Method for extracting total flavonoids in Chinese redbud by using ultrasonic-assisted eutectic solvent

Abstract

The invention discloses a method for extracting total flavonoids in Chinese redbud flowers by using an ultrasonic-assisted deep eutectic solvent (DES), belongs to the technical field of extraction and separation of natural products, and simultaneously detects the antioxidant activity of the extracted flavonoids. On the basis of screening out the optimal DES, the influence of the molar ratio, the liquid-material ratio, the water content, the ultrasonic time and the ultrasonic temperature on the Chinese redbud total flavone extraction rate is determined through a single-factor test, and the process is further optimized through a Box-Behnken response surface method. The test result shows that the extraction rate of the total flavonoids obtained by taking L-proline-lactic acid as the extraction solvent under the conditions that the molar ratio is 2: 1, the liquid-material ratio is 15mL / g, the water content is 32%, the ultrasonic time is 31 minutes and the ultrasonic temperature is 36 DEG C is up to 9.4%, which is about 7 times of the extraction efficiency of the traditional organic solvent. SEM results show that in the extraction process, DES enables the surface morphology of the Chinese redbud to be greatly changed, so that the Chinese redbud has a stronger cell wall breaking effect, and flavonoid compounds are easier to dissolve out.

Description

Technical Field

[0001] The present invention belongs to the field of extraction and separation of natural products, and particularly relates to a method for ultrasonic-assisted extraction of total flavonoids from Bauhinia flowers using a deep eutectic solvent. Background Art

[0002] Flavonoids, as secondary metabolites widely present in natural plants, have attracted much attention due to their biological activities such as antioxidant, anti-inflammatory, antibacterial, and cardiovascular protection. Bauhinia flowers are the flowers of Bauhinia chinensis, a plant of the genus Bauhinia in the legume family, and have the effects of clearing heat and cooling blood, and removing wind and detoxifying. They can be used to treat symptoms such as rheumatic pain in the muscles and bones, and nasal sores. As a traditional medicinal plant, Bauhinia flowers are rich in total flavonoids. Existing research has shown that they have significant antioxidant activity, as well as pharmacological effects of antiplatelet aggregation and antithrombosis, showing important potential in the fields of functional foods and medicine.

[0003] Traditional flavonoid extraction methods (such as hot water extraction and organic solvent extraction) have problems such as low efficiency, high energy consumption, and solvent residues (DIAS M C, PINTO D C G A, SILVA A M S. Plant flavonoids: Chemical characteristics and biological activity[J]. Molecules, 2021, 26: 5377.). In recent years, ultrasonic-assisted extraction technology has been widely used in the optimization of extraction of plant active ingredients due to its characteristic of accelerating cell breakage and component release through cavitation effects. For example, the study on the ultrasonic water extraction process of total flavonoids from Bauhinia flowers showed that under the conditions of a solid-liquid ratio of 1:8, an ultrasonic power of 70 W, and a time of 20 min, the extraction rate could reach 3.73 mg / g (Yuan Ling, Jiang Yiquan. Study on the technology of ultrasonic water extraction of total flavonoids from Bauhinia flowers[J]. Journal of Henan Agricultural Sciences, 2013, 42(3): 154 - 156.). However, the insufficient selectivity of traditional solvents (such as water or ethanol) may limit their further application. Deep Eutectic Solvents (DES), as a new type of green and designable solvent, have gradually become a research hotspot in the field of natural product extraction due to their low toxicity, high solubility, and recyclability. Although ultrasonic-assisted DES extraction technology has made progress in the extraction of flavonoids from other plants (such as Ginkgo biloba leaves and Inula japonica), there is still no report on the DES system for the extraction of total flavonoids from Bauhinia flowers.

[0004] This application aims to combine ultrasonic-assisted technology with the DES system to optimize the extraction process of total flavonoids from Bauhinia blakeana and evaluate their in vitro antioxidant activity. By screening the types and ratios of DES and ultrasonic parameters, it aims to establish an efficient and environmentally friendly extraction method and clarify the scavenging ability of the extract on PTIO free radicals. This application can provide a theoretical basis for the high-value utilization of Bauhinia blakeana resources and reference for the development of green preparation technologies for natural antioxidants. Summary of the Invention

[0005] The object of the present invention is to provide a method for extracting total flavonoids from Bauhinia blakeana by ultrasonic-assisted deep eutectic solvent.

