Method for extracting flavonoid compounds from sophora alopecuroides matrine production waste liquid

Abstract

The invention relates to the technical field of natural active ingredient extraction, and particularly discloses a method for extracting flavonoid compounds from sophora alopecuroide matrine production waste liquid, and the method comprises the following steps: sequentially adding a reducing agent and a chelating agent into alkaline water waste liquid, uniformly stirring, standing, and filtering to obtain pretreated filtrate; dropwise adding organic acid into the pretreated filtrate to adjust the pH to obtain a suspension; standing and curing the suspension, centrifugally collecting precipitate, washing, and freeze-drying to obtain flavone powder. According to the method, the reducing agent and the chelating agent are combined for use, the precipitation efficiency and purity of the flavonoid compounds are remarkably improved and the impurity content is reduced through the dual effects of chemical reduction and physical adsorption, the yield of the flavonoid compounds is improved through the synergistic reaction of the reducing agent and the chelating agent, and the dosage of chemicals and the complexity of waste liquid treatment in the production process are effectively reduced; and energy conservation, environmental protection and economic benefits are improved.

Description

Technical Field

[0001] This invention relates to the field of natural active ingredient extraction technology, and more specifically, to a method for extracting flavonoids from the waste liquid produced by the production of matrine from Sophora flavescens. Background Technology

[0002] *Sophora alopecuroides* L., belonging to the genus *Sophora* in the legume family, is a perennial herbaceous plant that often becomes woody at the base, resembling a subshrub. It is an important ecological grass in the ecosystem of Northwest my country, exhibiting strong drought and salt tolerance. The *Compendium of Materia Medica* records that the whole plant and seeds of *Sophora alopecuroides* can be used medicinally, possessing effects such as clearing heat and detoxifying, antibacterial and anti-inflammatory properties, diuresis and heat-clearing, analgesic and sedative effects. Modern pharmacological studies have further confirmed that *Sophora alopecuroides* contains abundant alkaloids and flavonoids, exhibiting a wide range of pharmacological effects including anticancer, antiviral, anti-inflammatory, and antibacterial activity. Among the many effective compounds in *Sophora alopecuroides*, quinolone alkaloids such as matrine and oxymatrine have been the most extensively studied and utilized.

[0003] Flavonoids, as an important class of plant secondary metabolites, exhibit significant biological activities due to their unique C6-C3-C6 basic carbon skeleton structure and polyphenolic groups. To date, 57 flavonoids have been isolated and identified from bitter bean, demonstrating important medicinal and biological activities in antioxidation, anti-inflammation, antiviral, antibacterial, and hypoglycemic and lipid-lowering effects. Therefore, the development of flavonoids from bitter bean has enormous economic and health value.

[0004] Currently, processing enterprises extract matrine monomers of varying purities (up to 97% or higher) from bitter bean seeds. The resulting matrine and total alkaloids from bitter bean seeds can be used in medical, pesticide, fruit and vegetable preservation, and daily necessities industries. Statistics show that since 2016, the demand for bitter bean seed raw materials in this industry chain has exceeded 2,000 tons annually, with an annual output value of 200 million yuan.

[0005] Although the extraction of matrine has reached a certain industrial scale, existing processes still face several challenges. One key factor hindering the development of this industry chain is the relatively singular focus on the target product. Current production primarily concentrates on the extraction of matrine monomers, failing to fully explore other valuable active ingredients in bitter beans. High raw material costs result in relatively low unit output value, impacting the overall competitiveness of the industry. Furthermore, existing matrine extraction processes generate substantial amounts of process waste liquid. (See attached...) Figure 1Taking the schematic diagram of the process for extracting matrine from bitter beans as an example, after steps such as ethanol extraction, concentration, acid extraction, alkali precipitation, and toluene extraction, a large amount of alkaline wastewater is generated. This alkaline wastewater is large in volume (approximately 0.68 tons per ton of raw material) and has a complex composition. Currently, it mainly relies on wastewater treatment plants for treatment before discharge, which not only increases the operating costs of enterprises but also brings environmental pollution pressure. Based on the above, this invention proposes a method for extracting flavonoids from the wastewater generated during the production of matrine from bitter beans. Summary of the Invention

[0006] To address the problems of single target product, low unit output value, and high wastewater treatment costs in the existing bitter bean matrine extraction industry chain, this invention provides a method for extracting flavonoids from the wastewater of bitter bean matrine production. This invention utilizes the principle of alkali extraction and acid precipitation to reuse the existing process wastewater to extract effective flavonoids from bitter bean, effectively realizing the resource utilization of process wastewater, reducing pollutant emissions, extending the bitter bean industry chain, and significantly increasing the industry's added value.

[0007] This invention provides a method for extracting flavonoids from waste liquid produced from the production of matrine in Sophora flavescens, employing the following technical solution:

[0008] A method for extracting flavonoids from the production waste liquid of matrine from Sophora flavescens includes the following steps:

[0009] S1. Under stirring, add reducing agent and chelating agent to alkaline waste liquid in sequence, stir evenly, let stand, and then filter to obtain pretreated filtrate.

[0010] S2. Under stirring, add organic acid dropwise to the pretreated filtrate to adjust the pH to 3-4. A precipitate will form on the surface of the liquid, resulting in a suspension.

[0011] S3. Place the suspension at 1-6℃ and let it stand for 8-12 hours. Then, centrifuge to collect the precipitate, wash it, and freeze-dry it to obtain flavonoid powder.

[0012] Preferably, the alkaline waste liquid in step S1 is generated by the process of extracting matrine from bitter beans, specifically by extracting bitter beans with ethanol, followed by concentration, acid extraction, alkali precipitation, and toluene extraction of bitter bean alkaloids.

[0013] Preferably, the process for extracting matrine from bitter beans includes the following steps:

[0014] (1) After crushing the bitter bean seeds, sieve them through a 30-mesh sieve to obtain coarse bitter bean powder. Control the mass-volume ratio of coarse bitter bean powder to ethanol solution to be 1:6-8, the stirring speed to be 200-300 rpm, and the soaking temperature to be 25-35℃. Soak the coarse bitter bean powder in ethanol solution with a mass fraction of 60-65% for 10-15 hours. Extract by percolation, control the flow rate of the percolate to be 3-5 mL / min, and continue to add ethanol solution with a mass fraction of 60-65% until the mass-volume ratio of coarse bitter bean powder to ethanol solution is 1:8-10 to obtain total alkali percolate. Control the vacuum degree to be -0.06 MPa to -0.08 MPa and the temperature to be 60-70℃. Concentrate the total alkali percolate under reduced pressure to 0.1-0.3 times the original volume to obtain total alkali concentrate.

[0015] (2) Control the stirring speed to 500-700 rpm and the dropping rate to 0.1-0.3 mL / min. Add sulfuric acid solution with a mass concentration of 10-20% to the total alkali concentrate to adjust the pH to 1-2. Heat to 50-60℃ and continue stirring for 30-60 min. Then cool to 4-10℃ and let stand for 6-10 h to separate the layers. Take the upper layer containing matrine sulfate to obtain the total alkali supernatant.

[0016] (3) Control the stirring speed to 500-700 rpm and the dropping rate to 0.4-0.6 mL / min. Add 20-30% sodium hydroxide solution to the total alkali supernatant to adjust the pH to 10.5-11.5 for alkali precipitation treatment to obtain alkalized solution. Add 0.3-0.5 times the volume of toluene to the alkalized solution. Stir at 800-1000 rpm for 15-25 min. Let stand for separation and collect the upper extract. Add toluene to the lower alkaline aqueous phase and extract 3-5 times to obtain the lower alkaline aqueous phase (i.e., alkali waste liquid) and the combined extract. Control the stirring speed to 500-700 rpm and the dropping rate to 0.1-0.3 mL / min. Add 5-10% sulfuric acid solution to the combined extract to adjust the pH to 3-4. Let stand for separation and take the lower layer of bitter bean total alkali solution.

