Combined preparation for improving microcirculation of diabetic foot and preparation method thereof

Abstract

The invention belongs to the field of preparation of diabetic complication relieving preparations, and particularly relates to a combined preparation for improving diabetic foot microcirculation and a preparation method of the combined preparation. The exosome containing rich growth factors and other active substances is obtained through stimulation of calcium gluconate and the like by taking platelet-rich plasma as a starting point, protamine nanoparticles are loaded, and the obtained bioactive substance has relatively strong antibacterial ability and can resist the development process of diabetic foot ulcer. According to the invention, bioactive glass, hydroxyethyl cellulose and the like are adopted to stabilize bioactive substances, the in-vitro life of the bioactive substances is prolonged, and modified carboxymethyl cellulose provides a better biodegradation effect. The prepared combined preparation is small in cell toxicity, good in antibacterial effect and capable of rapidly promoting wound healing, and microcirculation indexes show that the combined preparation can stimulate foot blood flow volume changes, improve foot artery blockage conditions and the like.

Description

Technical Field

[0001] The invention belongs to the field of preparation of diabetic complication alleviating preparations, and specifically relates to a combined preparation for improving diabetic foot microcirculation 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 and severe metabolic disorders may occur when the condition is severe or under stress. Long-term metabolic disorders of carbohydrates, fats and proteins can cause multi-system damage, leading to chronic progressive lesions, functional impairment and failure of tissues and organs such as the eyes, kidneys, nerves, heart and blood vessels. Acute complications of diabetes include diabetic ketoacidosis, hypoglycemia, hyperosmolar hyperglycemic state, lactic acidosis, etc. Chronic complications of diabetes include macrovascular disease: can cause coronary heart disease, ischemic or hemorrhagic cerebrovascular disease, renal artery sclerosis, limb arteriosclerosis, etc.; microvascular disease: mainly manifests in the retina, kidney, nerve and myocardial tissue, among which diabetic nephropathy and diabetic retinopathy are particularly important; neurological complications: peripheral neuropathy is the most common; autonomic neuropathy can affect the gastrointestinal, cardiovascular, and urogenital system functions, resulting in abnormal sweating, gastroparesis, diarrhea, constipation, etc.; another chronic complication of diabetes that needs attention is diabetic foot. In mild cases, diabetic foot causes foot deformities, dry and cold skin, and calluses; in severe cases, foot ulcers and gangrene may occur. Diabetic foot is the main cause of disability and death in diabetic patients.

[0003] The pathogenic factors of diabetic foot are complex, difficult to cure, high disability, high mortality and recurrence rate seriously affect the health and life of diabetic patients. In the pathogenesis of diabetic foot, ischemia, neuropathy and infection are the three main factors leading to foot ulcers and tissue necrosis. Microcirculatory disorder is one of the important physiological bases for the occurrence of diabetic peripheral neuropathy. Microcirculatory disorder manifests as changes in vascular structure, vasodilation dysfunction, and abnormal blood rheology, which can lead to energy metabolism disorders, sorbitol accumulation and inositol depletion, and oxidative stress. Obviously, improving microcirculation can help alleviate the development of diabetic neuropathy.

[0004] Prostaglandins and prostate analogs (prostaglandin E1, beraprost sodium), pentoxifylline, and drugs such as pancreatic kinase and phosphodiesterase inhibitors can improve microcirculatory disorders through different mechanisms, increase blood perfusion of nerve tissue, and thus reduce ischemic and hypoxic damage to nerve tissue. Some drugs will bring side effects of varying degrees, increasing the psychological burden of diabetic patients. Summary of the invention

[0005] Based on the above problems, the present invention takes platelet-rich plasma as the starting point, obtains exosomes containing active substances such as rich growth factors through stimulation by 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 to stabilize the bioactive substances and extend the in vitro life of the bioactive substances. The final prepared combination preparation has high safety, less cytotoxicity, better antibacterial effect, can promote wound healing faster, and microcirculation indicators show that it can stimulate changes in foot blood flow and improve foot artery blockage.

