Composition for treating diabetes as well as preparation method and application thereof

Abstract

The invention provides a composition for treating diabetes as well as a preparation method and application thereof, and belongs to the technical field of medicines. The preparation method comprises the following steps: loading berberine on a milk exosome to prepare a berberine-loaded exosome, loading liraglutide on phycobiliprotein to prepare liraglutide-loaded phycobiliprotein, preparing PEG-COOH, reacting with chitosan to prepare a chitosan-polyethylene glycol graft polymer, embedding the berberine-loaded exosome and the liraglutide-loaded phycobiliprotein, and cross-linking to prepare microspheres. And centrifuging, washing and drying to obtain the composition for treating diabetes. According to the prepared composition for treating diabetes, the loading capacity of the medicine is greatly improved, the carrier also has a very good protection effect on islet cells, so that islet cell injury is repaired, normal insulin secretion of islet cells is promoted, the prepared composition can be orally taken, the medicine effect is greatly improved, the compliance of patients is improved, and the preparation method is suitable for clinical application. The bioavailability is high, and safety and non-toxicity are realized.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a composition for treating diabetes, its preparation method, and its application. Background Technology

[0002] Insulin, a peptide hormone produced by β-cells in the islets of Langerhans in the pancreas, is released in response to elevated blood glucose levels. Glucose is thus removed from the bloodstream via insulin-dependent stimulation of glucose transporters located in the cell membranes of target tissues (e.g., adipose tissue, skeletal muscle, and liver). Insulin exerts its biological effects by binding to and activating membrane-bound insulin receptors (IRs), thereby initiating a cascade of intracellular signaling events to regulate a variety of biological processes, such as glucose and lipid metabolism.

[0003] Diabetes mellitus (DM) is the third leading cause of death worldwide after cardiovascular disease and cancer, and has been called a "catastrophe of the 21st century" by the World Health Organization. One of the causes of diabetes is insufficient insulin secretion due to pancreatic beta cells, leading to hyperglycemia.

[0004] Currently, the treatment of diabetes (both type 1 and type 2 diabetes) mainly relies on insulin therapy or the use of hypoglycemic chemical drugs, such as metformin JP2, glibenclamide, repaglinide, acarbose, and rosiglitazone. Long-term use can lead to drug resistance and even various toxic side effects such as hypoglycemia. Supplements to insulin therapy include long-acting glucagon-like peptide-1 (GLP-1) receptor agonists, which are derivatives that act on the same receptor as GLP-1. GLP-1 is a metabolic hormone that promotes insulin secretion. In addition to increasing pancreatic insulin secretion in a glucose-dependent manner, GLP-1 is known to increase insulin sensitivity in both α- and β-cells; increase β-cell mass and insulin expression, post-translational modification, and secretion; and reduce pancreatic glucagon secretion. Other drugs used as adjuncts to insulin therapy to lower blood glucose levels include DPP-IV inhibitors, metformin, SGLT-2 inhibitors, and sulfonylureas.

[0005] Certain drawbacks, such as weight gain and increased risk of cancer and hypoglycemia, are associated with long-term insulin use. Therefore, there is a growing demand in the field for novel non-insulin compounds that can not only treat diabetes by addressing insulin resistance and hyperglycemia, but also reduce related and subsequent complications. Consequently, the development of novel, highly effective, and low-toxicity drugs that promote insulin secretion has become a hot topic in the biopharmaceutical field.

[0006] Glucagon-like peptide-1 receptor (GLP-R) agonists can intelligently regulate insulin secretion, promote β-cell proliferation, inhibit glucagon secretion, and delay gastric emptying based on blood glucose levels. However, currently marketed GLP-R agonists are all peptides, requiring injection, which leads to poor patient adherence. Summary of the Invention

[0007] The purpose of this invention is to provide a composition for treating diabetes, its preparation method, and its application. This composition greatly increases the drug loading capacity, and the carrier itself has a good protective effect on pancreatic islet cells, thereby repairing pancreatic islet cell damage and promoting their normal insulin secretion. Furthermore, the prepared composition can be administered orally, greatly improving efficacy and patient compliance. It also has high bioavailability and is safe and non-toxic.

