A method for extracting phenolic compounds from sea buckthorn leaves using a natural deep eutectic solvent

Abstract

The present invention discloses a method for extracting phenolic ketone compounds from sea buckthorn leaves with an ultrasound-assisted natural low eutectic solvent. The sea buckthorn leaf powder is mixed with a natural low eutectic solution, ultrasonically extracted, and the supernatant is collected by centrifugation. The supernatant is concentrated, frozen, and dried to obtain the phenolic ketone compounds. The phenolic ketone compounds extracted by the present invention are diverse in type and high in content. By optimizing the type of natural low eutectic solvent, the extracted phenolic ketone compounds can have a better inhibitory effect on the activity of related enzymes, making them promising natural compounds for treating and preventing hyperglycemia, hyperlipidemia, hypertension, and gout.

Description

Technical Field

[0001] The invention belongs to the technical field of active substance extraction, and particularly relates to a method for extracting phenolic ketone compounds from seabuckthorn leaves with the assistance of an ultrasound-assisted natural deep eutectic solvent. Background Art

[0002] Sea buckthorn (Hippophaerhamnoides L.), a perennial deciduous shrub belonging to the Elaeagnaceae family, is rich in a variety of nutrients and bioactive compounds, including flavonoids, phenolic acids, proanthocyanidins, fatty acids, triterpenes, vitamins, and phytosterols. Accumulating research indicates that sea buckthorn leaves possess a wide range of health benefits, including antioxidant, anticancer, hypolipidemic, anti-obesity, anti-inflammatory, antibacterial, antiviral, and hepatoprotective effects. While numerous reports exist on the use of sea buckthorn berries for wine and other beverage production, bioactive extracts, and various other applications, fewer studies have explored the extensive utilization of sea buckthorn berries, despite their aforementioned health benefits. However, given the demand for natural therapies for patients with metabolic syndrome and considerations of a circular economy, the efficient and environmentally friendly recovery of health-maintaining components from sea buckthorn leaves warrants further investigation.

[0003] Diabetes, hyperlipidemia, gout, and hypertension are common chronic diseases in both industrialized and developing countries. Due to their severe adverse effects on human health, these diseases have become global health concerns. Several enzymes, such as α-glucosidase, α-amylase, pancreatic lipase (PL), cholesterol esterase (CE), xanthine oxidase (XO), and angiotensin-converting enzyme (ACE), play crucial roles in the development and progression of these diseases. Therefore, the use of substances to inhibit the activity of these enzymes and thus hinder the progression of related diseases is an effective strategy in the pharmaceutical industry. However, many of these clinical enzyme inhibitors are non-natural chemical drugs, such as acarbose, orlistat, allopurinol, and alacepril, which exhibit certain intestinal side effects and toxicity. Therefore, the search for safer and more effective inhibitors from natural sources is a desirable option.

[0004] Numerous studies have shown that phenolics from medicinal plants and foods are ideal inhibitors of various metabolic enzymes. Some plant-derived phenolics, such as quercetin, kaempferol, chrysanthemin, and myricetin, can enter the active center of α-glucosidase and competitively inhibit its enzymatic activity. Catechins, on the other hand, reduce PL activity by simultaneously binding to PL and its substrate to form an enzyme-substrate-catechin complex, thereby exerting a non-competitive inhibitory effect on PL. Summary of the Invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for extracting phenolic ketone compounds from seabuckthorn leaves using an ultrasound-assisted natural deep eutectic solvent.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: comprising:

[0009] The seabuckthorn leaves are dried, crushed and sieved to obtain seabuckthorn leaf powder;

[0010] The natural deep eutectic solvent is added with water to reduce the viscosity to obtain a natural deep eutectic solution;

[0011] The sea buckthorn leaf powder is mixed with the natural eutectic solution and then subjected to ultrasonic extraction. The supernatant is collected by centrifugation and concentrated, frozen and dried to obtain phenolic ketone compounds.

[0012] The natural deep eutectic solvent comprises one of choline chloride-acetic acid, choline chloride-malic acid, lactate-D-(+)-glucose, lactate-choline chloride, and glycerol-choline chloride.

[0013] As a preferred embodiment of the method for extracting phenolic ketone compounds from seabuckthorn leaves using an ultrasound-assisted natural deep eutectic solvent according to the present invention, the water content of the natural deep eutectic solution is 20-40%.