[0006] Based on the above object, the present invention adopts the following technical solutions: A method for extracting total flavonoids from Bauhinia blakeana by ultrasonic-assisted deep eutectic solvent is as follows: 1) Preparation of DES: The hydrogen bond acceptor and the hydrogen bond donor are melted together to obtain DES, and then left to stand; the hydrogen bond acceptor is at least one of choline chloride, L-proline, and betaine; the hydrogen bond donor is at least one of levulinic acid, glycerol, ethylene glycol, urea, and lactic acid; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1.5 - 2.5):1. 2) DES extraction: The DES obtained in step 1) is mixed with water to form an extraction solvent, and then the dried Bauhinia blakeana powder is evenly mixed with the extraction solvent, and ultrasonic extraction is carried out to obtain an extraction solution of total flavonoids. 3) Separation of total flavonoids by macroporous adsorption resin: The total flavonoid extraction solution obtained in step 2) is loaded onto the macroporous adsorption resin, and eluted successively with distilled water and ethanol, and then concentrated to obtain total flavonoids.

[0007] Furthermore, the melting treatment conditions in step 1) are: stirring in a water bath at 50 - 70 °C until a homogeneous and transparent liquid is formed.

[0008] Furthermore, the particle size of the dried Bauhinia blakeana powder in step 2) is not less than 80 mesh.

[0009] Furthermore, in step 2), the DES and water are mixed to form an extraction solvent with a moisture content of 20 - 40 v%; the liquid-to-solid ratio of the extraction solvent to the Bauhinia blakeana powder is 15 - 30 mL / g; the extraction conditions are: extraction at 30 - 60 °C under ultrasonic waves of 100 - 500 W for 15 - 35 min.

[0010] Furthermore, in step 3), the macroporous adsorption resin model is ADS-17, XAD-76, DM301, NKA-9, HPD-1100, D101, or AB-8, and the adsorption and separation conditions of the macroporous adsorption resin are: the solid-to-liquid ratio is 0.55 - 2.5 g / mL.

[0011] Further, in the step 3), elute with 70v% - 90v% ethanol, collect the 70v% - 90v% ethanol eluate, and concentrate to obtain the total flavonoids.

[0012] Further, the DES is composed of L-proline and lactic acid with a water content of 30 - 32v%, wherein the molar ratio of L-proline to lactic acid is 2:1.

[0013] Further, the extraction conditions are: extract at 36 - 40 °C under ultrasonic wave of 100 - 500 W for 25 - 31 min.

[0014] This application has established a method for rapidly and efficiently extracting total flavonoids from Bauhinia flower powder. Seven kinds of DES were synthesized and their physical properties were characterized. The extraction yields of flavonoids from Bauhinia flower by traditional organic solvents (60% ethanol solution by volume) and different DES were compared. The DES synthesized from L-proline and lactic acid (molar ratio 2:1) had better extraction effect. Under the conditions of a water content of 32%, an extraction time of 31 min, a solid-liquid ratio of 15 mL / g, and an extraction temperature of 36 °C, the extraction yield of flavonoids was 9.40%, and the extraction effect was better than that of traditional organic solvents. The SEM images of Bauhinia flower before and after extraction showed that compared with traditional organic solvents, when DES was used as the solvent to extract flavonoids from Bauhinia flower, the morphological characteristics of the surface of Bauhinia flower changed greatly, resulting in the easier dissolution of flavonoids, thus improving the extraction efficiency. The total flavonoids of Bauhinia flower obtained by extraction with DES had an IC 50 value less than that of quercetin and L-ascorbic acid, indicating that it is expected to be developed into a natural antioxidant. This method not only avoids the use of toxic and harmful solvents, but also has the advantages of being green, efficient, and fast, providing a feasible basis for replacing traditional organic solvents with DES to extract natural products, and has broad application prospects in the fields of food, medicine, and chemical industry. Description of the Drawings

[0015] Figure 1 shows the extraction rates of total flavonoids from Bauhinia flower by different DES; Figure 2 shows the influence of molar ratio on the extraction rate of total flavonoids; Figure 3 shows the influence of water content on the extraction rate of total flavonoids; Figure 4 shows the influence of ultrasonic time on the extraction rate of total flavonoids; Figure 5 shows the influence of ultrasonic temperature on the extraction rate of total flavonoids; Figure 6 shows the influence of solid-liquid ratio on the extraction rate of total flavonoids; Figure 7 shows the correlation analysis between actual values and predicted values; Figure 83D response surface diagram and contour map of the interaction effects of various factors on the total flavonoid extraction rate; Figure 9 Middle: SEM images of Bauhinia flowers without treatment (a), after 60% ethanol extraction (b), and after DES-6 extraction (c) (magnification 5000 times); Figure 10 This is the result of scavenging PTIO free radicals. DETAILED DESCRIPTION

[0016] The following examples may provide a more comprehensive understanding of the present invention for those skilled in the art, but are not intended to limit the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional reagent companies.