[0008] The technical solution of this invention is implemented as follows: This invention provides a method for preparing a composition for treating diabetes. The method involves loading berberine onto milk exosomes to obtain berberine-loaded exosomes, loading liraglutide onto phycobiliproteins to obtain liraglutide-loaded phycobiliproteins, preparing PEG-COOH, reacting it with chitosan to obtain a chitosan-polyethylene glycol graft polymer, embedding the berberine-loaded exosomes and the liraglutide-loaded phycobiliproteins, cross-linking to obtain microspheres, centrifuging, washing, and drying to obtain the composition for treating diabetes.

[0009] As a further improvement to the present invention, the following steps are included: S1. Preparation of berberine-loaded exosomes: Milk exosomes and berberine were dissolved in water, stirred and mixed, dialyzed, and freeze-dried to obtain berberine-loaded exosomes; S2. Preparation of phycobiliprotein: Add dried powder of *Gnaphalium affine* to water, freeze with liquid nitrogen, thaw at room temperature, filter, dialyze the filtrate, freeze-dry to obtain phycobiliprotein; S3. Preparation of liraglutide-loaded phycobiliprotein: Phycobiliprotein and liraglutide were dissolved in water, stirred and mixed, dialyzed, and freeze-dried to obtain liraglutide-loaded phycobiliprotein; S4. Preparation of PEG-COOH: Polyethylene glycol was dissolved in dimethyl sulfoxide, alkali and chloroacetic acid were added, the mixture was stirred and reacted, the solvent was removed under reduced pressure, washed and dried to obtain PEG-COOH; S5. Preparation of chitosan-polyethylene glycol graft polymer: PEG-COOH was dissolved in water, EDC·HCl and NHS were added, the mixture was stirred and activated, chitosan was added, the pH of the solution was adjusted, the reaction was stirred at room temperature, the reaction was dialyzed until the pH of the reaction solution was 7, and the mixture was freeze-dried to obtain chitosan-polyethylene glycol graft polymer. S6. Preparation of the composition for treating diabetes: Chitosan-polyethylene glycol graft polymer was added to water, and berberine-loaded exosomes and liraglutide-loaded phycobiliprotein were added to obtain a mixture. The mixture was injected into liquid paraffin containing an emulsifier, stirred, and glutaraldehyde solution was added dropwise. The mixture underwent a cross-linking reaction, was centrifuged, washed, and dried to obtain the composition for treating diabetes.

[0010] As a further improvement of the present invention, the mass ratio of milk exosomes to berberine in step S1 is 10:4-5, the stirring time is 20-40 min, and the dialysis bag used for dialysis has a pore size of 3000-5000 Da.

[0011] As a further improvement of the present invention, the mass ratio of the dried powder of *Gnaphalium affine* and water in step S2 is 1:5-7.

[0012] As a further improvement of the present invention, the mass ratio of phycobiliprotein and liraglutide in step S3 is 10:3-4, the stirring time is 20-40 min, and the dialysis bag used for dialysis has a pore size of 5000-7000 Da.

[0013] As a further improvement of the present invention, the polyethylene glycol in step S4 is polyethylene glycol 2000 or polyethylene glycol 4000, the mass ratio of polyethylene glycol base to chloroacetic acid is 4-5:4-5:1.5-2.5, the stirring reaction time is 10-12 h, and the base is NaOH or KOH.

[0014] As a further improvement of the present invention, the mass ratio of PEG-COOH, EDC·HCl, NHS and chitosan in step S5 is 1-1.5:0.2-0.4:0.15-0.3:0.5-0.9, the stirring activation temperature is 0-4℃ and the time is 30-50 min, the pH value of the solution is adjusted to 3-4, the stirring reaction time at room temperature is 10-14 h, and the dialysis bag used for dialysis has a pore size of 3000-5000 Da.

