Eutectic solvent extraction process of blueberry leaf phenolic substances

Abstract

The invention relates to the technical field of green extraction of natural products, and discloses a deep-eutectic solvent extraction process for phenolic substances in blueberry leaves. The process comprises the following steps: S1, raw material pretreatment, S2, preparation of a eutectic solvent, S3, preparation of a standard curve for determining the content of total phenols, S4, phenolic substance extraction: accurately weighing 0.200 + / -0.002 g of the standby Lackxiang blueberry leaf powder in the step S1, and fully mixing the Lackxiang blueberry leaf powder with the eutectic solvent prepared in the step S2, and S5, separation of an extracting solution and determination of the content of the total phenols. A single factor experiment is combined with a Box-Behnken response surface method, four key process parameters influencing the total phenol extraction amount are systematically optimized, a highly significant quadratic multiple regression model is established, an optimal process combination is determined, under the condition, the total phenol extraction amount of the Lackxia blueberry leaves is highly matched with a model predicted value, and the optimal process combination is determined. The extraction efficiency is far higher than that of some traditional methods.

Description

Technical Field

[0001] This invention relates to the field of green extraction technology of natural products, specifically a low-eutectic solvent extraction process for phenolic substances from blueberry leaves. Background Technology

[0002] Blueberry (Vaccinium spp.) leaves are a major byproduct of blueberry cultivation and processing, often discarded, resulting in resource waste. Studies have shown that blueberry leaves are rich in phenolic substances (including flavonoids, anthocyanins, and phenolic acids), which possess excellent antioxidant activity, endowing them with potential functions such as lowering blood lipids, anti-aging, anti-inflammation, and preventing cardiovascular diseases. Therefore, the efficient extraction and utilization of phenolic substances from blueberry leaves is of great significance for increasing the added value of the blueberry industry.

[0003] Currently, traditional solvents such as water, ethanol, or methanol are mostly used to extract phenolic compounds from plant materials. However, these methods have limitations in extraction efficiency, and organic solvents may be toxic or cause environmental pollution. Deep eutectic solvents (DESs), as a novel green solvent, have attracted widespread attention due to their advantages such as low toxicity, biodegradability, and simple preparation. In recent years, researchers have attempted to apply DESs to the extraction of active plant components, such as extracting chlorogenic acid from Artemisia argyi and polyphenols from Greek medicinal plants.

[0004] Existing technologies show that DESs are more efficient than traditional solvents in extracting certain active substances. However, when DESs are applied to extract phenolic substances from the leaves of a specific 'Lexie' blueberry variety, the extraction efficiency is significantly affected by the viscosity and polarity of the DESs themselves, as well as extraction process parameters (such as water content, solid-liquid ratio, number of extractions, and ultrasonic time). Existing technologies lack a systematically optimized DESs extraction process for this specific raw material, resulting in the total phenol extraction rate not reaching the optimal level, which limits the high-value development and utilization of this resource. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a low-eutectic solvent extraction process for phenolic substances from blueberry leaves, solving the problems of resource waste and inefficient extraction.

[0006] To achieve the above objectives, the present invention provides a low-eutectic solvent extraction process for phenolic substances from blueberry leaves, wherein the blueberry leaves are Lexi blueberry leaves, and the process includes the following steps:

[0007] S1. Raw material pretreatment: Freshly picked Lexi blueberry leaves are placed in a forced-air drying oven and baked at 45°C until constant weight. After being crushed, they are passed through an 80-mesh sieve and stored at -18°C for later use.

[0008] S2. Preparation of eutectic solvent: Choline chloride and glycerol are mixed in a 1:2 molar ratio and placed in a round-bottom flask. The mixture is stirred at 80°C for 8 hours in a constant-temperature magnetic stirrer. After stirring is completed and the mixture is cooled to room temperature, a colorless, stable and transparent eutectic solvent is obtained.

[0009] S3. Preparation of standard curve for determination of total phenol content: Using gallic acid as standard, plot the standard curve equation of gallic acid concentration-absorbance, specifically: y=14.214x+0.0282, R²=0.9953;

[0010] S4. Extraction of phenolic substances: Accurately weigh 0.200±0.002g of the Lexi blueberry leaf powder prepared in step S1, and mix it thoroughly with the eutectic solvent prepared in step S2. The water content of the eutectic solvent is 20%, and the material-liquid ratio is 1:50g / mL. Perform ultrasonic extraction twice, with each ultrasonic extraction lasting 50.00min.

