Preparation method of film spraying agent for treating diabetic foot ulcer

Abstract

The invention relates to a preparation method of a film spraying agent, in particular to a preparation method of a film spraying agent for treating diabetic foot ulcer. The problems that an existing film spraying agent is long in film forming time, poor in toughness, not prone to being torn off and low in anti-inflammatory degree are solved. The preparation method comprises the following steps: 1, medicine extraction; 2, preparing a film forming material; and 3, preparing the film spraying agent. The preparation method is used for preparing the film spraying agent for treating the diabetic foot ulcer.

Description

Technical Field

[0001] The invention relates to a method for preparing a film spraying agent. Background Art

[0002] The incidence of type 2 diabetes has been rising year by year in recent years, and diabetes and its various related complications are increasingly becoming a major threat to human health. Diabetic foot ulcers, which are skin infections, suppuration, and ulcers caused by peripheral neuropathy and peripheral vascular disease, are one of the major chronic complications of diabetes.

[0003] Currently, patients with diabetic foot ulcers use two approaches: oral medications such as epalrestat, methylcobalamin, and α-lipoic acid to slow the progression of diabetic peripheral neuropathy. Alternatively, medications such as erythromycin ointment, silver sulfadiazine cream, and sodium fusidate ointment are applied to the affected area for anti-inflammatory and antibacterial effects. All of these Western medicines suffer from poor efficacy, lack of specificity, and significant toxic side effects. Most importantly, these medications are topical, requiring a bandage to be applied to the wound site. This bandage, however, can reduce permeability and can cause inflammation.

[0004] In response to the needs of this intractable clinical disease, a film spray is a new type of preparation with polymer materials as the main raw material. This type of preparation is stored in liquid form and is sprayed on the skin surface to form a composite drug film with a certain structure. The composite drug film increases the release of various growth factors in the ulcer surface and creates a closed and moist environment for the ulcer surface, thereby achieving the effects of removing dead flesh, eliminating swelling and pain, healing the wound surface, and preventing scar formation. It can shorten the healing time and effectively improve clinical efficiency. However, existing film sprays take a long time to form and have poor toughness and are difficult to remove, making debridement and dressing changes difficult and the anti-inflammatory effect is not high. Summary of the Invention

[0005] The present invention aims to solve the problems of existing spray film agents such as long film forming time, poor toughness and difficulty in peeling off, and low anti-inflammatory effect, and further provides a method for preparing a spray film agent for treating diabetic foot ulcers.

[0006] A method for preparing a film spray for treating diabetic foot ulcers is carried out according to the following steps:

[0007] 1. Drug extraction:

[0008] Grinding a Chinese medicinal material for treating diabetic foot ulcer into powder, then soaking it in an ethanol solution and refluxing it, filtering it after refluxing to obtain a medicinal solution, concentrating the medicinal solution into a viscous state, and then freeze-drying it to obtain a medicinal powder;

[0009] 2. Preparation of film-forming materials:

[0010] Chitosan hydrochloride, para-aminosalicylic acid, fish collagen, and polyvinyl alcohol (PVA1788) are added into ultrapure water for swelling, and then mixed uniformly under stirring conditions to obtain a film-forming material.

[0011] 3. Preparation of spray film:

[0012] The drug powder is added to a mixed solution of anhydrous ethanol and ultrapure water, and then added to a film-forming material. Finally, a moisturizer, a solubilizer, a thickener and a penetration enhancer are added under stirring and mixed evenly to obtain a spray film for treating diabetic foot ulcers.

[0013] The beneficial effects of the present invention are:

[0014] The present invention addresses the current problems in the treatment of diabetic foot ulcers and provides a novel drug spray film formulation. Para-aminobenzoic acid is added to the spray film formulation as a crosslinker for the spray film material. The crosslinking reaction can transform the molecular structure of the polymer from a linear to a three-dimensional network structure. This structural transformation significantly improves the tensile strength of the spray film material, making the film-forming material tougher and more durable. The spray film formulation precisely applies the drug to the affected area through spraying, and a protective film is formed 5 to 7 minutes after the solvent evaporates. The film has good toughness, high uniformity, and excellent anti-inflammatory properties.