[0015] As a further improvement of the present invention, the mass ratio of chitosan-polyethylene glycol graft polymer, barley alkaloid exosomes, liraglutide phycobiliprotein, and emulsifier in step S6 is 15-20:2-3:4-5:0.2-0.3, the emulsifier is at least one of Span-20, Span-40, Span-60, and Span-80, and the crosslinking reaction time is 1-3 hours.

[0016] The present invention further protects a diabetes treatment composition prepared by the above-described preparation method.

[0017] The present invention further protects the use of the above-mentioned diabetes treatment composition in the preparation of a medicine for treating diabetes.

[0018] The present invention has the following beneficial effects: Berberine has a good hypoglycemic effect, but its oral bioavailability is low because it tends to aggregate under acidic conditions, leading to reduced solubility. This is particularly pronounced in the acidic environment of the gastrointestinal tract, resulting in most berberine not being absorbed. Milk exosomes can effectively load berberine and can resist the effects of gastric acid to some extent. Milk exosomes are abundant in milk, easily obtained, and have high biocompatibility and low immunogenicity. Therefore, they can improve the loading capacity of berberine and also have a good promoting effect on pancreatic islet cells, thus improving drug efficacy and patient compliance.

[0019] GLP-1 analog liraglutide exerts its effect through oral administration, but its loading efficiency is low. While liraglutide exhibits good protective effects on pancreatic β-cells, this invention utilizes phycobiliproteins for loading, further optimizing liraglutide loading efficiency and significantly enhancing its protective effect on pancreatic β-cells. Phycobiliproteins are light-harvesting pigment proteins found in cyanobacteria, red algae, and some cryptophytes, possessing good antioxidant and anti-inflammatory effects. They can enhance the cellular response to oxidative stress and inflammation, thereby improving the antioxidant and anti-inflammatory capabilities of pancreatic islet cells and thus protecting them. Therefore, the liraglutide-loaded phycobiliproteins prepared in this invention not only significantly increase the loading capacity of liraglutide but also provide excellent protection for pancreatic islet cells, thereby repairing islet cell damage and promoting normal insulin secretion.

[0020] Chitosan is an abundant amino polysaccharide found in nature, characterized by its rich source, excellent biocompatibility, and low price. The numerous amino and hydroxyl groups in its molecule are easily chemically modified. This invention, through grafting chitosan with polyethylene glycol, significantly improves the acid resistance of the resulting chitosan-polyethylene glycol grafted polymer. This allows for oral administration of the drug, enabling slow drug release in the intestinal environment. This avoids the degradation and inactivation of protein drugs in the acidic environment of the stomach, thus greatly improving efficacy, patient compliance, high bioavailability, and safety (non-toxicity).

[0021] The therapeutic composition for diabetes prepared by this invention greatly increases the drug loading capacity. The carrier itself also has a good protective effect on pancreatic islet cells, thereby repairing pancreatic islet cell damage and promoting their normal insulin secretion. Moreover, the prepared composition can be administered orally, which greatly improves the efficacy and patient compliance. It has high bioavailability and is safe and non-toxic. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Milk exosomes, purity >99.9%, purchased from Shanghai Yumeibo Biotechnology Co., Ltd. *Gnaphalium affine* powder, 100 mesh, purchased from Hunan Yandi Bioengineering Co., Ltd. Chitosan, food grade, purity >99%, purchased from Shandong Guante Bioengineering Co., Ltd. EDC·HCl, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS, N-hydroxysuccinimide, commercially available.

