A method for extracting flavones from hawthorn leaves

Abstract

The present application belongs to the technical field of extraction of natural bioactive substances, and particularly relates to a method for extracting flavonoids from hawthorn leaves. The method comprises the following steps: mixing hawthorn leaves with a hydrophobic eutectic solvent, performing ultrasonic treatment, and then centrifuging to obtain an extraction solution. The total flavonoid content extracted by the method is 3.69 times that of ethyl acetate extraction solution and 6.11 times that of ethanol extraction solution. The average DPPH free radical scavenging rate of the extracted extract is 74.35% of the same concentration of vitamin C, the alpha-glucosidase activity inhibition rate is 1.24 times the inhibition rate of the same concentration of acarbose, and the alpha-amylase activity inhibition rate is 2.80 times the inhibition rate of the same concentration of acarbose.

Description

Technical Field

[0001] This invention belongs to the field of extraction technology of natural bioactive substances, specifically relating to a method for extracting flavonoids from hawthorn leaves. Background Technology

[0002] This invention relates to the field of plant active ingredient extraction technology, specifically to a method for extracting flavonoids from hawthorn leaves. Hawthorn leaves are the dried leaves of the Rosaceae plant *Crataegus pinnatifida* or *Crataegus pinnatifida*, which are rich in flavonoids, triterpenoids, trace elements, organic acids, and organic amines, among other secondary metabolites. Flavonoids are the most abundant bioactive substances in hawthorn leaves. Related studies have confirmed that flavonoids not only have hepatoprotective, hepatotoxic, antifungal, and therapeutic effects on acute and chronic hepatitis and cirrhosis, but also possess significant antioxidant properties. Furthermore, they exhibit good activity in relieving cough, expectorating phlegm, relieving asthma, and antibacterial effects. Therefore, efficient extraction of flavonoids from hawthorn leaves is of great significance for realizing the high-value utilization of hawthorn leaves, enhancing their resource value, and promoting the application of flavonoid active ingredients in the fields of medicine, food, and health products.

[0003] Currently, traditional extraction processes for hawthorn leaf flavonoids mostly involve prolonged soaking in water or organic solvents. While various extraction schemes have been disclosed in existing technologies, all exhibit significant technical drawbacks. Zhang Liming et al. used water extraction to extract hawthorn leaf flavonoids. Under optimal conditions of a material-to-liquid ratio of 1:20 (g / mL), an extraction time of 120 min, and two extractions, the extracted flavonoid content was only 21.97 mg / g dry sample. Ma Juanjuan et al. used water as the extractant. Under optimal conditions of a material-to-liquid ratio of 1:35, an extraction time of 60 min, an extraction temperature of 60℃, a solution pH of 3.6, and three extractions, the extracted flavonoid content was approximately 51.01 mg / g dry sample. This demonstrates that using water as a solvent generally results in problems such as high extraction temperatures, long extraction times, the need for multiple extraction cycles, and low extraction rates. Zhou Zhiqiang et al. used 70% ethanol aqueous solution as the extractant and employed ultra-high pressure extraction to extract flavonoids from hawthorn leaves. Under the optimal conditions of a material-to-liquid ratio of 1:20 g / mL, an extraction pressure of 350 MPa, and an extraction temperature of 60℃, the actual extraction rate of flavonoids was only 7.068%. This indicates that using ethanol aqueous solution as the solvent has drawbacks such as excessively high operating pressure, high extraction temperature, easy solvent volatility, and certain biological toxicity, making it difficult to meet the requirements of high efficiency, greenness, and safety for large-scale industrial production.

[0004] Hydrophobic eutectic solvents (HES) are stable liquid systems formed by mixing hydrogen bond donors and hydrogen bond acceptors in a specific ratio and through intermolecular hydrogen bonding forces. They have advantages such as melting point lower than that of a single component, simple composition, low toxicity, biodegradability, and acceptable drug toxicity. They also have the property of repelling water and show good application prospects in the field of extraction and separation of plant active ingredients. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a method for extracting flavonoids from hawthorn leaves.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides a method for extracting flavonoids from hawthorn leaves. The extraction method includes: mixing hawthorn leaves with a hydrophobic eutectic solvent, subjecting the mixture to ultrasonic treatment, and then centrifuging to obtain the extract.

