A method for extracting or isolating salvianic acid and / or tanshinone from salvia miltiorrhiza

Abstract

The application provides a method for extracting or separating Danshensuan and / or Danshenshuan from Danshen, comprising the following steps: adding a eutectic solvent cellulase system into Danshen powder, extracting, centrifuging, and then extracting or separating the Danshensuan and / or Danshenshuan in situ; the eutectic solvent cellulase system comprises a eutectic solvent, cellulase and a H3C6H5O7 / Na3C6H5O7 buffer solution; the eutectic solvent is composed of a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond donor is levulinic acid or 1,4-butanediol, the hydrogen bond acceptor is choline chloride, L-proline or triethylbenzylammonium chloride, and the molar ratio of the hydrogen bond donor and the hydrogen bond acceptor is 3:1-1:3; the system is sensitive to pH response, and high-efficiency separation of two substances can be realized by adjusting pH, and the recovery rates of target components are all above 80%. The method is suitable for comprehensive utilization and industrialized separation of multi-component active substances in natural medicines.

Description

Technical Field

[0001] This invention relates to a method for extracting or separating tanshinone and / or tanshinone from tanshinone, belonging to the field of natural product active ingredient extraction and separation technology. Background Technology

[0002] Deep eutectic solvents (DES), as a new generation of green solvents, have attracted widespread attention due to their excellent physicochemical properties. Their advantages, such as non-flammability, good thermal stability, low toxicity, simple preparation, and low raw material cost, have shown great potential in many fields. Compared with water and traditional organic solvents, DESs have stronger solubility for both hydrophilic and hydrophobic compounds. Furthermore, DESs exhibit good biocompatibility. DESs are eutectic mixtures composed of hydrogen-bond donors (HBDs) and hydrogen-bond acceptors (HBAs) in a specific stoichiometric ratio.

[0003] Danshen, a traditional Chinese medicine, primarily contains hydrophilic acids (such as tanshinone) and lipophilic tanshinone, which are significantly different and cannot be simultaneously and efficiently extracted and separated using conventional solvents. Traditional methods, such as hot reflux extraction with water, ethanol, or methanol, and ultrasonic-assisted extraction, not only consume large amounts of solvent and impose heavy environmental burdens, but also involve cumbersome and inefficient separation and adsorption processes. Furthermore, prolonged high-temperature treatment can easily lead to the degradation of heat-sensitive active ingredients, hindering the industrial-scale processing and utilization of Danshen. Therefore, there is an urgent need to develop green, sustainable, and efficient separation strategies. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a method for extracting or separating tanshinone and / or tanshinone from tanshinone, so as to solve the problems of low extraction efficiency, large amount of organic solvent used, and inability to achieve simultaneous and efficient extraction and separation of polar and non-polar components in the prior art.

[0005] Technical Solution: This invention provides a method for extracting or separating tanshinone and / or tanshinone from Salvia miltiorrhiza, comprising the following steps: adding a eutectic solvent cellulase system to Salvia miltiorrhiza powder, extracting, centrifuging, and performing chromatographic analysis to extract or separate the tanshinone and / or tanshinone in situ; the eutectic solvent cellulase system comprises a eutectic solvent, cellulase, and an H3C6H5O7 / Na3C6H5O7 buffer solution; the eutectic solvent is composed of a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond donor is levulinic acid or 1,4-butanediol; the hydrogen bond acceptor is choline chloride, L-proline, or triethylbenzylammonium chloride; and the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 3:1 to 1:3.

[0006] Preferably, the hydrogen bond donor is levulinic acid (Lac); the hydrogen bond acceptor is choline chloride (CC); and the molar ratio of choline chloride to levulinic acid is 2:1 to 1:2.

[0007] Preferably, the molar ratio of choline chloride to acetylpropionic acid is 1:1 to 1:2.

[0008] More preferably, the molar ratio of choline chloride to acetylpropionic acid is 2:1.

[0009] The liquid-to-solid ratio of the DES enzyme system to the danshen powder is 5:1-40:1 (mL / g).

[0010] Preferably, the liquid-to-solid ratio of the DES enzyme system to the danshen powder is 20:1 (mL / g).

[0011] The water content of the eutectic solvent cellulase system is 10% to 50%.

[0012] The extraction time is 10-60 minutes; the extraction temperature is 20-60℃.

[0013] Preferably, the extraction time is 30-40 min; the extraction temperature is 30-40℃.

[0014] The volume ratio of the eutectic solvent to the H3C6H5O7 / Na3C6H5O7 buffer solution is 60-80:100; the mass ratio of the cellulase in the eutectic solvent cellulase system is 1-4 mg / g.

[0015] The pH value of the H3C6H5O7 / Na3C6H5O7 buffer solution is 3 to 7.

[0016] Preferably, the pH of the H3C6H5O7 / Na3C6H5O7 buffer solution is 4-6.

[0017] More preferably, the pH of the H3C6H5O7 / Na3C6H5O7 buffer solution is 4.8.

[0018] The content of the H3C6H5O7 / Na3C6H5O7 buffer solution is 20-40%.