[0011] S5. Separation of extract and determination of total phenol content: The mixture after ultrasonic extraction in step S4 was centrifuged at 8000 rpm for 10 min, and the supernatant was taken and diluted to 4 mL to obtain the extract. A certain amount of extract was diluted to obtain the test solution, and the total phenol content was determined according to the standard curve equation in step S3. The total phenol content was expressed as gallic acid equivalent per clexi blueberry leaf powder. At the same time, a blank experiment was performed using the corresponding eutectic solvent.

[0012] Preferably, the formula for calculating the total phenol content in step S5 is:

[0013]

[0014] In the formula:

[0015] The total phenol content in the sample solution is expressed in mg / g.

[0016] The total phenol content in the test solution is expressed in mg / mL.

[0017] The dilution factor for the test sample;

[0018] The total volume of the sample solution after dilution is expressed in mL.

[0019] The sample mass is expressed in grams (g).

[0020] Preferably, in step S1, the prepared Lexi blueberry leaf powder further includes a low-temperature plasma pretreatment step before mixing in step S4:

[0021] Low-temperature plasma with air medium was used to treat Lexi blueberry leaf powder for 2-5 minutes at a power of 10-30W.

[0022] Preferably, after taking the supernatant and adjusting the volume to obtain the extract in step S5, the step further includes an in-situ regeneration-ultrasonic activation step using a eutectic solvent:

[0023] The phenolic substances in the extract were separated from the eutectic solvent by low-temperature vacuum distillation at 40-50℃ and 0.08MPa. The recovered eutectic solvent was ultrasonically treated at 20-30kHz for 10min. The treated eutectic solvent was then used in the next step S4 to extract the Lexi blueberry leaf powder with the eutectic solvent.

[0024] Preferably, during the ultrasonic extraction process in step S4, a modified Fe3O4 magnetic adsorbent with surface-modified hydroxyl groups is also added simultaneously.

[0025] After ultrasonic extraction in step S4, the modified Fe3O4 magnetic adsorbent and the eutectic solvent system are first separated by an external magnetic field. Then, the enriched phenolic substances are eluted from the modified Fe3O4 magnetic adsorbent using a weakly polar eluent. The eluted modified Fe3O4 magnetic adsorbent is regenerated and reused for the adsorption of phenolic substances in the ultrasonic extraction process.

[0026] This invention provides a low-eutectic solvent extraction process for phenolic substances from blueberry leaves. It offers the following advantages:

[0027] 1. This invention combines single-factor experiments with Box-Behnken response surface methodology to systematically optimize four key process parameters affecting total phenol extraction. A highly significant quadratic multinomial regression model was established and the optimal process combination was determined. Under these conditions, the total phenol extraction yield of Lexi blueberry leaves is highly consistent with the model prediction, and the extraction efficiency is much higher than some traditional methods.

[0028] 2. This invention uses a eutectic solvent composed of choline chloride and glycerol as the extraction medium. This solvent has the characteristics of low toxicity and biodegradability, which is in line with the principles of green chemistry. The DESs in-situ regeneration-ultrasonic activation step can realize the efficient recovery and recycling of the solvent, significantly reducing production costs and environmental impact, and improving the sustainability of the process.

[0029] 3. This invention utilizes Fe3O4 magnetic adsorbent with surface-modified hydroxyl groups to selectively adsorb phenolic substances, and achieves rapid and efficient solid-liquid separation through an external magnetic field. Eluting is then carried out using a weakly polar eluent, achieving efficient enrichment and purification of phenolic substances and simplifying subsequent separation processes.

[0030] 4. This invention effectively disrupts the cell wall structure of leaves through physical means, increasing its permeability. This, combined with subsequent ultrasonic extraction of DESs, creates a synergistic effect, providing an effective way to further improve the dissolution efficiency of the target substance. Attached Figure Description

[0031] Figure 1 This is a graph showing the effect of water content on the total phenolic content of blueberry leaves in this invention.

[0032] Figure 2 This is a graph showing the effect of the material-to-liquid ratio on the total phenolic content of blueberry leaves in this invention.

[0033] Figure 3 This is a graph showing the effect of the number of extractions on the total phenolic content of blueberry leaves in this invention;

[0034] Figure 4 This is a graph showing the effect of ultrasound time on the total phenolic content of blueberry leaves in this invention;

[0035] Figure 5 The response surface and contour plots show the effect of water content and extraction times on total phenol content in this invention.