[0014] As a preferred embodiment of the method for extracting phenolic ketone compounds from sea buckthorn leaves with the aid of ultrasound-assisted natural deep eutectic solvent according to the present invention, the mass-to-volume ratio of the sea buckthorn leaf powder to the natural deep eutectic solvent is 1:20-40.

[0015] As a preferred embodiment of the method for extracting phenolic ketone compounds from sea buckthorn leaves using an ultrasound-assisted natural deep eutectic solvent according to the present invention, the ultrasonic power of the ultrasonic extraction is 100 to 600W.

[0016] As a preferred embodiment of the method for extracting phenolic ketone compounds from sea buckthorn leaves using an ultrasound-assisted natural deep eutectic solvent according to the present invention, the extraction time of the ultrasound extraction is 8 to 20 minutes.

[0017] As a preferred embodiment of the method for extracting phenolic ketone compounds from sea buckthorn leaves with the aid of ultrasound-assisted natural deep eutectic solvent according to the present invention, the extraction temperature of the ultrasonic extraction is 30-50°C.

[0018] As a preferred embodiment of the method for extracting phenolic ketone compounds from seabuckthorn leaves using an ultrasound-assisted natural deep eutectic solvent according to the present invention, the duty cycle of the ultrasound extraction is 20 to 100%.

[0019] As a preferred embodiment of the method for extracting phenolic ketone compounds from sea buckthorn leaves with an ultrasound-assisted natural deep eutectic solvent according to the present invention, the natural deep eutectic solvent comprises choline chloride-acetic acid or lactic acid-choline chloride.

[0020] Another object of the present invention is to provide a phenolic ketone compound extracted from sea buckthorn leaves by an ultrasound-assisted natural deep eutectic solvent, wherein the compound includes hydrolyzable tannins, flavonols, flavan-3-ols, flavanones, phenolic acids, and flavonoid organic compounds.

[0021] Another object of the present invention is to provide a use of phenolic ketone compounds in seabuckthorn leaves in the preparation of medicines for treating and preventing hyperglycemia, hyperlipidemia, hypertension and gout.

[0022] Beneficial effects of the present invention:

[0023] The present invention uses ultrasound-assisted natural deep eutectic solvents to extract phenolic ketone compounds from sea buckthorn leaves. The phenolic ketone compounds in the final extract are diverse in type and high in content. By optimizing the type of natural deep eutectic solvent, the extracted phenolic ketone compounds can better inhibit the activity of related enzymes, making it possible to become a natural compound for treating and preventing hyperglycemia, hyperlipidemia, hypertension and gout. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0025] Figure 1 (a) is the total ion chromatogram (TIC) of the phenolic compounds of Example 1 in negative ion mode.

[0026] Figure 1 (b) is the inhibitory ability of the phenolic compounds extracted from Example 1 and Comparative Examples 1 and 2 on α-glucosidase.

[0027] Figure 1(c) is the inhibitory ability of the phenolic ketone compounds extracted in Example 1 and Comparative Examples 1 and 2 on α-amylase.

[0028] Figure 1 (d) The inhibitory ability of the phenolic compounds extracted from Example 1 and Comparative Examples 1 and 2 on pancreatic lipase.

[0029] Figure 1 (e) The inhibitory ability of the phenolic compounds extracted from Example 1 and Comparative Examples 1 and 2 on cholesterol esterase.

[0030] Figure 1 (f) is the inhibitory ability of the phenolic compounds extracted from Example 1 and Comparative Examples 1 and 2 on xanthine oxidase.

[0031] Figure 1 (g) The inhibitory ability of the phenolic compounds extracted from Example 1 and Comparative Examples 1 and 2 on angiotensin converting enzyme. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0033] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0035] The total phenol content (TPC) in the present invention is determined by the Folin-Ciocalteau method.

[0036] The total flavonoid content (TFC) determination method of the present invention is as follows:

[0037] 400 μL of the diluted extract was reacted with 200 μL of NaNO2 solution (5%, w / v) at room temperature for 6 min, and then 200 μL of Al(NO3)3 solution (10%, w / v) was added and reacted at room temperature for 6 min. Finally, 2 mL of NaOH (4%, w / v) was added and the total volume was made up to 4 mL with deionized water. The reaction was continued for 15 min, and the absorbance was measured at 510 nm. Rutin (10-100 μg / mL) was used as the standard, and the standard curve equation was Y=0.008854X-0.0074(R 2 =0.9994, n=7). The results are expressed as milligrams of rutin equivalent per gram of dry weight (mgRE / gDW).