[0015] The film spray of the present invention forms a resilient active drug film upon application to the skin, achieving long-lasting, anti-inflammatory, and wound-healing benefits. Chitosan hydrochloride, a chitosan derivative modified to impart excellent water solubility, is used as the primary film-forming material. This property allows chitosan hydrochloride to dissolve more easily in solvents, forming a uniform solution, facilitating the preparation of a traditional Chinese medicine film spray. Chitosan hydrochloride carries a positive charge, and this cationic nature enables it to interact with negatively charged substances (such as certain active ingredients in traditional Chinese medicine or the negative charge on the skin surface), thereby enhancing drug adsorption and permeability. Furthermore, chitosan hydrochloride exhibits excellent biocompatibility and biodegradability, being degraded by enzymes in the body into non-toxic, natural metabolites. Furthermore, the addition of fish collagen allows the film spray to form a continuous, uniform film layer, enhancing its integrity and density. The resulting film can be removed intact, facilitating film shedding. The addition of a humectant (glycerol) absorbs and retains moisture from the air, providing long-lasting moisturization to the skin. This effectively alleviates dryness and tightness, enhancing the user experience and increasing comfort. The addition of solubilizers (polyvinyl pyrrolidone) and polyvinyl alcohol significantly increases the solubility of poorly soluble ingredients in traditional Chinese medicine sprays, allowing them to be more evenly dispersed in the solvent. These two substances, combined with other excipients (such as chitosan hydrochloride), form a stable film. This film protects the drug from environmental influences, improving its bioavailability and efficacy. Cytotoxicity studies demonstrated that this spray had no significant effect on the proliferation of human umbilical vein endothelial cells (HUVECS), demonstrating that the resulting film is non-toxic to humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The film-forming effect diagram and film-shedding effect diagram of the film-spraying agents prepared in Examples 1, 2, 3, 4, 7 and Comparative Experiment 1 in 5 minutes are shown. 1 is a film-forming effect diagram, a is Example 1, b is Example 2, c is Example 3, d is Example 4, e is Example 7, and 2 is a physical diagram of the film-spraying agent prepared in Example 1 after film formation and peeling off.

[0017] Figure 2 This is the infrared spectrum of the film-spraying agent prepared in Example 1 after film formation;

[0018] Figure 3 This is a comparison chart of the tensile properties of the spray film prepared in Example 1 and Comparative Experiments 2 to 4 after film formation;

[0019] Figure 4 The effects of the spray film prepared in Examples 1 to 6 on the proliferation of human umbilical vein endothelial cell line (HUVECS), 1 to 6 are Examples 1 to 6;

[0020] Figure 5 The effects of the spray films prepared in Examples 1 to 6 on inflammatory factors in RAW264.7 cells are shown in Figures 1 to 6.

[0021] Figure 6 The effects of the spray films prepared in Examples 2, 5 and 6 on the migration of HUVECS cells are shown in Figures 2, 5 and 6. DETAILED DESCRIPTION

[0022] Specific embodiment 1: This embodiment is a method for preparing a spray film for treating diabetic foot ulcers, which is carried out according to the following steps:

[0023] 1. Drug extraction:

[0024] Grinding a Chinese medicinal material for treating diabetic foot ulcer into powder, then soaking it in an ethanol solution and refluxing it, filtering it after refluxing to obtain a medicinal solution, concentrating the medicinal solution into a viscous state, and then freeze-drying it to obtain a medicinal powder;

[0025] 2. Preparation of film-forming materials:

[0026] Chitosan hydrochloride, para-aminosalicylic acid, fish collagen, and polyvinyl alcohol (PVA1788) are added into ultrapure water for swelling, and then mixed uniformly under stirring conditions to obtain a film-forming material.

[0027] 3. Preparation of spray film:

[0028] The drug powder is added to a mixed solution of anhydrous ethanol and ultrapure water, and then added to a film-forming material. Finally, a moisturizer, a solubilizer, a thickener and a penetration enhancer are added under stirring and mixed evenly to obtain a spray film for treating diabetic foot ulcers.

[0029] The spray film prepared in this specific embodiment for treating diabetic foot ulcers is used to thoroughly clean and disinfect the wound before treatment to ensure that the spray film is evenly covered on the wound surface and to avoid wound contamination during the treatment process.

[0030] This specific embodiment, through reflux extraction experiments, produces a traditional Chinese medicine powder with optimal efficacy. This powder is non-toxic and can improve the treatment of diabetic foot ulcers. The spray film can be sprayed directly onto the ulcer wound, forming a protective film that protects the wound from external contamination while promoting drug penetration and absorption.

