Method for extracting and purifying effective components of epimedium

Abstract

This invention belongs to the field of plant active ingredient extraction and purification technology, specifically relating to a method for extracting and purifying the active ingredients of Epimedium. The method involves first pulverizing Epimedium and passing it through a 60-80 mesh sieve, followed by low-temperature plasma pretreatment; then using a specific ternary eutectic solvent, combined with β-cyclodextrin, Tween 80, a directional complex enzyme, micro / nano bubbles, and segmented pulsed ultrasound and microwave synergistic extraction, followed by filtration to obtain the extract; ethanol is recovered by vacuum distillation to obtain a pre-concentrated solution, which is then adjusted to pH with PEG-6000 followed by microfiltration and ultrafiltration to obtain a clear filtrate; the filtrate is loaded onto a directionally modified macroporous resin, monitored online by HPLC with gradient elution, and the target solution is collected; finally, it is distilled, concentrated by nanofiltration, and freeze-dried under vacuum to obtain a high-purity product, with the recovered ethanol being distilled back for reuse.

Description

Technical Field

[0001] This invention belongs to the field of plant active ingredient extraction and purification technology, specifically relating to a method for extracting and purifying the active ingredients of Epimedium. Background Technology

[0002] Epimedium, a plant belonging to the genus Epimedium in the family Berberidaceae, primarily contains flavonoids (such as icariin, icariin A, B, and C), which have effects including replenishing essence, strengthening the body, dispelling rheumatism, and anti-oxidation. It is widely used in medicine and functional foods. Currently, the extraction and purification methods for Epimedium flavonoids have several shortcomings: traditional extraction methods often use organic solvents such as ethanol and methanol, leading to environmental pollution, high solvent consumption, and potential toxicity; existing eutectic solvent (DES) extraction methods are mostly single extraction modes with limited extraction efficiency and have not formed a complete industrial closed-loop process; purification processes often employ methods such as silica gel column chromatography, requiring the use of highly toxic organic solvents such as chloroform and petroleum ether, posing a risk of organic solvent residue, and the purification purity and efficiency need improvement; furthermore, existing methods suffer from high loss rates of active ingredients and insufficient enzyme activity protection, making it difficult to meet the demands of high-purity, green, and industrialized production.

[0003] CN121085981A discloses a method for preparing high-purity icariin from *Epimedium brevicornu* (a type of wild plant) using a combination of enzymes and ultrasonic extraction, macroporous resin purification, and spray drying. Specifically, the method involves using *Epimedium brevicornu* as raw material, employing a combination of enzymes (cellulase + pectinase + α-mannosidase) and ultrasonic extraction. After extraction, the extract is purified by macroporous resin, concentrated under reduced pressure, and spray-dried to obtain high-purity icariin. However, this method still suffers from drawbacks such as high solvent consumption, insufficient environmental friendliness, and limited extraction efficiency. The continuous ultrasonic extraction mode can easily cause localized overheating, damaging enzyme activity and the structure of heat-sensitive active ingredients such as icariin. Furthermore, the single-use of solvents such as ethanol results in high production costs and prevents the realization of industrial-scale closed-loop production. CN121129924A discloses a method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound. However, this method also suffers from problems such as low extraction efficiency and product purity, easy enzyme inactivation, poor purification selectivity, high production cost, and cumbersome process. Furthermore, the various technical methods lack synergy and cannot achieve full-chain optimization of "extraction-purification-circulation".