[0038] The abbreviations of the natural deep eutectic solvents in the present invention correspond to their Chinese names as follows:

[0039]

[0040] The raw materials used in the present invention and the related synthesis and determination methods are all routinely available to those skilled in the art.

[0041] Example 1

[0042] This example provides a method for extracting phenolic compounds from seabuckthorn leaves using Chcl-AA assisted by ultrasound, specifically:

[0043] 1) Pick and remove the seabuckthorn leaves from the branches, dry them in a forced air drying oven at 45°C for 4 hours, grind them with a universal grinder, and pass them through a 60-mesh sieve to obtain seabuckthorn leaf powder;

[0044] 2) Chcl-AA (the molar ratio of Chcl to AA is 1:2) is heated at 80°C with constant stirring until a stable transparent liquid is formed, and water is added to reduce the viscosity to obtain a natural eutectic solution with a water content of 30%;

[0045] 3) The sea buckthorn leaf powder and the natural low eutectic solution were mixed in a solid-liquid ratio of 1:30, and ultrasonic extraction was performed for 14 minutes at an ultrasonic temperature of 40°C, an ultrasonic power of 400 W, and an ultrasonic duty cycle of 67%. The supernatant was centrifuged, concentrated, frozen, and dried to obtain the phenolic ketone compound of this example.

[0046] Example 2

[0047] The difference between this embodiment and embodiment 1 is that the natural deep eutectic solvent in step 2) is adjusted to Chcl-MA (the molar ratio of Chcl to MA is 1:1), specifically:

[0048] 1) Pick and remove the seabuckthorn leaves from the branches, dry them in a forced air drying oven at 45°C for 4 hours, grind them with a universal grinder, and pass them through a 60-mesh sieve to obtain seabuckthorn leaf powder;

[0049] 2) Chcl-MA (the molar ratio of Chcl to MA is 1:1) is heated at 80°C with constant stirring until a stable transparent liquid is formed, and water is added to reduce the viscosity to obtain a natural eutectic solution with a water content of 30%;

[0050] 3) The sea buckthorn leaf powder and the natural low eutectic solution were mixed in a solid-liquid ratio of 1:30, and ultrasonic extraction was performed for 14 minutes at an ultrasonic temperature of 40°C, an ultrasonic power of 400 W, and an ultrasonic duty cycle of 67%. The supernatant was centrifuged, concentrated, frozen, and dried to obtain the phenolic ketone compound of this example.

[0051] Example 3

[0052] The difference between this embodiment and embodiment 1 is that the natural deep eutectic solvent in step 2) is adjusted to LA-Glu (the molar ratio of LA to Glu is 5:1), specifically:

[0053] 1) Pick and remove the seabuckthorn leaves from the branches, dry them in a forced air drying oven at 45°C for 4 hours, grind them with a universal grinder, and pass them through a 60-mesh sieve to obtain seabuckthorn leaf powder;

[0054] 2) LA-Glu (LA to Glu molar ratio of 5:1) was heated at 80°C with constant stirring until a stable transparent liquid was formed, and water was added to reduce the viscosity to obtain a natural eutectic solution with a water content of 30%;

[0055] 3) The sea buckthorn leaf powder and the natural low eutectic solution were mixed in a solid-liquid ratio of 1:30, and ultrasonic extraction was performed for 14 minutes at an ultrasonic temperature of 40°C, an ultrasonic power of 400 W, and an ultrasonic duty cycle of 67%. The supernatant was centrifuged, concentrated, frozen, and dried to obtain the phenolic ketone compound of this example.

[0056] Example 4

[0057] The difference between this embodiment and embodiment 1 is that the natural deep eutectic solvent in step 2) is adjusted to LA-Chcl (the molar ratio of LA to Chcl is 3:1), specifically:

[0058] 1) Pick and remove the seabuckthorn leaves from the branches, dry them in a forced air drying oven at 45°C for 4 hours, grind them with a universal grinder, and pass them through a 60-mesh sieve to obtain seabuckthorn leaf powder;

[0059] 2) LA-Chcl (LA to Chcl molar ratio of 3:1) was heated at 80°C with constant stirring until a stable transparent liquid was formed, and water was added to reduce the viscosity to obtain a natural eutectic solution with a water content of 30%;

[0060] 3) The sea buckthorn leaf powder and the natural low eutectic solution were mixed in a solid-liquid ratio of 1:30, and ultrasonic extraction was performed for 14 minutes at an ultrasonic temperature of 40°C, an ultrasonic power of 400 W, and an ultrasonic duty cycle of 67%. The supernatant was centrifuged, concentrated, frozen, and dried to obtain the phenolic ketone compound of this example.