[0006] A combined preparation for improving diabetic foot microcirculation comprises 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 gamma-aminobutyric acid, and 0.8-2 parts of glycerol.

[0007] The bioactive substance is obtained by mixing protamine with exosomes derived from platelet-rich plasma, and the specific preparation method 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 a speed of 150-200 rpm at room temperature for 15-20 hours. After the end, remove the anhydrous ethanol by vacuum distillation to obtain liquid 1, add glutaraldehyde aqueous solution, stir and disperse, let it stand for 5-10 minutes, 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 for 10-15 minutes, 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. Mix substance 1 obtained in step M1 and substance 2 obtained in step M2 in a mass ratio of 1:8-10, fully precool at 4°C, perform electroporation at 1000-1200 kV for 5-8 ms, and then incubate at 37°C for 30-45 min to obtain the bioactive substance.

[0008] Preferably, the amount of anhydrous ethanol added in step M1 is 0.2-0.3 times the volume of the protamine aqueous solution, the amount of glutaraldehyde aqueous solution added is 0.1-0.2 times the volume of liquid 1, the mass percentage concentration of the glutaraldehyde aqueous solution is preferably 3-4%, and the high-speed centrifugation is carried out at 4°C, and the centrifugation is preferably carried out at a centrifugal force of 8000-10000g.

[0009] Preferably, in step M2, the volume ratio of platelet-rich plasma: calcium gluconate solution is 10:1-1.5, the mass percentage concentration of 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.

[0010] Preferably, the pH of the PBS buffer solution used in steps M1 and M2 is 7.0, and both need to be pre-cooled at 4° C. before use.

[0011] The preparation method of the modified carboxymethyl cellulose is as follows: L1. Add carboxymethyl cellulose to pyridine, stir until uniformly dispersed, add betaine chlorochloride, and stir at room temperature at a speed of 120-180 rpm for 24-36 hours to obtain substance 3; L2. The substance 3 obtained in step L1 is precipitated with acetone, the precipitate is fully washed with anhydrous ethanol, and dried under reduced pressure to a constant weight to obtain modified carboxymethyl cellulose.

[0012] Preferably, in step L1, the mass volume ratio of carboxymethyl cellulose:pyrrole:betaine chloride is 1:50-60:0.2-0.3.

[0013] Preferably, the amounts of acetone and anhydrous ethanol used in step L2 are not limited and conventional operating amounts are sufficient. The reduced pressure drying is preferably performed at 55-60°C.

[0014] The preparation method of the combined preparation for improving diabetic foot microcirculation is 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 add component 2 obtained in step V2 to component 1 obtained in step V1 to obtain a combined preparation.

[0015] 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 mass of modified carboxymethyl cellulose.

[0016] The beneficial effects of the present invention are as follows: Existing studies have shown that platelet-rich plasma has a better effect on relieving wound ulcers. The advantage is that after platelets are activated, they will release platelet-derived growth factor, vascular endothelial growth factor, transfer growth factor, epidermal growth factor, basic fibroblast growth factor, fibronectin and other nutrients through degranulation. These nutrients can promote ulcer healing and angiogenesis. In addition, after platelets are activated, they will release a variety of active substances with antibacterial effects such as antimicrobial peptides, chemokines, and bactericidal proteins, which have certain anti-infection capabilities. Platelet-rich plasma derived from the body can also prevent patients from immune rejection due to the use of exogenous substances and will not cause serious adverse reactions. However, platelet-rich plasma also has certain application limitations, such as its therapeutic effect in patients with neuropathic diabetic foot with better local blood supply is better than that in patients with ischemic ulcers, and its in vitro life is shorter. In view of the application advantages of platelet-rich plasma, the present invention uses calcium gluconate as an agonist on this basis to stimulate platelet-rich plasma to release a large amount of active substances such as growth factors, and then further obtains its exosomes, uses phosphatase and silk protein to stabilize the exosomes, and prolongs the existence time of the active substances. In addition, the present invention specially makes protamine into nanoparticles and loads them into exosomes by electroporation, so that the biologically active substances finally obtained have strong antibacterial ability and can resist the development process of diabetic foot ulcers. Bioactive glass and hydroxyethyl cellulose can prolong the in vitro life of biologically active substances, promote the therapeutic effect of biologically active substances, and the modified carboxymethyl cellulose has better biodegradability than unmodified carboxymethyl cellulose, so that the overall combination preparation is safer. The combination preparation obtained by the present invention has less cytotoxicity, better antibacterial effect, can promote wound healing faster, and microcirculation indicators show that it can stimulate changes in foot blood flow and improve foot artery blockage. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 The results of the combined preparation on cell viability are shown in FIG. Figure 2 This is a test chart of the antibacterial effect of the combined preparation; Figure 3 This is a graph showing the results of a diabetic wound healing evaluation experiment; Figure 4 This is the result diagram of the blood perfusion change rate during treatment; Figure 5 The results of ankle-brachial index before and after treatment. DETAILED DESCRIPTION