[0007] Preferably, in the above extraction method, the hydrophobic eutectic solvent is selected from any one of the following: (i) the hydrogen bond acceptor is DL-menthol, the hydrogen bond donor is decanoic acid, and the molar ratio of DL-menthol to decanoic acid is 1:1; (ii) the hydrogen bond acceptor is thymol, the hydrogen bond donor is decanoic acid, and the molar ratio of thymol to decanoic acid is 1:(1-3); (iii) the hydrogen bond acceptor is thymol, the hydrogen bond donor is undecenoic acid, and the molar ratio of thymol to undecenoic acid is 1:(1-4); (iv) the hydrogen bond acceptor is tetrabutylammonium bromide, the hydrogen bond donor is lauric acid, and the molar ratio of tetrabutylammonium bromide to lauric acid is 1:1.2.

[0008] More preferably, in the above extraction method, the hydrophobic eutectic solvent is selected from any one of the following: the hydrogen bond acceptor is thymol, the hydrogen bond donor is decanoic acid, and the molar ratio of thymol to decanoic acid is 3:1 or 1:2; or the hydrogen bond acceptor is thymol, the hydrogen bond donor is undecenoic acid, and the molar ratio of thymol to undecenoic acid is 3:2, 1:1, 1:2 or 1:4.

[0009] Preferably, in the above extraction method, the hawthorn leaves are in powder form.

[0010] More preferably, in the above extraction method, the liquid-to-solid ratio of the hydrophobic eutectic solvent and the hawthorn leaves is 10 mL / g to 30 mL / g.

[0011] More preferably, in the above extraction method, the liquid-to-solid ratio of the hydrophobic eutectic solvent and hawthorn leaves is 22.06 mL / g.

[0012] Preferably, in the above extraction method, the conditions for ultrasonic treatment include: ultrasonic power of 144W to 240W; ultrasonic temperature of 30℃ to 50℃; and ultrasonic time of ≥40min.

[0013] More preferably, in the above extraction method, the ultrasonic time is 40 min.

[0014] More preferably, in the above extraction method, the ultrasonic power is 198.7 W and the ultrasonic temperature is 50℃.

[0015] The beneficial effects of this invention include at least the following: the total flavonoid content extracted by the hawthorn leaf flavonoid extraction method provided by this invention is 3.69 times that of the ethyl acetate extract and 6.11 times that of the ethanol extract; and the extracted extract has an average DPPH free radical scavenging rate of 74.35% of that of vitamin C at the same concentration, an inhibition rate of α-glucosidase activity of 1.24 times that of acarbose at the same concentration, and an inhibition rate of α-amylase activity of 2.80 times that of acarbose at the same concentration. Attached Figure Description

[0016] Figure 1 The total flavonoid content of different HES extracts; Figure 2 The results are from a single-factor experiment; where (a) is the ultrasonic power, (b) is the liquid-to-solid ratio, (c) is the ultrasonic time, and (d) is the ultrasonic temperature. Detailed Implementation

[0017] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0018] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0019] This invention provides a method for extracting flavonoids from hawthorn leaves. The extraction method includes: mixing hawthorn leaves with a hydrophobic eutectic solvent, subjecting the mixture to ultrasonic treatment, and then centrifuging to obtain the extract.

[0020] It should be noted that hawthorn leaf flavonoids are natural flavonoid compounds from hawthorn leaves that possess antioxidant and lipid-lowering biological activities. The hydrophobic eutectic solvent is a low-melting-point, low-viscosity, green solvent formed by mixing hydrogen bond acceptors and hydrogen bond donors in a specific ratio. It is non-volatile, non-toxic, and environmentally friendly, and has excellent solubility for hydrophobic flavonoid components. After mixing hawthorn leaves with this solvent, ultrasonic treatment can disrupt the cell wall structure of hawthorn leaves through the mechanical and cavitation effects generated by high-frequency vibration, accelerating the release of flavonoid components from the cells and promoting the dissolution of flavonoids by the solvent. Subsequent centrifugation can quickly separate the extract from the residue. The entire extraction method is simple, convenient, requires no organic solvents, is environmentally friendly and pollution-free, has high extraction efficiency, and can retain the biological activity of flavonoid components to the greatest extent, making it suitable for large-scale production.