[0017] 1 Materials and Methods 1.1 Reagents and Instruments Bauhinia flowers were collected from the Bauhinia cultivation base of Yanling Zhonglin Garden Engineering Co., Ltd.; all other chemical reagents were of analytical grade and purchased from Nanjing Angel Biotechnology Co., Ltd.

[0018] GENESYS 50 UV spectrometer, Thermo Fisher Scientific; Nova NanoSEM450 scanning electron microscope (SEM), FEI Company, USA; MS7-H550-Pro magnetic stirrer, Dalong Xingchuang Experimental Instrument (Beijing) Co., Ltd.; YL1622-4080120 ultrasonic cleaner, Yunyi Ultrasonic (Shenzhen) Co., Ltd.; Thermo Fisher Heraeus Pico 17 centrifuge, Thermo Fisher Scientific; PHS-23C digital display benchtop pH meter, Shaoxing Supo Instrument Co., Ltd.; NDJ-8S digital rotational viscometer, Shanghai Yutong Instrument Factory; Multiskan FC microplate reader, Thermo Fisher Scientific.

[0019] 1.2 Synthesis of DES Seven DESs were synthesized using choline chloride, L-proline and betaine as hydrogen bond acceptors (HBAs) and levulinic acid, glycerol, ethylene glycol, lactic acid and urea as hydrogen bond donors (HBDs).

[0020] As shown in Table 1, appropriate amounts of HBA and HBD were weighed according to a specific molar ratio, placed in a round-bottom flask, heated in an oil bath at 60°C with magnetic stirring until a homogeneous transparent liquid was formed, and then cooled to room temperature and stored in a vacuum desiccator for later use.

[0021] Table 1 Seven kinds of DES prepared 1.3 Characterization viscosity of DES: The viscosity test was carried out at room temperature using a digital viscometer. Rotors 1#, 2#, 3#, and 4# were selected and measured at rotation speeds of 6, 12, 30, and 60 rpm respectively.

[0022] Polarity: Nile red dye was used as a solvent chromogenic probe. 10 mg of nile red dye was dissolved in an ethanol solution and made up to 10 mL in a volumetric flask. 1 mL of the stock solution was diluted to 100 mL to obtain a 0.01 mg / mL nile red ethanol solution. 0.3 mL of the nile red ethanol solution was mixed with 2.4 mL of DES, shaken well, and its maximum absorption wavelength was scanned in the wavelength range of 200 - 800 nm using a UV-visible spectrophotometer. The polarity parameter (ENR) of nile red was calculated according to formula (1): ENR = hcNA / λ max (1) Where: ENR is the polarity parameter of nile red, kcal / mol; h is Planck's constant; c is the speed of light in a vacuum, m / s; NA is Avogadro's constant; λ max is the maximum absorption wavelength in the UV-visible spectrogram, nm.

[0023] 1.4 Preparation of test solution The Bauhinia flowers were dried to a constant weight at 85 °C, pulverized by a pulverizer, passed through an 80-mesh sieve, and placed in a sealed bag for later use. 0.125 g of Bauhinia flower powder was weighed into a stoppered conical flask, 4 mL of the DES listed in Table 1 was added, and ultrasonic extraction (power 250 W) was carried out for 20 min. The extraction conditions were: moisture content 20%, liquid-to-solid ratio 30 mL / g, extraction temperature 60 °C. Then it was centrifuged at 6000 r / min for 5 min, the supernatant was recovered and filtered through a 0.22 μm polyethersulfone (PES) microporous membrane. The obtained test solution was used for the determination of total flavonoids.

[0024] 1.5 Preparation of control solution 0.125 g of Bauhinia flower powder was weighed into a stoppered conical flask, 4 mL of 60% (v / v) ethanol solution was added, and the subsequent operations were the same as those for the preparation of the test solution.