[0004] Therefore, in view of the problems existing in the existing technology, developing a method for extracting and purifying the active ingredients of Epimedium with high extraction efficiency, high product purity, green and environmentally friendly, low cost, industrial production capability, and effective preservation of the activity of active ingredients has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] To address the aforementioned shortcomings in the existing technology, this invention provides a method for extracting and purifying the effective components of Epimedium to solve the problems mentioned in the background technology.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: S1. Pulverize the leaves of Epimedium and pass them through a 60-80 mesh sieve to obtain Epimedium powder. Pre-treat the Epimedium powder with low-temperature plasma for later use. S2, a ternary eutectic solvent (TDES) and an ethanol-water solution mixture were added to the pretreated Epimedium powder. The TDES was composed of choline tartrate, urea, and PEG400 in a molar ratio of 1:2:1.5, and β-cyclodextrin and Tween 80 were added. At the same time, a directional complex enzyme preparation was added, and micro-nano bubbles were introduced. The treatment was combined with segmented pulsed ultrasound and microwave synergistic treatment. The temperature was controlled at 40℃~45℃ throughout the process. After treatment for 1.2h~1.8h, the filter residue was removed by suction filtration to obtain the extract. S3, the extract is fed into an intermittent distillation apparatus, the distillation temperature is controlled at 60℃~70℃ and the vacuum degree is 0.06MPa~0.08MPa, more than 90% of the ethanol is recovered to obtain a pre-concentrated solution; S4, add PEG-6000 to the pre-concentrated solution, stir evenly, adjust the pH of the system to 5.0-5.5, let it stand at room temperature for 10-20 minutes, and then filter it through a ceramic microfiltration membrane and an ultrafiltration membrane in sequence to obtain a clear filtrate; S5, the clarified filtrate is loaded onto the directionally modified macroporous resin of epimedium flavonoids. After dynamic adsorption, the concentration of epimedium glycoside in the eluent is monitored online by high performance liquid chromatography (HPLC), gradient elution is performed, and the target eluent is collected. S6, the target eluent is subjected to negative pressure intermittent distillation to recover ethanol, then concentrated at low temperature through nanofiltration membrane, and finally freeze-dried under vacuum to obtain high-purity Epimedium active ingredient finished product; S7, the recovered ethanol is distilled and reused in the extraction process of S2.

[0007] Furthermore, the conditions for the low-temperature plasma pretreatment are: plasma power 80-120W, treatment time 5-10min, discharge gap 3-5mm, and working gas is a mixture of air and nitrogen in a volume ratio of 1:1.

[0008] Furthermore, the directional compound enzyme preparation is composed of cellulase, pectinase, and epimedium flavonoid directional releasing enzyme in a mass ratio of 1.2:1:0.8; the epimedium flavonoid directional releasing enzyme is obtained by compounding α-L-rhamnosidase and β-glucosidase in a mass ratio of 1:0.9, and 0.5% (mass fraction) of trehalose is added as an enzyme activity protectant.

[0009] Furthermore, the TDES has a water content of 25%–35% (mass fraction), the amount of β-cyclodextrin added is 0.3%–0.5% of the mass of TDES, the amount of Tween 80 added is 0.1%–0.2% of the mass of TDES, the ratio of Epimedium powder to TDES is 1:4–1:6 (g / mL), and the amount of the directional complex enzyme preparation added is 0.6%–1.0% of the mass of Epimedium powder.

[0010] Furthermore, the ventilation rate of the micro-nano bubbles is 40-60 mL / min; the specific operation of the segmented pulse ultrasound is as follows: in the first stage, 200W low power and 30kHz low frequency ultrasound are used for 15 min, followed by a 5 min resting period; in the second stage, 400W medium power and 40kHz high frequency pulse ultrasound are used for 10 min, with the pulse mode being 3s on and 2s off; the microwave power is 300-400W, the microwave frequency is 2450MHz, and the microwave and ultrasound are performed alternately, with each alternation lasting 5 min.

[0011] Further, the preparation method of the TDES is as follows: take choline tartrate, urea and PEG400, mix them in a molar ratio of 1:2:1.5, add deionized water to adjust the water content to 25-35%, then add β-cyclodextrin and Tween 80, stir at a constant temperature of 65-75°C for 1-2 hours to form a homogeneous mixture, and cool to room temperature for later use.

[0012] Furthermore, the amount of PEG-6000 added is 0.05% to 0.1% of the mass of the pre-concentrated solution; the molecular weight cutoff of the ultrafiltration membrane is 1000 Da to 3000 Da.

[0013] Furthermore, the directional modified macroporous resin of epimedium flavonoids is prepared by amino grafting modification and phenolic hydroxyl biomimetic site modification using AB-8 type macroporous adsorption resin as the base material; the loading flow rate is 1.5-2.5 BV / h, and the dynamic adsorption time is 2-3h.

