Lonicera caerulea polyphenol extraction method based on deep-eutectic solvent and application of Lonicera caerulea polyphenol extraction method

Abstract

The invention discloses a lonicera caerulea polyphenol extraction method based on a deep-eutectic solvent and application of lonicera caerulea polyphenol extraction method, and belongs to the technical field of plant extraction. A method for extracting lonicera caerulea polyphenol by using a deep-eutectic solvent comprises the following steps: S1, freeze-drying fresh lonicera caerulea, and crushing to obtain lonicera caerulea freeze-dried powder; s2, uniformly mixing a hydrogen bond acceptor and a hydrogen bond donor reagent, heating and stirring until transparent and uniform liquid is formed, and naturally cooling to room temperature to obtain DES; s3, mixing the lonicera caerulea raw material with DES according to a ratio of 1: (10-50) g / mL, putting the mixture into an ultrasonic instrument, extracting at set temperature, time and ultrasonic power, and centrifuging to obtain supernate, namely a lonicera caerulea polyphenol extracting solution. The yield of the lonicera caerulea polyphenol extracted by the method is obviously higher than that of the lonicera caerulea polyphenol extracted by a traditional water solvent and an alcohol solvent, and the obtained extract has excellent antioxidant activity and antithrombotic activity.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically, it relates to the optimization of process conditions for extracting polyphenols from honeysuckle fruit using a eutectic solvent and its application. Background Technology

[0002] Honeysuckle berry, scientifically known as *Lonicera caerulea* L., is a deciduous shrub belonging to the genus *Lonicera* in the family Caprifoliaceae. It is also known as black bear fruit, blue fruit, and goat's milk fruit. In my country, honeysuckle berry is mainly distributed in North and Northwest China, including Heilongjiang, Jilin, and Inner Mongolia. It has a long history of consumption in Northeast China and is known as the "King of Anthocyanins." Research has found that honeysuckle berry contains abundant bioactive components, such as organic acids, iridoids, flavonoids, polyphenols, and polysaccharides, which have anti-inflammatory, antioxidant, and eye-fatigue-relieving effects. Currently, the application of honeysuckle berry is mainly concentrated in the food industry, such as processing it into honeysuckle berry juice and jam. However, the discovery of various active ingredients has enhanced its value as a medicinal plant.

[0003] Polyphenols are one of the important active components of honeysuckle berries, and have attracted widespread attention due to their anti-inflammatory and antioxidant effects. Currently, extraction methods for honeysuckle berry polyphenols (LCPs) include solvent extraction, enzymatic-assisted extraction, and ultrasound-assisted extraction. Traditional solvent extraction methods, mainly using alcohol or water extraction, often suffer from low yields or weak extract activity. Therefore, developing a novel and efficient extraction solvent is an urgent problem to be solved in this field.

[0004] Deep eutectic solvents (DES) are green, biodegradable solvents, typically composed of two or more components (hydrogen bond donors and hydrogen bond acceptors). They exhibit properties similar to ionic liquids, are low-cost and easy to prepare, and possess unique advantages in the extraction of bioactive substances. Therefore, screening a suitable DES solvent for extracting honeysuckle berry polyphenols and optimizing the extraction process are of great significance for improving the yield and utilization rate of honeysuckle berry polyphenols. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for extracting honeysuckle fruit polyphenols using a eutectic solvent, which has the advantages of being environmentally friendly and having a high polyphenol yield.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of the present invention provides a method for extracting honeysuckle fruit polyphenols using a eutectic solvent, comprising the following steps: S1. After freeze-drying fresh honeysuckle berries, pulverize them to obtain honeysuckle berry freeze-dried powder; S2. Mix the hydrogen bond acceptor and hydrogen bond donor reagent, heat and stir until a transparent and homogeneous liquid is formed, and cool naturally to room temperature to obtain DES. S3. Mix the honeysuckle berry raw material with DES at a ratio of 1:(10~50) g / mL, place it in an ultrasonic instrument and set the temperature, time and ultrasonic power for extraction. After centrifugation, obtain the supernatant, which is the product. Preferably, in step S2, the hydrogen bond acceptor is selected from choline chloride and betaine, and the hydrogen bond donor is selected from ethylene glycol, glycerol, 1,4-butanediol, 1,3-butanediol, lactic acid, urea, glucose, and fructose.

