An antibacterial and hemostatic drug composition for skin healing and its preparation method

Abstract

This invention belongs to the field of pharmaceutical combination technology, specifically relating to an antibacterial and hemostatic pharmaceutical composition for skin healing and its preparation method. The pharmaceutical composition is a dual-gel, consisting of an oleogel and a hydrogel in a mass ratio of 3-5:5-7. The oleogel uses medium-chain triglycerides and *Ipomoea aquatica* wax as a matrix and is loaded with astragaloside A, while the hydrogel uses chitosan and sodium alginate as a matrix and is loaded with the antimicrobial peptide GL13K. The preparation method includes preparing a CS-SA-GL13K hydrogel and a medium-chain triglyceride *Ipomoea aquatica* wax-astragaloside A oleogel, then mixing the two in a specific ratio, homogenizing and emulsifying, and rapidly cooling to obtain the dual-gel. Testing showed that the pharmaceutical composition exhibits a bactericidal rate of over 95% against Staphylococcus aureus and Escherichia coli, good hemostatic effect, promotes skin wound healing, and demonstrates good stability.

Description

Technical Field

[0001] This invention belongs to the field of drug combination technology, specifically relating to an antibacterial and hemostatic drug composition for skin healing and its preparation method. Background Art

[0002] As the largest organ in the human body, the skin performs core functions such as protection, barrier function, and immune defense. However, skin injuries are extremely common in daily life and various accidents. After an injury occurs, a series of serious problems follow, among which bleeding, infection, and delayed healing are the most prominent and urgent challenges to be addressed.

[0003] Trauma-induced bleeding presents in various forms, ranging from minor superficial abrasions to severe bleeding in deep tissues or organs. Traditional hemostatic materials, such as gauze, primarily rely on physical pressure to slow bleeding, but their effectiveness is often unsatisfactory for oozing or deep wounds. While biomaterials like fibrin glue possess some hemostatic properties, their high cost and complex manufacturing processes limit their widespread clinical application. Furthermore, most of these traditional hemostatic materials lack antibacterial properties, failing to effectively prevent infection during hemostasis and posing risks to subsequent treatment.

[0004] Open wounds are highly susceptible to pathogens in exposed environments, with Staphylococcus aureus and Escherichia coli being common causative agents. Once these bacteria proliferate at the wound site, they can cause severe infection symptoms, not only delaying the wound healing process but also potentially leading to life-threatening complications such as chronic infection and even sepsis.

[0005] Traditional methods of anti-infective treatment have many limitations. Long-term use of antibiotics easily leads to bacterial resistance, causing initially effective drugs to gradually lose their efficacy. Furthermore, antibiotics have poor penetration into biofilms, making it difficult to completely eliminate pathogens hidden within them. Chemical disinfectants such as povidone-iodine and ethanol, while possessing broad-spectrum bactericidal properties, exhibit significant cytotoxicity. They inhibit fibroblast migration and collagen deposition, both crucial for tissue repair and regeneration during wound healing. Therefore, the use of chemical disinfectants often delays wound healing, hindering patient recovery.

[0006] For certain types of wounds, such as those in diabetic patients or burn wounds, the healing failure rate is even higher. Diabetic patients, due to metabolic disorders, experience microcirculatory disturbances and neuropathy, leading to insufficient local nutrient supply and reduced nerve sensation in the wound area, making them more susceptible to injury and hindering healing. Burn wounds, on the other hand, suffer from impaired skin barrier function, significant fluid loss, and are accompanied by inflammatory reactions and tissue necrosis, further increasing the difficulty of healing.

[0007] Currently, methods to promote wound healing mainly include the use of growth factors and cell therapy. While growth factors can stimulate cell proliferation and differentiation and promote tissue repair, they have poor stability, are easily inactivated in the complex environment of wounds, and are expensive. Cell therapy, on the other hand, faces many challenges such as limited cell sources, ethical issues, and immune rejection, which restricts its widespread clinical application.

[0008] In summary, most existing methods for hemostasis, antibacterial treatment, and wound healing promotion are single-function therapies, lacking a comprehensive treatment approach that can simultaneously achieve rapid hemostasis, broad-spectrum antibacterial activity, high biocompatibility, and tissue regeneration promotion. Furthermore, some synthetic materials may trigger inflammatory reactions during application, hindering tissue regeneration and further increasing the complexity and risks of wound treatment.

