Method for extracting boxthorn leaf total flavonoids by using ultrasonic-assisted deep-eutectic solvent and application of boxthorn leaf total flavonoids

Abstract

The invention belongs to the technical field of active substance extraction, and discloses a method for extracting boxthorn leaf total flavonoids through an ultrasonic-assisted deep-eutectic solvent and application of the boxthorn leaf total flavonoids. Heating and mixing the hydrogen bond acceptor and the hydrogen bond donor, stirring to be transparent, and adding water to reduce viscosity to obtain the eutectic solvent; mixing the boxthorn leaf powder with a deep-eutectic solvent, performing ultrasonic extraction and centrifugation, and collecting supernate; adsorbing by using macroporous resin, washing and desorbing to obtain an eluent; and performing vacuum freeze drying to constant weight. According to the method disclosed by the invention, the eutectic solvent with low melting point and strong solubility is selected, the flavonoid compounds in the boxthorn leaves are green and efficiently extracted, and the total flavonoid content of the boxthorn leaves obtained by taking the eutectic solvent as an extraction solvent is 123.94 + / -1.04 mg / g and is far higher than the total flavonoid content of the boxthorn leaves obtained by traditional ethanol extraction by 64.60 + / -0.87 mg / g; the method is good in safety, short in time consumption and high in extraction rate.

Description

Technical Field

[0001] This invention belongs to the field of active substance extraction technology, and in particular to a method and application of ultrasound-assisted eutectic solvent extraction of total flavonoids from wolfberry leaves. Background Technology

[0002] Lycium barbarum L. leaves are the leaves of the Lycium barbarum plant (Lycium barbarum L. leaves) belonging to the Solanaceae family and the Lycium genus. Lycium Chinese Mill ) and Ningxia wolfberry ( Lycium barbarum L. The dried tender leaves of wolfberry (Goji berry). Wolfberry leaves and wolfberries belong to the same root group, and their influencing and active components are essentially the same. Traditional medicine believes that wolfberry leaves are sweet and bitter in taste and cold in nature, possessing the effects of strengthening the heart, dispersing sores and swellings, dispelling wind and improving eyesight, and clearing heat and toxins. Modern pharmacological research reports that wolfberry leaves contain abundant functional substances, exhibiting various biological activities such as antioxidant, blood pressure lowering, blood lipid lowering, and blood sugar lowering. Studies have shown that wolfberry leaves are rich in flavonoids and phenolic acids, with significantly higher levels of phenolic acids and flavonoids than wolfberry fruits. Furthermore, the flavonoids in wolfberry leaves possess various physiological and pharmacological effects, including antioxidant, anti-inflammatory, antibacterial, and gastrointestinal function improvement. Therefore, the isolation and extraction of flavonoid compounds from wolfberry leaves has broad market prospects and application value.

[0003] The extraction of flavonoids from wolfberry leaves mainly relies on traditional methods (such as hot water extraction and organic solvent extraction), which suffer from low extraction efficiency and solvent residue, limiting their application in pharmaceuticals, food, and cosmetics. Compared to traditional organic solvents, deep eutectic solvents (DES) possess excellent thermal stability, a lower melting point, and are easier to operate at room temperature. Furthermore, the synthesis process of DES is simple, the raw materials are inexpensive, non-toxic, and biodegradable, effectively reducing environmental pollution. To further improve the extraction efficiency of DES, ultrasound-assisted extraction is commonly used. Ultrasonic extraction technology utilizes the cavitation, mechanical, and thermal effects generated when ultrasound waves propagate in a liquid to accelerate the disruption of plant cell walls, thereby accelerating the release and dissolution of intracellular active substances. Therefore, ultrasound-assisted deep eutectic solvent extraction is a green and efficient extraction method.

[0004] The search revealed the following patent publications related to this invention's patent application: 1. Patents CN102000208A and CN106177170A disclose a method for extracting and preparing flavonoids from wolfberry leaves, including drying and pulverizing wolfberry leaves, followed by ethanol extraction, and using a combination of extraction, adsorption, elution, and concentration techniques to prepare flavonoids, resulting in wolfberry leaf flavonoid powder. However, both patents use traditional organic solvent extraction, which is time-consuming and results in low extraction rates and purity of wolfberry leaf flavonoids.

[0005] 2. Patent CN108785449A discloses an extraction process for flavonoids from wolfberry using a combination of ultrasound and microwave. This invention involves drying and pulverizing wolfberries, then extracting them with a 70% ethanol solution under specific conditions using a combination of ultrasound and microwave, followed by filtration to obtain the filtrate. Although this patent uses microwave-assisted extraction to shorten the extraction time, it uses traditional ethanol as the solvent, which has reagent toxicity.

[0006] 3. Patent CN110934922A discloses a method for extracting total flavonoids from black goji berries, and its applications. It uses a choline chloride-type eutectic solvent as the extractant to extract flavonoids from black goji berries. The flavonoid extract is then adsorbed onto a macroporous resin, followed by the addition of polyethylene glycol, and finally freeze-dried under vacuum to obtain the final product. However, this patent does not include component analysis or activity testing of the extracted goji berry flavonoids.

[0007] 4. Patent CN120305336A discloses a method for extracting phenolic compounds from wolfberry leaves and its application. This invention uses choline chloride-lactic acid as a eutectic solvent to extract phenolic compounds from wolfberry leaves. After purification and separation of the crude extract using macroporous resin, the obtained wolfberry leaf phenolic compounds contain compounds such as rutin, chlorogenic acid, and quercetin. This patent uses a eutectic solvent to extract the target components, but it does not compare the activity differences between wolfberry leaf polyphenols extracted using traditional extraction methods and eutectic solvent methods.

[0008] In summary, a comparison of the above patents reveals that current extraction methods for total flavonoids from wolfberry leaves primarily rely on traditional ethanol extraction, but there is still room for improvement. The technology for extracting total flavonoids from wolfberry leaves using eutectic solvents is currently lacking. To improve the extraction efficiency of flavonoids from wolfberry leaves, reduce application toxicity, and expand application scenarios, this invention proposes to use an ultrasound-assisted eutectic solvent method to optimize the extraction process of total flavonoids from wolfberry leaves. This method combines macroporous resin to separate and purify flavonoids from wolfberry leaves, evaluate their antioxidant activity, and compare the activity differences between traditional extraction methods and eutectic solvent extraction, laying the foundation for its application. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and application for extracting total flavonoids from wolfberry leaves using ultrasound-assisted eutectic solvent.

[0010] The technical solution adopted by this invention to solve its technical problem is: A method for extracting total flavonoids from wolfberry leaves using an ultrasound-assisted eutectic solvent includes the following steps: (1) After drying, wolfberry leaves are pulverized and sieved to obtain wolfberry leaf powder; (2) Heat and mix the hydrogen bond acceptor and hydrogen bond donor at 80°C, stir until transparent, and add water to reduce viscosity to obtain a eutectic solvent; (3) After mixing the wolfberry leaf powder prepared in step (1) with the eutectic solvent prepared in step (2), extract by ultrasonication, centrifuge, collect the supernatant, and obtain the total flavonoid extract of wolfberry leaves; (4) The total flavonoid extract of wolfberry leaves obtained in step (3) was adsorbed using macroporous resin. The adsorbed macroporous resin was washed and desorbed to obtain the eluent. (5) The eluent obtained in the concentration step (4) is freeze-dried under vacuum to constant weight to obtain Lycium barbarum leaf flavonoid freeze-dried powder.

