Method for extracting flavone and DNJ from mulberry leaves

Abstract

The invention relates to the technical field of natural product extraction and purification, in particular to a method for extracting flavone and DNJ from mulberry leaves, and the method comprises the following steps: pretreating raw materials, and performing microwave-assisted enzymolysis by using a compound enzyme preparation; performing multi-mode extraction, namely performing two-stage extraction by using a pulse type ultrasonic-counter-current extraction device; gradient separation is carried out, and AB-8 and D101 type resin composite chromatographic column elution is carried out; performing targeted purification, adsorbing and eluting the DNJ through ion exchange resin, and removing impurities from flavone by adopting an ultrafiltration membrane and Zeta potential regulation and control; preparing a finished product, namely performing vacuum concentration and low-temperature spray drying to obtain a high-purity finished product. According to the method, efficient extraction and high-purity preparation of flavone and DNJ are achieved through the steps of finely-controlled raw material pretreatment, multi-mode extraction, gradient separation and purification, finished product preparation, quality monitoring and the like, the extraction process is optimized, the product quality and stability are greatly improved, and the method is suitable for industrial production. Powerful support is provided for comprehensive development and utilization of mulberry leaf resources.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction and purification of natural products, and specifically to a method for extracting flavonoids and DNJ from mulberry leaves. Background Art

[0002] In the prior art, there are significant defects in the methods for extracting flavonoids and DNJ from mulberry leaves. Traditional enzymatic hydrolysis processes mostly use single enzyme preparations or composite enzymes with fixed ratios, and the enzyme activity ratios are unreasonable, resulting in incomplete cell wall fragmentation and low release efficiency of active ingredients. In addition, the conventional enzymatic hydrolysis process lacks microwave-assisted technology and only relies on constant-temperature reactions, with an enzymatic hydrolysis time of more than 60 minutes, high energy consumption, and easy degradation of heat-sensitive components. At the same time, mechanical vibration strengthening means are not introduced in the enzymatic hydrolysis stage of the prior art, and the contact between the enzyme and the material is uneven, restricting the further improvement of the extraction rate.

[0003] In terms of the extraction process, traditional methods mostly use single-frequency ultrasound or static countercurrent extraction, and cannot achieve multi-modal synergistic effects. The ultrasonic field distribution of a fixed frequency is uneven, the effect of destroying cell structure is limited, and the solvent dispersion is poor, resulting in insufficient contact area between ethanol and the material and low extraction efficiency. Existing ultrasonic devices lack a phase modulation module and cannot dynamically regulate the field strength, making it difficult to maintain a stable ultrasonic energy output and affecting the consistency of extraction quality. In addition, the solvent is not subjected to high-pressure homogenization in the conventional pre-treatment before extraction, the ethanol dispersion is insufficient, and the nano-level dispersion effect is lacking, further restricting the dissolution rate and extraction rate of active ingredients.

[0004] The above defects lead to problems such as low extraction efficiency, high energy consumption, and unstable product purity in the prior art, which urgently need to be improved. Therefore, a method for extracting flavonoids and DNJ from mulberry leaves is proposed to solve the above problems. Summary of the Invention

[0005] Technical Problems to be Solved

[0006] In view of the above-mentioned drawbacks of the prior art, the present invention provides a method for extracting flavonoids and DNJ from mulberry leaves. Through steps such as precisely controlled raw material pretreatment, multi-modal extraction, gradient separation and purification, and finished product preparation and quality monitoring, the present invention realizes the high-efficiency extraction and high-purity preparation of flavonoids and DNJ. This method not only optimizes the extraction process but also greatly improves the quality and stability of the product, providing strong support for the comprehensive development and utilization of mulberry leaf resources.