[0007] Preferably, in step S2, the moisture content of the DES is 25% to 50%.

[0008] More preferably, in step S2, the optimal moisture content of the DES is 29%.

[0009] More preferably, in step S2, the hydrogen bond acceptor of the DES is choline chloride, and the hydrogen bond donor is 1,3-butanediol.

[0010] More preferably, in step S2, the molar ratio of choline chloride to 1,3-butanediol in the DES is 1:(2~6). More preferably, in step S2, the optimal molar ratio of choline chloride to 1,3-butanediol in the DES is 1:5.

[0011] Preferably, in step S3, the ultrasonic power is 120~240W.

[0012] Preferably, in step S3, the extraction temperature is 30~60℃.

[0013] Preferably, in step S3, the extraction time is 35-55 minutes.

[0014] Preferably, in step S3, the centrifugation speed is 2000~5000 r / min and the time is 10~30 min.

[0015] The second aspect of the present invention provides a honeysuckle polyphenol prepared by the method described in the first aspect of the present invention, which has superior antioxidant activity compared to traditional solvent extraction.

[0016] The third aspect of the present invention provides the application of the honeysuckle berry polyphenols described in the second aspect of the present invention in the preparation of antioxidant and antithrombotic products, providing a new direction for the application of honeysuckle berry polyphenols.

[0017] In a specific embodiment of the present invention, the present invention measures the effects of DPPH·, OH· and ABTS. +The scavenging rate demonstrates the antioxidant capacity of the honeysuckle polyphenols prepared by the method described in the first aspect of this invention.

[0018] In a specific embodiment of the present invention, zebrafish were used to demonstrate that the honeysuckle berry polyphenols prepared by the method described in the first aspect of the present invention can, to a certain extent, inhibit phenylhydrazine-induced thrombus formation in zebrafish and restore the zebrafish's ability to supply blood to the heart. The research results confirm that honeysuckle berry polyphenols have an antithrombotic effect, and this research finding provides a theoretical basis for developing honeysuckle berry polyphenols into a natural antithrombotic drug.

[0019] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention provides a eutectic solvent system and a method for extracting honeysuckle berry polyphenols. The system uses choline chloride and other similar substances as hydrogen bond acceptors and 1,3-butanediol and other similar substances as hydrogen bond donors. Compared with traditional solvents, this method significantly improves the extraction yield of honeysuckle berry polyphenols compared to traditional aqueous and alcoholic solvent extraction, greatly enhancing the extraction efficiency. Furthermore, the eutectic solvent system provided by this invention uses non-toxic, environmentally friendly, and biocompatible raw materials. In summary, the eutectic solvent system provided by this invention has strong practicality.

[0020] The antioxidant capacity of the extract obtained under the optimal extraction conditions of this invention was verified by in vitro antioxidant studies, and its antioxidant activity was superior to that of extracts obtained by traditional methods. Furthermore, this invention demonstrated the antithrombotic effect of honeysuckle berry polyphenols using a zebrafish thrombosis model, providing new ideas and directions for the development and utilization of honeysuckle berry polyphenols. Attached Figure Description

[0021] Figure 1 This is a standard curve of the absorbance value of gallic acid versus its concentration.

[0022] Figure 2 The results of the DES filtering are shown.

[0023] Figure 3 The results of single-factor investigation of DES solvent extraction are shown in the figure. A represents the effect of the molar ratio of choline chloride and 1,3-butanediol on the polyphenol yield; B represents the effect of DES water content on the polyphenol yield; C represents the effect of solid-liquid ratio on the polyphenol yield; D represents the effect of extraction time on the polyphenol yield; and E represents the effect of extraction temperature on the polyphenol yield.

[0024] Figure 4 This is a three-dimensional response surface plot of the interaction between different extraction factors. In the plot, A represents the DES molar ratio and DES water content, B represents the DES molar ratio and solid-liquid ratio, C represents the DES molar ratio and extraction time, D represents the DES water content and solid-liquid ratio, E represents the DES water content and extraction time, and F represents the solid-liquid ratio and extraction time.