[0025] 1.6 Determination of flavonoid extraction rate The NaNO2-Al(NO3)3-NaOH colorimetric method in the reference (Saru Li, Muqier, Wang Cuifang, et al. Study on the total flavonoid content and its in vitro antioxidant and antibacterial activities of extracts from different parts of Allium mongolicum Regel [J]. Science and Technology of Food Industry, 2014, 35(22): 124-127+134.) was used for determination. The specific operation steps are as follows: Accurately weigh 6.0 mg of rutin reference substance, dissolve it with absolute ethanol, and make up the volume to 30 mL in a volumetric flask, shake well to obtain a standard solution to be measured with a concentration of 0.2 mg / mL, and store it in a refrigerator at 4 °C for later use; Absorb 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mL of rutin standard product into 1-8th 1-mL volumetric flasks respectively, add 60 v% ethanol to 1 mL, and the blank control group does not add the standard product; Add 30 μL of 20 wt% NaNO2 solution, fully mix and react for 3 min, add 40 μL of 40 wt% Al(NO3)3 solution, fully mix and react for 3 min, then add 100 μL of 20 wt% NaOH solution, fully mix and react for 10 min, and pipette 200 μL from each tube to measure the absorbance value of each solution at 510 nm. Taking the mass concentration of rutin as the abscissa (X) and the absorbance value as the ordinate (Y) to draw a standard curve, and its linear regression equation is: Y = 9.28561X + 0.04328, where R 2 = 0.99212.

[0026] The method for determining the total flavonoid content in the sample is: Absorb 0.1 mL of the extract into a test tube, after carrying out the color reaction according to the above steps, add 1 mL of 60 v% ethanol for dilution, pipette 200 μL of the solution to measure the absorbance at 510 nm, and combine the standard curve equation and formula (2) to obtain the total flavonoid extraction rate (%): Total flavonoid extraction rate (%) = [(CVN × 10 -3 ) / M] × 100% (2) In the formula: C is the concentration of flavonoids in the sample solution, mg / mL; V is the volume of the test solution, mL; N is the dilution factor; M is the mass of the Bauhinia blakeana Dunn powder, g.

[0027] 1.7 Optimization of flavonoid extraction conditions with DES On the basis of determining L-proline-lactic acid combination as the target DES, referring to "1.4 Preparation of test solution", the molar ratio of L-proline-lactic acid (1:3, 1:2, 1:1, 2:1, 3:1), the water content of DES (0, 10, 20, 30, 40, 50, 60 v%), the liquid-to-solid ratio of DES to Bauhinia blakeana Dunn powder (5, 10, 15, 20, 25, 30 mL / g) and the ultrasonic extraction time (15, 20, 25, 30, 35, 40 min), and the ultrasonic extraction temperature (30, 40, 50, 60, 70 °C) were screened in turn to determine the optimal extraction process parameters.

[0028] 1.8 Response surface experimental design On the basis of single-factor experiments, with extraction temperature, extraction time, and DES moisture content as influencing factors and flavonoid extraction rate as the evaluation index, a three-factor and three-level response surface experiment was designed using Design-Expert 13 with Box-Behnken design.

[0029] 1.9 Determination of PTIO free radical scavenging activity The PTIO free radical scavenging activity experiment was carried out according to the method reported in the reference (Saruli, Muqier, Wang Cuifang, et al. Study on the total flavonoid content and in vitro antioxidant and antibacterial activities of extracts from different parts of Allium mongolicum Regel [J]. Science and Technology of Food Industry, 2014, 35(22): 124 - 127 + 134.).

[0030] 1.9.1 Solution preparation PTIO free radical working solution: Take 3 mg of PTIO solid and dissolve it in 18 - 20 mL of methanol to control the absorbance value at 0.2 - 0.3, obtaining a dark blue PTIO free radical working solution. Store it in the dark for later use.

[0031] Sample solution: Take 1 mg of each compound and dissolve it in methanol to make 0.25 mg / mL solutions. Then, through serial dilution, prepare 0.125, 0.0625, 0.03125, and 0.015625 mg / mL sample solutions from the 0.25 mg / mL sample solution.

[0032] Positive control (L-ascorbic acid, quercetin) solution: Take 1 mg of L-ascorbic acid or quercetin and dissolve it in deionized water to make 0.25 mg / mL solutions. Then, through serial dilution, prepare 0.125, 0.0625, 0.03125, and 0.015625 mg / mL sample solutions from the 0.25 mg / mL sample solution.