[0036] Figure 6 This is a response surface and contour plot showing the effect of moisture content and ultrasonic time on total phenol content in this invention. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example:

[0039] Please see the appendix Figure 1-6 This invention provides a process for extracting phenolic substances from blueberry leaves using a eutectic solvent, comprising:

[0040] I. Experimental Materials and Instruments

[0041] 1.1 Materials and Reagents

[0042] Raw materials: Mature leaves of Lexi blueberries that are growing well, free from pests and diseases, and without mechanical damage were randomly selected and collected in mid-October 2021 from the Lexi blueberry variety cultivated for 3 years at the Hunan Botanical Garden Blueberry Base.

[0043] Reagent: Folin-Ciocalteu (Hefei Bomei Biotechnology Co., Ltd.);

[0044] Gallic acid (Tianjin Guangfu Fine Chemical Research Institute);

[0045] Glycerol (Sinopharm Chemical Reagent Co., Ltd.);

[0046] Choline chloride (Sinopharm Chemical Reagent Co., Ltd.);

[0047] Sodium carbonate (Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.);

[0048] All the above reagents are of analytical grade;

[0049] The water used in the experiment was distilled water.

[0050] 1.2 Main Instruments and Equipment

[0051] UV1200 UV-Vis spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.);

[0052] HJ-2B Dual Digital Display Temperature Control Magnetic Stirrer (Xicheng Xinrui Instrument Factory, Jintan District).

[0053] H-1650 centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., maximum speed 16000rpm);

[0054] Forced-air drying oven (standard laboratory use, for drying leaves);

[0055] High-speed pulverizer (standard laboratory use, for crushing blades);

[0056] Ultrasonic extraction instrument (for routine laboratory use, used for the extraction of phenolic substances);

[0057] Low-temperature vacuum distillation apparatus (for routine laboratory use, used for regeneration of eutectic solvents);

[0058] Low-temperature plasma treatment device (for routine laboratory use, used for pretreatment of leaf powder).

[0059] Thermostatic magnetic stirrer (same model as HJ-2B dual digital display temperature control magnetic stirrer, used for the preparation of eutectic solvents).

[0060] II. Implementation of Extraction Process

[0061] This embodiment uses Lexi blueberry leaves as raw material and employs a low eutectic solvent (DESs) combined with ultrasound-assisted extraction of phenolic substances. The specific steps are as follows:

[0062] Step S1: Raw material pretreatment

[0063] Freshly picked Lexi blueberry leaves were placed in a forced-air drying oven at 45°C and dried until the leaves reached a constant weight. The dried leaves were then removed and pulverized using a high-speed pulverizer, and passed through an 80-mesh standard sieve. The sieved leaf powder was collected and placed in a sealed container. The sieved leaf powder was then spread evenly on the sample stage of a low-temperature plasma treatment device (1-2 mm thick), and air was introduced. The treatment power was set to 10-30 W and the treatment time to 2-5 min. After pretreatment, the powder was stored at -18°C for later use.

[0064] Step S2, Preparation of eutectic solvent (DESs)

[0065] Accurately weigh choline chloride and glycerol, mix them in a 1:2 molar ratio, and transfer the mixture to a round-bottom flask. Place the round-bottom flask on an HJ-2B dual digital display temperature-controlled magnetic stirrer, set the stirring temperature to 80℃, turn on the stirrer and maintain the constant temperature for 8 hours. After stirring, turn off the stirrer and allow the mixture to cool naturally to room temperature to obtain a colorless, stable, and transparent liquid, which is the eutectic solvent (DESs). Seal and store for later use.

[0066] Step S3: Preparation of standard curve for total phenol content determination

[0067] Includes the following sub-steps:

[0068] S3.1 Preparation of standard solution:

[0069] Accurately weigh 0.0100 g of gallic acid standard and place it in a 100 mL volumetric flask. Add distilled water to dissolve and dilute to the mark. Shake well to obtain a gallic acid standard stock solution with a concentration of 0.1 mg / mL. Measure 0.5 mL, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL of the standard stock solution and place them in a 10 mL volumetric flask. Dilute to the mark with distilled water and shake well to obtain a series of gallic acid standard working solutions with concentrations of 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, and 0.05 mg / mL, respectively.