[0018] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided 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 technical features well known in the art are not described.

[0019] Example 1: This example provides a method for preparing a biologically active substance, the specific contents are as follows: M1. Dissolve protamine in distilled water to obtain a 1% protamine aqueous solution, let it stand overnight (10 hours) and then heat it to 40°C, add anhydrous ethanol in an amount of 0.2 times the volume of the protamine aqueous solution, and continue stirring at 150 rpm for 15 hours at room temperature. After the end, remove the anhydrous ethanol by vacuum distillation to obtain liquid 1, add 3% glutaraldehyde aqueous solution in an amount of 0.1 times the volume of liquid 1, stir and disperse, let it stand for 5 minutes, centrifuge at 8000g at 4°C to obtain a precipitate, wash the precipitate with PBS buffer solution precooled at 4°C and pH=7.0, and centrifuge again to obtain the precipitate, which is substance 1; M2. Obtain platelet-rich plasma, weigh platelet-rich plasma and calcium gluconate solution with a mass percentage concentration of 6% in a volume ratio of 10:1, mix the two, incubate in a 37°C water bath for 10 minutes, and then obtain exosomes by ultracentrifugation. The exosomes are resuspended in PBS buffer with a pH of 7.0 after precooling at 4°C, and then mixed with phosphatase and silk fibroin. The mass ratio of exosomes: PBS buffer: phosphatase: silk fibroin is 1:400:80:50. After the end, substance 2 is obtained; M3. Mix substance 1 obtained in step M1 and substance 2 obtained in step M2 in a mass ratio of 1:8, fully precool at 4°C (24h), place in an electroporation cuvette that has also been precooled at 4°C, perform electroporation at 1000kV for 8ms using an electroporator, and incubate at 37°C for 30min to obtain the bioactive substance.

[0020] Example 2: This example provides a method for preparing a biologically active substance, the specific contents are as follows: M1. Dissolve protamine in distilled water to obtain a protamine aqueous solution with a mass percentage concentration of 1.2%, let it stand overnight (10 hours) and then heat it to 45°C, add anhydrous ethanol in an amount of 0.25 times the volume of the protamine aqueous solution, and continue stirring at 180 rpm for 17 hours at room temperature. After the end, remove the anhydrous ethanol by vacuum distillation to obtain liquid 1, add glutaraldehyde aqueous solution with a mass percentage concentration of 3.5% in an amount of 0.15 times the volume of liquid 1, stir and disperse, let it stand for 8 minutes, and centrifuge at 9000g at 4°C to obtain a precipitate. The precipitate is fully washed with PBS buffer solution precooled at 4°C and pH=7.0, and centrifuged again at high speed to obtain the precipitate, which is substance 1; M2. Obtain platelet-rich plasma, weigh platelet-rich plasma and calcium gluconate solution with a mass percentage concentration of 7% in a volume ratio of 10:1.2, mix the two, incubate in a 37°C water bath for 12 minutes, and then obtain exosomes by ultracentrifugation. The exosomes are resuspended in PBS buffer with a pH of 7.0 after precooling at 4°C, and then mixed with phosphatase and silk fibroin. The mass ratio of exosomes: PBS buffer: phosphatase: silk fibroin is 1:500:100:60. After the end, substance 2 is obtained; M3. Mix substance 1 obtained in step M1 and substance 2 obtained in step M2 in a mass ratio of 1:9, fully precool at 4°C (24h), place in an electroporation cuvette that has also been precooled at 4°C, perform electroporation at 1100kV for 6ms using an electroporator, and then incubate at 37°C for 38min to obtain the bioactive substance.

