Antibacterial hemostatic pharmaceutical composition for skin healing and preparation method thereof

Abstract

The invention belongs to the technical field of pharmaceutical compositions, and particularly relates to an antibacterial and hemostatic pharmaceutical composition for skin healing and a preparation method thereof. The pharmaceutical composition is double gel and is composed of oleogel and hydrogel according to the mass ratio of (3-5): (5-7), the oleogel takes medium-chain triglyceride and Xiawei wax as matrixes and loads astragaloside, and the hydrogel takes chitosan and sodium alginate as matrixes and loads antibacterial peptide GL13K. The preparation method comprises the following steps: preparing the CS-SA-GL13K hydrogel and the medium-chain triglyceride small single-tree wax-astragaloside oil gel, mixing the CS-SA-GL13K hydrogel and the medium-chain triglyceride small single-tree wax-astragaloside oil gel in proportion, homogenizing and emulsifying, and quickly cooling to obtain the double gel. Tests show that the sterilization rates of the pharmaceutical composition on staphylococcus aureus and escherichia coli are both greater than 95%, the hemostatic effect is good, skin wound healing can be promoted, and the stability is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug combinations, and in particular relates to an antibacterial and hemostatic drug composition for skin healing and a preparation method thereof. Background Art

[0002] As the body's largest organ, the skin undertakes core functions such as protection, barrier function, and immune defense. However, skin trauma is extremely common in daily life and various accidents. Following trauma, a series of serious problems follow, among which bleeding, infection, and delayed healing are the most prominent and urgently need to be addressed.

[0003] Bleeding caused by trauma varies, ranging from minor superficial abrasions to severe bleeding in deep tissues or organs. Traditional hemostatic materials, such as gauze, rely mainly on physical compression to slow bleeding, but their hemostatic effects are often unsatisfactory for oozing or deep wounds. Although biomaterials such as fibrin glue have certain hemostatic functions, they have problems such as high cost and complex preparation process, which limit their widespread clinical application. In addition, most of these traditional hemostatic materials lack antibacterial function and cannot effectively prevent infection during the hemostasis process, posing hidden dangers to subsequent treatment.

[0004] Open wounds are highly susceptible to pathogens in exposed environments, with Staphylococcus aureus and Escherichia coli being common pathogens. Once these bacteria multiply in wounds, they can cause severe infection, delaying wound healing and potentially leading to chronic infection or even life-threatening complications such as sepsis.

[0005] Traditional methods of anti-infection treatment have many limitations. Long-term use of antibiotics can easily lead to bacterial resistance, causing previously effective drugs to gradually lose their effectiveness. Moreover, antibiotics have poor permeability to pathogens within biofilms, making it difficult to completely eliminate pathogens hidden in biofilms. Chemical disinfectants such as povidone-iodine and ethanol, while broad-spectrum bactericidal, are also highly cytotoxic. They inhibit fibroblast migration and collagen deposition, both of which are crucial for tissue repair and regeneration during wound healing. Therefore, the use of chemical disinfectants often delays wound healing and is not conducive to patient recovery.

[0006] For some special wounds, such as those of diabetic patients and burn wounds, the healing failure rate is even higher. Diabetic patients suffer from metabolic disorders, leading to microcirculatory impairment and neuropathy. This leads to insufficient local nutrient supply and decreased nerve sensation in the wound, making it susceptible to trauma and difficult to heal. Burn wounds, on the other hand, experience a disrupted skin barrier, massive fluid loss, and are accompanied by inflammation and tissue necrosis, further increasing the difficulty of healing.

[0007] Currently, methods for promoting wound healing primarily include the use of growth factors and cell therapy. While growth factors can stimulate cell proliferation and differentiation, promoting tissue repair, they are relatively unstable, prone to inactivation in the complex environment of wounds, and expensive. Cell therapy, on the other hand, faces numerous challenges, including limited cell sources, ethical concerns, and immune rejection, limiting its widespread clinical application.

[0008] In summary, existing methods for hemostasis, antibacterial treatment, and wound healing promotion are mostly single-function treatments, lacking a comprehensive approach that simultaneously delivers rapid hemostasis, broad-spectrum antibacterial properties, high biocompatibility, and tissue regeneration. Some synthetic materials can also trigger inflammatory responses during application, hindering tissue regeneration and further increasing the complexity and risk of wound treatment.