[0009] Therefore, developing a novel antibacterial and hemostatic drug composition for skin healing to overcome the aforementioned deficiencies in existing technologies and meet the clinical needs for comprehensive wound treatment is of significant practical importance and has broad application prospects. Summary of the Invention

[0010] To solve the above-mentioned technical problems, this invention discovered through compounding studies that the combination of antimicrobial peptides and astragaloside A can enhance the antimicrobial effect. Further through formulation research, a chitosan double gel antimicrobial and hemostatic drug composition containing the combination of antimicrobial peptides and astragaloside A was developed. Experimental verification showed that this antimicrobial and hemostatic drug composition can promote the healing of skin wounds.

[0011] On one hand, the present invention provides a pharmaceutical composition for skin healing and antibacterial hemostasis, the pharmaceutical composition being a dual gel, the dual gel being composed of an oleogel and a hydrogel in a mass ratio of 3~5:5~7, the oleogel being loaded with astragaloside A, and the hydrogel being loaded with an antimicrobial peptide, the antimicrobial peptide being 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 uses chitosan and sodium alginate as the hydrogel matrix, and the mass ratio of chitosan to sodium alginate is 1.5~4:1.

[0014] Furthermore, in the pharmaceutical composition, the mass percentage of astragaloside A in the oleogel is 0.004% to 0.06%.

[0015] Furthermore, in the pharmaceutical composition, the oleogel uses medium-chain triglycerides and small-leaf eugenol wax as the oleogel matrix.

[0016] Furthermore, a method for preparing the pharmaceutical composition described in this invention is also provided, comprising the following steps:

[0017] S1. Preparation of CS-SA-GL13K hydrogel:

[0018] Chitosan solution and sodium alginate solution were mixed at a volume ratio of 1:1 to 2:1, and GL13K solution was added to prepare a mixed solution with a final concentration of GL13K of 0.1 to 2 mg / mL. The mixed solution was then immersed in 1% to 1.5% CaCl2 solution for crosslinking for 10 to 15 min, and washed to obtain CS-SA-GL13K hydrogel.

[0019] S2. Preparation of medium-chain triglyceride *Solanum lyratum* wax-astragaloside A oil gel:

[0020] Astragaloside A was dispersed in medium-chain triglycerides to obtain an astragaloside A suspension;

[0021] Medium-chain triglycerides and *Ipomoea aquatica* wax were melt-mixed at a mass ratio of 90-91:4-8, and astragaloside A suspension was added. After stirring evenly and cooling to solidify, medium-chain triglyceride *Ipomoea aquatica* wax-astragaloside A oil gel was obtained.

[0022] S3. Preparation of the double gel:

[0023] The medium-chain triglyceride *Solanum lyratum* wax-astragaloside A oil gel was heated to 50-60°C and mixed with CS-SA-GL13K hydrogel at a volume ratio of 3:7-5:5. After homogenization and emulsification, the mixture was rapidly cooled to obtain the pharmaceutical composition.

[0024] In the further described preparation method, the chitosan in the CS-SA-GL13K hydrogel has a mass-volume percentage of 1.5% to 3.0%, and the sodium alginate has a mass-volume percentage of 1.0% to 1.5%.

[0025] Furthermore, in the preparation method, the concentration of astragaloside A in the oleogel is 2~5 mg / mL.

[0026] Furthermore, in the preparation method, the homogenization emulsification speed is 8000~12000 rpm and the time is 5~15 min.

[0027] Finally, the invention also provides the use of the pharmaceutical composition described herein in the preparation of hemostatic or antibacterial drugs for skin wounds.

[0028] Compared with the prior art, the present invention has at least the following advantages or beneficial effects:

[0029] (1) The antimicrobial peptide GL13K loaded in the pharmaceutical composition provided by the present invention works synergistically with astragaloside A to kill more than 95% of common pathogens such as Staphylococcus aureus and Escherichia coli, effectively reducing the risk of wound infection.

