactivate the enzyme. Filter to obtain the enzymatic hydrolysate. S3. A ceramic membrane with a molecular weight cutoff of 800 Da is laid at the bottom of the extraction chamber for extraction. The primary membrane permeate and the secondary membrane permeate are collected and combined. S4. The combined membrane permeate was pumped into the dihydromyricetin molecularly imprinted column using a high-pressure pump. After sample loading, rinsing solution was introduced into the column. After rinsing, eluent was introduced into the column. The column was monitored using a UV detector with a detection wavelength of 290 nm, a flow cell path length of 10 mm, a time constant of 1.0 s, and a sampling rate of 10 pt / s. Eluent collection was started when the absorbance was ≥0.05 AU and stopped when the absorbance was ≤0.05 AU. S5. Transfer the eluent to a rotary evaporator and concentrate it to 1 / 3 of its original volume. Then transfer it to a crystallization apparatus for cooling and crystallization, centrifugation, and vacuum drying to obtain dihydromyricetin.

[0026] In step S2, the compound enzyme preparation consists of cellulase, pectinase, and xylanase in a mass ratio of 2.5:1.5:1, and the amount of compound enzyme preparation added is 0.4% of the mass of the berry tea powder.

[0027] Step S3 extraction process involves uniformly pumping the 88°C enzymatic hydrolysate into the extraction chamber at a pressure of 15 MPa. After the enzymatic hydrolysate extraction is completed, the primary membrane permeate is collected, and the filter residue is retained. The filter residue is then further extracted with 85°C deionized water at a pressure of 15 MPa for 30 min. The secondary membrane permeate is then collected, and the two membrane permeates are combined.

[0028] In step S4, the dihydromyricetin molecularly imprinted chromatographic column is packed with dihydromyricetin molecularly imprinted polymer microspheres. The preparation method of the dihydromyricetin molecularly imprinted polymer microspheres includes the following steps: A1. Take 2 parts of dihydromyricetin and add it to a three-necked flask. Add 50 parts of anhydrous ethanol and stir to dissolve. Add 6 parts of methacrylic acid and continue stirring for 20 min. Add 25 parts of ethylene glycol dimethacrylate and stir evenly. Add 0.6 parts of azobisisobutyronitrile and ultrasonically disperse for 10 min at a power of 250 W and a frequency of 30 kHz to obtain an oil-phase prepolymer solution. A2. Take 150 parts of deionized water, add it to the reaction vessel, add 5 parts of polyvinyl alcohol, heat to 65°C, stir until completely dissolved, cool to 35°C, add 0.02 parts of sodium dodecyl sulfate, stir for 10 min to form an aqueous dispersion. A3. Under a nitrogen atmosphere, the oil phase prepolymer solution is slowly added dropwise to the aqueous phase dispersion at a dropping rate of 5 mL / min. During the dropping process, the stirring speed is maintained at 700 r / min. The temperature is raised to 55℃ and reacted for 3.5 h. Then the temperature is raised to 70℃ and reacted at a constant temperature for 7 h. A4. After the reaction is complete, stop stirring and allow it to cool naturally to room temperature. Filter the reaction solution through a 110-mesh stainless steel sieve, wash it twice with deionized water, collect the precipitate, add a mixture of ethanol and 1 mol / L hydrochloric acid at a volume ratio of 3.5:1 (4 times the volume of the precipitate), reflux and heat with stirring for 3 h, wash with deionized water until neutral, and dry in a 70℃ forced-air drying oven for 10 h to obtain dihydromyricetin molecularly imprinted polymer microspheres.

[0029] In step S4, the loading parameters are: loading flow rate 1.5 BV / h, loading volume 18 L / column, and loading time 1.5 h.

[0030] In step S4, the rinsing solution is deionized water, and the pH is adjusted to 3.2 in advance with a 9% citric acid solution. The rinsing parameters are: rinsing flow rate of 5 BV / h, rinsing volume of 4 BV, and rinsing time of 1.5 h.