[0021] In some specific examples, the hydrophobic eutectic solvent in the above extraction method is selected from any of the following: (i) the hydrogen bond acceptor is DL-menthol, the hydrogen bond donor is decanoic acid, and the molar ratio of DL-menthol to decanoic acid is 1:1; (ii) the hydrogen bond acceptor is thymol, the hydrogen bond donor is decanoic acid, and the molar ratio of thymol to decanoic acid is 1:(1-3); (iii) the hydrogen bond acceptor is thymol, the hydrogen bond donor is undecenoic acid, and the molar ratio of thymol to undecenoic acid is 1:(1-4); (iv) the hydrogen bond acceptor is tetrabutylammonium bromide, the hydrogen bond donor is lauric acid, and the molar ratio of tetrabutylammonium bromide to lauric acid is 1:1.2.

[0022] It should be noted that DL-menthol, thymol, and tetrabutylammonium bromide are all commonly used hydrogen bond acceptors, while decanoic acid, undecenoic acid, and lauric acid are hydrogen bond donors. These form a eutectic solvent through hydrogen bonding, which can significantly lower the solvent's melting point and improve the solubility of hawthorn leaf flavonoids. A 1:1 molar ratio of DL-menthol to decanoic acid forms a stable and homogeneous eutectic system with excellent solubility and flowability. For thymol and decanoic acid, intermediate molar ratios of 1:1.5, 1:2, and 1:2.5 can be selected, as can for thymol and undecenoic acid, which can be selected at intermediate molar ratios of 1:1.5, 1:2.5, and 1:3.5. This range allows for flexible control of solvent hydrophobicity and solubility to adapt to different extraction conditions. A 1:1.2 molar ratio of tetrabutylammonium bromide to lauric acid forms a solvent system with strong stability and high solubility. All four solvents are non-toxic, environmentally friendly, easy to prepare, and highly selective for flavonoid components, effectively reducing impurities and improving the purity of the extract.

[0023] In some specific examples, in the above extraction method, the hydrophobic eutectic solvent is selected from any of the following: the hydrogen bond acceptor is thymol, the hydrogen bond donor is decanoic acid, and the molar ratio of thymol to decanoic acid is 3:1 or 1:2; or the hydrogen bond acceptor is thymol, the hydrogen bond donor is undecenoic acid, and the molar ratio of thymol to undecenoic acid is 3:2, 1:1, 1:2, or 1:4.

[0024] It should be noted that the fixed molar ratios of thymol to decanoic acid (3:1, 1:2) and thymol to undecenoic acid (3:2, 1:1, 1:2, 1:4) are all optimal ratios selected through experimental optimization. The hydrophobic eutectic solvents formed under these ratios have moderate viscosity and good fluidity, achieving peak solubility for hawthorn leaf flavonoids. Furthermore, the solvents exhibit strong stability, are not prone to stratification or deterioration. Among these ratios, 3:1 and 3:2 are more focused on enhancing solvent hydrophobicity, facilitating the efficient dissolution of hydrophobic flavonoid components. The 1:1, 1:2, and 1:4 ratios balance solvent solubility and fluidity, facilitating subsequent ultrasonic extraction and centrifugation. Fixed ratios improve the consistency of extraction results between batches, reduce the difficulty of process control, and are suitable for industrialized and standardized production.

[0025] In some specific examples, the hawthorn leaves are in powder form in the above extraction method.

[0026] It should be noted that pulverizing hawthorn leaves into powder significantly increases the contact area between the leaves and the hydrophobic eutectic solvent, reducing the mass transfer resistance of flavonoids released from cells. Simultaneously, it facilitates the cavitation effect generated by ultrasonic vibration to penetrate the cell wall, accelerating the dissolution of flavonoids. Compared to whole or chopped hawthorn leaves, powdered hawthorn leaves significantly improve extraction efficiency, shorten extraction time, and allow for more complete extraction of flavonoids, reducing raw material waste. Furthermore, powdered hawthorn leaves are easier to mix evenly with the solvent, avoiding uneven extraction caused by localized raw material agglomeration. Subsequent centrifugation also allows for rapid separation of residues and extract, improving process smoothness.

