A combined preparation for improving microcirculation of diabetic foot and its preparation method

Abstract

The present invention belongs to the field of preparation of diabetes complication remission agents, and particularly relates to a combined preparation for improving microcirculation of diabetic foot and a preparation method thereof. Starting from platelet-rich plasma, the present invention obtains exosomes containing rich active substances such as growth factors through stimulation with calcium gluconate and the like, and loads protamine nanoparticles to obtain bioactive substances with strong antibacterial ability, which can resist the development process of diabetic foot ulcers. The present invention uses bioactive glass, hydroxyethyl cellulose and the like to stabilize the bioactive substances, prolong the in vitro lifespan of the bioactive substances, and modified carboxymethyl cellulose provides better biodegradation effect. The combined preparation prepared by the present invention has less cytotoxicity, better antibacterial effect, can promote wound healing quickly, and the microcirculation index shows that it can stimulate the change of foot blood flow and improve the condition of foot artery blockage, etc.

Description

Technical Field

[0001] The present invention belongs to the field of preparation of agents for relieving diabetic complications, and particularly relates to a combined preparation for improving microcirculation of diabetic foot and a preparation method thereof. Background Art

[0002] Diabetes is a group of metabolic diseases characterized by chronic hyperglycemia caused by genetic and environmental factors. Acute severe metabolic disorders may occur in severe cases or under stress. Long-term disorders of carbohydrate, fat, and protein metabolism can cause damage to multiple systems, leading to chronic progressive lesions, functional decline, and failure of tissues and organs such as the eyes, kidneys, nerves, heart, and blood vessels. Acute diabetic complications such as diabetic ketoacidosis, hypoglycemia, hyperglycemic hyperosmolar state, lactic acidosis, etc., and chronic diabetic complications such as macrovascular lesions: can cause coronary heart disease, ischemic or hemorrhagic cerebrovascular diseases, renal arteriosclerosis, limb arteriosclerosis, etc. Another example is microvascular lesions: mainly manifested in the retina, kidneys, nerves, and myocardial tissues, among which diabetic nephropathy and diabetic retinopathy are particularly important. Another example is neurological complications: peripheral neuropathy is the most common, and autonomic neuropathy can affect the functions of the gastrointestinal, cardiovascular, and urogenital systems, resulting in abnormal sweating, gastroparesis, diarrhea, constipation, etc. In addition, a chronic diabetic complication that needs attention is diabetic foot. In mild cases of diabetic foot, there are foot deformities, dry and cool skin, and calluses. In severe cases, foot ulcers and gangrene can occur. Diabetic foot is the main cause of disability and death in diabetic patients.

[0003] The pathogenic factors of diabetic foot are complex. The fact that it is difficult to cure, has a high disability rate, a high fatality rate, and a high recurrence rate seriously affects the health and life of diabetic patients. Among the pathogenesis of diabetic foot, ischemia, neuropathy, and infection are the three main factors leading to foot ulcers and tissue necrosis. Microcirculation disorder is one of the important physiological bases for the occurrence of diabetic peripheral neuropathy. Microcirculation disorder is manifested as changes in blood vessel structure, blood vessel dilation dysfunction, and abnormal hemorheology, which will lead to energy metabolism disorder, sorbitol accumulation, inositol depletion, and oxidative stress, etc. Obviously, improving microcirculation helps to relieve the development process of diabetic neuropathy.

[0004] Prostaglandin and prostaglandin analog preparations (prostaglandin E1, beraprost sodium), pentoxifylline, etc., as well as drugs such as pancreatinogenase and phosphodiesterase inhibitors can improve microcirculation disorder through different mechanisms, increase blood perfusion of nerve tissues, and thus reduce ischemic and hypoxic injury of nerve tissues. Some drugs will bring different degrees of side effects and increase the psychological burden of diabetic patients. Summary of the Invention

[0005] Based on the above problems, the present invention starts from platelet-rich plasma, obtains exosomes containing rich active substances such as growth factors through the stimulation of calcium gluconate, etc., and loads protamine nanoparticles, and the obtained bioactive substances have strong antibacterial ability. The present invention uses bioactive glass and hydroxyethyl cellulose, etc. to stabilize the bioactive substances and extend the in vitro lifespan of the bioactive substances. The finally prepared combined preparation has high safety, less cytotoxicity, better antibacterial effect, can promote wound healing quickly, and the microcirculation index shows that it can stimulate the change of foot blood flow and improve the condition of foot artery occlusion, etc.