[0019] Preferably, the content of the H3C6H5O7 / Na3C6H5O7 buffer solution is 30%.

[0020] The concentration of the cellulase is 0.5~4 mg / mL.

[0021] Preferably, the concentration of the cellulase is 2.0 mg / mL.

[0022] The tanshinone includes tanshinone (DSS) and / or tanshinone B (SAB); the tanshinone includes dihydrotanshinone I (DHT), cryptotanshinone (CTS) and / or tanshinone IIA (TIIA).

[0023] The method further includes adjusting the pH of the extracted system to 4.5-5.1, so that the system forms two phases: a DES phase and an aqueous phase. Tanshinone components are enriched in the upper DES phase, and tanshinone components are enriched in the lower aqueous phase.

[0024] Preferably, the pH value is adjusted to 4.8.

[0025] This invention also provides the application of a eutectic solvent cellulase system in enhancing the neuroprotective bioactivity of Salvia miltiorrhiza. The eutectic solvent cellulase system comprises a eutectic solvent, cellulase, and an H3C6H5O7 / Na3C6H5O7 buffer solution. The eutectic solvent consists of a hydrogen bond donor and a hydrogen bond acceptor. The hydrogen bond donor is levulinic acid, and the hydrogen bond acceptor is choline chloride. The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 3:1 to 1:3.

[0026] This invention also provides the application of a eutectic solvent cellulase system in enhancing the antioxidant activity of Salvia miltiorrhiza. The eutectic solvent cellulase system comprises a eutectic solvent, cellulase, and an H3C6H5O7 / Na3C6H5O7 buffer solution. The eutectic solvent is composed of a hydrogen bond donor and a hydrogen bond acceptor. The hydrogen bond donor is levulinic acid, and the hydrogen bond acceptor is choline chloride. The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 3:1 to 1:3.

[0027] This invention utilizes the COSMO-SAC machine learning model to efficiently screen the optimal solvent CC / Lac from a variety of candidate DESs using the solubility coefficient and activity coefficient of the target compound as indicators.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention uses green, low-toxicity, biodegradable and recyclable DES to replace traditional organic solvents, which significantly reduces environmental and safety risks; 2. It demonstrates the simultaneous and efficient extraction of tanshinone and tanshinone with huge polarity differences, and the extraction efficiency is significantly higher than that of conventional solvents such as methanol and ethanol. The optimized CC / Lac cellulase system achieves a total extraction rate of 133.56 mg / g, which is more than twice that of traditional methanol extraction. 3. Utilizing the pH-responsive characteristics of the DES enzyme system, phase separation can be induced simply by adjusting the pH value, achieving in-situ efficient separation of highly polar tanshinone and low-polarity tanshinone in one step. The recovery rate of all target compounds can reach over 80%, greatly simplifying the operation unit and improving separation efficiency. 4. This DES enzyme system has good recyclability and cycling stability. After five cycles, the extraction efficiency can still be maintained at over 85% of the initial efficiency, reducing production costs and showing broad industrial application prospects. 5. The extracted tanshinone extract exhibits significant antioxidant activity and neuroprotective effects, with efficacy superior to traditional solvent extracts. Attached Figure Description