[0011] Further, the hydrogen bond acceptor in step (2) is at least one of choline chloride and betaine; the hydrogen bond donor is at least one of glycerol, ethylene glycol, lactic acid, p-toluenesulfonic acid, 1,2-propylene glycol, 1,3-butanediol, glucose, and urea; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2-5. Further, in step (2), the eutectic solvent is betaine-ethylene glycol, wherein the molar ratio between betaine and ethylene glycol is 1:1~8, and the water content of the eutectic solvent is 0~50%.

[0012] Further, the conditions for ultrasonic extraction in step (3) are: the material-to-liquid ratio is 10~70 mL / g; the ultrasonic time is 10~70 min; the ultrasonic temperature is 50℃; the ultrasonic power is 500 W; the centrifugation speed in step (3) is 10000 xg / min; and the centrifugation time is 10 min.

[0013] Further, in step (4), D101 type macroporous resin is selected to adsorb the active substances of wolfberry leaves. After adsorption, the D101 type macroporous resin is washed with distilled water until it is colorless and then eluted with 70% ethanol by volume. The purpose is to purify wolfberry leaf flavonoids and remove eutectic solvents and other water-soluble impurities. Alternatively, in step (4), the total flavonoid extract of wolfberry leaves is diluted with Wahaha water before purification to break the hydrogen bonds between the eutectic solvent and the active substances of wolfberry leaves, thereby enhancing the adsorption effect of macroporous resin on total flavonoids of wolfberry leaves.

[0014] Further, in step (2), the eutectic solvent is betaine-ethylene glycol, wherein the molar ratio between betaine and ethylene glycol is 1:4.48, and the water content of the eutectic solvent is 22%; in step (3), the ultrasonic extraction conditions are: ultrasonic time 40 min, material-liquid ratio 41.80 mL / g, under which the total flavonoid extraction rate of wolfberry leaves is 123.94±1.04 mg / g.

[0015] The application of the method described above in the production of total flavonoids from wolfberry leaves.

[0016] Total flavonoids from wolfberry leaves were prepared using the method described above.

[0017] Furthermore, the main components of the Lycium barbarum leaf flavonoid freeze-dried powder include rutin, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid.

[0018] The applications of total flavonoids from wolfberry leaves as described above in the preparation of pharmaceuticals and / or food and / or cosmetics.

[0019] The advantages and positive effects of this invention are as follows: 1. The method of this invention uses a low melting point and high solubility eutectic solvent to extract flavonoids from wolfberry leaves in a green and efficient manner. The total flavonoid content of wolfberry leaves obtained by using a eutectic solvent (betaine-ethylene glycol) as the extraction solvent is 123.94±1.04 mg / g, which is much higher than the total flavonoid content of 64.60±0.87 mg / g obtained by traditional ethanol extraction. Moreover, this method has the advantages of good safety, short time consumption and high extraction rate.

[0020] 2. The method of this invention extracts flavonoids from wolfberry leaves using an ultrasound-assisted eutectic solvent. After purification with macroporous resin, the extraction rate of wolfberry leaf flavonoids is high (9.00%) and the purity is high (70%-85%). The total flavonoids obtained from wolfberry leaves through the optimized extraction process have better antioxidant bioactivity compared with traditional extraction methods. The safety and high extraction efficiency of the eutectic solvent make it more promising for application in pharmaceuticals, food and cosmetics.

[0021] 3. The method of this invention involves mixing wolfberry leaves with a natural eutectic solvent and then performing ultrasonic extraction. The mixture is centrifuged, the supernatant is collected, purified using D101 macroporous resin, and freeze-dried to obtain wolfberry leaf flavonoids. This invention uses betaine-ethylene glycol as a eutectic solvent, achieving an extraction rate of up to 123.94 ± 1.04 mg / g for flavonoids from wolfberry leaves. After macroporous resin purification, the flavonoid extraction rate is 9.00%, with a purity exceeding 70%. These flavonoids are rich in rutin, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid, among other compounds. Furthermore, the total flavonoids extracted using the eutectic solvent exhibit superior antioxidant bioactivity compared to traditional extraction methods, demonstrating excellent antioxidant properties and promising applications in biomedicine, food, and cosmetics. Attached Figure Description

[0022] Figure 1 This is a graph showing the effect of different types of DES solvents on the extraction rate of total flavonoids from wolfberry leaves in this invention; Figure 2 This is a graph showing the effect of different liquid-to-solid ratios on the extraction rate of total flavonoids from wolfberry leaves in this invention. Figure 3 This is a graph showing the effect of different ultrasound times on the extraction rate of total flavonoids from wolfberry leaves in this invention; Figure 4 This is a graph showing the effect of different DES moisture contents on the extraction rate of total flavonoids from wolfberry leaves in this invention. Figure 5 This is a graph showing the effect of different DES molar ratios on the extraction rate of total flavonoids from wolfberry leaves in this invention. Figure 6 This is a 3D response surface plot showing the effects of material-liquid ratio, ultrasonic time, DES water content, and DES molar ratio on the extraction rate of total flavonoids from wolfberry leaves in this invention. Figure 7 This is a contour plot showing the effects of material-liquid ratio, ultrasonic time, DES water content, and DES molar ratio on the extraction rate of total flavonoids from wolfberry leaves in this invention. Figure 8 This is a graph showing the DPPH free radical scavenging capacity of different concentrations of total flavonoids from wolfberry leaves prepared in this invention; Figure 9 The graph shows the ABTS free radical scavenging capacity of different concentrations of total flavonoids from wolfberry leaves prepared in this invention. Figure 10 The graph shows the total VC reducing power of different concentrations of total flavonoids from wolfberry leaves prepared in this invention. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0024] The various experimental operations involved in the specific embodiments are all conventional techniques in the art. For parts not specifically annotated herein, those skilled in the art can refer to various commonly used reference books, scientific and technological literature, or related instructions and manuals prior to the filing date of this invention for implementation. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0025] A method for extracting total flavonoids from wolfberry leaves using an ultrasound-assisted eutectic solvent includes the following steps: (1) After drying, wolfberry leaves are pulverized and sieved to obtain wolfberry leaf powder; (2) Heat and mix the hydrogen bond acceptor and hydrogen bond donor at 80°C, stir until transparent, and add water to reduce viscosity to obtain a eutectic solvent; (3) After mixing the wolfberry leaf powder prepared in step (1) with the eutectic solvent prepared in step (2), extract by ultrasonication, centrifuge, collect the supernatant, and obtain the total flavonoid extract of wolfberry leaves; (4) The total flavonoid extract of wolfberry leaves obtained in step (3) was adsorbed using macroporous resin. The adsorbed macroporous resin was washed and desorbed to obtain the eluent. (5) The eluent obtained in the concentration step (4) is freeze-dried under vacuum to constant weight to obtain Lycium barbarum leaf flavonoid freeze-dried powder.

[0026] Preferably, the hydrogen bond acceptor in step (2) is at least one of choline chloride and betaine; the hydrogen bond donor is at least one of glycerol, ethylene glycol, lactic acid, p-toluenesulfonic acid, 1,2-propylene glycol, 1,3-butanediol, glucose, and urea; and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2-5. Preferably, in step (2), the eutectic solvent is betaine-ethylene glycol, wherein the molar ratio between betaine and ethylene glycol is 1:1~8, and the water content of the eutectic solvent is 0~50%.