[0031] The beneficial effects of this embodiment are:

[0032] This embodiment addresses the current challenges in treating diabetic foot ulcers by providing a novel drug spray film formulation. Para-aminobenzoic acid is added to the spray film formulation as a crosslinker. The crosslinking reaction transforms the polymer's molecular structure from a linear to a three-dimensional network. This structural transformation significantly increases the tensile strength of the spray film, making the film-forming material more robust and durable. The spray film precisely applies the drug to the affected area via spraying, forming a protective film 5-7 minutes after the solvent evaporates. The film exhibits excellent toughness, uniformity, and anti-inflammatory properties.

[0033] The film spray described in this embodiment forms a resilient active drug film upon application to the skin, achieving long-lasting, anti-inflammatory, and wound-healing benefits. Chitosan hydrochloride, a chitosan derivative modified to impart excellent water solubility, is used as the primary film-forming material. This property allows chitosan hydrochloride to dissolve more easily in solvents, forming a uniform solution, facilitating the preparation of a traditional Chinese medicine film spray. Chitosan hydrochloride carries a positive charge, and this cationic nature enables it to interact with negatively charged substances (such as certain active ingredients in traditional Chinese medicine or the negative charge on the skin surface), thereby enhancing drug adsorption and permeability. Furthermore, chitosan hydrochloride exhibits excellent biocompatibility and biodegradability, being degraded by enzymes in the body into non-toxic, natural metabolites. Furthermore, the addition of fish collagen allows the film spray to form a continuous, uniform film layer, enhancing its integrity and density. The resulting film can be removed intact, facilitating film removal. The addition of a humectant (glycerol) absorbs and retains moisture from the air, providing long-lasting moisturization to the skin. This effectively alleviates dryness and tightness, enhancing the user experience and increasing comfort. The addition of solubilizers (polyvinyl pyrrolidone) and polyvinyl alcohol significantly increases the solubility of poorly soluble ingredients in traditional Chinese medicine sprays, allowing them to be more evenly dispersed in the solvent. These two substances, combined with other excipients (such as chitosan hydrochloride), form a stable film. This film protects the drug from environmental influences, improving its bioavailability and efficacy. Cytotoxicity studies demonstrated that this spray had no significant effect on the proliferation of human umbilical vein endothelial cells (HUVECS), demonstrating that the resulting film is non-toxic to humans.

[0034] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that the Chinese medicinal materials for treating diabetic foot ulcers described in step 1 include, by weight, 0.5-10 parts of water chestnuts, 0.5-5 parts of raw rhubarb, 0.2-5 parts of Panax notoginseng, 1.5-5 parts of sanguisorba officinalis, 0.3-2 parts of geranium, 0.1-1 part of Achyranthes bidentata, and 0.2-1 part of Astragalus. Other aspects are the same as those in specific embodiment 1.

[0035] Specific embodiment 3: This embodiment differs from either specific embodiment 1 or 2 in that the mass percentage of the ethanol solution in step 1 is 60% to 100%; and the volume ratio of the Chinese medicinal material for treating diabetic foot ulcers in step 1 to the ethanol solution is 1g:(8-10)mL. Other steps are the same as specific embodiments 1 or 2.

[0036] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the reflux in step 1 is carried out at a temperature of 60° C. to 80° C. for 2 to 3 hours. Other aspects are the same as specific embodiments 1 to 3.

[0037] Specific embodiment 5: This embodiment differs from Specific embodiments 1 to 4 in that the concentration to a viscous state in step 1 is performed using a rotary evaporator at a temperature of 40°C to 60°C and a rotation speed of 300 to 700 rpm for 1 to 3 hours; and the freeze-drying in step 1 is performed at a temperature of -40°C to -60°C for 36 to 48 hours. Other aspects are the same as Specific embodiments 1 to 4.

[0038] Specific embodiment 6: This embodiment differs from Specific embodiments 1 to 5 in that, in step 2, 0.5 to 1.5 parts chitosan hydrochloride, 0.1 to 1.5 parts p-aminosalicylic acid, 0.2 to 1 part fish collagen, 0 to 1.5 parts polyvinyl alcohol (PVA1788), and 10 to 30 parts ultrapure water are weighed by weight; the fish collagen in step 2 is tilapia fish gelatin. Otherwise, the same as Specific embodiments 1 to 5.

[0039] Specific embodiment 7: This embodiment differs from specific embodiments 1 to 6 in that the swelling in step 2 is carried out at room temperature for 24 to 48 hours. Other aspects are the same as specific embodiments 1 to 6.