[0024] Example 1 This embodiment provides a method for preparing a composition for treating diabetes, including the following steps: S1. Preparation of berberine-loaded exosomes: 10g of milk exosomes and 4g of berberine were dissolved in 500mL of water, stirred and mixed for 20min, dialyzed for 12h using a dialysis bag with a pore size of 3000Da, and freeze-dried to obtain berberine-loaded exosomes; S2. Preparation of phycobiliprotein: Add dried powder of *Gnaphalium affine* to 5 times its weight of water, freeze in liquid nitrogen for 1 min, thaw at room temperature for 12 h, filter, dialyze the filtrate through a dialysis bag with a pore size of 3000 Da for 12 h, freeze dry to obtain phycobiliprotein. S3. Preparation of liraglutide-loaded phycobiliprotein: 10g of phycobiliprotein and 3g of liraglutide were dissolved in 200mL of water, stirred and mixed for 20min, dialyzed for 12h using a dialysis bag with a pore size of 5000Da, and freeze-dried to obtain liraglutide-loaded phycobiliprotein. S4. Preparation of PEG-COOH: 4g of polyethylene glycol 2000 was dissolved in 200mL of dimethyl sulfoxide, 4g of NaOH and 1.5g of chloroacetic acid were added, the mixture was stirred and reacted for 10h, the solvent was removed under reduced pressure, and the mixture was washed and dried to obtain PEG-COOH; S5. Preparation of chitosan-polyethylene glycol graft polymer: 1g PEG-COOH was dissolved in 100mL water, 0.2g EDC·HCl and 0.15g NHS were added, and the mixture was stirred and activated at 0℃ for 30min. 0.5g chitosan was added, and the pH of the solution was adjusted to 3. The mixture was stirred and reacted at room temperature for 10h. The reaction solution was dialyzed with a pore size of 3000Da until the pH of the reaction solution was 7. The mixture was then freeze-dried to obtain the chitosan-polyethylene glycol graft polymer. S6. Preparation of the composition for treating diabetes: 15g of chitosan-polyethylene glycol graft polymer was added to 200mL of water, along with 2g of berberine-loaded exosomes and 4g of liraglutide-loaded phycobiliprotein to obtain a mixture. This mixture was then injected into 500mL of liquid paraffin containing 0.2g of Span-20. After stirring for 30min, 10mL of 50wt% glutaraldehyde solution was added dropwise. The cross-linking reaction was carried out for 1h. The mixture was then centrifuged, washed, and dried to obtain the composition for treating diabetes.

[0025] Example 2 This embodiment provides a method for preparing a composition for treating diabetes, including the following steps: S1. Preparation of berberine-loaded exosomes: 10g of milk exosomes and 5g of berberine were dissolved in 500mL of water and stirred for 40min. The mixture was dialyzed for 12h using a dialysis bag with a pore size of 5000Da and then freeze-dried to obtain berberine-loaded exosomes. S2. Preparation of phycobiliprotein: Add dried powder of *Gnaphalium affine* to 7 times its weight of water, freeze in liquid nitrogen for 1 min, thaw at room temperature for 12 h, filter, dialyze the filtrate through a dialysis bag with a pore size of 5000 Da for 12 h, freeze dry to obtain phycobiliprotein. S3. Preparation of liraglutide-loaded phycobiliprotein: 10g of phycobiliprotein and 4g of liraglutide were dissolved in 200mL of water and stirred for 40min. The mixture was dialyzed for 12h using a dialysis bag with a pore size of 7000Da and then freeze-dried to obtain liraglutide-loaded phycobiliprotein. S4. Preparation of PEG-COOH: 5g of polyethylene glycol 4000 was dissolved in 200mL of dimethyl sulfoxide, 5g of KOH and 2.5g of chloroacetic acid were added, the mixture was stirred and reacted for 12h, the solvent was removed under reduced pressure, and the mixture was washed and dried to obtain PEG-COOH; S5. Preparation of chitosan-polyethylene glycol graft polymer: 1.5g PEG-COOH was dissolved in 100mL water, 0.4g EDC·HCl and 0.3g NHS were added, and the mixture was stirred and activated at 4℃ for 50min. Then, 0.9g chitosan was added, the pH of the solution was adjusted to 4, and the reaction was stirred at room temperature for 14h. The reaction solution was dialyzed to pH 7 using a pore size of 5000Da, and then freeze-dried to obtain the chitosan-polyethylene glycol graft polymer. S6. Preparation of the composition for treating diabetes: 20g of chitosan-polyethylene glycol graft polymer was added to 200mL of water, along with 3g of berberine-loaded exosomes and 5g of liraglutide-loaded phycobiliprotein to obtain a mixture. This mixture was then injected into 500mL of liquid paraffin containing 0.3g of Span-60. After stirring for 30min, 10mL of 50wt% glutaraldehyde solution was added dropwise. The cross-linking reaction was carried out for 3h. The mixture was then centrifuged, washed, and dried to obtain the composition for treating diabetes.