[0021] Example 3: This example provides a method for preparing a biologically active substance, the specific contents are as follows: M1. Dissolve protamine in distilled water to obtain a protamine aqueous solution with a mass percentage concentration of 1.5%, let it stand overnight (10 hours), then heat it to 50°C, add anhydrous ethanol in an amount of 0.3 times the volume of the protamine aqueous solution, and continue stirring at 200 rpm for 20 hours at room temperature. After the end, remove the anhydrous ethanol by vacuum distillation to obtain liquid 1, add glutaraldehyde aqueous solution with a mass percentage concentration of 4% in an amount of 0.2 times the volume of liquid 1, stir and disperse, let it stand for 10 minutes, and centrifuge it at 10000g at 4°C to obtain a precipitate. The precipitate is fully washed with PBS buffer solution precooled at 4°C and pH=7.0, and centrifuged again at high speed to obtain the precipitate, which is substance 1; M2. Obtain platelet-rich plasma, weigh platelet-rich plasma and calcium gluconate solution with a mass percentage concentration of 8% in a volume ratio of 10:1.5, mix the two, incubate in a 37°C water bath for 15 minutes, and then obtain exosomes by ultracentrifugation. The exosomes are resuspended in PBS buffer with a pH of 7.0 after precooling at 4°C, and then mixed with phosphatase and silk fibroin. The mass ratio of exosomes: PBS buffer: phosphatase: silk fibroin is 1:600:120:80. After the end, substance 2 is obtained; M3. Mix substance 1 obtained in step M1 and substance 2 obtained in step M2 in a mass ratio of 1:10, fully precool at 4°C (24h), place in an electroporation cuvette that has also been precooled at 4°C, perform electroporation at 1200kV for 5ms using an electroporator, and incubate at 37°C for 45min to obtain the bioactive substance.

[0022] In addition, when the source of platelet-rich plasma is human, it is obtained as follows: Collect 10 ml of venous blood, add it into an anticoagulant test tube and mix evenly, centrifuge it at 2500rpm for 10 minutes, and the sample is divided into three layers after centrifugation, the top layer is plasma, the middle layer is concentrated platelets, and the bottom layer is red blood cells. Remove the top plasma and the bottom red blood cell layer, take the middle layer and centrifuge it again at 2200rpm for 10 minutes, discard part of the supernatant, and the remaining part is platelet-rich plasma.

[0023] When the source of platelet-rich plasma is rat, the intermediate layer centrifugation is preferably maintained at 2000 rpm for 10 minutes to obtain platelet-rich plasma.

[0024] In Example 1-3, the specific steps of the ultracentrifugation method in step M2 are as follows: Centrifuge at 300g for 10 min, at 2000g for 10 min, at 10000g for 30 min, and at 150000g for ultracentrifugation for 65 min.

