Extraction process of apocynum venetum flavonoid compound

Abstract

The invention discloses an apocynum venetum flavonoid compound extraction technology, and relates to the technical field of natural product effective component extraction, and the technology comprises the following steps: raw material pretreatment, pulsed electric field treatment, double-solvent gradient extraction, antioxidant concentration, crystal form regulation crystallization, and residue treatment; according to the method, a polysaccharide-flavone dynamic complexing barrier is broken through cooperation of pulsed electric field pretreatment and double-solvent gradient extraction, the mass transfer efficiency is improved in combination with a surfactant interface activation effect, and the problems of low yield and purification load caused by secondary complexing of effective components in a traditional process are solved; degradation of heat-sensitive components is inhibited through cooperation of an antioxidant and a low-temperature vacuum concentration technology, the paradox of low-temperature and low-efficiency-high-temperature degradation is broken through, and the structural stability of flavonoid compounds is guaranteed; crystal form directional induction is realized by virtue of specific solvent molecule recognition characteristics and crystallization kinetics control, the recessive influence of the crystal form on bioavailability is solved, and the product quality consistency is ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of extracting active ingredients from natural products, and specifically to a process for extracting flavonoid compounds from Apocynum venetum Background Art

[0002] Apocynum venetum leaves are rich in flavonoid compounds such as hyperoside, isoquercitrin, quercitrin, etc., which have various pharmacological activities such as antioxidant, anti-inflammatory, and lipid regulation, and are important raw materials for preparing cardiovascular drugs and functional foods. Currently, commonly used industrial extraction methods include ethanol reflux extraction, ultrasonic-assisted extraction, microwave-assisted extraction, etc., and their core goal is to increase the yield of flavonoid compounds and reduce the subsequent purification cost

[0003] However, the existing extraction processes face significant technical bottlenecks: through high-performance liquid chromatography (HPLC) for component analysis of Apocynum venetum raw materials, the theoretical yield of flavonoid compounds (calculated based on quercitrin) can reach 85%-90%, but the actual extraction yield using the ethanol reflux method (solvent concentration 60%, extraction temperature 70°C, extraction time 2h) is only 60%-70%, with a yield gap of 20%-30%. Through size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) for separation and detection of the extract, it is found that there are polysaccharide-flavonoid nanocomposites with a molecular weight of 10-50 kDa, and their content accounts for 25%-35% of the total flavonoid compounds. Further research shows that polysaccharide components such as arabinogalactan contained in Apocynum venetum form a reversible complex with the phenolic hydroxyl groups of flavonoid compounds through hydrogen bonding between the hydroxyl and carboxyl groups on the molecular chain. This complexation reaction occurs rapidly in the initial stage of solid-liquid mass transfer after raw material fragmentation (within 5-10 minutes), forming a dynamic equilibrium barrier, resulting in the obstruction of the mass transfer process of flavonoid compounds to the solvent phase

[0004] The existing technology mainly destroys the plant cell wall structure through cellulase hydrolysis (such as the enzyme-assisted extraction method disclosed in CN108948765A) or microwave fragmentation (such as the microwave-assisted extraction method disclosed in CN111205341A) to promote the release of active ingredients. However, such methods only address the "initial release" problem in the solid-liquid mass transfer process and do not solve the problem of secondary complexation of released flavonoid compounds and polysaccharides in the solution phase. From the perspective of the mechanism of action, the traditional extraction theory is based on a one-way mass transfer model of "cell wall fragmentation - component dissolution", ignoring the influence of intermolecular interactions in the solution phase on the free degree of active ingredients and not establishing a kinetic regulation mechanism for polysaccharide-flavonoid complexation reactions. In terms of process parameter design, the existing technology has not been combined with the dissociation constant of the polysaccharide-flavonoid complexation reaction (Kd = 1.2×10 -4(M) and the dynamic balance law, key parameters such as solvent concentration, extraction time, and stirring rate cannot effectively break the complexation equilibrium, resulting in insufficient decomplexation efficiency, ultimately leading to low extraction yield and a decrease in resin adsorption efficiency during subsequent purification (more than 40% lower than the theoretical adsorption capacity), significantly increasing production costs.

[0005] Therefore, in view of the dynamic complexation inhibition effect of polysaccharides and flavonoid compounds in Apocynum venetum raw materials, it is urgent to develop an extraction process that can precisely regulate the complexation-decomplexation equilibrium to break through the bottlenecks in yield improvement and purification efficiency of the existing technology.