[0030] (2) The combination of oleogel and hydrogel in the pharmaceutical composition provided by the present invention has a synergistic hemostatic effect, significantly reduces the amount of bleeding in the liver bleeding model, and the hemostatic effect is better than that of a single gel.

[0031] (3) In a mouse full-thickness skin wound model, the drug composition provided by the present invention can accelerate wound healing, especially the drug composition with a specific ratio has the best effect.

[0032] (4) The results of accelerated testing showed that the pharmaceutical composition provided by the present invention did not exhibit stratification after 6 months of accelerated testing and its performance remained stable, indicating that it has good stability. Attached Figure Description

[0033] Figure 1 Graphs showing the hemostatic effects of different drug combinations.

[0034] Figure 2 The graph shows the wound healing rate results for different drug combinations. Detailed Implementation

[0035] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0036] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

[0037] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0038] Chitosan (CS): Medical grade.

[0039] Sodium alginate (SA): Medical grade.

[0040] GL13K: purity ≥95%, purchased from Nanjing Yuantai Biotechnology Co., Ltd., the sequence of which is GKIIKLKASLKLL.

[0041] Medium-chain triglycerides (MCTs); pharmaceutical grade, wherein the medium-chain triglycerides have a viscosity of 20~50 mPa·s and an iodine value ≤1gI2 / 100g.

[0042] Astragaloside A: purity ≥98%, purchased from Nanjing Bencao Yikang Biotechnology Co., Ltd.

[0043] Small single-tree wax: CAS number is 8006-44-8.

[0044] Example 1

[0045] This example demonstrates the preparation of an antibacterial and hemostatic drug composition for skin healing.

[0046] 1.1 Preparation of CS-SA-GL13K hydrogel

[0047] Preparation of chitosan (CS) solution:

[0048] Accurately weigh 1.5g of chitosan and add it to 100mL of 1% acetic acid solution (i.e., 1mL of acetic acid dissolved in 99mL of deionized water). Place the solution on a magnetic stirrer and stir at 300~400rpm for 4 hours at room temperature to allow the chitosan to dissolve completely.

[0049] Use a pH meter to measure the pH value of the solution, and slowly adjust the pH to 6.5 with 1 mol / L sodium hydroxide solution. Continue stirring for 10-15 minutes to make the solution homogeneous.

[0050] Preparation of sodium alginate (SA) solution:

[0051] Accurately weigh 1g of sodium alginate, add it to 100 mL of deionized water, place it in a constant temperature water bath at 60℃, and stir at a stirring speed of 200~300rpm for 1 hour until the sodium alginate is completely dissolved, resulting in a clear and transparent solution.

[0052] Preparation of GL13K solution:

[0053] Accurately weigh GL13K and dissolve it in PBS with a pH of 7.4 to prepare a GL13K stock solution with a concentration of 5 mg / mL.

[0054] Preparation of crosslinking agent solution:

[0055] Accurately weigh 1g of calcium chloride, add it to 100mL of deionized water, and stir until completely dissolved to obtain a 1% (w / v) CaCl2 solution.

[0056] A mixture of CS and SA:

[0057] Mix the prepared CS solution and SA solution at a volume ratio of 1:1, place them on a magnetic stirrer, and stir at 300 rpm for 30 minutes to ensure that the two solutions are fully mixed.

[0058] Load GL13K:

[0059] Slowly add the GL13K stock solution to the mixed CS-SA solution while gently stirring to ensure uniform dispersion of GL13K in the solution, until a final concentration of GL13K of 0.1 mg / mL is achieved. Then continue stirring gently for 10 minutes.

[0060] Ionic crosslinking:

[0061] Slowly inject the above mixture into a silicone mold (be careful to avoid generating air bubbles), then immerse the silicone mold containing the mixture in a 1% CaCl2 solution and crosslink it at room temperature for 10 minutes to allow sodium alginate to undergo an ionic crosslinking reaction with calcium ions to form a hydrogel.

[0062] After crosslinking, the silicone mold was removed from the CaCl2 solution and gently washed three times with PBS buffer for 10 minutes each time to remove unreacted calcium ions and other impurities, thus obtaining CS-SA-GL13K hydrogel.