[0029] Figure 1 Solubility (A) and activity coefficient (B) of different DESs predicted based on the COSMO-SAC model for DSS, SAB, DHT, CTS and TIIA. Figure 2 High-performance liquid chromatography (HPLC) of a representative tanshinone extract (A) and a mixture of five standards (B), and chemical structures of five control compounds (DSS, SAB, DHT, CTS and TIIA) (C). Figure 3 The total extraction rate of five analytes (DSS, SAB, DHT, CTS, and TIIA) from Salvia miltiorrhiza was determined using eight of the most effective DES strains selected from screening: (A) absence of cellulase; (B) presence of cellulase. Figure 4 The effects of various extraction parameters on the extraction content of five analytes (DSS, SAB, DHT, CTS, and TIIA) were investigated: (A) the molar ratio of choline chloride to levulinic acid; (B) the extraction time. Figure 5 The effects of various extraction parameters on the extraction content of five analytes (DSS, SAB, DHT, CTS, and TIIA) were investigated: (A) extraction temperature; (B) solid-liquid ratio of choline chloride and levulinic acid. Figure 6 The effects of various extraction parameters on the extraction content of five analytes (DSS, SAB, DHT, CTS, and TIIA) were investigated: (A) buffer pH; (B) cellulase concentration; (C) water content. Figure 7 The three-dimensional response surface plot shows the interactions between significant variables; Figure 8 The effects of different extraction methods (A) and different solvents (B) on the extraction efficiency of five analytes (DSS, SAB, DHT, CTS and TIIA) in Salvia miltiorrhiza were investigated. Figure 9 The antioxidant activity and neuroprotective activity of different tanshinone extracts were evaluated by DPPH (A) and ABTS (B) assays (C). Figure 10 To separate tanshinone and tanshinone from Salvia miltiorrhiza in situ using a pH-responsive CC / Lac matrix cellulase system: (A) Effect of pH on the phase transition behavior of the CC / Lac phase (red line: two-phase system; blue line: single-phase solution); (B) High-performance liquid chromatography (HPLC): (a) total extract, (b) aqueous phase (lower layer), (c) CC / Lac phase (upper layer), where peaks 1-5 correspond to DSS, SAB, DHT, CTS, and TIIA, respectively; (C) Separation and recovery rates of the five target analytes under different pH conditions; Figure 11 To assess the recyclability of the optimized CC / Lac cellulase system: (A) FT-IR spectra of CC / Lac after 0–5 extraction cycles (DES-C0 to DES-C5); (B) extraction yields of five analytes (DSS, SAB, DHT, CTS, and TIIA) over five consecutive cycles. Figure 12 (A) FT-IR spectra of CC, Lac, CC / Lac, and CC / Lac with added cellulase; (B) Representative SEM images: (a) raw Salvia miltiorrhiza powder; (b) powder extracted with methanol; (c) powder extracted with ethanol; (d) powder extracted using the CC / Lac cellulase system. Figure 13 The distribution of ESP on the surfaces of different compounds: (A) BDO; (B) TBAC; (C) DSS; (D) SAB; (E) DHT; (F) CTS; and (G) TIIA; Figure 14 Molecular dynamics simulations for five target analytes in the CC / Lac system: (A) snapshots of solvation spatial distribution at 0 ns and 100 ns; (B) changes in analyte-solvent interaction energy over time using the DSS model; (C) dynamic stability of intermolecular hydrogen bonds between CC and Lac; (D) average number of hydrogen bonds between analytes and solvent. Figure 15To assess the environmental friendliness of the ultrasonic-DES-enzyme extraction method using AGREE and GAPI evaluation tools. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0031] 1. Experimental materials and reagents Salvia miltiorrhiza was purchased from Yangzhou Municipal Hospital of Traditional Chinese Medicine (Yangzhou, Jiangsu Province, China) in March 2025. The herb was ground into powder using a commercial grinder and sieved through a 60-mesh sieve before use. Tanshinone B (SAB), tanshinone (DSS), tanshinone IIA (TIIA), cryptotanshinone (CTS), and dihydrotanshinone I (DHT) were provided by Chengdu Pusi Biotechnology Co., Ltd. Betaine (Bet), L-carboxylic acid (Lac), 1,2,4-butanetriol (BT), choline chloride (CC), L-proline (L-Pro), triethylphenylammonium chloride (TEBAC), glycerol (Gly), and urea (Ure) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Cellulase was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Methanol, acetonitrile, and formic acid were provided by ROE Scientific Inc. (Newark, Delaware, USA).

[0032] 2. Extraction of tanshinone and tanshinone from Salvia miltiorrhiza To preliminarily screen the optimal eutectic solvent (DES), 1.0 g of Salvia miltiorrhiza powder was accurately weighed and then 20 mL of DES aqueous solution (water to DES volume ratio of 3:7) was added. The mixture was then ultrasonically extracted at 30 °C (400 W, 40 kHz) for 60 minutes. Finally, the sample was centrifuged at 13000 rpm for 10 minutes, and the supernatant was injected into a high-performance liquid chromatography (HPLC) system for analysis.

[0033] In addition, cellulase was added to determine the optimal DES-cellulase extraction solvent. This DES-based cellulase system consisted of 30% H3C6H5O7 / Na3C6H5O7 buffer solution and 70% DES, with a cellulase concentration of 2 mg / mL. The extraction process and quantitative analysis were completely consistent with the methods used in the preliminary screening.

[0034] Example 1: Methodological Investigation and Chromatographic Conditions 1. Chromatographic conditions Quantitative analysis was performed using a Waters 2695 system equipped with a binary pump, VWD detector, and column incubator. Chromatographic separation was performed using a Waters Symmetry C18 column (4.6 × 250 mm, 5 μm). The mobile phase consisted of solvent A (water containing 0.1% (v / v) formic acid) and solvent B (acetonitrile). The gradient elution program was set as follows: 0–8 min, 2–3% B; 8–10 min, 3–5% B; 10–16 min, 5–50% B; 16–21 min, 50–60% B; 21–36 min, 60–70% B; 36–44 min, 2–3% B; 44–46 min, 80–100% B; 46–47 min, 100%–2% B; 47–54 min, 2% B. The flow rate, injection volume, column temperature, and detection wavelength were set to 0.8 mL / min, 5 μL, 25 ℃, and 280 nm, respectively.

[0035] 2. Methodological Validation Under the chromatographic conditions described above, tanshinone (DSS), tanshinone B (SAB), dihydrotanshinone I (DHT), cryptotanshinone (CTS), and tanshinone IIA (TIIA) were quantitatively analyzed. A series of standard solutions were prepared, and linear regression was performed on peak area against concentration to obtain standard curves for each compound. The precision, repeatability, stability, and recovery of the method were investigated. The results (Table 1) show that the method exhibits good linearity (R²). 2 It exhibits high precision (RSD < 3.13%), good repeatability (RSD < 2.23%), strong stability (RSD < 1.98%), and a recovery rate (95.16%-100.11%) that meets the requirements, making it suitable for subsequent quantitative analysis.