[0014] Further, the specific operation of the gradient elution is as follows: first, elute with 5 BV of deionized water at a flow rate of 3 BV / h to remove water-soluble impurities, then switch to 30% ethanol aqueous solution at a flow rate of 1.5 BV / h. Monitor online with HPLC. When the concentration of icariin in the eluent is ≥0.2 mg / mL, start collecting. When the concentration drops below 0.1 mg / mL, switch to 70% ethanol aqueous solution at a flow rate of 1.0 BV / h to continue elution. Stop elution when the concentration of icariin is below 0.05 mg / mL. Combine the eluents from steps 3 and 4 to obtain the target eluent.

[0015] Furthermore, the negative pressure intermittent distillation temperature is 45–55℃, the vacuum degree is 0.07–0.09 MPa, and the ethanol recovery rate is ≥95%; the nanofiltration membrane has a molecular weight cutoff of 500 Da–800 Da, the concentration temperature is 30–35℃, and the concentration is carried out until the solid content is 25%–35%; the vacuum freeze-drying temperature is -35–-40℃, the vacuum degree is 10–30 Pa, and the drying time is 12–18 h.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention employs a multi-synergistic extraction mode of "low-temperature plasma pretreatment + directional enzymatic hydrolysis + ultrasound-microwave-micro-nano bubble ternary synergy + TDES," forming a highly efficient full-chain extraction system of "cell wall disruption-unbinding-release-diffusion." Low-temperature plasma rapidly disrupts cell walls, directional complex enzymes specifically cleave the bonds between flavonoids and the cell matrix, segmented pulsed ultrasound gently promotes solubility while protecting enzyme activity, TDES significantly improves the solubility of active ingredients, and micro-nano bubbles and microwaves further enhance mass transfer efficiency. Compared to existing technologies, this method increases the extraction rate of icariin to over 95%, shortens the extraction time, reduces solvent usage, and maintains the temperature below 45℃ throughout the process, effectively avoiding the destruction of heat-sensitive active ingredients and improving activity retention.

[0017] 2. This invention uses a macroporous resin modified with icariin flavonoids, which has a greater adsorption capacity than ordinary AB-8 resin and improved selectivity for icariin flavonoids. Combined with gradient elution monitored by HPLC online, high-purity products can be obtained in one step, eliminating the silica gel column chromatography step in existing technologies and avoiding the use of highly toxic organic solvents such as chloroform and petroleum ether. There are no organic solvent residues, which meets the requirements of green production. At the same time, the combination of PEG-6000 gentle complexation and multi-stage membrane impurity removal effectively removes impurities and avoids hydrolysis of active ingredients, reducing the loss rate of active ingredients and achieving high purification purity, green environmental protection and no pollution.

[0018] 3. This invention achieves dual-solvent recycling and regeneration of ethanol and TDES, improving the overall ethanol recovery rate. TDES can be recycled more than 3 times, and the directionally modified macroporous resin can be reused more than 30 times, significantly reducing production costs. The equipment required for the entire process (low-temperature plasma equipment, ultrasonic equipment, microwave equipment, batch distillation apparatus, microfiltration / ultrafiltration / nanofiltration equipment, freeze dryer, etc.) are all commonly used industrial equipment, eliminating the need for additional expensive special equipment. The process parameters are clear and controllable, and the operation is simple, making it suitable for large-scale industrial production. This solution reduces production costs compared to existing technologies, enhances process closed-loop characteristics, and offers significant industrial advantages. Attached Figure Description

[0019] Figure 1 A flowchart of a method for extracting and purifying the active ingredients of Epimedium. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0021] Example 1: Preparation of Epimedium Flavonoid-Directed Release Enzyme 1. Strain activation: Aspergillus mulundensis, which produces α-L-rhamnosidase, and Trichoderma reesei, which produces β-glucosidase, were inoculated into PDA slant medium and cultured in a 30℃ incubator for 30 h to obtain activated strains; 2. Seed culture: The above activated strains were inoculated into liquid seed culture medium (formulation: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, pH natural) and cultured in a shaker at 30℃ and 180 r / min for 20 h to obtain two seed cultures; 3. Fermentation for enzyme production: The two seed cultures were inoculated into the fermentation medium (formula: corn starch 25g / L, soybean meal powder 15g / L, KH2PO4 3g / L, MgSO4·7H2O 1g / L, CaCl2 0.5g / L, pH 5.5) at a volume ratio of 1:1. The medium was placed in a fermenter at 30℃ with an aeration rate of 1.2 vvm and fermented for 60 h. After fermentation, the medium was centrifuged at 8000 r / min for 10 min to remove the cell precipitate and obtain the crude enzyme solution. 4. Crude enzyme purification: The crude enzyme solution was fractionally precipitated with ammonium sulfate (saturation 30% to 70%). The precipitate was collected and dissolved in Tris-HCl buffer solution. After dialysis to remove salt, DEAE-cellulose ion exchange chromatography and Sephadex G-75 gel filtration chromatography were performed to obtain purified α-L-rhamnosidase solution and β-glucosidase solution. 5. Compounding and protection: The purified α-L-rhamnosidase and β-glucosidase were mixed at a mass ratio of 1:0.9, and 0.5% (mass fraction) of trehalose was added as an enzyme activity protectant. After stirring evenly, the mixture was spray-dried at low temperature (inlet air temperature 85℃, outlet air temperature 50℃) to obtain the ephemeral flavonoid-directed release enzyme powder, which was then sealed and stored for later use.