[0009] Therefore, developing a novel antibacterial hemostatic pharmaceutical composition for skin healing to address the above-mentioned defects in the prior art and meet the clinical demand for comprehensive wound treatment has important practical significance and broad application prospects. Summary of the Invention

[0010] To solve the above technical problems, the present invention discovered through compounding research that the combination of antimicrobial peptides and astragaloside IV can enhance the antibacterial effect. Further through formulation research, a chitosan double-gel antibacterial hemostatic pharmaceutical composition containing the combination of antimicrobial peptides and astragaloside IV was developed. Experiments have verified that the antibacterial hemostatic pharmaceutical composition can promote skin wound healing.

[0011] On the one hand, the present invention provides an antibacterial and hemostatic pharmaceutical composition for skin healing, wherein the pharmaceutical composition is a double gel, which is composed of an oil gel and a hydrogel in a mass ratio of 3-5:5-7, the oil gel is loaded with astragalus methyl glycosides, and the hydrogel is loaded with an antimicrobial peptide, wherein the antimicrobial peptide is GL13K.

[0012] Furthermore, in the pharmaceutical composition, the mass percentage of GL13K in the hydrogel is 0.1% to 2%.

[0013] Furthermore, in the pharmaceutical composition, the hydrogel contains chitosan and sodium alginate as hydrogel matrices, and the mass ratio of chitosan to sodium alginate is 1.5-4:1.

[0014] Furthermore, in the pharmaceutical composition, the mass percentage of astragalus membranaceus in the oil gel is 0.004%~0.06%.

[0015] Furthermore, in the pharmaceutical composition, the oil gel uses medium-chain triglycerides and scutellaria baicalensis wax as the oil gel matrix.

[0016] In another aspect, a method for preparing the pharmaceutical composition of the present invention is provided, comprising the following steps: S1. Preparation of CS-SA-GL13K hydrogel: Chitosan solution and sodium alginate solution were mixed in a volume ratio of 1:1 to 2:1, and GL13K solution was added to prepare a mixed solution, wherein the final concentration of GL13K in the mixed solution was 0.1 to 2 mg / mL; the mixed solution was immersed in a 1% to 1.5% CaCl2 solution for cross-linking for 10 to 15 minutes, and then washed to obtain a CS-SA-GL13K hydrogel; S2. Preparation of medium-chain triglyceride Dictyophora japonica wax-astragaloside oil gel: dispersing astragaloside IV in medium chain triglycerides to obtain an astragaloside IV suspension; The medium chain triglyceride and the wax of the Chinese yew tree are melted and mixed at a mass ratio of 90-91:4-8, and the astragaloside IV suspension is added. After stirring uniformly, the mixture is cooled and solidified to prepare the medium chain triglyceride and the wax of the Chinese yew tree-astragaloside IV oil gel; S3. Preparation of double gel: The chain triglyceride Dictyophora japonica wax-astragaloside oleogel is heated to 50-60° C., mixed with the CS-SA-GL13K hydrogel in a volume ratio of 3:7-5:5, homogenized and emulsified, and then rapidly cooled to obtain the pharmaceutical composition.

[0017] Furthermore, in the preparation method, in the CS-SA-GL13K hydrogel, the mass volume percentage of the chitosan is 1.5% to 3.0%, and the mass volume percentage of the sodium alginate is 1.0% to 1.5%.

[0018] Furthermore, in the preparation method, the concentration of astragaloside IV in the oil gel is 2-5 mg / mL.

[0019] Furthermore, in the preparation method, the speed of the homogenization and emulsification is 8000-12000 rpm, and the time is 5-15 minutes.

[0020] Finally, the present invention also provides the use of the pharmaceutical composition described in the present invention in the preparation of hemostatic drugs or antibacterial drugs for skin wounds.

[0021] Compared with the prior art, the present invention has at least the following advantages or beneficial effects: (1) The antimicrobial peptide GL13K loaded in the pharmaceutical composition provided by the present invention works synergistically with astragaloside IV, achieving a sterilization rate exceeding 95% against common pathogens such as Staphylococcus aureus and Escherichia coli, effectively reducing the risk of wound infection.