[0025] Example 4: This example provides a method for preparing a combined preparation for improving diabetic foot microcirculation, the specific contents are as follows: The combined preparation for improving diabetic foot microcirculation in this embodiment comprises the following raw materials in parts by mass: 3 parts of bioactive substances, 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, and 0.8 parts of glycerol. The bioactive substances involved are from Example 1, and the preparation method of the modified carboxymethyl cellulose involved is as follows: L1. Weigh carboxymethyl cellulose, pyrrole and betaine chloride according to the mass volume ratio of 1g:50mL:0.2g, add carboxymethyl cellulose to pyridine, stir until uniformly dispersed, add betaine chloride, stir at room temperature at a speed of 120rpm for 24h, and 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 at 55°C to constant weight to obtain modified carboxymethyl cellulose; The steps for preparing the combined preparation are as follows: V1. Weigh hydroxyethyl cellulose and dissolve it in water. The amount of water added is 20 times the mass of hydroxyethyl cellulose. Mix thoroughly. Add bioactive glass 45S5 and bioactive glass 1393. Stir until evenly dispersed to obtain component 1. V2. Weigh γ-aminobutyric acid, glycerol, and modified carboxymethyl cellulose, add water and stir until uniformly dispersed, the amount of water added is 10 times that of the modified carboxymethyl cellulose, add bioactive substances, and obtain component 2; V3. Slowly add component 2 obtained in step V2 to component 1 obtained in step V1 to obtain a combined preparation.

[0026] Example 5: This example provides a method for preparing a combined preparation for improving diabetic foot microcirculation, the specific contents are as follows: The combined preparation for improving diabetic foot microcirculation in this embodiment comprises 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 are from Example 2, and the preparation method of the modified carboxymethyl cellulose involved is as follows: L1. Weigh carboxymethyl cellulose, pyrrole and betaine chloride according to the mass volume ratio of 1g:55mL:0.25g, add carboxymethyl cellulose to pyridine, stir until uniformly dispersed, add betaine chloride, stir at room temperature at a speed of 140rpm for 30h, and 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 at 58°C to constant weight to obtain modified carboxymethyl cellulose; The steps for preparing the combined preparation are as follows: V1. Weigh hydroxyethyl cellulose and dissolve it in water. The amount of water added is 24 times the mass of hydroxyethyl cellulose. Mix thoroughly. Add bioactive glass 45S5 and bioactive glass 1393. Stir until evenly dispersed to obtain component 1. V2. Weigh γ-aminobutyric acid, glycerol, and modified carboxymethyl cellulose, add water and stir until uniformly dispersed, the amount of water added is 16 times that of the modified carboxymethyl cellulose, add bioactive substances, and obtain component 2; V3. Slowly add component 2 obtained in step V2 to component 1 obtained in step V1 to obtain a combined preparation.

[0027] Example 6: This example provides a method for preparing a combined preparation for improving diabetic foot microcirculation, the specific contents are as follows: The combined preparation for improving diabetic foot microcirculation in this embodiment comprises the following raw materials 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 are from Example 3. The preparation method of the modified carboxymethyl cellulose involved is as follows: L1. Weigh carboxymethyl cellulose, pyrrole and betaine chloride according to the mass volume ratio of 1g:60mL:0.3g, add carboxymethyl cellulose to pyridine, stir until uniformly dispersed, add betaine chloride, stir at room temperature at a speed of 180rpm for 36h, and 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 at 60°C to constant weight to obtain modified carboxymethyl cellulose; The steps for preparing the combined preparation are as follows: V1. Weigh hydroxyethyl cellulose and dissolve it in water. The amount of water added is 30 times the mass of hydroxyethyl cellulose. Mix thoroughly. Add bioactive glass 45S5 and bioactive glass 1393. Stir until evenly dispersed to obtain component 1. V2. Weigh γ-aminobutyric acid, glycerol, and modified carboxymethyl cellulose, add water and stir until uniformly dispersed, the amount of water added is 20 times that of the modified carboxymethyl cellulose, add bioactive substances, and obtain component 2; V3. Slowly add component 2 obtained in step V2 to component 1 obtained in step V1 to obtain a combined preparation.

[0028] Comparative Example 1: This comparative example provides a combined preparation, the specific contents are as follows: The difference between the combination preparation of this comparative example and the combination preparation obtained in Example 5 is that the combination preparation of this comparative example does not contain biologically active substances, and other raw materials and preparation steps are consistent with those in Example 5.