[0007] Technical Solutions

[0008] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0009] A method for extracting flavonoids and DNJ from mulberry leaves, the method comprising the following steps:

[0010] (a) Raw material pretreatment: Crush mulberry leaves with a water content ≤ 8% to a particle size of 80 - 120 mesh, mix them with a citric acid - disodium hydrogen phosphate buffer solution with a pH of 4.5 - 5.5 at a mass ratio of 1:12 - 15, add a composite enzyme preparation composed of cellulase, pectinase, and β - glucosidase, where the enzyme activity ratio of cellulase, pectinase, and β - glucosidase is 2:1:0.5, and carry out microwave - assisted enzymatic hydrolysis at 45 - 50 °C for 30 - 40 minutes, with the microwave power density controlled at 0.5 - 0.8 W / g;

[0011] (b) Multimodal extraction: Add a 65% ethanol solution containing 0.2% L - ascorbic acid to the enzymatic hydrolysate, adjust the ethanol concentration of the system to 45 - 50%, and use a pulsed ultrasound - countercurrent extraction combined device to carry out two - stage extraction at 60 - 65 °C and a pressure of 0.08 - 0.12 MPa. Among them, the ultrasound frequency in the first stage is 28 kHz and the power is 250 W, and in the second stage, it is switched to 40 kHz and the power is 180 W, with the total extraction time ≤ 60 minutes;

[0012] (c) Gradient separation: Pass the extract through a composite chromatography column filled with AB - 8 type and D101 type resins, where the mass ratio of AB - 8 type and D101 type resins is 1:2. First, elute DNJ with 25% ethanol at a flow rate of 1.2 BV / h for 4 - 6 times the column volume, and then elute flavonoids with 70% ethanol at a flow rate of 2.0 BV / h for 8 - 10 times the column volume;

[0013] (d) Targeted purification: After the DNJ eluate is adsorbed by 001×7 type strong - acid cation - exchange resin, it is eluted with a gradient of 0.1 mol / L ammonia water. The flavonoid eluate uses a combined technology of ultrafiltration membrane and Zeta potential regulation to remove polyphenol impurities, where the cut - off molecular weight of the ultrafiltration membrane is 5000 Da;

[0014] (e) Finished product preparation: The purified solution is subjected to three - stage vacuum concentration and low - temperature spray drying respectively to obtain a finished product with a DNJ purity ≥ 98.5% and a total flavonoid content ≥ 92%. Among them, the temperature in the three - stage vacuum concentration process is gradually decreased according to 50 °C, 45 °C, and 40 °C, and the inlet air temperature in the low - temperature spray drying process is 110 °C and the outlet air temperature is 55 °C.

[0015] Furthermore, the addition amount of the composite enzyme preparation in step (a) is 1.2 - 1.8% of the dry weight of mulberry leaves, where the cellulase activity ≥ 5000 U / g, the pectinase activity ≥ 3000 U / g, and the β - glucosidase activity ≥ 800 U / g;

[0016] The microwave treatment adopts an intermittent irradiation mode, specifically working for 10 s and then pausing for 5 s, and simultaneously applying mechanical vibration at 20 - 30 kHz to enhance the cell wall breaking efficiency.

[0017] Furthermore, the pulsed ultrasound-countercurrent extraction device in step (b) is equipped with a phase modulation module to make the ultrasonic field intensity between 120-180 W / cm 2 The range fluctuates according to the law of sine waves, and the ethanol solution is treated with high-pressure homogenization before entering the extraction system, with a pressure of 60-80MPa and 3 cycles to achieve nano-scale dispersion.

[0018] Furthermore, the composite chromatography column in step (c) is activated by alternating 0.1 mol / L NaOH and anhydrous ethanol before filling, and the change of the dielectric constant of the eluent is monitored in real time during the chromatography process, and the eluent ratio is switched when the dielectric constant drops to 40-45.

[0019] Furthermore, in step (d), the Zeta potential regulation is achieved by adding 0.05% chitosan quaternary ammonium salt, controlling the solution pH value at 4.0-4.5 to form floccules, and combining electric field assisted sedimentation to increase the impurity removal rate to more than 98%, specifically, the voltage is 15V / cm and the action time is 20min.

[0020] Furthermore, the method also includes step (f) quality monitoring: using near-infrared spectroscopy combined with HPLC to establish a process analysis technology, real-time detection of DNJ characteristic peak (retention time 6.8±0.2min) and flavonoid characteristic absorption band (second-order derivative spectrum peak at λ=510nm), and dynamic adjustment of elution parameters to make RSD≤2.5%.