[0033] 1.9.2 Activity determination Add 40 μL of the sample solution and 160 μL of the PTIO solution to a 96-well microplate as the sample group. Add 40 μL of the L-ascorbic acid solution or quercetin solution and 160 μL of the PTIO solution to a 96-well microplate as the positive control. Add 40 μL of methanol solution and 40 μL of deionized water respectively and 160 μL of the PTIO solution to a 96-well microplate as the negative control. After incubating at 37 °C in the dark for 30 min, measure the absorbance at 585 nm using a Multiskan FC microplate reader. Set 3 replicates for each sample concentration. The PTIO free radical scavenging activity is calculated using formula (3).

[0034] [[ID= Where: As is the absorbance of the sample group; Ac is the absorbance of the negative control group.

[0035] 2 Results 2.1 Characterization of DES The polarity parameters, pH, and viscosity of 7 kinds of DES are shown in Table 2. Polarity is caused by the uneven distribution of charges in covalent bonds or covalent molecules. The DES with choline chloride and betaine as hydrogen bond acceptors has slightly lower polarity, while the DES with L-proline as the hydrogen bond acceptor has slightly higher polarity, which is 0.0211 kcal / mol. The viscosity of DES is regulated by intermolecular forces (such as hydrogen bonds and van der Waals forces), which directly affects the mass transfer efficiency. Among the 7 kinds of DES prepared, the DES with L-proline as the hydrogen bond acceptor has the highest viscosity, which is 699 mPa·s. From the perspective of pH, all 7 kinds of DES are slightly acidic, and DES-1 has the strongest acidity with a pH of 1.45.

[0036] Table 2 Polarity parameters, viscosity, and pH of DES 2.2 Screening of DES Taking the total flavonoid extraction rate as the evaluation index, the DES with the best extraction effect on Bauhinia blakeana flavonoids was screened. Under the same extraction conditions (ultrasonic extraction at 250 W for 20 min, water content of 20%, liquid-solid ratio of 30 mL / g, extraction temperature of 60 °C), the extraction effect is as Figure 1 shown.

[0037] 2.3 Optimization of DES extraction conditions 2.3.1 Molar ratio The molar ratio can significantly affect the physical and chemical properties of DES, such as density, intermolecular forces, viscosity, and polarity. In this study, 5 different molar ratios (1:3, 1:2, 1:1, 2:1, 3:1) were set to explore the effect of different molar ratios on the total flavonoid extraction rate. As Figure 2 can be seen, when the content of L-proline gradually increases, the flavonoid extraction rate first increases and then decreases. When the molar ratio is 2:1, the flavonoid extraction rate increases to 8.77%. If the proportion of L-proline continues to increase, the flavonoid extraction rate will decrease significantly. This may be because the number of hydrogen bonds formed has reached the maximum at this time, and the competition of too many hydrogen bond acceptors makes the formation of hydrogen bonds unstable, resulting in a decrease in the extraction amount. Therefore, the optimal molar ratio of L-proline: lactic acid is selected as 2:1, and further single-factor experiments such as water content are carried out.

[0038] 2.3.2 Water content There is a strong hydrogen bond interaction between the two eutectic components. An appropriate water content can reduce the viscosity of the solvent, thereby increasing its solubility for the extract. In this study, the effects of different water contents (0, 10, 20, 30, 40, 50, 60%) on the extraction efficiency were investigated. Under the same extraction conditions, only the water content of the DES was changed. The results are as Figure 3 shown. When the water content was 0 - 30%, the extraction rate of total flavonoids increased with the increase of water content. When the water content was 30 - 60%, the extraction rate of total flavonoids decreased with the increase of water content. When the water content was 30%, the extraction rate of total flavonoids reached the maximum value of 10.02%. The reason may be that when the water content increased, the viscosity of the eutectic solvent decreased, which was beneficial to the extraction of total flavonoids. However, with the further increase of water content, the interaction between hydrogen bonds gradually weakened, so the extraction rate of total flavonoids decreased. Therefore, the optimal water content was 30%.