[0040] Specific embodiment 8: This embodiment differs from Specific embodiments 1 to 7 in that the mixing under stirring conditions described in step 2 is specifically carried out at a stirring speed of 30 to 100 r / min and a temperature of 25° C. to 60° C. for 20 to 60 minutes. Other aspects are the same as Specific embodiments 1 to 7.

[0041] Specific embodiment 9: This embodiment differs from any one of specific embodiments 1 to 8 in that: the moisturizing agent in step 3 is glycerol; the solubilizing agent in step 3 is polyvinyl pyrrolidone; the thickening agent in step 3 is gelatin; and the penetration enhancer in step 3 is borneol or Tween 80. Other aspects are the same as specific embodiments 1 to 8.

[0042] Specific embodiment 10: This embodiment differs from specific embodiments 1 to 9 in that the volume ratio of ultrapure water to anhydrous ethanol in the mixed solution of anhydrous ethanol and ultrapure water described in step 3 is 1:(1-1.5); in step 3, 1 to 3 parts of drug powder, 30 to 50 parts of the mixed solution of anhydrous ethanol and ultrapure water, 1 to 3 parts of film-forming material, 0.5 to 1 part of moisturizing agent, 0.5 to 1 part of solubilizing agent, 0.2 to 1 part of thickening agent, and 0 to 0.5 part of penetration enhancer are weighed in parts by mass. Other aspects are the same as specific embodiments 1 to 9.

[0043] The following examples are used to verify the beneficial effects of the present invention:

[0044] Example 1:

[0045] A method for preparing a film spray for treating diabetic foot ulcers is carried out according to the following steps:

[0046] 1. Drug extraction:

[0047] Grind a Chinese medicinal material used for treating diabetic foot ulcers into powder, then soak it in an ethanol solution, reflux it at 60°C for 2 hours, filter it after reflux to obtain a medicinal solution, use a rotary evaporator at 60°C and 700 r / min to evaporate the medicinal solution for 1.5 hours to concentrate it into a viscous state, and then freeze-dry it at -50°C for 48 hours to obtain a medicinal powder;

[0048] The Chinese medicinal materials for treating diabetic foot ulcers are composed of 10 parts of scutellaria baicalensis, 0.5 parts of raw rhubarb, 5 parts of Panax notoginseng, 1.5 parts of Sanguisorba officinalis, 2 parts of Geranium herb, 1 part of Achyranthes bidentata and 1 part of Astragalus membranaceus in parts by mass;

[0049] The mass percentage of the ethanol solution is 60%; the volume ratio of the mass of the Chinese medicinal materials for treating diabetic foot ulcers to the ethanol solution is 1g:10mL;

[0050] 2. Preparation of film-forming materials:

[0051] Weigh 0.5 parts of chitosan hydrochloride, 0.2 parts of p-aminosalicylic acid, 0.2 parts of fish collagen, 1.5 parts of polyvinyl alcohol PVA1788 and 15 parts of ultrapure water by mass, add chitosan hydrochloride, p-aminosalicylic acid, fish collagen and polyvinyl alcohol PVA1788 to ultrapure water, swell at room temperature for 24 hours, and then stir and mix for 60 minutes at a stirring speed of 40 r / min and a temperature of 40°C to obtain a film-forming material;

[0052] The fish collagen is tilapia fish glue;

[0053] 3. Preparation of spray film:

[0054] Weigh 1.5 parts of drug powder, 50 parts of a mixed solution of anhydrous ethanol and ultrapure water, 2 parts of a film-forming material, 0.8 parts of a humectant, 0.6 parts of a solubilizer, 0.4 parts of a thickener, and 0.2 parts of a penetration enhancer, by weight. Add the drug powder to the mixed solution of anhydrous ethanol and ultrapure water, then add it to the film-forming material. Finally, add the humectant, solubilizer, thickener, and penetration enhancer under stirring and mix thoroughly to obtain a spray film (CVA / GE / PAS) for treating diabetic foot ulcers.

[0055] The moisturizing agent is glycerol; the solubilizing agent is polyvinyl pyrrolidone; the thickening agent is gelatin; and the penetration enhancer is borneol.

[0056] The volume ratio of ultrapure water to anhydrous ethanol in the mixed solution of anhydrous ethanol and ultrapure water is 1:1.5.

