Method for synergistically extracting flavonoid compounds in epimedium wushanense by combining ternary eutectic solvent with in-situ ultrasound

Abstract

The invention discloses a method for synergistically extracting flavonoid compounds in epimedium wushanense by combining a ternary eutectic solvent with in-situ ultrasound, which is characterized in that choline bitartrate-urea-glycerol (Chb: Ure: Gly) is used as the ternary eutectic solvent, and the flavonoid compounds in epimedium wushanense are synergistically extracted by the in-situ ultrasound. The method is high in extraction speed and good in repeatability, solvent consumption can be reduced, operation steps are simplified, and efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of comprehensive utilization of plant resources, and relates to a method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound. Background Technology

[0002] Epimedium wushanense is a perennial herb belonging to the Berberidaceae family, mainly distributed in southwestern and northwestern China, including Chongqing, Sichuan, Guizhou, and Guangxi. This plant is widely used in dietary supplements and clinical treatment, possessing the effects of replenishing essence, strengthening the body, and dispelling wind and dampness. Phytochemical studies have shown that Epimedium wushanense is rich in flavonoids, phenolic acids, organic acids, phenylpropanoids, nucleosides, terpenes, and alkaloids.

[0003] Traditional solvents such as ethanol and methanol have been widely used for the extraction of flavonoids, but organic reagents have obvious limitations such as environmental pollution, large solvent consumption, and potential toxicity.

[0004] Eutectic solvents (DESs) are a novel class of solvents composed of hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) in an appropriate molar ratio. They offer significant advantages such as low cost, ease of preparation, environmental friendliness, good biocompatibility, sustainability, and customizability. Currently, DESs have become a mainstream technology for the efficient extraction of target bioactive components from natural matrices, and their application in phytochemical extraction is gaining increasing recognition due to their superior selectivity compared to traditional solvent systems.

[0005] Currently, there are no reported methods for extracting flavonoids from Epimedium wushanense. This invention presents a novel ternary DES system (TDES) based on hydrocholine tartrate, which exhibits superior extraction efficiency compared to other DESs. This method not only offers rapid extraction and good reproducibility but also reduces solvent consumption, simplifies operational steps, and improves efficiency. It is an environmentally friendly, efficient, and reproducible method, which is of great significance for the sustainable utilization of medicinal plant resources and the development of green extraction technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound. This method enhances the cell wall disruption effect and improves the solubility of flavonoids in the solvent, providing an environmentally friendly, efficient, and reproducible method for the extraction of bioactive flavonoids from Epimedium wushanense. This method is of great significance for the sustainable utilization of medicinal plant resources and the development of green extraction technology.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound includes the following steps: (1) Preparation of TDES: Take tartrate choline, urea and glycerol, add water and mix continuously to form a homogeneous mixture. The mixture is a ternary eutectic solvent for mixing hydrogen bond acceptors and hydrogen bond donors, and TDES is obtained for later use. (2) After crushing the Epimedium puerariae, sieve it, add TDES, vortex mix it, sonicate it, filter the sonicated mixture through a 0.22μm filter membrane, centrifuge the filtrate, collect the supernatant, and obtain the Epimedium puerariae extract.

[0008] In step (1) above, tartrate choline is a mixed hydrogen bond acceptor, urea and glycerol are hydrogen bond donors, and the molar ratio of tartrate choline, urea and glycerol is 1:2:2.

[0009] In step (1) above, the temperature for continuous mixing is 65~75℃ and the time is 0.5~2.5h.

[0010] Specifically, in the aforementioned step (1), the mixing and stirring temperature is 70°C and the time is 1~2 hours.

[0011] In step (1) above, the water content of TDES is 20-40%.

[0012] Specifically, in step (1) above, the water content of TDES is 32%.

[0013] In step (2) above, the powdered Epimedium glutamate is passed through a 30-50 mesh sieve, and the ratio of Epimedium glutamate powder to TDES is 1g:20-40mL.

[0014] Specifically, in step (2) above, the Wushan Epimedium is pulverized and passed through a 40-mesh sieve, and the ratio of Wushan Epimedium powder to TDES is 1g:34mL.

[0015] In step (2) above, the vortex mixing time is 8~12min; the ultrasonic treatment time is 25~35min, the ultrasonic power is 240~300W, the ultrasonic frequency is 35~45kHz, the ultrasonic temperature is 32~34℃; the centrifugation speed is 3500~4500r / min, and the centrifugation time is 8~12min.

[0016] Specifically, in step (2) above, the vortex mixing time is 10 min; the ultrasonic treatment time is 30 min, the ultrasonic power is 270 W, the ultrasonic frequency is 40 kHz, the ultrasonic temperature is 33 ℃; the centrifugation speed is 4000 r / min, and the centrifugation time is 10 min.

[0017] Compared with the prior art, the present invention has the following advantages: 1. This invention is the first to propose a dual-mechanism extraction strategy combining in-situ extraction and ultrasound to extract flavonoids from Epimedium wushanense. Ultrasound technology, through the mechanical shear force generated by cavitation, can improve mass transfer efficiency, playing a key role in promoting sustainable green chemistry and extraction processes. This method not only offers rapid extraction and good repeatability but also reduces solvent consumption, simplifies operation steps, and improves efficiency. Its core lies in the cavitation effect—the phenomenon of alternating compression caused by ultrasound-induced pressure oscillations. This pressure fluctuation leads to the formation and rupture of microbubbles in the liquid medium, generating localized high-energy jets and shear forces, disrupting the cell's ultrastructure, thereby promoting the dissolution and release of the target bioactive components. Therefore, this synergistic extraction mechanism can achieve rapid and efficient release of phytochemicals from the plant matrix.

[0018] 2. Through systematic screening, tartrate choline-urea-glycerol (Chb:Ure:Gly) was determined to be the optimal TDES. Optimization using the RSM-ANN-GA framework determined the optimal extraction conditions to be: 32% water content, 10 min vortexing time, 34:1 mL / g liquid-to-solid ratio, 30 min sonication time, and 270 W sonication power. Under these conditions, the total flavonoid yield was 26.39 ± 0.61 mg / g. The total flavonoid yields of ISUSE (22.24 ± 1.21 mg / g) and UAE (16.21 ± 1.37 mg / g) are both lower than those of the ternary eutectic solvent combined with in-situ ultrasonic method (TDES-ISUSE) of this invention. Therefore, compared with traditional extraction techniques, TDES-ISUSE exhibits significantly superior extraction efficiency.