[0027] Preferably, the conditions for ultrasonic extraction in step (3) are: a material-to-liquid ratio of 10-70 mL / g; an ultrasonic time of 10-70 min; an ultrasonic temperature of 50℃; an ultrasonic power of 500 W; and a centrifugation speed of 10000 xg / min and a centrifugation time of 10 min.

[0028] Preferably, in step (4), D101 type macroporous resin is used to adsorb the active substances of wolfberry leaves. After adsorption, the D101 type macroporous resin is washed with distilled water until it is colorless and then eluted with 70% ethanol by volume. The purpose is to purify wolfberry leaf flavonoids and remove eutectic solvents and other water-soluble impurities. Alternatively, in step (4), the total flavonoid extract of wolfberry leaves is diluted with Wahaha water before purification to break the hydrogen bonds between the eutectic solvent and the active substances of wolfberry leaves, thereby enhancing the adsorption effect of macroporous resin on total flavonoids of wolfberry leaves.

[0029] Preferably, in step (2), the eutectic solvent is betaine-ethylene glycol, wherein the molar ratio between betaine and ethylene glycol is 1:4.48, and the water content of the eutectic solvent is 22%; the ultrasonic extraction conditions in step (3) are: ultrasonic time 40 min, material-liquid ratio 41.80 mL / g, under which the total flavonoid extraction rate of wolfberry leaves is 123.94±1.04 mg / g.

[0030] The application of the method described above in the production of total flavonoids from wolfberry leaves.

[0031] Total flavonoids from wolfberry leaves were prepared using the method described above.

[0032] Preferably, the main components of the lyophilized wolfberry leaf flavonoid powder include rutin, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid.

[0033] The applications of total flavonoids from wolfberry leaves as described above in the preparation of pharmaceuticals and / or food and / or cosmetics.

[0034] Further optional techniques for extracting total flavonoids from wolfberry leaves include: the total flavonoid content in the wolfberry leaf extract obtained in step (3) is detected by the sodium nitrite-aluminum nitrate colorimetric method, and the total flavonoid content is expressed as the rutin equivalent per gram of dry weight sample (mg / g).

[0035] Specifically, the relevant preparation and testing methods are as follows: In this invention, the total flavonoid content was determined using the sodium nitrite-aluminum nitrate method. The specific method is as follows: Using rutin as a standard, 1 mL of each sample was placed in a 10 mL clean tube, 0.3 mL of NaNO2 solution (5%, w / v, mass concentration) was added, mixed, and allowed to stand at room temperature for 6 min. Then, 0.3 mL of Al(NO3)3 solution (10%, w / v, mass concentration) was added, mixed, and allowed to stand at room temperature for 6 min. Finally, 4.0 mL of NaOH solution (4%, w / v, mass concentration) was added, mixed, and allowed to stand for 15 min. The absorbance was measured at 510 nm. A standard curve was plotted with rutin concentration on the x-axis (mg / mL) and absorbance on the y-axis. The regression equation was y = 1.3216x - 0.0005 (R² / 2π × 10⁻¹² ... 2 =0.9997), the result is expressed as rutin equivalents (mg / g) per gram of dry weight sample.

[0036] Example 1: This embodiment provides a method for extracting total flavonoids from wolfberry leaves using an ultrasound-assisted eutectic solvent (choline chloride-ethylene glycol). Specifically: The dried wolfberry leaves were selected, pulverized, and passed through a 60-mesh sieve to obtain coarse wolfberry leaf powder. 1 g of the coarse wolfberry leaf powder was mixed thoroughly with 20 mL of a eutectic solvent (choline chloride-ethylene glycol, where the molar ratio of choline chloride to ethylene glycol was 1:2, and the water content was 30%). The mixed sample solution was then ultrasonically treated in an ultrasonic cleaner. The extraction conditions were: eutectic solvent water content 30%, solid-liquid ratio 1:20 g / mL, ultrasonic power 500 W, ultrasonic time 30 min, and ultrasonic temperature 50℃. After extraction, the sample was centrifuged at 10000 x g / min for 10 min, and the supernatant was collected to obtain a wolfberry leaf extract containing flavonoids. The obtained wolfberry leaf extract was diluted with methanol and filtered through a 0.45 μm organic filter membrane. The resulting sample solution was used for the determination of total flavonoid content.

[0037] Example 2: The difference between this embodiment and Embodiment 1 is that the eutectic solvent in Embodiment 1 is replaced with choline chloride-urea, and the molar ratio of choline chloride to urea is 1:2, specifically: The dried wolfberry leaves were selected, pulverized, and passed through a 60-mesh sieve to obtain coarse wolfberry leaf powder. 1 g of the coarse wolfberry leaf powder was mixed thoroughly with 20 mL of a eutectic solvent (choline chloride-urea, where the molar ratio of choline chloride to urea was 1:2, and the water content was 30%). The mixed sample solution was then ultrasonically treated in an ultrasonic cleaner. The extraction conditions were: eutectic solvent water content 30%, solid-liquid ratio 1:20 g / mL, ultrasonic power 500 W, ultrasonic time 30 min, and ultrasonic temperature 50℃. After extraction, the sample was centrifuged at 10000 x g / min for 10 min, and the supernatant was collected to obtain a wolfberry leaf extract containing flavonoids. The obtained wolfberry leaf extract was diluted with methanol and filtered through a 0.45 μm organic filter membrane. The resulting sample solution was used for the determination of total flavonoid content.

[0038] Example 3: The difference between this embodiment and Embodiment 1 is that the eutectic solvent in Embodiment 1 is replaced with choline chloride-glucose, and the molar ratio of choline chloride to glucose is 1:2. Specifically: The dried wolfberry leaves were selected, pulverized, and passed through a 60-mesh sieve to obtain coarse wolfberry leaf powder. 1 g of coarse wolfberry leaf powder was mixed with 20 mL of a eutectic solvent (choline chloride-glucose, where the molar ratio of choline chloride to glucose was 1:2, and the water content was 30%). The mixed sample solution was then ultrasonically treated in an ultrasonic cleaner. The extraction conditions were: eutectic solvent water content 30%, solid-liquid ratio 1:20 g / mL, ultrasonic power 500 W, ultrasonic time 30 min, and ultrasonic temperature 50℃. After extraction, the sample was centrifuged at 10000 x g / min for 10 min, and the supernatant was collected to obtain a wolfberry leaf extract containing flavonoids. The obtained wolfberry leaf extract was diluted with methanol and filtered through a 0.45 μm organic filter membrane. The resulting sample solution was used for the determination of total flavonoid content.

[0039] Example 4: The difference between this embodiment and Embodiment 1 is that choline chloride is replaced with betaine, and the molar ratio of betaine to ethylene glycol is 1:2. Specifically: The dried wolfberry leaves were selected, pulverized, and passed through a 60-mesh sieve to obtain coarse wolfberry leaf powder. 1 g of coarse wolfberry leaf powder was mixed with 20 mL of a eutectic solvent (betaine-ethylene glycol, where the molar ratio of betaine to ethylene glycol was 1:2, and the water content was 30%). The mixed sample solution was then ultrasonically treated in an ultrasonic cleaner. The extraction conditions were: eutectic solvent water content 30%, solid-liquid ratio 1:20 g / mL, ultrasonic power 500 W, ultrasonic time 30 min, and ultrasonic temperature 50℃. After extraction, the sample was centrifuged at 10000 x g / min for 10 min, and the supernatant was collected to obtain a wolfberry leaf extract containing flavonoids. The obtained wolfberry leaf extract was diluted with methanol and filtered through a 0.45 μm organic filter membrane. The resulting sample solution was used for the determination of total flavonoid content.