[0006] In view of this, a process for extracting flavonoid compounds from Apocynum venetum is provided to overcome the above problems. Summary of the Invention

[0007] The purpose of the present invention is to provide a process for extracting flavonoid compounds from Apocynum venetum to solve the problems raised in the above background technology.

[0008] To solve the above technical problems, a process for extracting flavonoid compounds from Apocynum venetum provided by the present invention includes the following steps:

[0009] S1. Raw material pretreatment: After drying and crushing Apocynum venetum leaves, mix them with a 30%-65% ethanol solution to obtain a raw material mixture.

[0010] S2. Pulsed electric field treatment: Place the raw material mixture in a pulsed electric field device and treat it under the conditions of a pulsed voltage of 15-25 kV / cm and a frequency of 30-70 Hz for 200-400 ms.

[0011] S3. Double-solvent gradient extraction:

[0012] In the first stage, use 30%-40% ethanol as the solvent, add 0.1%-0.3% Tween-80, and perform ultrasonic-assisted extraction at 45-65°C for 30-50 minutes.

[0013] In the second stage, gradually increase the ethanol concentration to 60%-70% and switch to microwave-assisted extraction for 20-40 minutes.

[0014] S4. Antioxidant concentration: After filtering the extract, add 0.03%-0.07% ascorbic acid and perform vacuum concentration at a vacuum degree of 0.07-0.09 MPa and a temperature of 40-50°C.

[0015] S5. Crystal form regulation crystallization: After cooling the concentrated solution to 25-35°C, add 1-3 times the volume of pre-cooled methanol, cool it at a rate of 0.3-0.7°C / min to 2-8°C, and maintain the stirring rate at 80-120 rpm.

[0016] S6. Residue treatment: The extracted residue is mixed with cellulase, enzymatically hydrolyzed, and then fermented to produce ethanol.

[0017] Further, in S2, the pulsed voltage for pulsed electric field treatment is 20 kV / cm, the frequency is 50 Hz, and the treatment time is 300 ms.

[0018] Further, in S3, the ethanol concentration in the first stage is 30%, the addition amount of Tween-80 is 0.2%, the extraction temperature is 55 ± 2 °C, the ultrasonic power is 400 W, the frequency is 25 kHz, and the extraction time is 40 minutes.

[0019] Further, in S3, the ethanol concentration in the second stage is 65%, the power of microwave-assisted extraction is 300 W, and the extraction time is 30 minutes.

[0020] Further, in S4, the addition amount of ascorbic acid is 0.05%, the vacuum degree of vacuum concentration is 0.08 MPa, the temperature is 45 °C, concentrated to a relative density of 1.10, and measured at 60 °C.

[0021] Further, in S5, the temperature of precooled methanol is -10 °C, the cooling rate is 0.5 °C / min, and the stirring rate is 100 rpm.

[0022] Further, in S6, the enzyme activity of cellulase is 10000 U / g, the addition amount is 2%, the enzymatic hydrolysis conditions are enzymatic hydrolysis at 50 °C and pH 4.8 for 8 hours, and the fermentation inoculation amount is 5%.

[0023] Further, the Apocynum venetum leaves are harvested in summer, the polysaccharide content is 8.5%, the weight-average molecular weight is 150 kDa, dried in a blast dryer at 60 °C until the water content ≤ 8%, and pulverized through a 40-mesh sieve.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] First, breaking through the dynamic complexation inhibition effect and significantly improving the flavonoid yield and purification efficiency:

[0026] Pulsed electric field combined with double-solvent gradient extraction: Through the combination of a pulsed voltage of 20 kV / cm and an ethanol gradient of 30% - 65%, using the electrophoretic force to break the hydrogen bond between polysaccharide and flavonoid (the dissociation constant Kd is increased to 3.5×10 -4 M), combined with the interfacial activation of Tween-80 (the interfacial tension is reduced to 22 mN / m, and the protein adsorption amount is reduced by 40%), the cumulative dissolution rate of flavonoid reaches 92%, which is 22% higher than that of traditional single-solvent extraction, and the dissolution amount of polysaccharide is reduced by 35%. It fundamentally solves the mass transfer barrier problem caused by "secondary complexation of released components", and reduces the subsequent resin purification load (the adsorption capacity is increased by 53%).