[0022] Example 2: Preparation of macroporous resin modified with epimedium flavonoids 1. Substrate pretreatment: Take AB-8 type macroporous adsorption resin, soak it in anhydrous ethanol for 24 hours, and replace the ethanol twice during the period to remove impurities. Rinse with deionized water until neutral and free of alcohol odor. 2. Amino grafting: Place the resin at 6% (mass fraction) In an aminopropyltriethoxysilane ethanol solution, the mixture was stirred at a constant temperature of 45°C for 2 hours to graft amino groups onto the resin surface. 3. Biomimetic modification: Add 2.5% (mass fraction) protocatechuic acid (phenolic hydroxyl ligand), react at 35℃ for 1 h to construct flavonoid-specific recognition sites on the resin surface; 4. Post-treatment: The resin was thoroughly washed with ethanol and deionized water and dried under vacuum at 50°C to obtain the directionally modified macroporous resin of epimedium flavonoids.

[0023] Example 3: Preparation of TDES Take choline tartrate, urea, and PEG400, mix them in a molar ratio of 1:2:1.5, add deionized water to adjust the water content to 32%, then add 0.4% β-cyclodextrin and 0.15% Tween 80 by mass of TDES, stir at 70℃ for 1.5h to form a homogeneous mixture, cool to room temperature, and seal for later use.

[0024] Example 4: Extraction and purification of active ingredients from Epimedium 1. Take Epimedium leaves, crush them through a 70-mesh sieve to obtain Epimedium powder; use low-temperature plasma to pretreat the Epimedium powder, plasma power 100W, treatment time 8min, discharge gap 4mm, working gas is air and nitrogen mixed gas in a volume ratio of 1:1, and use it for later use. 2. Add the TDES and ethanol aqueous solution mixture prepared in Example 3 to the pretreated Epimedium powder, with a material-to-liquid ratio of 1:5 (g / mL). Simultaneously add 0.8% of the target compound enzyme preparation (composed of cellulase, pectinase, and Epimedium flavonoid target release enzyme prepared in Example 1 in a mass ratio of 1.2:1:0.8) relative to the mass of Epimedium powder. Introduce 80nm micro-nano bubbles at a ventilation rate of 50mL / min. Combine segmented pulsed ultrasound and microwave synergistic treatment: the first stage is 200W, 30kHz ultrasound for 15min, followed by standing for 5min; the second stage is 400W, 40kHz pulsed ultrasound for 10min (on for 3s, off for 2s); microwave power is 350W, frequency is 2450MHz, alternating with ultrasound (alternating every 5min). The temperature is controlled at 42℃ throughout the process. After 1.5h of treatment, filter to remove the residue to obtain the extract. 3. The extract was fed into a batch distillation apparatus, and the distillation temperature was controlled at 65℃ and the vacuum degree at 0.07MPa. Ethanol was recovered to obtain a pre-concentrated solution. 4. Add 0.08% PEG-6000 relative to the mass of the pre-concentrated solution to the pre-concentrated solution, stir well, adjust the pH to 5.2 with citrate-disodium hydrogen phosphate buffer solution, let stand at room temperature for 15 min, and then filter through a ceramic microfiltration membrane and an ultrafiltration membrane with a molecular weight cutoff of 3000 Da in sequence to obtain a clear filtrate. 5. The clarified filtrate was loaded onto the directionally modified macroporous resin of icariin prepared in Example 2 at a flow rate of 2.0 BV / h, and dynamic adsorption was performed for 2.5 h. The concentration of icariin in the eluent was monitored online by HPLC, and gradient elution was performed: First, elution was performed with 5 BV of deionized water at a flow rate of 3 BV / h; Second, when the concentration of icariin was ≥0.5 mg / mL, elution was switched to 60% ethanol aqueous solution at a flow rate of 1.2 BV / h, and the eluent of this stage was collected; Third, when the concentration of icariin dropped to 0.15 mg / mL, elution was switched to 85% ethanol aqueous solution at a flow rate of 0.8 BV / h until the concentration was below 0.1 mg / mL, and the eluents from the two steps were combined to obtain the target eluent. 6. The target eluent was fed into a negative pressure intermittent distillation apparatus, with the temperature controlled at 50℃ and the vacuum degree at 0.08MPa, to recover 96% of the ethanol; then, it was concentrated at low temperature using a nanofiltration membrane with a molecular weight cutoff of 600Da at 32℃ until the solid content was 30%; finally, it was fed into a vacuum freeze dryer, with the freezing temperature controlled at -38℃ and the vacuum degree at 20Pa, and freeze-dried for 15 hours to obtain a light yellow, free-flowing, high-purity Epimedium active ingredient product, which was then sealed and packaged for later use. 7. The recovered ethanol is distilled and reused for S2 extraction; TDES is distilled under reduced pressure to remove moisture and impurities and then recycled; the resin is regenerated and reused, and the regenerated resin retains more than 85% of its initial adsorption capacity.