[0040] Example 5: The difference between this embodiment and Example 1 is that the eutectic solvent in Example 1 is replaced with betaine-1,3-butanediol, and the molar ratio of betaine to 1,3-butanediol is 1:5, specifically: The dried wolfberry leaves were selected, pulverized, and passed through a 60-mesh sieve to obtain coarse wolfberry leaf powder. 1 g of coarse wolfberry leaf powder was mixed with 20 mL of a eutectic solvent (betaine-1,3-butanediol, with a molar ratio of 1:5 and a water content of 30%). The mixed sample solution was then ultrasonically treated in an ultrasonic cleaner. The extraction conditions were: eutectic solvent water content 30%, solid-liquid ratio 1:20 g / mL, ultrasonic power 500 W, ultrasonic time 30 min, and ultrasonic temperature 50℃. After extraction, the sample was centrifuged at 10000 x g / min for 10 min, and the supernatant was collected to obtain a wolfberry leaf extract containing flavonoids. The obtained wolfberry leaf extract was diluted with methanol and filtered through a 0.45 μm organic filter membrane. The resulting sample solution was used for the determination of total flavonoid content.

[0041] Example 6: The difference between this embodiment and Example 1 is that the eutectic solvent in Example 1 is replaced with betaine-lactic acid, and the molar ratio of betaine to lactic acid is 1:2, specifically: The dried wolfberry leaves were selected, pulverized, and passed through a 60-mesh sieve to obtain coarse wolfberry leaf powder. 1 g of coarse wolfberry leaf powder was mixed with 20 mL of a eutectic solvent (betaine-lactic acid, where the molar ratio of betaine to lactic acid was 1:2, and the water content was 30%). The mixed sample solution was then ultrasonically treated in an ultrasonic cleaner. The extraction conditions were: eutectic solvent water content 30%, solid-liquid ratio 1:20 g / mL, ultrasonic power 500 W, ultrasonic time 30 min, and ultrasonic temperature 50℃. After extraction, the sample was centrifuged at 10000 x g / min for 10 min, and the supernatant was collected to obtain a wolfberry leaf extract containing flavonoids. The obtained wolfberry leaf extract was diluted with methanol and filtered through a 0.45 μm organic filter membrane. The resulting sample solution was used for the determination of total flavonoid content.

[0042] Comparative Example 1: The difference between this comparative example and Example 1 is that the eutectic solvent was adjusted to 70% ethanol by volume, while the remaining steps and conditions were the same as in Example 1, thus obtaining the total flavonoid extract of wolfberry leaves in this comparative example.

[0043] Comparative Example 2: The difference between this comparative example and Example 1 is that the eutectic solvent was changed to pure water, while the remaining steps and conditions were the same as in Example 1, thus obtaining the total flavonoid extract of wolfberry leaves in this comparative example.

[0044] Figure 1 The effect of different eutectic solvents, including those used in the examples and comparative examples, on the total flavonoid extraction rate from wolfberry leaf extract was investigated.

[0045] The results of determining the total flavonoid content of wolfberry leaves in the supernatant of Examples 1-6 and Comparative Examples 1 and 2 are shown in Table 1.

[0046] Table 1. Effects of different extraction conditions on the extraction rate of total flavonoids from wolfberry leaves.

[0047] As shown in Table 1, the flavonoid extraction rate of the extract obtained by using polyols as hydrogen bond donors in eutectic solvents was significantly higher than that of the ethanol and water extracts in the comparative examples. This is because eutectic solvents can increase the extraction degree of flavonoids from wolfberry leaves by forming hydrogen bonds with the target compounds. Furthermore, compared to ethanol, which has toxicity and high volatility, DESs are more suitable for use in pharmaceutical, food, and cosmetic research.

[0048] Comparative Example 3 The difference between this comparative example and Example 4 is that the eutectic solvent in Example 1 is adjusted to betaine-urea, wherein the molar ratio of betaine to urea is 1:2. The remaining steps are the same as in Example 1, thus obtaining the total flavonoid extract of wolfberry leaves in this comparative example.

[0049] Comparative Example 4 The difference between this comparative example and Example 4 is that the eutectic solvent in Example 1 is adjusted to betaine-1,2-propylene glycol, wherein the molar ratio of betaine to 1,2-propylene glycol is 1:4. The remaining steps are the same as in Example 1, thus obtaining the total flavonoid extract of wolfberry leaves in this comparative example.

[0050] Comparative Example 5 The difference between this comparative example and Example 4 is that the eutectic solvent in Example 1 is adjusted to betaine-1,3-butanediol-lactic acid, wherein the molar ratio of betaine, 1,3-butanediol and lactic acid is 1:2:1. The remaining steps are the same as in Example 1, thus obtaining the total flavonoid extract of wolfberry leaves in this comparative example.

[0051] Comparative Example 6 The difference between this comparative example and Example 4 is that the eutectic solvent in Example 1 is adjusted to betaine-1,3-butanediol-lactic acid, wherein the molar ratio of betaine, 1,3-butanediol and lactic acid is 1:1:2. The remaining steps are the same as in Example 1, thus obtaining the total flavonoid extract of wolfberry leaves in this comparative example.

[0052] The total flavonoid content of wolfberry leaves in the supernatant of comparative examples 3-6 was determined and compared with that of example 4. The results are shown in Table 2.

[0053] Table 2. Extraction rate of total flavonoids from wolfberry leaves under different comparative ratios

[0054] Table 2 shows that the content of flavonoids in the extracts obtained by ultrasonic extraction assisted by eutectic solvents with different hydrogen bond donors varies significantly. This is due to the different strengths of hydrogen bond formation between the different eutectic solvents and the target compounds, as well as the different polarities of the resulting eutectic solvents. Betaine eutectic solvents are more effective at extracting flavonoids than DES containing choline chloride or acids. The DES composed of betaine and ethylene glycol in Example 4 exhibits the strongest ability to form hydrogen bonds with the target compounds, resulting in the highest flavonoid extraction rate. Therefore, betaine-ethylene glycol was selected as the DES combination for subsequent optimization of the total flavonoid extraction process from wolfberry leaves.

[0055] Meanwhile, by comparing Examples 1, 4, and Comparative Examples 3 to 6, it can be seen that betaine and ethylene glycol have a synergistic effect in the method of the present invention, which can synergistically improve the extraction rate of total flavonoids from wolfberry leaves.

[0056] Next, based on Example 4, we studied the optimal process for extracting total flavonoids from wolfberry leaves using eutectic solvents.