[0025] Figure 5 This is a two-dimensional contour plot showing the interaction of different extraction factors. In the plot, A represents the DES molar ratio and DES water content, B represents the DES molar ratio and solid-liquid ratio, C represents the DES molar ratio and extraction time, D represents the DES water content and solid-liquid ratio, E represents the DES water content and extraction time, and F represents the solid-liquid ratio and extraction time.

[0026] Figure 6 Results of DPPH· scavenging rates of honeysuckle berry polyphenols extracted with different solvents.

[0027] Figure 7 The results show the scavenging rate of OH· by honeysuckle fruit polyphenols extracted with different solvents.

[0028] Figure 8 The effect of honeysuckle fruit polyphenols extracted with different solvents on ABTS + The clearance rate results.

[0029] Figure 9 The image shows the staining and analysis results of erythrocytes in the heart of zebrafish. In the figure, A is the staining effect of the heart of each group of zebrafish, and B is the analysis result of the heart staining intensity. Detailed Implementation

[0030] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0031] The honeysuckle berries used in the following examples are honeysuckle berries purchased from a certain region.

[0032] The following describes the determination of total polyphenol content using the Folin-Ciocalteu method, the specific method of which is as follows: 1. Plotting the standard curve Accurately weigh 10 mg of gallic acid standard, dissolve it in purified water, and dilute to 50 mL to prepare a 200 μg / mL gallic acid standard stock solution. Measure 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 mL of the standard stock solution into 10 mL volumetric flasks, and dilute to the mark with purified water to prepare gallic acid standard solutions with concentrations of 20, 30, 40, 50, 60, 70, 80, and 90 μg / mL, respectively. Accurately measure 1.0 mL of each gallic acid standard solution into a colorimetric tube, then add 4 mL of purified water and 2 mL of Folin-Ciocalteu reagent, mix well, and then add 3 mL of 7.5% Na₂CO₃ solution over 1–8 min. Mix thoroughly and react at room temperature in the dark for 1 h. Using purified water treated in the same way as a blank, measure the absorbance at 765 nm using a UV-Vis spectrophotometer. A standard curve was plotted with gallic acid concentration on the x-axis and absorbance on the y-axis, as shown below. Figure 1 As shown.

[0033] 2. Determination of Polyphenol Content in Honeysuckle Fruit Extract The polyphenol content in honeysuckle berry extract was determined using the Folin-Ciocalteu method. The specific procedure was as follows: 1.0 ml of each extract was accurately measured into a colorimetric tube, followed by the addition of 4 mL of purified water and 2 mL of Folin-Ciocalteu reagent. The mixture was thoroughly mixed, and then 3 mL of 7.5% Na₂CO₃ solution was added over 1–8 min. The mixture was allowed to react at room temperature in the dark for 1 h. The absorbance was then measured at 765 nm using a UV-Vis spectrophotometer. The absorbance value was then used to calculate the polyphenol content of the honeysuckle berry extract using a standard curve.

[0034] Preparation Examples 1-10: In this preparation example, 10 eutectic solvents were prepared, and their flowability and appearance were observed.

[0035] Choline chloride (ChCl) and betaine (Bet) were used as hydrogen bond acceptors (HBA), and one of the following was used as a hydrogen bond donor (HBD): ethylene glycol (EG), glycerol (Gly), 1,4-butanediol (1,4-BuD), 1,3-butanediol (1,3-BuD), lactic acid (LA), urea (Ur), glucose (Glu), and fructose (Fru). The mixture was prepared according to the combinations and molar ratios in Table 1, placed in a beaker, and heated and stirred until a clear, homogeneous liquid was formed. The mixture was then removed and allowed to cool naturally to room temperature. If no precipitate formed, the DES synthesis was successful. According to Table 1, different hydrogen bond donors and hydrogen bond acceptors were mixed in different molar ratios and reacted at 80°C until the system became a clear and transparent liquid. The mixture was then removed and allowed to cool naturally to room temperature. If no precipitate formed, the DES synthesis was successful. At the same time, their fluidity and appearance were observed.