[0019] 3. The present invention identified 15 compounds in the extract of Epimedium wushanense, of which 9 were flavonoids. Three in vitro antioxidant experiments showed that the flavonoids extracted from Epimedium wushanense by the present invention have strong antioxidant activity, suggesting their potential in the development of functional food ingredients or drugs. Attached Figure Description

[0020] Figure 1 Chromatographic peaks of the mixed standard and four flavonoids in the extract of Epimedium wushanense (a is the chromatographic peak of EA, EB, EC and IA in the mixed standard; b is the chromatographic peak of EA, EB, EC and IA in the extract of Epimedium wushanense; peak 1 is icariin A (EA); peak 2 is icariin B (EB); peak 3 is icariin C (EC); peak 4 is icariin (IA)). Figure 2 : BP neural network model structure diagram; Figure 3 The yield of total flavonoids (TF) extracted from Epimedium wushanense using different eutectic solvents (DESs); Figure 4Results of a single-factor experiment; Figure 5 Response surface diagram; Figure 6 The training, validation, testing, and overall data fitting results of the BP-ANN model; Figure 7 Comparison of total flavonoid yields using four extraction methods; Figure 8 UHPLC-Q-TOF-MS extraction ion chromatogram of Epimedium wushanense extract (A is positive ion mode; B is negative ion mode). Figure 9 FT-IR spectra of tartrate choline, urea, glycerol and TDES and 1 HNMR spectrum (A is FT-IR spectrum; B is HNMR spectrum) 1 (H NMR spectrum) Figure 10 Microstructures of Epimedium wushanense powder treated by different methods (A: TDES-ISUSE treatment group; B: ISUSE treatment group; C: UAE treatment group; D: ME treatment group). Figure 11 : Snapshot of EC dissolution, diagram of the number of hydrogen bonds between EC and solvent, and diagram of the hydrogen bond lifetime between EC and solvent (A is a snapshot of EC dissolution in TDES solution; B is a snapshot of EC dissolution in ethanol solution; C is a diagram of the number of hydrogen bonds between EC and TDES and EC and ethanol; D is a diagram of the hydrogen bond lifetime between EC and TDES and EC and ethanol). Figure 12 Antioxidant activity of Epimedium wushanense extract (A is DPPH free radical scavenging rate; B is ABTS free radical scavenging rate; C is ferric ion reducing ability). Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The present invention will be further described below with reference to the embodiments. The embodiments are merely further supplements and explanations of the present invention, and are not intended to limit the invention.

[0022] Example 1: Extraction of flavonoids from Epimedium wushanense TDES preparation: Take tartrate choline, urea and glycerol (molar ratio of tartrate choline, urea and glycerol is 1:2:2), add water and mix at 70°C for 1.5 h to form a homogeneous mixture with a water content of 32%. The mixture is a ternary eutectic solvent for mixing hydrogen bond acceptors and hydrogen bond donors, which is TDES, for later use. (2) After crushing the Epimedium wushanense into powder, pass it through a 40-mesh sieve. Weigh 3.0g of Epimedium wushanense powder and add it to 102mL of TDES aqueous solution containing 32% (v / v) water. Vortex mix for 10min, then sonicate for 30min. The sonication power is 270W, the sonication frequency is 40kHz, and the sonication temperature is 33℃. Filter the mixture after sonication, centrifuge the filtrate at 4000r / min for 10min, and collect the supernatant to obtain the Epimedium wushanense extract.

[0023] Example 2: Extraction of flavonoids from Epimedium wushanense TDES preparation: Take tartrate choline, urea and glycerol (molar ratio of tartrate choline, urea and glycerol is 1:2:2), add water and mix at 65°C for 2.5 h to form a homogeneous mixture with a water content of 40%. The mixture is a ternary eutectic solvent for mixing hydrogen bond acceptors and hydrogen bond donors, which is TDES, for later use. (2) After crushing the Epimedium wushanense into powder, pass it through a 30-mesh sieve. Weigh 3.0g of Epimedium wushanense powder and add it to 120mL of TDES aqueous solution containing 40% (v / v) water. Vortex mix for 12min, then sonicate for 25min. The sonication power is 240W, the sonication frequency is 45kHz, and the sonication temperature is 34℃. Filter the mixture after sonication, centrifuge the filtrate at 4500r / min for 8min, and collect the supernatant to obtain Epimedium wushanense extract.

[0024] Example 3: Extraction of flavonoids from Epimedium wushanense TDES preparation: Take tartrate choline, urea and glycerol (molar ratio of tartrate choline, urea and glycerol is 1:2:2), add water and mix at 75°C for 0.5 h to form a homogeneous mixture with a water content of 20%. The mixture is a ternary eutectic solvent for mixing hydrogen bond acceptors and hydrogen bond donors, which is TDES, for later use. (2) After crushing the Epimedium wushanense, pass it through a 50-mesh sieve. Weigh 3.0g of Epimedium wushanense powder and add it to 60mL of TDES aqueous solution containing 20% ​​(v / v) water. Vortex mix for 8min, then sonicate for 35min. The sonication power is 300W, the sonication frequency is 35kHz, and the sonication temperature is 32℃. Filter the mixture after sonication, centrifuge the filtrate at 3500r / min for 12min, and collect the supernatant to obtain Epimedium wushanense extract.

[0025] Example 4: Extraction of flavonoids from Epimedium wushanense TDES preparation: Take tartrate choline, urea and glycerol (molar ratio of tartrate choline, urea and glycerol is 1:2:2), add water and mix at 70°C for 2 hours to form a homogeneous mixture with a water content of 30%. The mixture is a ternary eutectic solvent for mixing hydrogen bond acceptors and hydrogen bond donors, which is TDES, for later use. (2) After crushing the Epimedium wushanense, pass it through a 40-mesh sieve. Weigh 3.0g of Epimedium wushanense powder and add it to 80mL of TDES aqueous solution containing 30% (v / v) water. Vortex mix for 8-12min, then sonicate for 32min. The sonication power is 270W, the sonication frequency is 40kHz, and the sonication temperature is 33℃. Filter the mixture after sonication, centrifuge the filtrate at 4000r / min for 11min, and collect the supernatant to obtain the Epimedium wushanense extract.

[0026] This invention has undergone extensive experimental research, and the results of this experimental research are as follows: 1. Materials and Methods 1.1 Materials and Reagents Epimedium glutinosa from Wushan County, Chongqing Municipality, was purchased in July 2024 from Guandu Town, Wushan County, Chongqing Municipality, and was identified. The dried Epimedium glutinosa was pulverized and passed through a 40-mesh sieve. 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH), 2,2'-adiazon-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydrogen choline tartrate, dihydrocholine citrate, 1,4-butanediol, xylitol, malic acid, ethylene glycol, lactic acid, urea, icariin A (EA), icariin B (EB), icariin C (EC), and icariin (IA) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0027] 1.2 Preparation and Screening of Eutectic Solvents (DESs) Preparation of eutectic solvents: Twenty different DESs were prepared. Each DES was prepared by mixing hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD), adding water at 70°C, and stirring continuously for 1–2 hours until a homogeneous mixture was formed; a measured amount of water was added if necessary. The HBAs included dihydrocholine citrate and hydrogen choline tartrate; the HBDs included malic acid, lactic acid, glycerol, xylitol, acetic acid, urea, 1,4-butanediol, propylene glycol, and ethylene glycol. Detailed compositions and molar ratios of each DES are shown in Table 1.