[0061] Example 5

[0062] The difference between this embodiment and embodiment 1 is that the natural deep eutectic solvent in step 2) is adjusted to Gly-Chcl (the molar ratio of Gly to Chcl is 3:1), specifically:

[0063] 1) Pick and remove the seabuckthorn leaves from the branches, dry them in a forced air drying oven at 45°C for 4 hours, grind them with a universal grinder, and pass them through a 60-mesh sieve to obtain seabuckthorn leaf powder;

[0064] 2) Gly-Chcl (molar ratio of Gly to Chcl is 3:1) was heated at 80°C with constant stirring until a stable transparent liquid was formed, and water was added to reduce the viscosity to obtain a natural eutectic solution with a water content of 30%;

[0065] 3) Sea buckthorn leaf powder and natural eutectic solution were mixed at a solid-liquid ratio of 1:30 and ultrasonically extracted for 14 min at an ultrasonic temperature of 40°C, an ultrasonic power of 400 W, and an ultrasonic duty cycle of 67%. The supernatant was centrifuged, concentrated, frozen, and dried to obtain the phenolic ketone compound of this example, which was recorded as SLNP.

[0066] Comparative Example 1

[0067] The difference between this comparative example and Example 1 is that the natural deep eutectic solvent in step 2) is adjusted to ethanol, and the remaining steps are the same as Example 1, that is, the phenolic ketone compound of this comparative example is obtained, which is recorded as SLEP.

[0068] Comparative Example 2

[0069] The difference between this comparative example and Example 1 is that the natural deep eutectic solvent in step 2) is adjusted to water, and the remaining steps are the same as Example 1, that is, the phenolic ketone compound of this comparative example is obtained, which is recorded as SLWP.

[0070] Figure 1 (a) is the total ion chromatogram (TIC) of phenolic compounds of Example 1 in negative ion mode, in which 25 compounds were identified, which can be roughly divided into hydrolyzable tannins, flavonols, flavan-3-ols, flavanones, phenolic acids and flavonoid organic compounds. Figure 1 (b)~ Figure 1(g) The inhibitory ability of the phenolic compounds extracted from Example 1 and Comparative Examples 1 and 2 on α-glucosidase (b), α-amylase (c), pancreatic lipase (d), cholesterol esterase (e), xanthine oxidase (f) and angiotensin-converting enzyme (g).

[0071] from Figure 1 (b)~ Figure 1 (g) It can be seen that the phenolic compounds extracted by each solution have inhibitory effects on all tested enzymes, with the most prominent inhibitory activities against α-glucosidase, pancreatic lipase, and angiotensin-converting enzyme. Compared with other common solvent extracts, the extract of Example 1 has a better inhibitory effect on α-glucosidase, α-amylase, pancreatic lipase, cholesterol esterase, and xanthine oxidase.

[0072] The total contents of total phenolic compounds (TPC) and total ketone compounds (TFC) in the supernatants of Examples 1 to 5 and Comparative Examples 1 and 2 were measured. The results are shown in Table 1.

[0073] Table 1

[0074]

[0075] As can be seen from Table 1, the contents of TPC and TFC in the extracts obtained by ultrasonic extraction assisted by natural deep eutectic solvents were significantly higher than those in the ethanol extract and water extract of the comparative example. This is because the natural deep eutectic solvent can increase the extraction capacity by forming hydrogen bonds between it and the target compound.

[0076] Comparative Example 3

[0077] The difference between this comparative example and Example 1 is that the natural deep eutectic solvent in step 2) is adjusted to Chcl-CA-Gly (the molar ratio of Chcl, CA, and Gly is 1:1:1), and the remaining steps are the same as in Example 1, thereby obtaining the phenolic ketone compound of this comparative example.

[0078] Comparative Example 4

[0079] The difference between this comparative example and Example 1 is that the natural deep eutectic solvent in step 2) is adjusted to Chcl-Glu (the molar ratio of Chcl and Glu is 3:2), and the remaining steps are the same as Example 1, that is, the phenolic ketone compound of this comparative example is obtained.

[0080] Comparative Example 5

[0081] The difference between this comparative example and Example 1 is that the natural deep eutectic solvent in step 2) is adjusted to LA-Gly (the molar ratio of LA, Gly, and water is 3:1:3), and the remaining steps are the same as in Example 1, that is, the phenolic ketone compound of this comparative example is obtained.