[0036] Table 1. Calibration curves, limits of quantitation (LOQ), limits of detection (LOD), repeatability, precision, and recovery rates for DSS, SAB, DHT, CTS, and TIIA.

[0037] Example 2: DESs Screening and Experimental Validation Based on the COSMO-SAC Model 1. Calculations were performed using the Dmol3 module in Materials Studio 7.0 (Accelrys, USA). Tanshinone B, tanshinone, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I were used as model solutes in the simulation. Eutectic solvents (DESs) were treated as single molecular entities, and structure and energy optimizations were performed to generate their σ-profiles. Furthermore, the interaction energies between the five analytes and the DESs were calculated. σ-profile analysis was further performed using the COSMO-SAC model. After final optimization, COSMO data for these analytes were obtained. Subsequently, polarity information for each analyte was generated. Finally, the solubility and infinite dilution activity coefficients of the target analytes in various types of DESs (Table 2) were obtained, which can be used to predict extraction efficiency.

[0038] Table 2. Composition of DES used for COSMO-SAC prediction

[0039] The results showed that ( Figure 1 The DES using choline chloride (CC) and triethylbenzylammonium chloride (TEBAC) as hydrogen bond acceptors (HBAs) exhibited high solubility and low activity coefficients for five target compounds, predicting optimal extraction efficiency. Based on the feasibility of practical operation, eight DES based on different HBAs were finally selected for experimental verification, and their compositions are shown in Table 3.

[0040] Table 3 Composition of 8 Selected DES

[0041] 2. Preparation and preliminary screening of DES Based on the prediction results of the COSMO-SAC model, eight DES with the highest solubility coefficients were selected for further validation. The mixture of HBA and HBS from Table 3 was placed in a flask and heated to 80°C with continuous magnetic stirring until a homogeneous and transparent solution was obtained. To reduce viscosity, 30% deionized water was added to each prepared DES. 1.0 g of Danshen powder was accurately weighed and then 20 mL of DES aqueous solution (DES to water volume ratio of 3:7) was added. Subsequently, the mixture was ultrasonically extracted at 30°C (400 W, 40 kHz) for 60 minutes. Finally, the sample was centrifuged at 13000 rpm for 10 minutes, and the supernatant was injected into a high-performance liquid chromatography system for analysis under the above conditions. The results are as follows: Figure 3 As shown in Figure A, without the addition of cellulase, the total extraction rates of the three DES types—CC / Lac, L-Pro / Lac, and TEBAC / BDO—were significantly better than the methanol control. Among them, the CC / Lac system had the highest total extraction efficiency, which was approximately twice that of traditional methanol extraction.

[0042] 3. Determination of the optimal DES enzyme system In the above eight DES systems, cellulase (final concentration 2 mg / mL) and 30% H3C6H5O7 / Na3C6H5O7 buffer (pH 5) were added respectively to construct DES enzyme systems for extraction experiments. The results are as follows: Figure 3 As shown in Figure B, the overall extraction efficiency of the CC / Lac and cellulase combination for the five target compounds was still significantly better than other DES enzyme systems, indicating that CC / Lac and cellulase have excellent biocompatibility and synergistic effects. Therefore, CC / Lac based on choline chloride and levulinic acid was ultimately determined as the optimal DES, and the system formed by CC / Lac and cellulase (CC / Lac-cellulase system) is the most suitable DES enzyme system.

[0043] Example 3: Optimization of Key Extraction Process Parameters 1. Single-factor experiment Single-factor experiments were designed using a selected DES (choline chloride / levulinic acid) as the extraction solvent to investigate the effects of multiple parameters on the extraction yields of tanshinone and tanshinone from *Salvia miltiorrhiza*. These parameters included the molar ratio of choline chloride to levulinic acid (3:1, 2:1, 1:1, 1:2, and 1:3), extraction time (10, 20, 30, 40, 50, and 60 min), extraction temperature (20, 30, 40, 50, and 60 °C), solid-liquid ratio of *Salvia miltiorrhiza* powder to the DES enzyme system (1:5, 1:10, 1:20, 1:30, and 1:40), moisture content (10%, 20%, 30%, 40%, and 50%), buffer pH (3, 4, 5, 6, and 7), and cellulase concentration (0.5, 1, 2, 3, and 4 mg / mL). Table 4 summarizes the range of extraction parameters used in this invention.

[0044] Table 4 Single-factor experimental design

[0045] like Figure 4 As shown in Figure A, the total extraction rates of salvianolic acid and tanshinone reached a maximum of 116.52 ± 4.78 mg / g when the molar ratio of CC to Lac changed from 3:1 to 1:2. The initial increase in the solubility of these analytes was attributed to the presence of strong hydrogen-bonding donors at the lower ratio, which promoted solute dissolution. However, excess Lac induced intramolecular hydrogen bonding with CC, leading to disruption of the eutectic network and a decrease in extraction efficiency. Therefore, the optimal molar ratio was determined to be 1:2.