[0027] Cross-scale mass transfer regulation: Pulse electric field pretreatment increases the cell breakage rate to 85%, and the proportion of free flavonoids increases from 35% to 60%, laying the foundation for gradient extraction; Two-stage ultrasound-microwave assistance (power 400W / 300W) achieves the kinetic matching of decoordination and enrichment extraction, avoiding the problems of excessive energy consumption or insufficient efficiency of traditional single methods (such as only microwave crushing).

[0028] II. Innovate the protection mechanism of heat-sensitive components to ensure the stability of active ingredients:

[0029] Antioxidant synergistic concentration technology: 0.05% ascorbic acid and vacuum concentration at 45°C (vacuum degree 0.08MPa) work together to scavenge 85% of O2-· free radicals through the hydrogen donor mechanism, control the degradation rate of hyperoside at 4.2%, reduce the degradation rate by 10% compared with traditional concentration at 60°C, break through the paradox of "low temperature inefficiency - high temperature degradation", and meet the quality requirements of pharmaceutical-grade flavonoids (degradation products ≤ 1%).

[0030] Precise temperature control and free radical capture: The concentration of ascorbic acid is accurately controlled at 0.05% (non-empirical and extensive addition), combined with plate-frame filtration (5μm pore size) to remove solid impurities, forming a three-level protection system of "filtration - antioxidant - low-temperature concentration" to ensure the structural integrity of heat-sensitive flavonoid glycosides.

[0031] III. Achieve crystal form orientation control to ensure product quality consistency:

[0032] Solvent-induced and rate-controlled crystallization: Utilize the hydrogen bond acceptor property of methanol solvent (dielectric constant 32.7) and slow cooling at 0.5°C / min to promote the oriented growth of β crystal form (the intensity ratio of (110) crystal plane diffraction peak is 92%), the crystal form purity ≥ 95%, which is significantly improved compared with conventional crystallization (the proportion of mixed crystal forms ≥ 40%).

[0033] Precise kinetic control: The critical stirring rate of 100rpm avoids the disordered growth of crystal nuclei, and the crystallization yield reaches 85%, solving the hidden influence of crystal form on bioavailability that has not been concerned in the existing technology, meeting the ICHQ6B crystal form control guidelines, and providing high-purity raw materials for high-end preparations (such as nanocrystal carriers).

[0034] IV. Build a residue resource utilization system to improve the environmental protection and economy of the process:

[0035] Cellulase hydrolysis coupled fermentation technology: 2% cellulase (enzyme activity 10000U / g) hydrolyzes the residue for 8 hours at 50°C and pH 4.8, combined with fermentation with a yeast inoculation amount of 5%, and the ethanol yield reaches 22g / kg residue, which is 10 times higher than that of the traditional composting method, realizing the high-value utilization of extraction residues (accounting for 60 - 70% of the raw materials), reducing solid waste emissions while creating additional value (the expected income per ton of residue is 200 yuan).

[0036] Circular economy closed loop: Residue treatment is linked to the optimization of main process parameters (such as pulse electric field intensity and enzymatic hydrolysis pH) to form an environmentally friendly "extraction-purification-resource utilization" full-process system, which complies with the strict requirements of the EU REACH regulations on solvent residues (methanol residue ≤100ppm) and by-product treatment.

[0037] 5. Dynamic adaptation of process parameters to ensure stability of different raw materials and scale:

[0038] Intelligent matching of raw material characteristics: For summer high-polysaccharide raw materials (such as those produced in Xinjiang, Mw=150kDa), an initial ethanol concentration of 30% and an electric field of 20kV / cm are set, while spring low-polysaccharide raw materials (produced in Gansu, Mw=80kDa) can be dynamically adjusted to 25% ethanol and an electric field of 18kV / cm. Through the polysaccharide molecular weight-solvent polarity mapping model, the yield fluctuation of different batches of raw materials is ensured to be ≤2% (the fluctuation of existing technology is ≥20%).

[0039] Feasibility verification of scale-up: The pulsed electric field parameters (electrode spacing 2cm, processing time 300ms) and the dual solvent gradient logic were verified by CFD simulation. 3 The flow field uniformity error in industrial-grade reactors is ≤5%, solving the problem of mass and heat transfer instability during scale-up of traditional processes (an industry pain point where the yield rate in small-scale trials is 85% but drops sharply to 65% in pilot trials). BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The present invention is a schematic diagram of the extraction process of apocynum equisetum ketone compounds. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] See also Figure 1 , the present invention provides a technical solution:

[0043] See Figure 1 As shown, an embodiment of a process for extracting apocynum equisetum ketone compounds is as follows:

[0044] Example 1: Pulsed electric field assisted dual solvent gradient extraction process:

[0045] Raw materials: Apocynum venetum leaves produced in Xinjiang and harvested in summer (polysaccharide content 8.5%, weight-average molecular weight 150 kDa, total flavonoid compound content 3.2%), dried in a forced-air oven at 60°C until the water content ≤ 8%, and then crushed and sieved through a 40-mesh sieve.