[0006] A combined preparation for improving microcirculation of diabetic foot, comprising the following raw materials in parts by mass: 3-6 parts of bioactive substances, 10-15 parts of bioactive glass 45S5, 5-8 parts of bioactive glass 1393, 3-5 parts of hydroxyethyl cellulose, 6-10 parts of modified carboxymethyl cellulose, 1-3 parts of γ-aminobutyric acid, 0.8-2 parts of glycerol.

[0007] The bioactive substance is obtained by mixing and loading protamine and exosomes derived from platelet-rich plasma. The specific preparation method is as follows:

[0008] M1. Dissolve protamine in distilled water to obtain a protamine aqueous solution with a mass percentage concentration of 1-1.5%, heat it to 40-50 °C after standing overnight, add absolute ethanol, continuously stir at a speed of 150-200 rpm at room temperature for 15-20 h, after completion, remove absolute ethanol by vacuum distillation to obtain Liquid 1, add glutaraldehyde aqueous solution, stir and disperse, then stand for 5-10 min, centrifuge at high speed to obtain a precipitate, wash the precipitate thoroughly with PBS buffer solution, and centrifuge at high speed again to obtain the precipitate, which is Substance 1;

[0009] M2. Obtain platelet-rich plasma, mix the platelet-rich plasma with calcium gluconate solution, incubate in a 37 °C water bath for 10-15 min, after completion, obtain exosomes by ultracentrifugation method, resuspend the exosomes with PBS buffer solution, and then mix with phosphatase and silk fibroin to obtain Substance 2;

[0010] M3. Mix the Substance 1 obtained in step M1 with the Substance 2 obtained in step M2 according to a mass ratio of 1:8-10, pre-cool thoroughly at 4 °C, perform electroporation at 1000-1200 kV for 5-8 ms, and after completion, incubate at 37 °C for 30-45 min to obtain the bioactive substance.

[0011] Preferably, the addition amount of absolute ethanol in step M1 is 0.2 - 0.3 times the volume of the protamine aqueous solution, the addition amount of the glutaraldehyde aqueous solution is 0.1 - 0.2 times the volume of liquid 1, the mass percentage concentration of the glutaraldehyde aqueous solution is preferably 3 - 4%, the high-speed centrifugation is carried out at 4 °C, and the centrifugation is preferably carried out at a centrifugal force of 8000 - 10000 g.

[0012] Preferably, in step M2, the volume ratio of platelet-rich plasma to calcium gluconate solution is 10:1 - 1.5, the mass percentage concentration of the calcium gluconate solution is preferably 6 - 8%, and the mass ratio of exosomes: PBS buffer: phosphatase: silk fibroin is 1:400 - 600:80 - 120:50 - 80.

[0013] Preferably, the pH of the PBS buffer solution used in steps M1 and M2 is 7.0, and it needs to be pre-cooled at 4 °C before use.

[0014] The preparation method of the modified carboxymethyl cellulose is as follows:

[0015] L1. Add carboxymethyl cellulose to pyridine, stir until evenly dispersed, add chlorobetaine acyl chloride, and stir at a speed of 120 - 180 rpm at room temperature for 24 - 36 h to obtain substance 3;

[0016] L2. Precipitate the substance 3 obtained in step L1 with acetone, wash the precipitate thoroughly with absolute ethanol, and obtain the modified carboxymethyl cellulose after drying under reduced pressure to constant weight.

[0017] Preferably, in step L1, the mass-volume ratio of carboxymethyl cellulose: pyrrole: chlorobetaine acyl chloride is 1:50 - 60:0.2 - 0.3.

[0018] Preferably, in step L2, the amounts of acetone and absolute ethanol are not limited and can be used in conventional amounts, and the drying under reduced pressure is preferably carried out at 55 - 60 °C.

[0019] The preparation method of the combined preparation for improving microcirculation of diabetic foot is as follows:

[0020] V1. Weigh hydroxyethyl cellulose and dissolve it in water, mix well, add bioactive glass 45S5 and bioactive glass 1393, and stir until evenly dispersed to obtain component 1;

[0021] V2. Weigh γ-aminobutyric acid, glycerol, and modified carboxymethyl cellulose, add water and stir until evenly dispersed, and then add bioactive substances to obtain component 2;

[0022] V3. Slowly add the component 2 obtained in step V2 to the component 1 obtained in step V1 to obtain the combined preparation.

[0023] Preferably, the amount of water added in step V1 is 20 - 30 times the mass of hydroxyethyl cellulose, and the amount of water added in step V2 is 10 - 20 times the modified carboxymethyl cellulose.