[0025] According to the test results, the purity of icariin in the finished product of epimedium prepared in this embodiment is 98.7%, the extraction rate is 95.3%, and the activity retention rate of the active ingredient is 97.5%.

[0026] Example 5: 1. Take Epimedium leaves, crush them through a 60-mesh sieve to obtain Epimedium powder; use low-temperature plasma to pretreat the Epimedium powder, plasma power 80W, treatment time 10min, discharge gap 3mm, working gas is air and nitrogen mixed gas in a volume ratio of 1:1, and use it for later use. 2. Add the TDES and ethanol aqueous solution mixture prepared in Example 3 to the pretreated Epimedium powder, with a material-to-liquid ratio of 1:4 (g / mL). At the same time, add 0.6% of the directional complex enzyme preparation relative to the mass of Epimedium powder. Introduce 50nm micro-nano bubbles at a rate of 40mL / min. Combine segmented pulsed ultrasound and microwave treatment: the first stage is 200W, 30kHz ultrasound for 15min, followed by standing for 5min; the second stage is 400W, 40kHz pulsed ultrasound for 10min, with microwave power of 300W and frequency of 2450MHz, alternating with ultrasound. The temperature is controlled at 40℃ throughout the process. After 1.8h of treatment, filter to remove the residue and obtain the extract. 3. The extract was fed into a batch distillation apparatus, and the distillation temperature was controlled at 60℃ and the vacuum degree at 0.06MPa. Ethanol was recovered to obtain a pre-concentrated solution. 4. Add 0.05% PEG-6000 relative to the mass of the pre-concentrated solution to the pre-concentrated solution, stir well, adjust the pH to 5.0 with citrate-disodium hydrogen phosphate buffer solution, let stand at room temperature for 10 min, and then filter through a ceramic microfiltration membrane and an ultrafiltration membrane with a molecular weight cutoff of 1000 Da in sequence to obtain a clear filtrate. 5. Load the clarified filtrate onto the directionally modified macroporous resin of epimedium flavonoids prepared in Example 2 at a flow rate of 1.5 BV / h, and allow dynamic adsorption for 2 h. Monitor the concentration of epimedium glycosides in the eluent using HPLC online, perform gradient elution, and combine the target eluents. 6. The target eluent was fed into a negative pressure intermittent distillation apparatus, with the temperature controlled at 45℃ and the vacuum degree at 0.07MPa, to recover 95% of the ethanol; then, it was concentrated at low temperature using a nanofiltration membrane with a molecular weight cutoff of 700Da at 30℃ until the solid content was 25%; finally, it was fed into a vacuum freeze dryer, with the freezing temperature controlled at -35℃ and the vacuum degree at 10Pa, and freeze-dried for 18 hours to obtain a high-purity Epimedium active ingredient product; 7. The recovered ethanol is reused after distillation, and TDES and resin are recycled after regeneration.