[0046] Step 1: Pulsed electric field pretreatment (uncomplexation initiation):

[0047] Mix the raw materials with 30% ethanol (solid-liquid ratio 1:10, g / mL), and place them in a pulsed electric field treatment device (electrode distance 2 cm, pulsed voltage 20 kV / cm, frequency 50 Hz, treatment time 300 ms).

[0048] Mechanism of action: The transmembrane potential difference generated by the pulsed electric field (≥1 kV / cm) destroys the lipid bilayer structure of the plant cell wall, and at the same time, the hydrogen bonds of the polysaccharide-flavonoid complex are broken by the electrophoretic force (verification of the negative correlation between the electric field strength and the dissociation constant Kd: when the electric field strength > 15 kV / cm, the dissociation rate of the complex increases to 65%).

[0049] After pretreatment, the cell breakage rate of the raw materials reaches 85%, and the proportion of free flavonoids in the extract increases from the initial 35% to 60%, laying a foundation for subsequent uncomplexation.

[0050] Step 2: Dual-solvent gradient extraction (dynamic complexation regulation):

[0051] The first stage (uncomplexation extraction): Maintain the system temperature at 55 ± 2°C, use 30% ethanol as the solvent, add 0.2% Tween-80 (v / v), and perform ultrasonic-assisted extraction (power 400 W, frequency 25 kHz) for 40 minutes.

[0052] Interface chemical action: The hydrophilic group (polyoxyethylene group) of Tween-80 binds to the polysaccharide hydroxyl group, and the hydrophobic group (sorbitan ester) wraps the flavonoid molecule, reducing the complexation probability through steric hindrance (the interfacial tension decreases from 35 mN / m to 22 mN / m, and the protein adsorption amount decreases by 40%).

[0053] The second stage (flavonoid enrichment extraction): Gradually increase the ethanol concentration to 65%, maintain the temperature at 55 ± 2°C, and switch to microwave-assisted (power 300 W) extraction for 30 minutes.

[0054] Solvent polarity regulation: Low-concentration ethanol (30%) preferentially destroys the polysaccharide-flavonoid complex (ethanol molecules insert into the hydrogen bond sites, increasing the Kd from 1.2×10 -4 M to 3.5×10 -4 M), and high-concentration ethanol (65%) selectively dissolves free flavonoids, avoiding excessive dissolution of polysaccharides (the polysaccharide solubility decreases from 8% to 3%).

[0055] Two-stage extraction enables the cumulative dissolution rate of flavonoids to reach 92%, which is 22% higher than that of single-solvent extraction (60% ethanol, 2 hours), and the dissolution amount of polysaccharides is reduced by 35%, reducing the subsequent purification load.

[0056] Step 3: Antioxidant synergistic concentration (thermal sensitivity protection):

[0057] After the extract is filtered through a plate and frame filter (filter cloth pore size 5 μm), 0.05% ascorbic acid (w / v) is added, and it is concentrated under reduced pressure to a relative density of 1.10 (measured at 60 °C) under the conditions of a vacuum degree of 0.08 MPa and a temperature of 45 °C.

[0058] Degradation inhibition mechanism: Ascorbic acid, as a hydrogen donor, captures O2-· free radicals in the extract (ESR detection shows that the free radical scavenging rate reaches 85%), combined with low-temperature concentration (reducing the degradation rate by 10% compared with traditional 60 °C concentration), controls the degradation rate of hyperoside at 4.2%.

[0059] Step 4: Crystal form regulation crystallization (quality consistency guarantee):

[0060] After the concentrated solution is cooled to 30 °C, 2 volumes of pre-cooled methanol (-10 °C) are added, and the temperature is lowered to 5 °C at a rate of 0.5 °C / min, while maintaining the stirring rate at 100 rpm (critical crystal form induction rotation speed).

[0061] Crystal form control principle: The hydrogen bond acceptor property of the methanol solvent (dielectric constant 32.7) promotes the formation of the β crystal form of flavonoid molecules (XRD detection shows that the diffraction peak intensity ratio of the (110) crystal plane accounts for 92%). Slow cooling avoids the formation of the metastable α crystal form, and the crystallization yield reaches 85%, and the crystal form purity ≥ 95%.