[0057] Example 7 This embodiment provides a single-factor optimization experiment for the extraction of total flavonoids from wolfberry leaves using an ultrasound-assisted eutectic solvent (Example 4): The difference between this embodiment and Example 4 is that the molar ratio of betaine to ethylene glycol in the eutectic solvent of Example 4 is adjusted to 1:2, the water content is 30%, the material-to-liquid ratio is 10~70 mL / g, and the ultrasonic time is 30 min. All other conditions are the same as in Example 4. Specifically: The dried wolfberry leaves were selected, pulverized, and passed through a 60-mesh sieve to obtain coarse wolfberry leaf powder. 1 g of the coarse wolfberry leaf powder was mixed evenly with different volumes of eutectic solvent (betaine-ethylene glycol, molar ratio 1:2, water content 30%). The mixed sample solution was then ultrasonically treated in an ultrasonic cleaner. The extraction conditions were: eutectic solvent water content 30%, solid-liquid ratio 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70 g / mL, ultrasonic power 500 W, ultrasonic time 30 min, ultrasonic temperature 50℃. After extraction, the sample was centrifuged at 10000 x g / min for 10 min, and the supernatant was collected to obtain a wolfberry leaf extract containing flavonoids. The obtained wolfberry leaf extract was diluted with methanol and filtered through a 0.45 μm organic filter membrane. The resulting sample solution was used for the determination of total flavonoid content.

[0058] The total flavonoid content of wolfberry leaves in the supernatant of Example 7 was determined and compared with that of Example 4. The results are shown in Table 3.

[0059] Table 3. Effect of different solid-liquid ratios on the extraction rate of total flavonoids from wolfberry leaves.

[0060] The solvent-to-sample ratio affects the mass transfer of the target compound in the solvent. A low solid-liquid ratio may result in ineffective solvent wetting of the material, easy local saturation of the sample and solvent concentrations, high mass transfer resistance, and incomplete extraction of the target compound from the sample, leading to low extraction efficiency. Conversely, an excessively high solid-liquid ratio results in a too-small concentration gradient, weakened mass transfer driving force, slow diffusion rate, and affects the extraction rate and final extraction yield, while also wasting extraction solvent. Figure 2 As shown, the extraction rate of total flavonoids from wolfberry leaves increased significantly when the solid-liquid ratio increased from 1:10 to 1:40. However, with further increases in the solid-liquid ratio, the extraction rate of total flavonoids from wolfberry leaves began to decrease. Excessive solvent reduces the extraction rate of total flavonoids from wolfberry leaves and increases extraction costs. Therefore, the optimal solid-liquid ratio of 1:40 was selected for subsequent optimized extraction.

[0061] Example 8 This embodiment provides a single-factor optimization experiment for the extraction of total flavonoids from wolfberry leaves using an ultrasound-assisted eutectic solvent (Example 4): The difference between this embodiment and Example 4 is that the molar ratio of betaine to ethylene glycol in the eutectic solvent of Example 4 is adjusted to 1:2, the water content is 30%, the material-to-liquid ratio is 1:20 g / mL, and the ultrasonic time is 10-70 min. All other conditions are the same as in Example 4. Specifically: The dried wolfberry leaves were selected, pulverized, and passed through a 60-mesh sieve to obtain coarse wolfberry leaf powder. 1 g of coarse wolfberry leaf powder was mixed thoroughly with 20 mL of a eutectic solvent (betaine-ethylene glycol, molar ratio 1:2, water content 30%). The mixed sample solution was then ultrasonically treated in an ultrasonic cleaner for different times. The extraction conditions were: eutectic solvent water content 30%, solid-liquid ratio 1:20 g / mL, ultrasonic power 500 W, ultrasonic times of 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, and 70 min, and ultrasonic temperature 50℃. After extraction, the sample was centrifuged at 10000 x g / min for 10 min, and the supernatant was collected to obtain a wolfberry leaf extract containing flavonoids. The obtained wolfberry leaf extract was diluted with methanol and filtered through a 0.45 μm organic filter membrane. The resulting sample solution was used for the determination of total flavonoid content.

[0062] The total flavonoid content of wolfberry leaves in the supernatant of Example 8 was determined and compared with that of Example 4. The results are shown in Table 4.

[0063] Table 4. Effects of different ultrasound times on the extraction rate of total flavonoids from wolfberry leaves.

[0064] Ultrasonic extraction technology utilizes the cavitation, mechanical, and thermal effects generated by ultrasound waves propagating in liquids to accelerate the disruption of plant cell walls, thereby accelerating the release and dissolution of intracellular active substances. Appropriate ultrasound can improve solvent extraction rates, but excessive ultrasound may destroy the target compounds, resulting in a reduced extraction rate. For example... Figure 3 As shown, when the material-to-liquid ratio was fixed at 1:20 g / mL and the ultrasonic power was 500 W, the extraction rate of total flavonoids from wolfberry leaves increased with increasing ultrasonic time from 10 min to 40 min. However, with the extension of ultrasonic time, the target extract may be affected by the ultrasound, leading to a decrease in extraction rate. Therefore, we selected ultrasonic time levels of 30 min, 40 min, and 50 min for subsequent experiments.

[0065] Example 9 This embodiment provides a single-factor optimization experiment for the extraction of total flavonoids from wolfberry leaves using an ultrasound-assisted eutectic solvent (Example 4): The difference between this embodiment and Example 4 is that the molar ratio of betaine to ethylene glycol in the eutectic solvent of Example 4 is adjusted to 1:4, the water content is 0-50%, the material-to-liquid ratio is 1:20 g / mL, and the ultrasonic time is 30 min. All other conditions are the same as in Example 4. Specifically: The dried wolfberry leaves were selected, pulverized, and passed through a 60-mesh sieve to obtain coarse wolfberry leaf powder. 1 g of coarse wolfberry leaf powder was mixed thoroughly with 20 mL of a eutectic solvent (betaine-ethylene glycol, molar ratio 1:4, water content 0-50%). The mixed sample solution was then ultrasonically treated in an ultrasonic cleaner. Extraction conditions were as follows: eutectic solvent water content of 0%, 10%, 20%, 30%, 40%, and 50%; solid-liquid ratio of 1:20 g / mL; ultrasonic power of 500 W; ultrasonic time of 30 min; and ultrasonic temperature of 50℃. After extraction, the sample was centrifuged at 10000 x g / min for 10 min, and the supernatant was collected to obtain a wolfberry leaf extract containing flavonoids. The obtained wolfberry leaf extract was diluted with methanol and filtered through a 0.45 μm organic filter membrane. The resulting sample solution was used for the determination of total flavonoid content.

[0066] The total flavonoid content of wolfberry leaves in the supernatant of Example 9 was determined and compared with that of Example 4. The results are shown in Table 5.

[0067] Table 5. Effects of different DES moisture contents on the extraction rate of total flavonoids from wolfberry leaves.

[0068] The unique extraction ability of DES stems from its strong hydrogen bond acceptor or donor properties. HBA and HBD form a dense hydrogen bond network through hydrogen bonding, resulting in DES with extremely high viscosity, severely hindering molecular diffusion and limiting solvent penetration into plant cell walls and solute mass transfer efficiency. Adding an appropriate amount of water can significantly reduce viscosity, improve flowability and permeability, and enhance mass transfer rate while maintaining the DES hydrogen bond network, thereby improving the extraction efficiency of the target compound. The effect of different water contents on the extraction rate of total flavonoids from wolfberry leaves is as follows: Figure 4 As shown, the extraction rate of total flavonoids from wolfberry leaves reached its maximum when the moisture content of DES increased from 0 to 20%. With further increases in moisture content, the hydrogen bonding of DES weakened, the hydrogen bond structure was disrupted, and the extraction rate of total flavonoids from wolfberry leaves began to decrease. Therefore, moisture contents of 10%, 20%, and 30% were selected as the optimal conditions for extracting total flavonoids from wolfberry leaves using DES.

