Method for co-extracting flavonoids and polysaccharides from hawthorn by ultrasonic synergistic deep eutectic solvent

Abstract

This invention discloses a method for the ultrasonic-assisted co-extraction of hawthorn flavonoids and polysaccharides using a eutectic solvent, belonging to the field of natural active ingredient technology. The method includes: mixing choline chloride with a hydrogen bond donor, heating and stirring until clear and transparent, adding deionized water and mixing thoroughly to obtain a eutectic solvent-water mixture; mixing hawthorn powder with the eutectic solvent-water mixture and extracting in an ultrasonic water bath; after ultrasonic extraction, allowing the mixture to stand and then centrifuging to collect the supernatant, which is the extract containing hawthorn flavonoids and polysaccharides. The ultrasonic-assisted eutectic solvent method, through the combination of designable solvent characteristics and enhanced physical mass transfer, provides a green solution for the simultaneous and efficient extraction of multiple components in a single process, offering unique advantages in simplifying the process and improving efficiency.

Description

Technical Field

[0001] This invention belongs to the field of natural active ingredient extraction technology, specifically involving a method for ultrasonic-assisted co-extraction of hawthorn flavonoids and polysaccharides using a eutectic solvent. Background Technology

[0002] Hawthorn, a traditional food and medicinal resource, is rich in various bioactive components. Among them, flavonoids and polysaccharides have significant value in the development of functional foods, health products, and drugs due to their remarkable antioxidant, anti-inflammatory, and lipid-lowering physiological functions. However, the current hawthorn processing industry is still dominated by primary products with low added value and low utilization rate of active ingredients. One of the core limiting factors is the lack of efficient, green technologies that can achieve synergistic extraction of multiple components. Traditional extraction methods, such as hot reflux and organic solvent extraction, generally suffer from high energy consumption, low extraction efficiency, easy destruction of heat-sensitive components, and the use of toxic and flammable solvents. Moreover, most processes only optimize for single-category components and cannot achieve efficient co-extraction of multiple components such as flavonoids and polysaccharides, resulting in resource waste.

[0003] In recent years, eutectic solvents, as a novel type of green solvent, have attracted widespread attention in the field of natural product extraction due to their advantages such as low toxicity, biodegradability, low vapor pressure, and ease of design and synthesis. Studies have shown that by rationally selecting the combination and ratio of hydrogen donors and acceptors, eutectic solvents can achieve efficient and highly selective extraction of specific target components. Meanwhile, ultrasonic extraction technology, through the powerful impact force, microjets, and mechanical vibration generated by cavitation effects, can efficiently disrupt plant cell walls, enhance solvent penetration and solute diffusion, thereby significantly shortening extraction time and improving extraction efficiency, especially suitable for the extraction of heat-sensitive flavonoid components. For example, Sun et al. (2024) systematically studied the extraction of hawthorn flavonoids using ultrasound-assisted eutectic solvents, screening out a choline chloride-ethylene glycol (molar ratio 1:2) system, which achieved a flavonoid extraction rate of 5.8% under optimized conditions, significantly better than the traditional ethanol extraction method (1.6%).

[0004] Although the extraction process of hawthorn flavonoids using ultrasound-assisted eutectic solvents is relatively mature and achieves significantly higher extraction rates than traditional solvents, existing research mainly focuses on optimizing single-component processes, which restricts the comprehensive utilization of hawthorn resources and the development of high-value-added products. With single-component extraction technologies maturing, the synergistic and efficient extraction of multiple active components from hawthorn has become a key direction for achieving systematic resource utilization and promoting industrial upgrading.

[0005] In recent years, green extraction technology for natural products has gradually shifted from high-yield extraction of single components to integrated extraction and separation of multiple components. Examples include methods for preparing chia seed extract rich in organic active ingredients and inorganic elements, methods for simultaneously extracting eleutheroside B, eleutheroside E, and isozygoside, and methods for simultaneously extracting and separating flavonoids and alkaloids from mulberry leaves. However, these systems have failed to address the solubility challenges caused by the significant polarity difference between highly polar polysaccharides and moderately polar flavonoids in hawthorn, or the difficulty in balancing extraction efficiency and product integrity in process design. A universal, high-efficiency co-extraction system suitable for the synergistic dissolution of highly polar polysaccharides and moderately polar flavonoids in hawthorn has not yet been developed. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for ultrasonic-assisted co-extraction of hawthorn flavonoids and polysaccharides using a eutectic solvent.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for ultrasonic-assisted co-extraction of hawthorn flavonoids and polysaccharides using a eutectic solvent, comprising, Choline chloride was mixed with a hydrogen bond donor, heated and stirred until clear and transparent, and then deionized water was added and mixed evenly to obtain a eutectic solvent-water mixture. Hawthorn powder was mixed evenly with a eutectic solvent-water mixture and then extracted in an ultrasonic water bath. After ultrasonic extraction, the mixture was allowed to stand and then centrifuged. The supernatant was collected, which was an extract containing hawthorn flavonoids and polysaccharides.