[0046] Figure 4B shows that the extraction rates of the five analytes significantly increased when the extraction time was extended from 10 minutes to 30 minutes. However, when the extraction time was further extended to 50 minutes, the overall extraction rate decreased. This decrease was attributed to the degradation of the target analytes caused by prolonged ultrasonic and heat treatment. The results indicate that 30 minutes is the optimal extraction time, effectively disrupting cell walls to improve extraction efficiency while preventing the degradation of target components.

[0047] Temperature significantly affects the extraction efficiency of natural products by altering the density and viscosity of the DES enzyme system. For example... Figure 5 As shown in Figure A, within the temperature range of 20-60°C, the total extraction rate of the five analytes initially increased and then decreased with increasing temperature. The extraction rate increased significantly at 40°C. Further heating led to a decrease in the extraction rate, with the lowest yield observed at 60°C. Therefore, 40°C was determined to be the optimal extraction temperature.

[0048] Sufficient solvent volume facilitates full contact with the plant matrix and promotes dissolution, while over-diluted systems may increase the co-extraction of impurities. Figure 5 Figure B shows that the total extraction rate of the five analytes was positively correlated with the liquid-to-solid ratio, reaching a peak at 20 mL / g. However, the total extraction rate decreased slightly after further increasing the liquid-to-solid ratio. Therefore, a liquid-to-solid ratio of 20:1 mL / g was determined to be the optimal parameter.

[0049] like Figure 6 As shown in Figure A, the total extraction rates of SAB, DSS, TIIA, CTS, and DHT differed significantly across the tested pH range, reaching a maximum at pH 5.0. When the pH increased from 5 to 7, the extraction rates of all five components decreased significantly. These results indicate that excessively acidic or alkaline conditions lead to enzyme inactivation. Enzyme inactivation weakens the enzyme's ability to degrade plant cell walls, thereby reducing the extraction rate. Therefore, pH 5.0 was selected as the optimal pH value.

[0050] Sufficient enzyme levels can promote plant cell wall degradation and the release of active compounds. When the enzyme concentration increased from 0.5 mg / mL to 2.0 mg / mL, the total extraction rates of SAB, DSS, TIIA, CTS, and DHT significantly increased. Figure 6 B). However, no significant difference in extraction rate was found when the enzyme concentration exceeded 2.0 mg / mL. Therefore, the optimal enzyme concentration is 2.0 mg / mL.

[0051] Adding a small amount of water to the DES enzyme system is necessary to reduce its viscosity and enhance mass transfer, but excessive water will disrupt the hydrogen bond network within the solvent, reducing extraction efficiency. This invention uses a CC / Lac mixture with a water content of 10% to 50% (w / w) to extract tanshinone and salvianolic acid from *Salvia miltiorrhiza*. Figure 6 As shown in C, the maximum extraction rates of the five analytes were obtained at a water content of 30%.

[0052] In summary, the optimal extraction results are achieved when the molar ratio is 2:1, the extraction time is 30 min, the temperature is 40 ℃, the liquid-to-solid ratio is 20:1 mL / g, the buffer pH is 5, the enzyme concentration is 2.0 mg / mL, and the water content is 30%.

[0053] 2. Response Surface Methodology Optimization Within the framework of response surface methodology (RSM), a Box-Behnken design (BBD) with three factors, each with three levels, was employed to optimize the extraction parameters. Extraction temperature (A), buffer pH (B), and water content of the DES enzyme system (C) were used as independent variables, while the total extraction rate (Y) was defined as the unique response variable.

[0054] Table 5 Total extractable yield of target analytes under 17 different conditions in the Box-Behnken design.

[0055] Table 6. Analysis of variance results of the regression equation using the response surface methodology (RSM).

[0056] The experimental results are summarized in Table 5, and the analysis of variance (ANOVA) for the regression model is detailed in Table 6. The ANOVA results show that the regression model has high significance (…). p <0.0001), and the model fit term is not statistically significant ( p = 0.1287), indicating a good model fit and reliable predictive ability. All linear terms (A, B, and C) and interaction terms (AB, BC, and AC) were statistically significant, indicating that these factors had a significant impact on the extraction response. Based on F-values ​​and p-values, the relative influence of each factor on extraction efficiency was ranked as follows: buffer pH > extraction temperature > water content of the DES enzyme system. The regression equations for these three variables on the extraction yield are as follows: Y=6.43+0.0038×A-0.0039×B-0.0032×C+0.0041×AB-0.0077×AC-0.0001×BC-0.0488×A 2 -0.0585×B 2 -0.0591×C 2 .

[0057] Three-dimensional (3D) response surface plot Figure 7The interaction effects of independent variables on the extraction rates of five analytes were demonstrated. With the increase of two variables, the extraction rate initially increased and then decreased, indicating that the synergistic effect among these three variables should be considered when optimizing the extraction rate. Generally, elliptical contour lines indicate a significant interaction effect between the two factors. Compared to the AB and AC interactions, the BC interaction has a steeper slope and greater ellipticity, indicating that the interaction between buffer pH and the water content of the DES enzyme system significantly affects the extraction yield. The fitted equations show that the optimized extraction parameters are: extraction temperature 30.2℃, buffer pH 4.8, and DES enzyme system water content 30.1%, under which the total extraction rate is 132.79±3.53 mg / g. Under these optimal conditions, the total extraction rate of the target analyte is 133.56±4.18 mg / g, close to the expected extraction rate. These results demonstrate that the developed RSM model is reliable for optimizing the extraction parameters of active ingredients in natural products.