[0029] Comparative Example 2: This comparative example provides a combined preparation, the specific contents are as follows: The difference between the combination preparation of this comparative example and the combination preparation obtained in Example 5 is that sodium carboxymethyl cellulose is used to replace the modified sodium carboxymethyl cellulose in the combination preparation of this comparative example, and other raw materials and preparation steps are consistent with those in Example 5.

[0030] Comparative Example 3: This comparative example provides a combined preparation, the specific contents are as follows: The difference between the combination preparation of this comparative example and the combination preparation obtained in Example 5 is that the combination preparation of this comparative example does not contain γ-aminobutyric acid, and other raw materials and preparation steps are consistent with those in Example 5.

[0031] Comparative Example 4: This comparative example provides a combined preparation, the specific contents are as follows: The difference between the combination preparation of this comparative example and the combination preparation obtained in Example 5 is that the combination preparation of this comparative example contains only component 2.

[0032] Comparative Example 5: This comparative example provides a combined preparation, the specific contents are as follows: The difference between the combination preparation of this comparative example and the combination preparation obtained in Example 5 is that the combination preparation of this comparative example uses platelet-rich plasma of the same mass and source, and the platelet-rich plasma and hyaluronic acid are mixed in a mass ratio of 1:3. The obtained thick mixture is stored in an environment of 4°C to obtain the combination preparation of this comparative example.

[0033] Experimental test: 1. Experiment on the effect of combined preparations on cell viability: 100 μL L929 cell suspension (density 1×10 4 cells / mL) were inoculated in the wells of a 96-well plate. After culturing for 24 hours, the combined preparations prepared in Examples 4-6 and Comparative Examples 1-5 (pre-shaped into discs with a diameter of 5 mm and a thickness of 1 mm) were placed in the wells and co-cultured for another 24 hours. Then, the combined preparations were removed and the wells were washed three times with PBS buffer. A culture 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% CO2 for 1 hour. Subsequently, the cell viability was measured at an absorbance of 450nm using an ELISA reader, and the cell viability was calculated according to the following formula: Cell viability = (OD1-OD0) / (OD2-OD0) × 100%; OD1 is the OD value measured in the experimental group, which corresponds to the combined preparation obtained in Examples 4-6; OD0 is the OD value of the blank group, which has no combined preparation but CCK-8 solution; and OD2 is the OD value measured without any treatment.

[0034] The results are as follows Figure 1 As shown by Figure 1It can be seen that the combined preparations prepared in Examples 4-6 and Comparative Examples 1-5 have little effect on cell viability.

[0035] 2. Antibacterial effect test of combined preparations: The combined preparations obtained in Examples 4-6 and Comparative Examples 1-5 were tested. 150 μL of the combined preparations were incubated with 5 mL of a 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 the incubation, 0.1 mL of bacterial suspension was drawn onto the LB medium, evenly spread, and incubated at 37°C. After 24 hours of incubation, photos were taken to record the contamination situation.

[0036] The results are as follows Figure 2 As shown by Figure 2 It can be seen that the combination preparation obtained in comparative example 1 has a poor inhibitory effect on Staphylococcus aureus, followed by the combination preparation obtained in comparative example 4. The combination preparations used in other experimental groups have good inhibitory effects on Staphylococcus aureus at the dosages used in the experiment. The experiment confirmed that the bioactive substances play an important role in the antibacterial effect of the combination preparation, and the combination preparation needs to be mixed for better effect. Component 2 alone cannot completely inhibit the growth of Staphylococcus aureus.

[0037] 3. Diabetes wound healing evaluation experiment: 100 SPF healthy SD rats were used, with a body weight of 230-270g. They were adaptively fed for one week before the experiment, with a temperature of 20±3℃ and a relative humidity of 55±10%, and free access to food and water. They were fasted for 12h before modeling, and each rat was given a 1% STZ solution (prepared with citrate buffer) at a dose of 50mg / kg. After 30min of injection, the rats were no longer restricted from food and water intake. Blood glucose was measured in the tail vein 72h after injection. Blood glucose ≥16.7mM indicated successful modeling, and rats with unsuccessful modeling were not included in the experiment.