[0039] 2.3.3 Extraction time The ultrasonic time has a great influence on the extraction of active ingredients. If the time is too long, the components may decompose. If the time is too short, the components may not be completely dissolved. In this study, the effects of different ultrasonic times (15, 20, 25, 30, 35, 40 min) on the extraction efficiency were investigated. Under the same extraction conditions, only the ultrasonic time was changed. The results are as Figure 4 shown. When the ultrasonic time was 15 - 30 min, the extraction rate of total flavonoids increased with the increase of ultrasonic time. When the ultrasonic time was 30 - 40 min, the extraction rate of total flavonoids decreased with the increase of ultrasonic time. When the ultrasonic time was 30 min, the extraction rate of total flavonoids reached the maximum value of 6.94%. The reason may be that when the ultrasonic time increased, the total flavonoids were more completely dissolved. However, with the further increase of ultrasonic time, the ultrasonic waves destroyed the structure of total flavonoids and caused their decomposition, so the extraction rate of total flavonoids decreased. Therefore, the optimal ultrasonic time was 30 min.

[0040] 2.3.4 Extraction temperature Increasing the temperature may reduce the viscosity of the DES, thereby increasing its solubility for the extract. In this study, the effects of different ultrasonic temperatures (30, 40, 50, 60, 70 °C) on the extraction efficiency were investigated. Under the same extraction conditions, only the ultrasonic temperature was changed. The results are as Figure 5 shown.

[0041] When the ultrasonic temperature is 30 - 40 °C, the extraction rate of total flavonoids increases with the increase of ultrasonic temperature. When the ultrasonic temperature is 40 - 70 °C, the extraction rate of total flavonoids decreases with the increase of ultrasonic temperature. When the ultrasonic temperature is 40 °C, the extraction rate of total flavonoids reaches the maximum of 6.58%. The reason may be that when the ultrasonic temperature increases, the viscosity of the deep eutectic solvent decreases, thus increasing the solubility of total flavonoids. However, with the increase of ultrasonic temperature, on the one hand, the interaction between hydrogen bonds is disrupted, and on the other hand, the structure of total flavonoids may be damaged, so the extraction rate of total flavonoids decreases. Therefore, the optimal ultrasonic temperature is selected as 40 °C.

[0042] 2.3.5 Solid-liquid ratio In this study, the effects of different solid-liquid ratios (5, 10, 15, 20, 25, 30 mL / g) on the extraction effect were investigated. Under the same extraction conditions, only the solid-liquid ratio was changed. The results are as Figure 6 shown. When the solid-liquid ratio is 5 - 15 mL / g, the extraction rate of total flavonoids increases with the increase of the solid-liquid ratio. When the solid-liquid ratio is 15 - 30 mL / g, the extraction rate of total flavonoids decreases with the increase of the solid-liquid ratio. When the solid-liquid ratio is 15 mL / g, the extraction rate of total flavonoids reaches the maximum of 5.51%. The reason may be that when the solid-liquid ratio increases, the contact area between the extraction solvent and the Bauhinia blakeana flower powder increases, thus increasing the solubility of total flavonoids. However, with the increase of the solid-liquid ratio, on the one hand, the concentration of the extraction solution decreases, and on the other hand, other substances may be dissolved out, resulting in a decrease in the total flavonoid content, so the extraction rate of total flavonoids decreases. Therefore, the optimal solid-liquid ratio is selected as 15 mL / g.

[0043] 2.4 Response surface methodology On the basis of the single-factor experiments, with the solid-liquid ratio fixed at 15 mL / g, the ultrasonic temperature (A), ultrasonic time (B), and moisture content (C), which have relatively significant effects, were taken as the investigated factors, and the extraction rate of total flavonoids was taken as the investigated index. The Box-Behnken method was used to design a response surface experiment with 3 factors and 3 levels. The relevant levels and factors are shown in Table 3, and the results are shown in Table 4.

[0044] Table 3 Factors and levels selected for the response surface experiment Table 4 Design and results of the response surface optimization experiment 2.4.1 Establishment and analysis of the total flavonoid content model The Design expert software was used to perform multiple regression fitting on the experimental data. The results showed that the fitting degree of the quadratic model was good. Using the quadratic model to analyze the data, the following quadratic multiple regression model was obtained: Y = -94.03875 + 1.65837A + 3.17875B + 1.559C - 0.02145AB - 0.0055AC + 0.0025BC - 0.011462A 2 - 0.04025B 2 - 0.022638C 2 。

[0045] Through analysis of variance, it was found that the P - value of the model was less than 0.0001, indicating a highly significant difference. At the same time, the P - value of the lack - of - fit term was 0.5349, which was not significant, indicating that the goodness of fit of the model was good. As can be seen from Table 5, the quadratic - term interaction AB had a highly significant effect on the total flavonoid content, AC had a significant effect on the total flavonoid content, and BC had an insignificant effect on the total flavonoid content. The order of influence magnitude was AB > AC > BC. The determination coefficient R 2 of the model was 0.9794, indicating a high significance of the model. At the same time, R 2 Ad j = 0.9529, which could explain 95.29% of the response value variation in the experiment, and the difference from the predicted correlation coefficient R 2 Pre = 0.8520 was within 0.2.