[0017] (4) Place the total alkaline solution of bitter bean in a high-pressure reactor, add 1-5% of the total alkaline solution of bitter bean on carbon catalyst, seal the reactor and replace the air with nitrogen, then introduce hydrogen to a pressure of 0.5-1.5 MPa, stir the reaction at 40-60℃ for 2-6 hours to obtain the conversion liquid, filter the conversion liquid to recover the catalyst, and obtain the hydrogenated solution of total alkaline solution of bitter bean;

[0018] (5) Control the stirring speed to 500-700 rpm and the dropping rate to 0.1-0.3 mL / min. Add concentrated sulfuric acid with a mass fraction of 70-80% to the hydrogenated solution of total alkaloids of Sophora flavescens to adjust the pH to 4-5 to obtain an acidified solution. Concentrate the acidified solution under reduced pressure to near saturation under vacuum conditions of -0.06 MPa to -0.08 MPa and temperature of 40-50℃ to obtain a concentrated solution. Allow the concentrated solution to cool naturally to room temperature and stand until crystal nuclei are formed. Then transfer it to a temperature of 0-4℃ for crystallization for 12-24 hours. Centrifuge the solid-liquid mixture after crystallization at a speed of 800-1200 rpm to remove the solid. Dry it under vacuum at a temperature of 50-60℃ to constant weight to obtain matrine (monomer).

[0019] Preferably, the amount of reducing agent added in step S1 is 1-3% of the total mass of the alkaline waste liquid.

[0020] Preferably, the reducing agent in step S1 is prepared by the following method:

[0021] Step 1: Add alkaline lignin to sodium hydroxide solution, stir to dissolve, filter, and collect the lignin alkaline solution;

[0022] Step II: Add the lignin alkali solution to the high-pressure reactor, add D-xylose and stir until homogeneous, then heat to react. After the reaction is complete, allow it to cool naturally to room temperature to obtain the reaction solution.

[0023] Step III: Under nitrogen protection and stirring, add sodium sulfide nonahydrate to the reaction solution and heat to react. After the reaction is completed, cool naturally to room temperature to obtain the reaction mixture.

[0024] Step IV: In an ice-water bath, add hydrochloric acid dropwise to the reaction mixture to adjust the pH to neutral. After standing at room temperature for 1-2 hours, centrifuge, collect the precipitate, wash, dry, and grind to obtain the reducing agent.

[0025] Preferably, in step I, the mass ratio of alkaline lignin to sodium hydroxide solution is 1:10-15, and the mass fraction of sodium hydroxide solution is 2-5%.

[0026] Preferably, in step I, a 40-60μm filter screen is used for filtration.

[0027] Preferably, in step II, the amount of D-xylose added is 20-25% of the alkaline lignin content.

[0028] Preferably, in step II, the heating rate is 5-8℃ / min, the reaction temperature is 170-190℃, and the reaction time is 2-4h.

[0029] Preferably, in step III, the stirring speed is 300-500 rpm.

[0030] Preferably, in step III, the amount of sodium sulfide nonahydrate added is 28-32% of the alkaline lignin content, and the sodium sulfide nonahydrate is added in three batches with an interval of 10-15 minutes between each batch, and the temperature during the feeding process is controlled not to exceed 40°C.

[0031] Preferably, in step III, the heating rate is 1-2℃ / min, the reaction temperature is 80-90℃, and the reaction time is 4-6h.

[0032] Preferably, in step IV, the hydrochloric acid concentration is 5-6M.

[0033] Preferably, in step IV, the centrifugation speed is 5000-6000 rpm and the centrifugation time is 10-20 min.

[0034] Preferably, in step IV, drying refers to drying in a vacuum oven at 60-70°C until constant weight.

[0035] Preferably, in step IV, grinding refers to grinding through an 80-100 mesh sieve.

[0036] Preferably, the amount of chelating agent added in step S1 is 4-8% of the total mass of the alkaline waste liquid.

[0037] Preferably, the chelating agent in step S1 is prepared by the following method:

[0038] Step 1: Dissolve chitosan in acetic acid solution to obtain chitosan solution;

[0039] Step 2: Add the chitosan solution dropwise to the sodium hydroxide solution. After the addition is complete, let it stand and solidify for 20-40 minutes, then filter and wash to obtain chitosan microspheres.

[0040] Step 3: Immerse the chitosan microspheres in sodium alginate solution, allow them to stand for 20-40 minutes for adsorption, then filter to obtain the adsorbed microspheres.

[0041] Step 4: Immerse the adsorbed microspheres in calcium chloride solution and allow the cross-linking reaction to proceed for 50-60 minutes. Filter and wash to obtain core-shell microspheres.

[0042] Step 5: Add the core-shell microspheres to the carbonate buffer solution, add glutaraldehyde solution to carry out the cross-linking reaction. After the reaction is completed, filter and wash to obtain the cross-linked microspheres.

[0043] Step 6: Immerse the cross-linked microspheres in sodium tripolyphosphate solution, add sodium hydroxide solution to adjust the pH to 10-11, react, filter, and wash to obtain phosphorylated microspheres;

[0044] Step 7: Disperse the phosphorylated microspheres in deionized water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir and activate at room temperature for 20-40 min, add ethylenediamine and stir evenly, add dilute hydrochloric acid dropwise to adjust the pH to 4.8-5.5, continue the reaction for 12-16 h, filter, wash and dry to obtain the chelating agent.

[0045] Preferably, in step 1, the mass ratio of chitosan to acetic acid solution is 1:42-46, and the mass fraction of acetic acid solution is 2-4%.

[0046] Preferably, in step 2, the stirring speed is 300-500 rpm and the dropping rate is 1-3 mL / min.

[0047] Preferably, in step 2, the volume ratio of chitosan solution to sodium hydroxide solution is 1:15-20, and the mass fraction of sodium hydroxide solution is 8-15%.

[0048] Preferably, in step 3, the mass ratio of chitosan microspheres to sodium alginate solution is 1:10-12, and the mass fraction of sodium alginate solution is 3-5%.

[0049] Preferably, in step 4, the mass ratio of the adsorbed microspheres to the calcium chloride solution is 1:10-12, and the mass fraction of the calcium chloride solution is 3-8%.

[0050] Preferably, in step 5, the mass ratio of core-shell microspheres, carbonate buffer solution, and glutaraldehyde solution is 1:10-12:1-2, the carbonate buffer solution is a sodium carbonate-sodium bicarbonate buffer solution with a pH of 9.5-10.5, and the mass fraction of glutaraldehyde solution is 0.5-1%.

[0051] Preferably, in step 5, the crosslinking reaction temperature is 25-35℃ and the crosslinking reaction time is 2-3h.

[0052] Preferably, in step 6, the mass ratio of the cross-linked microspheres to the sodium tripolyphosphate solution is 1:10-12, and the mass fraction of the sodium tripolyphosphate solution is 8-15%.

[0053] Preferably, in step 6, the concentration of the sodium hydroxide solution is 1-2M.

[0054] Preferably, in step 6, the reaction temperature is 50-60℃ and the reaction time is 3-5h.

[0055] Preferably, in step 7, the mass ratio of phosphorylated microspheres, deionized water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and ethylenediamine is 8-10:20-22:0.4-0.6:0.2-0.4:1-3.

[0056] Preferably, in step 7, the concentration of dilute hydrochloric acid is 0.1-0.2M.

[0057] Preferably, the drying in step 7 refers to drying in an oven at 50-60℃ for 3-5 hours.

[0058] Preferably, during the preparation of the chelating agent, a 150-200μm filter screen is used for filtration.

[0059] Preferably, in step S1, the stirring speed is 200-400 rpm, the stirring time after adding the reducing agent is 10-30 min, and the stirring time after adding the chelating agent is 20-30 min.

[0060] Preferably, in step S1, "standing still" means standing still at room temperature for 2-3 hours.

[0061] Preferably, in step S1, filtration refers to filtration through a 50-100μm filter.

[0062] Preferably, in step S2, the stirring speed is 300-500 rpm and the dropping rate is 4-6 mL / min.

[0063] Preferably, the organic acid in step S2 is a lactic acid solution with a mass fraction of 40-50%.

[0064] Preferably, centrifugation in step S3 refers to centrifugation at a speed of 3000-5000 rpm for 10-20 minutes.

[0065] Preferably, in step S3, washing refers to: adding 2-4 times its mass of pre-cooled deionized water at 1-6℃ to the precipitate, stirring evenly, resuspending the precipitate, and centrifuging again to obtain the washed precipitate.

[0066] Preferably, in step S3, freeze drying refers to freeze drying to constant weight under conditions of vacuum of 3-8 Pa and temperature of -30 to -50°C, away from light.