[0028] Extraction of flavonoid compounds from Epimedium wushanense: Weigh 3.0g of Epimedium wushanense powder, add 60mL of DES or TDES containing 30% (v / v) water, vortex mix for 10min, and then sonicate for 30min (ultrasonic power 270W, frequency 40kHz, temperature 33℃); finally, filter the mixture, centrifuge it using a centrifuge (80-2 type, Jiangsu Jincheng Guosheng Co., Ltd.) (4000 r / min, 10min), and collect the supernatant.

[0029] Table 1 Formulations of different eutectic solvents Serial number Abbreviation HBA:HBD Molar ratio DES-1 Cdc:Mal Dihydrogen citrate choline: malic acid 1:2 DES-2 Cdc:Lac Dihydrogen citrate choline: lactic acid 1:2 DES-3 Cdc:Gly Dihydrogen citrate choline: glycerol 1:2 DES-4 Cdc:XyL Dihydrogen citrate choline: xylitol 1:2 DES-5 Cdc:Ace Dihydrogen citrate choline: acetic acid 1:2 DES-6 Chb:Ure Choline hydrogen tartrate: urea 1:2 DES-7 Chb:Lac Choline hydrogen tartrate: lactic acid 1:2 DES-8 Chb:Mal Choline hydrogen tartrate: malic acid 1:2 DES-9 Chb:Ace Choline hydrogen tartrate: acetic acid 1:2 DES-10 Chb:Xyl Choline hydrogen tartrate: xylitol 1:2 DES-11 Cdc:But:Gly Dihydrogen citrate choline: 1,4-butanediol: glycerol 1:2:2 DES-12 Cdc:Pro:Gly Dihydrogen citrate choline: propylene glycol: glycerol 1:2:2 DES-13 Cdc:Eth:Gly Dihydrogen citrate choline: ethylene glycol: glycerol 1:2:2 DES-14 Cdc:Mal:Ure Dihydrogen citrate choline: malic acid: urea 1:2:2 DES-15 Cdc:Lac:Gly Dihydrogen citrate choline: lactic acid: glycerol 1:2:2 DES-16 Cdc:Ace:Gly Dihydrogen citrate choline: acetic acid: glycerol 1:2:2 DES-17 Chb:Ure:Gly Choline hydrogen tartrate: urea: glycerol 1:2:2 DES-18 Chb:Eth:Lac Choline hydrogen tartrate: ethylene glycol: lactic acid 1:2:2 DES-19 Chb:But:Gly Choline hydrogen tartrate: 1,4-butanediol: glycerol 1:2:2 DES-20 Chb:But:Lac Choline hydrogen tartrate: 1,4-butanediol: lactic acid 1:2:2 1.3 Analytical Methods 1.3.1 Determination of total flavonoid content The total flavonoid (TF) content in Epimedium wushanense was determined by ultraviolet spectrophotometry. A standard curve (Y = 12.124X - 0.0316, R0) was plotted using rutin as a standard in the concentration range of 40–200 μg / mL. 2 =0.9994), the curve shows a good linear relationship. The total flavonoid content is expressed as milligrams (mg / g) of rutin equivalents per gram of dried Epimedium wushanense.

[0030] 1.3.2 High Performance Liquid Chromatography (HPLC) Analysis Four flavonoid components (EA, EB, EC, IA) were separated using an Agilent 1220 high-performance liquid chromatograph equipped with an Elite ODS-B C18 column (5μm, 250mm×4.6mm). The gradient elution program is shown in Table 2. The chromatographic conditions were: column temperature 25℃, flow rate 1 mL / min, and injection volume 10 μL. Figure 1 The chromatographic peaks of EA, EB, EC and IA are shown respectively.

[0031] Table 2 HPLC gradient elution program Time (min) Ultra-pure water (%) Acetonitrile (%) 0 76 24 40 74 26 1.3.3 Component Identification of Epimedium Wushanense Extract The chemical constituents of Epimedium wushanense extract were analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The analytical instrument was an Agilent 1290UHPLC system coupled with an Agilent 6546Q-TOF high-resolution mass spectrometer, equipped with a Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The mobile phase was acetonitrile (A) and 0.1% formic acid aqueous solution (B), and the gradient elution program is detailed in Table 3. Chromatographic separation conditions: column temperature 35℃, detection wavelength 254 nm, injection volume 1 μL. Electrospray ionization (ESI) sources were operated in both positive and negative ion modes.

[0032] Table 3 UHPLC-Q-TOF-MS conditions Time (min) A(%) B(%) 0 5 95 30 25 75 45 55 45 55 95 5 60 95 5 60.01 5 95 66 5 95 1.4 Optimization of Extraction Process 1.4.1 Single-factor experiment To ensure efficiency, only the main factors affecting the extraction of bioactive components were examined first. As shown in Table 4, this study systematically investigated five key factors to determine the optimal combination of conditions for maximizing the yield of total flavonoids. In the single-factor experiment, the optimal DES was added to 3.0g of Epimedium wushanense powder to investigate the effect of each factor on the total flavonoid content; only one variable was changed during the experiment, while the other conditions remained unchanged.

[0033] Table 4 Single-factor experiments 1.4.2 Response Surface Methodology (RSM) Experiment A Box-Behnken design (BBD) was used to examine the interactions among key variables. A three-factor, two-level BBD experiment was conducted to optimize extraction conditions and maximize the total flavonoid yield. The main influencing variables were water content (20%–30%–40%), vortexing time (8–10–12 min), and liquid-to-solid ratio (20–30–40 mL / g). The 17 variable levels are shown in Table 3. The regression equation is as follows: (1) In the formula: Y is the total flavonoid yield; β0 is a constant term; βᵢⱼ is a coefficient; X is an influencing factor.

[0034] 1.5 Response Surface Methodology-Artificial Neural Network-Genetic Algorithm (RSM-ANN-GA) Modeling Although Regression-Solution Modeling (RSM) is a core method for optimizing extraction processes, its quadratic regression-based modeling approach struggles to capture the crucial and complex nonlinear interactions in ultrasound-assisted systems. To address this, a hybrid RSM-ANN-GA framework was constructed: training data for the system was generated using Box-Behnken design under RSM; a multilayer perceptron structure based on artificial neural networks (ANNs) was employed to model the total flavonoid yield with higher nonlinear fidelity; and a genetic algorithm (GA) was used to search for the global optimum on the response surface predicted by the ANN. This integration retains the experimental rigor of RSM while significantly improving prediction accuracy and optimization robustness.