[0031] In step S4, the eluent is a 42% (v / v) aqueous ethanol solution, and the pH is adjusted to 3.2 with a 9% (w / w) citric acid solution. The elution parameters are: elution flow rate of 1.5 BV / h, elution volume of 2.5 BV, and elution time of 1 h.

[0032] In step S5, the parameters for the rotary evaporator are: temperature 45℃, vacuum degree -0.09 MPa, and rotation speed 250 r / min; the parameters for the crystallization equipment are: stirring speed 180 r / min, stirring at 45℃ for 10 min, cooling to 20℃, holding for 1 h, cooling to 2℃, and holding for 7 h; the centrifugation parameters are: centrifugation at 9000 r / min for 10 min; and the vacuum drying parameters are: temperature 55℃, vacuum degree -0.085 MPa, and drying time 7 h. Example

[0033] A method for isolating and purifying dihydromyricetin from berry tea includes the following steps: S1. Grind the dried berry tea into 60 mesh, sieve it, and then dry it in a 70℃ forced-air drying oven for 3 hours to obtain berry tea powder. S2. Add 20 times the mass of deionized water to the berry tea powder, adjust the pH to 5.5 with 10% citric acid solution, add the compound enzyme preparation, and hydrolyze at 55℃ for 3 h. Then raise the temperature to 95℃ and keep warm for 20 min to inactivate the enzyme. Filter to obtain the enzymatic hydrolysate. S3. A ceramic membrane with a molecular weight cutoff of 1000 Da is laid at the bottom of the extraction chamber for extraction. The primary membrane permeate and the secondary membrane permeate are collected and combined. S4. The combined membrane permeate was pumped into the dihydromyricetin molecularly imprinted column using a high-pressure pump. After sample loading, rinsing solution was introduced into the column. After rinsing, eluent was introduced into the column. The column was monitored using a UV detector with a detection wavelength of 290 nm, a flow cell path length of 10 mm, a time constant of 1.0 s, and a sampling rate of 10 pt / s. Eluent collection was started when the absorbance was ≥0.05 AU and stopped when the absorbance was ≤0.05 AU. S5. Transfer the eluent to a rotary evaporator and concentrate it to 1 / 4 of its original volume. Then transfer it to a crystallization apparatus for cooling and crystallization, centrifugation, and vacuum drying to obtain dihydromyricetin.

[0034] In step S2, the compound enzyme preparation consists of cellulase, pectinase, and xylanase in a mass ratio of 3:2:1, and the amount of compound enzyme preparation added is 0.5% of the mass of the berry tea powder.

[0035] Step S3 extraction process involves uniformly pumping the 90°C enzymatic hydrolysate into the extraction chamber at a pressure of 20 MPa. After the enzymatic hydrolysate extraction is completed, the primary membrane permeate is collected, and the filter residue is retained. The filter residue is then further extracted with 90°C deionized water at a pressure of 20 MPa for 40 min. The secondary membrane permeate is then collected, and the two membrane permeates are combined.

[0036] In step S4, the dihydromyricetin molecularly imprinted chromatographic column is packed with dihydromyricetin molecularly imprinted polymer microspheres. The preparation method of the dihydromyricetin molecularly imprinted polymer microspheres includes the following steps: A1. Take 3 parts of dihydromyricetin and add it to a three-necked flask. Add 60 parts of anhydrous ethanol and stir to dissolve. Add 8 parts of methacrylic acid and continue stirring for 30 min. Add 30 parts of ethylene glycol dimethacrylate and stir evenly. Add 0.8 parts of azobisisobutyronitrile and ultrasonically disperse for 15 min at a power of 300 W and a frequency of 40 kHz to obtain an oil-phase prepolymer solution. A2. Take 200 parts of deionized water, add it to the reaction vessel, add 6 parts of polyvinyl alcohol, heat to 70°C, stir until completely dissolved, cool to 40°C, add 0.03 parts of sodium dodecyl sulfate, stir for 15 min to form an aqueous dispersion. A3. Under a nitrogen atmosphere, the oil phase prepolymer is slowly added dropwise to the aqueous phase dispersion at a rate of 10 mL / min. During the addition process, the stirring speed is maintained at 800 r / min. The temperature is raised to 60℃ and reacted for 4 h. Then the temperature is raised to 75℃ and reacted at a constant temperature for 8 h. A4. After the reaction is complete, stop stirring and allow it to cool naturally to room temperature. Filter the reaction solution through a 120-mesh stainless steel sieve, wash it repeatedly with deionized water three times, collect the precipitate, add a 4:1 volume ratio of ethanol-1 mol / L hydrochloric acid mixture (5 times the volume of the precipitate), reflux and heat with stirring for 4 h, wash with deionized water until neutral, and dry in an 80℃ forced-air drying oven for 12 h to obtain dihydromyricetin molecularly imprinted polymer microspheres.