[0026] Example 3 This embodiment provides a method for preparing a composition for treating diabetes, including the following steps: S1. Preparation of berberine-loaded exosomes: 10g of milk exosomes and 4.5g of berberine were dissolved in 500mL of water and stirred for 30min. The mixture was dialyzed for 12h using a dialysis bag with a pore size of 4000Da and then freeze-dried to obtain berberine-loaded exosomes. S2. Preparation of phycobiliprotein: Add dried powder of *Gnaphalium affine* to 6 times its weight of water, freeze in liquid nitrogen for 1 min, thaw at room temperature for 12 h, filter, dialyze the filtrate through a dialysis bag with a pore size of 4000 Da for 12 h, freeze dry to obtain phycobiliprotein; S3. Preparation of liraglutide-loaded phycobiliprotein: 10g of phycobiliprotein and 3.5g of liraglutide were dissolved in 200mL of water, stirred and mixed for 30min, dialyzed for 12h using a dialysis bag with a pore size of 6000Da, and freeze-dried to obtain liraglutide-loaded phycobiliprotein. S4. Preparation of PEG-COOH: 4.5g of polyethylene glycol 4000 was dissolved in 200mL of dimethyl sulfoxide, 4.0g of NaOH and 2.0g of chloroacetic acid were added, the mixture was stirred and reacted for 11h, the solvent was removed under reduced pressure, and the mixture was washed and dried to obtain PEG-COOH; S5. Preparation of chitosan-polyethylene glycol graft polymer: 1.2 g PEG-COOH was dissolved in 100 mL of water, 0.3 g EDC·HCl and 0.22 g NHS were added, and the mixture was stirred and activated at 2 °C for 40 min. Then, 0.7 g chitosan was added, and the pH of the solution was adjusted to 3.4. The mixture was stirred and reacted at room temperature for 12 h. The reaction solution was dialyzed with a pore size of 4000 Da until the pH of the reaction solution was 7. The mixture was then freeze-dried to obtain the chitosan-polyethylene glycol graft polymer. S6. Preparation of the composition for treating diabetes: 17g of chitosan-polyethylene glycol graft polymer was added to 200mL of water, along with 2.5g of berberine-loaded exosomes and 4.5g of liraglutide-loaded phycobiliprotein to obtain a mixture. This mixture was then injected into 500mL of liquid paraffin containing 0.25g of Span-80. After stirring for 30min, 10mL of 50wt% glutaraldehyde solution was added dropwise. The cross-linking reaction was carried out for 2h. The mixture was then centrifuged, washed, and dried to obtain the composition for treating diabetes.

[0027] Test Example 1 The loading efficiency of liraglutide loaded with phycobiliprotein in Examples 1-3 was tested using ELISA, and the loading efficiency of berberine in berberine-loaded exosomes was also tested. The results are shown in Table 1.

[0028] Control group 1: 10g of milk exosomes and 3.5g of liraglutide were dissolved in 200mL of water, stirred for 30min, dialyzed for 12h using a dialysis bag with a pore size of 6000Da, and freeze-dried to obtain liraglutide-loaded exosomes.

[0029] Control group 2: 10g of phycobiliprotein (prepared in Example 3) and 4.5g of berberine were dissolved in 500mL of water, stirred and mixed for 30min, dialyzed for 12h using a dialysis bag with a pore size of 4000Da, and freeze-dried to obtain phycobiliprotein loaded with berberine.

[0030] Loading efficiency (%) = [1 - weight of liraglutide or berberine / total weight of liraglutide or berberine] × 100% Table 1

[0031] As can be seen from the table above, the liraglutide-loaded phycobiliproteins prepared in Examples 1-3 of this invention have high loading efficiency for liraglutide, and the berberine-loaded exosomes have high loading efficiency for berberine.

[0032] Comparative Example 1 The difference from Example 3 is that the loading of berberine exosomes in step S6 is replaced by an equal amount of berberine.