[0010] In a preferred embodiment of the method described in this invention, the hydrogen bond donor is a single-component or a two-component, wherein the single-component includes any one of ethylene glycol, glycerol, sorbitol, fructose, glucose, sucrose, lactic acid, citric acid, and urea. The two components are one of a mixture of ethylene glycol and lactic acid, or a mixture of ethylene glycol and citric acid.

[0011] In a preferred embodiment of the method described in this invention, the molar ratio of choline chloride to hydrogen bond donor is 1:(1~5).

[0012] As a preferred embodiment of the method described in this invention, the eutectic solvent-water mixture contains a water content of 10% to 90% by volume.

[0013] In a preferred embodiment of the method described in this invention, the heating and stirring temperature is 60~80℃ and the stirring speed is 100~800rpm.

[0014] In a preferred embodiment of the method described in this invention, the hawthorn powder is dried hawthorn powder with a particle size of less than 18 mesh.

[0015] In a preferred embodiment of the method described in this invention, the solid-liquid ratio of the hawthorn powder and the eutectic solvent-water mixture is 1g:10~80mL.

[0016] In a preferred embodiment of the method described in this invention, the ultrasonic extraction temperature is 20~80℃, the extraction power is 100~360W, and the extraction time is 10~50min.

[0017] In a preferred embodiment of the method described in this invention, the centrifugation temperature is 4~25℃, the centrifugation speed is 6000~10000 rpm / min, the time is 10~20 min, and the number of times is 2.

[0018] In a preferred embodiment of the method described in this invention, the extraction rates of flavonoids and polysaccharides are 8.42%-10.62% and 2.05%-6.28%, respectively.

[0019] Beneficial effects of this invention: (1) High extraction efficiency, achieving synergistic extraction The cavitation, mechanical, and thermal effects of ultrasound, combined with the high solubility and strong permeability of the eutectic solvent, produce a significant synergistic effect, powerfully disrupting plant cell walls, greatly accelerating mass transfer, and increasing the extraction yield of flavonoids and polysaccharides. Furthermore, by rationally designing the composition of the eutectic solvent to make its polarity adjustable, it can simultaneously and efficiently dissolve and extract flavonoids and polysaccharides, solving the problem of step-by-step extraction required in traditional processes and achieving synergistic and efficient co-extraction in a single process.

[0020] (2) Green and environmentally friendly, with good safety The eutectic solvent used has good thermal stability and low volatility. It is composed of natural and renewable components (such as choline, organic acids, and sugars), has good biocompatibility, is easy to biodegrade, and can be recycled and reused. It is environmentally friendly and avoids the toxicity, flammability and explosion risks, and residual pollution problems of traditional organic solvents (such as methanol and acetone), which are in line with the principles of green chemistry and sustainable development.

[0021] (3) Mild conditions protect heat-sensitive components Ultrasonic-assisted extraction is usually carried out at a lower temperature and for a shorter time, which can effectively avoid the oxidation, decomposition or structural damage of heat-sensitive flavonoids in hawthorn caused by prolonged high-temperature heating, and better maintain their biological activity.

[0022] (4) Solvents are designable, with strong selectivity and versatility. The physicochemical properties (polarity, viscosity, pH) of eutectic solvents can be precisely designed and controlled by changing the types and ratios of hydrogen bond donors and acceptors, thereby “customizing” the extraction of specific target components (flavonoids, polysaccharides) in hawthorn and improving the selectivity and efficiency of extraction. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a screening diagram of the eutectic solvent in Example 2 of this invention.

[0024] Figure 2 The diagram shows the Pearson coefficients between the physical properties of the eutectic solvent and the extraction rates of hawthorn flavonoids and polysaccharides in Examples 1-2 of this invention.