[0036] Table 1 Combinations and Molar Ratios of DES Example 1: This embodiment prepares a honeysuckle fruit polyphenol extract using the following method: (1) Take fresh honeysuckle fruit, freeze-dry it, and then pulverize it into powder to obtain honeysuckle fruit dried powder; (2) Mix the dried honeysuckle fruit powder with an aqueous solution of a eutectic solvent (the eutectic solvent is the sample from Preparation Example 1, which has a water content of 30%) at a solid-liquid ratio of 1:20 (g / mL). Under the condition of a water bath temperature of 40°C, extract with ultrasonic assistance for 60 min. After centrifuging the extract at 4000 r / min for 20 min, take the supernatant to obtain the product.

[0037] Example 2-10: In this embodiment, nine kinds of honeysuckle fruit polyphenol extracts were prepared. The only difference between this method and Example 1 is that the eutectic solvent was replaced with the samples of Preparation Examples 2-9 in turn. The water content of each sample was 30%, and all other preparation conditions remained unchanged.

[0038] Comparative Example 1: This comparative example provides a honeysuckle fruit polyphenol extract, the method of which is as follows: (1) Take fresh honeysuckle fruit, freeze-dry it, and then pulverize it into powder to obtain honeysuckle fruit dried powder; (2) Mix the dried honeysuckle fruit powder with purified water at a solid-liquid ratio of 1:20 (g / mL), and extract with ultrasonic assistance at a water bath temperature of 40℃ for 60 min. After centrifuging the extract at 4000 r / min for 20 min, take the supernatant to obtain the product.

[0039] Comparative Example 2: This comparative example provides a honeysuckle fruit polyphenol extract, the method of which is as follows: (1) Take fresh honeysuckle fruit, freeze-dry it, and then pulverize it into powder to obtain honeysuckle fruit dried powder; (2) Mix the dried honeysuckle fruit powder with 50% (v / v) ethanol at a solid-liquid ratio of 1:20 (g / mL), and extract with ultrasonic assistance at a water bath temperature of 40℃ for 60 min. After centrifuging the extract at 4000 r / min for 20 min, take the supernatant to obtain the product.

[0040] Example 11 Based on Example 7, five portions of honeysuckle fruit lyophilized powder were accurately weighed and extracted by ultrasonication for 60 min under the following conditions: solid-liquid ratio of 1:20 (g / mL), extraction temperature of 40℃, DES water content of 30%, and molar ratios of choline chloride and 1,3-butanediol of 1:2, 1:3, 1:4, 1:5, and 1:6, respectively. The polyphenol content of the extract was determined and the polyphenol yield was calculated.

[0041] Example 12: Based on Example 7, five portions of honeysuckle fruit lyophilized powder were accurately weighed and extracted by ultrasonication for 60 min under the following conditions: solid-liquid ratio of 1:20 (g / mL), extraction temperature of 40℃, molar ratio of choline chloride to 1,3-butanediol of 1:5, and DES water content of 10%, 20%, 30%, 40%, and 50%, respectively. The polyphenol content of the extract was determined and the polyphenol yield was calculated.

[0042] Example 13: Based on Example 7, five portions of honeysuckle fruit lyophilized powder were accurately weighed and extracted under the following conditions: extraction temperature of 40°C, molar ratio of choline chloride to 1,3-butanediol of 1:5, DES water content of 30%, and solid-liquid ratios of 1:10, 1:20, 1:30, 1:40, and 1:50 (g / mL), respectively. The extract was ultrasonically extracted for 60 min, the polyphenol content was determined, and the polyphenol yield was calculated.

[0043] Example 14: Based on Example 7, five portions of honeysuckle fruit lyophilized powder were accurately weighed and extracted by ultrasonication for 30, 40, 50, 60 and 70 min respectively under the conditions of solid-liquid ratio of 1:40 (g / mL), extraction temperature of 40℃, molar ratio of choline chloride and 1,3-butanediol of 1:5 and DES water content of 30%. The polyphenol content of the extract was determined and the polyphenol yield was calculated.

[0044] Example 15: Based on Example 7, five portions of honeysuckle fruit lyophilized powder were accurately weighed and ultrasonically extracted for 60 min at the following conditions: solid-liquid ratio of 1:20 (g / mL), molar ratio of choline chloride to 1,3-butanediol of 1:5, DES water content of 30%, and extraction temperatures of 20, 30, 40, 50, and 60°C. The polyphenol content of the extract was determined and the polyphenol yield was calculated.

