Cherry-gourd extract, method for preparing the same and use thereof

Abstract

The application discloses a preparation method of cherimoya leaf extract and belongs to the technical field of natural medicine extraction and preparation. The conventional preparation method of the cherimoya leaf extract in the prior art has limited bioavailability of the extract, and lacks a systematic method for effectively enriching specific active components of the extract and stably encapsulating the specific active components in a suitable delivery system. The preparation method provided by the application comprises the following steps: crushing dried cherimoya leaves, refluxing and extracting the dried cherimoya leaves with an ethanol aqueous solution, and concentrating the extract; adding ethyl acetate to the concentrated solution to perform extraction; mixing the obtained ethyl acetate extraction phase with a phospholipid aqueous dispersion liquid composed of phosphatidylcholine and phosphatidylethanolamine in a specific ratio; performing high-pressure homogenization treatment to obtain a nanoliposome suspension liquid; and finally performing spray drying to obtain an extract powder. The extract prepared by the method can be used for preparing a medicine for treating type 2 diabetes.

Description

Technical Field

[0001] This invention relates to the field of natural drug extraction and preparation technology. More specifically, this invention relates to an extract of the leaves of the soursop fruit, its preparation method, and its application. Background Technology

[0002] Annona muricata L., a plant with potential medicinal value, has had its extracts from different parts studied for anti-diabetic applications. Current techniques often involve ethanol extraction, pH adjustment with acid, and ethyl acetate extraction to obtain Annona muricata root extract.

[0003] However, existing research on soursop leaf extract, especially its preparation process, still has some limitations. On the one hand, extracts obtained by conventional extraction methods (such as simple ethanol reflux extraction, concentration and drying) may contain active ingredients in crude extract form, resulting in limited bioavailability, which to some extent restricts the full realization of its efficacy. On the other hand, if the goal is to develop the extract into a more effective formulation, such as improving its stability and delivery efficiency through formulation methods, existing technologies lack a systematic approach to effectively enrich specific active ingredients (especially lipophilic components) from soursop leaves and simultaneously prepare them into stable carrier systems suitable for improving bioavailability. Specifically, how to selectively enrich lipophilic components that may be related to antidiabetic effects from soursop leaves and further stably encapsulate these components in a delivery system that can improve their solubility, stability, and in vivo behavior is a technical challenge. Directly mixing conventional extracts with simple phospholipids often fails to obtain nanoscale formulations with uniform particle size, stable encapsulation, and good reproducibility, which affects the controllability of the final product quality and the predictability of its efficacy. Summary of the Invention

[0004] One object of the present invention is to provide a method for preparing an extract from the leaves of the soursop, comprising the following steps: The dried soursop leaves are crushed to obtain soursop leaf powder. The powder of soursop leaf and branch was mixed with an ethanol aqueous solution with a volume concentration of 50% to 95% at a material-to-liquid ratio of 1 g: 5 to 15 mL, and refluxed at 60 to 85°C for 1 to 3 hours. The extract was then collected. One object of the present invention is to provide a method for preparing an extract from the leaves of *Annona squamosa* sp., comprising the following steps: [The extract is subjected to a process where the extract is heated at 40-65°C.] The dried soursop leaves are crushed to obtain soursop leaf powder. The powder of soursop leaf and branch was mixed with an ethanol aqueous solution with a volume concentration of 50% to 95% at a material-to-liquid ratio of 1 g: 5 to 15 mL, and refluxed at 60 to 85°C for 1 to 3 hours. The extract was then collected. The extract was concentrated under vacuum at 40-65°C to obtain a concentrated solution. Add 0.1 to 0.5 times the volume of the concentrated solution of ethyl acetate to the concentrated solution and extract 2 to 4 times to obtain the ethyl acetate extract phase. The ethyl acetate extract phase is mixed with a phospholipid aqueous dispersion with a mass concentration of 0.5% to 5% at a volume ratio of 1:1 to 5. The mixed solution is homogenized under high pressure at 50 to 150 MPa 1 to 5 times to obtain a nanoliposome suspension. The phospholipid aqueous dispersion includes phosphatidylcholine and phosphatidylethanolamine in a mass ratio of 7:3 to 9:1. The nanoliposome suspension was spray-dried to obtain an extract powder of soursop leaf.

[0005] Preferably, before the step of spray drying the nanoliposome suspension, the step further includes adding a lyophilization protectant to the nanoliposome suspension. The lyophilization protectant includes one or more of trehalose, mannitol, and sucrose, and the amount of the lyophilization protectant added is 5% to 20% of the total mass of phospholipids in the nanoliposome suspension.

[0006] Preferably, the freeze-drying protectant is a mixture of trehalose and mannitol, with a mass ratio of trehalose to mannitol of 1 to 3:1. After adding the freeze-drying protectant, the process further includes a pre-freezing step at -40°C to -20°C for 2 to 6 hours, followed by the spray drying step.

[0007] Preferably, the step of mixing the ethyl acetate extract phase with the phospholipid aqueous dispersion further includes the step of adding cholesterol, wherein the amount of cholesterol added is 10% to 30% of the total mass of phospholipids in the phospholipid aqueous dispersion.

[0008] Preferably, the step of mixing the ethyl acetate extract phase with the phospholipid aqueous dispersion further includes the step of adding bile salts, wherein the bile salts include one or more of sodium taurocholate, sodium glycocholate, and sodium deoxycholate, and the amount of bile salts added is 1% to 10% of the total mass of phospholipids in the phospholipid aqueous dispersion.

[0009] Preferably, before the step of mixing the ethyl acetate extract phase with the phospholipid aqueous dispersion, a step of dehydrating the ethyl acetate extract phase is further included: Anhydrous sodium sulfate is added to the ethyl acetate extract phase, wherein the amount of anhydrous sodium sulfate added is 1% to 5% of the mass of the ethyl acetate extract phase. After stirring for 10 to 60 minutes, the mixture is filtered to obtain the dehydrated ethyl acetate extract phase.

[0010] An extract of soursop leaf prepared by the method described above is provided.

[0011] An antidiabetic pharmaceutical composition is provided, comprising a therapeutically effective amount of the soursop leaf extract and a pharmaceutically acceptable carrier.

[0012] This invention provides the use of the soursop leaf extract in the preparation of a medicament for treating type 2 diabetes.

[0013] The present invention has at least the following beneficial effects: First, this invention, through the process route of "ethanol reflux extraction - ethyl acetate selective extraction - specific phospholipid nanoliposome encapsulation - spray drying", effectively enriches and stably encapsulates a group of lipophilic active ingredients suitable for nano-encapsulation in the leaves of custard apple, and solves the technical problems of low bioavailability of extracts and lack of stable delivery systems in the prior art.