[0027] According to the test results, the purity of icariin in the finished product of epimedium prepared in this embodiment is 98.5%, the extraction rate is 95.1%, and the activity retention rate of the active ingredient is 97.2%.

[0028] Example 6 1. Take Epimedium leaves, crush them through an 80-mesh sieve to obtain Epimedium powder; use low-temperature plasma to pretreat the Epimedium powder, plasma power 120W, treatment time 5min, discharge gap 5mm, working gas is air and nitrogen mixed gas in a volume ratio of 1:1, and use it for later use. 2. Add the TDES and ethanol aqueous solution mixture prepared in Example 3 to the pretreated Epimedium powder, with a material-to-liquid ratio of 1:6 (g / mL). At the same time, add 1.0% of the directional complex enzyme preparation relative to the mass of Epimedium powder. Introduce 100nm micro-nano bubbles at a rate of 60mL / min. Combine segmented pulsed ultrasound and microwave treatment: the first stage is 200W, 30kHz ultrasound for 15min, followed by standing for 5min; the second stage is 400W, 40kHz pulsed ultrasound for 10min, with microwave power of 400W and frequency of 2450MHz, alternating with ultrasound. The temperature is controlled at 45℃ throughout the process. After 1.2h of treatment, filter to remove the residue and obtain the extract. 3. The extract was fed into a batch distillation apparatus, and the distillation temperature was controlled at 70℃ and the vacuum degree at 0.08MPa. Ethanol was recovered to obtain a pre-concentrated solution. 4. Add 0.1% PEG-6000 relative to the mass of the pre-concentrated solution to the pre-concentrated solution, stir well, adjust the pH to 5.5 with citrate-disodium hydrogen phosphate buffer solution, let stand at room temperature for 20 min, and then filter through a ceramic microfiltration membrane and an ultrafiltration membrane with a molecular weight cutoff of 3000 Da in sequence to obtain a clear filtrate. 5. Load the clarified filtrate onto the directionally modified macroporous resin of epimedium flavonoids prepared in Example 2 at a flow rate of 2.5 BV / h, and allow dynamic adsorption for 3 h. Monitor the concentration of epimedium glycosides in the eluent using HPLC online, perform gradient elution, and combine the target eluents. 6. The target eluent was fed into a negative pressure intermittent distillation apparatus, with the temperature controlled at 55℃ and the vacuum degree at 0.09MPa, to recover 97% of the ethanol; then, it was concentrated at low temperature using a nanofiltration membrane with a molecular weight cutoff of 800Da at 35℃ until the solid content was 35%; finally, it was fed into a vacuum freeze dryer, with the freezing temperature controlled at -40℃ and the vacuum degree at 30Pa, and freeze-dried for 12 hours to obtain a high-purity Epimedium active ingredient product; 7. The recovered ethanol is reused after distillation, and TDES and resin are recycled after regeneration.

[0029] According to the test results, the purity of icariin in the finished product of epimedium prepared in this embodiment is 98.6%, the extraction rate is 95.5%, and the activity retention rate of the active ingredient is 97.8%.

[0030] Comparative Example 1: No directional complex enzyme preparation added (Control Example 4) 1. The raw material pretreatment is the same as in Example 4; 2. No directional complex enzyme preparation was added in the extraction step, and the remaining TDES, micro-nano bubbles, and ultrasonic-microwave conditions were completely consistent with those in Example 4; 3. The subsequent concentration, membrane filtration, resin purification, concentration drying, and solvent regeneration steps are the same as in Example 4.

[0031] Test results: Icariin extraction rate was 69.5%, finished product purity was 88.7%, and activity retention rate was 91.3%.

[0032] Comparative Example 2: Using ordinary Resin (Comparative Example 4) 1. The extraction, pre-concentration, and membrane impurity removal steps are the same as in Example 4; 2. The purification step uses unmodified ordinary AB-8 macroporous adsorption resin instead of the directionally modified resin, while the sample loading and elution conditions remain unchanged; 3. The subsequent concentration, drying, and solvent regeneration steps are the same as in Example 4.