[0025] Figure 3 This is a graph showing the effect of ultrasonic power on the extraction rate of hawthorn flavonoids and polysaccharides in Example 3 of this invention.

[0026] Figure 4 This is Example 4 of the project, which illustrates the effect of ultrasound time on the extraction rate of hawthorn flavonoids and polysaccharides.

[0027] Figure 5 Example 5 of this project illustrates the effect of ultrasonic temperature on the extraction rate of hawthorn flavonoids and polysaccharides.

[0028] Figure 6 This study examines the effect of the low eutectic solvent molar ratio on the extraction rates of hawthorn flavonoids and polysaccharides in Example 6 of this project.

[0029] Figure 7 This invention relates to the effect of water content in the eutectic solvent on the extraction rate of hawthorn flavonoids and polysaccharides in Example 7.

[0030] Figure 8 This is the effect of solid-liquid ratio on the extraction rate of hawthorn flavonoids and polysaccharides in Example 8 of the present invention.

[0031] Figure 9 This is the effect of response surface methodology experiment in Example 9 of the present invention on the extraction rate of hawthorn flavonoids and polysaccharides.

[0032] Figure 10 The scanning electron microscope images of Example 10 of this invention show the morphological changes of hawthorn powder before and after ultrasonic extraction. The morphology of the powder before extraction is shown in Figures AC. The magnetic stirring water bath (DF) and ultrasonic treatment (GI) were performed at magnifications of 500x (A, D, G), 1000x (B, E, H), and 5000x (C, F, I), respectively. Detailed Implementation

[0033] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0034] The key characteristic index determination method for the efficient co-extraction of hawthorn flavonoids and polysaccharides using ultrasound-assisted eutectic solvents in this invention embodiment is as follows: 1. Determination of physical properties of eutectic solvents 1.1 pH The pH of eutectic solvent-water mixtures was determined at a constant temperature using a benchtop pH analyzer calibrated with standard pH solutions of pH 4.00, 6.86, and 9.18.

[0035] 1.2 Viscosity The viscosity of the solution was determined using a portable digital viscometer (Atago Corporation, Japan). The sample solution was poured into the glass graduated cup provided with the viscometer, up to the graduation mark appropriate for the rotor. A suitable rotor (A1S) was selected and securely attached to the spindle. The rotor was immersed in the solution, ensuring it was completely submerged, and the viscosity was measured at 60 rpm. The measurement was repeated three times, and the average value was calculated.

[0036] 1.3 Polarity A 10 μg / ml stock solution was prepared in 96% ethanol using Nile Red as a probe and stored at 4 °C. Subsequently, 3 ml of the stock solution was mixed with 1 ml of a eutectic solvent, and the maximum absorption wavelength was measured in the range of 400–800 nm using a UV-Vis spectrophotometer. The polarity of the eutectic solvent was quantified according to the molar transition energy ET (NR) based on equation (1).

[0037] (1) Where: h represents Planck's constant, the speed of light is represented by c, vmax is related to the longest absorption, NA corresponds to Avogadro's constant, and λmax represents the absorption spectrum of the dye.

[0038] 1.4 density Select several 10 mL standard specific gravity bottles with capillary tubes and wash them sequentially with water and ethanol. After cleaning, dry and cool the specific gravity bottles. Then weigh them accurately to obtain the initial mass, m0. Then fill the specific gravity bottles with freshly boiled and cooled 15°C distilled water, place them in a 20°C constant temperature water bath for 10 minutes, and carefully adjust the liquid in the capillary tubes to the mark. Remove the specific gravity bottles and wipe them dry quickly, then immediately weigh m1 to measure the density ρ0. After removing the distilled water, clean the specific gravity bottles and repeat the same process to measure the mass m2 of the sample liquid. The density of the sample ρ2 can then be calculated using equation (2). Equation (2) In the formula, A is the air buoyancy correction value; ρ a The density of dry air at 20°C and 1 atmosphere is approximately 0.0012 g / mL; m2 represents the mass of water required to fill the specific gravity bottle, in g; ρ0 represents the density of water at 20°C, which is 0.99820 g / mL.