[0037] In step S4, the loading parameters are: loading flow rate 2 BV / h, loading volume 20 L / column, and loading time 2 h.

[0038] In step S4, the rinsing solution is deionized water, and the pH is adjusted to 3.5 beforehand with a 10% citric acid solution. The rinsing parameters are: rinsing flow rate of 6 BV / h, rinsing volume of 5 BV, and rinsing time of 2 h.

[0039] In step S4, the eluent is a 45% (v / v) aqueous ethanol solution, and the pH is adjusted to 3.5 with a 10% (w / w) citric acid solution. The elution parameters are: elution flow rate of 2 BV / h, elution volume of 3 BV, and elution time of 2 h.

[0040] In step S5, the parameters for the rotary evaporator are: temperature 50℃, vacuum degree -0.09 MPa, and rotation speed 300 r / min; the parameters for the crystallization equipment are: stirring speed 200 r / min, stirring at 50℃ for 15 min, cooling to 25℃, holding for 2 h, cooling to 5℃, and holding for 8 h; the centrifugation parameters are: centrifugation at 10000 r / min for 15 min; and the vacuum drying parameters are: temperature 60℃, vacuum degree -0.09 MPa, and drying time 8 h.

[0041] Comparative Example 1: Step S2 was replaced by adding 15 times the mass of deionized water to an equal mass of berry tea powder, adjusting the pH to 4.5 with 8% citric acid, directly heating to 90°C and holding for 30 min, and filtering to obtain the water extract; the remaining processes were the same as in Example 1.

[0042] Comparative Example 2: In step S4, the dihydromyricetin molecularly imprinted chromatographic column was replaced with an AB-8 macroporous adsorption resin column, and the rest of the process was the same as in Example 1.

[0043] Comparative Example 3: Step S3 was omitted. Step S4 was performed using the enzymatic hydrolysate from Step S2. The remaining processes were the same as in Example 1.

[0044] Test items: 1. Product Appearance Evaluation Visual inspection: Record the product's color, state (powder / crystal), and uniformity. The absence of obvious lumps or discoloration indicates that the product is qualified.

[0045] 2. Dihydromyricetin purity test Instrument: High performance liquid chromatograph (HPLC, equipped with C18 column and UV detector); Detection conditions: mobile phase was methanol-0.1% formic acid aqueous solution (50:50, v / v), flow rate was 1.0 mL / min, column temperature was 30℃, detection wavelength was 290 nm, and injection volume was 20 μL; Calculation method: The external standard method was used. A standard curve was plotted using dihydromyricetin standard (purity ≥99.9%), and the mass fraction of dihydromyricetin in the sample was calculated.

[0046] 3. Overall Yield Calculation Yield formula: Total yield (%) = (Final product quality × Product purity) / (Raw berry tea quality × Initial dihydromyricetin content in raw material) × 100%; Initial content detection of raw materials: Take the pretreated berry tea powder, extract it three times with 70% ethanol using ultrasound, combine the extracts, and detect the total amount of dihydromyricetin by HPLC.