[0067] In summary, the present invention has the following beneficial effects:

[0068] This invention extracts flavonoids from the alkaline wastewater generated during the extraction of matrine from bitter beans, achieving efficient resource recycling, reducing wastewater discharge, and being environmentally friendly. Through the combined application of reducing agents and chelating agents, the extraction efficiency and purity of flavonoids are improved, ensuring full recovery of effective components from the wastewater. The process is simple to operate, with mild extraction conditions, guaranteeing the structure and activity of the flavonoids, resulting in high-quality products suitable for industrial-scale mass production, and promoting resource recycling and green development.

[0069] This invention involves mixing alkaline lignin and D-xylose, then treating with sodium sulfide nonahydrate to obtain a reducing agent. This agent promotes the reduction and stability of flavonoids by breaking down impurity complexes in alkaline wastewater, thereby improving their solubility and recovery rate. This significantly enhances the extraction rate and purity of flavonoids while reducing impurity interference.

[0070] This invention prepares a chelating agent by modifying chitosan-based microspheres through multi-step cross-linking and phosphorylation. The chelating agent synergistically chelates metal ions and multivalent impurities in waste liquid through chitosan and alginate groups, and increases chemical stability through cross-linking structure. It effectively captures and removes impurities, significantly reduces the interference of impurities on flavonoid precipitation, improves the purity and stability of flavonoids, and achieves the effect of enhancing the selective extraction and high recovery rate of flavonoid compounds.

[0071] This invention employs a combination of reducing and chelating agents. Through the dual effects of chemical reduction and physical adsorption, it significantly improves the precipitation efficiency and purity of flavonoids and reduces impurity content. The synergistic reaction of the two not only increases the yield of flavonoids but also effectively reduces the amount of chemicals used in the production process and the complexity of wastewater treatment, achieving a dual improvement in energy conservation, environmental protection, and economic benefits. Attached Figure Description

[0072] Figure 1 This is a flowchart illustrating the process of extracting matrine from bitter beans in the background art of this invention.

[0073] Figure 2 This is a state diagram of the alkaline waste liquid in step S1 and the suspension in step S2 in Embodiment 1 of the present invention.

[0074] Figure 3 This is a state diagram of the flavonoid powder prepared in Example 1 of the present invention. Detailed Implementation

[0075] The present invention will be further described in detail below with reference to the embodiments.

[0076] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0077] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0078] Among them, the palladium-on-carbon catalyst has the following characteristics: CAS No. 7440-05-3, active palladium metal content of 3% w / w, activated carbon particle size of 20-50 μm, and specific surface area of ​​900-1200 m². 2 / g, density approximately 0.5-0.7g / cm³ 3 The palladium particles, with a diameter of 5-10 nm, were purchased from Hubei Chengfeng Chemical Co., Ltd.

[0079] Alkaline lignin: CAS No. 8068-05-1, content 98%, brand Lanabai, purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.

[0080] Chitosan: CAS No. 9012-76-4, content 99%, brand Youtai, purchased from Anhui Youtai Bioengineering Co., Ltd.

[0081] Sodium alginate: CAS No. 9005-38-3, purity 99%, brand Changxinghang, purchased from Hebei Changxinghang Biotechnology Co., Ltd.

[0082] The alkaline waste liquid is generated by the following process of extracting matrine from bitter beans. Specifically, it is generated after the bitter bean seeds (i.e., bitter bean seeds) are crushed and screened, and then subjected to steps such as ethanol extraction, concentration, acid extraction, alkali precipitation, and toluene extraction. That is, the alkaline waste liquid obtained by step (3) of the following process of extracting matrine from bitter beans.

[0083] The process for extracting matrine from bitter beans includes the following steps:

[0084] (1) After crushing the bitter bean seeds, sieve them through a 30-mesh sieve to obtain coarse bitter bean powder. Control the mass-volume ratio of coarse bitter bean powder to ethanol solution to be 1:7, the stirring speed to be 250 rpm, and the soaking temperature to be 30℃. Soak the coarse bitter bean powder in 65% ethanol solution for 12 hours. Extract by percolation, control the flow rate of the percolate to be 4 mL / min, and continue to add 65% ethanol solution until the mass-volume ratio of coarse bitter bean powder to ethanol solution is 1:8 to obtain total alkali percolate. Control the vacuum degree to be -0.08 MPa and the temperature to be 60℃. Concentrate the total alkali percolate under reduced pressure to 0.3 times the original volume to obtain total alkali concentrate.

[0085] (2) Control the stirring speed to 500 rpm and the dropping rate to 0.2 mL / min. Add 15% sulfuric acid solution to the total alkali concentrate to adjust the pH to 1.5. Heat to 60℃, continue stirring for 30 min, cool to 4℃, let stand for 6 h to separate the layers, take the liquid containing matrine sulfate in the upper layer to obtain the total alkali supernatant.

[0086] (3) Control the stirring speed to 500 rpm and the dropping rate to 0.5 mL / min. Add 25% sodium hydroxide solution to the total alkali supernatant to adjust the pH to 11 for alkali precipitation treatment to obtain alkalized solution. Add 0.4 times the volume of toluene to the alkalized solution. Stir at 1000 rpm for 20 min. Let stand for separation and collect the upper layer extract. Add toluene to the lower alkaline aqueous phase and extract 4 times to obtain the lower alkaline aqueous phase (i.e., alkali waste liquid) and the combined extract. Control the stirring speed to 500 rpm and the dropping rate to 0.2 mL / min. Add 8% sulfuric acid solution to the combined extract to adjust the pH to 3.5. Let stand for separation and take the lower layer of bitter bean total alkali solution.

[0087] (4) Place the total alkaline solution of bitter bean in a high-pressure reactor, add 3% of the total alkaline solution of bitter bean on carbon catalyst, seal the reactor and replace the air with nitrogen, then introduce hydrogen to a pressure of 1.0 MPa, stir the reaction at 50°C for 4 hours to obtain the conversion liquid, filter the conversion liquid to recover the catalyst, and obtain the hydrogenated solution of total alkaline solution of bitter bean.

[0088] (5) Control the stirring speed to 500 rpm and the dropping rate to 0.2 mL / min. Add concentrated sulfuric acid with a mass fraction of 75% to the hydrogenated solution of total alkaloids of Sophora flavescens to adjust the pH to 4.5 to obtain an acidified solution. Concentrate the acidified solution under reduced pressure to near saturation under vacuum conditions of -0.06 MPa and 50°C to obtain a concentrated solution. Allow the concentrated solution to cool naturally to room temperature and stand until crystal nuclei are formed. Then, transfer it to a temperature of 4°C for crystallization for 20 h. Centrifuge the solid-liquid mixture after crystallization at a speed of 1000 rpm to remove the solid. Dry it under vacuum at 60°C to constant weight to obtain matrine (monomer).

[0089] Spectrophotometric analysis revealed that the flavonoid content in the alkaline wastewater obtained in step (3) was 5.26 g / kg, a decrease of only 1.5 g / kg compared to the 6.76 g / kg flavonoid content in the total alkali concentrate obtained in step (1). Therefore, the alkaline wastewater obtained in step (3) possesses significant potential for the development and reuse of flavonoids from bitter bean.

[0090] Example 1

[0091] A method for extracting flavonoids from the production waste liquid of matrine from Sophora flavescens includes the following steps:

[0092] S1. Control the stirring speed to 300 rpm, add 2% of the reducing agent by mass to 1 kg of alkaline waste liquid, stir for 20 min, add 6% of the chelating agent by mass and continue stirring for 25 min until uniformly dispersed, let stand at room temperature for 2.5 h, and then filter through a 60 μm filter to obtain the pretreated filtrate.

[0093] S2. Control the stirring speed at 400 rpm and the dropping rate at 5 mL / min. Add 45% lactic acid solution (w / w) to the pretreated filtrate to adjust the pH to 3.5. A golden precipitate will form, resulting in a suspension (see Appendix for details). Figure 2 );

[0094] S3. After allowing the suspension to stand and mature at 4℃ for 10 hours, centrifuge at 4000 rpm for 15 minutes, collect the precipitate, add three times its mass of pre-cooled deionized water at 4℃ to the precipitate, stir thoroughly and resuspend, and centrifuge again at 4000 rpm for 15 minutes to obtain the washed precipitate. Place the washed precipitate under a vacuum of 5 Pa and a temperature of -40℃, and freeze-dry it in the dark until constant weight to obtain the golden yellow flavonoid powder (see appendix for details). Figure 3 ).