[0070] S3.2, Colorimetric reaction and absorbance measurement:

[0071] Take 1 mL of each of the above concentration standard working solutions and place them in stoppered test tubes. Add 1 mL of Folin-Ciocalteu reagent, shake well, and let stand for 5 min. Then add 2 mL of 10% sodium carbonate solution, shake well, and place in a dark place to react at room temperature for 60 min. After the reaction, use a UV1200 UV-Vis spectrophotometer to measure the absorbance of each solution at a wavelength of 765 nm (the conventional wavelength for the determination of phenolic substances, ensuring that it matches the standard curve data in the disclosure document). Use distilled water as a blank control and subtract the blank absorbance.

[0072] S3.3, Plotting the Standard Curve:

[0073] A standard curve was plotted using linear regression with the concentration of gallic acid standard working solution (unit: mg / mL) as the abscissa (x) and the corresponding absorbance as the ordinate (y). The standard curve equation was y = 14.214x + 0.0282, and the correlation coefficient R² = 0.9953 was calculated. This curve was used for subsequent calculation of total phenol content.

[0074] Step S4: Extraction of phenolic substances

[0075] Accurately weigh 0.200 ± 0.002 g of the Lexi blueberry leaf powder prepared in step S1 using an analytical balance, and place it in a stoppered conical flask. Add the eutectic solvent (DESs) prepared in step S2 to the conical flask, and add 0.01-0.03 g of modified Fe3O4 magnetic adsorbent with surface-modified hydroxyl groups per 0.200 g of leaf powder. Stir with a glass rod for 1 min to ensure uniform dispersion of the adsorbent. The water content of the eutectic solvent is adjusted to 20% (achieved by adding distilled water to the DESs and stirring evenly). The ratio of leaf powder to DESs is controlled at 1:50 g / mL (i.e., 10 mL of DESs is added per 0.200 g of leaf powder). Place the conical flask in an ultrasonic extractor. Set the ultrasonic power to the standard extraction power (ensuring it matches the extraction effect described in the manual), and perform two ultrasonic extractions, each lasting 50.00 min. No separation is required between extractions; the operation is continuous. After ultrasonic extraction, attach a permanent magnet with a magnetic field strength ≥0.5T to the outside of the conical flask and hold it for 5 min to allow the modified Fe3O4 magnetic adsorbent to adhere to the flask wall. Pour off the upper DESs solution for later use. Add 2 mL of ethyl acetate-ethanol mixed eluent (volume ratio 1:1) to the conical flask, shake for 5 min, and then adsorb again with a magnet. Collect the eluent (i.e., the enriched phenolic substances). The eluted adsorbent is then dried in an 80℃ forced-air drying oven for 1 h to regenerate it, allowing it to be reused for subsequent ultrasonic extractions.

[0076] Step S5: Extraction and determination of total phenol content

[0077] It also includes the following sub-steps:

[0078] S5.1 Transfer the mixture (leaf powder + DESs + phenolic substances) extracted by ultrasound in step S4 to a centrifuge tube. Place the centrifuge tube in an H-1650 centrifuge, set the speed to 8000 rpm, and the centrifugation time to 10 min. After centrifugation, carefully aspirate the supernatant with a pipette and place it in a 4 mL volumetric flask. Dilute to the mark with distilled water and shake well to obtain the extract of phenolic substances from Lexi blueberry leaves. To ensure the reliability of the results, three parallel experiments were set up for each extracted sample to obtain three parallel extracts.

[0079] S5.2 Preparation and determination of the test solution:

[0080] Take 0.5 mL of the above extract and place it in a 10 mL volumetric flask. Dilute to the mark with distilled water and shake well to obtain the test solution. Refer to the colorimetric reaction method in step 2.3, take 1 mL of the test solution to perform the colorimetric reaction and measure its absorbance. At the same time, take 1 mL of the eutectic solvent (DESs) prepared in step 2.2, perform the colorimetric reaction in the same way and measure the absorbance as a blank control to subtract the interference of blank absorbance on the measurement results.

[0081] S5.3 Calculation of total phenol content:

[0082] Based on the standard curve equation obtained in step S3, substitute the absorbance of the test solution to calculate the concentration of total phenols in the test solution (c, unit: mg / mL); then calculate the total phenol content (W, unit: mg / g) in the Lexi blueberry leaf powder using the following formula:

[0083] In the formula:

[0084] The total phenol content in the sample solution;

[0085] The total phenol content in the test solution;

[0086] The dilution factor for the test sample is 20 in this step, which is 10 mL / 0.5 mL.