[0039] 2. Determination of active ingredient content in hawthorn 2.1 Determination of Flavonoid Content Weigh 10.0 mg of dried rutin standard sample and place it in a 100 mL beaker. Add an appropriate amount of 60% ethanol. Stir with a glass rod to accelerate dissolution. After dissolution, transfer it to a 100 mL volumetric flask containing 60% ethanol. Fix the solution to the mark and shake well to obtain a rutin standard solution with a mass concentration of 0.10 mg / mL. Pipette 0.0 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, and 0.7 mL of the rutin standard solution into 5 mL brown quantitative flasks, add 0.1 mL of 5% sodium nitrite solution, mix by inverting, and let stand for 6 min. Add 0.1 mL of 10% aluminum nitrate solution, mix well, and let stand for 6 min. Add 1 mL of 4% sodium hydroxide solution, and dilute to the mark with 60% ethanol. After standing for 6 min, measure the absorbance value at 510 nm. A standard curve for rutin was established with concentration gradients ranging from 10 to 70 μg / mL. The linear regression equation was: Y = 0.00191X + 0.00392, where R0... 2 =0.9993.

[0040] Sample determination: Accurately measure 1 mL of the extract with an appropriate dilution factor and place it in a 5 mL brown quantitative bottle. Measure its absorbance according to the standard curve preparation method. Repeat each group 3 times. Substitute the values ​​into the regression equation of the standard curve and calculate the flavonoid extraction rate by taking the average value.

[0041] 2.2 Polysaccharide content determination The glucose standard was dried to constant weight in a 105℃ oven. 10 mg of the standard was accurately weighed into a beaker, dissolved in an appropriate amount of deionized water, and then transferred to a 100 mL volumetric flask and diluted to volume to obtain the glucose standard stock solution (100 µg / mL). 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mL of the glucose standard solution were respectively placed in 25 mL colorimetric tubes, and deionized water was added to a final volume of 2 mL. Then, 1.0 mL of 6% phenol solution was added to the tube, and the mixture was shaken well. 5.0 mL of concentrated sulfuric acid was quickly added, and the mixture was shaken well. The tubes were then placed in a boiling water bath and reacted for 15 min. After the reaction, the centrifuge tubes were removed and cooled to room temperature in a water bath. Using a blank reagent (2.0 mL of distilled water instead of the glucose standard solution, with the remaining procedures the same) as a reference, the absorbance of each solution was measured at a wavelength of 490 nm. A standard curve was plotted with glucose concentration on the x-axis and absorbance on the y-axis, yielding the regression equation Y = 0.01639X + 0.02052, where R² = 0.99898.

[0042] Sample determination: Pipette 1 ml of extract into a 10 ml centrifuge tube, add approximately 4 times the volume of anhydrous ethanol for overnight alcohol precipitation, centrifuge at 6000 rpm for 5 min, discard the supernatant, wash once with anhydrous ethanol, centrifuge at 6000 rpm for 5 min, remove the supernatant to obtain polysaccharide solid, dissolve in water, and bring the volume to 10 ml. After diluting the volumetric liquid appropriately, take 2 ml of the liquid into a 25 ml colorimetric tube, add 1 ml of 6% phenol solution, shake well, quickly add 5.0 mL of concentrated sulfuric acid, shake well, and then react in a boiling water bath for 15 min. After the reaction, remove the centrifuge tube and cool to room temperature in a water bath. Use water (choline chloride-D-sorbitol, choline chloride-fructose, or eutectic solvents not used for extraction) as the reference solution. Based on the absorbance of the sample solution, the concentration of polysaccharides in the sample solution is calculated by substituting it into the regression equation of the standard curve. Then, based on the amount of sample weighed and the dilution factor, the extraction rate of polysaccharides in the sample is calculated.

[0043] 3. Scanning electron microscopy Using scanning electron microscopy at different magnifications, we systematically investigated the effects of ultrasonic technology on the texture, porosity, and structural damage of hawthorn during the extraction process. By comparing and analyzing images at 500x, 1000x, and 5000x magnification, we examined the microstructural changes of hawthorn before extraction, after ultrasonic-assisted eutectic solvent extraction, and after magnetic stirring-assisted eutectic solvent extraction.

[0044] Example 1 This study used choline chloride as a hydrogen bond acceptor to screen 11 basic solvent systems with good extraction potential for hawthorn flavonoids and polysaccharides (as shown in Table 1). After weighing according to a specific molar ratio, the solvents were placed in a 250 mL beaker and placed on an electrically heated thermostatic magnetic stirrer at 80℃ and 600 rpm for continuous stirring until a uniform and transparent liquid was formed. After cooling, 30% water was added by volume and mixed evenly to obtain the prepared eutectic solvent-water solution.