[0014] Secondly, the extract of soursop leaf (such as AME-N-5) obtained by the method of the present invention is a nano-sized powder with an average particle size of less than 200 nm, uniform distribution (PDI<0.2), high encapsulation efficiency (up to 85.7% or more), and significant sustained-release characteristics. These characteristics together ensure the high stability and quality control of the product.

[0015] Third, the experiments of this invention demonstrate that the ethyl acetate extraction step is crucial for subsequent nano-encapsulation. Direct mixing without this step will lead to process failure. At the same time, ethyl acetate has a unique selectivity for the target active ingredient group compared to other organic solvents, which can achieve a higher encapsulation rate. The specific ratio of phosphatidylcholine and phosphatidylethanolamine is the basis for the formation of stable nanoliposomes. High-pressure homogenization is the key to obtaining nanoparticles with uniform particle size. Optimized steps such as adding cholesterol, bile salts, dehydration, and lyophilization protection can further reduce particle size, lower PDI, and improve encapsulation rate and stability.

[0016] Fourth, pharmacodynamic experiments of this invention show that, under the same amount of raw drug, the nanoliposome extract AME-N-5 prepared in this invention can significantly reduce fasting blood glucose, postprandial blood glucose peak, and total glycemic load (AUC) in diabetic mice, with effects equivalent to the positive control drug metformin; it can effectively reduce glycated hemoglobin (HbA1c) levels, indicating its sustained glycemic regulation ability; it can significantly reduce the insulin resistance index and regulate abnormal blood lipid levels, improving metabolic syndrome through a dual pathway of glucose and lipid metabolism; it can significantly reduce the levels of liver damage markers (ALT, AST) and effectively reverse liver steatosis and inflammation from a histopathological perspective; at the same time, it can alleviate pancreatic islet atrophy and structural damage, protect pancreatic β-cell function, correct diabetes-related energy metabolism disorders, and promote liver glycogen synthesis.

[0017] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0018] Figure 1 This is an in vitro cumulative release curve of the extract and control sample in the embodiments of the present invention, where Time (h) represents the time of the release experiment in hours, and Cumulative Release Rate (%) represents the percentage of drug or active ingredient released cumulatively over time. Figure 2 HE-stained liver tissue micrographs (×200x) of diabetic mice in each group are shown. A is the blank group, B is the diabetic model group, C is the metformin group, D is the high-dose AME-N-5 group, E is the medium-dose AME-N-5 group, and F is the low-dose AME-N-5 group. Yellow arrows indicate fatty degeneration, black arrows indicate Kupffer cell proliferation, blue arrows indicate inflammatory cell infiltration, and green arrows indicate focal hepatocyte regeneration. Figure 3 HE-stained sections of pancreatic tissue from diabetic mice in each group are micrographs (×20 and partial ×200 magnification). Among them, A is the blank group, B is the diabetic model group, C is the metformin group, D is the high-dose AME-N-5 group, E is the medium-dose AME-N-5 group, and F is the low-dose AME-N-5 group. Yellow arrows indicate vacuolar degeneration of cells, blue arrows indicate mild fibrosis, and black arrows indicate inflammatory cells. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0020] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0021] <Example 1> The preparation method of custard apple leaf extract includes the following steps: Take 1 kg of dried custard apple leaves, crush them with a grinder, and pass them through a 40-mesh sieve to obtain custard apple leaf powder. The above powder was mixed with a 70% (v / v) ethanol aqueous solution at a ratio of 1 g:10 mL, and placed in a reflux extraction apparatus. The mixture was refluxed at 75°C for 2 hours, filtered, and the extract was collected. The residue was extracted again under the same conditions, and the two extracts were combined. The combined extracts were concentrated under reduced pressure at 50°C and 0.08 MPa to recover ethanol until the volume of the concentrate was approximately 100 mL, thus obtaining the concentrate. Add 0.3 times the volume of ethyl acetate to the above concentrate, shake thoroughly in a separatory funnel for 5 minutes, let stand until completely separated, collect the upper ethyl acetate phase, and repeat the extraction twice with the same volume of ethyl acetate on the lower aqueous phase. Combine the three ethyl acetate extracts. Weigh 16 g of phosphatidylcholine and 4 g of phosphatidylethanolamine (mass ratio 8:2), totaling 20 g, and slowly disperse them in 980 mL of purified water. Stir on a magnetic stirrer at room temperature for 2 hours until a uniform, translucent dispersion is formed, which yields a phosphatidyl aqueous dispersion with a mass concentration of approximately 2%. The obtained ethyl acetate extract phase was mixed with the prepared phospholipid aqueous dispersion at a volume ratio of 1:3, placed on a magnetic stirrer, and stirred at 500 rpm for 30 minutes to form a uniform pre-emulsion. The pre-emulsion was transferred to a high-pressure homogenizer and homogenized three times at 100 MPa pressure to obtain a milky white, slightly opalescent nanoliposome suspension. The nanoliposome suspension was spray-dried with an inlet air temperature of 135°C, an outlet air temperature of 60°C, and a feed rate of 5 mL / min. The powder at the bottom of the drying tower was collected to obtain the soursop leaf extract, denoted as AME-N-1.

[0022] <Example 2> The preparation method of custard apple leaf extract includes the following steps: Take 1 kg of dried custard apple leaves, crush them with a grinder, and pass them through a 40-mesh sieve to obtain custard apple leaf powder. The above powder was mixed with a 70% (v / v) ethanol aqueous solution at a ratio of 1 g:10 mL, and placed in a reflux extraction apparatus. The mixture was refluxed at 75°C for 2 hours, filtered, and the extract was collected. The residue was extracted again under the same conditions, and the two extracts were combined. The combined extracts were concentrated under reduced pressure at 50°C and 0.08 MPa to recover ethanol until the volume of the concentrate was approximately 100 mL, thus obtaining the concentrate. Add 0.3 times the volume of ethyl acetate to the above concentrate, shake thoroughly in a separatory funnel for 5 minutes, let stand until completely separated, collect the upper ethyl acetate phase, and repeat the extraction twice with the same volume of ethyl acetate on the lower aqueous phase. Combine the three ethyl acetate extracts. Weigh 16 g of phosphatidylcholine and 4 g of phosphatidylethanolamine (mass ratio 8:2), totaling 20 g, and slowly disperse them in 980 mL of purified water. Stir on a magnetic stirrer at room temperature for 2 hours until a uniform, translucent dispersion is formed, which yields a phosphatidyl aqueous dispersion with a mass concentration of approximately 2%. The obtained ethyl acetate extract phase was mixed with the prepared phospholipid aqueous dispersion at a volume ratio of 1:3, placed on a magnetic stirrer, and stirred at 500 rpm for 30 minutes to form a uniform pre-emulsion. The pre-emulsion was transferred to a high-pressure homogenizer and homogenized three times at 100 MPa pressure to obtain a milky white, slightly opalescent nanoliposome suspension. Add a lyophilization protectant to the above nanoliposome suspension. The lyophilization protectant is a mixture of trehalose and mannitol (mass ratio 2:1). The amount added is 15% of the total mass of phospholipids in the suspension (20 g), i.e., 3 g. Stir at room temperature for 1 hour to completely dissolve the protectant. The above-mentioned suspension with added protective agent was dispensed into stainless steel freeze-drying trays with a liquid thickness of about 1 cm. The freeze-drying trays were then placed in an ultra-low temperature freezer at -30°C for 4 hours to pre-freeze the samples until they were completely frozen. The pre-frozen sample was taken out and immediately spray-dried. The inlet air temperature was set to 135℃, the outlet air temperature to 60℃, and the feed rate to 5 mL / min. The powder at the bottom of the drying tower was collected to obtain the soursop leaf extract, denoted as AME-N-2.