[0082] Comparative Example 6

[0083] The difference between this comparative example and Example 1 is that the natural deep eutectic solvent in step 2) is adjusted to Gly-MA (the molar ratio of Gly and MA is 1:1), and the remaining steps are the same as Example 1, that is, the phenolic ketone compound of this comparative example is obtained.

[0084] Comparative Example 7

[0085] The difference between this comparative example and Example 1 is that the natural deep eutectic solvent in step 2) is adjusted to Gly-SA (the molar ratio of Gly and SA is 1:1), and the remaining steps are the same as in Example 1, that is, the phenolic ketone compound of this comparative example is obtained.

[0086] The total contents of total phenolic compounds (TPC) and total ketone compounds (TFC) in the supernatants of Comparative Examples 3 to 7 were measured and compared with those in Example 1. The results are shown in Table 2.

[0087] Table 2

[0088]

[0089] As can be seen from Table 2, the contents of TPC and TFC in the extracts obtained by ultrasonic extraction assisted by different types of natural deep eutectic solvents also vary significantly. This is because different natural deep eutectic solvents have different abilities to form hydrogen bonds with the target compounds. Chcl-AA in Example 1 has the strongest ability to form hydrogen bonds with the target compound and has the highest extraction rate.

[0090] Example 6

[0091] This example is used to explore the effect of the water content of different natural eutectic solutions on the extraction effect. The difference from Example 1 is that the water content of the natural eutectic solution in step 2) is adjusted to 10%, 20%, 30%, 40%, and 50%, respectively. The remaining steps and processes are the same as in Example 1. The extraction amount of phenolic compounds under different water contents is measured, and the results are shown in Table 3.

[0092] Table 3

[0093]

[0094] As can be seen in Table 3, TPC and TFC increased significantly as the water content increased from 10% to 30%. This is because the increase in water can reduce the viscosity of NADES, promote the diffusion of the solvent in the biomass, and increase the polarity of the solvent, thereby improving the possibility of extracting more polar phenolic compounds. When the water content further increased to 50%, TPC and TFC began to decline. This may be because the hydrogen bonds between the components in NADES were broken at this time, causing them to be released from the NADES, resulting in a decrease in extraction ability.

[0095] Example 7

[0096] This example is used to explore the effect of different solid-liquid ratios of sea buckthorn powder and natural low eutectic solution on the extraction effect. The difference from Example 1 is that the solid-liquid ratio of sea buckthorn powder and natural low eutectic solution in step 3) is adjusted to 1:10, 1:20, 1:30, 1:40, and 1:50, respectively. The remaining steps and processes are the same as in Example 1. The extraction amount of phenolic compounds under different solid-liquid ratios is measured, and the results are shown in Table 4.

[0097] Table 4

[0098]

[0099] As shown in Table 4, when the liquid-to-solid ratio increases from 10 mL / g to 30 mL / g, both TPC and TFC increase significantly. This is because the increase in the liquid-to-solid ratio increases the contact area between the solvent and the solid sample, thereby enhancing the formation of hydrogen bonds between the natural deep eutectic solvent and the target compound. Furthermore, the increase in the liquid-to-solid ratio lowers the threshold of the ultrasonic cavitation effect, making cavitation more likely to occur, thereby helping to improve the extraction yield. When the liquid-to-solid ratio exceeds 30 mL / g, the extraction yield gradually decreases, which may be due to the limited effect of the liquid-to-solid ratio on the concentration gradient during the extraction process. An excessively low cavitation threshold increases the duration of transient high temperatures, resulting in a decrease in the extraction yield.

[0100] Example 8

[0101] This example is used to explore the effect of different extraction temperatures on the extraction effect. The difference from Example 1 is that the ultrasonic temperature in step 3) is adjusted to 20, 30, 40, 50, and 60°C, respectively. The remaining steps and processes are the same as those in Example 1. The extraction amount of phenolic compounds at different extraction temperatures is measured, and the results are shown in Table 5.