[0022] (2) The pharmaceutical composition provided by the present invention comprises a combination of oleogel and hydrogel in a specific ratio, which has a synergistic hemostatic effect and significantly reduces the amount of bleeding in a liver bleeding model. The hemostatic effect is better than that of a single gel.

[0023] (3) In a mouse full-thickness skin wound model, the pharmaceutical composition provided by the present invention can accelerate wound healing, especially the pharmaceutical composition with a specific ratio has the best effect.

[0024] (4) The results of the accelerated test showed that the pharmaceutical composition provided by the present invention had no stratification phenomenon after 6 months of accelerated testing and had stable performance, indicating that it had good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The diagram shows the hemostatic effects of different drug compositions.

[0026] Figure 2 This is a graph showing the wound healing rate results of different drug compositions. DETAILED DESCRIPTION

[0027] The technical solutions of the present invention are described below with reference to the following embodiments; however, the present invention is not limited to the following embodiments.

[0028] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

[0029] The experimental methods and detection methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0030] Chitosan (CS): medical grade.

[0031] Sodium Alginate (SA): Medical grade.

[0032] GL13K: purity ≥95%, purchased from Nanjing Yuanpeptide Biotechnology Co., Ltd., the sequence of GL13K is GKIIKLKASLKLL.

[0033] Medium-chain triglycerides (MCT); pharmaceutical grade, having a viscosity of 20-50 mPa·s and an iodine value ≤1 gI² / 100 g.

[0034] Astragaloside IV: purity ≥98%, purchased from Nanjing Bencao Yikang Biotechnology Co., Ltd.

[0035] Cera alba: CAS number is 8006-44-8.

[0036] Example 1 This example is about preparing an antibacterial hemostatic pharmaceutical composition for skin healing.

[0037] 1.1 Preparation of CS-SA-GL13K hydrogel Preparation of chitosan (CS) solution: Accurately weigh 1.5 g of chitosan and add it to 100 mL of 1% acetic acid solution (i.e., 1 mL of acetic acid dissolved in 99 mL of deionized water). Place it on a magnetic stirrer and stir at 300-400 rpm at room temperature for 4 h to fully dissolve the chitosan.

[0038] Use a pH meter to measure the pH value of the solution, slowly adjust the pH to 6.5 with 1 mol / L sodium hydroxide solution, and continue stirring for 10-15 minutes to make the solution uniform.

[0039] Preparation of sodium alginate (SA) solution: Accurately weigh 1 g of sodium alginate and add it to 100 mL of deionized water. Place the mixture in a constant temperature water bath at 60°C and stir at 200-300 rpm for 1 h until the sodium alginate is completely dissolved to obtain a clear and transparent solution.

[0040] Preparation of GL13K solution: GL13K was accurately weighed and dissolved in PBS with a pH of 7.4 to prepare a GL13K stock solution with a concentration of 5 mg / mL.

[0041] Preparation of crosslinker solution: Accurately weigh 1 g of calcium chloride, add it to 100 mL of deionized water, and stir until completely dissolved to obtain a 1% (w / v) CaCl2 solution.

[0042] Mixture of CS and SA: The prepared CS solution and SA solution were mixed in a volume ratio of 1:1, placed on a magnetic stirrer, and stirred at a stirring speed of 300 rpm for 30 min to fully mix the two solutions.

[0043] Load GL13K: Slowly add the GL13K stock solution to the mixed CS-SA solution, stirring gently as you add to evenly disperse the GL13K in the solution to a final GL13K concentration of 0.1 mg / mL. Continue stirring gently for 10 minutes.

[0044] Ionic crosslinking: The above mixture was slowly injected into the silicone mold (be careful to avoid generating bubbles), and then the silicone mold containing the mixture was immersed in a 1% CaCl2 solution. Cross-linking was carried out at room temperature for 10 minutes to allow sodium alginate to undergo ionic cross-linking reaction with calcium ions to form a hydrogel.

[0045] After cross-linking was completed, the silicone mold was removed from the CaCl2 solution and gently washed three times with PBS buffer, each washing time was 10 min to remove unreacted calcium ions and other impurities to obtain CS-SA-GL13K hydrogel.