[0023] <Example 3> The preparation method of custard apple leaf extract includes the following steps: Take 1 kg of dried custard apple leaves, crush them with a grinder, and pass them through a 40-mesh sieve to obtain custard apple leaf powder. The above powder was mixed with a 70% (v / v) ethanol aqueous solution at a ratio of 1 g:10 mL, and placed in a reflux extraction apparatus. The mixture was refluxed at 75°C for 2 hours, filtered, and the extract was collected. The residue was extracted again under the same conditions, and the two extracts were combined. The combined extracts were concentrated under reduced pressure at 50°C and 0.08 MPa to recover ethanol until the volume of the concentrate was approximately 100 mL, thus obtaining the concentrate. Add 0.3 times the volume of ethyl acetate to the above concentrate, shake thoroughly in a separatory funnel for 5 minutes, let stand until completely separated, collect the upper ethyl acetate phase, and repeat the extraction twice with the same volume of ethyl acetate on the lower aqueous phase. Combine the three ethyl acetate extracts. Weigh out 16 g of phosphatidylcholine and 4 g of phosphatidylethanolamine (mass ratio 8:2), totaling 20 g. Weigh out 4 g of cholesterol. Slowly disperse all components together in 976 mL of purified water. Stir on a magnetic stirrer at room temperature for 2 hours until a uniform, semi-transparent dispersion is formed, which is a cholesterol-containing phospholipid aqueous dispersion with a mass concentration of about 2%. The obtained ethyl acetate extract phase was mixed with the prepared cholesterol phospholipid aqueous dispersion at a volume ratio of 1:3, placed on a magnetic stirrer, and stirred at 500 rpm for 30 minutes to form a uniform pre-emulsion. The pre-emulsion was transferred to a high-pressure homogenizer and homogenized three times at 100 MPa pressure to obtain a milky white, slightly opalescent nanoliposome suspension. Add a lyophilization protectant to the above nanoliposome suspension. The lyophilization protectant is a mixture of trehalose and mannitol (mass ratio 2:1). The amount added is 15% of the total mass of phospholipids in the suspension (20 g), i.e., 3 g. Stir at room temperature for 1 hour to completely dissolve the protectant. The above-mentioned suspension with added protective agent was dispensed into stainless steel freeze-drying trays with a liquid thickness of about 1 cm. The freeze-drying trays were then placed in an ultra-low temperature freezer at -30°C for 4 hours to pre-freeze the samples until they were completely frozen. The pre-frozen sample was taken out and immediately spray-dried. The inlet air temperature was set to 135℃, the outlet air temperature to 60℃, and the feed rate to 5 mL / min. The powder at the bottom of the drying tower was collected to obtain the soursop leaf extract, denoted as AME-N-3.

[0024] <Example 4> The preparation method of custard apple leaf extract includes the following steps: Take 1 kg of dried custard apple leaves, crush them with a grinder, and pass them through a 40-mesh sieve to obtain custard apple leaf powder. The above powder was mixed with a 70% (v / v) ethanol aqueous solution at a ratio of 1 g:10 mL, and placed in a reflux extraction apparatus. The mixture was refluxed at 75°C for 2 hours, filtered, and the extract was collected. The residue was extracted again under the same conditions, and the two extracts were combined. The combined extracts were concentrated under reduced pressure at 50°C and 0.08 MPa to recover ethanol until the volume of the concentrate was approximately 100 mL, thus obtaining the concentrate. Add 0.3 times the volume of ethyl acetate to the above concentrate, shake thoroughly in a separatory funnel for 5 minutes, let stand until completely separated, collect the upper ethyl acetate phase, and repeat the extraction twice with the same volume of ethyl acetate on the lower aqueous phase. Combine the three ethyl acetate extracts. Weigh out 16 g of phosphatidylcholine and 4 g of phosphatidylethanolamine (mass ratio 8:2), totaling 20 g. Weigh out 4 g of cholesterol and 1 g of sodium taurocholate. Slowly disperse all components together in 975 mL of purified water. Stir on a magnetic stirrer at room temperature for 2 hours until a uniform, translucent dispersion is formed, which is a phospholipid dispersion containing cholesterol and bile salts with a mass concentration of approximately 2%. The obtained ethyl acetate extract phase was mixed with the prepared aqueous dispersion of cholesterol and bile salt phospholipids at a volume ratio of 1:3. The mixture was placed on a magnetic stirrer and stirred at 500 rpm for 30 minutes to form a uniform pre-emulsion. The pre-emulsion was then transferred to a high-pressure homogenizer and homogenized three times at 100 MPa pressure to obtain a milky white, slightly opalescent nanoliposome suspension. Add a lyophilization protectant to the above nanoliposome suspension. The lyophilization protectant is a mixture of trehalose and mannitol (mass ratio 2:1). The amount added is 15% of the total mass of phospholipids in the suspension (20 g), i.e., 3 g. Stir at room temperature for 1 hour to completely dissolve the protectant. The above-mentioned suspension with added protective agent was dispensed into stainless steel freeze-drying trays with a liquid thickness of about 1 cm. The freeze-drying trays were then placed in an ultra-low temperature freezer at -30°C for 4 hours to pre-freeze the samples until they were completely frozen. The pre-frozen sample was taken out and immediately spray-dried. The inlet air temperature was set to 135℃, the outlet air temperature to 60℃, and the feed rate to 5 mL / min. The powder at the bottom of the drying tower was collected to obtain the soursop leaf extract, denoted as AME-N-4.