[0021] Furthermore, molecular imprinting polymers (MIPs) were introduced in the DNJ purification stage. The preparation method included: using DNJ as a template molecule, acrylamide as a functional monomer, and ethylene glycol dimethacrylate as a cross-linking agent, polymerizing under supercritical CO2 conditions to form specific recognition sites with an adsorption capacity of more than 35 mg / g.

[0022] Furthermore, in step (e), the low-temperature spray drying adopts a two-fluid nozzle and electrostatic atomization synergistic technology to control the surface charge density of the droplets to 0.8-1.2 C / m 2 A vortex stabilizer is installed in the drying tower to ensure that the moisture content of the product is ≤3% and the particle size D90 is ≤25μm.

[0023] Beneficial Effects

[0024] Compared with the known public technology, the technical solution provided by the present invention has the following beneficial effects:

[0025] 1. A method for extracting flavonoids and DNJ from mulberry leaves proposed by the present invention significantly improves the extraction efficiency and purity of flavonoids and DNJ through precisely controlled raw material pretreatment, multi-modal extraction, and gradient separation steps. Especially in the raw material pretreatment stage, the use of a composite enzyme preparation and microwave-assisted enzymatic hydrolysis technology, combined with an intermittent irradiation mode and mechanical vibration, effectively strengthens the cell wall breaking effect, improves the extraction rate of flavonoids and DNJ, not only optimizes the extraction process, but also greatly improves the utilization rate of raw materials and reduces production costs.

[0026] 2. In the multi-modal extraction stage of the present invention, a pulsed ultrasound-countercurrent extraction combined device is used. Through the synergistic effect of ultrasound with different frequencies and powers in two stages, the cell structure is further damaged, promoting the dissolution of flavonoids and DNJ. At the same time, the phase modulation module and high-pressure homogenization treatment equipped in the device make the ultrasound field act more uniformly on the material, increasing the contact area between the ethanol solution and the mulberry leaf material, thereby achieving a significant increase in the extraction rate of flavonoids and DNJ, not only improving the extraction efficiency, but also ensuring the quality stability of the extract.

[0027] 3. The present invention adopts a three-stage vacuum concentration and low-temperature spray drying technology, combined with gradient cooling and the synergistic technology of two-fluid nozzles and electrostatic atomization, effectively avoiding the damage of flavonoids and DNJ by high temperature, while improving the drying efficiency and shortening the drying time. In addition, by introducing molecularly imprinted polymers (MIPs) for DNJ purification, the purity and recovery rate of DNJ are further improved. At the same time, the establishment of a quality monitoring system ensures the controllability of the production process and the stability of product quality, providing consumers with a safer and more reliable product guarantee. Detailed implementation manners

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] The present invention will be further described below with reference to the embodiments.

[0030] I. Raw material pretreatment

[0031] (I) Raw material selection and preliminary treatment

[0032] Select fresh mulberry leaves without pests and diseases as the starting raw material. After picking, wash the mulberry leaves in time to remove surface soil, impurities, etc. They can be naturally air-dried or dried in a low-temperature environment not exceeding 40°C to reduce the water content of the mulberry leaves to ≤8%. Controlling this water content can avoid affecting the operation effect or causing microbial contamination due to excessive water during subsequent crushing, enzymatic hydrolysis, and extraction processes, resulting in the loss of active ingredients.

[0033] (II) Crushing operation

[0034] Use a professional crusher to crush the dried mulberry leaves to a particle size of 80 - 120 mesh. A suitable particle size can significantly increase the contact area between the mulberry leaves and the enzymatic hydrolysis solution and extraction solvent, improving the dissolution efficiency of active ingredients. Through experimental comparison, under the same enzymatic hydrolysis and extraction conditions, the flavonoid extraction rate of 80-mesh mulberry leaf powder is 12% higher than that of 60-mesh mulberry leaf powder, and the DNJ extraction rate is 10% higher. If the particle size is too large, the contact area is small and the extraction efficiency is low; if the particle size is too small, it will increase the filtration difficulty and raise the production cost.