[0062] Step 5: Resource treatment of residues

[0063] After the extraction residues are centrifuged and dehydrated, they are mixed with cellulase (enzyme activity 10000 U / g, addition amount 2%), and enzymatically hydrolyzed at 50 °C and pH 4.8 for 8 hours. The obtained sugar solution is fermented by yeast (inoculation amount 5%) to produce ethanol (yield 22 g / kg residue, 10 times higher than the traditional composting method).

[0064] Example 2: Changing the pulsed electric field voltage:

[0065] Raw materials: The same as in Example 1.

[0066] Step 1: Pulsed electric field pretreatment:

[0067] The raw materials are mixed with 30% ethanol (solid-liquid ratio 1:10, g / mL) and placed in a pulsed electric field treatment device (electrode spacing 2 cm, pulsed voltage 15 kV / cm, frequency 50 Hz, treatment time 300 ms).

[0068] Considerations for selection of changes: Explore the effect of lower electric field voltage on the decoordination effect of polysaccharide-flavonoid complexes and determine the lower limit value of the pulsed electric field voltage.

[0069] Mechanism of action: The transmembrane potential difference and electrophoretic force generated by the lower electric field voltage are relatively weak, resulting in a decrease in the ability to break the hydrogen bonds of polysaccharide-flavonoid complexes (when the electric field strength is 15 kV / cm, the dissociation rate of the complex is 50%).

[0070] The cell disruption rate of the raw material after pretreatment reaches 75%, and the proportion of free flavonoids in the extract increases to 50%. Although the decoordination effect is slightly weaker compared to Example 1, it still has a certain promoting effect on the subsequent extraction.

[0071] Subsequent steps:

[0072] Steps 2-5 are the same as in Example 1. The cumulative dissolution rate of flavonoids is 85%, which is 7% lower than that in Example 1, indicating that a pulsed electric field voltage of 20 kV / cm has a better effect on decoordination.

[0073] Example 3: Changing the pulsed electric field frequency:

[0074] Raw material: The same as in Example 1.

[0075] Step 1: Pulsed electric field pretreatment:

[0076] Mix the raw material with 30% ethanol (solid-liquid ratio 1:10, g / mL) and place it in a pulsed electric field treatment device (electrode distance 2 cm, pulsed voltage 20 kV / cm, frequency 30 Hz, treatment time 300 ms).

[0077] Considerations for selection of changes: Study the effect of pulsed electric field frequency on the decoordination process and cell disruption effect, and explore the reasonable range of frequency parameters.

[0078] Mechanism of action: The lower pulsed electric field frequency enhances the intermittency of the electric field action, reducing the number of times of action on polysaccharide-flavonoid complexes and cell walls per unit time, and affecting the decoordination and cell disruption efficiency. [[ID=3,2]]

[0079] The cell disruption rate of the raw material after pretreatment is 78%, and the proportion of free flavonoids in the extract reaches 52%. The subsequent steps are the same as in Example 1, and the cumulative dissolution rate of flavonoids is 86%, indicating that the 50 Hz frequency in Example 1 is more conducive to improving the flavonoid yield in the overall process.

[0080] Example 4: Changing the ethanol concentration in the first stage:

[0081] Raw material: The same as in Example 1.

[0082] Step 2: Double-solvent gradient extraction:

[0083] The first stage (complex dissociation extraction): Maintain the system temperature at 55 ± 2 °C, use 25% ethanol as the solvent, add 0.2% Tween-80 (v / v), and perform ultrasonic-assisted extraction (power 400 W, frequency 25 kHz) for 40 minutes.

[0084] Considerations for the change: Try to reduce the ethanol concentration in the first stage, observe its impact on the disruption of the polysaccharide-flavonoid complex and the dissolution amount of polysaccharides, and seek a more optimal initial solvent concentration.

[0085] Mechanism of action: A 25% ethanol concentration has a relatively weak ability to disrupt the polysaccharide-flavonoid complex. The efficiency of ethanol molecules inserting into the hydrogen bond sites decreases, but at the same time, the initial dissolution amount of polysaccharides also decreases.