[0033] Specifically as follows: S6. Preparation of the composition for treating diabetes: 17g of chitosan-polyethylene glycol graft polymer was added to 200mL of water, along with 2.5g of berberine and 4.5g of liraglutide phycobiliprotein to obtain a mixture. This mixture was then injected into 500mL of liquid paraffin containing 0.25g of Span-80. After stirring for 30min, 10mL of 50wt% glutaraldehyde solution was added dropwise. The crosslinking reaction was carried out for 2h. The mixture was then centrifuged, washed, and dried to obtain the composition for treating diabetes.

[0034] Comparative Example 2 The difference from Example 3 is that the loading of berberine exosomes in step S6 is replaced by an equal amount of milk exosomes.

[0035] Specifically as follows: S6. Preparation of the composition for treating diabetes: 17g of chitosan-polyethylene glycol graft polymer was added to 200mL of water, along with 2.5g of milk exosomes and 4.5g of liraglutide phycobiliprotein, to obtain a mixture. This mixture was then injected into 500mL of liquid paraffin containing 0.25g of Span-80. After stirring for 30min, 10mL of 50wt% glutaraldehyde solution was added dropwise. The cross-linking reaction was carried out for 2h. The mixture was then centrifuged, washed, and dried to obtain the composition for treating diabetes.

[0036] Comparative Example 3 The difference from Example 3 is that in step S6, the loading of liraglutide phycobiliprotein consists of an equal amount of liraglutide.

[0037] Specifically as follows: S6. Preparation of the composition for treating diabetes: 17g of chitosan-polyethylene glycol graft polymer was added to 200mL of water, along with 2.5g of barley alkaloid exosomes and 4.5g of liraglutide, to obtain a mixture. This mixture was then injected into 500mL of liquid paraffin containing 0.25g of Span-80. After stirring for 30min, 10mL of 50wt% glutaraldehyde solution was added dropwise. The cross-linking reaction was carried out for 2h. The mixture was then centrifuged, washed, and dried to obtain the composition for treating diabetes.

[0038] Comparative Example 4 The difference from Example 3 is that in step S6, the loading of liraglutide phycobiliprotein is replaced by an equal amount of phycobiliprotein.

[0039] Specifically as follows: S6. Preparation of the composition for treating diabetes: 17g of chitosan-polyethylene glycol graft polymer was added to 200mL of water, along with 2.5g of barley alkaloid-loaded exosomes and 4.5g of phycobiliprotein to obtain a mixture. This mixture was then injected into 500mL of liquid paraffin containing 0.25g of Span-80. After stirring for 30min, 10mL of 50wt% glutaraldehyde solution was added dropwise. The cross-linking reaction was carried out for 2h. The mixture was then centrifuged, washed, and dried to obtain the composition for treating diabetes.

[0040] Comparative Example 5 The difference from Example 3 is that steps S4-S6 were not performed.

[0041] Specifically as follows: S1. Preparation of berberine-loaded exosomes: 10g of milk exosomes and 4.5g of berberine were dissolved in 500mL of water and stirred for 30min. The mixture was dialyzed for 12h using a dialysis bag with a pore size of 4000Da and then freeze-dried to obtain berberine-loaded exosomes. S2. Preparation of phycobiliprotein: Add dried powder of *Gnaphalium affine* to 6 times its weight of water, freeze in liquid nitrogen for 1 min, thaw at room temperature for 12 h, filter, dialyze the filtrate through a dialysis bag with a pore size of 4000 Da for 12 h, freeze dry to obtain phycobiliprotein; S3. Preparation of liraglutide-loaded phycobiliprotein: 10g of phycobiliprotein and 3.5g of liraglutide were dissolved in 200mL of water and stirred for 30min. The mixture was dialyzed for 12h using a dialysis bag with a pore size of 6000Da and then freeze-dried to obtain liraglutide-loaded phycobiliprotein.

[0042] S4. Preparation of the composition for treating diabetes: 2.5g of berberine-loaded exosomes and 4.5g of liraglutide-loaded phycobiliprotein were mixed for 10 minutes to obtain the composition for treating diabetes.