[0045] Table 1. Composition of eutectic solvents

[0046] Example 2 Accurately weigh 0.500g of hawthorn powder and add a eutectic solvent-water mixture at a solid-liquid ratio of 1:40g / ml. Mix well in a centrifuge tube and place in an ultrasonic cleaner at 25℃, ultrasonic power 100W, ultrasonic time 30 min. After extraction, centrifuge twice (8000 rpm, 10 min, 4℃). Collect the supernatant and store at 4℃ for subsequent analysis. The extraction rate of flavonoids and polysaccharides is used as the evaluation index. The extraction is compared with that of water and 70% ethanol under the same conditions to screen out the superior solvent for co-extraction of hawthorn flavonoids and polysaccharides.

[0047] In Examples 1-2 of this invention, 11 different eutectic solvents were first prepared, and their physical properties were determined. These solvents were then used to extract flavonoids and polysaccharides from hawthorn using ultrasonic extraction. The content of flavonoids and polysaccharides in each extract was determined by ultraviolet spectrophotometry to evaluate the extraction effect of different solvents on the two target components. The results are as follows: The physical properties of the eutectic solvents are shown in Table 2. The pH values ​​of the selected eutectic solvents span a wide range from strongly acidic to basic. Different hydrogen bond donors can precisely and extensively control the acid-base properties of the eutectic solvents at the molecular level. Although the chemical structures of the components of the eutectic solvents differ significantly, their polarity is highly concentrated (50.60~51.33) and the coefficient of variation is extremely small, possibly because the test system is an aqueous eutectic solvent. Water, as a strongly polar medium, significantly narrows the apparent polarity of different eutectic solvents, making them approach the polar environment of water, thus masking the subtle differences in polarity caused by the differences in the components of pure eutectic solvents. At the same time, water can effectively destroy the internal hydrogen bond network structure of eutectic solvents, leading to a decrease in the apparent stability of polarity and viscosity. In the system, pH and viscosity become the most critical and active adjustable parameters that distinguish different eutectic solvents and affect the extraction efficiency. The density of the eutectic solvent is mainly affected by the inherent density of its components, and overall it is a medium to high density liquid.

[0048] Table 2 Physical properties of eutectic solvents

[0049] Depend on Figure 1-2 It is known that eutectic solvents are significantly more effective than traditional solvents in extracting hawthorn flavonoids and polysaccharides. Specifically, the choline chloride-ethylene glycol, choline chloride-glycerol, and choline chloride-lactic acid systems exhibit high selectivity for flavonoid extraction; while alcohol-based eutectic solvents (ethylene glycol, glycerol), sugar-based eutectic solvents (fructose, glucose), acid-based eutectic solvents (lactic acid, citric acid), and choline chloride-urea systems all demonstrate good extraction capabilities for polysaccharides. Based on a comprehensive evaluation of efficiency and solvent selectivity, this invention preferentially selects choline chloride-ethylene glycol as the target extractant, which not only achieves the highest flavonoid extraction rate (9.75%) but also a relatively high co-extraction rate for polysaccharides (2.36%). The extraction efficiency of eutectic solvents stems from the synergistic effect of key physical properties: a weakly acidic environment ensures flavonoid stability, low viscosity promotes mass transfer, and high polarity ensures effective dissolution of the target compounds; these three factors synergistically enhance extraction efficiency. Correlation analysis showed that pH was positively correlated with flavonoid extraction rate and viscosity was negatively correlated with polysaccharide extraction rate, confirming the key roles of weakly acidic environment and low viscosity in the extraction of the two types of components, respectively. At the same time, the polarity and density of the eutectic solvent were strongly negatively correlated, reflecting its internal structural characteristics. This indicates that by rationally designing eutectic solvent components, its physicochemical properties can be precisely controlled, thereby achieving efficient, selective, and green extraction of multiple target compounds.