[0035] A sophisticated backpropagation artificial neural network (BP-ANN) model was constructed using a genetic algorithm (GA) system within the Toolbox™ of MATLAB R2024a software. The model was trained on a dataset generated by RSM, which encompasses the multivariate relationships between extraction parameters and the yield of the target component. The training scheme included three key input variables: water content (w / w, %), vortexing time (min), and liquid-to-solid ratio (mL / g), aiming to predict the key output variable—total flavonoid extraction yield.

[0036] By minimizing the mean squared error (MSE) function, a 3-9-1 ANN architecture (containing 3 input neurons, 9 hidden neurons, and 1 output neuron) was determined, as follows: Figure 2 As shown, the architecture was determined through system sensitivity analysis to balance model complexity and generalization ability. The Levenberg-Marquardt algorithm was used for network training due to its higher computational efficiency in error surface search. To achieve robust model construction, the total samples generated by RSM were allocated proportionally: 70% for training, allowing the network to learn the inherent patterns and nonlinear correlations in the data; 15% for validation; and the remaining 15% for testing. The optimal ANN model was selected based on the lowest MSE and the highest correlation coefficient (R), and then the model parameters were further improved through GA optimization to enhance the model's prediction accuracy and stability.

[0037] 1.6 Comparison with traditional extraction methods 1.6.1 Ethanol-assisted in-situ ultrasonic-assisted extraction (ISUSE) Weigh 3.0g of Epimedium wushanense powder, add 60mL of ethanol, vortex mix for 10min, and then sonicate for 30min (ultrasonic power 270W, frequency 40kHz, temperature 33℃); centrifuge (4000r / min, 10min) using a centrifuge (80-2 type, Jiangsu Jincheng Guosheng Co., Ltd.), and collect the supernatant; determine the total flavonoid content in the extract by ultraviolet spectrophotometry.

[0038] 1.6.2 Ultrasonic-assisted ethanol extraction (UAE) Weigh 3.0g of Epimedium wushanense powder, add 60mL of ethanol, and extract with ultrasonic assistance at 270W power for 30min (frequency 40kHz, temperature 33℃). After centrifugation (4000r / min, 15min), collect the supernatant. Determine the total flavonoid content in the extract by ultraviolet spectrophotometry.

[0039] 1.6.3 Impregnation Extraction (ME) Weigh 3.0g of Epimedium wushanense powder, place it in a 100mL flask, add 60mL of ethanol, soak and extract at 25℃ for 8h, centrifuge (4000 r / min, 15min) and collect the supernatant; determine the total flavonoid content in the extract by ultraviolet spectrophotometry.

[0040] 1.7 Research on Extraction Mechanism 1.7.1 Fourier Transform Infrared Spectroscopy (FT-IR) Analysis To investigate the hydrogen bond formation characteristics of novel TDES, Fourier transform infrared spectroscopy was used in the range of 4000–400 cm⁻¹. 1 The optimal DES formulation was analyzed within the wavenumber range.

[0041] 1.7.2 Scanning Electron Microscopy (SEM) Analysis The morphological characteristics of Epimedium wushanense powder after treatment were observed by SEM, and the powders under four experimental conditions were compared and analyzed: ① TDES-ISUSE treatment group; ② ISUSE treatment group; ③ UAE treatment group; ④ ME treatment group.

[0042] 1.7.3 Molecular Dynamics (MD) Analysis Molecular dynamics simulations are a core technique in computational chemistry, used to investigate the time evolution and intermolecular interactions of atomic-scale systems. This simulation, based on the classical mechanics framework, uses Newton's equations of motion to predict particle trajectories under a predefined energy potential field. GROMACS, a powerful molecular dynamics package specifically designed for simulating large molecular systems, was used for the simulations. The aim of this simulation was to investigate the dynamic behavior and interactions of molecules in two different solvents over a 50 nanosecond (ns) timescale. The OPLS-AA force field was used to accurately simulate intermolecular interactions within the system: for the solvents, the TIP3P water model was used for the ethanol system, while a custom solvent model was used for the TDES system. The model parameters were optimized to ensure the realism of the simulation conditions.

[0043] The simulations were conducted at a temperature of 300 K and a pressure of 1 standard atmosphere (atm). Each production simulation phase lasted 50 ns with a time step of 2 femtoseconds (fs). The simulation process began with system construction: the molecular and solvent structures were first combined, followed by energy minimization to eliminate steric hindrance conflicts and optimize the initial configuration; then, a 100 picosecond (ps) NVT ensemble (constant particle number, volume, and temperature) equilibrium simulation was performed to achieve temperature stabilization, followed by a 100 picosecond NPT ensemble (constant particle number, pressure, and temperature) equilibrium simulation to achieve pressure and volume stabilization; finally, the production simulation was performed under the NPT ensemble, with a data acquisition frequency of 1 ps / time throughout the entire simulation.

[0044] 1.8 Determination of antioxidant activity 1.8.1 DPPH free radical scavenging experiment The extract of Epimedium wushanense (prepared in Example 1) was prepared into five sample solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, respectively. Two mL of each sample solution was mixed with two mL of freshly prepared DPPH solution (0.15 mmol / L, dissolved in methanol), and incubated in the dark for 30 min. The absorbance was then measured at 517 nm. The clearance rate was calculated using the following formula: (2) In the formula: Ac is the absorbance of the blank control group; As is the absorbance of the sample group.

[0045] 1.8.2 ABTS Free Radical Scavenging Experiment Mix 250 mL of ABTS solution (7 mmol / L) with 250 mL of potassium persulfate solution (2.45 mmol / L) and incubate in the dark for 16 h. Using a UV-Vis spectrophotometer, dilute the stock solution with ethanol to a absorbance of 0.70 ± 0.02 at 734 nm to prepare the ABTS⁺ working solution. Mix 0.5 mL of each of five sample solutions (0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL) with 3 mL of ABTS⁺ working solution, incubate in the dark for 6 min, and then measure the absorbance at 734 nm. The clearance rate is calculated using the following formula: (3) In the formula: A0 represents the absorbance of the blank control group; A1 represents the absorbance of the sample group.