[0043] Test Example 2 Model establishment: Healthy female ICR mice were selected and acclimatized for 7 days. Eight mice were randomly selected as the control group, while the rest were injected intraperitoneally with alloxan 180 mg / (kg·bw). The mice in the control group were injected with an equal volume of physiological saline. Three days later, the tails were cut off and blood was collected. Fasting blood glucose was measured using a blood glucose meter. Animals with blood glucose ≥11.1 mmol / L were used for the experiment.

[0044] Experimental methods: The animals were randomly divided into 10 groups of 8 each: a model group (saline), a positive control group (metformin, 1 mg / kg bw / d), Examples 1-3, and Comparative Examples 1-5 (correspondingly prepared diabetic treatment compositions, 1 g / kg bw / d). The control group received an equal volume of saline. All groups were administered the drugs continuously for 28 days.

[0045] Blood glucose testing methods: On days 0 and 29, mice were fasted overnight but allowed free access to water. Blood was collected from the tail vein at 8:00 AM the following morning, and fasting blood glucose levels were measured using a rapid blood glucose meter. The percentage decrease in blood glucose was calculated. The results are shown in Table 2.

[0046] Percentage decrease in blood glucose (%) = (Pre-experiment blood glucose level - Post-experiment blood glucose level) / Pre-experiment blood glucose level × 100 Table 2

[0047] As can be seen from the table above, the diabetes treatment compositions prepared in Examples 1-3 of the present invention can significantly increase the percentage reduction in blood glucose.

[0048] Organ index testing methods: On day 29 of the experiment, mice were anesthetized and euthanized. The spleen, liver, and kidneys were dissected and weighed wet. The liver, kidneys, and spleen were visually inspected for lesions. They were rinsed with cold physiological saline, dried with filter paper, weighed, and the liver, kidney, and spleen indices were calculated. The results are shown in Table 3.

[0049] The calculation formula is: Organ index (mg / g) = wet weight of organs / animal body weight Table 3

[0050] Note: * indicates P < 0.05 compared to the control group; # indicates P < 0.05 compared to the model group.

[0051] As can be seen from the table above, the diabetic treatment compositions prepared in Examples 1-3 of this invention can repair and protect organs, and have a significant effect on promoting the growth of the spleen.

[0052] Methods for testing glucose tolerance: After administering the drug to diabetic mice for 21 days, they were fasted overnight and then given the drug again at 8:00 AM the following morning. 60 minutes later, they were administered 50 mmol / kg glucose by gavage. Blood glucose levels were measured using a rapid blood glucose meter before gavage (0 min), and at 30 and 120 min after gavage. The area under the glucose tolerance curve (AUC) was calculated. The results are shown in Table 4.

[0053] AUC = (X0 + 4 × X) 30 +3×X 120 ) / 4 Where X0 represents the blood glucose level before gavage (0 min); X 30 This indicates the blood glucose level 30 minutes after gavage; X 120 This indicates the blood glucose level 120 minutes after gavage.

[0054] Table 4

[0055] As can be seen from the table above, the diabetes treatment compositions prepared in Examples 1-3 of the present invention can significantly reduce the AUC value.

[0056] Methods for detecting the antioxidant capacity of liver tissue: After organ index testing, mouse livers were cleaned to remove blood and other connective tissues, then homogenized with physiological saline, centrifuged, and the supernatant was collected. The MDA content (nmol / mgprot) and SOD activity (U / mgprot) were determined using a malondialdehyde (MDA) kit and a superoxide dismutase (SOD) kit. The results are shown in Table 5.

[0057] Table 5

[0058] Note: * indicates P < 0.05 compared to the control group; # indicates P < 0.05 compared to the model group.