[0024] The beneficial effects of the present invention are as follows:

[0025] Existing studies have shown that platelet - rich plasma has a better effect on relieving wound ulcers. The advantages are that after platelet activation, through degranulation, it releases various nutritional components such as platelet - derived growth factor, vascular endothelial growth factor, transforming growth factor, epidermal growth factor, basic fibroblast growth factor, fibronectin, etc. These nutritional components can promote ulcer healing and blood vessel regeneration, etc. In addition, after platelet activation, it also releases various antibacterial active substances such as antimicrobial peptides, chemotactic factors, and bactericidal proteins, having a certain anti - infection ability. Platelet - rich plasma derived from the body can also avoid immune rejection caused by the use of exogenous substances and does not bring serious adverse reactions. However, platelet - rich plasma also has certain application limitations. For example, its treatment effect is better in patients with neuropathic diabetic foot with better local blood supply than in patients with ischemic ulcers, and its in - vitro lifespan is relatively short, etc. In view of the application advantages of platelet - rich plasma, on this basis, the present invention uses calcium gluconate as an agonist to stimulate platelet - rich plasma to release a large amount of growth factors and other active substances, and then further obtains its exosomes. Phosphatase and silk fibroin are used to stabilize the exosomes and extend the existence time of the active substances. In addition, the present invention specifically makes protamine into nanoparticles and loads them into exosomes by electroporation, so that the finally obtained bioactive substances have strong antibacterial ability and can resist the development process of diabetic foot ulcers. Bioactive glass and hydroxyethyl cellulose, etc. can extend the in - vitro lifespan of bioactive substances and can promote the bioactive substances to exert their therapeutic effects. Modified carboxymethyl cellulose has better biodegradability than unmodified carboxymethyl cellulose, making the overall combined preparation safer. The combined preparation obtained by the present invention has less cytotoxicity, better antibacterial effect, can promote wound healing quickly, and microcirculation indicators show that it can stimulate changes in foot blood flow and improve the condition of foot artery occlusion, etc. Brief Description of the Drawings

[0026] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation of the present application. In the drawings:

[0027] Figure 1 It is a graph showing the effect of the combined preparation on cell viability;

[0028] Figure 2 It is a test chart of the antibacterial effect of the combined preparation;

[0029] Figure 3 It is a graph of the experimental results for evaluating diabetic wound healing;

[0030] Figure 4 It is a graph of the change rate of blood perfusion during treatment;

[0031] Figure 5 It is a graph of the ankle-brachial index before and after treatment. Specific implementation mode

[0032] In order to more clearly explain the overall concept of this application, the following will be described in detail by way of examples in conjunction with the accompanying drawings of the specification. In the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some well-known technical features in the art are not described.

[0033] Example 1: This example provides a preparation method of a bioactive substance, and the specific content is as follows:

[0034] M1. Dissolve protamine in distilled water to obtain a protamine aqueous solution with a mass percentage concentration of 1%. After standing overnight (10 h), heat it to 40 °C, add anhydrous ethanol with a volume 0.2 times that of the protamine aqueous solution, and continuously stir at a speed of 150 rpm at room temperature for 15 h. After completion, remove the anhydrous ethanol by vacuum distillation to obtain Liquid 1. Add an aqueous glutaraldehyde solution with a mass percentage concentration of 3% and a volume 0.1 times that of Liquid 1, stir and disperse, then stand for 5 min, and centrifuge at a high speed of 8000 g at 4 °C to obtain a precipitate. The precipitate is thoroughly washed with a PBS buffer solution pre-cooled to 4 °C and with a pH of 7.0, and after high-speed centrifugation again, the obtained precipitate is Substance 1;

[0035] M2. Obtain platelet-rich plasma, weigh platelet-rich plasma and an aqueous calcium gluconate solution with a mass percentage concentration of 6% according to a volume ratio of 10:1, mix the two, incubate in a 37 °C water bath for 10 min, and after completion, obtain exosomes by ultracentrifugation. The exosomes are resuspended with a PBS buffer solution pre-cooled to 4 °C and with a pH of 7.0, and then mixed with phosphatase and silk fibroin. The mass ratio of exosomes:PBS buffer solution:phosphatase:silk fibroin is 1:400:80:50, and after completion, Substance 2 is obtained;

[0036] M3. Mix Substance 1 obtained in step M1 and Substance 2 obtained in step M2 according to a mass ratio of 1:8, and fully pre-cool at 4 °C (24 h). Place it in an electroporation cuvette pre-cooled to the same 4 °C, and perform electroporation at 1000 kV for 8 ms using an electroporator. After completion, incubate at 37 °C for 30 min to obtain the bioactive substance.