[0057] Example 2: This example differs from Example 1 in that in step 2, 0.8 parts of chitosan hydrochloride, 0.2 parts of p-aminosalicylic acid, 0.5 parts of fish collagen, 1.2 parts of polyvinyl alcohol (PVA1788), and 15 parts of ultrapure water are weighed by mass. Other steps are the same as in Example 1.

[0058] Example 3: This example differs from Example 1 in that: in step 2, 0.8 parts of chitosan hydrochloride, 0.5 parts of para-aminosalicylic acid, 0.5 parts of fish collagen, 1 part of polyvinyl alcohol (PVA1788), and 15 parts of ultrapure water are weighed in parts by mass; in step 3, 1.5 parts of drug powder, 40 parts of a mixed solution of anhydrous ethanol and ultrapure water, 2 parts of a film-forming material, 0.8 parts of a moisturizer, 0.8 parts of a solubilizer, 0.4 parts of a thickener, and 0.2 parts of a penetration enhancer are weighed in parts by mass. Other components are the same as in Example 1.

[0059] Example 4: This example differs from Example 1 in that in step 2, 1 part chitosan hydrochloride, 0.5 part p-aminosalicylic acid, 0.8 part fish collagen, 1 part polyvinyl alcohol PVA1788, and 15 parts ultrapure water are weighed by mass. Other steps are the same as in Example 1.

[0060] Example 5: This example differs from Example 1 in that: in step 2, 1 part chitosan hydrochloride, 0.1 part para-aminosalicylic acid, 0.8 part fish collagen, 0.8 part polyvinyl alcohol (PVA1788), and 15 parts ultrapure water are weighed in parts by mass; in step 3, 1.5 parts of drug powder, 35 parts of a mixed solution of anhydrous ethanol and ultrapure water, 3 parts of a film-forming material, 0.8 part of a moisturizer, 0.8 part of a solubilizer, 0.4 part of a thickener, and 0.2 part of a penetration enhancer are weighed in parts by mass. Other components are the same as in Example 1.

[0061] Example 6: This example differs from Example 1 in that in step 2, 1.5 parts of chitosan hydrochloride, 0.15 parts of p-aminosalicylic acid, 1 part of fish collagen, 0.5 parts of polyvinyl alcohol (PVA1788), and 15 parts of ultrapure water are weighed by mass. Other steps are the same as in Example 1.

[0062] Example 7: This example differs from Example 1 in that, in step 2, 1.5 parts of chitosan hydrochloride, 0.3 parts of para-aminosalicylic acid, 1 part of fish collagen, 0.3 parts of polyvinyl alcohol (PVA1788), and 15 parts of ultrapure water are weighed in parts by mass; in step 3, 1.5 parts of drug powder, 30 parts of a mixed solution of anhydrous ethanol and ultrapure water, 3 parts of a film-forming material, 0.8 parts of a moisturizer, 0.8 parts of a solubilizer, 0.4 parts of a thickener, and 0.2 parts of a penetration enhancer are weighed in parts by mass. Other components are the same as in Example 1.

[0063] Comparative Experiment 1: This embodiment differs from the first embodiment in that the addition of the drug powder is omitted in step 3, and a blank spray film is obtained in step 3. The rest is the same as the first embodiment.

[0064] Comparative Experiment 2: This embodiment differs from the first embodiment in that the addition of p-aminosalicylic acid and fish collagen is omitted in step 2, and the CVA spray film is obtained in step 3. Other steps are the same as those in the first embodiment.

[0065] Comparative Experiment 3: This embodiment differs from the first embodiment in that the addition of p-aminosalicylic acid and chitosan hydrochloride is omitted in step 2, and the GE spray agent is obtained in step 3. The rest is the same as the first embodiment.

[0066] Comparative Experiment 4: This embodiment differs from the first embodiment in that the addition of p-aminosalicylic acid is omitted in step 2, and the CVA / GE spray film is obtained in step 3. The rest is the same as the first embodiment.

[0067] The spray effect and film-forming time are used as rating indicators. The specific evaluation criteria are as follows: put the product into a commercially available 50mL spray bottle, spray it at a distance of 10 cm from a white glass plate, and observe the spray effect; after the spray is completed, put it in a 37℃ oven, and consider the film to be formed after the glass surface is completely dry. Record the film-forming time, observe the film-forming effect diagram and the film-shedding effect. The specific results are shown in Table 1 and Figure 1 . Figure 1 The film-forming effect diagram and film-shedding effect diagram of the film-spraying agents prepared in Examples 1, 2, 3, 4, 7 and Comparative Experiment 1 in 5 minutes are shown. 1 is a film-forming effect diagram, a is Example 1, b is Example 2, c is Example 3, d is Example 4, e is Example 7, and 2 is a physical diagram of the film-spraying agent prepared in Example 1 after film formation and peeling off.