[0063] 1.2 Preparation of MCT-Small Tree Wax-Astragaloside A Oil Gel

[0064] Preparation of astragaloside A suspension:

[0065] Accurately weigh astragaloside A and add it to pharmaceutical-grade MCT. Disperse the astragaloside A in a 60°C water bath for 30 minutes using ultrasonication to initially disperse the astragaloside A in the MCT. The concentration of astragaloside A is 2 mg / mL.

[0066] Melt mixing:

[0067] Accurately weigh 90g of pharmaceutical-grade MCT and 8g of small tree wax, put them into a container, and then place the container in a constant temperature water bath at 65℃ to heat until the small tree wax is completely melted.

[0068] During heating, continuously stir the mixture of pharmaceutical-grade MCT and small tree wax to ensure thorough and uniform mixing. Once the mixture is completely melted and homogeneous, add 2g of pre-prepared astragaloside A suspension to the molten pharmaceutical-grade MCT-small tree wax mixture, and continue stirring with a magnetic stirrer at a speed of 300-400 rpm until the solution is homogeneous, ensuring that astragaloside A is uniformly dispersed in the oil phase.

[0069] Cooling and curing:

[0070] Remove the well-stirred oleogel precursor solution from the constant temperature water bath and let it stand at room temperature for 30 minutes to allow the solution to gradually cool and solidify, forming a semi-solid oleogel.

[0071] 1.3 Preparation of bigels

[0072] Pre-emulsification:

[0073] The prepared olegel is heated to 50°C to give it a certain degree of fluidity.

[0074] Accurately measure the oleogel and hydrogel at a volume ratio of 3:7. Slowly add the oleogel to the hydrogel while simultaneously starting a homogenizer and homogenizing at 10,000 rpm for 5 minutes. During homogenization, ensure the two gels are thoroughly mixed to form a preliminary emulsion.

[0075] Rapid cooling:

[0076] The pre-emulsified mixture was rapidly transferred to an ice bath apparatus and stirred at 200-300 rpm to rapidly cool the mixture. This yielded an antibacterial and hemostatic drug composition for skin healing, labeled as #1.

[0077] Example 2

[0078] This example demonstrates the preparation of an antibacterial and hemostatic drug composition for skin healing.

[0079] 2.1 Preparation of CS-SA-GL13K hydrogel

[0080] Preparation of chitosan (CS) solution:

[0081] Accurately weigh 2.0g of chitosan and add it to 100mL of 1% acetic acid solution (i.e., 1mL of acetic acid dissolved in 99mL of deionized water). Place the solution on a magnetic stirrer and stir at 300~400rpm for 4 hours at room temperature to allow the chitosan to dissolve completely.

[0082] 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 15 minutes to make the solution homogeneous.

[0083] Preparation of sodium alginate (SA) solution:

[0084] Accurately weigh 1g of sodium alginate, add it to 100 mL of deionized water, place it in a constant temperature water bath at 60℃, and stir at 250 rpm for 1.5 h until the sodium alginate is completely dissolved, resulting in a clear and transparent solution.

[0085] Preparation of GL13K solution:

[0086] Accurately weigh GL13K and dissolve it in PBS with a pH of 7.4 to prepare a GL13K stock solution with a concentration of 5 mg / mL.

[0087] Preparation of crosslinking agent solution:

[0088] Accurately weigh 1g of calcium chloride, add it to 100mL of deionized water, and stir until completely dissolved to obtain a 1% (w / v) CaCl2 solution.

[0089] A mixture of CS and SA:

[0090] Mix the prepared CS solution and SA solution at a volume ratio of 2:1, place them on a magnetic stirrer, and stir at 400 rpm for 20 minutes to ensure that the two solutions are fully mixed.

[0091] Load GL13K:

[0092] Slowly add the GL13K stock solution to the mixed CS-SA solution while gently stirring to ensure uniform dispersion of GL13K in the solution, until a final concentration of GL13K of 0.5 mg / mL is achieved. Then continue stirring gently for 15 min.

[0093] Ionic crosslinking:

[0094] Slowly inject the above mixture into a silicone mold (be careful to avoid generating air bubbles), then immerse the silicone mold containing the mixture in a 1% CaCl2 solution and crosslink it at room temperature for 10 minutes to allow sodium alginate to undergo an ionic crosslinking reaction with calcium ions to form a hydrogel.