[0086] After the first stage of extraction, the solubility of polysaccharides is 6%, which is lower than that in Example 1, but the degree of flavonoid dissociation is not as high as that in Example 1. The second stage is the same as in Example 1. The final cumulative dissolution rate of flavonoids is 88%, and the dissolution amount of polysaccharides is reduced by 40%, indicating that the 30% ethanol concentration in Example 1 is more optimal in terms of the comprehensive extraction effect.

[0087] Example 5: Change the addition amount of Tween-80 in the first stage:

[0088] Raw materials: The same as in Example 1.

[0089] Step 2: Dual-solvent gradient extraction:

[0090] The first stage (complex dissociation extraction): Maintain the system temperature at 55 ± 2 °C, use 30% ethanol as the solvent, add 0.1% Tween-80 (v / v), and perform ultrasonic-assisted extraction (power 400 W, frequency 25 kHz) for 40 minutes.

[0091] Considerations for the change: Study the impact of the addition amount of Tween-80 on reducing the complexation probability and improving the mass transfer effect, and determine the optimal addition ratio.

[0092] Mechanism of action: A lower addition amount of Tween-80 weakens the ability of its hydrophilic groups to bind to polysaccharide hydroxyl groups and its hydrophobic groups to encapsulate flavonoid molecules. The steric hindrance effect decreases, and the interfacial tension only drops from 35 mN / m to 28 mN / m, and the protein adsorption amount decreases by 25%.

[0093] After the first stage of extraction, the increase in the mass transfer coefficient is relatively small. The second stage is the same as in Example 1. The final cumulative dissolution rate of flavonoids is 89%, which is 3% lower than that in Example 1, indicating that the 0.2% addition amount of Tween-80 in Example 1 has a better effect in promoting extraction.

[0094] Example 6: Change the ethanol concentration in the second stage:

[0095] Raw materials: The same as in Example 1.

[0096] Step 2: Double-solvent gradient extraction:

[0097] Second stage (flavonoid enrichment extraction): Gradually increase the ethanol concentration to 70%, maintain the temperature at 55 ± 2 °C, and switch to microwave-assisted (power 300 W) extraction for 30 minutes.

[0098] Considerations for the change: Increase the ethanol concentration in the second stage and examine its effects on the dissolution of free flavonoids and the risk of excessive polysaccharide dissolution, and optimize the solvent gradient.

[0099] Mechanism of action: The dissolution ability of 70% ethanol concentration for free flavonoids is enhanced, but at the same time, the solubility of polysaccharides will also increase to some extent, and too high a concentration may lead to the co-dissolution of some flavonoids with other impurities.

[0100] After the second-stage extraction, the solubility of polysaccharides increases to 5%, the cumulative dissolution rate of flavonoids is 90%, which is 2% lower than that in Example 1, and the difficulty of removing impurities in the subsequent purification process increases, indicating that the 65% ethanol concentration in Example 1 has more advantages in balancing extraction efficiency and purification difficulty.

[0101] Example 7: Change the type of antioxidant:

[0102] Raw materials: The same as those in Example 1.

[0103] Step 3: Antioxidant synergistic concentration:

[0104] After the extract is filtered through a plate-and-frame filter (filter cloth pore size 5 μm), add 0.05% tea polyphenols (w / v), and concentrate it under reduced pressure to a relative density of 1.10 (measured at 60 °C) under a vacuum of 0.08 MPa and a temperature of 45 °C.

[0105] Considerations for the change: Compare the protective effects of different antioxidants on heat-sensitive flavonoid components and explore the diversity of antioxidant selection.

[0106] Mechanism of action: Tea polyphenols mainly react with free radicals through phenolic hydroxyl groups, but its scavenging ability for O2-· free radicals is different from that of ascorbic acid, and its effects on other components in the extract are also different.

[0107] ESR detection shows that the free radical scavenging rate is 75%, which is 10% lower than that in Example 1, and the degradation rate of hyperoside is 6.5%, which is higher than that in Example 1, indicating that the antioxidant synergistic effect of ascorbic acid is better in this process.

[0108] Example 8: Change the cooling rate:

[0109] Raw materials: The same as those in Example 1.

[0110] Step 4: Crystal form control crystallization:

[0111] After the concentrate was cooled to 30°C, 2 volumes of pre-cooled methanol (-10°C) were added and the temperature was lowered to 5°C at a rate of 1°C / min while maintaining a stirring rate of 100 rpm (critical crystal induction speed).

[0112] Considerations for choosing changes: Explore the effect of cooling rate on flavonoid crystal formation and identify appropriate crystallization kinetic conditions.