[0046] 2.8.3 Experiment on the reducing power of iron ions The extract of Epimedium wushanense (prepared in Example 1) was prepared into 5 concentrations (0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, respectively). 3 mL of sample solution was taken, and 2 mL of sodium phosphate buffer (0.2 mol / L, pH 6.6) and 2 mL of potassium ferricyanide solution (1%, w / v) were added. The mixture was incubated in a 50°C water bath for 30 min. After the reaction was completed, 2 mL of trichloroacetic acid (10%, w / v) was added, and the mixture was centrifuged (3000 r / min, 10 min). 2 mL of the supernatant was taken, and 0.1 mL of ferric chloride solution (0.1%, w / v) and 2 mL of water were added. After reacting for 10 min, the absorbance was measured at a wavelength of 700 nm.

[0047] 1.9 Statistical Analysis One-way ANOVA was used to perform statistical analysis on the RSM results. Data are expressed as mean ± standard deviation (SD), and significance levels are marked as ***p < 0.001, **p < 0.01, and *p < 0.05.

[0048] 2 Results and Discussion 2.1 Screening of the Optimal Eutectic Solvent (DES) Using dihydrocholine citrate and hydrogen tartrate as key hydrogen bond acceptors (HBAs), 20 DESs (including 10 binary DESs and 10 ternary DESs) were designed and prepared for the extraction of total flavonoids from Epimedium wushanense. Figure 3 As shown, through systematic screening of 20 DESs (with a water content of 30%), it was found that the total flavonoid extraction efficiencies varied among different DESs, with the tartrate choline-urea-glycerol system (Chb:Ure:Gly) exhibiting the best extraction effect. Notably, the total flavonoid extraction yield from ethanol was 22.24 ± 1.21 mg / g, a result consistent with the principle of "like dissolves like." In conclusion, Chb:Ure:Gly is the optimal solvent for the extraction of total flavonoids from Epimedium wushanense, and this solvent will be used in subsequent process optimization and mechanism studies.

[0049] 2.2 Results of Single-Factor Experiment 2.2.1 Effect of Moisture Content The rheological properties (especially viscosity) of DESs significantly affect their extraction performance by modulating mass transfer kinetics. Generally, the viscosity of DESs decreases with increasing water content, and an optimal water content can increase the total flavonoid content. Therefore, this study investigated the effect of different water contents in the optimal DES on the extraction of total flavonoids.

[0050] After adjusting the moisture content, it was found that the moisture content had a non-linear relationship with the yield of total flavonoids from Epimedium wushanense. For example... Figure 4As shown in Figure A, when the water content in DES increases from 20% to 30%, the total flavonoid yield increases significantly. This may be because the increased water content reduces the solution viscosity, thereby improving the extraction efficiency of flavonoid components. However, when the water content further increases to 40%~50%, the total flavonoid yield decreases significantly. It is speculated that the excessive water in the DES system weakens the intermolecular interaction between flavonoid components and DES components, thus limiting the dissolution of flavonoid components.

[0051] 2.2.2 Effects of vortex time, liquid-to-solid ratio, ultrasonic time, and ultrasonic power Vortexing promotes rapid and uniform mixing of medicinal powder and solvent, accelerating the dissolution of target components and thus achieving a high extraction yield in a short time. For example... Figure 4 As shown in B, the total flavonoid yield increases with the extension of vortexing time, reaching a peak at 10.0 min. This indicates that the target component diffuses rapidly in the early stage of extraction. If the vortexing time is further extended, a large number of impurities may be washed away, interfering with the dissolution of flavonoid components and leading to a decrease in the total flavonoid content.

[0052] The liquid-to-solid ratio can regulate the diffusion behavior of solutes in a solvent system: increasing the liquid-to-solid ratio can enhance extraction efficiency by increasing the driving force, but it also increases solvent consumption and promotes the co-extraction of impurities. Figure 4 As shown in C, the total flavonoid yield increases with the increase of the liquid-to-solid ratio, reaching a maximum at 30 mL / g. If the liquid-to-solid ratio is further increased, the total flavonoid yield decreases instead. This may be due to excessive contact between the solvent and the medicinal matrix, leading to the dissolution of a large number of interfering impurities. In addition, excessive solvent will weaken the propagation of ultrasonic energy, reduce the degree of damage to the plant cell wall, and ultimately lead to a decrease in extraction efficiency.

[0053] Ultrasound time also has a crucial impact on the total flavonoid yield, because ultrasound requires sufficient time to adequately disrupt plant cell walls. For example... Figure 4 As shown in D, the total flavonoid yield gradually increased with the extension of ultrasonic time, reaching a relatively high plateau at 30 min. If the ultrasonic time was further extended, the total flavonoid yield did not change significantly. This is because the plant cell wall structure had been sufficiently destroyed, and the solvent and target components reached mass transfer equilibrium. Therefore, extending the ultrasonic time could not significantly improve the extraction efficiency.

[0054] The intensity of ultrasonic cavitation depends on the applied power; a suitable power range can efficiently and directionally disrupt the complex plant cell wall structure. As shown in Figure 4E, the total flavonoid yield increases with increasing ultrasonic power, reaching a maximum at 270W. If the power is further increased, the yield does not change significantly because the ultrasonic power has reached the saturation threshold, at which point the cavitation intensity has achieved maximum disruption of the cell wall structure and maximum mass transfer of flavonoid components.

[0055] In this experiment, all other experimental parameters remained constant.Figure 4 As shown in A~E, the above variables all have a significant impact on the total flavonoid yield. Therefore, water content (20%~40%), vortex time (8~12min), and liquid-to-solid ratio (20~40mL / g) were selected for subsequent experiments.

[0056] Note: A represents the effect of water content; B represents the effect of vortex time; C represents the effect of liquid-to-solid ratio; D represents the effect of ultrasonic time; E represents the effect of ultrasonic power.

[0057] 2.3 Response Surface Method (RSM) Analysis The Box-Behnken design (BBD) was used to optimize three key variables: water content, vortex time, and liquid-to-solid ratio. The experimental design was performed using Design-Expert software.

[0058] The variables and response values ​​are shown in Table S3, and the results of the analysis of variance (ANOVA) for the model predictions are shown in Table 3. The results show that the model is statistically significant (p < 0.001); the coefficient of determination (R²) for the response variable is... 2 The adjusted coefficient of determination (Adj. R) is 0.9718. 2 The p-value was 0.9498, indicating that the model has high accuracy in predicting future results; the lack of fit test result was not significant (p>0.05), indicating that the predictive model can fully represent the experimental data. To improve the regression performance of the optimized model, non-significant terms with p>0.1 were removed from the model.