[0046] Table 5 Regression analysis of the total flavonoid extraction rate model and regression coefficients Note: * indicates significant difference (P < 0.05); ** indicates highly significant difference (P < 0.01); ns indicates insignificant.

[0047] The experimental data was subjected to multiple - regression fitting, and the correlation analysis (see Figure 7 ) showed that all points in the two models were close to the regression line (blue points: extraction rate was 5.14 - 5.7%; green points: extraction rate was 6.39 - 8.01%; black points: extraction rate was 8.63 - 9.79%), indicating a high correlation between the experimental values and the predicted values. Therefore, this model could be used to analyze and predict the optimal process for the total flavonoid extraction rate. According to the magnitude of the F - value, the order of influence of each factor on the total flavonoid extraction rate of Bauhinia blakeana was: ultrasonic temperature (A) > moisture content (C) > ultrasonic time (B).

[0048] 2.4.2 Interaction of factors The steepness of the response surface and the elliptical shape of the contour plot can intuitively reflect the influence of the interaction between factors on the total flavonoid extraction rate of Bauhinia blakeana. The steeper the response surface and the more elliptical the contour plot, the more obvious the interaction between the two factors. Conversely, it is not obvious. The interaction between factors is shown in Figure 8 . As shown by Figure 8It can be seen that the response surface slope of ultrasonic temperature and ultrasonic time is the largest, and the contour map is closest to the ellipse, indicating that the interaction between ultrasonic temperature and ultrasonic time has the greatest effect on the extraction rate of total flavonoids from Bauhinia flowers, which is consistent with the variance analysis results in Table 5.

[0049] 2.5 Verification Experiment Using Design Expert software, we determined the optimal extraction parameters for total flavonoids from Bauhinia flowers: ultrasonic temperature of 35.753°C, ultrasonic time of 30.951 minutes, and a moisture content of 31.791%. Under these conditions, the theoretical total flavonoid extraction yield reached 9.584%. Considering operational convenience and feasibility, we adjusted the parameters appropriately, ultimately settling on an ultrasonic temperature of 36°C, an ultrasonic time of 31 minutes, and a moisture content of 32%.

[0050] Three parallel experiments were conducted on 20g of dried Bauhinia flower (80-mesh) powder using the optimal extraction parameters (ultrasonic extraction temperature 36°C, ultrasonic extraction time 31 minutes, moisture content 32% by volume, solid-liquid ratio 15mL / g, L-proline-lactic acid eutectic solution molar ratio 2:1). The results showed an actual total flavonoid yield of 9.4%, which was very close to the theoretically predicted value. This result demonstrates that the model has a good fit and can accurately guide the optimized extraction of total flavonoids from Bauhinia flower, providing reliable technical support and theoretical basis for related production practices.

[0051] The extract was purified on AB-8 macroporous adsorption resin (solid-to-liquid ratio of macroporous adsorption resin to extract: 2.5 g / mL). Elution was performed sequentially with distilled water (total volume approximately 6 times the volume of the sample solution) and then with 90% ethanol (total volume approximately 15 times the volume of the sample solution). The 90% ethanol eluate was collected and concentrated to yield 1.83 g of total flavonoids from Bauhinia chinensis (extraction yield: 9.15%).

[0052] 2.6 SEM analysis Natural products such as flavonoids are usually distributed in cells (such as cell walls) and subcellular (such as vacuoles) tissues. Destroying the cell walls helps flavonoids to flow out faster and improve the extraction efficiency. This study used SEM to observe the changes in the morphology and structure of Bauhinia flower powder before and after extraction. The results are as follows Figure 9 As shown. Figure 9 The surface texture of the Bauhinia flower, untreated with an extraction solvent, is clearly visible; however, after ultrasonic extraction with a 60% ethanol solution, the texture becomes noticeably distorted. After ultrasonic treatment with DES-6 (L-proline-lactic acid), the texture structure is almost completely eliminated. This is likely due to the DES's enhanced cell wall disruption during the extraction of flavonoids from Bauhinia flower, which facilitates the dissolution of the flavonoids and significantly improves extraction efficiency.