[0095] The reducing agent is prepared by the following method:

[0096] Step 1: Add alkaline lignin to a 3.5% sodium hydroxide solution at a mass ratio of 1:12, stir to dissolve for 2.5 hours, filter through a 50μm filter to remove insoluble impurities, and collect the lignin alkaline solution.

[0097] Step II: Add the lignin alkaline solution to the high-pressure reactor, add 22% D-xylose (by weight of alkaline lignin) and stir until homogeneous. Then, heat the mixture to 180°C at a rate of 6.5°C / min and maintain the temperature for 3 hours. After the reaction is complete, allow it to cool naturally to room temperature to obtain the reaction solution.

[0098] Step III: Under nitrogen protection, control the stirring speed at 400 rpm, add sodium sulfide nonahydrate (30% of the mass of alkaline lignin) to the reaction solution in three equal batches, with an interval of 12 min between each batch. Control the temperature during the addition process to not exceed 40℃ to avoid side reactions. After the addition is completed, raise the temperature to 85℃ at a rate of 1.5℃ / min and keep the temperature for 5 hours. After the reaction is completed, let it cool naturally to room temperature to obtain the reaction mixture.

[0099] Step IV: In an ice-water bath, add 5.5M hydrochloric acid dropwise to the reaction mixture to adjust the pH to neutral. After standing at room temperature for 1.5 hours, centrifuge at 5500 rpm for 15 minutes, collect the precipitate, wash the precipitate repeatedly with deionized water 5 times to remove residual impurities, dry it in a vacuum oven at 65℃ until constant weight, take it out, grind it through a 90-mesh sieve to obtain the reducing agent.

[0100] Chelating agents are prepared by the following methods:

[0101] Step 1: Dissolve chitosan in a 3% (w / w) acetic acid solution at a mass ratio of 1:44 to obtain a chitosan solution.

[0102] Step 2: Control the stirring speed to 400 rpm, the dropping rate to 2 mL / min, and the volume ratio of chitosan solution to sodium hydroxide solution to 1:18. Add the chitosan solution dropwise to the 12% sodium hydroxide solution. After the addition is complete, let it stand and solidify for 30 min. Filter it with an 180 μm filter and wash it repeatedly with deionized water until the filtrate is neutral to obtain chitosan microspheres.

[0103] Step 3: Control the mass ratio of chitosan microspheres to sodium alginate solution to 1:11. Immerse the chitosan microspheres in a 4% sodium alginate solution and allow them to stand for 30 minutes for adsorption. Then filter the solution using an 180μm filter to obtain the adsorbed microspheres.

[0104] Step 4: Control the mass ratio of the adsorbed microspheres to the calcium chloride solution to 1:11. Immerse the adsorbed microspheres in a 5% (w / w) calcium chloride solution and allow the cross-linking reaction to proceed for 55 min. Filter the solution using an 180 μm filter and wash with deionized water to ensure the removal of excess calcium salts and impurities, thus obtaining core-shell microspheres.

[0105] Step 5: Control the mass ratio of core-shell microspheres, sodium carbonate-sodium bicarbonate buffer, and glutaraldehyde solution to 1:11:1.5. Add the core-shell microspheres to the sodium carbonate-sodium bicarbonate buffer at pH 10, add 0.8% glutaraldehyde solution, and allow the cross-linking reaction to proceed at 30°C for 2.5 hours. Filter the mixture through an 180μm filter and wash thoroughly with deionized water to avoid glutaraldehyde residue, thus obtaining the cross-linked microspheres.

[0106] Step 6: Control the mass ratio of cross-linked microspheres to sodium tripolyphosphate solution to 1:11. Immerse the cross-linked microspheres in a 12% sodium tripolyphosphate solution, add 1.5M sodium hydroxide solution to adjust the pH to 10.5, react at 55℃ for 4 hours, filter with a 180μm filter, wash with deionized water until neutral, and obtain phosphorylated microspheres.

[0107] Step 7: Control the mass ratio of phosphorylated microspheres, deionized water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and ethylenediamine to 9:21:0.5:0.3:2. Disperse the phosphorylated microspheres in deionized water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir and activate at room temperature for 30 min, add ethylenediamine and stir evenly, add 0.15M dilute hydrochloric acid dropwise to adjust the pH to 5.2, continue the reaction for 14 h, filter with a 180 μm filter, wash with 0.15M dilute hydrochloric acid to remove reaction residues, wash with deionized water until neutral, and dry in an oven at 55℃ for 4 h to obtain the chelating agent.

[0108] Example 2

[0109] A method for extracting flavonoids from the production waste liquid of matrine from Sophora flavescens includes the following steps:

[0110] S1. Control the stirring speed to 200 rpm, add 1% of the reducing agent by mass to 1 kg of alkaline waste liquid, stir for 10 min, add 8% of the chelating agent by mass and continue stirring for 30 min until uniformly dispersed, let stand at room temperature for 2 h, and then filter through a 50 μm filter to obtain the pretreated filtrate.

[0111] S2. Control the stirring speed to 300 rpm and the dropping rate to 4 mL / min. Add 40% lactic acid solution (by mass) to the pretreated filtrate to adjust the pH to 4. A golden yellow precipitate will precipitate, resulting in a suspension.

[0112] S3. Place the suspension at 1℃ and let it stand for 8 hours. Then, centrifuge at 3000 rpm for 20 minutes and collect the precipitate. Add twice the mass of the precipitate to pre-cooled deionized water at 1℃, stir thoroughly and resuspend. Centrifuge again at 3000 rpm for 20 minutes to obtain the washed precipitate. Place the washed precipitate under a vacuum of 3 Pa and a temperature of -30℃ and freeze-dry it in the dark until constant weight to obtain golden yellow flavonoid powder.

[0113] The reducing agent is prepared by the following method:

[0114] Step 1: Add alkaline lignin to a 5% sodium hydroxide solution at a mass ratio of 1:10, stir and dissolve for 2 hours, then filter through a 40μm filter to remove insoluble impurities and collect the lignin alkaline solution.

[0115] Step II: Add the lignin alkaline solution to the high-pressure reactor, add 20% D-xylose of alkaline lignin and stir evenly. Then, heat to 170℃ at a rate of 5℃ / min and keep the temperature for 4 hours. After the reaction is completed, let it cool naturally to room temperature to obtain the reaction solution.

[0116] Step III: Under nitrogen protection, control the stirring speed at 300 rpm, add sodium sulfide nonahydrate (28% by weight of alkaline lignin) to the reaction solution in three equal batches, with an interval of 10 min between each batch. Control the temperature during the addition process to not exceed 40℃ to avoid side reactions. After the addition is complete, raise the temperature to 80℃ at a rate of 1℃ / min and keep the reaction at this temperature for 6 hours. After the reaction is completed, allow it to cool naturally to room temperature to obtain the reaction mixture.

[0117] Step IV: In an ice-water bath, add 5M hydrochloric acid dropwise to the reaction mixture to adjust the pH to neutral. After standing at room temperature for 2 hours, centrifuge at 5000 rpm for 20 minutes, collect the precipitate, wash the precipitate repeatedly with deionized water 4 times to remove residual impurities, dry it in a vacuum oven at 60℃ until constant weight, take it out, grind it through an 80-mesh sieve to obtain the reducing agent.

[0118] Chelating agents are prepared by the following methods:

[0119] Step 1: Dissolve chitosan in a 4% (w / w) acetic acid solution at a mass ratio of 1:42 to obtain a chitosan solution.

[0120] Step 2: Control the stirring speed to 300 rpm, the dropping rate to 1 mL / min, and the volume ratio of chitosan solution to sodium hydroxide solution to 1:20. Add the chitosan solution dropwise to the 8% sodium hydroxide solution. After the addition is complete, let it stand and solidify for 40 min. Filter it with a 150 μm filter and wash it repeatedly with deionized water until the filtrate is neutral to obtain chitosan microspheres.

[0121] Step 3: Control the mass ratio of chitosan microspheres to sodium alginate solution to 1:10. Immerse the chitosan microspheres in a 5% sodium alginate solution and allow them to stand for 20 minutes for adsorption. Then filter the solution using a 150μm filter to obtain the adsorbed microspheres.

[0122] Step 4: Control the mass ratio of the adsorbed microspheres to the calcium chloride solution to 1:10. Immerse the adsorbed microspheres in an 8% (w / w) calcium chloride solution and allow the cross-linking reaction to proceed for 50 min. Filter the solution using a 150 μm filter and wash with deionized water to ensure the removal of excess calcium salts and impurities, thus obtaining core-shell microspheres.