[0045] Example 16: Based on Examples 11-15, response surface methodology (RSM) was conducted using four factors: DES molar ratio (A), water content (B), solid-liquid ratio (C), and extraction time (D). A four-factor, three-level combination experiment was designed using Design-Expert 13.0 software, with polyphenol yield as the response value, and the RSM was used for optimization. The factor and level design table is shown in Table 2.

[0046] Table 2 Factor and Level Design Table The data from the combined experiments were analyzed using Design-Expert 13.0 software, resulting in a multiple quadratic regression analysis to obtain the regression model equation and predict the optimal extraction conditions for honeysuckle berry polyphenols. Three extraction experiments were then conducted under the predicted optimal extraction conditions to calculate the polyphenol yield and analyze the statistical differences between the measured values ​​and the model predictions.

[0047] Test Example 1: The polyphenol content of the honeysuckle berry polyphenol extracts prepared in Examples 1-16 and Comparative Examples 1-2 was determined using the Folin-Ciocalteu method, and the results are as follows: Figures 2-5 As shown in Tables 3 and 4.

[0048] Depend on Figure 2 Data shows that the polyphenol yields of all 10 DES types were higher than those obtained by water extraction, and the polyphenol yields of 2 DES types were higher than those obtained by alcohol extraction. Among them, the polyphenol yield of DES-7 was significantly higher than that of other solvents. Therefore, subsequent optimization of the extraction process will be carried out based on DES-7 (choline chloride as hydrogen bond acceptor and 1,3-butanediol as hydrogen bond donor).

[0049] Depend on Figure 3As shown in A, within a certain range, the polyphenol yield increases with the increase of the molar ratio of 1,3-butanediol, reaching its highest value when the molar ratio of choline chloride to 1,3-butanediol is 1:5. Therefore, a molar ratio of 1:5 was chosen as the central level for the response surface methodology study, and 1:4 and 1:6 were chosen as the other two levels for the DES molar ratio in the response surface design. Figure 3 As shown in Figure B, the polyphenol yield first increases and then decreases with increasing DES moisture content, reaching its highest level at 30% moisture content, and decreasing further above 30%. Therefore, 20%, 30%, and 40% were selected as the three moisture content levels for DES in the response surface methodology. Furthermore, as... Figure 3 As shown in C, the polyphenol yield is highest when the solid-liquid ratio is 1:40 (g / mL). Therefore, 1:30, 1:40, and 1:50 (g / mL) are used as three levels of solid-liquid ratio in response surface methodology. Figure 3 The results of D showed that the polyphenol yield changed with extraction time. In the 30–50 min period, the polyphenol yield increased continuously with increasing extraction time, reaching its highest value at 50 min, and then decreased significantly. Therefore, extraction times of 40, 50, and 60 min were selected for response surface methodology analysis. Figure 3 The results showed that the polyphenol yield only significantly increased when the extraction temperature increased from 20℃ to 30℃, while the effect of extraction temperature on polyphenol yield was not significant in the range of 30~60℃. Therefore, in the subsequent response surface methodology design, 40℃ was directly selected as the fixed extraction temperature.

[0050] Based on the software design results, a total of 29 groups were set up for the four-factor, three-level combined experiment, with each group undergoing three parallel trials. The experimental results are shown in Table 3. The data in Table 3 were processed using Design-Expert 13.0 software. After fitting, the regression equation was obtained as follows: Y = 6.88 + 0.0097A - 0.0325B + 0.0609C - 0.0273C - 0.0263AB - 0.0066AC + 0.0365AD + 0.1205BC + 0.0466BD + 0.0607CD - 0.4284A 2 -0.1946B 2 -0.2162C 2 -0.1416D 2 The results of the analysis of variance for the response surface methodology are shown in Table 4. The p-value of the model is less than 0.0001, indicating that the model is highly significant; the p-value for the lack-of-fit term is 0.4302, which is greater than 0.05 and not significant, indicating that there are no obvious factors of lack of fit, and the predictive model is sufficient to accurately represent the test data; the adjusted coefficient of determination R... 2 adj =0.9705, indicating that the model can explain 97.05% of the changes in the response value; the coefficient of determination R... 2=0.9852, coefficient of variation (CV) = 0.6262%, indicating that the regression equation has a good fit, strong stability, and high accuracy, and the model is suitable for experimental analysis. The influence of each factor on the polyphenol yield is as follows: solid-liquid ratio (g / mL) > DES water content (%) > extraction time (min) > DES molar ratio.