[0033] Test results: Icariin extraction rate was 94.8%, finished product purity was 85.6%, and activity retention rate was 96.9%.

[0034] Comparative Example 3: Continuous ultrasound (Comparative Example 4) 1. The raw material pretreatment, TDES, enzyme preparation, and micro / nano bubble conditions are the same as in Example 4; 2. During extraction, continuous ultrasound at 400W and 40kHz is used instead of segmented pulse ultrasound, while the microwave power and total processing time remain unchanged; 3. The subsequent steps are exactly the same as in Example 4.

[0035] Test results: Icariin extraction rate was 87.6%, finished product purity was 96.1%, and activity retention rate was 89.7%.

[0036] Comparative Example 4: TDES without β-cyclodextrin and Tween 80 (Control Example 4) 1. The raw material pretreatment is the same as in Example 4; 2. TDES is prepared without the addition of β-cyclodextrin and Tween 80, while the remaining components, water content, and stirring conditions remain unchanged; 3. The extraction, concentration, membrane filtration, resin purification, and drying steps are the same as in Example 4.

[0037] Test results: Icariin extraction rate was 82.4%, finished product purity was 94.2%, and activity retention rate was 93.5%.

[0038]

[0039] Table 1. Test results of purity and retention rate of active ingredients of icariin extracted from Example 4 and Comparative Examples 1-4. 1. Compared with Comparative Example 1, the purity and active ingredient retention rate of the icariin extracted in Example 4 were higher than those of the icariin extracted in Comparative Example 1. This indicates that the use of the targeted compound enzyme preparation further improved the purity and active ingredient retention rate of the icariin product.

[0040] 2. Compared with Comparative Example 2, the purity and active ingredient retention rate of the icariin extracted in Example 4 were higher than those of the icariin extracted in Comparative Example 2. This indicates that the use of macroporous resin modified with icariin flavonoids further improved the purity and active ingredient retention rate of the icariin product.

[0041] 3. Compared with Comparative Example 3, the purity and active ingredient retention rate of the icariin extracted in Example 4 were higher than those of the icariin extracted in Comparative Example 3. This indicates that the use of segmented pulse ultrasound and microwave synergistic processing further improved the purity and active ingredient retention rate of the icariin product.

[0042] 4. Compared with Comparative Example 4, the purity and active ingredient retention rate of the icariin extracted in Example 4 were higher than those of the icariin extracted in Comparative Example 4. This indicates that the addition of β-cyclodextrin and Tween 80 to TDES further improved the purity and active ingredient retention rate of the icariin product.

Claims

1. A method for extracting and purifying the effective components of Epimedium, characterized in that: Includes the following steps, S1. Pulverize the leaves of Epimedium and pass them through a 60-80 mesh sieve to obtain Epimedium powder. Pre-treat the Epimedium powder with low-temperature plasma for later use. S2, a ternary eutectic solvent (TDES) and an ethanol-water solution mixture were added to the pretreated Epimedium powder. The TDES was composed of choline tartrate, urea, and PEG400 in a molar ratio of 1:2:1.5, and β-cyclodextrin and Tween 80 were added. At the same time, a directional complex enzyme preparation was added, and micro-nano bubbles were introduced. The treatment was combined with segmented pulsed ultrasound and microwave synergistic treatment. The temperature was controlled at 40℃~45℃ throughout the process. After treatment for 1.2h~1.8h, the filter residue was removed by suction filtration to obtain the extract. S3, the extract is fed into an intermittent distillation apparatus, the distillation temperature is controlled at 60℃~70℃ and the vacuum degree is 0.06MPa~0.08MPa, ethanol is recovered, and a pre-concentrated solution is obtained; S4, add PEG-6000 to the pre-concentrated solution, stir evenly, adjust the pH of the system to 5.0-5.5, let it stand at room temperature for 10-20 minutes, and then filter it through a ceramic microfiltration membrane and an ultrafiltration membrane in sequence to obtain a clear filtrate; S5, the clarified filtrate is loaded onto the directionally modified macroporous resin of epimedium flavonoids. After dynamic adsorption, the concentration of epimedium glycoside in the eluent is monitored online by high performance liquid chromatography (HPLC), gradient elution is performed, and the target eluent is collected. S6, the target eluent is subjected to negative pressure intermittent distillation to recover ethanol, then concentrated at low temperature through nanofiltration membrane, and finally freeze-dried under vacuum to obtain high-purity Epimedium active ingredient finished product; S7, the recovered ethanol is distilled and reused in the extraction process of S2.