[0123] Step 5: Control the mass ratio of core-shell microspheres, sodium carbonate-sodium bicarbonate buffer, and glutaraldehyde solution to 1:10:1. Add the core-shell microspheres to the sodium carbonate-sodium bicarbonate buffer at pH 9.5, add 0.5% glutaraldehyde solution, and perform the cross-linking reaction at 35°C for 2 hours. Filter the mixture through a 150μm filter and wash thoroughly with deionized water to avoid glutaraldehyde residue, thus obtaining the cross-linked microspheres.

[0124] Step 6: Control the mass ratio of cross-linked microspheres to sodium tripolyphosphate solution to 1:10. Immerse the cross-linked microspheres in a 15% sodium tripolyphosphate solution, add 1M sodium hydroxide solution to adjust the pH to 11, and react at 50°C for 5 hours. Filter the solution through a 150μm filter and wash with deionized water until neutral to obtain phosphorylated microspheres.

[0125] Step 7: Control the mass ratio of phosphorylated microspheres, deionized water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and ethylenediamine to 8:20:0.4:0.2:1. Disperse the phosphorylated microspheres in deionized water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir and activate at room temperature for 20 min, add ethylenediamine and stir evenly, add 0.1M dilute hydrochloric acid dropwise to adjust the pH to 5.5, continue the reaction for 16 h, filter with a 150 μm filter, wash with 0.1M dilute hydrochloric acid to remove reaction residues, wash with deionized water until neutral, and dry in an oven at 50℃ for 5 h to obtain the chelating agent.

[0126] Example 3

[0127] A method for extracting flavonoids from the production waste liquid of matrine from Sophora flavescens includes the following steps:

[0128] S1. Control the stirring speed to 400 rpm, add 3% of the reducing agent by mass to 1 kg of alkaline waste liquid, stir for 30 min, add 4% of the chelating agent by mass and continue stirring for 20 min until uniformly dispersed, let stand at room temperature for 3 h, and then filter through a 75 μm filter to obtain the pretreated filtrate.

[0129] S2. Control the stirring speed to 500 rpm and the dropping rate to 6 mL / min. Add 50% lactic acid solution (by mass) to the pretreated filtrate to adjust the pH to 3. A golden yellow precipitate will precipitate, resulting in a suspension.

[0130] S3. Place the suspension at 6℃ and let it stand for 12 hours. Then, centrifuge at 5000 rpm for 10 minutes and collect the precipitate. Add 4 times its mass of pre-cooled deionized water at 6℃ to the precipitate, stir thoroughly and resuspend. Centrifuge again at 5000 rpm for 10 minutes to obtain the washed precipitate. Place the washed precipitate under a vacuum of 8 Pa and a temperature of -50℃ and freeze-dry it in the dark until constant weight to obtain golden yellow flavonoid powder.

[0131] The reducing agent is prepared by the following method:

[0132] Step 1: Add alkaline lignin to a 2% sodium hydroxide solution at a mass ratio of 1:15. After stirring and dissolving for 3 hours, filter the solution through a 60μm filter to remove insoluble impurities and collect the lignin alkaline solution.

[0133] Step II: Add the lignin alkaline solution to the high-pressure reactor, add 25% D-xylose of alkaline lignin and stir evenly, then heat to 190℃ at a rate of 8℃ / min and keep the temperature for 2 hours. After the reaction is completed, let it cool naturally to room temperature to obtain the reaction solution.

[0134] Step III: Under nitrogen protection, control the stirring speed at 500 rpm, add sodium sulfide nonahydrate (32% by weight of alkaline lignin) to the reaction solution in three equal batches, with an interval of 15 min between each batch. Control the temperature during the addition process to not exceed 40℃ to avoid side reactions. After the addition is completed, raise the temperature to 90℃ at a rate of 2℃ / min and keep the temperature for 4 hours. After the reaction is completed, let it cool naturally to room temperature to obtain the reaction mixture.

[0135] Step IV: In an ice-water bath, add 6M hydrochloric acid dropwise to the reaction mixture to adjust the pH to neutral. After aging at room temperature for 1 hour, centrifuge at 6000 rpm for 10 minutes, collect the precipitate, wash the precipitate repeatedly with deionized water 5 times to remove residual impurities, dry it in a vacuum oven at 70℃ until constant weight, take it out, grind it through a 100-mesh sieve to obtain the reducing agent.

[0136] Chelating agents are prepared by the following methods:

[0137] Step 1: Dissolve chitosan in a 2% (w / w) acetic acid solution at a mass ratio of 1:46 to obtain a chitosan solution.

[0138] Step 2: Control the stirring speed to 500 rpm, the dropping rate to 3 mL / min, and the volume ratio of chitosan solution to sodium hydroxide solution to 1:15. Add the chitosan solution dropwise to the 15% sodium hydroxide solution. After the addition is complete, let it stand and solidify for 20 min. Filter it with a 200 μm filter and wash it repeatedly with deionized water until the filtrate is neutral to obtain chitosan microspheres.

[0139] Step 3: Control the mass ratio of chitosan microspheres to sodium alginate solution to 1:12. Immerse the chitosan microspheres in a 3% sodium alginate solution and allow them to stand for adsorption for 40 minutes. Then filter the solution using a 200μm filter to obtain the adsorbed microspheres.

[0140] Step 4: Control the mass ratio of the adsorbed microspheres to the calcium chloride solution to 1:12. Immerse the adsorbed microspheres in a 3% calcium chloride solution and allow the cross-linking reaction to proceed for 60 min. Filter the solution using a 200 μm filter and wash with deionized water to ensure the removal of excess calcium salts and impurities, thus obtaining core-shell microspheres.

[0141] Step 5: Control the mass ratio of core-shell microspheres, sodium carbonate-sodium bicarbonate buffer, and glutaraldehyde solution to 1:12:2. Add the core-shell microspheres to the sodium carbonate-sodium bicarbonate buffer at pH 10.5, add 1% glutaraldehyde solution, and perform the cross-linking reaction at 25°C for 3 hours. Filter the mixture through a 200μm filter and wash it thoroughly with deionized water to avoid glutaraldehyde residue, thus obtaining the cross-linked microspheres.

[0142] Step 6: Control the mass ratio of cross-linked microspheres to sodium tripolyphosphate solution to 1:12. Immerse the cross-linked microspheres in 8% sodium tripolyphosphate solution, add 2M sodium hydroxide solution to adjust the pH to 10, react at 60℃ for 3 hours, filter with a 200μm filter, wash with deionized water until neutral, and obtain phosphorylated microspheres.

[0143] Step 7: Control the mass ratio of phosphorylated microspheres, deionized water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and ethylenediamine to 10:22:0.6:0.4:3. Disperse the phosphorylated microspheres in deionized water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir and activate at room temperature for 40 min, add ethylenediamine and stir evenly, add 0.2M dilute hydrochloric acid dropwise to adjust the pH to 4.8, continue the reaction for 12 h, filter with a 200 μm filter, wash with 0.2M dilute hydrochloric acid to remove reaction residues, wash with deionized water until neutral, and dry in an oven at 60℃ for 3 h to obtain the chelating agent.

[0144] Comparative Example 1

[0145] Comparative Example 1 is the same as Example 1, except that D-xylose was not pre-treated with heat during the preparation of the reducing agent. Details are as follows:

[0146] The reducing agent is prepared by the following method:

[0147] Step 1: Add alkaline lignin to a 3.5% sodium hydroxide solution at a mass ratio of 1:12, stir to dissolve for 2.5 hours, filter through a 50μm filter to remove insoluble impurities, and collect the lignin alkaline solution.

[0148] Step II: Under nitrogen protection, control the stirring speed at 400 rpm, add sodium sulfide nonahydrate (30% by weight of alkaline lignin) to the lignin alkaline solution in three equal batches, with an interval of 12 min between each batch. Control the temperature during the addition process to not exceed 40℃ to avoid side reactions. After the addition is complete, raise the temperature to 85℃ at a rate of 1.5℃ / min and keep the temperature for 5 hours. After the reaction is completed, allow it to cool naturally to room temperature to obtain the reaction mixture.