[0051] Table 3 Response Surface Experimental Design and Results Table 4. Analysis of Variance for Response Surface Regression Model Note: "**" indicates extremely significant difference (P < 0.01); "*" indicates significant difference (P < 0.05); "ns" indicates no significant difference (P > 0.05).

[0052] 3D surface plots and 2D contour plots, such as Figure 4 and Figure 5 As shown in the figure, the three-dimensional response surface plot reflects the influence of the interaction between factors on the yield of honeysuckle berry polyphenols. The steeper the slope of the response surface, the greater the influence of the factor on the yield. The two-dimensional response contour plot can reflect both the interaction between independent factors and the significance of each independent factor. The closer the contour plot is to an ellipse, the more significant the interaction between the two factors. As can be seen from the figure, the curves of the interaction terms BC, BD, and CD are relatively steep, and the contour plots are close to ellipses, indicating that their interaction is significant; while the interaction of AB, AC, and AD is not obvious. Based on the obtained model, the optimal extraction process for honeysuckle berry polyphenols is predicted to be: a molar ratio of choline chloride to 1,3-butanediol of 1:5.009, a DES water content of 29.419%, a solid-liquid ratio of 1:41.132 (g / mL), an extraction time of 49.195 min, and a predicted polyphenol yield of 6.882%.

[0053] Based on practical operation, the prediction conditions were slightly modified: the molar ratio of choline chloride to 1,3-butanediol was 1:5, the water content of DES was 29%, the solid-liquid ratio was 1:41 (g / mL), and the extraction time was 50 min. Under these conditions, three parallel experiments were conducted to verify the model, and the average yield was 6.812%, which was not significantly different from the predicted value, indicating that the regression model is accurate and can be used to predict the extraction of honeysuckle fruit polyphenols.

[0054] Example 17: In vitro antioxidant activity of honeysuckle fruit polyphenols: The in vitro antioxidant activity of the honeysuckle fruit polyphenol extracts prepared in Example 16, Comparative Example 1, and Comparative Example 2 was studied. DPPH method, OH method and ABTS + • The method is a commonly used method for determining the in vitro antioxidant activity of substances.

[0055] (1) Evaluation of DPPH· scavenging activity Accurately weigh 5 mg of DPPH, dissolve it in anhydrous ethanol, and dilute to 100 ml to prepare a DPPH solution with a concentration of 50 μg / mL. Take an appropriate amount of honeysuckle berry polyphenols and prepare honeysuckle berry polyphenol solutions with concentrations of 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. Take 150 μL of honeysuckle berry polyphenol solution of different concentrations and mix it with 150 μL of DPPH solution. After reacting at 25℃ in the dark for 30 min, measure the absorbance at a wavelength of 517 nm and calculate the DPPH scavenging rate according to formula (1). At the same time, use ascorbic acid solution of the same concentration as a positive control.

[0056] DPPH clearance rate (%) = [1 - (A1 - A2) / A0] × 100% (1) In the formula, A0 is the absorbance of the system with purified water instead of the sample solution; A1 is the absorbance of the system with the sample solution; and A2 is the absorbance of the system with anhydrous ethanol instead of DPPH.

[0057] (2) Evaluation of OH· scavenging activity Take an appropriate amount of honeysuckle berry polyphenols and prepare solutions with concentrations of 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. Add 0.5 ml of 9 mmol / L FeSO4 solution, 0.5 ml of 9 mmol / L salicylic acid-anhydrous ethanol solution, 1.0 ml of different concentrations of honeysuckle berry polyphenol solution, and 0.5 ml of 8.8 mmol / L H2O2 solution to test tubes sequentially. After incubating in a water bath at 37℃ for 30 min, measure the absorbance at a wavelength of 510 nm and calculate the OH· scavenging rate according to formula (2). Simultaneously, use an ascorbic acid solution of the same concentration as a positive control.