[0037] Example 2: This example provides a method for preparing a bioactive substance, and the specific content is as follows:

[0038] M1. Dissolve protamine in distilled water to obtain a protamine aqueous solution with a mass percentage concentration of 1.2%. After standing overnight (10 h), heat it to 45 °C, add anhydrous ethanol with a volume 0.25 times that of the protamine aqueous solution, and continuously stir at a speed of 180 rpm at room temperature for 17 h. After completion, remove the anhydrous ethanol by vacuum distillation to obtain Liquid 1. Add an aqueous glutaraldehyde solution with a mass percentage concentration of 3.5% and a volume 0.15 times that of Liquid 1. After stirring and dispersing, let it stand for 8 min, and centrifuge at a high speed with a centrifugal force of 9000 g at 4 °C to obtain a precipitate. The precipitate is thoroughly washed with a PBS buffer solution pre-cooled to 4 °C and with a pH of 7.0. After centrifuging at a high speed again, the obtained precipitate is Substance 1;

[0039] M2. Obtain platelet-rich plasma. Weigh platelet-rich plasma and an aqueous calcium gluconate solution with a mass percentage concentration of 7% according to a volume ratio of 10:1.2. Mix the two, incubate in a 37 °C water bath for 12 min. After completion, obtain exosomes by ultracentrifugation. The exosomes are resuspended with a PBS buffer solution pre-cooled to 4 °C and with a pH of 7.0, and then mixed with phosphatase and silk fibroin. The mass ratio of exosomes:PBS buffer solution:phosphatase:silk fibroin is 1:500:100:60. After completion, obtain Substance 2;

[0040] M3. Mix Substance 1 obtained in step M1 and Substance 2 obtained in step M2 according to a mass ratio of 1:9, and pre-cool thoroughly at 4 °C (24 h). Place it in an electroporation cuvette pre-cooled to the same 4 °C, and perform electroporation at 1100 kV for 6 ms using an electroporator. After completion, incubate at 37 °C for 38 min to obtain the bioactive substance.

[0041] Example 3: This example provides a method for preparing a bioactive substance, and the specific content is as follows:

[0042] M1. Dissolve protamine in distilled water to obtain a protamine aqueous solution with a mass percentage concentration of 1.5%. After standing overnight (10 h), heat it to 50 °C, add anhydrous ethanol with a volume 0.3 times that of the protamine aqueous solution, and continuously stir at a speed of 200 rpm at room temperature for 20 h. After completion, remove the anhydrous ethanol by vacuum distillation to obtain Liquid 1. Add an aqueous glutaraldehyde solution with a mass percentage concentration of 4% and a volume 0.2 times that of Liquid 1. After stirring and dispersing, let it stand for 10 min, and centrifuge at a high speed with a centrifugal force of 10000 g at 4 °C to obtain a precipitate. The precipitate is thoroughly washed with a PBS buffer solution pre-cooled to 4 °C and with a pH of 7.0. After centrifuging at a high speed again, the obtained precipitate is Substance 1;

[0043] M2. Obtain platelet-rich plasma. Weigh platelet-rich plasma and calcium gluconate solution with a mass percentage concentration of 8% according to a volume ratio of 10:1.5. Mix the two, incubate at 37 °C in a water bath for 15 min. After that, obtain exosomes by ultracentrifugation. The exosomes are resuspended with pre-cooled PBS buffer at 4 °C with a pH of 7.0, and then mixed with phosphatase and silk fibroin. The mass ratio of exosomes:PBS buffer:phosphatase:silk fibroin is 1:600:120:80. After that, obtain Substance 2;

[0044] M3. Mix Substance 1 obtained in step M1 and Substance 2 obtained in step M2 according to a mass ratio of 1:10, and fully pre-cool at 4 °C (24 h). Place it in an electroporation cuvette pre-cooled at the same 4 °C, and perform electroporation at 1200 kV for 5 ms using an electroporator. After that, incubate at 37 °C for 45 min to obtain the bioactive substance.

[0045] In addition, when the source of platelet-rich plasma is human, the acquisition method is as follows:

[0046] Collect 10 ml of venous blood, add it to an anticoagulant test tube and mix evenly. Centrifuge at 2500 rpm for 10 min. After centrifugation, the sample is divided into three layers. The top layer is plasma, the middle layer is concentrated platelets, and the bottom layer is red blood cells. Remove the top layer plasma and the bottom layer red blood cell layer. Take the middle layer and centrifuge again at 2200 rpm for 10 min. Discard part of the supernatant, and the remaining part is platelet-rich plasma.

[0047] When the source of platelet-rich plasma is rat, the middle layer centrifugation should be carried out at 2000 rpm for 10 min to obtain platelet-rich plasma.