[0149] Step III: In an ice-water bath, add 5.5M hydrochloric acid dropwise to the reaction mixture to adjust the pH to neutral. After standing at room temperature for 1.5 hours, centrifuge at 5500 rpm for 15 minutes, collect the precipitate, wash the precipitate repeatedly with deionized water 5 times to remove residual impurities, dry it in a vacuum oven at 65℃ until constant weight, take it out, grind it through a 90-mesh sieve to obtain the reducing agent.

[0150] Comparative Example 2

[0151] Comparative Example 2 is the same as Example 1, except that sodium sulfide nonahydrate is not added during the preparation of the reducing agent. Details are as follows:

[0152] The reducing agent is prepared by the following method:

[0153] Step 1: Add alkaline lignin to a 3.5% sodium hydroxide solution at a mass ratio of 1:12, stir to dissolve for 2.5 hours, filter through a 50μm filter to remove insoluble impurities, and collect the lignin alkaline solution.

[0154] Step II: Add the lignin alkaline solution to the high-pressure reactor, add 22% D-xylose (by weight of alkaline lignin) and stir until homogeneous. Then, heat the mixture to 180°C at a rate of 6.5°C / min and maintain the temperature for 3 hours. After the reaction is complete, allow it to cool naturally to room temperature to obtain the reaction solution.

[0155] Step III: In an ice-water bath, add 5.5M hydrochloric acid dropwise to the reaction solution to adjust the pH to neutral. After standing and aging at room temperature for 1.5 hours, centrifuge at 5500 rpm for 15 minutes, collect the precipitate, wash the precipitate repeatedly with deionized water 5 times to remove residual impurities, dry it in a vacuum oven at 65℃ until constant weight, take it out, grind it through a 90-mesh sieve to obtain the reducing agent.

[0156] Comparative Example 3

[0157] Comparative Example 3 is the same as Example 1, except that sodium sulfide nonahydrate is added all at once during the preparation of the reducing agent. Details are as follows:

[0158] The reducing agent is prepared by the following method:

[0159] Step 1: Add alkaline lignin to a 3.5% sodium hydroxide solution at a mass ratio of 1:12, stir to dissolve for 2.5 hours, filter through a 50μm filter to remove insoluble impurities, and collect the lignin alkaline solution.

[0160] Step II: Add the lignin alkaline solution to the high-pressure reactor, add 22% D-xylose (by weight of alkaline lignin) and stir until homogeneous. Then, heat the mixture to 180°C at a rate of 6.5°C / min and maintain the temperature for 3 hours. After the reaction is complete, allow it to cool naturally to room temperature to obtain the reaction solution.

[0161] Step III: Under nitrogen protection, control the stirring speed at 400 rpm, add sodium sulfide nonahydrate (30% by weight of alkaline lignin) to the reaction solution, stir and mix for 24 min, heat to 85℃ at a rate of 1.5℃ / min, keep the temperature for 5 h, and after the reaction is completed, cool naturally to room temperature to obtain the reaction mixture.

[0162] Step IV: In an ice-water bath, add 5.5M hydrochloric acid dropwise to the reaction mixture to adjust the pH to neutral. After standing at room temperature for 1.5 hours, centrifuge at 5500 rpm for 15 minutes, collect the precipitate, wash the precipitate repeatedly with deionized water 5 times to remove residual impurities, dry it in a vacuum oven at 65℃ until constant weight, take it out, grind it through a 90-mesh sieve to obtain the reducing agent.

[0163] Comparative Example 4

[0164] Comparative Example 4 is the same as Example 1, except that glutaraldehyde was not added for secondary crosslinking during the preparation of the chelating agent. Details are as follows:

[0165] Chelating agents are prepared by the following methods:

[0166] Step 1: Dissolve chitosan in a 3% (w / w) acetic acid solution at a mass ratio of 1:44 to obtain a chitosan solution.

[0167] Step 2: Control the stirring speed to 400 rpm, the dropping rate to 2 mL / min, and the volume ratio of chitosan solution to sodium hydroxide solution to 1:18. Add the chitosan solution dropwise to the 12% sodium hydroxide solution. After the addition is complete, let it stand and solidify for 30 min. Filter it with an 180 μm filter and wash it repeatedly with deionized water until the filtrate is neutral to obtain chitosan microspheres.

[0168] Step 3: Control the mass ratio of chitosan microspheres to sodium alginate solution to 1:11. Immerse the chitosan microspheres in a 4% sodium alginate solution and allow them to stand for 30 minutes for adsorption. Then filter the solution using an 180μm filter to obtain the adsorbed microspheres.

[0169] Step 4: Control the mass ratio of the adsorbed microspheres to the calcium chloride solution to 1:11. Immerse the adsorbed microspheres in a 5% (w / w) calcium chloride solution and allow the cross-linking reaction to proceed for 55 min. Filter the solution using an 180 μm filter and wash with deionized water to ensure the removal of excess calcium salts and impurities, thus obtaining core-shell microspheres.

[0170] Step 5: Control the mass ratio of core-shell microspheres and sodium tripolyphosphate solution to 1:11. Immerse the core-shell microspheres in a 12% sodium tripolyphosphate solution, add 1.5M sodium hydroxide solution to adjust the pH to 10.5, react at 55℃ for 4 hours, filter with a 180μm filter, wash with deionized water until neutral, and obtain phosphorylated microspheres.

[0171] Step 6: Control the mass ratio of phosphorylated microspheres, deionized water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and ethylenediamine to 9:21:0.5:0.3:2. Disperse the phosphorylated microspheres in deionized water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir and activate at room temperature for 30 min, add ethylenediamine and stir evenly, add 0.15M dilute hydrochloric acid dropwise to adjust the pH to 5.2, continue the reaction for 14 h, filter with a 180 μm filter, wash with 0.15M dilute hydrochloric acid to remove reaction residues, wash with deionized water until neutral, and dry in an oven at 55℃ for 4 h to obtain the chelating agent.

[0172] Comparative Example 5

[0173] Comparative Example 5 is the same as Example 1, except that phosphorylation modification was not performed during the preparation of the chelating agent. Details are as follows:

[0174] Chelating agents are prepared by the following methods:

[0175] Step 1: Dissolve chitosan in a 3% (w / w) acetic acid solution at a mass ratio of 1:44 to obtain a chitosan solution.

[0176] Step 2: Control the stirring speed to 400 rpm, the dropping rate to 2 mL / min, and the volume ratio of chitosan solution to sodium hydroxide solution to 1:18. Add the chitosan solution dropwise to the 12% sodium hydroxide solution. After the addition is complete, let it stand and solidify for 30 min. Filter it with an 180 μm filter and wash it repeatedly with deionized water until the filtrate is neutral to obtain chitosan microspheres.

[0177] Step 3: Control the mass ratio of chitosan microspheres to sodium alginate solution to 1:11. Immerse the chitosan microspheres in a 4% sodium alginate solution and allow them to stand for 30 minutes for adsorption. Then filter the solution using an 180μm filter to obtain the adsorbed microspheres.

[0178] Step 4: Control the mass ratio of the adsorbed microspheres to the calcium chloride solution to 1:11. Immerse the adsorbed microspheres in a 5% (w / w) calcium chloride solution and allow the cross-linking reaction to proceed for 55 min. Filter the solution using an 180 μm filter and wash with deionized water to ensure the removal of excess calcium salts and impurities, thus obtaining core-shell microspheres.

[0179] Step 5: Control the mass ratio of core-shell microspheres, sodium carbonate-sodium bicarbonate buffer, and glutaraldehyde solution to 1:11:1.5. Add the core-shell microspheres to the sodium carbonate-sodium bicarbonate buffer at pH 10, add 0.8% glutaraldehyde solution, and allow the cross-linking reaction to proceed at 30°C for 2.5 hours. Filter the mixture through an 180μm filter and wash thoroughly with deionized water to avoid glutaraldehyde residue, thus obtaining the cross-linked microspheres.

[0180] Step 6: Control the mass ratio of cross-linked microspheres, deionized water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and ethylenediamine to 9:21:0.5:0.3:2. Disperse the cross-linked microspheres in deionized water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir and activate at room temperature for 30 min, add ethylenediamine and stir evenly, add 0.15M dilute hydrochloric acid to adjust the pH to 5.2, continue the reaction for 14 h, filter with a 180 μm filter, wash with 0.15M dilute hydrochloric acid to remove reaction residues, wash with deionized water until neutral, and dry in an oven at 55℃ for 4 h to obtain the chelating agent.