[0035] (III) Enzymatic hydrolysis process

[0036] Buffer solution preparation: Precisely prepare a citric acid - disodium hydrogen phosphate buffer solution with a pH of 4.5 - 5.5. This buffer solution creates a suitable pH environment for the enzymatic hydrolysis reaction, maintains the activity of the enzyme, and at the same time reduces the chemical damage to the active ingredients of the mulberry leaves. Enzymatic hydrolysis experiments were carried out under different pH conditions. The results show that when the pH value of the buffer solution is 5.0, the enzymatic hydrolysis extraction rates of flavonoids and DNJ are the highest. Compared with pH values of 4.0 and 6.0, the flavonoid extraction rate is 15% and 18% higher respectively, and the DNJ extraction rate also increases significantly accordingly.

[0037] Addition of compound enzyme preparation: The compound enzyme preparation is composed of cellulase, pectinase, and β-glucosidase, and the enzyme activity ratio is fixed at 2:1:0.5. The addition amount is 1.2 - 1.8% of the dry weight of the mulberry leaves. Among them, the cellulase activity ≥5000U / g, the pectinase activity ≥3000U / g, and the β-glucosidase activity ≥800U / g. These enzymes work together to effectively destroy the cell wall structure of the mulberry leaves and promote the release of flavonoids and DNJ. Experimental data show that when the addition amount of the compound enzyme preparation is 1.5%, compared with the non-added group, the flavonoid extraction rate increases by 35% and the DNJ extraction rate increases by 40%.

[0038] Microwave-assisted enzymatic hydrolysis conditions: After mixing the crushed mulberry leaves with the buffer solution at a mass ratio of 1:12 - 15, place them in a microwave reactor for enzymatic hydrolysis. The enzymatic hydrolysis temperature is controlled at 45 - 50 °C. This temperature range takes into account both enzyme activity and reaction rate. The microwave power density is 0.5 - 0.8 W / g. The intermittent irradiation mode is adopted, that is, working for 10 s and then pausing for 5 s, and mechanical vibration of 20 - 30 kHz is applied synchronously. This operation mode can strengthen the cell wall breaking effect and accelerate the release of active ingredients. Through experimental verification, under the synergistic effect of microwave and mechanical vibration, the extraction rates of flavonoids and DNJ are increased by 20% and 25% respectively compared with simple enzymatic hydrolysis. The enzymatic hydrolysis time is controlled within 30 - 40 minutes. If the time is too short, the enzymatic hydrolysis is insufficient; if it is too long, it may lead to the degradation of active ingredients. The influence of different enzymatic hydrolysis times on the extraction rate is shown in the following table:

[0039] Enzymolysis time (min) Flavonoid extraction rate (%) DNJ extraction rate (%) 20 35.6 28.5 30 48.2 39.8 40 52.1 43.6 50 49.5 41.2

[0040] II. Multimodal extraction

[0041] (I) Addition of extraction solvent

[0042] Add a 65% ethanol solution containing 0.2% L-ascorbic acid to the enzymatic hydrolysate, and adjust the ethanol concentration in the system to 45 - 50%. L-ascorbic acid, as an antioxidant, can prevent the oxidation of flavonoids and DNJ during the extraction process and ensure product quality. The ethanol concentration is crucial for the extraction effect. Too high or too low ethanol concentration will reduce the extraction rate. Experiments show that when the ethanol concentration is 48%, the extraction rates of flavonoids and DNJ are the best. Compared with ethanol concentrations of 40% and 55%, the extraction rate of flavonoids is increased by 10% and 12% respectively, and the extraction rate of DNJ also shows a similar change trend.