[0027] In some specific examples, the liquid-to-solid ratio of the hydrophobic eutectic solvent and hawthorn leaves in the above extraction method is 10 mL / g to 30 mL / g.

[0028] It should be noted that the liquid-to-solid ratio can be selected from the middle values ​​of 15mL / g, 20mL / g, and 25mL / g. The liquid-to-solid ratio is a key parameter affecting the extraction efficiency of flavonoids. When the liquid-to-solid ratio is too low, the amount of solvent used is insufficient, and the flavonoid components in the hawthorn leaf powder cannot be fully dissolved, resulting in a low extraction rate. When the liquid-to-solid ratio is too high, the amount of solvent used is excessive, which not only wastes solvent and increases production costs, but also increases the difficulty and energy consumption of subsequent flavonoid separation and purification. This range of liquid-to-solid ratio can balance extraction efficiency and production costs, ensuring that the solvent can fully dissolve the flavonoid components while avoiding excessive solvent, which is suitable for the extraction needs of powdered hawthorn leaves, and facilitates subsequent process control.

[0029] In some specific examples, the liquid-to-solid ratio of the hydrophobic eutectic solvent and hawthorn leaves in the above extraction method is 22.06 mL / g.

[0030] It should be noted that the liquid-to-solid ratio of 22.06 mL / g is the optimal value determined through extensive experimental optimization. At this ratio, the hydrophobic eutectic solvent can fully contact the powdered hawthorn leaves, which can completely dissolve the flavonoid active ingredients in the hawthorn leaves, achieving the highest extraction rate, while avoiding waste caused by excessive solvent and the burden of subsequent purification. At the same time, this liquid-to-solid ratio is suitable for ultrasonic extraction process, which can make full use of the cavitation effect and mechanical effect of ultrasonic vibration, accelerate the dissolution of flavonoid components, and ensure strong batch-to-batch stability of extraction effect. No additional adjustment of solvent volume is required, simplifying process operation and reducing the cost and difficulty of industrial production.

[0031] In some specific examples, the conditions for ultrasonic processing in the above extraction method include: ultrasonic power of 144W to 240W; ultrasonic temperature of 30℃ to 50℃; and ultrasonic time of ≥40min.

[0032] It should be noted that the ultrasonic power can be selected from the middle values ​​of 160W, 190W, and 220W. When the power is too low, the cavitation and mechanical effects are weak, which cannot effectively destroy the cell walls of hawthorn leaves, resulting in a slow flavonoid dissolution rate and insufficient extraction. When the power is too high, it is easy to generate too much heat, which will destroy the biological activity of flavonoid components and may also lead to solvent evaporation or system instability. The ultrasonic temperature can be selected from the middle values ​​of 35℃, 40℃, and 45℃. Too high a temperature will destroy the activity of flavonoids, while too low a temperature will reduce the solvent solubility and the flavonoid dissolution rate. The ultrasonic time is ≥40min, and 45min, 60min, and 80min can be selected. If the time is too short, the flavonoids will not dissolve sufficiently. This ultrasonic condition range is mild and controllable, which can balance the extraction efficiency, flavonoid activity, and process energy consumption, ensuring that the flavonoids are fully dissolved and retain their biological activity to the greatest extent, and is suitable for different liquid-solid ratios and solvent systems.

[0033] In some specific examples, the ultrasound time in the above extraction method is 40 minutes.

[0034] It should be noted that the flavonoid content does not change significantly after an ultrasound time of ≥40 min. Taking all factors into consideration, the ultrasound time of 40 min is the preferred time in this invention.

[0035] In some specific examples, the ultrasonic power in the above extraction method is 198.7 W and the ultrasonic temperature is 50℃.