[0102] Table 5

[0103]

[0104] Table 5 shows that TPC and TFC increase as the temperature increases from 20°C to 40°C. This increase in temperature increases the kinetic energy of the molecules in the natural deep eutectic solvent, reducing viscosity and thereby enhancing the interaction between the solvent and the solid particles. This increase in temperature also reduces the surface tension of the solvent, thereby lowering the cavitation threshold during ultrasonication, intensifying the cavitation effect, and improving the extraction yield. When the temperature exceeds 50°C, TPC decreases significantly. This decrease can be attributed to the temperature sensitivity of phenolics. Excessively high temperatures and the heat generated by the intense cavitation effect accelerate the degradation of phenolics, thereby offsetting the positive effects of the temperature increase.

[0105] Example 9

[0106] This example is used to explore the effect of different ultrasonic powers on the extraction effect. The difference from Example 1 is that the ultrasonic power in step 3) is adjusted to 100, 200, 300, 400, 500, and 600 W, respectively. The remaining steps and processes are the same as those in Example 1. The extraction amount of phenolic compounds under different ultrasonic powers is measured, and the results are shown in Table 6.

[0107] Table 6

[0108]

[0109]

[0110] As shown in Table 6, when the ultrasonic power increases from 100 W to 400 W, TPC and TFC initially increase, then decline. The positive effect of ultrasound on TFC is more pronounced, likely due to the release of flavonoids bound to the cell walls of seabuckthorn leaves. Increasing the ultrasonic power enhances the cavitation effect, intensifies the collapse of cavitation bubbles, and increases the mechanical shear force on the sample, thereby promoting the extraction process. Ultrasonic co-treatment with ChCl-AA results in significant surface rupture of the sample powder, with a noticeable roughness and loose structure observed after ultrasonic exposure. However, when the ultrasonic power exceeds a certain limit, the uncontrolled shear force induced by ultrasound, the sonochemical effects caused by bubble collapse, and the high temperature effect can lead to the degradation of phenolic compounds, negatively affecting the extraction process.

[0111] Example 10

[0112] This example is used to explore the effect of different ultrasonic duty cycles on the extraction effect. The difference from Example 1 is that the ultrasonic duty cycle in step 3) is adjusted to 20, 33, 67, and 100%, respectively. The remaining steps and processes are the same as Example 1, and the extraction amount of phenolic compounds under different ultrasonic duty cycles is measured.

[0113] When the ultrasonic duty cycle increased from 20% to 67%, TFC and TPC continued to increase. Above 67%, TFC and TPC decreased slightly, but not significantly. The continuous operation of the ultrasonic wave caused the extraction system to be in an ultrasonic state for a long time, generating excessive heat. Furthermore, the continuous cavitation and excessive mechanical shear may have caused phenolic degradation and weakened the hydrogen bonds between NADES and phenolic compounds.

[0114] In summary, the present invention uses ultrasound-assisted natural deep eutectic solvents to extract phenolic ketone compounds from sea buckthorn leaves. The phenolic ketone compounds in the final extract are diverse in type and high in content. By optimizing the type of natural deep eutectic solvent, the extracted phenolic ketone compounds can better inhibit the activity of related enzymes, making it possible to become a natural compound for the treatment and prevention of hyperglycemia, hyperlipidemia, hypertension and gout.

[0115] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for extracting phenolic compounds from seabuckthorn leaves using an ultrasound-assisted natural deep eutectic solvent, characterized in that: include, The seabuckthorn leaves are dried, crushed and sieved to obtain seabuckthorn leaf powder; A natural deep eutectic solvent composed of choline chloride and acetic acid in a molar ratio of 1:2 is added with water to reduce the viscosity, thereby obtaining a natural deep eutectic solution with a water content of 30%; Sea buckthorn leaf powder and natural eutectic solution are mixed in a solid-liquid ratio of 1:30, and ultrasonic extraction is performed under an ultrasonic power of 400 W. The ultrasonic extraction temperature is 40-50° C. and the duty cycle is 33-100%. The supernatant is collected by centrifugation and concentrated, frozen, and dried to obtain phenolic ketone compounds.

2. The method for extracting phenolic compounds from seabuckthorn leaves using an ultrasound-assisted acidic natural deep eutectic solvent according to claim 1, wherein: The extraction time of the ultrasonic extraction is 8 to 20 minutes.

3. The method for extracting phenolic compounds from seabuckthorn leaves using an ultrasound-assisted acidic natural deep eutectic solvent according to claim 2, wherein: The extraction temperature of the ultrasonic extraction is 40°C.

4. The method for extracting phenolic compounds from seabuckthorn leaves using an ultrasound-assisted acidic natural deep eutectic solvent according to claim 3, wherein: The duty cycle of the ultrasonic extraction is 67-100%.

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