[0025] <Example 5> The preparation method of custard apple leaf extract includes the following steps: Take 1 kg of dried custard apple leaves, crush them with a grinder, and pass them through a 40-mesh sieve to obtain custard apple leaf powder. The above powder was mixed with a 70% (v / v) ethanol aqueous solution at a ratio of 1 g:10 mL, and placed in a reflux extraction apparatus. The mixture was refluxed at 75°C for 2 hours, filtered, and the extract was collected. The residue was extracted again under the same conditions, and the two extracts were combined. The combined extracts were concentrated under reduced pressure at 50°C and 0.08 MPa to recover ethanol until the volume of the concentrate was approximately 100 mL, thus obtaining the concentrate. Add 0.3 times the volume of ethyl acetate to the above concentrate, shake thoroughly in a separatory funnel for 5 minutes, let stand until completely separated, collect the upper ethyl acetate phase, and repeat the extraction twice with the same volume of ethyl acetate on the lower aqueous phase. Combine the three ethyl acetate extracts. Add 3% (by weight) of anhydrous sodium sulfate (analytical grade) to the combined ethyl acetate extract phase, stir on a magnetic stirrer at room temperature for 30 minutes, filter with medium-speed qualitative filter paper to remove the solid anhydrous sodium sulfate, and collect the clear, dehydrated ethyl acetate extract phase; Weigh out 16 g of phosphatidylcholine and 4 g of phosphatidylethanolamine (mass ratio 8:2), totaling 20 g. Weigh out 4 g of cholesterol and 1 g of sodium taurocholate. Slowly disperse all components together in 975 mL of purified water. Stir on a magnetic stirrer at room temperature for 2 hours until a uniform, translucent dispersion is formed, which is a phospholipid dispersion containing cholesterol and bile salts with a mass concentration of approximately 2%. The dehydrated ethyl acetate extract phase was mixed with the prepared aqueous dispersion of cholesterol and bile salt phospholipids at a volume ratio of 1:3. The mixture was placed on a magnetic stirrer and stirred at 500 rpm for 30 minutes to form a uniform pre-emulsion. The pre-emulsion was then transferred to a high-pressure homogenizer and homogenized three times at 100 MPa pressure to obtain a milky white, slightly opalescent nanoliposome suspension. Add a lyophilization protectant to the above nanoliposome suspension. The lyophilization protectant is a mixture of trehalose and mannitol (mass ratio 2:1). The amount added is 15% of the total mass of phospholipids in the suspension (20 g), i.e., 3 g. Stir at room temperature for 1 hour to completely dissolve the protectant. The above-mentioned suspension with added protective agent was dispensed into stainless steel freeze-drying trays with a liquid thickness of about 1 cm. The freeze-drying trays were then placed in an ultra-low temperature freezer at -30°C for 4 hours to pre-freeze the samples until they were completely frozen. The pre-frozen sample was taken out and immediately spray-dried. The inlet air temperature was set to 135℃, the outlet air temperature to 60℃, and the feed rate to 5 mL / min. The powder at the bottom of the drying tower was collected to obtain the soursop leaf extract, denoted as AME-N-5.

[0026] <Control Group 1> The preparation method of custard apple leaf extract includes the following steps: Take 1 kg of dried custard apple leaves, crush them with a grinder, and pass them through a 40-mesh sieve to obtain custard apple leaf powder. The powder was mixed with a 70% ethanol aqueous solution at a ratio of 1 g: 10 mL, and placed in a reflux extraction device. The mixture was refluxed at 75°C for 2 hours, filtered, and the extract was collected. The residue was extracted again under the same conditions, and the two extracts were combined. The combined extracts were concentrated under reduced pressure at 50°C and 0.08 MPa to recover ethanol and obtain a thick paste concentrate. The paste was transferred to a vacuum drying oven and dried at 60°C to constant weight. The dried material was pulverized and passed through an 80-mesh sieve to obtain a dried crude extract powder of soursop leaf and branch, denoted as AME-A.

[0027] <Control Group 2> The preparation method of custard apple leaf extract includes the following steps: Take 1 kg of dried custard apple leaves, crush them with a grinder, and pass them through a 40-mesh sieve to obtain custard apple leaf powder. The above powder was extracted three times by reflux with 10 times the amount of 95% ethanol, each time for 1.5 hours. The extracts were combined and the ethanol was recovered. Add sulfuric acid dropwise to the aqueous solution after ethanol recovery to adjust the pH to 3.5, then extract three times with an equal volume of ethyl acetate, and combine the ethyl acetate extracts; The combined ethyl acetate extract was washed three times with pure water, and then the solvent was evaporated under reduced pressure to obtain a dry extract powder, denoted as AME-B.

[0028] <Comparative Example 1> The preparation method of custard apple leaf extract includes the following steps: Take 1 kg of dried custard apple leaves, crush them with a grinder, and pass them through a 40-mesh sieve to obtain custard apple leaf powder. The powder was mixed with a 70% ethanol aqueous solution at a ratio of 1 g: 10 mL, and placed in a reflux extraction device. The mixture was refluxed at 75°C for 2 hours, filtered, and the extract was collected. The residue was extracted again under the same conditions, and the two extracts were combined. The combined extracts were concentrated under reduced pressure at 50°C and 0.08 MPa to recover ethanol until the volume of the concentrate was approximately 100 mL, thus obtaining the concentrate. Weigh out 16 g of phosphatidylcholine and 4 g of phosphatidylethanolamine (mass ratio 8:2), totaling 20 g. Weigh out 4 g of cholesterol and 1 g of sodium taurocholate. Slowly disperse all components together in 975 mL of purified water. Stir on a magnetic stirrer at room temperature for 2 hours until a uniform, translucent dispersion is formed, which is a phospholipid dispersion containing cholesterol and bile salts with a mass concentration of approximately 2%. The obtained concentrate was mixed with the prepared aqueous dispersion containing cholesterol and bile salt phospholipids at a volume ratio of 1:3. The mixture was placed on a magnetic stirrer and stirred at 500 rpm. During mixing, the system quickly became turbid and flocculated, and a uniform primary emulsion could not be formed. The mixture was forcibly transferred to a high-pressure homogenizer and homogenized three times at a pressure of 100 MPa. The material was completely separated into an aqueous phase and an oil phase, and a uniform nanoliposome suspension could not be obtained, so the process failed.

[0029] <Comparative Example 2> The preparation method of custard apple leaf extract includes the following steps: Take 1 kg of dried custard apple leaves, crush them with a grinder, and pass them through a 40-mesh sieve to obtain custard apple leaf powder. The powder was mixed with a 70% ethanol aqueous solution at a ratio of 1 g: 10 mL, and placed in a reflux extraction device. The mixture was refluxed at 75°C for 2 hours, filtered, and the extract was collected. The residue was extracted again under the same conditions, and the two extracts were combined. The combined extracts were concentrated under reduced pressure at 50°C and 0.08 MPa to recover ethanol until the volume of the concentrate was approximately 100 mL, thus obtaining the concentrate. Add 0.3 times the volume of ethyl acetate to the above concentrate, shake thoroughly in a separatory funnel for 5 minutes, let stand until completely separated, collect the upper ethyl acetate phase, and repeat the extraction twice with the same volume of ethyl acetate on the lower aqueous phase. Combine the three ethyl acetate extracts. Add 3% (by weight) of anhydrous sodium sulfate (analytical grade) to the combined ethyl acetate extract phase, stir on a magnetic stirrer at room temperature for 30 minutes, filter with medium-speed qualitative filter paper to remove the solid anhydrous sodium sulfate, and collect the clear, dehydrated ethyl acetate extract phase; The dehydrated ethyl acetate extract phase was subjected to rotary evaporation under reduced pressure at 40°C to recover the ethyl acetate and obtain a viscous extract paste. The extract was transferred to a vacuum drying oven and dried at 40°C to constant weight. It was then pulverized to obtain a dried ethyl acetate extract powder, denoted as AME-C.