[0059] Based on the coded values, after removing non-significant terms, the quadratic regression model equation for the total flavonoid yield is as follows: Y = 25.73 + 0.5825A + 0.2B + 1.01C + 0.565BC - 0.2785A 2 -0.5635B 2 -1.68C 2 Table 3. Analysis of variance (ANOVA) results for the quadratic regression model. Source Sum of squares Degrees of freedom Mean square F value P value Significance Model 26.81 7 3.83 44.27 ＜ 0.0001 Significant A 2.71 1 2.71 31.38 0.0003 B 0.32 1 0.32 3.7 0.0866 C 8.08 1 8.08 93.4 ＜ 0.0001 BC 1.28 1 1.28 14.76 0.004 A 2 ]] 0.3266 1 0.3266 3.77 0.0839 B 2 ]]> 1.34 1 1.34 15.45 0.0035 C 2 ]]> 11.93 1 11.93 137.93 ＜ 0.0001 Residual error 0.7786 9 0.0865 Error of misfit 0.4943 5 0.0989 1.39 0.3856 Not significant Pure error 0.2843 4 0.0711 Corrected total 27.59 16 As shown in Figure 5, the 3D response surface methodology visually illustrates the influence of various variables on the total flavonoid yield. The figure indicates that the optimal extraction conditions are: water content 36.47%, vortexing time 11.13 min, and liquid-to-solid ratio 35.35:1 mL / g, with a theoretical total flavonoid yield of 26.23 mg / g. For ease of experimental operation, the actual extraction conditions were adjusted to: water content 36%, vortexing time 11 min, and liquid-to-solid ratio 35:1 mL / g.

[0060] As shown in Figure 5, the total flavonoid yield initially increased and then decreased with increasing vortex time and liquid-to-solid ratio, reaching its peak under optimal conditions. Moderately extending the vortex time and increasing the liquid-to-solid ratio can enhance the ultrasonic cavitation effect, promote cell wall disruption, reduce DES viscosity, and accelerate mass transfer kinetics, thereby promoting the dissolution of flavonoids. However, excessively extending the vortex time and using excessively high liquid-to-solid ratios can lead to the co-extraction of a large number of impurities, competitively inhibiting the elution of flavonoids. Notably, the liquid-to-solid ratio has a more significant impact on the yield than the vortex time, indicating that it plays a more crucial role in regulating extraction efficiency.

[0061] 2.4 RSM-ANN-GA Analysis Using the dataset obtained from RSM analysis as training samples, a BP-ANN model was constructed. The input variables were three key factors: water content, vortex time, and liquid-to-solid ratio. The output variable was the total flavonoid extraction yield. The hyperbolic tangent function (tansig) was used as the activation function between the input and hidden layers, and a linear function was used as the transfer function between the hidden and output layers. The Levenberg-Marquardt backpropagation algorithm (trainlm) was used for training. The network training parameters were set as follows: 1000 iterations, 25 steps per iteration, and a target training error of 0.0001. Figure 6 As shown, the correlation coefficients (R) of the training set, validation set, test set, and total dataset are all greater than 0.90, indicating that the BP-ANN model has good predictive ability for training, validation, and test samples.

[0062] To maximize the output, the network data generated by the RSM-ANN-GA integration framework is used as the objective function. The three independent variables are treated as matrix variables, and the boundary constraints are set as follows: [20;8;20]≤[X1;X2;X3]≤[40;12;40] Through RSM-ANN-GA integrated optimization, the optimal extraction parameters were determined to be: water content 31.75%, vortexing time 10.33 min, and liquid-to-solid ratio 34.12 mL / g, with a theoretical total flavonoid yield of 26.36 mg / g. To improve the operability of the experiment, the conditions were adjusted to: water content 32%, vortexing time 10 min, and liquid-to-solid ratio 34 mL / g in the verification experiment.

[0063] 2.5 Verification Experiment Table 4 shows that both the RSM model and the RSM-ANN-GA model have good predictive performance for the total flavonoid yield, with minimal deviation from the experimental values. The experimental yield after optimization of the traditional RSM model was 26.17 ± 1.01 mg / g, with a relative error of 0.19% and a relative standard deviation (RSD) of 0.69%. In contrast, the experimental yield of the RSM-ANN-GA hybrid model was 26.39 ± 0.61 mg / g, with an even lower relative error (0.12%) and a significantly reduced RSD (0.24%). These results indicate that the hybrid model has higher accuracy and stability compared to RSM alone, thanks to its stronger ability to capture complex nonlinear interactions.

[0064] Table 4 Validation Experiment Results Moisture content Vortex time Liquid-solid ratio Experimental value RSD RSM 36 11 35:1 26.17±1.01 0.69 RSM-ANN-GA 32 10 34:1 26.39±0.61 0.24 2.6 Comparison with traditional extraction methods To verify the advantages of the TDES-ISUSE extraction method in improving the yield of total flavonoids from Epimedium wushanense, this method was systematically compared with traditional extraction methods (ISUSE, UAE, ME). Figure 7 As shown, compared with ME, TDES-ISUSE not only significantly shortened the extraction time but also significantly increased the total flavonoid yield. Furthermore, the total flavonoid yield of TDES-ISUSE was higher than that of ISUSE (22.24±1.21 mg / g) and UAE (16.21±1.37 mg / g). These results indicate that TDES-ISUSE combines the advantages of rapid extraction, high efficiency, and environmental sustainability, demonstrating significant application potential in optimizing the extraction of total flavonoids from Epimedium wushanense.

[0065] 2.7 UHPLC-Q-TOF-MS Analysis UHPLC-Q-TOF-MS, with its high sensitivity, high-resolution separation capability, and accurate mass determination performance, has become a reliable high-throughput technique for identifying bioactive components in natural products. This study is the first to employ UHPLC-Q-TOF-MS to analyze the chemical composition of Epimedium wushanense extract, aiming to elucidate its bioactive component composition. For example... Figure 8 As shown, total ion current (TIC) chromatograms were obtained in both positive and negative ion modes. By matching mass spectrometry fragments with the PCDL MS database and referring to literature, a total of 15 compounds were identified, including 9 flavonoids, 3 organic acids, 1 terpene, 1 phenylpropanoid, and 1 alkaloid. Detailed information is shown in Table 5. Among them, compounds 8–11 are EA, EB, EC, and IA, respectively, with EC being the major component.