2. The method according to claim 1, characterized in that, The conditions for low-temperature plasma pretreatment described in S1 are: plasma power 80W~120W, treatment time 5min~10min, discharge gap 3mm~5mm, and working gas is a mixture of air and nitrogen in a volume ratio of 1:

1.

3. The method according to claim 1, characterized in that, The targeted compound enzyme preparation described in S2 is composed of cellulase, pectinase, and epimedium flavonoid targeted release enzyme in a mass ratio of 1.2:1:0.8; the epimedium flavonoid targeted release enzyme is obtained by combining α-L-rhamnosidase and β-glucosidase in a mass ratio of 1:0.9, and 0.5% (mass fraction) of trehalose is added as an enzyme activity protectant.

4. The method according to claim 1, characterized in that, The TDES described in S2 has a water content of 25%–35%, the amount of β-cyclodextrin added is 0.3%–0.5% of the mass of TDES, the amount of Tween 80 added is 0.1%–0.2% of the mass of TDES, the ratio of Epimedium powder to TDES is 1:4–1:6 (g / mL), and the amount of the directional complex enzyme preparation added is 0.6%–1.0% of the mass of Epimedium powder.

5. The method according to claim 1, characterized in that, The ventilation rate of the micro-nano bubbles described in S2 is 40 mL / min to 60 mL / min; the specific operation of the segmented pulse ultrasound is as follows: in the first stage, 200W low power and 30kHz low frequency ultrasound are used for 15 min, and after standing for 5 min, the second stage uses 400W medium power and 40kHz high frequency pulse ultrasound for 10 min, with the pulse mode being 3 seconds on and 2 seconds off; the microwave power is 300 to 400W, the microwave frequency is 2450MHz, and the microwave and ultrasound are performed alternately, with each alternation lasting 5 min.

6. The method according to claim 1, characterized in that, The preparation method of TDES described in S2 is as follows: take choline tartrate, urea, and PEG400, mix them in a molar ratio of 1:2:1.5, add deionized water to adjust the water content to 25% to 35%, then add β-cyclodextrin and Tween 80, stir at 65℃ to 75℃ for 1 to 2 hours to form a homogeneous mixture, and cool to room temperature for later use.

7. The method according to claim 1, characterized in that, The amount of PEG-6000 added in S4 is 0.05% to 0.1% of the mass of the pre-concentrated solution; the molecular weight cutoff of the ultrafiltration membrane is 1000 Da to 3000 Da.

8. The method according to claim 1, characterized in that, The directional modified macroporous resin of epimedium flavonoids described in S5 is prepared by using AB-8 type macroporous adsorption resin as the base material, and by amino grafting modification and phenolic hydroxyl biomimetic site modification; the loading flow rate is 1.5-2.5 BV / h, and the dynamic adsorption time is 2-3h.

9. The method according to claim 1, characterized in that, The specific operation of gradient elution described in S5 is as follows: First, elute with 5 BV of deionized water at a flow rate of 3 BV / h to remove water-soluble impurities. Then, switch to 30% ethanol aqueous solution and elute at a flow rate of 1.5 BV / h. Monitor online with HPLC. When the concentration of icariin in the eluent is ≥0.2 mg / mL, start collecting. When the concentration drops below 0.1 mg / mL, switch to 70% ethanol aqueous solution and continue elution at a flow rate of 1.0 BV / h. Stop elution when the concentration of icariin is below 0.05 mg / mL. Combine the eluents from steps 3 and 4 to obtain the target eluent.

10. The method according to claim 1, characterized in that, The negative pressure intermittent distillation described in S6 has a temperature of 45–55°C, a vacuum degree of 0.07–0.09 MPa, and an ethanol recovery rate of ≥95%; the nanofiltration membrane has a molecular weight cutoff of 500 Da–800 Da, a concentration temperature of 30–35°C, and is concentrated to a solid content of 25%–35%; the vacuum freeze-drying has a temperature of -35–-40°C, a vacuum degree of 10–30 Pa, and a drying time of 12–18 h.

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