[0030] <Comparative Example 3> The preparation method of custard apple leaf extract includes the following steps: Take 1 kg of dried custard apple leaves, crush them with a grinder, and pass them through a 40-mesh sieve to obtain custard apple leaf powder. The powder was mixed with a 70% ethanol aqueous solution at a ratio of 1 g: 10 mL, and placed in a reflux extraction device. The mixture was refluxed at 75°C for 2 hours, filtered, and the extract was collected. The residue was extracted again under the same conditions, and the two extracts were combined. The combined extracts were concentrated under reduced pressure at 50°C and 0.08 MPa to recover ethanol until the volume of the concentrate was approximately 100 mL, thus obtaining the concentrate. Add 0.3 times the volume of n-butanol to the above concentrate, shake and extract thoroughly in a separatory funnel for 5 minutes, let stand until complete separation, collect the upper n-butanol phase, and repeat the extraction twice with the same volume of n-butanol for the lower aqueous phase. Combine the three n-butanol extract phases. Add 3% (by weight) of anhydrous sodium sulfate (analytical grade) to the combined n-butanol extract phase, stir on a magnetic stirrer at room temperature for 30 minutes, filter with medium-speed qualitative filter paper to remove the solid anhydrous sodium sulfate, and collect the clear, dehydrated n-butanol extract phase. Weigh out 16 g of phosphatidylcholine and 4 g of phosphatidylethanolamine (mass ratio 8:2), totaling 20 g. Weigh out 4 g of cholesterol and 1 g of sodium taurocholate. Slowly disperse all components together in 975 mL of purified water. Stir on a magnetic stirrer at room temperature for 2 hours until a uniform, translucent dispersion is formed, which is a phospholipid dispersion containing cholesterol and bile salts with a mass concentration of approximately 2%. The dehydrated n-butanol extract phase was mixed with the prepared aqueous dispersion of cholesterol and bile salt phospholipids at a volume ratio of 1:3. The mixture was placed on a magnetic stirrer and stirred at 500 rpm for 30 minutes to form a uniform pre-emulsion. The pre-emulsion was then transferred to a high-pressure homogenizer and homogenized three times at 100 MPa pressure to obtain a nanoliposome suspension. Add a lyophilization protectant to the above nanoliposome suspension. The lyophilization protectant is a mixture of trehalose and mannitol (mass ratio 2:1). The amount added is 15% of the total mass of phospholipids in the suspension (20 g), i.e., 3 g. Stir at room temperature for 1 hour to completely dissolve the protectant. The above-mentioned suspension with added protective agent was dispensed into stainless steel freeze-drying trays with a liquid thickness of about 1 cm. The freeze-drying trays were then placed in an ultra-low temperature freezer at -30°C for 4 hours to pre-freeze the samples until they were completely frozen. The pre-frozen sample was taken out and immediately spray-dried. The inlet air temperature was set to 135℃, the outlet air temperature to 60℃, and the feed rate to 5 mL / min. The powder at the bottom of the drying tower was collected to obtain the soursop leaf extract, denoted as AME-D.

[0031] <Determination of Physicochemical Properties> Samples AME-N-1 ​​to AME-N-5, AME-A, AME-B, AME-C and AME-D were prepared according to the methods of Examples 1 to 5, Control Groups 1 to 2 and Comparative Examples 1 to 3, respectively. Among them, Comparative Example 1 did not obtain a sample that could be used for testing due to process failure.

[0032] The above samples were subjected to physicochemical property tests, including particle size and polydispersity index, encapsulation efficiency, and in vitro release rate. The specific determination methods are as follows: Particle size and polydispersity index (PDI): The nanoliposome suspensions prepared in each example and comparative example (or their powders were reconstituted with pure water to the same concentration) were used to determine their average particle size (Z-Average) and PDI using a dynamic light scattering particle size analyzer. Among them, the samples of control group 1, control group 2 and comparative example 2 (AME-D) were conventional powders, which were directly dispersed in water for measurement. Encapsulation efficiency determination: Ultrafiltration centrifugation was used. Specifically, an appropriate amount of nanoliposome suspension (or reconstituted solution) was placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa and centrifuged at 4000 rpm for 15 minutes. The filtrate (i.e., the unencapsulated free drug) was collected, and the content of the marker active ingredient in the filtrate and the total suspension after demulsification was determined by high performance liquid chromatography (HPLC). The encapsulation efficiency (EE%) was calculated as follows: EE% = (1 - drug content in filtrate / total drug content) × 100%.

[0033] In vitro release rate: The dialysis bag method was used. Specifically, each sample containing the same amount of crude drug was placed in a dialysis bag and immersed in simulated gastric fluid (pH 1.2) for 2 hours. Then, it was transferred to simulated intestinal fluid (pH 6.8) for further release. Samples were taken at different time points, and the cumulative amount of drug released in the release medium was determined by HPLC.

[0034] The obtained physicochemical property data, such as particle size, PDI, and encapsulation efficiency, are shown in Table 1 below. right Ratio 1 cannot form nanoliposomes, so there is no relevant data, and N / A indicates it is not applicable. AME-A, AME-B, and AME-C are conventional powders without encapsulation structures. Table 1. Results of physicochemical property determination for each sample As shown in Table 1, Examples 1-5 of this invention successfully prepared nanoliposomes with a particle size of less than 200 nm and uniform distribution (PDI < 0.2). With the addition of optimization steps including cholesterol, bile salts, and dehydration, the particle size was further reduced and the PDI decreased, indicating that the system was more stable. The failure of Comparative Example 1 directly proves that without selective extraction with ethyl acetate, the components in the aqueous concentrate are incompatible with the phospholipid system, making it impossible to implement the core encapsulation process of this invention, highlighting the necessity of this step.

[0035] The encapsulation rates of the extracts prepared from the leaves of *Annona squamosa* in Examples 1-5 of this invention are significantly higher than those of Comparative Example 3, which uses n-butanol for extraction and has an encapsulation rate of only 52.3%, significantly lower than that of Example 5, which uses ethyl acetate. This indicates that ethyl acetate has a unique selectivity for extracting active ingredients from *Annona squamosa* leaves that can efficiently bind to this specific phospholipid system and are suitable for nano-encapsulation. This effect cannot be achieved by other common solvents such as n-butanol.