[0043] (II) Pulse ultrasonic - countercurrent extraction combined device

[0044] Extraction conditions: Use a pulse ultrasonic - countercurrent extraction combined device for extraction operation. The extraction temperature is set at 60 - 65 °C, and the pressure is controlled at 0.08 - 0.12 MPa. This temperature and pressure environment can accelerate molecular movement and improve the mass transfer efficiency of active ingredients. The extraction is carried out in two stages. In the first stage, the ultrasonic frequency is 28 kHz and the power is 250 W; in the second stage, it is switched to an ultrasonic frequency of 40 kHz and a power of 180 W. The synergistic effect of ultrasonic waves with different frequencies and powers further destroys the cell structure and promotes the dissolution of components. The total extraction time should be ≤ 60 minutes to avoid the degradation of components caused by too long extraction time. The influence of different ultrasonic conditions on the extraction rate is shown in the following table:

[0045]

[0046] Device optimization: This device is equipped with a phase modulation module to make the ultrasonic field strength at 120 - 180 W / cm 2It fluctuates according to the sine wave law within a certain range to ensure that the ultrasonic field acts more uniformly on the materials and improve the extraction effect. At the same time, before the ethanol solution enters the extraction system, it undergoes high-pressure homogenization treatment at a pressure of 60 - 80 MPa for 3 cycles to achieve nano-level dispersion. The high-pressure homogenization treatment increases the contact area between the ethanol solution and the mulberry leaf materials and improves the extraction rate. After testing, the extraction rates of flavonoids and DNJ after treatment are increased by 15% and 18% respectively compared with those before treatment.

[0047] III. Gradient Separation

[0048] (I) Preparation of Composite Chromatography Column

[0049] The composite chromatography column is composed of AB-8 type and D101 type resins in a mass ratio of 1:2. Before packing, it is alternately activated with 0.1 mol / L NaOH and absolute ethanol. The NaOH solution can remove the impurities and unreacted groups on the resin surface, and absolute ethanol is used to clean and activate the resin to enhance its adsorption performance. Experiments show that the adsorption amounts of flavonoids and DNJ on the activated resin are increased by 20% and 25% respectively compared with those on the unactivated resin.

[0050] (II) Elution Process

[0051] DNJ Elution: After the extract is loaded onto the composite chromatography column, first use 25% ethanol with a volume 4 - 6 times that of the column to elute DNJ at a flow rate of 1.2 BV / h. This elution condition can effectively elute DNJ while reducing the elution of other impurities. During the elution process, the change in the dielectric constant of the eluent is monitored in real time. When the dielectric constant drops to 40 - 45, it indicates that the elution of DNJ is basically complete, and at this time, the eluent ratio is switched.

[0052] Flavonoid Elution: Switch to 70% ethanol with a volume 8 - 10 times that of the column to elute flavonoids at a flow rate of 2.0 BV / h. 70% ethanol can selectively elute flavonoids to further separate flavonoids from other impurities. The effects of different eluent concentrations and flow rates on the elution effect are shown in the following table:

[0053]

[0054] IV. Targeted Purification

[0055] (I) DNJ Purification

[0056] After the DNJ eluate is adsorbed by 001×7 type strong acidic cation exchange resin, it is eluted with a gradient of 0.1 mol / L ammonia water. The 001×7 type strong acidic cation exchange resin has a specific adsorption effect on DNJ. By controlling the gradient elution of ammonia water, the purity of DNJ can be further improved. During the elution process, the elution flow rate and ammonia water concentration are precisely controlled to achieve efficient purification of DNJ.

[0057] (II) Flavonoid Purification

[0058] The flavonoid eluate uses the combined technology of ultrafiltration membrane and Zeta potential regulation to remove polyphenol impurities. The molecular weight cut-off of the ultrafiltration membrane is 5000 Da, which can effectively remove macromolecular impurities. The Zeta potential regulation is achieved by adding 0.05% quaternary ammonium chitosan. When the solution pH value is controlled at 4.0 - 4.5, flocs are formed, and then combined with electric field-assisted sedimentation (voltage 15 V / cm, action time 20 min), the impurity removal rate is increased to over 98%. The effects of different treatment methods on the removal rate of polyphenol impurities in flavonoids are shown in the following table:

[0059]

[0060]

[0061] V. Preparation of Finished Product

[0062] (I) Three-stage vacuum concentration

[0063] The purified solutions are respectively subjected to three-stage vacuum concentration to remove the solvent and increase the concentration of the target components. During the concentration process, the temperature is decreased in a gradient of 50 °C, 45 °C, and 40 °C. This gradient temperature decrease method can avoid the destruction of flavonoids and DNJ at high temperatures and ensure the concentration effect at the same time. Experiments show that after gradient temperature decrease concentration, the retention rates of flavonoids and DNJ are respectively 15% and 18% higher than those of concentration at a constant temperature of 50 °C.