[0036] It should be noted that the optimal combination of ultrasonic power (198.7W) and ultrasonic temperature (50℃) is the best process combination. At this power, the cavitation and mechanical effects can effectively disrupt the cell walls of hawthorn leaves, accelerating the dissolution of flavonoids without generating excessive heat that could damage their activity. The temperature of 50℃ maximizes the solubility of flavonoids in the hydrophobic eutectic solvent, further promoting flavonoid dissolution. The synergistic effect of these two factors can achieve the highest extraction rate and efficiency while ensuring the bioactivity of flavonoids. Fixing these ultrasonic parameters can avoid instability in extraction results caused by fluctuations in process parameters, improve batch-to-batch product consistency, and adapt to standardized industrial production. At the same time, it eliminates the need for complex parameter adjustments and is easy to operate.

[0037] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0038] In the following examples, the preparation of different types of hydrophobic eutectic solvents (HES) is as follows: The hydrogen bond donor and acceptor components listed in Table 1 are accurately weighed according to a specific molar ratio and placed in a constant temperature water bath (DK-D, Jintan Baita Xinbao Instrument Factory) and maintained at 80°C. The system is stirred continuously until it changes from a turbid state to a homogeneous transparent liquid and no suspended matter is observed to be present. HES is then successfully prepared.

[0039] Table 1. HES used in the following examples

[0040] In the following example, the hawthorn leaf powder was prepared as follows: Hawthorn leaves were pulverized into powder using a grinder (MFJ-W317, Beijing Liren Technology Co., Ltd.), and large particles were removed by sieving through a 40-mesh sieve. The sieved hawthorn leaf powder was then placed in a sealed bag for storage.

[0041] Example 1: Comparison of extraction effects of different types of hydrophobic eutectic solvents Accurately weigh 1.0 g of hawthorn leaf powder and add it to 20 mL of different specific ratios of hydrophobic eutectic solvents (Table 1). After thorough mixing, transfer the mixture to an ultrasonic extraction device (SB-5200DTD, Ningbo Xinzhi Biotechnology Co., Ltd.), set the temperature to 40℃ and the power output to 240W, and perform extraction for 30 min. After ultrasonic treatment, transfer the mixture to a high-speed centrifuge (5810R, Shanghai Tianmei Biochemical Instrument Equipment Engineering Co., Ltd.) and centrifuge at 10000 rpm for 10 min. Collect the supernatant (extract) after centrifugation, and use spectrophotometry to determine the absorbance and calculate the total flavonoid content (TFC) using a detection wavelength of 405 nm. The specific test method is as follows: Take 1 mL of the extract and place it in a 25 mL volumetric flask. Add 5.0 mL of 0.1 mol / L aluminum trichloride solution and 2.5 mL of 0.2 mol / L acetate-sodium acetate buffer solution (pH 5.2). Dilute to volume with 60% ethanol solution and shake well. Heat in a 40℃ water bath for 15 min, cool to room temperature, and use a colorimetric reagent as a blank control (5.0 mL of 0.1 mol / L aluminum trichloride solution and 2.5 mL of 0.2 mol / L acetate-sodium acetate buffer solution (pH 5.2)). Measure the absorbance at 405 nm. Construct a standard curve using rutin as a standard substance. Finally, convert the measurement results into milligrams of rutin equivalents extracted per gram of hawthorn leaf powder, expressing the total flavonoid content (mg / g).

[0042] The total flavonoid content of extracts from different hydrophobic eutectic solvents is as follows: Figure 1 And as shown in Table 2 below. Through Figure 1 A comparison of the extraction effects of 29 hydrophobic eutectic solvents revealed significant differences in total flavonoid content. Solvent No. 29 (molar ratio of tetrabutylammonium bromide to lauric acid of 1:1.2) produced the highest total flavonoid content, but its high viscosity made it the preferred extraction solvent. In contrast, solvent No. 18 (molar ratio of thymol to undecenoic acid of 3:2) had a relatively high total flavonoid content while also exhibiting lower viscosity, making it the preferred extraction solvent.

[0043] Table 2. Total flavonoid content in extracts from different hydrophobic eutectic solvents

[0044] Example 2: Single-factor experiment This invention employs a single-factor variable control method to systematically investigate the effects of different process parameters on the yield of total flavonoids extracted using a hydrophobic eutectic solvent (specific steps are as described in Example 1). The specific experimental design is as follows: the effects of four key parameters on the yield of total flavonoids were investigated: ultrasonic time (10 min, 20 min, 30 min, and 40 min), ultrasonic temperature (30℃, 40℃, 50℃, and 60℃), ultrasonic power (160 W, 240 W, 320 W, and 360 W), and liquid-to-solid ratio (g:mL) (1:10, 1:20, 1:30, and 1:40). During the experiment, one operating parameter was changed while the other operating parameters remained constant (the other constant parameters are as described in Example 1).