[0058] Example 4: Comparative Evaluation with Traditional Methods and Solvents The optimized CC / Lac cellulase system extraction method was compared with the traditional reflux extraction method and different solvents (methanol, acetonitrile, ethanol, and water). The amounts of different solvents used were the same as those in the optimized CC / Lac cellulase system, i.e., a liquid-to-solid ratio of 20:1. The extraction method was the same as CC / Lac extraction: an appropriate amount of Salvia miltiorrhiza powder was accurately weighed, and different solvents with a liquid-to-solid ratio of 20:1 mL / g were added. The mixture was ultrasonically extracted for 30 min. Other parameters were the same as for the DES enzyme system extraction. After centrifugation at 13000 rpm / min, the supernatant was collected and analyzed by HPLC. The traditional reflux extraction method involved accurately weighing a certain amount of Salvia miltiorrhiza powder and placing it in a round-bottom flask. Extraction solvent was added at a material-to-liquid ratio of 1:20 g / mL. The flask was connected to a reflux condenser and placed in a constant-temperature water bath. The temperature was set to a state of slight boiling, and the mixture was heated and refluxed for 1.5 hours. After extraction, the mixture was filtered under reduced pressure while hot, and the filtrate was collected. To ensure complete extraction, the residue could be extracted once more under the same conditions. The two extracts were combined, centrifuged at 13000 rpm for 10 min, and the supernatant was collected for HPLC analysis. The results are as follows: Figure 8 As shown, the extraction efficiency of the optimized method of this invention is significantly higher than that of traditional methods. Specifically, its total extraction rate (approximately 132.69 mg / g) is 4.83 times that of water reflux extraction and 2.51 times that of methanol reflux extraction. Compared with ultrasound-assisted extraction using different solvents, the extraction efficiency is ranked as follows: CC / Lac cellulase system > methanol > acetonitrile > ethanol > water. This demonstrates that the method of this invention has an absolute advantage in extraction efficiency.

[0059] Example 5: Pharmacodynamic evaluation of the extract 1. Evaluation of Antioxidant Activity: Bioactive compounds in *Salvia miltiorrhiza* alleviate cellular oxidative damage by directly scavenging free radicals. The antioxidant activity of *Salvia miltiorrhiza* extracts extracted with different solvents (DES enzyme system, methanol, acetonitrile, water, and ethanol) was evaluated using DPPH and ABTS scavenging assays. Specifically, equal volumes of various *Salvia miltiorrhiza* extracts were dissolved before analysis. For the test samples, 5 μL of crude extract was mixed with 195 μL of ABTS or DPPH working solution. For the blank control, 5 μL of ethanol was mixed with 195 μL of the corresponding ABTS or DPPH working solution. The mixtures were vigorously shaken and incubated at room temperature in the dark for 30 minutes. Finally, the absorbance was measured at 517 nm for DPPH assays and at 734 nm for ABTS assays. The results of both DPPH and ABTS assays showed that the antioxidant activity increased in a concentration-dependent manner, and the CC / Lac-based cellulase system showed significantly higher efficacy than the conventional solvents. Figure 9 A and Figure 9 B). The ranking of free radical scavenging efficiency is consistent in both assays: CC / Lac > methanol > ethanol > acetonitrile > water.

[0060] 2. Evaluation of Neuroprotective Effect: Danshen (Salvia miltiorrhiza) has a significant neuroprotective effect against cerebral ischemia, thereby reducing neuronal damage and improving neurological prognosis. HT22 cells cultured in complete DMEM were washed with Earle's balanced salt solution and subjected to oxygen-glucose deprivation (OGD; 5% CO2, 94% N2, 1% O2) for 4 hours. During the subsequent 24-hour reoxygenation phase, cells were treated with complete medium containing different concentrations (200 ug / mL, 100 ug / mL) of Danshen extract (extracted with DES enzyme system or methanol). Cells were filtered through a 0.45 μm microporous membrane before administration. Cells cultured in serum-free medium not exposed to OGD / R served as a control group. Cell viability was assessed by incubating cells with Calcein-AM / PI solution at 37°C for 30 minutes, followed by fluorescence microscopy analysis. Figure 9As shown in Figure C, both the low-dose and high-dose groups of *Salvia miltiorrhiza* extract extracted using methanol and the optimal DES enzyme system exhibited dose-dependent neuroprotective effects. Specifically, DS-DES-H and DS-DES-L represent high-dose (200 ug / mL) and low-dose (100 ug / mL) *Salvia miltiorrhiza* extracts extracted using the DES enzyme system, respectively; DS-MET-H and DS-MET-L represent high-dose (200 ug / mL) and low-dose (100 ug / mL) *Salvia miltiorrhiza* extracts extracted using methanol, respectively. Notably, cells treated with the *Salvia miltiorrhiza* extract extracted using the optimal DES enzyme system showed significantly lower red fluorescence intensity than the methanol-extracted group. This indicates a significant reduction in neuronal apoptosis, thus confirming that the optimized DES enzyme system enhances the neuroprotective bioactivity of *Salvia miltiorrhiza* extract.