[0181] Comparative Example 6

[0182] Comparative Example 6 is the same as Example 1, except that ethylenediamine modification was not performed during the preparation of the chelating agent. Details are as follows:

[0183] Chelating agents are prepared by the following methods:

[0184] Step 1: Dissolve chitosan in a 3% (w / w) acetic acid solution at a mass ratio of 1:44 to obtain a chitosan solution.

[0185] Step 2: Control the stirring speed to 400 rpm, the dropping rate to 2 mL / min, and the volume ratio of chitosan solution to sodium hydroxide solution to 1:18. Add the chitosan solution dropwise to the 12% sodium hydroxide solution. After the addition is complete, let it stand and solidify for 30 min. Filter it with an 180 μm filter and wash it repeatedly with deionized water until the filtrate is neutral to obtain chitosan microspheres.

[0186] Step 3: Control the mass ratio of chitosan microspheres to sodium alginate solution to 1:11. Immerse the chitosan microspheres in a 4% sodium alginate solution and allow them to stand for 30 minutes for adsorption. Then filter the solution using an 180μm filter to obtain the adsorbed microspheres.

[0187] Step 4: Control the mass ratio of the adsorbed microspheres to the calcium chloride solution to 1:11. Immerse the adsorbed microspheres in a 5% (w / w) calcium chloride solution and allow the cross-linking reaction to proceed for 55 min. Filter the solution using an 180 μm filter and wash with deionized water to ensure the removal of excess calcium salts and impurities, thus obtaining core-shell microspheres.

[0188] Step 5: Control the mass ratio of core-shell microspheres, sodium carbonate-sodium bicarbonate buffer, and glutaraldehyde solution to 1:11:1.5. Add the core-shell microspheres to the sodium carbonate-sodium bicarbonate buffer at pH 10, add 0.8% glutaraldehyde solution, and allow the cross-linking reaction to proceed at 30°C for 2.5 hours. Filter the mixture through an 180μm filter and wash thoroughly with deionized water to avoid glutaraldehyde residue, thus obtaining the cross-linked microspheres.

[0189] Step 6: Control the mass ratio of cross-linked microspheres to sodium tripolyphosphate solution to 1:11. Immerse the cross-linked microspheres in a 12% sodium tripolyphosphate solution, add 1.5M sodium hydroxide solution to adjust the pH to 10.5, react at 55℃ for 4 hours, filter with a 180μm filter, wash with deionized water until neutral, and obtain phosphorylated microspheres.

[0190] Step 7: Control the mass ratio of phosphorylated microspheres to deionized water to 3:7, disperse the phosphorylated microspheres in deionized water, add 0.15M dilute hydrochloric acid to adjust the pH to 5.2, let stand for 1 hour, filter with a 180μm filter, wash with deionized water until neutral, and dry in an oven at 55℃ for 4 hours to obtain the chelating agent.

[0191] Comparative Example 7

[0192] Comparative Example 7 is the same as Example 1, except that in the preparation of the chelating agent, it is modified with ethylenediamine first and then modified with phosphorylation. Details are as follows:

[0193] Chelating agents are prepared by the following methods:

[0194] Step 1: Dissolve chitosan in a 3% (w / w) acetic acid solution at a mass ratio of 1:44 to obtain a chitosan solution.

[0195] Step 2: Control the stirring speed to 400 rpm, the dropping rate to 2 mL / min, and the volume ratio of chitosan solution to sodium hydroxide solution to 1:18. Add the chitosan solution dropwise to the 12% sodium hydroxide solution. After the addition is complete, let it stand and solidify for 30 min. Filter it with an 180 μm filter and wash it repeatedly with deionized water until the filtrate is neutral to obtain chitosan microspheres.

[0196] Step 3: Control the mass ratio of chitosan microspheres to sodium alginate solution to 1:11. Immerse the chitosan microspheres in a 4% sodium alginate solution and allow them to stand for 30 minutes for adsorption. Then filter the solution using an 180μm filter to obtain the adsorbed microspheres.

[0197] Step 4: Control the mass ratio of the adsorbed microspheres to the calcium chloride solution to 1:11. Immerse the adsorbed microspheres in a 5% (w / w) calcium chloride solution and allow the cross-linking reaction to proceed for 55 min. Filter the solution using an 180 μm filter and wash with deionized water to ensure the removal of excess calcium salts and impurities, thus obtaining core-shell microspheres.

[0198] Step 5: Control the mass ratio of core-shell microspheres, sodium carbonate-sodium bicarbonate buffer, and glutaraldehyde solution to 1:11:1.5. Add the core-shell microspheres to the sodium carbonate-sodium bicarbonate buffer at pH 10, add 0.8% glutaraldehyde solution, and allow the cross-linking reaction to proceed at 30°C for 2.5 hours. Filter the mixture through an 180μm filter and wash thoroughly with deionized water to avoid glutaraldehyde residue, thus obtaining the cross-linked microspheres.

[0199] Step 6: Control the mass ratio of cross-linked microspheres, deionized water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and ethylenediamine to 9:21:0.5:0.3:2. Disperse the cross-linked microspheres in deionized water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir and activate at room temperature for 30 min, add ethylenediamine and stir evenly, add 0.15M dilute hydrochloric acid to adjust the pH to 5.2, continue the reaction for 14 h, filter with a 180 μm filter, wash with 0.15M dilute hydrochloric acid to remove reaction residues, and then wash with deionized water until neutral to obtain ethylenediamine modified microspheres;

[0200] Step 7: Control the mass ratio of ethylenediamine-modified microspheres to sodium tripolyphosphate solution to 1:11. Immerse the ethylenediamine-modified microspheres in a 12% sodium tripolyphosphate solution. Add 1.5M sodium hydroxide solution to adjust the pH to 10.5. React at 55℃ for 4 hours. Filter the solution through a 180μm filter, wash with deionized water until neutral, and dry in an oven at 55℃ for 4 hours to obtain the chelating agent.

[0201] Performance testing

[0202] I. Determination of Flavonoid Powder Yield

[0203] The flavonoid powders extracted in Examples 1-3 and Comparative Examples 1-7 were placed in a desiccator (containing calcium chloride desiccant) and left to stand for 48 hours under constant temperature and humidity conditions (relative temperature 25±2℃, relative humidity 45±5%). They were then weighed using an electronic analytical balance with an accuracy of ±0.1mg. The weighing was repeated three times, with a 1-hour interval between each weighing. The weight was considered constant when the difference between two consecutive weighings was ≤0.5mg, and this was recorded as the yield of flavonoid powder.

[0204] II. Determination of flavonoid purity (NaNO2-Al(NO3)3-NaOH spectrophotometric method)

[0205] 1. Preparation of the standard curve: Accurately weigh 10.0 mg of rutin standard dried to constant weight at 60℃, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain a rutin standard stock solution with a concentration of 0.2 mg / mL. Accurately pipette 0.5, 1.0, 1.5, 2.0, and 2.5 mL of the stock solution into 10 mL volumetric flasks, add 0.3 mL of 5% NaNO2 solution to each, shake well, and let stand for 6 min; add 0.3 mL of 10% Al(NO3)3 solution, shake well, and let stand for 6 min; then add 4.0 mL of 4% NaOH solution, dilute to the mark with water, shake well, and let stand for 15 min. Use the corresponding reagent without rutin standard as a blank control, and measure the absorbance (A) at a wavelength of 510 nm. A standard curve was plotted with rutin concentration (C) on the x-axis and absorbance (A) on the y-axis, yielding the regression equation: A = 12.85C + 0.015, R0 2 =0.9996.

[0206] 2. Sample Determination: Accurately weigh 10.0 mg of flavonoid powder extracted from Examples 1-3 and Comparative Examples 1-7, prepare sample solutions according to the above method, and measure the absorbance. Substitute the measured absorbance into the regression equation to calculate the concentration of flavonoids in the sample solution, and then calculate the purity of the flavonoid powder.

[0207] ,in, The flavonoid concentration (mg / mL) is calculated from the regression equation. The final volume (mL) of the sample is determined. This is the dilution factor. The sample mass is measured in mg.

[0208] III. Determination and Calculation of Flavonoid Yield

[0209] The experiment used 1 kg (1000 g) of alkaline waste liquid as the starting material and determined the initial flavonoid content in the alkaline waste liquid using the above-mentioned NaNO2-Al(NO3)3-NaOH spectrophotometric method. The initial flavonoid content was 5.26 g / kg. Therefore, the theoretical initial flavonoid content = 1000 g × 5.26 g / kg / 1000 g / kg = 5.26 g.