[0045] The results of the single-factor experiment are as follows Figure 2As shown in the results, when the power is in the range of 144W to 180W, the flavonoid extraction rate shows a significant increasing trend. This is mainly due to the effective disruption of the hawthorn leaf cell wall structure by ultrasound, promoting the release of flavonoids. When the power is increased to the range of 180W to 240W, the extraction rate decreases significantly, presumably because the ultrasonic thermal effect leads to the oxidation of flavonoids. When the power is higher than 240W, the flavonoid extraction yield fluctuates to some extent, possibly due to the combined positive and negative effects of power on the extraction yield. Based on the above analysis, 144W to 240W is determined to be the optimal parameter range.

[0046] In addition, the yield of flavonoids gradually increases when the liquid-to-solid ratio is in the range of 10 mL / g to 20 mL / g, but the yield gradually decreases when the liquid-to-solid ratio exceeds 20 mL / g. Therefore, the liquid-to-solid ratio is preferably 10 mL / g to 30 mL / g.

[0047] In addition, the flavonoid content showed an upward trend within the extraction time range of 20 min to 40 min, but the content tended to stabilize after 40 min, indicating that the extraction reached a dynamic equilibrium at this time. Therefore, the optimal extraction time is 40 min.

[0048] In addition, temperature has a two-way effect on the extraction effect. Specifically, the flavonoid content increases from 30℃ to 50℃. Moderate heating can reduce solvent viscosity and promote solute diffusion. However, when the temperature exceeds 50℃, the flavonoid content decreases. This may be because high temperature causes the decomposition of flavonoids. The final optimized temperature range is set to 30℃~50℃.

[0049] Example 3: Response Surface Analysis and Model Validation Based on single-factor experimental data, this embodiment of the invention employs a response surface methodology to systematically optimize the extraction process. Using the total flavonoid yield as the objective function (specific testing methods follow those in Example 1), a three-factor, three-level Box-Behnken experimental design is constructed using the Design Expert 13.0 software platform. A quadratic regression mathematical model is established to analyze the influence of the interaction of various parameters on the flavonoid extraction yield. A numerical optimization algorithm is used to solve for the combination of process parameters corresponding to the maximum extraction yield. Extraction experiments are conducted under optimal conditions to verify the optimization results.

[0050] (1) Establishing a model This invention is based on the Box-Behnken Design experimental design principle, selecting liquid-to-solid ratio (A), ultrasonic temperature (B), and ultrasonic power (C) as key influencing factors, and constructing a three-factor, three-level experimental matrix with total flavonoid yield as the response value. As shown in Table 3, the experimental matrix covers 17 treatment combinations, where the ultrasonic temperature gradient is set to 40℃-60℃, the power adjustment range is 144W-240W, and the liquid-to-solid ratio is controlled at 10mL / g-30 mL / g.

[0051] Table 3 Experimental Design and Results

[0052] A predictive model is established using quadratic polynomial regression analysis: .

[0053] Table 4 shows the results of the analysis of variance, which indicate the predicted variance. R 2 (0.9456) and the adjusted R 2 The difference (0.9866) is only 0.041, far less than 0.2, indicating that the model has high predictive ability and stability. The CV value is 8.55%, less than 10%, indicating that the experimental results are reliable. The model's... p A value less than 0.0001 indicates a highly significant effect. (The term lacks a clear definition.) p The value of 0.3337 indicates a good fit of the model.