[0069] Example 10 This embodiment provides a single-factor optimization experiment for the extraction of total flavonoids from wolfberry leaves using an ultrasound-assisted eutectic solvent (Example 4): The difference between this embodiment and Example 4 is that the molar ratio of betaine to ethylene glycol in the eutectic solvent of Example 4 is adjusted to 1:1~8, the water content is 30%, the material-to-liquid ratio is 1:20 g / mL, and the ultrasonic time is 30 min. All other conditions are the same as in Example 4. Specifically: The dried wolfberry leaves were selected, pulverized, and passed through a 60-mesh sieve to obtain coarse wolfberry leaf powder. 1 g of coarse wolfberry leaf powder was mixed thoroughly with 20 mL of a eutectic solvent (betaine-ethylene glycol, molar ratio 1:1~1:8, water content 30%). The mixed sample solution was then ultrasonically treated in an ultrasonic cleaner. The extraction conditions were: eutectic solvent water content 30%, solid-liquid ratio 1:20 g / mL, ultrasonic power 500 W, ultrasonic time 30 min, and ultrasonic temperature 50℃. After extraction, the sample was centrifuged at 10000 x g / min for 10 min, and the supernatant was collected to obtain a wolfberry leaf extract containing flavonoids. The obtained wolfberry leaf extract was diluted with methanol and filtered through a 0.45 μm organic filter membrane. The resulting sample solution was used for the determination of total flavonoid content.

[0070] The total flavonoid content of wolfberry leaves in the supernatant of Example 10 was determined and compared with that of Example 4. The results are shown in Table 6.

[0071] Table 6. Effect of different DES molar ratios on the extraction rate of total flavonoids from wolfberry leaves.

[0072] The molar ratio of hydrogen bond donors (HBDs) to hydrogen bond acceptors (HBAs) in DES is a key factor affecting its physicochemical properties and extraction efficiency. Different molar ratios result in variations in the hydrogen bond network strength, polarity, viscosity, and affinity for the target compound in the DES solvent, thus significantly impacting the extraction rate of plant components. Example 10 investigated the effect of different molar ratios on the extraction rate of total flavonoids from wolfberry leaves. Figure 5 The results showed that the extraction rate initially increased and then decreased with increasing ethylene glycol content, reaching its highest value at a betaine to ethylene glycol molar ratio of 1:4. This is because an appropriate amount of ethylene glycol can form a stable and moderate hydrogen bond network with betaine, giving it the advantages of low viscosity and strong dissolution power, and increasing the fluidity of DES to promote the diffusion of the target product. Excessive HBA or HBD content can lead to poor hydrogen bond network stability and affect extraction efficiency; therefore, a DES molar ratio of 1:4 was selected for subsequent optimization experiments.

[0073] Example 11 This embodiment provides a response surface optimization experiment for the extraction of total flavonoids from wolfberry leaves using a eutectic solvent: The difference between this embodiment and Example 4 is that the ultrasonic power is fixed at 500 W, and the factor levels of the response surface methodology are designed as shown in Table 7. All other conditions are the same as in Example 4. Specifically, the molar ratio of the eutectic solvent (DES) in Example 4 was adjusted to 1:2, 1:4, and 1:6 for betaine and ethylene glycol, respectively; the water content was adjusted to 10%, 20%, and 30%, respectively; the material-to-liquid ratio was adjusted to 1:30, 1:40, and 1:50; and the ultrasonic time was adjusted to 30 min, 40 min, and 50 min. The effects of different conditions on the total flavonoid content of wolfberry leaves were investigated.

[0074] Table 7 Factor Levels in Box-Behnken Experimental Design

[0075] The extraction results of total flavonoids from Lycium barbarum leaves in Example 11 were statistically analyzed and are shown in Table 8. Based on the single-factor experiments, the extraction process parameters were further optimized using the BBD response surface methodology. Using the extraction rate of total flavonoids from Lycium barbarum leaves as the response value, optimizations were made in four aspects: ultrasonic time (30-50 min, A), DES water content (10%-30%, B), material-to-liquid ratio (30-50 mL / g, C), and DES molar ratio (1:2-1:6, D). The extraction rate of total flavonoids from Lycium barbarum leaves ranged from 96.47 mg / g to 123.87 mg / g. In the fixed-center composite experiment, any two terms of the multiple quadratic regression equation were at the 0 level. A 3D response surface methodology was then performed on the remaining two factors. Figure 6 ) and contour map ( Figure 7 To evaluate the pairwise interactions of different experimental factors on the total flavonoid content extracted from wolfberry leaves, in order to determine the optimal range of action for each factor.

[0076] Table 8. Response Surface Experiment Design and Results

[0077]

[0078] The above data were subjected to fit testing and multiple regression analysis using Design-Expert 13 to determine the relationship between the four variables and the total flavonoid extraction rate Y, in order to determine the optimal extraction conditions. The final second-order polynomial equation for the total flavonoid extraction rate of wolfberry leaves is as follows: Flavonoid content (mg / g) = 122.92 + 0.8970A + 1.78B + 3.36C + 1.98D - 4.20AB - 0.0047AC - 2.61AD + 2.36BC - 0.6479BD + 1.46CD - 10.28A 2-5.42B 2 - 11.36C 2 - 4.61D 2 A, B, C, and D are the four independent variables in the table above. The analysis of variance is shown in Table 9. The model's p-value is significant (<0.0001), the F-value is 61.86, and the lack-of-fit term is not significant, indicating that the model is suitable for optimizing the extraction process. In this experiment, R... 2 =0.9841, Corrected coefficient of determination R Adj 2 =0.9682, indicating a good model fit and the test value is close to the actual value. Predicted value R0 2 =0.9367, and R 2 R Adj 2 The difference is not significant, and there is a high correlation between the predicted and actual values. The model is representative and can well reflect the relationship between various factors and the response value. This model can effectively predict and analyze the total flavonoid extraction rate of wolfberry leaves.

[0079] Table 9. Significance of Experimental Results, Regression Model, and Analysis of Variance Results

[0080]

[0081] Example 12 This embodiment was used to verify the experimental results of the optimized model parameters. The optimal extraction conditions for total flavonoids from wolfberry leaves were: ultrasonic time 39.724 min, DES water content 22.002%, solid-liquid ratio 41.844 mL / g, and DES molar ratio 1:4.476. Under these conditions, the predicted extraction rate was 123.629 mg / g. Based on the feasibility of the experiment, the modified extraction parameters were: ultrasonic time 40 min, DES water content 22%, solid-liquid ratio 41.80 mL / g, and DES molar ratio 1:4.48. Verification experiments under these conditions yielded an extraction rate of 123.94 ± 1.04 mg / g for total flavonoids from wolfberry leaves, which was basically consistent with the predicted value, with a relative deviation of 0.25%. This indicates that the Box-Behnken response surface methodology for optimizing the process parameters for ultrasonic-assisted eutectic solvent extraction of total flavonoids from wolfberry leaves is accurate and reliable, and has practical application value.