[0061] Example 6: pH-responsive in-situ separation, solvent recycling, and mechanism investigation 1. pH-responsive in-situ separation: Under optimal conditions, the pH of the CC / Lac extract is changed by adjusting different amounts of H3C6H5O7 / Na3C6H5O7 buffer solution (4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or 5.1). For example... Figure 10 As shown in Figure A, the system exhibits a clear pH-dependent phase transition behavior. By examining the separation efficiency at different pH values ​​(4.5-5.1), pH 4.8 was determined to be the optimal separation condition. At pH 4.8, the system clearly separates into two phases: lipophilic tanshinones (DHT, CTS, TIIA) are highly enriched in the upper CC / Lac phase, while hydrophilic tanshinones (DSS, SAB) are mainly distributed in the lower aqueous phase. Figure 10 C). Under these conditions, the separation and recovery rates of all five target analytes exceeded 80% ( Figure 2 and Figure 10 (B) Successfully achieved efficient in-situ separation of two types of compounds.

[0062] 2. Recoverability Assessment of the CC / Lac-Based Cellulase System: The cyclic stability and recoverability of the CC / Lac-based cellulase system were assessed by evaluating changes in Fourier transform infrared (FT-IR) spectroscopy and the yields of tanshinone and salvianolic acid obtained from the extract. The recoverability of the optimal DES was evaluated by performing five consecutive extraction cycles under the same conditions. The physicochemical properties of the recoverable DES were characterized using FT-IR spectroscopy. After each extraction cycle, the recoveries of tanshinone and salvianolic acid in the aqueous phase and the DES phase were determined, respectively. FT-IR analysis showed that the system exhibited good structural stability during consecutive extraction cycles, with no significant shift in characteristic absorption peaks and no significant change in peak intensity. Figure 11A). This indicates that the basic chemical structure of the CC / Lac system and its inherent hydrogen bond network maintain structural integrity during repeated use. Figure 11 B demonstrates the extraction efficiency over five consecutive recovery cycles. After the first recovery cycle (DES-C1), the total extraction yield of the five target analytes was 131.14 mg / g, a slight decrease of 1.63% compared to the initial extraction (DES-C0, 133.31 mg / g). Although the extraction yield gradually decreased with increasing cycle number, the system exhibited excellent stability. After five cycles (DES-C5), the total extraction yield remained at 117.79 mg / g, retaining 88.36% of the initial extraction capacity. This good recyclability not only improves the utilization efficiency of the CC / Lac-based cellulase system but also reduces extraction costs, providing a cost-effective solution for industrial applications.

[0063] 3. Investigation into the extraction mechanism: 3.1 Fourier transform infrared spectroscopy characterization analysis: such as Figure 12 As shown in Figure A, the characteristic absorption bands of the CC, Lac, CC / LA, and CC / Lac / cellulase systems were analyzed to elucidate the hydrogen bond network underlying the extraction mechanism. (3244-3248 cm⁻¹) -1 The broad OH stretching band within the range is attributed to the hydroxyl groups in CC and Lac, showing a slight red shift (3248.01 cm⁻¹) in the CC / Lac system. -1 This indicates that intermolecular hydrogen bonds formed between CC and Lac. After the addition of cellulase, this peak shifted to 3246.16 cm⁻¹. -1 This indicates that the cellulase integrated into the hydrogen bond network of DES without disrupting its basic structure. The C=O stretching vibration peak of the carboxyl group in Lac is located at 1626-1628 cm⁻¹. -1 Furthermore, the consistency across all spectra confirms the preservation of the chemical integrity of the DES matrix. CH bending peaks (~1455-1459 cm⁻¹) -1 CO stretching peak (~1293.94 cm⁻¹) -1 ) and CN stretching peak (~1106-1108 cm⁻¹) -1 The displacement of the cells is negligible, thus confirming the structural stability of the CC / Lac system. These results indicate that the CC / Lac / cellulase system possesses a significant hydrogen bond network.

[0064] 3.2 Scanning Electron Microscopy (SEM) Analysis: Scanning electron microscopy (SEM) analysis provides deeper mechanistic insights into the efficient extraction of bioactive components from Salvia miltiorrhiza. For example... Figure 12As shown in B(a), the surface morphology of untreated Salvia miltiorrhiza powder is relatively dense, intact, and non-porous. After extraction, the powder exhibits varying degrees of morphological changes. After ethanol treatment ( Figure 12 B(b)) and methanol treatment ( Figure 12 Samples B(c) all exhibited slight surface erosion and formed shallow, dispersed pores. Notably, the powder extracted using the DES enzyme system showed more significant microstructural disruption, characterized by pronounced cavitation, extensive tissue fragmentation, and deeper porous channels. Figure 12 B(d)). These obvious structural changes facilitate the penetration of DES and the subsequent intracellular release of the target compound.