[0054] Table 4. Analysis of Variance Table

[0055] The effects of the liquid-to-solid ratio (A) and ultrasonic power (C) on the response value showed a high statistical significance. p <0.0001), its quadratic parameter A 2 B 2 The effect of temperature (B) was also highly significant. p =0.0232), and in particular, it shows a close correlation with the combined effect of ultrasonic power (C). p <0.0001). In terms of the order of influence of each parameter, the liquid-to-solid ratio (A) has the dominant influence with an F value of 73.89, followed by ultrasonic power (C), while temperature (B) has a relatively weaker influence. Furthermore, the quadratic term A... 2 and B 2 The F-values ​​reached 6.96 and 19.76, respectively, further confirming the significant influence of the nonlinear effect of the liquid-solid ratio on the results, as well as the effects of ultrasonic power and temperature.

[0056] (2) Optimal Condition Verification Response surface methodology (RSM) analysis revealed the optimal parameter combination for hawthorn leaf flavonoid extraction: temperature 50℃, liquid-to-solid ratio 22.06 mL / g, and power 198.7 W. To ensure accuracy, three parallel experiments were conducted under the optimized conditions, yielding an average total flavonoid content of 59.25 mg / g (tested according to Example 1). This represents a relative deviation of 7.48% from the model prediction of 54.82 mg / g, confirming the applicability of RSM in process optimization.

[0057] Example 4: Determination of antioxidant activity The antioxidant activity of the extract was evaluated using the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radical scavenging method. Specifically, the extract (50 μL) was mixed with 100 μmol / L DPPH-ethanol (400 μL) in a 2 mL centrifuge tube; the mixture was then incubated in the dark at 25°C for 20 minutes; absorbance A2 was measured at 517 nm; pure ethanol was used as a blank control; absorbance A1 was measured using anhydrous ethanol instead of the DPPH solution; absorbance A0 was measured using anhydrous ethanol instead of the sample solution; and the scavenging rate was calculated.

[0058] .

[0059] When the flavonoid concentration in the HES solvent system extract (with a thymol to undecenoic acid molar ratio of 3:2, and the extract was obtained under optimal conditions: temperature 50℃, liquid-to-solid ratio 22.06 mL / g, power 198.7 W, time 40 min, and other conditions the same as in Example 1) was 0.08 g / L, the average DPPH free radical scavenging rate of the extract was 64.50 ± 3.52%, reaching 74.35% of the DPPH free radical scavenging rate of vitamin C at the same concentration. This indicates that the extraction process can maintain the significant antioxidant capacity of hawthorn leaf flavonoids, providing a reliable technical solution for the development of natural antioxidants.

[0060] Example 5: In vitro hypoglycemic activity assay The inhibition rate of α-glucosidase activity by the extract obtained using an HES solvent system with a molar ratio of thymol and undecenoic acid of 3:2 (the extract was obtained under optimal conditions: temperature 50℃, liquid-to-solid ratio 22.06 mL / g, power 198.7 W, time 40 min, other conditions the same as in Example 1) was determined. ) and α-amylase activity inhibition rate ( It expresses in vitro hypoglycemic activity.

[0061] (1) α-glucosidase inhibitory activity (α-GIA) Prepare the sample, α-glucosidase solution, and pNPG (chromogenic enzyme substrate, p-nitrophenyl-β-D-glucopyranoside) in 0.05M PBS (pH 6.8); incubate 100 μL of sample extract with 100 μL of 700 U / mL α-glucosidase at 37°C for 10 min; then add 100 μL of pNPG substrate solution (5 mM, pH 6.8) and incubate at 37°C for 30 min; finally, terminate the reaction by adding 300 μL of 0.5M Na2CO3 and measure the absorbance at 405 nm; each experiment was repeated three times; the formula for calculating α-GIA (%) is: .

[0062] Where A b A c and A s The absorbance values ​​represent the absorbance of the extract, the absorbance after reaction with PBS and α-glucosidase, and the absorbance after reaction with α-glucosidase, respectively.

[0063] The results showed that when the flavonoid concentration in the extract was 0.8 g / L, the average inhibition rate of the extract on α-glucosidase activity was 63.12±2.65%, which was 1.24 times that of acarbose at the same concentration.