[0087] The total volume of the sample solution is adjusted to volume, in mL; in this step, it is 4 mL.

[0088] The mass of the Lexi blueberry leaf powder was measured.

[0089] Analysis based on the quadratic multivariate regression model established in the experiment revealed that the optimal extraction conditions were: moisture content of 16.80%, material-to-liquid ratio of 1:50 g / mL, extraction times of 1.73, and ultrasonic time of 50.00 min. Under these conditions, the theoretical total phenol content could reach 198.30 mg GAE / gDW. To verify the feasibility of the experiment and considering the operability in actual production, the optimal extraction conditions were changed: moisture content to 20%, extraction times to 2, and other conditions remained unchanged. A verification experiment on the extraction of phenolic substances from blueberry leaves was conducted with 3 parallel groups. The results showed that the total phenol content of blueberry leaves was 193.57 mg GAE / gDW, which was basically consistent with the predicted value, proving that this embodiment was stable and reliable.

[0090] Step S6: In-situ regeneration of eutectic solvent - ultrasonic activation

[0091] Collect the lower layer residue (containing residual DESs) after centrifugation in step S5 and the upper layer DESs solution poured out in step S4, combine them and transfer them to a distillation flask of a low-temperature vacuum distillation apparatus; set the distillation temperature to 40-50℃ and the vacuum degree to 0.08MPa, distill for 30min to separate the distillate (containing a small amount of phenols and water), and the residual liquid in the distillation flask is the recovered DESs; transfer the recovered DESs to an ultrasonic extractor, set the frequency to 20-30kHz and the power to 200W, and ultrasonically treat for 10min; the treated DESs does not need to be re-prepared and can be directly used for the next round of extraction of Lexi blueberry leaf powder and DESs in step S4.

[0092] III. Single-factor experimental verification

[0093] 1. Solvent water content screening test:

[0094] Accurately weigh 0.200±0.002g of leaf powder after pretreatment in step S1, fix the material-liquid ratio at 1:20g / mL, the ultrasonic time at 40min, the number of extractions at 1, and the amount of adsorbent at 0.02g. Only change the moisture content of DESs (10%, 20%, 30%, 40%, 50%), and perform the remaining operations as in steps S4-S5.

[0095] Please see Figure 1 The results showed that when the water content was 10%-20%, the total phenol extract increased from 152.3 mg / g to 193.57 mg / g as the water content increased; when the water content exceeded 20%, the total phenol extract gradually decreased to 145.2 mg / g (at 40% water content). Therefore, the optimal water content was determined to be 20%.

[0096] Material-to-liquid ratio screening test:

[0097] Accurately weigh 0.200±0.002g of leaf powder, fix the DESs solvent water content at 20%, the ultrasonic time at 40min, the number of extractions at 1, and the amount of adsorbent at 0.02g, only change the material-liquid ratio (1∶10, 1∶20, 1∶30, 1∶40, 1∶50g / mL), and perform the remaining operations as in steps S4-S5.

[0098] Please see Figure 2 The results showed that the total phenol content was 112.8 mg / g when the material-liquid ratio was 1:10, increased to 189.7 mg / g when it was 1:40, and reached 193.57 mg / g when it was 1:50 (an increase of only 2.0%). Further decreasing the material-liquid ratio did not yield significant gains. Therefore, the optimal material-liquid ratio was determined to be 1:50 g / mL.

[0099] Extraction frequency screening experiment:

[0100] Accurately weigh 0.200±0.002g of leaf powder, fix the DESs solvent water content at 20%, the material-liquid ratio at 1:40g / mL, the ultrasonic time at 40min, and the adsorbent dosage at 0.02g, only changing the number of extractions (1, 2, 3, 4 times), and the rest of the operation is the same as steps S4-S5.

[0101] Please see Figure 3 The results showed that the total phenol content was 144.2 mg / g after the first extraction, increased to 193.57 mg / g after the second extraction, and fluctuated by only ±1 mg / g after the third and fourth extractions. Therefore, the optimal number of extractions was determined to be 2.

[0102] Ultrasonic time screening test:

[0103] Accurately weigh 0.200±0.002g of leaf powder, fix the DESs solvent water content at 20%, the material-liquid ratio at 1:40g / mL, the number of extractions at 2, and the amount of adsorbent at 0.02g. Only change the ultrasonic time (30, 40, 50, 60, 70 min), and perform the other operations as in steps S4-S5.