[0036] Based on the cumulative release rate (%) of each sample at different time points obtained from the detection, in vitro release curves were plotted, such as... Figure 1 As shown, the samples (AME-N-1 ​​and AME-N-5) of the present invention exhibited obvious sustained-release characteristics throughout the release process. For example, at the end of the 2-hour simulated gastric fluid stage, their cumulative release rates were only 28.4% and 35.6%, respectively, far from achieving burst release. At 12 hours, the release tended to be complete, indicating that the nanoliposome structure effectively delayed the release of the active ingredient. Among them, the release rate of AME-N-5 was slightly slower than that of AME-N-1 ​​at most time points, especially in the early and middle stages, indicating that the optimized process further enhanced the stability of the nanoliposomes and delayed drug release. Furthermore, the release rate of AME-E in Comparative Example 3 was significantly faster than that in Examples 1-5 of this invention. It had already released more than 50% within 2 hours and was basically completely released within 12 hours, with a significant reduction in the sustained-release effect. This proves that ethyl acetate, as an extractant, can form a more stable and milder release system in synergy with the subsequent phospholipid nano-encapsulation process. This effect cannot be replaced by n-butanol. The release behavior of AME-D in Comparative Example 2 and AME-A in Control Group 1 was similar, both showing rapid release, with a release rate of more than 85% within 2 hours, followed by a plateau. This directly proves that the nanoliposome encapsulation step is the fundamental reason for endowing the extract with sustained-release properties.

[0037] <Pharmacodynamic Verification Experiment> To verify the antidiabetic effect of the soursop leaf extract (using AME-N-5 as an example) prepared by the method of the present invention, especially its advantages over conventional extracts, the following animal experiments were conducted.

[0038] 1. Experimental Materials Test sample: The extract of soursop leaf prepared in Example 5 of this invention, denoted as AME-N-5, was reconstituted with pure water before use; Positive control drug: Metformin hydrochloride tablets, prepared as a suspension of 0.02 g / mL using 0.5% CMC-Na solution; Laboratory animals: SPF-grade male ICR mice, weighing 18-22 g; Main reagents: streptozotocin (STZ), citric acid, sodium citrate, high-fat diet (60% of calories from fat), blood glucose test strips, glycated hemoglobin (HbA1c) test kit, etc.

[0039] 2. Experimental Methods 2.1 Establishment of a mouse model of type 2 diabetes mellitus (T2DM) Ninety mice were acclimatized to a normal maintenance diet for one week and then randomly divided into a control group (n=15) and a high-energy diet group (n=75) according to their body weight. After the groups were divided, the high-energy diet group was replaced with a 60% high-fat diet, while the control group continued to be fed the normal maintenance diet.

[0040] Four weeks later, mice in the high-energy diet group were fasted for 12 hours (but not water) and injected intraperitoneally with freshly prepared 1% STZ citrate buffer (pH 4.5) at a dose of 60 mg / kg body weight once a week for two consecutive weeks. The control group was injected intraperitoneally with an equal volume of citrate-sodium citrate buffer.

[0041] Within 4 weeks after the last STZ injection, blood was collected from the rat tail to measure 8-hour fasting blood glucose (FBG). If the FBG was ≥11.1 mmol / L for two consecutive times, the type 2 diabetes model was considered to have been successfully established.

[0042] 2.2 Animal grouping and administration The successfully induced diabetic mice were randomly divided into 4 groups, and a corresponding number of control mice were randomly selected, resulting in a total of 6 groups with 10 mice in each group: Control group: Normal mice were administered 0.5% CMC-Na solution (10 mL / kg) by gavage. Model group: T2DM mice, administered 0.5% CMC-Na solution (10 mL / kg) by gavage; Metformin group: T2DM mice were administered metformin suspension (0.2 g / kg) by gavage. AME-N-5 low-dose group: T2DM mice were administered AME-N-5 reconstituted solution (crude drug dose 0.5 g / kg) by gavage. AME-N-5 medium-dose group: T2DM mice were administered AME-N-5 reconstituted solution (1 g / kg crude drug) by gavage. AME-N-5 high-dose group: T2DM mice were administered AME-N-5 reconstituted solution (raw drug 1.5 g / kg) by gavage. All groups were administered the drug via gavage for 4 consecutive weeks. During the administration period, the control group was given a normal maintenance diet, while the other groups continued to be given a 60% high-fat diet.

[0043] 3. Detection indicators General condition and weight: Record the mice’s mental state, coat color, food and water intake and weight changes weekly.

[0044] Fasting blood glucose (FBG): Fasting blood glucose was measured at the tail tip after fasting for 8 hours before administration (week 0) and at weeks 2 and 4 after administration.

[0045] Oral glucose tolerance test (OGTT): Performed on week 4 of drug administration. Mice were fasted for 8 hours, and fasting blood glucose (0 h) was measured. Then, they were administered glucose solution (2.5 g / kg) by gavage. Blood glucose levels were measured at 0.5 h, 1 h, and 2 h after gavage, and the area under the blood glucose curve (AUC) was calculated. The blood glucose AUC was calculated using the formula... calculate.

[0046] Glycated hemoglobin (HbA1c): After drug administration, blood was collected, and the level of glycated hemoglobin was measured using an ELISA kit. After centrifugation, the supernatant was collected, and the plasma insulin content was measured according to the formula. Calculate the insulin resistance index.

[0047] Specimen collection: After the experiment, fasting for 3 hours was performed, blood was drawn from the orbital cavity, serum was separated, and the tissue was quickly dissected to obtain pancreas and part of liver tissue. After rinsing with physiological saline, the tissue was fixed with 4% paraformaldehyde for subsequent pathological examination.

[0048] 4. Experimental Results 4.1 Successful establishment of diabetic mouse model The final success rate of diabetic mice meeting the modeling criteria was 53.33%. Thirty mice were selected and randomly divided into five groups of six mice each, based on their blood glucose and body weight. This ensured that there were no significant differences in blood glucose and body weight among the groups before drug administration. These mice were then used for subsequent drug administration.

[0049] 4.2 Effects of Anelka arugula leaf extract on the general condition of diabetic mice Compared with the control group, the diabetic model group mice showed obvious obesity, shinier fur, sparse fur in some areas, lethargy, significantly reduced activity, polydipsia and polyuria, with a gradual decrease in food intake after an initial increase, yellowish urine, and soft, yellow, sticky stools. Compared with the model group, the mental state, diet and bowel and bladder conditions of all groups were improved. The metformin group and the high-dose AME-N-5 group showed more significant improvement in appearance.