[0064] (II) Low-temperature spray drying

[0065] Drying technology: Low-temperature spray drying uses the combined technology of two-fluid nozzle and electrostatic atomization. The two-fluid nozzle atomizes the concentrated solution into fine droplets, and electrostatic atomization makes the droplet surface charged, controlling the surface charge density of the droplets at 0.8 - 1.2 C / m 2 , this combined technology improves the drying efficiency of the droplets, shortens the drying time, and reduces the thermal damage to the target components.

[0066] Settings inside the drying tower: A vortex flow stabilizer is set inside the drying tower to make the air flow distribution in the tower uniform and ensure the full drying of the droplets. Through this setting, the water content of the product can be ≤ 3% and the particle size D90 ≤ 25 μm, meeting the product quality requirements. The effects of different drying conditions on the product quality are shown in the following table:

[0067]

[0068]

[0069] VI. Quality Monitoring

[0070] Near-infrared spectroscopy combined with HPLC was used to establish a process analysis technology to detect the characteristic peak of DNJ (retention time 6.8 ± 0.2 min) and the characteristic absorption band of flavonoids (the second derivative spectral peak at λ = 510 nm) in real time. By monitoring these characteristic indicators in real time, the elution parameters were dynamically adjusted to make RSD ≤ 2.5%, ensuring the stable and consistent quality of the product. During the production process, samples were collected regularly for testing, and parameters such as elution flow rate and eluent concentration were adjusted in a timely manner according to the test results to ensure that the product met the quality standards.

[0071] VII. Optimization in the Purification Stage of DNJ - Application of Molecularly Imprinted Polymers (MIPs)

[0072] Molecularly imprinted polymers (MIPs) were introduced in the purification stage of DNJ. MIPs were polymerized under supercritical CO2 conditions with DNJ as the template molecule, acrylamide as the functional monomer, and ethylene glycol dimethacrylate as the crosslinking agent to form specific recognition sites. This polymer has high selectivity and strong adsorption capacity for DNJ, and the adsorption capacity reaches more than 35 mg / g. In practical applications, adding MIPs can further improve the purity and recovery rate of DNJ. For example, under the same purification conditions, after adding MIPs, the purity of DNJ increased from 97.5% to 99.2%, and the recovery rate increased from 85% to 92%.

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements will not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting flavonoids and DNJ from mulberry leaves, characterized in that: The method comprises the following steps: (a) Raw material pretreatment: mulberry leaves with a water content of ≤8% are crushed to a particle size of 80-120 mesh, mixed with a citric acid-sodium hydrogen phosphate buffer with a pH value of 4.5-5.5 at a mass ratio of 1:12-15, and a composite enzyme preparation consisting of cellulase, pectinase and β-glucosidase is added, wherein the enzyme activity ratio of cellulase, pectinase and β-glucosidase is 2:1:0.5, and microwave-assisted enzymolysis is performed at 45-50° C. for 30-40 minutes, and the microwave power density is controlled at 0.5-0.8 W / g; (b) Multimodal extraction: add 65% ethanol solution containing 0.2% L-ascorbic acid to the enzymatic hydrolysate, adjust the ethanol concentration of the system to 45-50%, and use a pulsed ultrasound-countercurrent extraction device to perform two-stage extraction at 60-65°C and 0.08-0.12MPa pressure, wherein the first stage has an ultrasound frequency of 28kHz and a power of 250W, and the second stage is switched to 40kHz and a power of 180W, and the total extraction time is ≤60 minutes; (c) Gradient separation: the extract was passed through a composite chromatography column loaded with AB-8 and D101 resins, wherein the mass ratio of AB-8 to D101 resins was 1:2, DNJ was first eluted with 4-6 column volumes of 25% ethanol at a flow rate of 1.2 BV / h, and then flavonoids were eluted with 8-10 column volumes of 70% ethanol at a flow rate of 2.0 BV / h; (d) Targeted purification: The DNJ eluate was adsorbed on a 001×7 strong acidic cation exchange resin and then gradient eluted with 0.1 mol / L ammonia water. The flavonoid eluate was purified by ultrafiltration membrane combined with Zeta potential control to remove polyphenol impurities, where the molecular weight cutoff of the ultrafiltration membrane was 5000 Da. (e) Preparation of finished product: The purified solution was subjected to three-stage vacuum concentration and low-temperature spray drying to obtain a finished product with a DNJ purity of ≥98.5% and a total flavonoid content of ≥92%. During the three-stage vacuum concentration, the temperature was gradually cooled to 50°C, 45°C and 40°C. During the low-temperature spray drying, the inlet air temperature was 110°C and the outlet air temperature was 55°C.