[0046] 1.2 Preparation of MCT-Ceratonia suffruticosa-astragaloside oil gel Preparation of Astragaloside IV Suspension: Astragaloside IV was accurately weighed and added to pharmaceutical grade MCT and ultrasonically dispersed in a water bath at 60°C for 30 min to preliminarily disperse astragaloside IV in MCT. The concentration of astragaloside IV was 2 mg / mL.

[0047] Melt mixing: Accurately weigh 90g of medical-grade MCT and 8g of Cetearyl styraciflua wax, put them into a container, and then place the container in a constant temperature water bath at 65°C to heat the Cetearyl styraciflua wax completely.

[0048] During the heating process, the mixture of pharmaceutical-grade MCT and Cercidiphyllum serrata was continuously stirred to ensure that the two were thoroughly mixed. Once the mixture was completely melted and homogeneous, 2g of the pre-prepared astragaloside IV suspension was added to the melted pharmaceutical-grade MCT-Cercidiphyllum serrata mixture. The mixture was stirred using a magnetic stirrer at a speed of 300-400 rpm until the solution was homogeneous, ensuring that the astragaloside IV was evenly dispersed in the oil phase.

[0049] Cooling and solidification: The uniformly stirred oleogel precursor solution was taken out from the constant temperature water bath and allowed to stand at room temperature for 30 minutes to allow the solution to gradually cool and solidify to form a semi-solid oleogel.

[0050] 1.3 Preparation of double gel Pre-emulsification: The prepared oil gel was heated to 50°C to make it have a certain fluidity.

[0051] Accurately measure the oleogel and hydrogel in a volume ratio of 3:7. Slowly add the oleogel to the hydrogel and start the homogenizer at 10,000 rpm for 5 minutes. During the homogenization process, ensure that the two gels are thoroughly mixed to form a preliminary emulsion.

[0052] Rapid cooling: The pre-emulsified mixture was quickly transferred to an ice bath and stirred at 200-300 rpm in the ice bath to rapidly cool the mixture. This yielded an antibacterial hemostatic pharmaceutical composition for skin healing, designated as Antibacterial Hemostatic Pharmaceutical Composition for Skin Healing #1.

[0053] Example 2 This example is about preparing an antibacterial hemostatic pharmaceutical composition for skin healing.

[0054] 2.1 Preparation of CS-SA-GL13K hydrogel Preparation of chitosan (CS) solution: Accurately weigh 2.0 g of chitosan and add it to 100 mL of 1% acetic acid solution (i.e., 1 mL of acetic acid dissolved in 99 mL of deionized water). Place it on a magnetic stirrer and stir at 300-400 rpm at room temperature for 4 h to fully dissolve the chitosan.

[0055] The pH value of the solution was measured using a pH meter, and the pH was slowly adjusted to 6.5 with 1 mol / L sodium hydroxide solution. Stirring was continued for 15 min to make the solution uniform.

[0056] Preparation of sodium alginate (SA) solution: Accurately weigh 1 g of sodium alginate, add it to 100 mL of deionized water, place it in a constant temperature water bath at 60°C, and stir at a stirring speed of 250 rpm for 1.5 h until the sodium alginate is completely dissolved to obtain a clear and transparent solution.

[0057] Preparation of GL13K solution: GL13K was accurately weighed and dissolved in PBS with a pH of 7.4 to prepare a GL13K stock solution with a concentration of 5 mg / mL.

[0058] Preparation of crosslinker solution: Accurately weigh 1 g of calcium chloride, add it to 100 mL of deionized water, and stir until completely dissolved to obtain a 1% (w / v) CaCl2 solution.

[0059] Mixture of CS and SA: The prepared CS solution and SA solution were mixed in a volume ratio of 2:1, placed on a magnetic stirrer, and stirred at a stirring speed of 400 rpm for 20 min to fully mix the two solutions.

[0060] Load GL13K: Slowly add the GL13K stock solution to the mixed CS-SA solution, stirring gently to evenly disperse the GL13K in the solution, ultimately achieving a final GL13K concentration of 0.5 mg / mL. Continue stirring gently for 15 minutes.

[0061] Ionic crosslinking: The above mixture was slowly injected into the silicone mold (be careful to avoid generating bubbles), and then the silicone mold containing the mixture was immersed in a 1% CaCl2 solution. Cross-linking was carried out at room temperature for 10 minutes to allow sodium alginate to undergo ionic cross-linking reaction with calcium ions to form a hydrogel.