[0065] 3.3 Electrostatic Potential (ESP) and Molecular Dynamics Analysis: Analysis of Electrostatic Potential (ESP) at Molecular Surfaces ( Figure 13 This reveals the charge distribution characteristics between the solvent and the analyte.

[0066] Table 7. Number of hydrogen bonds in analytes determined using the CC / Lac system

[0067] Molecular dynamics (MD) simulations (Table 7 and Figure 14 The results show that throughout the simulation, lipophilic tanshinone diffuses stably in the solvent via hydrophobic interactions, while hydrophilic phenolic acids undergo significant diffusion and extend their molecular conformation, exposing a large number of hydroxyl and carboxyl groups, forming a broad and stable hydrogen bond network with the solvent. Quantitative analysis of the interaction energies indicates that Coulombic (electrostatic) interactions are the main thermodynamic driving force for the dissolution of phenolic acids.

[0068] Example 7: Environmental impact assessment of the developed CC / Lac matrix cellulase system The environmental friendliness of the developed CC / Lac-based cellulase system was comprehensively evaluated using AGREE and GAPI assessment tools. Figure 15 As shown in Figure A, this method achieved an overall score of 0.77, indicating a high degree of adherence to the fundamental principles of Green Analytical Chemistry (GAC). The visualization is predominantly green, with only one orange area representing Criterion 8, a situation typically attributed to limited sample throughput and offline analysis techniques. Notably, no red areas appear in the figure, highlighting the extremely low level of environmental and operational risk of the developed method. The GAPI assessment further confirms these results, with its color-coded spectrum consisting mainly of green and yellow areas, indicating compliance with key parameters such as low waste generation, minimal operator exposure risk, and effective solvent management. Figure 15(B) Furthermore, the assessment yielded a high green score of 88, highlighting the system's superior environmental performance. Compared to traditional extraction techniques, this CC / Lac-based cellulase system operates efficiently under mild conditions. Overall, the results demonstrate that the developed system not only offers high extraction efficiency but also provides a highly sustainable, low-energy, and environmentally friendly alternative for the extraction of natural products.

Claims

1. A method for extracting or separating tanshinone and / or tanshinone from Salvia miltiorrhiza, characterized in that, Includes the following steps: Adding a eutectic solvent cellulase system to tanshinone powder, followed by extraction, centrifugation, and chromatographic analysis, allows for the extraction or in-situ separation of tanshinone and / or tanshinone. The eutectic solvent cellulase system comprises a eutectic solvent, cellulase, and an H3C6H5O7 / Na3C6H5O7 buffer solution. The eutectic solvent consists of a hydrogen bond donor and a hydrogen bond acceptor; the hydrogen bond donor is levulinic acid or 1,4-butanediol; the hydrogen bond acceptor is choline chloride, L-proline, or triethylbenzylammonium chloride; and the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 3:1 to 1:

3.

2. The method according to claim 1, characterized in that, The water content of the eutectic solvent cellulase system is 10%~50%.

3. The method according to claim 1, characterized in that, The extraction time is 10-60 minutes; the extraction temperature is 20-60℃.

4. The method according to claim 1, characterized in that, The solid-liquid ratio of the danshen powder to the eutectic solvent cellulase system is 1:5~40 (g / mL).

5. The method according to claim 1, characterized in that, The volume ratio of the eutectic solvent to the H3C6H5O7 / Na3C6H5O7 buffer solution is 60~80:100; the mass ratio of the cellulase in the eutectic solvent cellulase system is 1~4 mg / g.

6. The method according to claim 1, characterized in that, The pH value of the H3C6H5O7 / Na3C6H5O7 buffer solution is 3~7.

7. The method according to claim 1, characterized in that, The tanshinone includes tanshinone and / or tanshinone B; the tanshinone includes one or more of dihydrotanshinone I, cryptotanshinone, or tanshinone IIA.

8. The method according to claim 1, characterized in that, The method further includes: adjusting the pH of the extracted system to 4.5-5.1, so that the system forms two phases: a DES phase and an aqueous phase, with tanshinone components concentrated in the upper DES phase and tanshinone components concentrated in the lower aqueous phase.

9. The application of a eutectic solvent cellulase system in enhancing the neuroprotective bioactivity of *Salvia miltiorrhiza*, characterized in that... The eutectic solvent cellulase system comprises an eutectic solvent, cellulase, and an H3C6H5O7 / Na3C6H5O7 buffer solution; the eutectic solvent consists of a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond donor is levulinic acid and the hydrogen bond acceptor is choline chloride; the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 3:1 to 1:

3.

10. The application of a eutectic solvent cellulase system in enhancing the antioxidant activity of Salvia miltiorrhiza, characterized in that, The eutectic solvent cellulase system comprises an eutectic solvent, cellulase, and an H3C6H5O7 / Na3C6H5O7 buffer solution; the eutectic solvent consists of a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond donor is levulinic acid and the hydrogen bond acceptor is choline chloride; the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 3:1 to 1:3.

Images