[0095] After crosslinking, the silicone mold was removed from the CaCl2 solution and gently washed three times with PBS buffer for 10 minutes each time to remove unreacted calcium ions and other impurities, thus obtaining CS-SA-GL13K hydrogel.

[0096] 2.2 Preparation of MCT-Small Tree Wax-Astragaloside A Oil Gel

[0097] Preparation of astragaloside A suspension:

[0098] Accurately weigh astragaloside A and add it to pharmaceutical-grade MCT. Disperse the astragaloside A in a 60°C water bath for 30 minutes using ultrasonication to initially disperse the astragaloside A in the MCT. The concentration of astragaloside A is 5 mg / mL.

[0099] Melt mixing:

[0100] Accurately weigh 91g of pharmaceutical-grade MCT and 5g of small tree wax, put them into a container, and then place the container in a constant temperature water bath at 65℃ to heat until the small tree wax is completely melted.

[0101] During the heating process, continuously stir the mixture of pharmaceutical-grade MCT and small tree wax to ensure thorough and uniform mixing. Once the mixture is completely melted and homogeneous, add 4g of pre-prepared astragaloside A suspension to the molten pharmaceutical-grade MCT-small tree wax mixture, and continue stirring with a magnetic stirrer at 400rpm until the solution is homogeneous, ensuring that astragaloside A is uniformly dispersed in the oil phase.

[0102] Cooling and curing:

[0103] Remove the well-stirred oleogel precursor solution from the constant temperature water bath and let it stand at room temperature for 40 minutes to allow the solution to gradually cool and solidify, forming a semi-solid oleogel.

[0104] 2.3 Preparation of bigels

[0105] Pre-emulsification:

[0106] The prepared olegel was heated to 55°C to give it a certain degree of fluidity.

[0107] Accurately measure the oleogel and hydrogel at a volume ratio of 4:6. Slowly add the oleogel to the hydrogel while simultaneously starting a homogenizer and homogenizing at 8000 rpm for 15 minutes. During homogenization, ensure the two gels are thoroughly mixed to form a preliminary emulsion.

[0108] Rapid cooling:

[0109] The pre-emulsified mixture was rapidly transferred to an ice bath apparatus and stirred at 300 rpm to rapidly cool the mixture. This yielded an antibacterial and hemostatic drug composition for skin healing, labeled as #2.

[0110] Example 3

[0111] This example demonstrates the preparation of an antibacterial and hemostatic drug composition for skin healing.

[0112] 3.1 Preparation of CS-SA-GL13K hydrogel

[0113] Preparation of chitosan (CS) solution:

[0114] Accurately weigh 3.0g of chitosan and add it to 100mL of 1% acetic acid solution (i.e., 1mL of acetic acid dissolved in 99mL of deionized water). Place the solution on a magnetic stirrer and stir at 500rpm for 6 hours at room temperature to allow the chitosan to dissolve completely.

[0115] Use a pH meter to measure the pH value of the solution, and slowly adjust the pH to 6.5 with 1 mol / L sodium hydroxide solution. Continue stirring for 25 minutes to make the solution homogeneous.

[0116] Preparation of sodium alginate (SA) solution:

[0117] Accurately weigh 1.5g of sodium alginate, add it to 100 mL of deionized water, place it in a constant temperature water bath at 60℃, and stir at 300 rpm for 2.0 h until the sodium alginate is completely dissolved, resulting in a clear and transparent solution.

[0118] Preparation of GL13K solution:

[0119] Accurately weigh GL13K and dissolve it in PBS with a pH of 7.4 to prepare a GL13K stock solution with a concentration of 5 mg / mL.

[0120] Preparation of crosslinking agent solution:

[0121] Accurately weigh 2g of calcium chloride, add it to 100mL of deionized water, and stir until completely dissolved to obtain a 2% (w / v) CaCl2 solution.

[0122] A mixture of CS and SA:

[0123] Mix the prepared CS solution and SA solution at a volume ratio of 1:2, place them on a magnetic stirrer, and stir at 300 rpm for 10 minutes to ensure that the two solutions are fully mixed.