[0038] A circle with a diameter of 2 cm was cut on the back of the rat to obtain skin lesions. The combination preparations obtained in Examples 4-6 and Comparative Examples 1-5 were applied to the skin lesions twice a day for four weeks. The control group was treated with a phosphate buffer solution with a pH of 7.0. The diameter of the skin lesions was measured to confirm the wound healing effect. The results are as follows Figure 3 As shown by Figure 3 It can be seen that the combined preparations prepared in Examples 4-6 have a good effect of promoting wound healing, and the wound healing rate reaches 93% after 28 days. Compared with the control group, the combined preparations prepared in Comparative Examples 1-5 can also promote wound healing, and the effects of the combined preparations obtained in Comparative Examples 2 and 3 are better than that of Comparative Example 5.

[0039] IV. Clinical application experiment: The combined preparations prepared in Examples 4-6 were used for patients with diabetic foot. The patients met the diagnostic criteria for diabetic foot and had clinical manifestations of lower limb ischemia. The Wagner classification was 1 to 4, that is, there were ulcers of varying degrees and infections of varying degrees. The patients had no verified heart, liver, or kidney diseases, no abnormal coagulation function, and were not pregnant or lactating women. There were 12 patients involved, the youngest was 38 years old and the oldest was 56 years old. The control group was given conventional treatment. In addition to daily medications, anti-infection drugs were intravenously dripped after foot debridement, and the wound was cared for as usual.

[0040] During the clinical application experiment, the patient's pro-inflammatory cytokines, blood sugar, blood perfusion and other indicators, as well as the wound recovery status (whether the wound area is reduced, wound exudate status, granulation growth status, etc.) are tested. The following shows the detection method and results of blood perfusion and ankle-brachial index: 1) PeriFlux5000 laser Doppler (Perimed) was used to detect blood perfusion. The patients were placed in supine position at room temperature for 15 minutes. The blood perfusion was detected on the dorsum of the foot 2 cm above the third metatarsophalangeal joint, avoiding large blood vessels, areas with a lot of hair, and areas with damaged skin. The blood perfusion was detected after a slight cleaning. The basal blood perfusion was recorded as PU0, and the blood perfusion after heating to 45°C was recorded as PU1. The blood perfusion change rate = (PU1-PU0) / PU0×100%.

[0041] The results of blood perfusion are as follows Figure 4 As shown by Figure 4 It can be seen that during the 28-day treatment period, compared with the control group, the application of the combined preparation prepared in Examples 4-6 significantly improved the change rate of blood perfusion volume in patients, proving that the microcirculation disorder in patients with diabetic foot was significantly improved.

[0042] 2) The patient lies supine, and Doppler ultrasound is used to measure the systolic blood pressure of the ankle (dorsalis pedis artery or posterior carotid artery) and upper arm. The ankle-brachial index = the highest systolic blood pressure of the posterior tibial artery or dorsalis pedis artery / the highest systolic blood pressure of both arms.

[0043] The results of the ankle-brachial index are as follows Figure 5 As shown by Figure 5 It can be seen that after 28 days of treatment, the ankle-brachial index of the patient increased significantly, proving that compared with the control group, the blockage of the patient's foot artery was improved after using the combination preparation prepared in Examples 4-6.

[0044] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in 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.

2. The combined preparation for improving diabetic foot microcirculation according to claim 1, characterized in that: 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. Mix substance 1 obtained in step M1 and substance 2 obtained in step M2 in a mass ratio of 1:8-10, fully precool at 4°C, perform electroporation at 1000-1200 kV, and incubate at 37°C to obtain the bioactive substance.

3. The combined preparation for improving diabetic foot microcirculation according to claim 2, 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.

4. The combined preparation for improving diabetic foot microcirculation according to claim 3, characterized in that: 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.

3.

5. A method for preparing the combined preparation for improving diabetic foot microcirculation according to any one of claims 1 to 4, 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.

Images