[0066] Table 5. Compounds of Epimedium wushanense identified by UHPLC-Q-TOF-MS Serial number Retention time (min) Mode Component Molecular formula Mass to charge ratio (m / z) Main fragment ion 1 8.870 + Chlorogenic acid C16H18O9 355.1029 163.0392,145.0282,135.0439,117.0333 2 9.696 - Neochlorogenic acid C16H18O9 353.0884 173.0457,135.0452,111.0451 3 12.601 - 3-O-p-coumaroylquinic acid C16H18O8 337.0935 191.0563,163.0402,119.0502 4 13.086 + Magnoflorine C20H24NO4 342.1707 297.1119,265.0855,191.0851 5 19.551 + Quercetin 3-O-glucoside C21H20O12 465.1032 302.0501,285.0389,145.0495 6 23.721 + Eugenol rutinoside C22H32O11 490.229 351.1022,349.1262,331.1007 7 28.694 + Diphylloside B C38H48O19 809.2878 663.2291,517.1710,335.1175,129.0547 8 33.853 + Epimedin A C39H50O20 839.2984 677.2442,531.1863,369.1329,129.0544 9 34.198 + Epimedin B C38H48O19 809.2888 677.2451,531.1871,369.1335,313.0706,255.0759 10 34.626 + Epmedin C C39H50O19 823.3036 677.2451,531.1872,369.1334,313.0704,129.0546 11 34.907 + Icariin C33H40O15 677.2468 531.1883,369.1342,313.0710,129.0549 12 42.613 - Baohuoside VII C33H40O15 675.2294 367.1187,351.0884,323.0926 13 43.743 + Epicornuin F C25H26O7 439.1759 421.1642,403.1541,367.1177 14 43.973 - Baohuoside I C27H30O10 513.1773 366.1111,323.0935,217.0512 15 48.473 + epimedokoreanin B C25H26O6 423.1804 405.1691,349.1060,261.2208,127.0388 2.8 Method Validation HPLC analysis revealed that *Epimedium wushanense* contains four flavonoids: EA, EB, EC, and IA. Quantitative analysis of these components was performed under optimized conditions to accurately evaluate the quality of the *Epimedium wushanense* extract. A calibration curve was plotted using standard solutions to examine the linear relationship and determine the linear range and correlation coefficient (R²). 2 ), to verify the analytical reliability of the method.

[0067] The standard curves for EA, EB, EC, and IA are as follows: Y_EA = 32455x + 36.861 (R 2 =0.9994), Y_EB=21976x+21.8214 (R 2 =0.9996), Y_EC=23452x+26.3687 (R 2 =0.9993), Y_IA=30547x+15.4277 (R 2 =0.9998). As shown in Table 6, the four flavonoid components showed good linearity in the concentration range of 3.12~100.00 μg / mL, R 2 All values ​​were greater than 0.999; their limits of detection (LOD) and limits of quantitation (LOQ) were 0.33~0.46 μg / mL and 1.01~1.37 μg / mL, respectively.

[0068] Table 6. Method validation results Analyte LOD LOQ Intraday precision Daytime precision stability Repeatability Recovery rate EA 0.38 1.14 0.16 1.72 1.65 1.03 99.06 EB 0.46 1.37 0.63 1.15 1.30 1.25 99.41 EC 0.41 1.21 0.39 1.09 1.47 1.72 98.20 IA 0.33 1.01 0.64 1.43 1.68 1.46 99.84 Intra-day precision was evaluated by performing six consecutive analyses of the same sample within 24 hours, while inter-day precision was determined by analyzing the sample over six consecutive days. The results showed that the RSDs for both intra-day and inter-day precision were less than 2%, indicating that the method has excellent precision. Stability was assessed at 0, 3, 6, 9, and 12 hours at 25°C. The results showed good sample stability within 12 hours, with RSDs all less than 2%.

[0069] The repeatability of the method was evaluated by analyzing six identical samples. The RSD ranged from 1.03% to 1.72%, indicating that the method has high repeatability. To investigate the recovery rate, known amounts of four flavonoid standards were added to 3g of raw material, and the recovery rate was calculated. The results are shown in Table 6.

[0070] 2.9 Research on Extraction Mechanism 2.9.1 Characterization of eutectic solvent (DES) Ternary eutectic solvents (TDESs) are eutectic mixtures composed of one hydrogen bond acceptor (HBA) and two hydrogen bond donors (HBD). In this study, Fourier transform infrared spectroscopy (FT-IR) was used to characterize the molecular interactions and structural features of each individual component (hydrocholine tartrate, urea, and glycerol) and the prepared TDESs.

[0071] FT-IR spectroscopy ( Figure 9 A) shows that the characteristic peaks of each functional group in the system are as follows: -OH (3500~3200cm⁻) 1 CH (3000~2500cm⁻) 1 CO (1112~1000cm⁻) 1 In the prepared TDESs, the CN stretching vibration peak of tartrate choline (originally located at 1085 cm⁻) was observed. 1 The deviation still exists, but has shifted slightly to 1044 cm⁻ 1 Furthermore, compared to tartrate choline, urea, and glycerol, the vibrational absorption peak of -OH in TDESs is broadened and exhibits a blue shift, indicating the formation of numerous hydrogen bonds in the system. In summary, the FT-IR spectral characteristics (CN peak shift, broadened and blue-shifted -OH peak, and retention of key functional group characteristic peaks) provide direct evidence for the successful preparation of TDESs.

[0072] like Figure 9 As shown in B, 1 1H NMR spectroscopy provides strong evidence for the formation of TDES: the TDES spectrum retains all the characteristic proton signals of each individual component, and no new characteristic chemical shift peaks appear (excluding the possibility of covalent bond formation). This spectroscopic feature indicates that TDES are constructed through non-covalent intermolecular interactions (mainly hydrogen bonds) between hydrogen bond donors and acceptors, rather than through chemical reactions that alter the inherent molecular structure of the components.

[0073] 2.9.2 Scanning Electron Microscopy (SEM) Analysis Vortex action can generate turbulent mixing at the solid-liquid interface, enhancing mass transfer kinetics and accelerating the detachment of target analytes from the plant matrix. Furthermore, the efficiency of ultrasonic extraction mainly stems from cavitation—caused by ultrasound-induced pressure oscillations, manifested as alternating compression and rarefaction cycles in the liquid medium. Therefore, this study combines vortex and ultrasonic extraction to achieve synergistic extraction: this combined method utilizes both the turbulent mixing of vortices (promoting solvent penetration) and the mechanical shearing of ultrasonic cavitation (enhancing cell rupture), thereby achieving more thorough cell disruption. This result can be achieved through… Figure 10 Microscopic observation of A provided direct confirmation.

[0074] Cellulose is a major structural component of plant cell walls. Desiccant-soluble solvents (DESs) can efficiently disrupt and dissolve cellulose through selective hydrogen bonding interactions, exhibiting superior solubility and biomass pretreatment effects compared to traditional solvents. DESs are composed of hydrogen ions (HBA) and hydrogen ions (HBD), which synergistically act on the cellulose chain, significantly enhancing the dissolution process. This synergistic effect disrupts the hydrogen bond network in the cellulose structure, loosening its dense crystalline framework and ultimately achieving complete dissolution. (Comparison) Figure 10 As shown in A~D, compared with traditional methods, TDES-ISUSE treatment has a more significant destructive effect on plant cell walls, thereby improving the extraction efficiency of marker bioactive components.