[0104] Please see Figure 4 The results showed that the total phenol content was 168.5 mg / g after 30 min of ultrasound, 187.3 mg / g after 40 min, and 193.57 mg / g after 50 min. The total phenol content dropped to below 185.2 mg / g due to the degradation of phenols caused by the heat generated by ultrasound between 60 and 70 min. Therefore, the ultrasound time of 50.00 min was determined to be the optimal value.

[0105] IV. Response Surface Methodology Optimization Process Validation

[0106] 1. Experimental Design and Factor Levels: Based on the results of single-factor experiments, response surface methodology was used to optimize process parameters according to the Box-Behnken central composite experimental design principle. The factor level design is shown in Table 1 below.

[0107] Table 1. Factor Levels in Box-Benhnken Experimental Design

[0108] Table1LevelsandfactorsinvestigatedwithBox-Benhnkenexperimentdesign

[0109] Factor Levels and Coding A. Water content (%) B. Solid-liquid ratio (g / ml) C Extraction times / time D. Ultrasound time (Time / min) -1 10 30 1 30 0 20 40 2 40 1 30 50 3 50

[0110] 2. Response Surface Experimental Design and Results

[0111] Based on the above factor levels, 27 groups of experiments were designed. The experimental design and results are shown in Table 2 below:

[0112] Table 2 Response Surface Experimental Design and Results

[0113] Table2Experimentaldesignandresultsforresponsesurfaceanalysis

[0114] Number A. Water content (%) B. Solid-liquid ratio (g / ml) C Extraction times / time D. Ultrasound time (min / Time) Total phenol content (mg GAE / g DW) 1 -1 -1 0 0 135.56 2 1 -1 0 0 161.31 3 -1 1 0 0 164.49 4 1 1 0 0 175.04 5 0 0 -1 -1 158.42 6 0 0 1 -1 137.97 7 0 0 -1 1 171.55 8 0 0 1 1 153.35 9 -1 0 0 -1 129.71 10 1 0 0 -1 171.55 11 -1 0 0 1 172.30 12 1 0 0 1 161.79 13 0 -1 -1 0 152.58 14 0 1 -1 0 181.37 15 0 -1 1 0 143.15 16 0 1 1 0 173.40 17 -1 0 -1 0 144.53 18 1 0 -1 0 144.16 19 -1 0 1 0 111.70 20 1 0 1 0 149.22 21 0 -1 0 -1 164.82 22 0 1 0 -1 189.34 23 0 -1 0 1 160.04 24 0 1 0 1 189.11 25 0 0 0 0 176.61 26 0 0 0 0 168.74 27 0 0 0 0 172.11

[0115] 3. Regression Model Establishment and Analysis of Variance

[0116] Regression fitting analysis was performed on the experimental results in Table 2, and the quadratic polynomial regression model equation for total phenol extraction (Y) and water content (A), material-liquid ratio (B), extraction times (C), and ultrasonic time (D) was obtained: Y = 172.49 + 8.73A + 12.94B - 6.98C + 4.69D - 3.80AB + 9.47AC - 13.09AD + 0.36BC + 1.14BD + 0.56CD - 16.76A² + 4.35B² - 16.75C² - 0.56D² was obtained.

[0117] To test the validity of the equation, an analysis of variance was performed on the model, and the results are shown in Table 3 below:

[0118] Table 3. Analysis of variance of the regression equations

[0119] Table3Analysis of variance for the fittedregression model

[0120]

[0121] Note: A: Moisture content, B: Material-to-liquid ratio, C: Number of ultrasonic treatments, D: Ultrasonic treatment time, * indicates significant difference (p < 0.05), ** indicates extremely significant difference (p < 0.01).

[0122] 4. Model Validation Conclusions: The analysis of variance results showed that the model's F-value was 17.17 (P<0.0001), which was highly significant; R²=0.9525, and the adjusted R²Adj=0.8970, indicating a good fit; the lack of fit term P=0.3199>0.05, indicating no significant lack of fit; the coefficient of variation CV=3.71%, indicating high model accuracy and repeatability, and it can be used to predict the total phenolic extraction amount of Lexi blueberry leaves.

[0123] Please see Figures 5-6 The factors affecting the total phenol extraction yield, from strongest to weakest, are: material-liquid ratio (B), water content (A), extraction times (C), and ultrasonic time (D). Among them, the interaction terms AC, AD, A², and C² (P<0.0001) are all highly significant simulation terms.