[0062] After cross-linking was completed, the silicone mold was removed from the CaCl2 solution and gently washed three times with PBS buffer, each washing time was 10 min to remove unreacted calcium ions and other impurities to obtain CS-SA-GL13K hydrogel.

[0063] 2.2 Preparation of MCT-Ceratonia suffruticosa-astragaloside oil gel Preparation of Astragaloside IV Suspension: Astragaloside IV was accurately weighed and added to pharmaceutical grade MCT and ultrasonically dispersed in a water bath at 60°C for 30 min to preliminarily disperse astragaloside IV in MCT. The concentration of astragaloside IV was 5 mg / mL.

[0064] Melt mixing: Accurately weigh 91g of medical-grade MCT and 5g of Cetearyl Alcohol, put them into a container, and then place the container in a constant temperature water bath at 65°C to heat the Cetearyl Alcohol completely.

[0065] During the heating process, the mixture of pharmaceutical-grade MCT and C. edulis wax was continuously stirred to ensure that the two were thoroughly mixed. When the mixture was completely melted and uniform, 4 g of the pre-prepared astragaloside IV suspension was added to the melted pharmaceutical-grade MCT-C. edulis wax mixture and stirred at 400 rpm using a magnetic stirrer until the solution was homogeneous, ensuring that the astragaloside IV was evenly dispersed in the oil phase.

[0066] Cooling and solidification: The uniformly stirred oleogel precursor solution was taken out from the constant temperature water bath and allowed to stand at room temperature for 40 minutes to allow the solution to gradually cool and solidify to form a semi-solid oleogel.

[0067] 2.3 Preparation of double gel Pre-emulsification: The prepared oil gel was heated to 55°C to make it have a certain fluidity.

[0068] Accurately measure the oleogel and hydrogel in a 4:6 volume ratio. Slowly add the oleogel to the hydrogel and start the homogenizer at 8000 rpm for 15 minutes. During the homogenization process, ensure that the two gels are thoroughly mixed to form a preliminary emulsion.

[0069] Rapid cooling: The pre-emulsified mixture was quickly transferred to an ice bath and stirred at 300 rpm in the ice bath to rapidly cool the mixture. Thus, an antibacterial hemostatic pharmaceutical composition for skin healing was prepared, which was labeled as 2# antibacterial hemostatic pharmaceutical composition for skin healing.

[0070] Example 3 This example is about preparing an antibacterial hemostatic pharmaceutical composition for skin healing.

[0071] 3.1 Preparation of CS-SA-GL13K hydrogel Preparation of chitosan (CS) solution: Accurately weigh 3.0 g of chitosan and add it to 100 mL of 1% acetic acid solution (i.e., 1 mL of acetic acid dissolved in 99 mL of deionized water). Place it on a magnetic stirrer and stir at 500 rpm at room temperature for 6 h to fully dissolve the chitosan.

[0072] The pH value of the solution was measured using a pH meter, and the pH was slowly adjusted to 6.5 with 1 mol / L sodium hydroxide solution. Stirring was continued for 25 min to make the solution uniform.

[0073] Preparation of sodium alginate (SA) solution: Accurately weigh 1.5 g of sodium alginate, add it to 100 mL of deionized water, place it in a constant temperature water bath at 60°C, and stir at 300 rpm for 2.0 h until the sodium alginate is completely dissolved to obtain a clear and transparent solution.

[0074] Preparation of GL13K solution: GL13K was accurately weighed and dissolved in PBS with a pH of 7.4 to prepare a GL13K stock solution with a concentration of 5 mg / mL.

[0075] Preparation of crosslinker solution: Accurately weigh 2 g of calcium chloride, add it to 100 mL of deionized water, and stir until completely dissolved to obtain a 2% (w / v) CaCl2 solution.

[0076] Mixture of CS and SA: The prepared CS solution and SA solution were mixed in a volume ratio of 1:2, placed on a magnetic stirrer, and stirred at a stirring speed of 300 rpm for 10 min to fully mix the two solutions.