[0082] It can also be seen that the ultrasonic time and DES moisture content in the method of the present invention have a synergistic effect, which can synergistically improve the extraction rate of total flavonoids from wolfberry leaves; the DES moisture content and material-liquid ratio in the method of the present invention have a synergistic effect, which can synergistically improve the extraction rate of total flavonoids from wolfberry leaves; the material-liquid ratio and DES molar ratio in the method of the present invention have a synergistic effect, which can synergistically improve the extraction rate of total flavonoids from wolfberry leaves. In particular, the ultrasonic time of 40 min and the DES water content of 22% in the method of this invention have a significant synergistic effect, which can significantly improve the extraction rate of total flavonoids from wolfberry leaves. The DES water content of 22% and the material-to-liquid ratio of 41.80 mL / g in the method of this invention have a significant synergistic effect, which can significantly improve the extraction rate of total flavonoids from wolfberry leaves. The material-to-liquid ratio of 41.80 mL / g and the molar ratio of DES (betaine and ethylene glycol) of 1:4.48 in the method of this invention have a significant synergistic effect, which can significantly improve the extraction rate of total flavonoids from wolfberry leaves.

[0083] The components in the supernatant of total flavonoids from wolfberry leaves prepared in Example 12 were separated and purified using macroporous resin. The specific method is as follows: Pretreated D101 macroporous adsorption resin was packed into a column using the wet packing method. The flavonoid extract was diluted 8-fold and loaded at a volume of 4 BV at a flow rate of 1 mL / min. After adsorption, the DES solvent and water-soluble impurities were washed away with distilled water, followed by elution with 70% ethanol at a constant flow rate. The eluent was collected. Lycium barbarum leaf flavonoids were obtained by rotary evaporation and vacuum freeze-drying. The extraction rate, purity, and composition of Lycium barbarum leaf flavonoids were then determined.

[0084] The extraction rate of flavonoids from wolfberry leaves (%) = mass of flavonoids from wolfberry leaves after freeze-drying / mass of crude wolfberry leaf powder × 100%, where the mass unit is mg.

[0085] The purity of Lycium barbarum leaf flavonoids (%) = (C1×N) / C0×100%, where C1 is the mass concentration of flavonoids detected after being substituted into the standard curve (mg / mL), N is the dilution factor, and C0 is the mass concentration of Lycium barbarum leaf flavonoids prepared (mg / mL).

[0086] Identification of flavonoid components in Lycium barbarum leaves: The composition of flavonoid components in Lycium barbarum leaves was analyzed by UHPLC. HPLC conditions: XDB-C18 column (2.1×150 mm, 1.8-Micron); mobile phase: deionized water and methanol solution containing 0.1% formic acid; column temperature: 30℃; flow rate: 0.2 mL / min; sample loading: 2 μL; detection at 290 nm; gradient elution conditions as follows: 0–3 min, 5% B; 3–10 min, 5%–30% B; 10–12 min, 30% B; 12–28 min, 30%–60% B; 28–31 min, 60% B; 31–40 min, 60%–100% B; 40–42 min, 100% B.

[0087] The flavonoid product from Lycium barbarum leaves prepared in Example 12 was tested, and its extraction rate was 9.00%, with a purity of 70%-85%. Using the peak area normalization method, the components and relative percentages corresponding to the main chromatographic peaks were calculated as follows: neochlorogenic acid approximately 2.6%, chlorogenic acid approximately 11.1%, cryptochlorogenic acid approximately 3.5%, and rutin approximately 45.6%. The total relative percentage of these four main components was approximately 63%.

[0088] Experimental Example: Determination of the antioxidant activity of total flavonoid extract from wolfberry leaves 1) DPPH free radical scavenging ability test: Preparation of DPPH solution: Weigh 19.7 mg of DPPH powder and dissolve it in 250 mL of anhydrous ethanol. Mix well to obtain a 78.8 mM DPPH ethanol solution. Store in the dark at 0-4℃.

[0089] Specific experimental procedures: The system was divided into four groups: system control group, system blank group, sample control group, and sample group. The system control group consisted of 50 μL of DPPH ethanol solution (78.8 mM) and 50 μL of sample solvent; the system blank group consisted of 50 μL of anhydrous ethanol and 50 μL of sample solvent; the sample control group consisted of 50 μL of anhydrous ethanol and 50 μL of samples at various concentrations (0.08 mg / mL, 0.252 mg / mL, 0.374 mg / mL, 0.794 mg / mL, 1.179 mg / mL, 2.5 mg / mL, 5 mg / mL); the sample group consisted of 50 μL of DPPH ethanol solution (78.8 mM) and 50 μL of samples at various concentrations (0.08 mg / mL, 0.252 mg / mL, 0.374 mg / mL, 0.794 mg / mL, 1.179 mg / mL, 2.5 mg / mL, 5 mg / mL). (mg / mL), each group was vortexed and incubated at 37℃ in the dark for 30 min. Finally, the absorbance at 540 nm was measured using a microplate reader to detect the DPPH free radical scavenging ability of the samples and calculate EC. 50 (The mass concentration of total flavonoids from wolfberry leaves required to achieve 50% of the maximum effect).

[0090] DPPH free radical scavenging rate (%) = [(system control group - system blank group) - (sample group - sample control group)] / (system control group - system blank group) × 100%.

[0091] 2) ABTS free radical scavenging ability test: Preparation of ABTS stock solution: Weigh 0.246 g of sodium acetate powder and dissolve it completely in 100 mL of water to prepare a 30 mM sodium acetate solution; accurately measure 0.1705 mL of glacial acetic acid and add it to 100 mL of water to prepare a 30 mM acetic acid solution; accurately measure 7.5 mL of the sodium acetate solution and mix it with 92.5 mL of the acetic acid solution, adjust the pH to approximately 3.6, and finally prepare a sodium acetate buffer solution. Then weigh 0.549 g of ABTS powder and dissolve it completely in 100 mL of sodium acetate buffer solution to bring the final concentration to 10 mM. Add 172.7 μL of 35% H2O2 to generate blue-green ABTS free radicals. Store in the dark at 0-4℃ and it can be used overnight.

[0092] Preparation of ABTS working solution: Dilute ABTS stock solution with sodium acetate buffer, and adjust the OD value to 0.7±0.02 at an absorbance of 650 nm before use.

[0093] Specific experimental procedures: The system was divided into four groups: system control group, system blank group, sample control group, and sample group. The system control group was supplemented with 130 μL of ABTS and 5.5 μL of sample solvent; the system blank group was supplemented with 130 μL of sodium acetate buffer and 5.5 μL of sample solvent; the sample control group was supplemented with 130 μL of sodium acetate buffer and 5.5 μL of samples of various concentrations (0.8 mg / mL, 2.52 mg / mL, 3.74 mg / mL, 7.94 mg / mL, 11.79 mg / mL, and 25 mg / mL); the sample group was supplemented with 130 μL of ABTS and 5.5 μL of samples of various concentrations (0.8 mg / mL, 2.52 mg / mL, 3.74 mg / mL, 7.94 mg / mL, 11.79 mg / mL, and 25 mg / mL). The mixture was vortexed for 30 s and reacted at room temperature in the dark for 10 min. Finally, the absorbance at 650 nm was measured using a microplate reader to detect the ABTS radical scavenging ability of the sample and calculate the EC50. 50 (The mass concentration of total flavonoids from wolfberry leaves required to achieve 50% of the maximum effect).

[0094] ABTS free radical scavenging rate (%) = [(System control group - System blank group) - (Sample group - Sample control group)] / (System control group - System blank group) × 100%.