[0124] V. Experimental Examples and Comparative Examples

[0125] The experimental materials, instruments, and methods for determining total phenols in the following test examples and comparative examples are completely consistent with those in the examples. Only the process parameters described below have been changed to ensure that the differences in results are caused solely by the process changes.

[0126] (a) Experimental Example: Verification of the absence of technical features in the embodiment

[0127] The experimental example verifies the necessity of each feature for the process effect by omitting one newly added technical feature in the embodiment. The specific scheme and results are as follows:

[0128] Experimental Example 1: Omitting Low-Temperature Plasma Pretreatment

[0129] Process differences: The low-temperature plasma pretreatment in step S1 of the example is deleted, that is, the leaf powder is dried and pulverized and then stored directly at -18°C without plasma treatment of 10-30W for 2-5min. The remaining steps (DESs preparation, magnetic adsorption extraction, DESs regeneration) are completely consistent with the example.

[0130] Key results: Total phenol extraction yield was 168.23 mg GAE / gDW, a decrease of 13.08% compared to the previous example; the extraction time needed to be extended to 65 min to achieve the dissolution effect of 50 min in the previous example (because the cell walls of Lexi blueberry leaves were not physically destroyed, increasing the resistance to phenol dissolution).

[0131] Experimental Example 2: Omitted DESs in-situ regeneration-ultrasonic activation

[0132] Process differences: DESs regeneration in step S6 of the example is not performed. Freshly prepared choline chloride-glycerol 1:2 DESs is used for each extraction. The remaining steps (plasma pretreatment, magnetic adsorption extraction) are the same as in the example.

[0133] Key results: The total phenol content of a single extraction was 192.89 mg GAE / gDW (similar to the example), but the solvent cost for three consecutive extractions increased by 85% compared to the example (the newly prepared DESs required repeated weighing and stirring, and the raw material loss rate was about 10%); during the third extraction, the overall process economy decreased significantly due to solvent waste.

[0134] Experimental Example 3: Magnetic adsorption enrichment omitted

[0135] Process differences: In step S4 of the example, the modified Fe3O4 magnetic adsorbent with surface-modified hydroxyl groups was not added. After ultrasonic extraction, it was directly centrifuged at 8000 rpm for 10 min. The remaining steps (plasma pretreatment, DESs regeneration) were the same as in the example.

[0136] Key results: The total phenolic content was 189.56 mg GAE / gDW (a decrease of 2.07% compared to the example), but the content of impurities (polysaccharides, chlorophyll) in the extract increased significantly. At the same time, the purity of phenolic products was only 62.3% (86.7% in the example) as detected by high performance liquid chromatography (HPLC, column: C18 column, mobile phase: methanol-0.1% phosphoric acid aqueous solution). An additional macroporous resin purification step is required to achieve the same purity, resulting in a 4-hour extension of the process time per batch.

[0137] (ii) Comparative Example: Verification by comparison with existing technical solutions

[0138] The advantages of the comparative examples are as follows: Existing technologies such as traditional solvent extraction and unoptimized DESs extraction were selected for comparison.

[0139] Comparative Example 1: Extraction with Traditional Ethanol Solvent

[0140] Process scheme: Refer to the conventional methods for extracting phenolic compounds from blueberry leaves in existing technologies:

[0141] The raw material pretreatment is the same as in the example (without plasma treatment);

[0142] The extraction solvent was 70% aqueous ethanol solution (analytical grade);

[0143] Extraction parameters: material-to-liquid ratio 1:50 g / mL, ultrasonic time 50 min, extraction twice; centrifugation (8000 rpm × 10 min) followed by volume adjustment, and total phenol content was measured.

[0144] Key results: The total phenol extraction yield was 82.35 mg GAE / gDW, which is only 42.5% of that in the example; moreover, ethanol needs to be recovered by distillation (distillation temperature 78℃, energy consumption is higher than that of DESs regeneration at 40-50℃ in the example), and the solvent toxicity is higher than that of DESs, which does not meet the requirements of green production.

[0145] Comparative Example 2 (Unoptimized DESs Extraction)

[0146] Process scheme: Use choline chloride-glycerol 1:2 DESs, but do not perform single-factor or response surface optimization, and set parameters randomly.

[0147] DESs with a water content of 15%, a solid-liquid ratio of 1:30 g / mL, was extracted once and sonicated for 45 min.