[0077] Load GL13K: Slowly add the GL13K stock solution to the mixed CS-SA solution, stirring gently as you add to evenly disperse the GL13K in the solution to a final GL13K concentration of 2 mg / mL. Continue stirring gently for 20 minutes.

[0078] Ionic crosslinking: The above mixture was slowly injected into the silicone mold (be careful to avoid generating bubbles), and then the silicone mold containing the mixture was immersed in a 1.5% CaCl2 solution and cross-linked at room temperature for 15 minutes to allow sodium alginate to undergo an ionic cross-linking reaction with calcium ions to form a hydrogel.

[0079] After cross-linking, the silicone mold was removed from the CaCl2 solution and gently washed three times with PBS buffer, each washing time for 15 min, to remove unreacted calcium ions and other impurities to obtain CS-SA-GL13K hydrogel.

[0080] 3.2 Preparation of MCT-Ceratonia suffruticosa-astragaloside oil gel Preparation of Astragaloside IV Suspension: Astragaloside IV was accurately weighed and added to pharmaceutical grade MCT and ultrasonically dispersed in a water bath at 60°C for 30 min to preliminarily disperse astragaloside IV in MCT. The concentration of astragaloside IV was 5 mg / mL.

[0081] Melt mixing: Accurately weigh 90g of medical-grade MCT and 4g of Cetearyl Alcohol, put them into a container, and then place the container in a constant temperature water bath at 65°C to heat the Cetearyl Alcohol completely.

[0082] During the heating process, the mixture of pharmaceutical-grade MCT and C. edulis wax was continuously stirred to ensure that the two were thoroughly mixed. When the mixture was completely melted and uniform, 6 g of the pre-prepared astragaloside IV suspension was added to the molten pharmaceutical-grade MCT-C. edulis wax mixture. The mixture was stirred at 350 rpm using a magnetic stirrer until the solution was homogeneous, ensuring that the astragaloside IV was evenly dispersed in the oil phase.

[0083] Cooling and solidification: The uniformly stirred oleogel precursor solution was taken out from the constant temperature water bath and allowed to stand at room temperature for 35 minutes to allow the solution to gradually cool and solidify to form a semi-solid oleogel.

[0084] 3.3 Preparation of double gel Pre-emulsification: The prepared oil gel was heated to 60°C to make it have a certain fluidity.

[0085] Accurately measure the oleogel and hydrogel in a 5:5 volume ratio. Slowly add the oleogel to the hydrogel and start the homogenizer at 12,000 rpm for 8 minutes. During the homogenization process, ensure that the two gels are thoroughly mixed to form a preliminary emulsion.

[0086] Rapid cooling: The pre-emulsified mixture was quickly transferred to an ice bath and stirred at 300 rpm in the ice bath to rapidly cool the mixture. Thus, an antibacterial hemostatic pharmaceutical composition for skin healing was prepared, which was labeled as antibacterial hemostatic pharmaceutical composition for skin healing #3.

[0087] Example 4 The only difference between this example and example 2 is that the volume ratio of the oil gel to the hydrogel is 6:4, and the final concentrations of the antimicrobial peptide and astragaloside IV are the same as those in example 2. The prepared antimicrobial hemostatic pharmaceutical composition for skin healing is labeled as 4# antimicrobial hemostatic pharmaceutical composition for skin healing.

[0088] Example 5 The only difference between this example and example 2 is that the volume ratio of the oil gel to the hydrogel is 5:5, and the final concentrations of the antimicrobial peptide and astragaloside IV are the same as those in example 2. The prepared antimicrobial hemostatic pharmaceutical composition for skin healing is labeled as 5# antimicrobial hemostatic pharmaceutical composition for skin healing.

[0089] Test Example 1 This example is to test the antibacterial properties of the antibacterial hemostatic pharmaceutical compositions for skin healing of Examples 1 to 5.

[0090] Test pathogens: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922).

[0091] Test samples: 1#~5# antibacterial hemostatic pharmaceutical compositions for skin healing (abbreviated as 1#~5# pharmaceutical compositions), the hydrogel and oil gel prepared in Example 3 were used as control group 1 and control group 2, respectively, and a liquid culture medium was set as a negative control.