[0068] Table 1 Spraying effect of film spraying agent and film forming timetable

[0069]

[0070] The spray film prepared in Example 1 formed a complete film in 5 minutes and 48 seconds, dried completely and was not sticky. After the film was completely dried, it could be peeled off intact. Compared with a blank film without drug added, the film formation time gradually increased with increasing drug concentration. Furthermore, with increasing drug concentration, the film's toughness deteriorated, resulting in damage and cracking during peeling.

[0071] Figure 2 This is the infrared spectrum of the film-forming spraying agent prepared in Example 1. As can be seen from the figure, the characteristic peak of the aromatic carboxyl group is at 1672 cm -1 disappeared, and both p-aminosalicylic acid and chitosan hydrochloride showed 3425 cm -1 The broad band at 1612 cm represents the stretching vibration of -OH. However, there is no clear distinction between the two peaks. In addition, the peak of NH bond at 1612 cm -1 、Amide bond 1305cm -1 The complex corresponding to CN stretching and NH in-plane bending disappears from the CVA at 1732 cm -1 A new absorption peak appeared at 40°C (at the C=O bending vibration of the carboxyl end). These results indicate that the amino group in CVA reacted with the carboxyl group.

[0072] The spraying agents prepared in Example 1 and Comparative Experiments 2 to 4 were tested according to GB / T 1040.3-2006. The specific film-forming process was as follows: the spraying agent was placed in a commercial spray bottle, sprayed 10 cm away from the glass plate, and placed in an oven at 37°C to dry. Figure 3 This is a comparison of the tensile properties of the spray films prepared in Example 1 and Comparative Experiments 2 to 4. As shown in Figure A, after adding p-aminosalicylic acid, the tensile strength was 33.7 MPa. Group C, containing chitosan hydrochloride, had a tensile strength of 17.2 MPa. Group D, containing fish collagen, had a tensile strength of 12.4 MPa. Groups C and D, containing chitosan hydrochloride / fish collagen, had a tensile strength of 16.8 MPa. This is because the amino and hydroxyl groups in p-aminosalicylic acid chemically crosslink with those in chitosan hydrochloride and fish collagen, increasing the tensile strength of the resulting films.

[0073] In the following experiments, human umbilical vein endothelial cell line (HUVECS) and RAW264.7 macrophage cell line were purchased from Wuhan Punosai Biotechnology Co., Ltd. IL-6 and TNF-α were purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0074] (1) Effects on HUVECS cytotoxicity:

[0075] HUVECS were cultured in DMEM complete medium supplemented with 10% fetal bovine serum and 100 mg / L penicillin-streptomycin in a 37°C, 5% CO2 incubator. Subsequent studies were performed when the cell confluence reached approximately 80%.

[0076] After treatment of each cell group, 100 μL of diluted CCK-8 solution was added to each well, and the cells were incubated for another 2 h. The absorbance at 450 nm was measured. For the biosafety evaluation of the six sprays, the sprays prepared in Examples 1 to 6 were added 48 h after induction. The cells were cultured for another 24 h. The absorbance (OD) of each group was measured by the MTT assay, and the cell survival rate was calculated. A control group without the spray was used. Figure 4 Figure 1 shows the effects of the spray films prepared in Examples 1 to 6 on the proliferation of human umbilical vein endothelial cells (HUVECS). Figures 1 to 6 represent Examples 1 to 6. As can be seen from the figure, Example 3 had no significant effect on HUVECS, while Examples 2, 5, and 6 showed a certain effect in promoting cell proliferation.