[0124] Load GL13K:

[0125] Slowly add the GL13K stock solution to the mixed CS-SA solution while gently stirring to ensure uniform dispersion of GL13K in the solution, until a final concentration of GL13K of 2 mg / mL is achieved. Then continue stirring gently for 20 min.

[0126] Ionic crosslinking:

[0127] Slowly inject the above mixture into a silicone mold (be careful to avoid generating air bubbles), then immerse the silicone mold containing the mixture in a 1.5% CaCl2 solution and crosslink it at room temperature for 15 minutes to allow sodium alginate to undergo an ionic crosslinking reaction with calcium ions to form a hydrogel.

[0128] After crosslinking, the silicone mold was removed from the CaCl2 solution and gently washed three times with PBS buffer for 15 minutes each time to remove unreacted calcium ions and other impurities, thus obtaining CS-SA-GL13K hydrogel.

[0129] 3.2 Preparation of MCT-Small Tree Wax-Astragaloside A Oil Gel

[0130] Preparation of astragaloside A suspension:

[0131] Accurately weigh astragaloside A and add it to pharmaceutical-grade MCT. Disperse the astragaloside A in a 60°C water bath for 30 minutes using ultrasonication to initially disperse the astragaloside A in the MCT. The concentration of astragaloside A is 5 mg / mL.

[0132] Melt mixing:

[0133] Accurately weigh 90g of pharmaceutical-grade MCT and 4g of small tree wax, put them into a container, and then place the container in a constant temperature water bath at 65℃ to heat until the small tree wax is completely melted.

[0134] During the heating process, continuously stir the mixture of pharmaceutical-grade MCT and small tree wax to ensure thorough and uniform mixing. Once the mixture is completely melted and homogeneous, add 6g of pre-prepared astragaloside A suspension to the molten pharmaceutical-grade MCT-small tree wax mixture, and continue stirring with a magnetic stirrer at 350rpm until the solution is homogeneous, ensuring that astragaloside A is uniformly dispersed in the oil phase.

[0135] Cooling and curing:

[0136] Remove the well-stirred oleogel precursor solution from the constant temperature water bath and let it stand at room temperature for 35 minutes to allow the solution to gradually cool and solidify, forming a semi-solid oleogel.

[0137] 3.3 Preparation of bigels

[0138] Pre-emulsification:

[0139] The prepared olegel was heated to 60°C to give it a certain degree of fluidity.

[0140] Accurately measure the oleogel and hydrogel at a volume ratio of 5:5. Slowly add the oleogel to the hydrogel while simultaneously starting a homogenizer and homogenizing at 12,000 rpm for 8 minutes. During homogenization, ensure the two gels are thoroughly mixed to form a preliminary emulsion.

[0141] Rapid cooling:

[0142] The pre-emulsified mixture was rapidly transferred to an ice bath apparatus and stirred at 300 rpm to rapidly cool the mixture. This yielded an antibacterial and hemostatic drug composition for skin healing, labeled as #3 Antibacterial and Hemostatic Drug Composition for Skin Healing.

[0143] Example 4

[0144] The only difference between this embodiment and Example 2 is that the volume ratio of oleogel to hydrogel is 6:4, and the final concentrations of antimicrobial peptide and astragaloside A are the same as in Example 2. The resulting antimicrobial hemostatic drug composition for skin healing is labeled as #4.

[0145] Example 5

[0146] The only difference between this embodiment and Example 2 is that the volume ratio of oleogel to hydrogel is 5:5, and the final concentrations of antimicrobial peptide and astragaloside A are the same as in Example 2. The resulting antimicrobial hemostatic drug composition for skin healing is labeled as 5# Antimicrobial hemostatic drug composition for skin healing.

[0147] Test Example 1

[0148] This embodiment tests the antibacterial properties of the antibacterial and hemostatic drug compositions for skin healing used in Examples 1 to 5.

[0149] Pathogens tested: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922).

[0150] Test samples: 1#~5# antibacterial and hemostatic drug compositions for skin healing (referred to as 1#~5# drug compositions), the hydrogel and oleogel prepared in Example 3 were used as control group 1 and control group 2, respectively, and liquid culture medium was set as a negative control.