[0048] In Examples 1-3, the specific steps of the ultracentrifugation method in step M2 are as follows:

[0049] Centrifuge at a centrifugal force of 300 g for 10 min, centrifuge at a centrifugal force of 2000 g for 10 min, centrifuge at a centrifugal force of 10000 g for 30 min, and ultracentrifuge at a centrifugal force of 150000 g for 65 min.

[0050] Example 4: This example provides a preparation method of a combined preparation for improving microcirculation of diabetic foot, and the specific content is as follows:

[0051] In this example, the combined preparation for improving microcirculation of diabetic foot contains the following raw materials in parts by mass: 3 parts of bioactive substance, 10 parts of bioactive glass 45S5, 5 parts of bioactive glass 1393, 3 parts of hydroxyethyl cellulose, 6 parts of modified carboxymethyl cellulose, 1 part of γ-aminobutyric acid, 0.8 part of glycerol. The bioactive substance involved comes from Example 1. The preparation method of the modified carboxymethyl cellulose involved is as follows:

[0052] L1. Weigh carboxymethyl cellulose, pyrrole and betaine chloride acyl chloride according to the mass-volume ratio of 1 g: 50 mL: 0.2 g. Add carboxymethyl cellulose to pyridine and stir until evenly dispersed. Then add betaine chloride acyl chloride and stir at a speed of 120 rpm at room temperature for 24 h to obtain Substance 3.

[0053] L2. Precipitate the Substance 3 obtained in Step L1 with acetone. Wash the precipitate thoroughly with absolute ethanol and dry it under reduced pressure at 55 °C until constant weight to obtain modified carboxymethyl cellulose.

[0054] The preparation steps of the combined preparation are as follows:

[0055] V1. Weigh hydroxyethyl cellulose and dissolve it in water. The amount of water added is 20 times the mass of hydroxyethyl cellulose. Mix well and add bioactive glass 45S5 and bioactive glass 1393, and stir until evenly dispersed to obtain Component 1.

[0056] V2. Weigh γ-aminobutyric acid, glycerol and modified carboxymethyl cellulose, add water and stir until evenly dispersed. The amount of water added is 10 times the mass of modified carboxymethyl cellulose. Then add bioactive substances to obtain Component 2.

[0057] V3. Slowly add Component 2 obtained in Step V2 to Component 1 obtained in Step V1 to obtain the combined preparation.

[0058] Example 5: This example provides a preparation method of a combined preparation for improving microcirculation of diabetic foot, and the specific content is as follows:

[0059] The combined preparation for improving microcirculation of diabetic foot in this example contains the following raw materials in parts by mass: 4 parts of bioactive substances, 12 parts of bioactive glass 45S5, 6 parts of bioactive glass 1393, 4 parts of hydroxyethyl cellulose, 7 parts of modified carboxymethyl cellulose, 2 parts of γ-aminobutyric acid, and 1.3 parts of glycerol. The bioactive substances involved come from Example 2, and the preparation method of the modified carboxymethyl cellulose involved is as follows:

[0060] L1. Weigh carboxymethyl cellulose, pyrrole and betaine chloride acyl chloride according to the mass-volume ratio of 1 g: 55 mL: 0.25 g. Add carboxymethyl cellulose to pyridine and stir until evenly dispersed. Then add betaine chloride acyl chloride and stir at a speed of 140 rpm at room temperature for 30 h to obtain Substance 3.

[0061] L2. Precipitate the Substance 3 obtained in Step L1 with acetone. Wash the precipitate thoroughly with absolute ethanol and dry it under reduced pressure at 58 °C until constant weight to obtain modified carboxymethyl cellulose.

[0062] The preparation steps of the combined preparation are as follows:

[0063] V1. Weigh hydroxyethyl cellulose and dissolve it in water. The amount of water added is 24 times the mass of hydroxyethyl cellulose. Mix well, add bioactive glass 45S5 and bioactive glass 1393, and stir until evenly dispersed to obtain Component 1;

[0064] V2. Weigh γ-aminobutyric acid, glycerol, and modified carboxymethyl cellulose. After adding water, stir until evenly dispersed. The amount of water added is 16 times that of the modified carboxymethyl cellulose. Add bioactive substances to obtain Component 2;

[0065] V3. Slowly add Component 2 obtained in Step V2 to Component 1 obtained in Step V1 to obtain the combined preparation.