[0047] 4. Detection of residual impurities Heavy metals (lead, cadmium, mercury): atomic absorption spectrometry (AAS), detection limit 0.01 mg / kg; Solvent residue (ethanol): Gas chromatography-FID, detection limit 0.01%; Polysaccharide residue: Phenol-sulfuric acid method, calculate the mass fraction of polysaccharides in the sample (≤1% is acceptable).

[0048] Table 1 Test Results

[0049] Table 2 Test Results

[0050] As shown in the table, the purity of dihydromyricetin in Examples 1-3 was all above 99.5%. Comparative Example 1 had a purity of 85.2% and a yield of 4.2%, indicating that the compound enzymatic hydrolysis could destroy the cell walls of the tea berries, promoting the release of the target component and reducing the dissolution of impurities. Comparative Example 2 had a purity of 92.5% and a yield of 6.3%, indicating that the dihydromyricetin molecularly imprinted polymer microspheres prepared in this invention have a specific recognition advantage for dihydromyricetin, resulting in better impurity removal. Comparative Example 3 had a purity of 93.8% and a yield of 7.0%, indicating that the ceramic membrane with a molecular weight cutoff of 600-1000 Da can effectively retain large molecular impurities, reducing the burden on subsequent separation. Examples 1-3 all presented as white needle-like crystals that were uniform and free of lumps, exhibiting excellent appearance quality. Comparative Example 1 was a white powder with slight lumps, Comparative Example 2 was yellowish, and Comparative Example 3 contained pale yellow impurity spots, all of which affected the appearance due to residual impurities. Impurity detection showed that the polysaccharide residue in Examples 1-3 was 0.5-0.8%, which was much lower than the 1.5-3.2% in Comparative Examples 1-3, and the ethanol residue was ≤0.03%, indicating that the process of the present invention has outstanding impurity removal capabilities.

[0051] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for separating and purifying dihydromyricetin from mulberry tea, characterized in that, Includes the following steps: S1. Grind the dried berry tea into powder, sieve and dry to obtain berry tea powder; S2. Add deionized water to the berry tea powder, adjust the pH, add a compound enzyme preparation for enzymatic hydrolysis, then heat to inactivate, and filter to obtain the enzymatic hydrolysate. S3. A ceramic membrane with a molecular weight cutoff of 600~1000 Da is laid at the bottom of the extraction chamber. The extraction process is carried out, and the primary membrane permeate and the secondary membrane permeate are collected and combined. S4. The combined membrane permeate was pumped into the dihydromyricetin molecularly imprinted column using a high-pressure pump. After loading, the column was flushed with rinsing solution. After flushing, the column was flushed with eluent. The column was monitored using a UV detector. The eluent was collected when the absorbance was ≥0.05 AU and collected when the absorbance was ≤0.05 AU. S5. Transfer the eluent to a rotary evaporator and concentrate it to 1 / 3 to 1 / 4 of its original volume. Then transfer it to a crystallization apparatus for cooling and crystallization, centrifugation, and vacuum drying to obtain dihydromyricetin.

2. The method for separating and purifying dihydromyricetin from mulberry tea according to claim 1, characterized in that, In step S2, the compound enzyme preparation consists of cellulase, pectinase, and xylanase in a mass ratio of 2~3:1~2:1, and the amount of compound enzyme preparation added is 0.3~0.5% of the mass of the berry tea powder.

3. The method for separating and purifying dihydromyricetin from mulberry tea according to claim 1, characterized in that, Step S3 extraction process involves uniformly pumping the enzymatic hydrolysate at 85-90℃ into the extraction chamber at a pressure of 10-20 MPa. After the enzymatic hydrolysate extraction is completed, the primary membrane permeate is collected, and the filter residue is retained. The filter residue is then extracted again with deionized water at 85-90℃ at a pressure of 10-20 MPa for 20-40 min. The secondary membrane permeate is then collected, and the two membrane permeates are combined.