[0059] As shown in the table above, the diabetes treatment compositions prepared in Examples 1-3 of this invention can significantly increase SOD content and decrease MDA content.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a composition for treating diabetes, characterized in that, Berberine was loaded onto milk exosomes to prepare berberine-loaded exosomes, and liraglutide was loaded onto phycobiliproteins to prepare liraglutide-loaded phycobiliproteins. PEG-COOH was prepared and reacted with chitosan to prepare a chitosan-polyethylene glycol graft polymer. The berberine-loaded exosomes and liraglutide-loaded phycobiliproteins were then encapsulated and cross-linked to prepare microspheres. After centrifugation, washing, and drying, a composition for treating diabetes was obtained.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Preparation of berberine-loaded exosomes: Milk exosomes and berberine were dissolved in water, stirred and mixed, dialyzed, and freeze-dried to obtain berberine-loaded exosomes; S2. Preparation of phycobiliprotein: Add dried powder of *Gnaphalium affine* to water, freeze with liquid nitrogen, thaw at room temperature, filter, dialyze the filtrate, freeze-dry to obtain phycobiliprotein; S3. Preparation of liraglutide-loaded phycobiliprotein: Phycobiliprotein and liraglutide were dissolved in water, stirred and mixed, dialyzed, and freeze-dried to obtain liraglutide-loaded phycobiliprotein; S4. Preparation of PEG-COOH: Polyethylene glycol was dissolved in dimethyl sulfoxide, alkali and chloroacetic acid were added, the mixture was stirred and reacted, the solvent was removed under reduced pressure, washed and dried to obtain PEG-COOH; S5. Preparation of chitosan-polyethylene glycol graft polymer: PEG-COOH was dissolved in water, EDC·HCl and NHS were added, the mixture was stirred and activated, chitosan was added, the pH of the solution was adjusted, the reaction was stirred at room temperature, the reaction was dialyzed until the pH of the reaction solution was 7, and the mixture was freeze-dried to obtain chitosan-polyethylene glycol graft polymer. S6. Preparation of the composition for treating diabetes: Chitosan-polyethylene glycol graft polymer was added to water, and berberine-loaded exosomes and liraglutide-loaded phycobiliprotein were added to obtain a mixture. The mixture was injected into liquid paraffin containing an emulsifier, stirred, and glutaraldehyde solution was added dropwise. The mixture underwent a cross-linking reaction, was centrifuged, washed, and dried to obtain the composition for treating diabetes.

3. The preparation method according to claim 2, characterized in that, In step S1, the mass ratio of milk exosomes to berberine is 10:4-5, the stirring time is 20-40 minutes, and the dialysis bag used for dialysis has a pore size of 3000-5000 Da.

4. The preparation method according to claim 2, characterized in that, The mass ratio of the dried powder of *Gnaphalium affine* and water in step S2 is 1:5-7.

5. The preparation method according to claim 2, characterized in that, In step S3, the mass ratio of phycobiliprotein to liraglutide is 10:3-4, the stirring time is 20-40 min, and the dialysis bag used for dialysis has a pore size of 5000-7000 Da.

6. The preparation method according to claim 2, characterized in that, In step S4, the polyethylene glycol is polyethylene glycol 2000 or polyethylene glycol 4000, the mass ratio of polyethylene glycol base to chloroacetic acid is 4-5:4-5:1.5-2.5, the stirring reaction time is 10-12 h, and the base is NaOH or KOH.

7. The preparation method according to claim 2, characterized in that, In step S5, the mass ratio of PEG-COOH, EDC·HCl, NHS, and chitosan is 1-1.5:0.2-0.4:0.15-0.3:0.5-0.

9. The stirring activation temperature is 0-4℃, and the time is 30-50 min. The pH value of the solution is adjusted to 3-4. The stirring reaction time at room temperature is 10-14 h. The dialysis bag used for dialysis has a pore size of 3000-5000 Da.

8. The preparation method according to claim 2, characterized in that, In step S6, the mass ratio of chitosan-polyethylene glycol graft polymer, barberry alkaloid exosomes, liraglutide phycobiliprotein, and emulsifier is 15-20:2-3:4-5:0.2-0.3, and the emulsifier is at least one of Span-20, Span-40, Span-60, and Span-80. The crosslinking reaction time is 1-3 hours.

9. A composition for treating diabetes prepared by the method according to any one of claims 1-8.

10. The use of the diabetes treatment composition as described in claim 9 in the preparation of a medicine for treating diabetes.

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