[0050] 4.3 Effects of Annona squamosa leaf extract on body weight in diabetic mice To quantitatively assess the changes in body weight of mice in each group, body weight data before and after drug administration were recorded. The specific results are shown in Table 2 below: Table 2. Effects of each group on body weight of diabetic mice ( ) Note: Compared with the blank group, # P<0.05.

[0051] As shown in Table 2, compared with the blank group, the initial body weight and final body weight of diabetic model mice were significantly increased before and after drug administration. Compared with the model group, there was no significant difference in initial body weight between the positive control group (metformin) and the various dose groups of AME-N-5 of this invention, and the final body weight showed a decreasing trend, but the difference was not statistically significant. The changes in body weight further confirmed that the model group mice had the characteristics of lipid accumulation in the early stage of T2DM, which is consistent with the metabolic disorder caused by insulin resistance. The AME-N-5 extract prepared in this invention, especially the high-dose group, showed an inhibitory trend on weight gain. Combined with the improvement in general condition, it indicates that AME-N-5 may control the abnormal weight gain caused by metabolic disorder to a certain extent by regulating glucose and lipid metabolism and improving insulin sensitivity.

[0052] 4.4 Effects of Annona squamosa leaf extract on fasting blood glucose and oral glucose tolerance in diabetic mice To systematically evaluate the hypoglycemic effect of AME-N-5, we measured fasting blood glucose (FBG) in mice during drug administration and performed an oral glucose tolerance test (OGTT). The dynamic changes in FBG are shown in Table 3 below: Table 3. Effects of each group on fasting blood glucose levels in diabetic mice during drug administration ( ) Note: Compared with the blank group, ## P<0.01, ### P < 0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0053] As shown in Table 3, compared with the control group, FBG was significantly increased in the diabetes model group before and after drug administration (P<0.001). Compared with the diabetes model group, there were no significant differences in FBG before drug administration and in the three dosage groups of AME-N-5 compared with the metformin group and week 1 of drug administration; FBG was significantly decreased in the metformin group at weeks 3 and 4 of drug administration (P<0.05, P<0.001); FBG was significantly decreased in the high-dose AME-N-5 group at weeks 2, 3, and 4 of drug administration (P<0.05, P<0.01, P<0.001); FBG was significantly decreased in the medium-dose AME-N-5 group at weeks 3 and 4 of drug administration (P<0.01); and FBG was significantly decreased in the low-dose AME-N-5 group at weeks 3 and 4 of drug administration (P<0.001, P<0.01). The OGTT results more directly reflect the body's ability to regulate glucose load, as shown in Table 4 below: Table 4. Effects of each group on OGTT in diabetic mice ( ) Note: Compared with the blank group, ### P < 0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0054] As shown in Table 4, the model group had severely impaired glucose tolerance. The high-dose AME-N-5 group significantly reduced blood glucose and AUC at each time point after glucose load, demonstrating outstanding improvement.

[0055] The above results indicate that AME-N-5 can effectively reduce fasting blood glucose and significantly improve glucose tolerance. Its high-dose group shows rapid onset and sustained effect, and performs excellently in reducing postprandial blood glucose peak and overall glycemic load. This suggests that the nanoliposome extract of the present invention may regulate the body's glucose uptake and utilization more stably and persistently through its sustained-release properties.

[0056] 4.5 Effects of Annona squamosa leaf extract on glycated hemoglobin levels in diabetic mice Glycated hemoglobin (HbA1c) is a key indicator reflecting long-term blood glucose control. The HbA1c test results of each group of mice after the experiment are shown in Table 5 below: Table 5. Effects of each group on HbA1c levels in diabetic mice ( ) Note: Compared with the blank group, ## P<0.01; compared with the model group, ** P<0.01.

[0057] As shown in Table 5, long-term hyperglycemia led to a significant increase in HbA1c levels in the model group mice. Both the high-dose and low-dose AME-N-5 groups significantly reduced HbA1c levels, bringing them close to the normal range. The effective reduction in HbA1c levels indicates that AME-N-5 can not only control instantaneous blood glucose but also achieve stable long-term blood glucose control. This is consistent with its sustained and mild hypoglycemic effect in FBG and OGTT, further demonstrating the potential application value of the extract of this invention in diabetes management.

[0058] 4.6 Effects of Annona squamosa leaf extract on plasma insulin levels and insulin resistance index in diabetic mice To investigate the potential mechanism by which AME-N-5 improves blood glucose, we measured plasma insulin levels and calculated the insulin resistance index. The results are shown in Table 6 below: Table 6. Effects of each group on insulin levels and insulin resistance index in diabetic mice ( ) Note: Compared with the control group, ###P<0.001; compared with the model group, **P<0.01,***P<0.001.

[0059] Table 6 shows that the model group mice exhibited insufficient insulin secretion and severe insulin resistance. Although none of the treatment groups significantly increased insulin levels, they all significantly reduced the insulin resistance index. All treatment groups significantly improved the insulin resistance index, indicating that AME-N-5, similar to metformin, primarily lowers blood glucose through improving peripheral tissue sensitivity to insulin, rather than directly stimulating insulin secretion. This provides an important direction for understanding its mechanism of action.

[0060] 4.7 Effects of Annona squamosa leaf extract on blood lipid levels in diabetic mice Diabetes mellitus is often accompanied by lipid metabolism disorders. This invention detected four key blood lipid indicators in serum, and the results are shown in Table 7 below: Table 7 Effects of each group on blood lipid levels in diabetic mice ( ) Note: Compared with the blank group, ### P < 0.001; compared with the model group, * P<0.05, ** P<0.01.

[0061] Table 7 shows that the model group had obvious lipid metabolism abnormalities. The medium dose group of AME-N-5 (1.0 g / kg) could significantly reduce the abnormally elevated total cholesterol (TC) level. The regulatory effect of AME-N-5 on TC level indicates that it not only improves glucose metabolism, but also has a positive regulatory effect on lipid metabolism disorder in diabetes. This dual improvement in glucose and lipid metabolism helps to alleviate metabolic syndrome as a whole and protect target organs.

[0062] 4.8 Effects of Annona squamosa leaf extract on liver damage in diabetic mice 4.8.1 Effects on liver ALT and AST levels Liver transaminases (ALT, AST) are sensitive markers of hepatocellular damage. The results of their activity assays are shown in Table 8 below: Table 8. Effects of each group on ALT / AST levels in diabetic mice ( ) Note: Compared with the blank group, # P<0.05; compared with the model group, ** P<0.01, *** P<0.001.

[0063] As shown in Table 8, diabetes leads to liver damage, manifested as elevated ALT and AST activity. All dose groups of AME-N-5 can significantly reduce the activity of these two transaminases, and its effect is even better than that of the positive control drug metformin.