[0151] Add 1 mL of liquid culture medium to each well of a 24-well plate, along with the gels of each experimental group, the control group, and the negative control group. Add 10 μL (10 μL of liquid culture medium) to the surface of each gel and control group. 6 Suspensions of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were incubated in petri dishes at 37°C for 24 hours. The bactericidal rate of each treatment was determined using a viable count method. The formula for calculating the bactericidal rate is as follows:

[0152] Sterilization rate = (Viable bacterial concentration in negative control group - Viable bacterial concentration in experimental group) / Viable bacterial concentration in negative control group × 100%

[0153] The sterilization rates of each treatment are shown in Table 1.

[0154] Table 1. Statistical results of sterilization rate of different gels

[0155]

[0156] As shown in Table 1, the antimicrobial peptide GL13K loaded in the hydrogel and astragaloside A loaded in the oleogel have a synergistic bactericidal effect. The bactericidal effect of drug composition #2 against Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) is much higher than that of hydrogel and oleogel. The antibacterial rate of drug compositions #1 to #5 against Staphylococcus aureus and Escherichia coli is greater than 95%.

[0157] Test Example 2

[0158] This example tests the hemostatic properties of the antibacterial hemostatic drug compositions for skin healing from Examples 1 to 5.

[0159] Test samples: 1#~5# antibacterial and hemostatic drug compositions for skin healing (referred to as 1#~5# drug compositions), the hydrogel and oleogel prepared in Example 3 were used as control group 1 and control group 2 respectively, and a blank control group with free bleeding was set up.

[0160] A liver hemorrhage model was used to assess the hemostatic ability of each test sample. Each treatment was performed in triplicate, and all animal experiments were conducted according to national guidelines for laboratory animal care and use. Mice were anesthetized and restrained on a surgical board tilted at 30°. The liver was exposed through an abdominal incision, and the tissue fluid surrounding the liver was carefully removed. The liver was then placed on pre-weighed filter paper (W0). A 0.5 cm incision was made in the left lobe of the liver, 0.3 cm from the lower edge. The hemorrhage wound was immediately covered with the test sample. The control group experienced free bleeding without treatment. Three minutes after hemostasis, the weight (W) of the filter paper that absorbed the blood was measured. Measurements were repeated three times for all groups. The amount of bleeding was calculated using the following formula:

[0161] Liver bleeding amount = W – W0

[0162] Test results are as follows Figure 1 As shown, the hemostatic effect of the antibacterial hemostatic drug combination for skin healing (1#~5#) is significantly better than that of the blank control group. The oleogel has a weak hemostatic effect compared with the blank control group. The hemostatic effect of the antibacterial hemostatic drugs for skin healing (1#, 2#, and 4#) is significantly better than that of hydrogel and oleogel alone, suggesting that the ratio of oleogel to hydrogel in the range of 3~4:6~7 has a synergistic hemostatic effect.

[0163] Test Example 3

[0164] This test case is to test the skin healing effect of the antibacterial and hemostatic drug compositions for skin healing in Examples 1 to 5.

[0165] Test samples: 1#~5# antibacterial and hemostatic drug compositions for skin healing (referred to as 1#~5# drug compositions). The hydrogel and oleogel prepared in Example 3 were used as control group 1 and control group 2, respectively. A blank control group was also set up (the blank control was natural healing).

[0166] A full-thickness skin wound model was used in mice to evaluate the in vivo wound healing effect of hydrogels. All animal experiments were conducted in accordance with the National Guidelines for the Care and Use of Laboratory Animals. Twenty-four 6-8 week old male ICR mice were selected for the wound healing study. The day before the experiment, the mice's back hair was completely removed using depilatory cream. On the day of the experiment, after anesthetizing the mice, a 7 mm diameter full-thickness excision wound was created on the back skin of each mouse. The mice were then randomly divided into eight groups: drug combination groups (1-5), hydrogel group, oleogel group, ...

[0167] In the control group, the test samples were used on days 0-2. Wound size was calculated using ImageJ software at 0, 3, 6, and 9 days post-injury. The wound healing rate was calculated using the following formula:

[0168] Wound healing rate = (A0–At) / A0

[0169] Where A0 represents the wound area, and At represents the wound area after a fixed time interval. The test results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the wound healing effect of the 1#~5# skin healing antibacterial hemostatic drug compositions is significantly better than that of the control group, indicating that the 1#~5# skin healing antibacterial hemostatic drug compositions provided by the present invention can be used for skin wound healing, especially the 2# skin healing antibacterial hemostatic drug composition has the best effect.