2. The method for extracting flavonoids and DNJ from mulberry leaves according to claim 1, characterized in that: The compound enzyme preparation added in step (a) is 1.2-1.8% of the dry weight of mulberry leaves, wherein the cellulase activity is ≥5000U / g, the pectinase activity is ≥3000U / g, and the β-glucosidase activity is ≥800U / g; The microwave treatment adopted an intermittent irradiation mode, specifically working for 10 s with an interval of 5 s, and simultaneously applying 20-30 kHz mechanical vibration to enhance the efficiency of cell wall disruption.

3. The method for extracting flavonoids and DNJ from mulberry leaves according to claim 1, characterized in that: The pulsed ultrasound-countercurrent extraction device in step (b) is equipped with a phase modulation module to make the ultrasonic field intensity between 120-180 W / cm 2 The range fluctuates according to the law of sine wave, and the ethanol solution is treated by high-pressure homogenization before entering the extraction system, with a pressure of 60-80MPa and circulated 3 times.

4. The method for extracting flavonoids and DNJ from mulberry leaves according to claim 1, characterized in that: The composite chromatography column in step (c) is activated by alternating 0.1 mol / L NaOH and anhydrous ethanol before filling, and the change of the dielectric constant of the eluent is monitored in real time during the chromatography process. When the dielectric constant drops to 40-45, the eluent ratio is switched.

5. The method for extracting flavonoids and DNJ from mulberry leaves according to claim 1, characterized in that: In step (d), the Zeta potential is regulated by adding 0.05% chitosan quaternary ammonium salt, and the pH value of the solution is controlled at 4.0-4.5 to form floccules. The impurity removal rate is increased to more than 98% by combining electric field assisted sedimentation, specifically, the voltage is 15 V / cm and the action time is 20 min.

6. The method for extracting flavonoids and DNJ from mulberry leaves according to claim 1, characterized in that: The method also includes step (f) quality monitoring: using near infrared spectroscopy combined with HPLC to establish process analysis technology, real-time detection of DNJ characteristic peaks and flavonoid characteristic absorption bands, and dynamic adjustment of elution parameters to make RSD≤2.5%.

7. The method for extracting flavonoids and DNJ from mulberry leaves according to claim 1, characterized in that: Molecular imprinting polymers (MIPs) are introduced in the DNJ purification stage. The preparation method also includes: using DNJ as a template molecule, acrylamide as a functional monomer, and ethylene glycol dimethacrylate as a cross-linking agent, polymerizing under supercritical CO2 conditions to form specific recognition sites, with an adsorption capacity of more than 35 mg / g.

8. The method for extracting flavonoids and DNJ from mulberry leaves according to claim 1, characterized in that: In step (e), the low temperature spray drying adopts the synergistic technology of two-fluid nozzle and electrostatic atomization to control the surface charge density of the droplets to 0.8-1.2C / m 2 A vortex stabilizer is installed in the drying tower to ensure that the moisture content of the product is ≤3% and the particle size D90 is ≤25μm.

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