[0092] Add 1 mL of liquid culture medium from each test group gel, control group gel and negative control group to the 24-well plate. Add 10 μL (10 6 ) Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) suspensions were placed in a 37°C incubator and incubated for 24 hours. The bactericidal rate of each treatment was calculated using the viable count method. The bactericidal rate was calculated as follows:

[0093] Bactericidal rate = (live bacteria concentration in negative control group - live bacteria concentration in test group) / live bacteria concentration in negative control group × 100% The sterilization rates of each treatment are shown in Table 1.

[0094] Table 1 Statistical results of sterilization rates of different gels

[0095] As shown in Table 1, the antimicrobial peptide GL13K loaded in the hydrogel and the astragaloside loaded in the oil gel have a synergistic bactericidal effect. The bactericidal effect of the 2# drug composition on Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) is much higher than that of the hydrogel and oil gel. The antibacterial rates of the 1#~5# drug compositions on Staphylococcus aureus and Escherichia coli are all greater than 95%.

[0096] Test Example 2 This example is to test the hemostatic performance of the antibacterial hemostatic pharmaceutical composition for skin healing of Examples 1 to 5.

[0097] Test samples: 1#~5# antibacterial hemostatic pharmaceutical compositions for skin healing (abbreviated as 1#~5# pharmaceutical compositions), the hydrogel and oil gel prepared in Example 3 were used as control group 1 and control group 2, respectively, and a free bleeding blank control group was also set up.

[0098] A liver bleeding model was used to evaluate the hemostatic ability of each test sample. Three replicates were set for each treatment, and all animal experiments were performed in accordance with the National Guide for the Care and Use of Laboratory Animals. After anesthesia, the mice were fixed on a surgical board tilted at 30°. The liver of the mouse was exposed through an abdominal incision, and the tissue fluid around the liver was carefully removed. The liver was then placed on a pre-weighed filter paper (W0). Use scissors to make a 0.5 cm long wound in the left lobe of the liver 0.3 cm away from the lower edge of the liver. The liver bleeding wound was then immediately covered with each test sample. The control group was free bleeding and did not require treatment. After 3 minutes of hemostasis, the weight of the filter paper that absorbed the blood was measured (W). All groups repeated the measurement three times. The amount of bleeding was calculated according to the following formula:

[0099] Liver bleeding volume = W – W0 The test results are as follows Figure 1 As shown, the hemostatic effect of the antibacterial hemostatic drug composition for skin healing 1#~5# was significantly better than that of the blank control group, the oil gel had a weak hemostatic effect compared with the blank control group, and the hemostatic effect of the antibacterial hemostatic drug for skin healing 1#, 2#, and 4# was significantly better than that of the hydrogel and oil gel alone, indicating that the oil gel and hydrogel have a synergistic hemostatic effect when the ratio is in the range of 3~4:6~7.

[0100] Test Example 3 This test example is to test the skin healing effects of the antibacterial hemostatic pharmaceutical compositions for skin healing of Examples 1 to 5.

[0101] Test samples: 1#~5# antibacterial hemostatic pharmaceutical compositions for skin healing (abbreviated as 1#~5# pharmaceutical compositions), the hydrogel and oil gel prepared in Example 3 were used as control group 1 and control group 2, respectively, and a blank control group was set up (the blank control was natural healing).

[0102] A mouse full-thickness skin wound model was used to evaluate the in vivo wound healing effect of the hydrogel. All animal experiments were conducted in accordance with the National Guidelines for the Care and Use of Laboratory Animals. Twenty-four male ICR mice aged 6 to 8 weeks were selected for wound healing studies. The day before the experiment, the hair on the back of the mice was completely removed using a depilatory cream. On the day of the experiment, after anesthetizing the mice, a full-thickness excision wound with a diameter of 7 mm was created on the back skin of each mouse. Secondly, the mice were randomly divided into 8 groups: 1#~5# drug composition group, hydrogel group, oil gel group,

[0103] In the control group, each test sample was used on days 0 to 2, and the wound size was calculated using ImageJ software at 0, 3, 6, and 9 post-injury. The wound healing rate was calculated using the following formula:

[0104] Wound healing rate = (A0–At) / A0 Where A0 is the wound area, At is the wound area after a fixed time interval. Figure 2 As shown. Figure 2 It can be seen that the wound healing effect of the antibacterial hemostatic pharmaceutical composition for skin healing 1#~5# is significantly better than that of the control group, indicating that the antibacterial hemostatic pharmaceutical composition for skin healing 1#~5# provided by the present invention can be used for skin wound healing, especially the antibacterial hemostatic pharmaceutical composition for skin healing 2# has the best effect.