[0148] No plasma pretreatment, no magnetic adsorption, and no DESs regeneration;

[0149] The remaining steps (centrifugation, total phenol determination) are the same as in the example.

[0150] Key results: The total phenol extraction yield was 127.64 mg GAE / gDW, only 65.97% of that in the previous example; and due to unoptimized parameters, the DESs viscosity was too high (insufficient water content), resulting in turbid supernatant after centrifugation, requiring additional filtration, and reducing process efficiency by 30%.

[0151] The unoptimized parameters (15% water content, 1:30 g / mL material-to-liquid ratio, etc.) were set with reference to the conventional empirical parameters for the extraction of plant phenols by DESs in existing technologies.

[0152] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A blueberry leaf phenolic eutectic solvent extraction process, characterized in that, The blueberry leaves are Laxi blueberry leaves, and the process comprises the following steps: S1, raw material pretreatment: put the freshly picked Laxi blueberry leaves into a forced air drying oven at 45 DEG C and bake until the weight is constant, crush and pass through an 80 mesh sieve, and store at -18 DEG C for standby; S2, eutectic solvent preparation: mix choline chloride and glycerol at a molar ratio of 1:2 in a round-bottom flask, and stir at 80 DEG C in a constant temperature magnetic stirrer for 8 hours. After cooling to room temperature, a colorless, stable and transparent eutectic solvent is obtained; S3, standard curve preparation for total phenol content determination: using gallic acid as a standard, a standard curve of gallic acid concentration-absorbance is drawn, specifically: y=14.214x+0.0282, R²=0.9953; S4, phenolic substance extraction: accurately weigh 0.200±0.002g of the standby Laxi blueberry leaf powder in step S1, and mix it with the eutectic solvent prepared in step S2. The water content of the eutectic solvent is 20%, and the solid-liquid ratio is 1:50 g / mL. Perform 2 times of ultrasonic extraction, each time for 50.00 min; S5, separation of the extract and determination of the total phenol content: centrifuge the mixture after ultrasonic extraction in step S4 at a speed of 8000 rpm for 10 min, take the supernatant, dilute to 4 mL to obtain the extract; take a certain amount of the extract to dilute to the test solution, and determine the total phenol content according to the standard curve equation in step S3. The total phenol content is expressed as the gallic acid equivalent per gram of Laxi blueberry leaf powder. At the same time, a blank experiment is performed with the corresponding eutectic solvent.

2. The process for extracting blueberry leaf phenolics by deep eutectic solvent according to claim 1, characterized in that, The calculation formula of the total phenol content in step S5 is: In the formula: Total phenol content in the sample solution, unit mg / g; Total phenol content in the sample solution, unit: mg / mL; Test sample dilution factor; Total volume, unit mL, was set for the sample solution; Sample mass, in g.

3. The process for extracting blueberry leaf phenolics by deep eutectic solvent according to claim 1, characterized in that, In step S1, the standby Laxi blueberry leaf powder before mixing in step S4 also includes a low-temperature plasma pretreatment step: Use air medium low-temperature plasma to treat Laxi blueberry leaf powder at a power of 10-30 W for 2-5 min.

4. The process for extracting blueberry leaf phenolics by deep eutectic solvent according to claim 1, characterized in that, After obtaining the extract by taking the supernatant and diluting to volume in step S5, a eutectic solvent in-situ regeneration-ultrasonic activation step is further included: Use low-temperature vacuum distillation at 40-50 DEG C and 0.08 MPa to separate the phenolic substances and the eutectic solvent in the extract. The recovered eutectic solvent is treated by ultrasonic at 20-30 kHz for 10 min. The treated eutectic solvent is used for mixing and extracting Laxi blueberry leaf powder with eutectic solvent in the next step S4.

5. The process for extracting blueberry leaf phenolics by deep eutectic solvent according to claim 1, characterized in that, During the ultrasonic extraction in step S4, a modified Fe3O4 magnetic adsorbent with surface modified hydroxyl groups is also added; After the ultrasonic extraction in step S4, the modified Fe3O4 magnetic adsorbent and the eutectic solvent system are first separated by an external magnetic field, and then a weak polar eluent is used to elute the enriched phenolic substances from the modified Fe3O4 magnetic adsorbent. The eluted modified Fe3O4 magnetic adsorbent is regenerated and then repeatedly used for adsorption of phenolic substances in the ultrasonic extraction process.

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