[0077] (2) Effects on inflammatory factors in RAW264.7 cells:

[0078] Take the RAW264.7 cell suspension in the logarithmic growth phase and inoculate it into a 6-well cell culture plate at 5×105 / mL for culture. After 24 hours, discard the supernatant and divide the cells into a blank control group, an LPS model group and Example 1 to 6 groups, with 3 replicates in each group. Add 2mL of complete culture medium containing the corresponding spray agent to each group of the Example group, add an equal volume of complete culture medium to the blank control group and the model group, and culture for 2 hours. Except for the blank control group, the other groups were added with LPS at a final concentration of 200ng / mL for 24 hours. Pipette 50μL of cell supernatant culture fluid into a 96-well cell culture plate, add 50μL of Griess reagent 1 and 50μL of Griess reagent 2 in sequence, mix well, and place at room temperature for 10 minutes. Use an enzyme marker to detect the optical density D(λ) group at a wavelength of 540nm, and calculate the content of NO; Figure 5 The effects of the sprays prepared in Examples 1 to 6 on inflammatory factors in RAW264.7 cells are shown in Figures 1 to 6. Compared with the normal group, the IL-6 and TNF-α levels in the model group (LPS group) were significantly increased (P < 0.001). Compared with the model group, the IL-6 and TNF-α levels in all drug-treated groups were decreased (P < 0.05, P < 0.01, and P < 0.001). This indicates that high concentrations of the drug extracted using the same method promote the expression of IL-6 and TNF-α, resulting in a significant anti-inflammatory effect.

[0079] (3) Effects on HUVECS cell migration:

[0080] According to the results of the previous experiments, Examples 2, 5 and 6 have low cytotoxicity and significant anti-inflammatory effects. Therefore, the cell migration ability of Examples 2, 5 and 6 was tested. HUVECS cells were seeded in 6-well plates (1×106 cells / well) for adherent culture and divided into a blank control group, an LPS model group and Example 2, 5 and 6 groups. A 200μL sterile pipette tip was used to scratch the wells, and then the scratched cells were treated with the spray prepared in Examples 2, 5 and 6 for 48h. The scratch area was captured at 0h and 48h using an optical microscope, and the scratch width was measured. The scratch healing rate was expressed as the difference between the scratch widths at 0h and 48h and the percentage of the scratch width at 0h; Figure 6 Figure 2 shows the effects of the sprays prepared in Examples 2, 5, and 6 on HUVECS cell migration. Figures 2, 5, and 6 are for Examples 2, 5, and 6. As can be seen from the figure, Examples 2, 5, and 6 all have the ability to promote cell migration, with Examples 2 and 5 showing the most significant effects.

[0081] (4) Clinical observation

[0082] This study, based on voluntary participation, recruited 10 patients with diabetic foot ulcers. Inclusion criteria included a confirmed diagnosis of diabetic foot ulcer; age range 40 to 60 years; and signed informed consent. Exclusion criteria included patients who had taken antibiotics for more than one week in the past three months; patients who had participated in other clinical trials in the past three months; patients with serious primary diseases of the heart, brain, liver, kidney, or hematopoietic system, or psychiatric disorders; patients with allergies or multiple drug allergies; and patients deemed unsuitable for clinical trial participation by the researchers.

[0083] Twenty patients were randomly divided into an experimental group and a control group, with 10 patients in each group, with an average age of 50.56 years. The two groups had no significant difference in average age, making them comparable. All patients had diabetic foot ulcers with an average wound area of 4.5 square centimeters.

[0084] The experimental sample was the spray film prepared in Example 2. The experimental method was as follows: After debridement, the experimental group applied the experimental sample (i.e., the spray film prepared in Example 2) directly to the wound, with a coating width of 4 to 5 mm on each side of the wound. After spraying, the experimental sample automatically formed a protective film. Application was performed twice daily, ensuring an even application each time. The control group applied the liquid (prepared in Step 1 of Example 2) without the addition of a film-forming material. All other treatment methods were the same as for the experimental group. Clinical observation was conducted on both groups.

[0085] Clinical observation indicators:

[0086] Effectiveness observation indicator: wound healing time.

[0087] Safety observation indicators: skin irritation, allergic reactions, wound dehiscence and other adverse reactions.

[0088] Using the above methods, this study aims to evaluate the efficacy and safety of the experimental samples in the treatment of diabetic foot ulcers.

[0089] Data Analysis:

[0090] Table 2 Wound healing time

[0091]

[0092] From the results in the table, it can be seen that the wound healing time of the experimental group was shorter than that of the control group.

[0093] Table 3 Skin irritation index

[0094]

[0095] Table 4 Skin allergy index

[0096]

[0097] Table 5 Wound dehiscence index

[0098]

[0099] Table 6 Other adverse reaction indicators

[0100]

[0101] It can be seen from the results in the table that the film spraying agent of the embodiment is highly safe and meets the standards of medical materials.

[0102] In summary, the Chinese medicine film spray of the embodiment has the effect of promoting wound healing, is highly safe, and has good application effect in diabetic foot care.