[0210] .

[0211] The test results are shown in Table 1 below:

[0212] Table 1: Performance test data of flavonoids extracted in Examples 1-3 and Comparative Examples 1-7 Flavonoid powder yield (g) Flavonoid purity (%) Flavonoid yield (%) Example 1 4.80 89.2 81.4 Example 2 4.50 88.5 75.7 Example 3 4.75 85.4 77.1 Comparative Example 1 3.20 65.1 39.6 Comparative Example 2 2.75 62.3 32.6 Comparative Example 3 4.10 75.5 58.8 Comparative Example 4 3.42 68.3 44.4 Comparative Example 5 4.15 76.1 60.0 Comparative Example 6 4.28 71.5 58.2 Comparative Example 7 4.20 72.8 58.1

[0213] As shown in Table 1 above, the flavonoids extracted in Examples 1-3 of this invention have high yield, high purity, and high recovery rate, and their overall performance is far superior to that of Comparative Examples 1-7.

[0214] As can be seen from Comparative Example 1 and Example 1, the lack of D-xylose pretreatment leads to a decrease in reduction efficiency, insufficient destruction of impurity complexes, and a significant decrease in the yield, purity, and productivity of flavonoid powder.

[0215] As can be seen from Comparative Example 2 and Example 1, the lack of sodium sulfide leads to insufficient reduction stability of flavonoids, with only mild reduction by D-xylose, resulting in severely insufficient reducing power. Consequently, the yield, purity, and efficiency of flavonoid powder all decrease significantly.

[0216] As can be seen from Comparative Example 3 and Example 1, the one-time addition of sodium sulfide will cause an instantaneous exothermic reaction, a sharp increase in local temperature, and the occurrence of high-temperature catalytic side reactions, resulting in a decrease in the reducing performance of the final reducing agent, which in turn leads to a certain degree of decrease in the purity and yield of flavonoids.

[0217] As can be seen from Comparative Example 4 and Example 1, glutaraldehyde crosslinking can enhance the mechanical strength and chemical stability of microspheres. The lack of glutaraldehyde crosslinking leads to a loose microsphere structure, affecting subsequent phosphorylation and ethylenediamine modification reactions. The obtained chelating agent cannot effectively adsorb and chelate metal ions, which in turn leads to a decrease in flavonoid powder yield, flavonoid purity and yield.

[0218] As can be seen from Comparative Example 5 and Example 1, the lack of phosphorylation causes the microspheres to mainly rely on the amino group (-NH2) of chitosan for adsorption, resulting in decreased chelation ability, increased metal ion residue, and incomplete removal of the complex formed with flavonoids, which in turn leads to a decrease in flavonoid powder yield, flavonoid purity and yield.

[0219] As can be seen from Comparative Example 6 and Example 1, the absence of ethylenediamine results in the chelating agent having only the negative charge of the phosphate group, which reduces the adsorption capacity for organic impurities, increases the residue of organic impurities, and consequently leads to a decrease in the yield, purity and efficiency of flavonoid powder.

[0220] As can be seen from Comparative Example 7 and Example 1, when ethylenediamine is first modified, the surface of the microspheres will be occupied by long-chain modified molecules, forming a "spatial barrier" on the surface. On the one hand, this hinders the subsequent phosphorylation reaction, resulting in a decrease in phosphorylation efficiency and insufficient phosphate content. On the other hand, the phosphate groups will compete with -NH2, producing side reactions, which reduces the performance of the obtained chelating agent, resulting in a decrease in the yield, purity and efficiency of flavonoid powder.

[0221] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for extracting flavonoids from the waste liquid produced from the production of sophoramine from Sophora alopecuroides L., characterized in that, The method comprises the following steps: S1, under stirring, a reducing agent and a chelating agent are sequentially added into the alkali liquor waste liquid and stirred uniformly, and then filtered after standing, to obtain a pretreated filtrate; S2, under stirring, an organic acid is added dropwise into the pretreated filtrate to adjust the pH value to 3-4, and a precipitate is separated out on the liquid surface, to obtain a suspension liquid; S3, the suspension liquid is placed at a temperature of 1-6℃, and the precipitate is collected by centrifugation after standing and aging for 8-12h, and then washed and freeze-dried, to obtain a flavone powder.

2. The method of claim 1, wherein the method is characterized by, The alkali liquor waste liquid in the step S1 is produced after ethanol extraction of bitter bean, concentration, acid extraction, alkali precipitation and toluene extraction of bitter bean alkaloids.

3. The method of claim 1, wherein the method is characterized by, The adding amount of the reducing agent in the step S1 is 1-3% of the total mass of the alkali liquor waste liquid, and the adding amount of the chelating agent is 4-8% of the total mass of the alkali liquor waste liquid.

4. The method of claim 1, wherein the method is characterized by, The reducing agent in the step S1 is prepared by the following method: Step I, basic lignin is added into a sodium hydroxide solution, and then filtered after stirring and dissolving, to collect a lignin alkali solution; Step II, the lignin alkali solution is added into a high-pressure reaction kettle, D-xylose is added and stirred uniformly, and then heated to react, and then naturally cooled to room temperature after the reaction, to obtain a reaction liquid; Step III, under nitrogen protection and stirring, sodium sulfide nine hydrate is added into the reaction liquid to react after heating, and then naturally cooled to room temperature after the reaction, to obtain a reaction mixture; Step IV, hydrochloric acid is added dropwise into the reaction mixture in an ice water bath to adjust the pH value to neutral, and then centrifuged after standing and aging for 1-2h at room temperature, to collect the precipitate, which is washed, dried, ground, to obtain the reducing agent.

5. The method of claim 4, wherein the method is characterized by, In the step I, the mass ratio of the basic lignin to the sodium hydroxide solution is 1:10-15, and the mass fraction of the sodium hydroxide solution is 2-5%.

6. The method of claim 4, wherein the method is characterized by, In the step II, the adding amount of the D-xylose is 20-25% of the mass of the basic lignin.

7. The method of claim 4, wherein the method is characterized by, In the step III, the adding amount of the sodium sulfide nine hydrate is 28-32% of the mass of the basic lignin, and the sodium sulfide nine hydrate is added in three batches with an interval of 10-15min, and the temperature is controlled to be not more than 40℃ during the adding process.

8. The method of claim 1, wherein the method is characterized by, The chelating agent in the step S1 is prepared by the following method: Step 1, chitosan is dissolved in an acetic acid solution to obtain a chitosan solution; Step 2, the chitosan solution is added dropwise into a sodium hydroxide solution, and then filtered and washed after standing and solidifying for 20-40min, to obtain chitosan microspheres; Step 3, the chitosan microspheres are immersed into a sodium alginate solution, and then filtered after standing and adsorbing for 20-40min, to obtain adsorbed microspheres; Step 4, the adsorbed microspheres are immersed into a calcium chloride solution, and then crosslinked for 50-60min, and then filtered and washed, to obtain core-shell microspheres; Step 5, the core-shell microspheres are added into a carbonate buffer solution, and then crosslinked by adding a glutaraldehyde solution, and then filtered and washed after the reaction, to obtain crosslinked microspheres; Step 6, the crosslinked microspheres are immersed into a sodium tripolyphosphate solution, and then the pH value is adjusted to 10-11 by adding a sodium hydroxide solution, and then reacted, and then filtered and washed, to obtain phosphatized microspheres; Step 7, disperse the phosphorylated microspheres in deionized water, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, stir and activate at room temperature for 20-40 min, add ethylenediamine and stir until uniform, drop in dilute hydrochloric acid to adjust the pH to 4.8-5.5, continue to react for 12-16 h, filter, wash, and dry to obtain the chelating agent.

9. The method of claim 8, wherein the method is characterized by, In the step 5, the mass ratio of the core-shell microspheres, the carbonate buffer, and the glutaraldehyde solution is 1:10-12:1-2, the carbonate buffer is a sodium carbonate-sodium bicarbonate buffer with a pH of 9.5-10.5, and the mass fraction of the glutaraldehyde solution is 0.5-1%.

10. The method of claim 8, wherein the method is characterized by, In the step 7, the mass ratio of the phosphorylated microspheres, deionized water, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, and ethylenediamine is 8-10:20-22:0.4-0.6:0.2-0.4:1-3.

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