[0066] Example 6: This example provides a method for preparing a combined preparation for improving microcirculation in diabetic foot, and the specific content is as follows:

[0067] The combined preparation for improving microcirculation in diabetic foot in this example contains the following raw materials in parts by mass: 6 parts of bioactive substances, 15 parts of bioactive glass 45S5, 8 parts of bioactive glass 1393, 5 parts of hydroxyethyl cellulose, 10 parts of carboxymethyl cellulose, 3 parts of γ-aminobutyric acid, and 2 parts of glycerol. The bioactive substances involved come from Example 3. The preparation method of the modified carboxymethyl cellulose involved is as follows:

[0068] L1. Weigh carboxymethyl cellulose, pyrrole, and betaine chloride in a mass-to-volume ratio of 1 g: 60 mL: 0.3 g. Add carboxymethyl cellulose to pyridine and stir until evenly dispersed. Then add betaine chloride and stir at a speed of 180 rpm at room temperature for 36 h to obtain Substance 3;

[0069] L2. Precipitate Substance 3 obtained in Step L1 with acetone. Wash the precipitate thoroughly with absolute ethanol and dry it under reduced pressure at 60 °C until a constant weight is obtained to obtain the modified carboxymethyl cellulose;

[0070] The preparation steps of the combined preparation are as follows:

[0071] V1. Weigh hydroxyethyl cellulose and dissolve it in water. The amount of water added is 30 times the mass of hydroxyethyl cellulose. Mix well, add bioactive glass 45S5 and bioactive glass 1393, and stir until evenly dispersed to obtain Component 1;

[0072] V2. Weigh γ-aminobutyric acid, glycerol, and modified carboxymethyl cellulose. After adding water, stir until evenly dispersed. The amount of water added is 20 times that of the modified carboxymethyl cellulose. Add bioactive substances to obtain Component 2;

[0073] V3. Slowly add Component 2 obtained in Step V2 to Component 1 obtained in Step V1 to obtain the combined preparation.

[0074] Comparative Example 1: This comparative example provides a combined preparation, and the specific content is as follows:

[0075] The difference between the combined preparation of this comparative example and the combined preparation obtained in Example 5 is that the combined preparation of this comparative example does not contain bioactive substances, and other raw materials and preparation steps are the same as those in Example 5.

[0076] Comparative Example 2: This comparative example provides a combined preparation, and the specific content is as follows:

[0077] The difference between the combined preparation of this comparative example and the combined preparation obtained in Example 5 is that sodium carboxymethylcellulose is used to replace the modified sodium carboxymethylcellulose in the combined preparation of this comparative example, and other raw materials and preparation steps are the same as those in Example 5.

[0078] Comparative Example 3: This comparative example provides a combined preparation, and the specific content is as follows:

[0079] The difference between the combined preparation of this comparative example and the combined preparation obtained in Example 5 is that the combined preparation of this comparative example does not contain γ-aminobutyric acid, and other raw materials and preparation steps are the same as those in Example 5.

[0080] Comparative Example 4: This comparative example provides a combined preparation, and the specific content is as follows:

[0081] The difference between the combined preparation of this comparative example and the combined preparation obtained in Example 5 is that the combined preparation of this comparative example only contains Component 2.

[0082] Comparative Example 5: This comparative example provides a combined preparation, and the specific content is as follows:

[0083] The difference between the combined preparation of this comparative example and the combined preparation obtained in Example 5 is that the combined preparation of this comparative example uses platelet-rich plasma of the same quality and source, mixes the platelet-rich plasma and hyaluronic acid according to a mass ratio of 1:3, and stores the obtained thick mixture at 4°C to obtain the combined preparation of this comparative example.

[0084] Experimental test:

[0085] I. Experiment on the effect of the combined preparation on cell viability:

[0086] Add 100 μL of L929 cell suspension (density of 1×10 4Cells / mL) were seeded into the wells of a 96-well plate. After culturing for 24 h, the combined preparations prepared in Examples 4-6 and Comparative Examples 1-5 (pre-shaped into circular sheets with a diameter of 5 mm and a thickness of 1 mm) were placed into the wells, and then co-cultured for another 24 h. Then, the combined preparations were removed, and the wells were washed three times with PBS buffer. A medium containing 10% CCK-8 solution was added to each well, and the 96-well plate was placed in an incubator and incubated at 37 °C and 5% CO 2 for 1 h. Subsequently, the cell viability was measured with a microplate reader at an absorbance of 450 nm, and then the cell viability was calculated according to the following formula:

[0087] Cell viability = (OD1 - OD0) / (OD2 - OD0) × 100%;

[0088] where OD1 is the OD value measured in the experimental group, that is, the combined preparation obtained in Examples 4-6, OD0 is the OD value in the blank group, the blank group is the CCK-8 solution without the combined preparation, and OD2 is the OD value measured after no treatment.