[0058] OH· removal rate (%) = [1 - (A1 - A3) / A0] × 100% (2) In the formula, A0 is the absorbance of the purified water system replacing the sample solution; A1 is the absorbance of the sample solution system; and A3 is the absorbance of the purified water system replacing the H2O2 solution.

[0059] (3) Regarding ABTS + Evaluation of scavenging activity Mix 7 mmol / L ABTS solution with an equal volume of 2.45 mmol / L potassium persulfate solution and let stand at room temperature in the dark for 12 h to prepare ABTS stock solution. Dilute the stock solution with anhydrous ethanol by a certain factor so that its absorbance at 734 nm is 0.70±0.02, and use this as ABTS working solution. Take an appropriate amount of honeysuckle berry polyphenol and prepare honeysuckle berry polyphenol solutions with concentrations of 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg / mL. Take a test tube and add 2 ml of ABTS working solution and 0.1 ml of honeysuckle berry polyphenol solution of different concentrations respectively. After reacting at room temperature in the dark for 10 min, measure the absorbance at a wavelength of 734 nm and calculate ABTS according to formula (3). + • Clearance rate. Meanwhile, an ascorbic acid solution of the same concentration was used as a positive control.

[0060] ABTS + • Clearance rate (%) = [1 - (A1 - A4) / A0] × 100% (3) In the formula, A0 is the absorbance of the system with purified water instead of the sample solution; A1 is the absorbance of the system with the sample solution; and A4 is the absorbance of the system with anhydrous ethanol instead of ABTS.

[0061] Depend on Figure 6 The results showed that, within the range of 0.05–1.0 mg / mL, the DPPH· scavenging ability of honeysuckle berry polyphenols continuously increased with increasing concentration, comparable to that of the positive control ascorbic acid. The DPPH· scavenging activity of honeysuckle berry polyphenols extracted with different solvents also varied. The DPPH· scavenging ability of honeysuckle berry polyphenols extracted with DES was higher than that extracted with 50% (v / v) ethanol and water, with the difference being more pronounced in the low concentration range (0.05–0.2 mg / mL). These results indicate that honeysuckle berry polyphenols possess strong DPPH· scavenging activity, and this activity is influenced to some extent by the extraction solvent.

[0062] Depend on Figure 7 It was found that the OH· scavenging activity of honeysuckle berry polyphenols increased with increasing concentration, but all were lower than that of the positive control ascorbic acid. At low concentrations (0.05–0.1 mg / mL), the OH· scavenging activity of the three honeysuckle berry polyphenols showed no significant difference. However, with increasing concentration, the honeysuckle berry polyphenols extracted by DES gradually showed stronger OH· scavenging activity. The results indicate that honeysuckle berry polyphenols extracted by different solvents all have certain OH· scavenging activity, with the DES-extracted polyphenols showing stronger activity than the other two solvents.

[0063] Depend on Figure 8 It can be seen that the effects of different solvents on ABTS-extracted honeysuckle fruit polyphenols are significant. +The scavenging activity of ABTS extracted from honeysuckle fruit polyphenols by DES increased with increasing concentration. + • The scavenging rate was significantly higher than that of water extraction and alcohol extraction. The ABTS scavenging rate of honeysuckle berry polyphenols extracted by DES was significantly higher at a lower concentration (0.4 mg / mL). + The ABTS+· scavenging rate of the first polyphenol was similar to that of ascorbic acid, while the ABTS+· scavenging rates of the other two honeysuckle berry polyphenols were consistently lower than those of ascorbic acid. These results indicate that honeysuckle berry polyphenols extracted with different solvents can effectively scavenge ABTS+·. + • It produces a scavenging effect, and the polyphenols extracted by DES are more effective against ABTS than those extracted by alcohol and water. + The · has a stronger cleaning effect.

[0064] Example 18 The hematopoietic genetic network of zebrafish is highly conserved in evolution and is suitable for evaluating thrombosis mechanisms and therapeutic drugs.