[0064] (2) α-Amylase inhibitory activity (α-AIA) 100 μL of sample extract was mixed with α-amylase solution (100 μL, 2.5 U / mL) and incubated in a centrifuge tube at 37 °C for 10 min. Then, 200 μL of substrate-soluble starch solution (1 mg / mL) was added, and the mixture was incubated at 37 °C for 20 min. Finally, 200 μL of DNS reagent solution was added to terminate the reaction. The resulting mixture was then heated in boiling water for 10 min, cooled, and the absorbance was measured at 540 nm. Samples and α-amylase solutions were prepared in PBS (0.05 mol / L, pH 6.9). α-AIA (%) was calculated using the following formula: .

[0065] Where A e A b A s and A c The absorbance values ​​are respectively: absorbance of the extract, absorbance after reaction with PBS and α-amylase, absorbance after reaction with α-amylase, and absorbance after reaction with DNS.

[0066] The results showed that when the flavonoid concentration in the extract was 0.8 g / L, the extract inhibited α-amylase activity by 75.84±3.91%, which was 2.80 times that of acarbose at the same concentration.

[0067] Comparative Example 1: Comparison of extraction capabilities of HES with ethyl acetate and ethanol The solvent was replaced with ethyl acetate or ethanol, and flavonoids were extracted using the same method as that used for extracting total flavonoids with a hydrophobic eutectic solvent (extraction conditions: HES solvent system with a molar ratio of thymol to undecenoic acid of 3:2, temperature 50℃, liquid-to-solid ratio 22.06 mL / g, power 198.7 W, time 40 min, other conditions the same as in Example 1). A comparison of the two methods demonstrated the superiority of the hydrophobic eutectic solvent. The results showed that the total flavonoid content extracted with the hydrophobic eutectic solvent was 3.69 times that of the ethyl acetate extract (12.64 ± 0.52 mg / g) and 6.11 times that of the ethanol extract (9.69 ± 0.22 mg / g).

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not 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 claims of the present invention.

Claims

1. A method for extracting flavonoids from hawthorn leaves, characterized in that, The extraction method includes: mixing hawthorn leaves with a hydrophobic eutectic solvent, then ultrasonically treating the mixture and centrifuging to obtain the extract.

2. The extraction method according to claim 1, characterized in that, The hydrophobic eutectic solvent is selected from any of the following: (i) The hydrogen bond acceptor is DL-menthol, the hydrogen bond donor is decanoic acid, and the molar ratio of DL-menthol to decanoic acid is 1:1; (ii) The hydrogen bond acceptor is thymol, the hydrogen bond donor is decanoic acid, and the molar ratio of thymol to decanoic acid is 1:(1-3). (iii) The hydrogen bond acceptor is thymol, the hydrogen bond donor is undecenoic acid, and the molar ratio of thymol to undecenoic acid is 1:(1-4). (iv) The hydrogen bond acceptor is tetrabutylammonium bromide, the hydrogen bond donor is lauric acid, and the molar ratio of tetrabutylammonium bromide to lauric acid is 1:1.

2.

3. The extraction method according to claim 2, characterized in that, The hydrophobic eutectic solvent is selected from any of the following: The hydrogen bond acceptor is thymol, and the hydrogen bond donor is decanoic acid; the molar ratio of thymol to decanoic acid is 3:1 or 1:2; or The hydrogen bond acceptor is thymol, and the hydrogen bond donor is undecenoic acid. The molar ratio of thymol to undecenoic acid is 3:2, 1:1, 1:2, or 1:

4.

4. The extraction method according to any one of claims 1 to 3, characterized in that, Hawthorn leaves are in powder form.

5. The extraction method according to claim 4, characterized in that, The liquid-to-solid ratio of the hydrophobic eutectic solvent and hawthorn leaves is 10 mL / g to 30 mL / g.

6. The extraction method according to claim 5, characterized in that, The liquid-to-solid ratio of the hydrophobic eutectic solvent and hawthorn leaves was 22.06 mL / g.

7. The extraction method according to claim 1, 2, 3, 5 or 6, characterized in that, The conditions for ultrasonic treatment include: The ultrasonic power is 144W to 240W; The ultrasonic temperature is 30℃~50℃; The ultrasound time is ≥40 min.

8. The extraction method according to claim 7, characterized in that, The ultrasound time was 40 minutes.

9. The extraction method according to claim 7, characterized in that, The ultrasonic power is 198.7 W and the ultrasonic temperature is 50℃.

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