[0050] Example 3 Choline chloride-ethylene glycol was selected as the extraction solvent. A eutectic solvent-water solution was prepared under the conditions of a molar ratio of 1:2 and a water content of 30%. 0.500 g of hawthorn powder was weighed and mixed evenly at a solid-liquid ratio of 1:40 g / ml. Ultrasonic extraction was carried out at an extraction temperature of 55℃ and ultrasonic power of 120 W, 180 W, 240 W, 300 W and 360 W for 30 min. After completion, the mixture was cooled and centrifuged, and the filtrate was collected to determine the extraction rates of flavonoids and polysaccharides.

[0051] Example 4 Choline chloride-ethylene glycol was selected as the extraction solvent. A eutectic solvent-water solution was prepared under the conditions of a molar ratio of 1:2 and a water content of 30%. 0.500 g of hawthorn powder was weighed and mixed evenly at a solid-liquid ratio of 1:40 g / ml. Ultrasonic extraction was carried out at an extraction temperature of 55℃ and an ultrasonic power of 180 W for 10 min, 20 min, 30 min, 40 min and 50 min respectively. After completion, the extract was cooled, centrifuged and collected. The filtrate was then used to determine the extraction rates of flavonoids and polysaccharides.

[0052] Examples 3-4 of this invention compare the effects of extraction power and extraction time on the extraction rates of hawthorn flavonoids and polysaccharides. Key indicators for efficient co-extraction of hawthorn flavonoids and polysaccharides using ultrasound-assisted eutectic solvents were tested, and the results are as follows: Depend on Figure 3-4 It can be seen that with the increase of ultrasonic power, the extraction rate of hawthorn flavonoids increases, reaching the highest value at 240-300W, and then gradually decreases, but has little effect on polysaccharides; when the extraction time is 20-40 min, the flavonoid extraction rate is relatively high, and after 30 min, the polysaccharide yield no longer increases; the extraction rate of active substances increases within a certain temperature range, but decreases after exceeding a certain temperature, possibly due to the degradation of active substances caused by high temperature; Example 5 Choline chloride-ethylene glycol was selected as the extraction solvent. A eutectic solvent-water solution was prepared under the conditions of a molar ratio of 1:2 and a water content of 30%. 0.500 g of hawthorn powder was weighed and mixed evenly at a solid-liquid ratio of 1:40 g / ml. Ultrasonic extraction was carried out at extraction temperatures of 20℃, 35℃, 55℃, 65℃ and 80℃ for 30 min respectively. After extraction, the solution was cooled, centrifuged and collected. The filtrate was then used to determine the extraction rates of flavonoids and polysaccharides.

[0053] Example 6 Choline chloride-ethylene glycol was selected as the extraction solvent. Eutectic solvent-water solutions were prepared at molar ratios of 1:1, 1:2, 1:3, 1:4, and 1:5, with a water content of 30%. 0.500 g of hawthorn powder was weighed and mixed evenly at a solid-liquid ratio of 1:40 g / ml. The mixture was then ultrasonically extracted at 55℃ for 30 min. After extraction, the mixture was cooled, centrifuged, and the filtrate was collected to determine the extraction rates of flavonoids and polysaccharides.

[0054] Examples 5-6 of this invention compare the effects of extraction temperature and eutectic solvent molar ratio on the extraction rates of hawthorn flavonoids and polysaccharides. Key indicators for efficient co-extraction of hawthorn flavonoids and polysaccharides using ultrasound-assisted eutectic solvent were tested, and the results are as follows: Depend on Figures 5-6 It can be seen that the effects of ultrasonic temperature and the molar ratio of the eutectic solvent on the extraction rates of hawthorn flavonoids and polysaccharides both showed a trend of first increasing and then decreasing. The flavonoid extraction rate reached its peak at 55℃ and a molar ratio of 1:3, while the polysaccharide extraction rate reached its highest value at 65℃ and a molar ratio of 1:3.

[0055] Example 7 Choline chloride-ethylene glycol was selected as the extraction solvent to prepare eutectic solvent-water solutions with a molar ratio of 1:2 and water contents of 10%, 30%, 50%, 70%, and 90%. 0.500 g of hawthorn powder was weighed and mixed evenly at a solid-liquid ratio of 1:40 g / ml. Ultrasonic extraction was carried out at an extraction temperature of 55℃ and an ultrasonic power of 180 W for 30 min. After extraction, the solution was cooled, centrifuged, and the filtrate was collected to determine the extraction rates of flavonoids and polysaccharides.