[0065] (1) Establishment of zebrafish thrombosis model and drug administration Sixty healthy zebrafish juveniles at 72 hpf were randomly divided into four groups and placed in six-well plates, with 20 fish per well. The groups included a blank control group (CTRL), a model control group (PHZ), a positive control group (ASP), and a honeysuckle polyphenol treatment group. Before modeling, the normal control group and the model control group were given fish culture water, the positive control group was given 20 μg / mL aspirin, and the honeysuckle polyphenol treatment group was given 2 mg / mL honeysuckle polyphenol. All treatments were pre-protected by incubation at 28℃ for 24 h. All drugs were diluted with fresh zebrafish culture water. When the embryos reached 96 hpf, all groups except the normal control group were given 1 µmol / L phenylhydrazine to induce thrombosis, and the modeling process continued for another 12 h.

[0066] (2) Zebrafish hemoglobin staining A certain amount of o-anisidine was weighed and dissolved in anhydrous ethanol, shaken for 10 h to obtain a stock solution with a concentration of approximately 1.4 mg / mL, which was stored at 4℃ in the dark for later use. The o-anisidine working solution consisted of 2 mL of o-anisidine stock solution, 0.5 mL of 0.1 mol / L sodium acetate solution (pH = 4.5), 2 mL of ultrapure water, and 0.1 mL of 30% (w / w) hydrogen peroxide solution, prepared according to the required proportions. The culture medium in the zebrafish well plates after modeling was aspirated and replaced with the staining solution. Staining was performed at room temperature in the dark for about 10 min. After observing that the embryos were generally white and the heart region was stained red, the embryos were washed three times with PBS. The embryos were fixed overnight with 4% (w / v) paraformaldehyde. The stained embryos were photographed under a microscope, and the images were processed using ImageJ. The staining intensity of the heart region was analyzed and statistically analyzed.

[0067] The results of staining and analysis of erythrocytes from the hearts of zebrafish in each group are as follows: Figure 9 As shown, compared with the blank control group, the staining intensity of erythrocytes in the heart of zebrafish in the model group was significantly reduced (p<0.0001). This is because blood flow obstruction occurred in the zebrafish, leading to a decrease in the amount of blood returning to the heart, which in turn resulted in a decrease in the staining intensity of the heart region after staining. This indicates that phenylhydrazine successfully induced thrombus formation in zebrafish. After intervention with aspirin and honeysuckle berry polyphenols, the staining intensity of the zebrafish heart showed significant improvement. This indicates that honeysuckle berry polyphenols can inhibit PHZ-induced thrombus formation to a certain extent, restore blood supply to the zebrafish heart, and have a good antithrombotic effect.

[0068] The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for extracting blueberry polyphenols based on a deep eutectic solvent, characterized by, The method comprises the following steps: S1, freeze-drying fresh Lonicera caerulea fruits and crushing to obtain Lonicera caerulea freeze-dried powder; S2, mixing a hydrogen bond acceptor and a hydrogen bond donor reagent, heating and stirring until a transparent homogeneous liquid is formed, and naturally cooling to room temperature to obtain a DES; S3, mixing Lonicera caerulea raw materials and the DES at a ratio of 1: (10-50) g / mL, placing in an ultrasonic instrument, setting temperature, time and ultrasonic power for extraction, and centrifuging to obtain supernatant.

2. The method according to claim 1, wherein the method is characterized by, In the S2 step, the hydrogen bond acceptor is selected from one of choline chloride and betaine, and the hydrogen bond donor is selected from one of ethylene glycol, glycerol, 1,4-butanediol, 1,3-butanediol, lactic acid, urea, glucose and fructose.

3. The method according to claim 1, wherein the method is characterized by, The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1: (2-6).

4. The method according to claim 2, wherein the method is characterized by, The hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is 1,3-butanediol.

5. The method according to claim 4, wherein the method is characterized by, The molar ratio of the choline chloride to the 1,3-butanediol is 1:

5.

6. The method according to claim 1, wherein the method is characterized by: The water content of the DES solvent is 20%-40%.

7. The method according to claim 1, wherein the method is characterized by: The extraction temperature is 30-60℃.

8. The method according to claim 1, wherein the method is characterized by: The extraction time is 40-60 min.

9. Lonicera caerulea polyphenols obtained by the method of any one of claims 1-8.

10. Use of the Lonicera caerulea polyphenols of claim 9 in the preparation of antioxidants and antithrombotics.

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