[0075] 2.9.3 Molecular Dynamics (MD) Simulation Analysis To elucidate the mechanism of TDES-ISUSE extraction of flavonoids from Epimedium wushanense, this study used the most effective TDES (Chb:Ure:Gly) and a traditional solvent (ethanol) as research subjects. Molecular dynamics simulations were conducted to investigate the molecular interactions between these solvent molecules and EC, the main flavonoid component in Epimedium wushanense. The molecular interactions between solvent molecules and the target compound have a crucial impact on solubility parameters, thus affecting extraction efficiency.

[0076] Figure 11 Figures A and B show the spatial distribution of EC in the TDES and ethanol (EtOH) systems at 50 ns, respectively. The results indicate that EC exhibits a uniform dispersion in TDES, while local aggregation occurs in ethanol, suggesting that TDES has better solubility for EC, which stems from stronger intermolecular interactions. Figure 11 C shows the temporal evolution of hydrogen bond interactions between EC and TDES, and between EC and ethanol within 0–50 ns: the number of hydrogen bonds between EC and TDES is significantly greater than that between EC and ethanol, and the number of hydrogen bonds in the TDES system fluctuates less and exhibits higher stability. Furthermore, as… Figure 11 As shown in Figure D, the hydrogen bond lifetime between EC and TDES is significantly longer than that between EC and ethanol, indicating that the hydrogen bond interactions in the DES system are more stable. In summary, molecular dynamics (MD) simulations confirm that TDES has superior solubility for EC.

[0077] 2.10 Antioxidant activity To explore natural antioxidants, this study employed three oxidation assays to comprehensively evaluate the in vitro antioxidant activity of Epimedium wushanense extract. For example... Figure 12As shown in A and 12B, within the test concentration range (0.1~0.5 mg / mL), the extract exhibited significant scavenging ability against both DPPH⁺ and ABTS⁺ free radicals: at a concentration of 0.5 mg / mL, the maximum scavenging rates of *Epimedium wushanense* extract against DPPH⁺ and ABTS⁺ free radicals were 86.66%±1.84% and 98.61%±1.31%, respectively; furthermore, the free radical scavenging rate of the positive control (ascorbic acid) exceeded 90%. The ferric ion reducing power experiment is commonly used to determine the ability of ferric complexes to be reduced to ferrous ions, such as... Figure 12 As shown in Figure C, when the sample concentration reaches its maximum value (0.5 mg / mL), the absorbance of the extract is 0.558 ± 0.011. In conclusion, the extract of Epimedium wushanense possesses potential antioxidant activity and can be further developed into an antioxidant.

[0078] 3. Conclusion This invention prepares a novel natural ternary eutectic solvent (TDES) and develops a novel extraction method (ISUSE) for the extraction of total flavonoids (TF) from Epimedium wushanense. Through systematic screening, tartrate choline-urea-glycerol (Chb:Ure:Gly) was determined to be the optimal TDES. Optimization using the RSM-ANN-GA framework determined the optimal extraction conditions to be: 32% water content, 10 min vortexing time, 34:1 mL / g liquid-to-solid ratio, 30 min sonication time, and 270 W sonication power, resulting in a total flavonoid yield of 26.39 ± 0.61 mg / g. Compared with traditional extraction techniques, the TDES-ISUSE method demonstrates significantly superior extraction efficiency.

[0079] This invention employed UHPLC-Q-TOF-MS to identify the chemical constituents of Epimedium wushanense extract, clearly identifying 15 compounds, 9 of which were flavonoids. A validated HPLC method was also established for the simultaneous determination of four major flavonoids in Epimedium wushanense. Furthermore, FT-IR spectroscopy verified the successful preparation of the novel TDES; SEM analysis of the extracted plant residue provided structural evidence for the efficient extraction using the TDES-ISUSE method; and MD simulation further confirmed the superior solubility of TDES for EC. Three in vitro antioxidant experiments demonstrated that the extract possesses potent antioxidant activity, suggesting its potential in the development of functional food ingredients or drugs. In summary, this study successfully established a novel green extraction method (TDES-ISUSE) for the efficient extraction of total flavonoids from Epimedium wushanense.

Claims

1. A method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound, characterized in that: Includes the following steps: (1) Preparation of TDES: Take tartrate choline, urea and glycerol, add water and mix continuously to form a homogeneous mixture. The mixture is a ternary eutectic solvent for mixing hydrogen bond acceptors and hydrogen bond donors, and TDES is obtained for later use. (2) After crushing the Epimedium puerariae, sieve it, add TDES, vortex mix it, sonicate it, filter the sonicated mixture through a 0.22μm filter membrane, centrifuge the filtrate, collect the supernatant, and obtain the Epimedium puerariae extract.

2. The method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound as described in claim 1, characterized in that: In step (1), tartrate choline is a mixed hydrogen bond acceptor, urea and glycerol are hydrogen bond donors, and the molar ratio of tartrate choline, urea and glycerol is 1:2:

2.

3. The method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound as described in claim 1, characterized in that: In step (1), the mixing and stirring temperature is 65~75℃ and the time is 0.5~2.5h.

4. The method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound as described in claim 3, characterized in that: In step (1), the mixing and stirring temperature is 70°C and the time is 1~2h.

5. The method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound as described in claim 1, characterized in that: In step (1), the water content of TDES is 20-40%.

6. The method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound as described in claim 5, characterized in that: In step (1), the water content of TDES is 32%.

7. The method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound as described in claim 1, characterized in that: In step (2), the Wushan Epimedium is pulverized and passed through a 30-50 mesh sieve. The ratio of Wushan Epimedium powder to TDES is 1g:20-40mL.

8. The method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound as described in claim 7, characterized in that: In step (2), the Epimedium glutamate of Wushan is pulverized and passed through a 40-mesh sieve. The ratio of Epimedium glutamate powder to TDES is 1g:34mL.

9. The method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound as described in claim 1, characterized in that: In step (2), the vortex mixing time is 8~12min; the ultrasonic treatment time is 25~35min, the ultrasonic power is 240~300W, the ultrasonic frequency is 35~45kHz, the ultrasonic temperature is 32~34℃; the centrifugation speed is 3500~4500r / min, and the centrifugation time is 8~12min.

10. The method for synergistic extraction of flavonoids from Epimedium wushanense using a ternary eutectic solvent combined with in-situ ultrasound as described in claim 9, characterized in that: In step (2), the vortex mixing time is 10 min; the ultrasonic treatment time is 30 min, the ultrasonic power is 270 W, the ultrasonic frequency is 40 kHz, the ultrasonic temperature is 33 ℃; the centrifugation speed is 4000 r / min, and the centrifugation time is 10 min.

Images