[0064] 4.8.2 Effects on liver morphology The results of HE staining of liver sections showed the following: Figure 2 As shown, the liver tissue structure of mice in the blank group was normal, while the liver tissue structure of mice in the diabetic model group was disordered, with extensive hepatocyte swelling, accompanied by obvious fatty degeneration, Kupffer cell proliferation and inflammatory cell infiltration. Compared with the model group, the liver structure of mice in each dose group of AME-N-5 was significantly improved, and the hepatocyte swelling was reduced. Among them, fatty degeneration was basically not observed in the high dose group, and the degree of fatty degeneration decreased in the medium and low dose groups respectively. Focal hepatocyte regeneration was observed in all three groups.

[0065] It is evident that the biochemical indicators and histopathological results are highly consistent. The diabetic model group exhibited typical liver damage and steatosis. AME-N-5 not only significantly reduced the levels of liver damage markers but also effectively reversed steatosis and inflammatory infiltration in the liver from a tissue morphology perspective. The high-dose group showed the most significant effect, indicating that the extract of this invention has a strong protective effect against liver damage caused by diabetes. Its nano-formulation may help the active ingredients act more effectively on the liver.

[0066] 4.9 Effects of Annona squamosa leaf extract on pancreatic morphology in diabetic mice Results of HE staining of pancreas sections as follows Figure 3 As shown, the pancreatic tissue structure in the blank group was normal. Compared with the blank group, the pancreatic islets in the endocrine part of the diabetic model group were unevenly distributed, irregular in shape, smaller in size, and fewer in number, accompanied by some cells with vacuolar degeneration and mild fibrosis. Compared with the model group, the morphology and number of islets in each drug-treated group were improved to varying degrees. A small number of inflammatory cells were observed in the medium-dose AME-N-5 group.

[0067] Histological observations showed that AME-N-5 could alleviate islet atrophy and structural damage caused by diabetes and improved islet morphology in all treatment groups. This suggests that its hypoglycemic effect may be partly due to direct protection of islet tissue, thereby helping to maintain β-cell function.

[0068] 4.10 Effects of Annona squamosa leaf extract on energy metabolism in diabetic mice Energy metabolism disorder is one of the characteristics of diabetes. This invention detected indicators reflecting cell membrane function and glucose storage, and the data are shown in Table 9: Table 9 Effects of each group on energy metabolism in diabetic mice ( ) Note: Compared with the blank group, ## P<0.01, ### P < 0.001; compared with the model group, ** P<0.01, *** P<0.001.

[0069] Table 9 shows that Na in the model group mice + -K + - Abnormally elevated ATPase activity and excessive accumulation of liver glycogen; high and medium doses of AME-N-5 can significantly reduce Na+. + -K + -ATPase activity, and all dosage groups further promoted the synthesis and storage of liver glycogen, Na + -K + Elevated ATPase activity is often associated with oxidative stress and cellular dysfunction, while abnormal accumulation of liver glycogen reflects impaired glucose utilization under insulin resistance. AME-N-5 can correct these abnormalities, suggesting that it may promote the conversion of glucose to glycogen by alleviating oxidative stress, repairing cell membrane function, and improving insulin sensitivity, thereby comprehensively regulating energy metabolism.

[0070] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A method for preparing an extract from the leaves and branches of the soursop, characterized in that, Includes the following steps: The dried soursop leaves are crushed to obtain soursop leaf powder. The powder of soursop leaf and branch was mixed with an ethanol aqueous solution with a volume concentration of 50% to 95% at a material-to-liquid ratio of 1 g: 5 to 15 mL, and extracted by reflux at 60 to 85°C for 1 to 3 hours. The extract was then collected. The extract was concentrated under vacuum at 40-65°C to obtain a concentrated solution. Add 0.1 to 0.5 times the volume of the concentrated solution of ethyl acetate to the concentrated solution and extract 2 to 4 times to obtain the ethyl acetate extract phase. The ethyl acetate extract phase is mixed with a phospholipid aqueous dispersion with a mass concentration of 0.5% to 5% at a volume ratio of 1:1 to 5. The mixed solution is homogenized under high pressure at 50 to 150 MPa 1 to 5 times to obtain a nanoliposome suspension. The phospholipid aqueous dispersion includes phosphatidylcholine and phosphatidylethanolamine in a mass ratio of 7:3 to 9:

1. The nanoliposome suspension was spray-dried to obtain an extract powder of soursop leaf.

2. The method for preparing the extract of Annona squamosa leaf and branch as described in claim 1, characterized in that, Before the step of spray drying the nanoliposome suspension, the method further includes adding a lyophilization protectant to the nanoliposome suspension. The lyophilization protectant includes one or more of trehalose, mannitol, and sucrose. The amount of the lyophilization protectant added is 5% to 20% of the total mass of phospholipids in the nanoliposome suspension.

3. The method for preparing the extract of Annona squamosa leaf and branch as described in claim 2, characterized in that, The freeze-drying protectant is a mixture of trehalose and mannitol, with a mass ratio of trehalose to mannitol of 1 to 3:

1. After adding the freeze-drying protectant, the process further includes a pre-freezing step at -40°C to -20°C for 2 to 6 hours, followed by the spray drying step.

4. The method for preparing the extract of Annona squamosa leaf and branch as described in claim 1, characterized in that, The step of mixing the ethyl acetate extract phase with the phospholipid aqueous dispersion further includes the step of adding cholesterol, wherein the amount of cholesterol added is 10% to 30% of the total mass of phospholipids in the phospholipid aqueous dispersion.

5. The method for preparing the extract of Annona squamosa leaf and branch as described in claim 1, characterized in that, The step of mixing the ethyl acetate extract phase with the phospholipid aqueous dispersion further includes the step of adding bile salts, wherein the bile salts include one or more of sodium taurocholate, sodium glycocholate, and sodium deoxycholate, and the amount of bile salts added is 1% to 10% of the total mass of phospholipids in the phospholipid aqueous dispersion.

6. The method for preparing the extract of Annona squamosa leaf and branch as described in claim 1, characterized in that, Prior to the step of mixing the ethyl acetate extract phase with the phospholipid aqueous dispersion, a step of dehydrating the ethyl acetate extract phase is also included: Anhydrous sodium sulfate is added to the ethyl acetate extract phase, wherein the amount of anhydrous sodium sulfate added is 1% to 5% of the mass of the ethyl acetate extract phase. After stirring for 10 to 60 minutes, the mixture is filtered to obtain the dehydrated ethyl acetate extract phase.

7. The soursop leaf extract prepared by any one of claims 1 to 6.

8. An antidiabetic pharmaceutical composition, characterized in that, The extract comprises a therapeutically effective amount of the soursop leaf extract of claim 7 and a pharmaceutically acceptable carrier.

9. The use of the senna leaf extract as described in claim 7 in the preparation of a medicament for treating type 2 diabetes.

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