[0113] Mechanism of action: A faster cooling rate will rapidly increase the supersaturation of the solution, which may lead to the rapid formation of a large number of crystal nuclei, which is not conducive to the directional growth of the β crystal form and is prone to the formation of mixed crystals.

[0114] XRD detection showed that the (110) crystal plane diffraction peak intensity accounted for 80%, the crystallization yield was 75%, and the crystal purity was 85%, all of which were lower than Example 1, indicating that the cooling rate of 0.5°C / min in Example 1 was more conducive to obtaining high-purity β crystal.

[0115] Example 9: Changing the stirring rate:

[0116] Raw materials: the same as in Example 1.

[0117] Step 4: Crystallization control:

[0118] After the concentrate was cooled to 30°C, 2 volumes of pre-cooled methanol (-10°C) were added and the temperature was lowered to 5°C at a rate of 0.5°C / min while maintaining a stirring rate of 150 rpm.

[0119] Reasons for choosing the change: Study the effect of stirring rate on crystal induction and solution uniformity during crystallization to determine the optimal stirring conditions.

[0120] Mechanism of action: Excessively high stirring rates will increase the turbulence of the solution, which may destroy the orderly growth of the crystal nuclei and affect the formation of the crystal form and the crystallization yield.

[0121] XRD detection showed that the (110) crystal plane diffraction peak intensity accounted for 88%, the crystallization yield was 80%, and the crystal purity was 90%, all of which were lower than Example 1, indicating that the stirring rate of 100 rpm in Example 1 was more effective in controlling the crystal form.

[0122] Example 10: Changing the amount of cellulase added:

[0123] Raw materials: the same as in Example 1.

[0124] Step 5: Residue resource processing:

[0125] After centrifugal dehydration, the extracted residue was mixed with cellulase (enzyme activity 10,000 U / g, addition amount 1%), and enzymolysis was carried out at 50 °C and pH 4.8 for 8 hours. The obtained sugar solution was fermented by yeast (inoculation amount 5%) to produce ethanol.

[0126] Considerations for the selected change: Examine the effects of the addition amount of cellulase on the enzymolysis effect of the residue and the ethanol yield, and optimize the process parameters for resource utilization.

[0127] Mechanism of action: A lower addition amount of cellulase will reduce the decomposition efficiency of cellulose, resulting in a decrease in the amount of fermentable sugars produced, and thus affecting the ethanol yield.

[0128] The ethanol yield was 15 g / kg of residue, lower than that in Example 1, indicating that the addition amount of 2% cellulase in Example 1 was more capable of achieving efficient conversion in the resource utilization of the residue.

[0129] Example 11: Change the fermentation inoculation amount:

[0130] Raw materials: The same as in Example 1.

[0131] Step 5: Resource treatment of the residue:

[0132] After centrifugal dehydration, the extracted residue was mixed with cellulase (enzyme activity 10,000 U / g, addition amount 2%), and enzymolysis was carried out at 50 °C and pH 4.8 for 8 hours. The obtained sugar solution was fermented by yeast (inoculation amount 3%) to produce ethanol.

[0133] Considerations for the selected change: Study the effects of the yeast fermentation inoculation amount on the fermentation efficiency of the sugar solution and the ethanol production, and determine the optimal fermentation conditions.

[0134] Mechanism of action: A lower inoculation amount will slow down the growth and metabolism rate of yeast in the sugar solution, extend the fermentation cycle, and reduce the ethanol conversion rate.

[0135] The ethanol yield was 18 g / kg of residue, lower than that in Example 1, indicating that the inoculation amount of 5% in Example 1 had an advantage in ensuring fermentation efficiency and ethanol production.

[0136] Summary:

[0137] Through the comparison of the above 11 examples with Example 1, the superiority of Example 1 in all aspects was fully demonstrated.

[0138] In terms of breaking through the dynamic complexation inhibition effect, the parameter settings such as pulsed electric field voltage, frequency, and double-solvent gradient in Example 1 can more effectively disrupt the polysaccharide-flavonoid complexation compared to other examples with changed parameters, increase the proportion of free flavonoids, and achieve a flavonoid yield of over 92%, significantly higher than that of other examples. The addition amount of Tween-80 also shows the best interfacial activation effect in Example 1, with the largest increase in the mass transfer coefficient.

[0139] In terms of innovating the protection of heat-sensitive components, the synergistic effect of ascorbic acid and low-temperature vacuum concentration in Example 1 can more effectively capture free radicals compared to other examples with antioxidant replacements, control the degradation rate at 4.2%, lower than the degradation rates in other examples, and maximize the protection of heat-sensitive flavonoid components.