[0053] The scavenging activity of 2.7PTIO free radicals is Figure 10 It can be seen that the scavenging ability of total flavonoids from Bauhinia blakeana Dunn on PTIO free radicals increases with the increase of concentration. In the concentration range of 0.015625 - 0.125 mg / mL, the scavenging rate of total flavonoids on PTIO free radicals is higher than that of the positive controls quercetin and L-ascorbic acid. At 0.125 mg / mL, the scavenging rate of total flavonoids on PTIO free radicals is 65.25%, higher than that of the positive controls quercetin (63.23%) and L-ascorbic acid (64.31%). Using Graphpad Pism9.5 software to fit the curve, the IC 50 values of total flavonoids from Bauhinia blakeana Dunn, quercetin and L-ascorbic acid on PTIO free radicals are 39.41, 52.78 and 76.01 μg / mL respectively, indicating that the antioxidant ability of total flavonoids from Bauhinia blakeana Dunn is relatively strong.

Claims

1. A method for ultrasonic-assisted extraction of total flavonoids from Bauhinia blakeana flowers using a deep eutectic solvent, characterized in that, The process is as follows: 1) Preparation of DES: The hydrogen bond acceptor and the hydrogen bond donor are melted together to obtain DES, and then left standing; the hydrogen bond acceptor is at least one of choline chloride, L-proline, and betaine; the hydrogen bond donor is at least one of levulinic acid, glycerol, ethylene glycol, urea, and lactic acid; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1.5~2.5):1; 2) DES extraction: The DES obtained in step 1) is mixed with water to form an extraction solvent, and then the dried Bauhinia flower powder is mixed evenly with the extraction solvent and extracted by ultrasound to obtain an extract of total flavonoids; 3) Separation of total flavonoids by macroporous adsorption resin: The total flavonoid extract obtained in step 2) is loaded onto the macroporous adsorption resin, and is eluted successively with distilled water and ethanol, and concentrated to obtain total flavonoids.

2. The method for ultrasonic-assisted extraction of total flavonoids from Bauhinia blakeana Dunn using deep eutectic solvents according to claim 1, wherein, The melting treatment conditions in step 1) are: stirring in a water bath at 50~70°C until a homogeneous and transparent liquid is formed.

3. The method for extracting total flavonoids from Bauhinia blakeana flowers by ultrasonic-assisted deep eutectic solvent according to claim 1, characterized in that The particle size of the dried Bauhinia flower powder in step 2) is not less than 80 mesh.

4. The method for ultrasonic-assisted extraction of total flavonoids from Bauhinia blakeana Dunn with deep eutectic solvents according to claim 1, characterized in that, In step 2), the DES and water are made into an extraction solvent with a moisture content of 20~40 v%; the liquid-solid ratio of the extraction solvent to the Bauhinia flower powder is 15~30 mL / g; the extraction conditions are: 30~60°C, extraction for 15~35 min under ultrasound of 100~500 W.

5. The method for ultrasonic-assisted extraction of total flavonoids from Bauhinia blakeana Dunn with deep eutectic solvents according to claim 1, characterized in that, In step 3), the macroporous adsorption resin model is ADS-17, XAD-76, DM301, NKA-9, HPD-1100, D101, or AB-8, and the adsorption and separation conditions of the macroporous adsorption resin are: the solid-liquid ratio is 0.55~2.5 g / mL.

6. The method for ultrasonic-assisted extraction of total flavonoids from Bauhinia blakeana Dunn using deep eutectic solvents according to claim 1, wherein In step 3), elution is carried out with 70 v%~90 v% ethanol, the 70 v%~90 v% ethanol eluate is collected, and concentrated to obtain total flavonoids.

7. The method for ultrasonic-assisted extraction of total flavonoids from Bauhinia blakeana Dunn using deep eutectic solvents according to claim 4, wherein The DES is composed of L-proline and lactic acid with a moisture content of 30~32 v%, and the molar ratio of L-proline to lactic acid is 2:

1.

8. The method for ultrasonic-assisted extraction of total flavonoids from Bauhinia blakeana Dunn with deep eutectic solvents according to claim 4, wherein The extraction conditions are: 36~40°C, extraction for 25~31 min under ultrasound of 100~500 W.

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