[0095] 3) Detection of total VC reducing power: Take 10 μL of sample solutions of various concentrations (0.8 mg / mL, 2.52 mg / mL, 3.74 mg / mL, 7.94 mg / mL, 11.79 mg / mL, and 25 mg / mL, respectively), add 25 μL of 1×PBS and 25 μL of 1% potassium ferricyanide solution, vortex to mix, incubate at 50℃ for 20 min, and then rapidly cool on ice. Next, add 25 μL of 10% trichloroacetic acid, vortex to mix, centrifuge at 3000 rpm / min, and collect the supernatant. Then add 25 μL of distilled water and 25 μL of 10% trichloroacetic acid, vortex thoroughly to mix, and let stand for 10 min. Measure the absorbance at 650 nm. Substitute the measured absorbance into the VC standard curve (regression equation: y = 0.0018x + 0.0016, R²). 2 =0.9991), the total reducing power of the sample is expressed as VC equivalent (μg VCE / mL).

[0096] Results Analysis: The antioxidant capacity of different mass concentrations of total flavonoids from wolfberry leaves prepared in Example 12 was evaluated through DPPH free radical scavenging experiments, ABTS free radical scavenging experiments, and VC total reducing power determination experiments. Figures 8-10As shown, the total flavonoids from wolfberry leaves obtained by DES extraction, 70% ethanol extraction, and water extraction all exhibited increased antioxidant capacity with increasing concentration. At all tested concentrations, the overall antioxidant efficacy of the three methods followed a consistent pattern: DES extraction > 70% ethanol extraction > water extraction. The antioxidant capacity was further enhanced by determining EC... 50 A quantitative comparison of free radical scavenging abilities showed that DES extract had an EC50-95% scavenging capacity against DPPH free radicals. 50 The EC50 concentration for ABTS free radicals is 0.13 mg / mL. 50 The concentration was 12.38 mg / mL; in contrast, the 70% ethanol extract showed an EC50 concentration against DPPH and ABTS radicals. 50 The concentrations were 0.19 mg / mL and 19.70 mg / mL, respectively; the EC50 of the water extract against DPPH free radicals... 50 The EC50 concentration for ABTS free radicals is 0.46 mg / mL. 50 The concentration is higher than 25 mg / mL. The above data demonstrate that the DES extraction method used in this invention significantly enhances the free radical scavenging ability of total flavonoids from wolfberry leaves compared to traditional alcohol or water extraction methods, with an efficiency increase exceeding 30%. In the determination of total VC reducing power (… Figure 10 At the same mass concentration, the sample obtained by DES extraction showed a higher VC equivalent, further confirming its stronger antioxidant activity. Based on the combined results of the above various antioxidant activity tests, the total flavonoids from wolfberry leaves obtained by DES extraction exhibited the best performance in both free radical scavenging ability and total reducing power. Therefore, DES is the preferred solvent for extracting highly active flavonoids from wolfberry leaves.

[0097] Patent CN117100789B discloses a method for extracting lemon eucalyptus leaves using a eutectic solvent and its application. Although the comparative example (patent CN117100789B) and the example of this invention use different plant materials, their core technical issues (high efficiency, naturalness, and low cost) and application areas highly overlap. However, the total flavonoid content and relative extraction efficiency (total flavonoid content by DES extraction / total flavonoid content by traditional alcohol extraction) of this invention are significantly higher than those of the comparative example, and the antioxidant activity of this invention is also significantly superior to that of the comparative example in terms of DPPH (Table 10). Therefore, this invention provides a novel technical solution that is more effective and cost-efficient.

[0098] Table 10. EC based on the same antioxidant index, as in this invention and patent CN117100789B 50 Comparison with extraction efficiency

[0099] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for ultrasonic-assisted deep eutectic solvent extraction of total flavonoids from Lycium chinense leaves, characterized by comprising the following steps: It comprises the following steps: ​ (1) After drying the leaves of Lycium barbarum, crush and sieve to obtain Lycium barbarum leaf powder; (2) Heat and mix the hydrogen bond acceptor and the hydrogen bond donor at 80℃, stir until transparent, add water to reduce viscosity to obtain a deep eutectic solvent; (3) Mix the Lycium barbarum leaf powder prepared in step (1) with the deep eutectic solvent prepared in step (2) and ultrasonically extract, centrifuge, collect the supernatant to obtain a Lycium barbarum leaf total flavonoid extract; (4) Use a macroporous resin to adsorb the Lycium barbarum leaf total flavonoid extract obtained in step (3), wash and desorb the macroporous resin after adsorption to obtain an eluate; (5) Concentrate the eluate obtained in step (4), vacuum freeze-dry to constant weight to obtain a Lycium barbarum leaf flavonoid freeze-dried powder.

2. The method of claim 1, wherein: The hydrogen bond acceptor in step (2) is at least one of choline chloride and betaine; the hydrogen bond donor is at least one of glycerol, ethylene glycol, lactic acid, p-toluenesulfonic acid, 1,2-propanediol, 1,3-butanediol, glucose, and urea; and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2-5.

3. The method of claim 1, wherein: The deep eutectic solvent in step (2) is betaine-ethylene glycol, wherein the molar ratio between betaine and ethylene glycol is 1:1-8, and the water content of the deep eutectic solvent is 0-50%.

4. The method of claim 1, wherein: The ultrasonic extraction conditions in step (3) are as follows: the solid-liquid ratio is 10-70 mL / g; the ultrasonic time is 10-70 min; the ultrasonic temperature is 50℃; and the ultrasonic power is 500 W; the centrifugation speed in step (3) is 10000 x g / min, and the centrifugation time is 10 min.

5. The method according to any one of claims 1 to 4, characterized in that: In step (4), a D101 type macroporous resin is used to adsorb the Lycium barbarum leaf active substance, and the D101 type macroporous resin after adsorption is washed with distilled water until colorless, and then eluted with ethanol with a volume fraction of 70%, so as to purify the Lycium barbarum leaf flavonoids and remove the deep eutectic solvent and other water-soluble impurities; Alternatively, in step (4), the Lycium barbarum leaf total flavonoid extract is diluted with Wahaha water before purification to destroy the hydrogen bonds between the deep eutectic solvent and the Lycium barbarum leaf active substance, thereby enhancing the adsorption effect of the macroporous resin on the Lycium barbarum leaf total flavonoids.

6. The method of claim 1, wherein: The deep eutectic solvent in step (2) is betaine-ethylene glycol, wherein the molar ratio between betaine and ethylene glycol is 1:4.48, and the water content of the deep eutectic solvent is 22%; and the ultrasonic extraction conditions in step (3) are as follows: the ultrasonic time is 40 min, and the solid-liquid ratio is 41.80 mL / g; under these conditions, the total flavonoid extraction rate of Lycium barbarum leaf is 123.94±1.04 mg / g.

7. The method according to any one of claims 1 to 6 for use in the production of Lycium barbarum leaf total flavonoids.

8. Lycium barbarum leaf total flavonoids prepared by the method according to any one of claims 1 to 6.

9. The total flavones of Lycium leaf according to claim 8, characterized in that: The main components of the Lycium barbarum leaf flavonoid freeze-dried powder include rutin, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid.

10. Use of the Lycium barbarum leaf total flavonoids according to claim 8 or 9 in the preparation of pharmaceuticals, foodstuffs, and / or cosmetics.

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