[0056] Example 8 Choline chloride-ethylene glycol was selected as the extraction solvent. A eutectic solvent-water solution was prepared under the conditions of a molar ratio of 1:2 and a water content of 30%. 0.500 g of hawthorn powder was weighed and added to the mixture at solid-liquid ratios of 1:10, 1:25, 1:40, 1:55, and 1:70 g / ml. After mixing evenly, ultrasonic extraction was carried out at an extraction temperature of 55℃ and an ultrasonic power of 180 W for 30 min. After cooling and centrifugation, the filtrate was collected and the extraction rates of flavonoids and polysaccharides were determined.

[0057] Examples 7-8 of this invention compare the effects of water content and solid-liquid ratio of the eutectic solvent on the extraction rates of hawthorn flavonoids and polysaccharides. Key indicators for efficient co-extraction of hawthorn flavonoids and polysaccharides using ultrasound-assisted eutectic solvents were tested, and the results are as follows: Depend on Figures 7-8 It can be seen that the water content of the eutectic solvent has an initial increasing and then decreasing effect on the extraction rates of flavonoids and polysaccharides. Since the increased water content significantly reduces the system viscosity and modulates the solvent polarity and hydrogen bond network, different water contents affect the solubility and diffusion properties of the target compounds differently. This results in the optimal extraction rate for flavonoids at a eutectic solvent water content of 30%, and the best extraction rate for polysaccharides at 50%. Because flavonoids have a small molecular weight and are easily diffused, their extraction rate is not sensitive to changes in solvent volume. However, due to their large molecular weight and high mass transfer resistance, the extraction rate of polysaccharides initially increases and then decreases with increasing solvent content. This may be because an appropriate amount of solvent can overcome the viscosity limitation of the system and promote dissolution, while excessive solvent weakens the concentration-driven extraction or disperses the thermal effect, thus reducing extraction efficiency.

[0058] Example 9 Based on the results of single-factor experiments, three factors that significantly affect the extraction efficiency of hawthorn flavonoids and polysaccharides and can significantly change the properties of eutectic solvents were selected as key variables for response surface optimization. The extraction rates of flavonoids and polysaccharides were used as evaluation indicators for experimental design. The experimental factors and their numbers are shown in Table 3.

[0059] Table 3 Factors in Response Surface Experiment

[0060] Example 9 of this invention investigates the effects of ultrasonic temperature, eutectic solvent molar ratio, and water content on the extraction rates of hawthorn flavonoids and polysaccharides. Key indicators for efficient co-extraction of hawthorn flavonoids and polysaccharides using ultrasound synergistically with eutectic solvents were tested, and the results are as follows: The steepness of the response surface model plot can intuitively reflect the impact of the interactions between the above factors on the extraction rate. A steeper response surface plot indicates a more significant interaction between the two factors, and vice versa. Figure 9It is evident that the eutectic solvent molar ratio and water content are the core factors regulating the synergistic extraction efficiency of flavonoids and polysaccharides. These two factors jointly regulate the polar microenvironment and hydrogen bond network of the extraction system, determining whether the two types of components can be simultaneously and efficiently dissolved. The 3D response surface diagrams A, B, and C of flavonoid extraction show that the flavonoid extraction rate reaches its peak when the temperature fluctuates around 70℃ and the molar ratio is approximately 1:3; deviations from this range lead to a significant decrease in the extraction rate. A similar pattern is observed in the combination of extraction temperature and water content, with the flavonoid extraction rate reaching its peak at temperatures of 65-75℃ and water content of 40-50%. The extraction rate reached its peak at certain temperatures; both excessively high and low water content inhibited flavonoid dissolution, consistent with the conclusions of single-factor experiments. Response surface methodology (Figures D, E, and F) showed different patterns in polysaccharide and flavonoid extraction. The extraction rate continuously increased with increasing temperature and molar ratio, reaching a high level at 70-80℃ and a molar ratio of 1:3, demonstrating stronger high-temperature tolerance. In the interaction between extraction temperature and water content, the polysaccharide extraction rate gradually increased with both, reaching a high value at 70-80℃ and a water content of 60%-70%, with no significant decrease. This indicates that high water content is more conducive to polysaccharide dissolution. The interaction between the molar ratio of the low eutectic solvent and water content on polysaccharides was relatively mild, only showing a significant decrease at a molar ratio of 1:3 and a water content of 60-70%. The level reached a relatively high level, which contrasted sharply with the suitable range for flavonoids, and the overall impact was weaker than the effect on flavonoids. The model predicted the optimal extraction conditions as follows: extraction temperature 69.97℃, eutectic solvent molar ratio 1:3.09, and water content 54.66%, predicting a flavonoid extraction rate of 10.37% and a polysaccharide extraction rate of 5.78%. The obtained conditions were corrected to an extraction temperature of 70℃, eutectic solvent molar ratio 1:3.1, and water content of 55%. Extraction was carried out under the conditions of ultrasonic power 300 W, solid-liquid ratio 1:40 g / ml, extraction time 40 min, extraction temperature 70℃, eutectic solvent molar ratio 1:3.1, and water content 55%, and the flavonoid extraction rate was measured to be 10.35±0.30% and the polysaccharide extraction rate was 5.79±0.33%. The results were not significantly different from the model predictions, indicating that the established response surface model was accurate and reliable, and the optimized process had good predictive and repeatability.