4. The method for separating and purifying dihydromyricetin from mulberry tea according to claim 1, characterized in that, In step S4, the dihydromyricetin molecularly imprinted chromatographic column is filled with dihydromyricetin molecularly imprinted polymer microspheres.

5. The method for separating and purifying dihydromyricetin from berry tea according to claim 4, characterized in that, The preparation method of dihydromyricetin molecularly imprinted polymer microspheres includes the following steps: A1. Take 1-3 parts of dihydromyricetin and add it to a three-necked flask. Add 40-60 parts of anhydrous ethanol and stir to dissolve. Add 4-8 parts of methacrylic acid and continue stirring for 10-30 min. Add 20-30 parts of ethylene glycol dimethacrylate and stir evenly. Add 0.5-0.8 parts of azobisisobutyronitrile and ultrasonically disperse for 10-15 min at a power of 200-300 W and a frequency of 20-40 kHz to obtain an oil-phase prepolymer solution. A2. Take 100-200 parts of deionized water, add it to the reaction vessel, add 4-6 parts of polyvinyl alcohol, heat to 60-70℃, stir until completely dissolved, cool to 30-40℃, add 0.02-0.03 parts of sodium dodecyl sulfate, stir for 10-15 min to form an aqueous dispersion. A3. Under a nitrogen atmosphere, slowly add the oil phase prepolymer to the aqueous phase dispersion at a dropping rate of 5-10 mL / min. During the dropping process, maintain a stirring speed of 600-800 r / min, raise the temperature to 50-60℃, react for 3-4 h, then raise the temperature to 70-75℃ and keep the temperature constant for 6-8 h. A4. After the reaction is complete, stop stirring and allow it to cool naturally to room temperature. Filter the reaction solution through a 100-120 mesh stainless steel sieve, wash it repeatedly with deionized water 1-3 times, collect the precipitate, add a mixture of ethanol and 1 mol / L hydrochloric acid at a volume ratio of 3-4:1 (3-5 times the volume of the precipitate), reflux and heat with stirring for 2-4 hours, wash with deionized water until neutral, and dry in a 60-80℃ forced-air drying oven for 8-12 hours to obtain dihydromyricetin molecularly imprinted polymer microspheres.

6. The method for separating and purifying dihydromyricetin from mulberry tea according to claim 1, characterized in that, In step S4, the loading parameters are: loading flow rate of 1~2 BV / h, loading volume of 15~20 L / column, and loading time of 1.5~2 h.

7. The method for separating and purifying dihydromyricetin from mulberry tea according to claim 1, characterized in that, In step S4, the rinsing solution is deionized water, and the pH is adjusted to 3-3.5 in advance with 8-10% citric acid solution. The rinsing parameters are: rinsing flow rate of 4-6 BV / h, rinsing volume of 3-5 BV, and rinsing time of 1-2 h.

8. The method for separating and purifying dihydromyricetin from mulberry tea according to claim 1, characterized in that, In step S4, the eluent is an aqueous solution of ethanol with a volume fraction of 40-45%, and the pH is adjusted to 3-3.5 with a citric acid solution with a mass fraction of 8-10%. The elution parameters are: elution flow rate of 1-2 BV / h, elution volume of 2-3 BV, and elution time of 1-2 h.

9. The method for separating and purifying dihydromyricetin from mulberry tea according to claim 1, characterized in that, In step S5, the parameters for the rotary evaporator are: temperature 45~50℃, vacuum degree -0.08~-0.09 MPa, and rotation speed 200~300 r / min; the parameters for the crystallization equipment are: stirring speed 150~200 r / min, stirring at 45~50℃ for 10~15 min, cooling to 20~25℃, holding for 1~2 h, cooling to 0~5℃, and holding for 6~8 h; the centrifugation parameters are: centrifugation at 8000~10000 r / min for 10~15 min; and the vacuum drying parameters are: temperature 50~60℃, vacuum degree -0.085~-0.09 MPa, and drying time 6~8 h.

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