[0170] Test Example 4

[0171] This test assesses the accelerated stability of antibacterial and hemostatic drug compositions for skin healing (compositions #1 to #5).

[0172] The accelerated stability of the antibacterial and hemostatic drug compositions for skin healing in Examples 1#~5# was tested. Samples were taken at 0, 1, 2, 3 and 6 months respectively. The appearance of each test sample was observed, the uniformity of the test sample was visually inspected, and the presence of stratification was checked. The antibacterial activity was determined and the coagulation performance was tested by in vitro coagulation test. The results of the accelerated stability test are shown in Table 2.

[0173] Table 2. Accelerated stability test results of antibacterial and hemostatic drug compositions for skin healing

[0174]

[0175] As shown in Table 2, the antibacterial and hemostatic drug compositions for skin healing (1#~5#) remained uniform in appearance without stratification after 6 months of accelerated testing, and their antibacterial and coagulation properties showed no significant decline, indicating that the antibacterial and hemostatic drug compositions for skin healing (1#~5#) have good stability.

[0176] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications 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 protection scope defined by the present invention.

Claims

1. A pharmaceutical composition for skin healing with antibacterial and hemostatic properties, characterized in that, The pharmaceutical composition is a bigel, which is composed of an oleogel and a hydrogel in a mass ratio of 3-5:5-7. The oleogel uses medium-chain triglycerides and small-chain eugenol wax as its oleogel matrix, and loads astragaloside A in the oleogel at a mass percentage of 0.004%-0.06%. The hydrogel loads an antimicrobial peptide, GL13K, in the hydrogel at a mass percentage of 0.1%-2%. The hydrogel uses chitosan and sodium alginate as its hydrogel matrix, with a mass ratio of chitosan to sodium alginate of 1.5-4:

1.

2. The method for preparing the pharmaceutical composition according to claim 1, characterized in that, The following steps are involved: S1. Preparation of CS-SA-GL13K hydrogel: Chitosan solution and sodium alginate solution were mixed at a volume ratio of 1:1 to 2:1, and GL13K solution was added to prepare a mixed solution with a final concentration of GL13K of 0.1 to 2 mg / mL. The mixed solution was then immersed in 1% to 1.5% CaCl2 solution for crosslinking for 10 to 15 min, and washed to obtain CS-SA-GL13K hydrogel. S2. Preparation of medium-chain triglyceride *Solanum lyratum* wax-astragaloside A oil gel: Astragaloside A was dispersed in medium-chain triglycerides to obtain an astragaloside A suspension; Medium-chain triglycerides and *Ipomoea aquatica* wax were melt-mixed at a mass ratio of 90-91:4-8, and astragaloside A suspension was added. After stirring evenly and cooling to solidify, medium-chain triglyceride *Ipomoea aquatica* wax-astragaloside A oil gel was obtained. S3. Preparation of the double gel: The medium-chain triglyceride *Solanum lyratum* wax-astragaloside A oil gel was heated to 50-60°C and mixed with CS-SA-GL13K hydrogel at a volume ratio of 3:7-5:

5. After homogenization and emulsification, the mixture was rapidly cooled to obtain the pharmaceutical composition.

3. The preparation method according to claim 2, characterized in that, In the CS-SA-GL13K hydrogel, the chitosan has a mass-volume percentage of 1.5% to 3.0%, and the sodium alginate has a mass-volume percentage of 1.0% to 1.5%.

4. The preparation method according to claim 2, characterized in that, The concentration of astragaloside A in the oleogel is 2-5 mg / mL.

5. The preparation method according to claim 2, characterized in that, The homogenization emulsification speed is 8000~12000 rpm, and the time is 5~15 min.

6. The use of the pharmaceutical composition of claim 1 in the preparation of a hemostatic agent for skin wounds.

7. The use of the pharmaceutical composition of claim 1 in the preparation of a medicament for treating Staphylococcus aureus and / or Escherichia coli infections.

Images