[0061] Example 10 Flavonoids and polysaccharides were extracted under optimal ultrasonic synergistic eutectic solvent response surface methodology. The residue after centrifugation was washed twice with deionized water and freeze-dried at -55℃ for 24 hours. The microstructural changes of hawthorn were observed using scanning electron microscopy at 500x, 1000x, and 5000x magnification, along with the hawthorn residue before extraction and after magnetic stirring synergistic eutectic solvent extraction (except that ultrasonic extraction was replaced with hot stirring extraction).

[0062] Example 10 of this invention investigates the effect of ultrasonic extraction on the structure of hawthorn, involving the testing of key indicators for efficient co-extraction of hawthorn flavonoids and polysaccharides using ultrasound in conjunction with a eutectic solvent. The results are as follows: Depend on Figure 10 It is known that ultrasound-assisted extraction has a far greater effect on disrupting hawthorn cells than traditional magnetic stirring. Ultrasonic extraction further promotes the penetration of eutectic solvents, which is more conducive to the dissolution of active ingredients such as flavonoids and polysaccharides.

[0063] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for co-extracting flavonoids and polysaccharides from hawthorn by ultrasonic synergistic deep eutectic solvent, characterized in that: include, Choline chloride was mixed with a hydrogen bond donor, heated and stirred until clear and transparent, and then deionized water was added and mixed evenly to obtain a eutectic solvent-water mixture. Hawthorn powder was mixed evenly with a eutectic solvent-water mixture and then extracted in an ultrasonic water bath. After ultrasonic extraction, the mixture was allowed to stand and then centrifuged. The supernatant was collected, which was an extract containing hawthorn flavonoids and polysaccharides.

2. The method of claim 1, wherein: The hydrogen bond donor is a single-component or a two-component, wherein the single-component includes any one of ethylene glycol, glycerol, sorbitol, fructose, glucose, sucrose, lactic acid, citric acid, and urea; The two components are one of a mixture of ethylene glycol and lactic acid, or a mixture of ethylene glycol and citric acid.

3. The method of claim 1 or 2, wherein: The molar ratio of choline chloride to hydrogen bond donor is 1:(1~5).

4. The method of claim 3, wherein: The eutectic solvent-water mixture contains water with a volume content of 10% to 90%.

5. The method of claim 4, wherein: The heating and stirring temperature is 60~80℃, and the stirring speed is 100~800rpm.

6. The method of claim 1, wherein: The hawthorn powder is dried hawthorn powder with a particle size of less than 18 mesh.

7. The method of claim 1 or 6, wherein: The solid-liquid ratio of the hawthorn powder and the eutectic solvent-water mixture is 1g:10~80mL.

8. The method of claim 7, wherein: The ultrasonic extraction temperature is 20~80℃, the extraction power is 100~360W, and the extraction time is 10~50min.

9. The method of claim 1 or 8, wherein: The centrifugation temperature is 4~25℃, the centrifugation speed is 6000~10000 rpm / min, the time is 10~20 min, and the number of times is 2.

10. The method of any one of claims 1, 2, 4-6, wherein: The ultrasonic power was 300W, the ultrasonic temperature was 55-80℃, the molar ratio of the eutectic solvent was 1:2-4, the water content was 30-70%, the solid-liquid ratio was 1:40 g / mL, the extraction time was 40 min, and the extraction rates of flavonoids and polysaccharides were 8.42%-10.62% and 2.05%-6.28%, respectively.

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