[0140] For the technological innovation of crystal form controllability, the combination of methanol solvent induction, specific cooling rate, and stirring rate in Example 1 achieved a β-crystal form proportion of over 95%. The crystallization yield and crystal form purity are both better than those of other examples with changed relevant parameters, ensuring the consistency and stability of product quality.

[0141] In terms of ensuring the feasibility of process scale-up, the pulsed electric field treatment parameters and double-solvent gradient switching logic verified by CFD simulation in Example 1 demonstrated more stable process performance and higher production efficiency among the comparative examples with different parameter changes, overcoming many difficulties in the traditional scale-up process.

[0142] In summary, the process parameter combination of Example 1 was obtained through in-depth research and optimization of multiple key factors, with outstanding substantive features and significant progress.

Claims

1. A process for extracting flavonoid compounds from Apocynum venetum, characterized in that, It includes the following steps: S1. Raw material pretreatment: After drying and pulverizing Apocynum venetum leaves, mix them with 30%- 65% ethanol solution to obtain a raw material mixture. S2. Pulsed electric field treatment: Place the raw material mixture in a pulsed electric field device and treat it under the conditions of a pulsed voltage of 15-25 kV / cm, a frequency of 30-70 Hz for 200-400 ms. S3. Double-solvent gradient extraction: In the first stage, use 30%-40% ethanol as the solvent, add 0.1%-0.3% Tween-80, and perform ultrasonic-assisted extraction at 45-65 °C for 30-50 minutes. In the second stage, gradually increase the ethanol concentration to 60%-70%, and switch to microwave-assisted extraction for 20-40 minutes. S4. Antioxidant concentration: After filtering the extract, add 0.03%- 0.07% ascorbic acid, and perform vacuum concentration at a vacuum degree of 0.07-0.09 MPa and a temperature of 40-50 °C. S5. Crystal form control crystallization: After cooling the concentrated solution to 25-35 °C, add 1- 3 times the volume of precooled methanol, cool it at a rate of 0.3-0.7 °C / min to 2-8 °C, and maintain the stirring rate at 80-120 rpm. S6. Residue treatment: Mix the extraction residue with cellulase for enzymatic hydrolysis and then ferment to produce ethanol.

2. The extraction process of a flavonoid compound from Apocynum venetum as claimed in claim 1, characterized in that: In S2, the pulsed voltage of the pulsed electric field treatment is 20 kV / cm, the frequency is 50 Hz, and the treatment time is 300 ms.

3. The extraction process of a flavonoid compound from Apocynum venetum as described in claim 1, characterized in that: In S3, the ethanol concentration in the first stage is 30%, and the addition amount of Tween- 80 is 0.2%, the extraction temperature is 55±2 °C, the ultrasonic power is 400 W, the frequency is 25 kHz, and the extraction time is 40 minutes.

4. The extraction process of a flavonoid compound from Apocynum venetum as claimed in claim 1, wherein: In S3, the ethanol concentration in the second stage is 65%, the power of microwave-assisted extraction is 300 W, and the extraction time is 30 minutes.

5. The extraction process of a flavonoid compound from Apocynum venetum as claimed in claim 1, characterized in that: In S4, the addition amount of ascorbic acid is 0.05%, the vacuum degree of vacuum concentration is 0.08 MPa, the temperature is 45 °C, and it is concentrated to a relative density of 1.10, measured at 60 °C.

6. The extraction process of a flavonoid compound from Apocynum venetum as claimed in claim 1, characterized in that: In S5, the temperature of the precooled methanol is - 10 °C, the cooling rate is 0.5 °C / min, and the stirring rate is 100 rpm.

7. The extraction process of a flavonoid compound from Apocynum venetum as described in claim 1, characterized in that: In S6, the cellulase activity is 10000 U / g, the addition amount is 2%, the enzymatic hydrolysis conditions are 50 °C and pH 4.8 for 8 hours, and the fermentation inoculation amount is 5%.

8. Such as claims 1- The extraction process of a flavonoid compound from Apocynum venetum according to any one of claims 7, characterized in that: The Apocynum venetum leaves are harvested in summer, with a polysaccharide content of 8.5%, a weight-average molecular weight of 150 kDa, dried in a blast dryer at 60 °C until the water content ≤8%, and pulverized through a 40-mesh sieve.

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