[0089] The results are as Figure 1 shown, and it can be seen from Figure 1 that the combined preparations prepared in Examples 4-6 and Comparative Examples 1-5 have little effect on cell viability.

[0090] II. Antibacterial effect test of the combined preparation:

[0091] The combined preparations obtained in Examples 4-6 and Comparative Examples 1-5 were tested. 150 μL of the combined preparation was incubated with 5 mL of Staphylococcus aureus suspension at 37 °C for 24 h. The concentration of the Staphylococcus aureus suspension was 1×10 6 cfu / mL, and the control group was not treated with the combined preparation. After incubation, 0.1 mL of the bacterial suspension was pipetted onto the LB medium, evenly spread, incubated at 37 °C, and the contamination situation was photographed and recorded after culturing for 24 h.

[0092] The results are as Figure 2 shown, and it can be seen from Figure 2 that the combined preparation obtained in Comparative Example 1 has a poor inhibitory effect on Staphylococcus aureus, followed by the combined preparation obtained in Comparative Example 4. The combined preparations used in other experimental groups have good inhibitory effects on Staphylococcus aureus at the experimental doses used. The experiment confirmed that the bioactive substances play an important role in the antibacterial effect of the combined preparation, and the combined preparation has a better effect after mixing. Component 2 alone cannot completely inhibit the growth of Staphylococcus aureus.

[0093] III. Diabetic wound healing evaluation experiment:

[0094] One hundred SPF - level healthy SD rats with body weights in the range of 230 - 270 g were used. One week before the start of the experiment, they were adaptively fed. The breeding temperature was 20 ± 3°C, and the relative humidity was 55 ± 10%. They had free access to food and water. Twelve hours before modeling, the rats were fasted. Each rat was given a 1% STZ solution (prepared with citrate buffer) at a dose of 50 mg / kg. After 30 minutes of injection, the rats' food and water intake were no longer restricted. Seventy - two hours after injection, tail vein blood was taken to measure blood glucose. A blood glucose level ≥ 16.7 mM indicated successful modeling. Rats with unsuccessful modeling did not participate in the experiment.

[0095] A circle with a diameter of 2 cm was cut on the back of the rats to obtain skin injuries. The combined preparations obtained in Examples 4 - 6 and Comparative Examples 1 - 5 were applied to the skin injuries twice a day for four weeks. The control group was treated with a phosphate - buffered solution with pH = 7.0. The diameter of the skin injury was measured to confirm the wound - healing effect.

[0096] The results are as Figure 3 shown. As Figure 3 can be seen, the combined preparations prepared in Examples 4 - 6 had a good effect on promoting wound healing. The wound - healing rate reached 93% after 28 days. Compared with the control group, the combined preparations prepared in Comparative Examples 1 - 5 could also promote wound healing. The effects of the combined preparations obtained in Comparative Example 2 and Comparative Example 3 were better than those of Comparative Example 5.

[0097] IV. Clinical application experiment:

[0098] The combined preparations prepared in Examples 4 - 6 were used for patients with diabetic foot. The patients met the diagnostic criteria for diabetic foot, had clinical manifestations of lower - limb ischemia, and the Wagner grade was 1 - 4, that is, there were ulcers of varying degrees and signs of infection of varying degrees. The patients had no verified heart, liver, or kidney diseases, normal blood - coagulation function, and were non - pregnant and non - lactating women. Twelve patients participated, with the youngest age of 38 years and the oldest age of 56 years. The control group was given conventional treatment. In addition to daily medications, anti - infective drugs were intravenously infused after foot debridement, and the wounds were nursed routinely.

[0099] During the clinical application experiment, indicators such as pro - inflammatory cytokines, blood glucose, and blood perfusion volume of the patients were detected, as well as the wound - recovery situation (whether the wound area was reduced, the wound exudation situation, and the granulation growth situation, etc.). The following shows the detection methods and results of blood perfusion volume and ankle - brachial index:

[0100] 1) The PeriFlux 5000 laser Doppler flowmeter (Perimed) was used to detect the blood perfusion volume. The patient was in the supine position at room temperature. After lying quietly for 15 minutes, the dorsum of the patient's foot was taken as the detection site, and the blood perfusion volume at 2 cm above the third metatarsophalangeal joint was detected. Avoid the sites with large blood vessels, more hair, and skin damage. After slightly cleaning, the detection was carried out, and the basic blood perfusion volume was recorded as PU0, and the blood perfusion volume after heating to 45 °C was recorded as PU1. The change rate of blood perfusion volume = (PU1 - PU0) / PU0 × 100%.