[0105] Test Example 4 This test is to test the accelerated stability of 1#~5# antibacterial hemostatic pharmaceutical compositions for skin healing.

[0106] The accelerated stability of the antibacterial hemostatic pharmaceutical compositions for skin healing described in Examples 1# to 5# was tested. Samples were collected at 0, 1, 2, 3, and 6 months, respectively. The appearance of each test sample was observed for uniformity and the presence of stratification. The antibacterial activity was determined, and the coagulation performance was tested using an in vitro coagulation assay. The results of the accelerated stability test are shown in Table 2.

[0107] Table 2 Accelerated stability test results of the antibacterial hemostatic pharmaceutical composition for skin healing

[0108] As shown in Table 2, the appearance of the antibacterial hemostatic pharmaceutical compositions for skin healing #1~5# remained uniform and free of stratification after 6 months of accelerated testing, and the antibacterial and coagulation properties did not show significant attenuation, indicating that the antibacterial hemostatic pharmaceutical compositions for skin healing #1~5# had good stability.

[0109] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. A pharmaceutical composition for antibacterial hemostasis for skin healing, characterized in that: The pharmaceutical composition is a double gel, which is composed of an oil gel and a hydrogel in a mass ratio of 3-5:5-7. The oil gel is loaded with astragalus methyl ester, and the hydrogel is loaded with an antimicrobial peptide, and the antimicrobial peptide is GL13K.

2. The pharmaceutical composition according to claim 1, characterized in that The mass percentage of GL13K in the hydrogel is 0.1% to 2%.

3. The pharmaceutical composition according to claim 1, characterized in that The hydrogel uses chitosan and sodium alginate as hydrogel matrices, and the mass ratio of the chitosan to the sodium alginate is 1.5-4:

1.

4. The pharmaceutical composition according to claim 1, characterized in that The mass percentage of astragalin in the oil gel is 0.004% to 0.06%.

5. The pharmaceutical composition according to claim 1, characterized in that The oil gel uses medium-chain triglyceride and euphorbia cerifera wax as the oil gel matrix.

6. The method for preparing the pharmaceutical composition according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Preparation of CS-SA-GL13K hydrogel: Chitosan solution and sodium alginate solution were mixed in a volume ratio of 1:1-2:1, and GL13K solution was added to prepare a mixed solution, wherein the final concentration of GL13K in the mixed solution was 0.1-2 mg / mL; the mixed solution was immersed in a 1%-1.5% CaCl2 solution for cross-linking for 10-15 min, and then washed to obtain a CS-SA-GL13K hydrogel; S2. Preparation of medium-chain triglyceride Dictyophora japonica wax-astragaloside oil gel: dispersing astragaloside IV in medium chain triglycerides to obtain an astragaloside IV suspension; The medium chain triglyceride and the wax of the Chinese yew tree are melted and mixed at a mass ratio of 90-91:4-8, and the astragaloside IV suspension is added. After stirring uniformly, the mixture is cooled and solidified to prepare the medium chain triglyceride and the wax of the Chinese yew tree-astragaloside IV oil gel; S3. Preparation of double gel: The chain triglyceride Dictyophora japonica wax-astragaloside oleogel is heated to 50-60° C., mixed with the CS-SA-GL13K hydrogel in a volume ratio of 3:7-5:5, homogenized and emulsified, and then rapidly cooled to obtain the pharmaceutical composition.

7. The preparation method according to claim 6, characterized in that In the CS-SA-GL13K hydrogel, the mass volume percentage of the chitosan is 1.5% to 3.0%, and the mass volume percentage of the sodium alginate is 1.0% to 1.5%.

8. The preparation method according to claim 6, characterized in that The concentration of astragaloside IV in the oil gel is 2-5 mg / mL.

9. The preparation method according to claim 6, characterized in that The homogenization and emulsification speed is 8000-12000 rpm, and the time is 5-15 min.

10. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of hemostatic drugs or antibacterial drugs for skin wounds.

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