Claims

1. A method for preparing a film spray for treating diabetic foot ulcers, characterized in that It is carried out in the following steps:

1. Drug extraction: Grinding a Chinese medicinal material for treating diabetic foot ulcer into powder, then soaking it in an ethanol solution and refluxing it, filtering it after refluxing to obtain a medicinal solution, concentrating the medicinal solution into a viscous state, and then freeze-drying it to obtain a medicinal powder; 2. Preparation of film-forming materials: Chitosan hydrochloride, para-aminosalicylic acid, fish collagen, and polyvinyl alcohol (PVA1788) are added into ultrapure water for swelling, and then mixed uniformly under stirring conditions to obtain a film-forming material.

3. Preparation of spray film: The drug powder is added to a mixed solution of anhydrous ethanol and ultrapure water, and then added to a film-forming material. Finally, a moisturizer, a solubilizer, a thickener and a penetration enhancer are added under stirring and mixed evenly to obtain a spray film for treating diabetic foot ulcers.

2. The method for preparing a film spray for treating diabetic foot ulcers according to claim 1, characterized in that The Chinese medicinal materials for treating diabetic foot ulcers described in step 1 are composed of 0.5 to 10 parts of scutellaria baicalensis, 0.5 to 5 parts of raw rhubarb, 0.2 to 5 parts of Panax notoginseng, 1.5 to 5 parts of sanguisorba officinalis, 0.3 to 2 parts of geranium, 0.1 to 1 part of Achyranthes bidentata and 0.2 to 1 part of Astragalus membranaceus in parts by mass.

3. The method for preparing a film spray for treating diabetic foot ulcer according to claim 1, characterized in that The mass percentage of the ethanol solution described in step 1 is 60%~100%; the volume ratio of the mass of the traditional Chinese medicine for treating diabetic foot ulcer described in step 1 to the ethanol solution is 1g:(8~10)mL.

4. The method for preparing a film spray for treating diabetic foot ulcers according to claim 1, characterized in that The reflux in step 1 is specifically performed at a temperature of 60° C. to 80° C. for 2 h to 3 h.

5. The method for preparing a film spray for treating diabetic foot ulcer according to claim 1, characterized in that The concentration to a viscous state in step 1 is specifically performed by using a rotary evaporator at a temperature of 40°C to 60°C and a rotation speed of 300r / min to 700r / min for 1h to 3h; the freeze-drying in step 1 is specifically performed at a temperature of -40°C to -60°C for lyophilization for 36h to 48h.

6. The method for preparing a film spray for treating diabetic foot ulcers according to claim 1, characterized in that In step 2, 0.5 to 1.5 parts of chitosan hydrochloride, 0.1 to 1.5 parts of p-aminosalicylic acid, 0.2 to 1 part of fish collagen, 0 to 1.5 parts of polyvinyl alcohol (PVA1788) and 10 to 30 parts of ultrapure water are weighed in parts by mass; the fish collagen described in step 2 is tilapia fish gelatin.

7. The method for preparing a film spray for treating diabetic foot ulcers according to claim 1, characterized in that The swelling in step 2 is specifically carried out at room temperature for 24 to 48 hours.

8. The method for preparing a film spray for treating diabetic foot ulcers according to claim 1, characterized in that The mixing under stirring conditions in step 2 specifically comprises stirring and mixing for 20 minutes to 60 minutes at a stirring speed of 30 r / min to 100 r / min and a temperature of 25° C. to 60° C.

9. The method for preparing a film spray for treating diabetic foot ulcers according to claim 1, characterized in that The moisturizing agent in step 3 is glycerol; the solubilizing agent in step 3 is polyvinyl pyrrolidone; the thickening agent in step 3 is gelatin; and the penetration enhancer in step 3 is borneol or Tween 80.

10. The method for preparing a film spray for treating diabetic foot ulcers according to claim 1, characterized in that The volume ratio of ultrapure water to anhydrous ethanol in the mixed solution of anhydrous ethanol and ultrapure water described in step 3 is 1:(1~1.5); in step 3, 1~3 parts of drug powder, 30~50 parts of a mixed solution of anhydrous ethanol and ultrapure water, 1~3 parts of film-forming material, 0.5~1 part of moisturizing agent, 0.5~1 part of solubilizer, 0.2~1 part of thickener and 0~0.5 part of penetration enhancer are weighed in parts by mass.

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