[0101] The results of the blood perfusion volume are as Figure 4 shown. It can be seen from Figure 4 that during the 28-day treatment period, compared with the control group, after the application of the combined preparations prepared in Examples 4-6, the change rate of the patient's blood perfusion volume was significantly increased, proving that the microcirculation disorder of diabetic foot patients was significantly improved.

[0102] 2) The patient was in the supine position, and a Doppler ultrasound diagnostic instrument was used to detect the systolic blood pressure of the ankle (dorsalis pedis artery or posterior tibial artery) and the upper arm. The ankle-brachial index = the highest value of the systolic blood pressure of the posterior tibial artery or dorsalis pedis artery / the highest value of the systolic blood pressure of both arms.

[0103] The results of the ankle-brachial index are as Figure 5 shown. It can be seen from Figure 5 that after 28 days of treatment, the ankle-brachial index of the patient was significantly increased, proving that compared with the control group, after using the combined preparations prepared in Examples 4-6, the arterial occlusion condition of the patient's foot was improved.

[0104] The above are only the embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A combined preparation for improving diabetic foot microcirculation, characterized in that: The combined preparation comprises the following raw materials in parts by mass: 3-6 parts of biologically active substances, 10-15 parts of bioactive glass 45S5, 5-8 parts of bioactive glass 1393, 3-5 parts of hydroxyethyl cellulose, 6-10 parts of modified carboxymethyl cellulose, 1-3 parts of gamma-aminobutyric acid and 0.8-2 parts of glycerol; The preparation method of the biologically active substance is as follows: M1. Dissolve protamine in distilled water to obtain a protamine aqueous solution with a mass percentage concentration of 1-1.5%, let it stand overnight and then heat it to 40-50°C, add anhydrous ethanol, and continue stirring at room temperature. After the end, remove the anhydrous ethanol by vacuum distillation to obtain liquid 1, add glutaraldehyde aqueous solution, stir and disperse, let it stand, and obtain a precipitate by high-speed centrifugation. The precipitate is fully washed with PBS buffer, and the precipitate obtained after high-speed centrifugation again is substance 1; M2. Obtain platelet-rich plasma, mix the platelet-rich plasma with calcium gluconate solution, incubate in a 37°C water bath, and then obtain exosomes by ultracentrifugation. The exosomes are resuspended in PBS buffer and then mixed with phosphatase and silk fibroin to obtain substance 2; M3. The substance 1 obtained in step M1 and the substance 2 obtained in step M2 are mixed in a mass ratio of 1:8-10, fully precooled at 4°C, electroporated at 1000-1200 kV, and incubated at 37°C to obtain the bioactive substance; The preparation method of the modified carboxymethyl cellulose is as follows: L1. Add carboxymethyl cellulose to pyridine, stir until uniformly dispersed, add betaine chlorochloride, stir at room temperature to obtain substance 3; L2. The substance 3 obtained in step L1 was precipitated with acetone, the precipitate was fully washed with anhydrous ethanol, and dried under reduced pressure to constant weight to obtain modified carboxymethyl cellulose; In step L1, the mass volume ratio of carboxymethyl cellulose:pyrrole:betaine chloride is 1:50-60:0.2-0.

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2. The combined preparation for improving diabetic foot microcirculation according to claim 1, characterized in that: In step M1, the amount of anhydrous ethanol added is 0.2-0.3 times the volume of the protamine aqueous solution, and the amount of glutaraldehyde aqueous solution added is 0.1-0.2 times the volume of liquid 1; In step M2, the volume ratio of platelet-rich plasma: calcium gluconate solution is 10:1-1.5, and the mass ratio of exosomes: PBS buffer: phosphatase: silk fibroin is 1:400-600:80-120:50-80.

3. A method for preparing the combined preparation for improving diabetic foot microcirculation according to claim 1 or 2, characterized in that: The specific steps are as follows: V1. Weigh hydroxyethyl cellulose and dissolve it in water, mix thoroughly, add bioactive glass 45S5 and bioactive glass 1393, stir until evenly dispersed, and obtain component 1; V2. Weigh γ-aminobutyric acid, glycerol, and modified carboxymethyl cellulose, add water and stir until uniformly dispersed, add bioactive substances, and obtain component 2; V3. Slowly adding component 2 obtained in step V2 to component 1 obtained in step V1 to obtain a combined preparation; The amount of water added in step V1 is 20-30 times the mass of hydroxyethyl cellulose, and the amount of water added in step V2 is 10-20 times the mass of modified carboxymethyl cellulose.

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