An antimicrobial peptide spray, its preparation method, and its application in the prevention and treatment of peristomal skin infections.

Abstract

This invention provides an antimicrobial peptide spray, its preparation method, and its application in the prevention and treatment of peristomal skin infections. The spray comprises, by weight percentage, 0.05%-0.5% of a complex antimicrobial peptide, 0.1%-5% of a moisturizing and repairing film-forming matrix, 0.05%-0.2% of a buffer, 0.8%-1.2% of an isotonic regulator, 0.01%-0.5% of a stabilizer, and 0.005%-0.05% of a metal ion chelating agent, with the balance being water for injection. The complex antimicrobial peptide is composed of bovine lactoferrin antimicrobial peptide, sericin oligopeptide, and buckwheat-derived anti-inflammatory active peptide in a weight ratio of (3-5):(2-3):1. This spray possesses four core functions: broad-spectrum antibacterial activity, inhibition of bacterial biofilm, anti-inflammatory and soothing effects, and skin barrier repair. It is less likely to induce bacterial resistance, does not irritate damaged skin around the stoma, and can effectively prevent and treat peristomal skin infections.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical manufacturing technology, and in particular to an antimicrobial peptide spray, its preparation method, and its application in the prevention and treatment of peristomal skin infections. Background Technology

[0002] Following an ileostomy or colostomy, the diversion of waste leads to prolonged and direct exposure of the skin around the stoma to irritants such as fecal matter containing digestive enzymes and alkaline urine. This highly susceptible to fecal dermatitis, causing damage to the skin barrier. The damaged skin then becomes a breeding ground for bacteria and fungi, easily leading to secondary infections and the formation of difficult-to-remove bacterial biofilms. This results in persistent erythema, erosion, pain, and even ulcers, severely impacting the patient's quality of life and significantly increasing the difficulty of care and medical costs.

[0003] Current clinical approaches to the prevention and treatment of peristomal skin infections have significant shortcomings: antibiotic ointments are first-line treatments, but long-term topical use can easily induce bacterial resistance and is extremely ineffective against biofilm-mediated refractory infections, while also posing a risk of disrupting the normal skin flora and causing secondary infections; disinfectants such as iodine and alcohol can quickly kill bacteria, but they are highly irritating to broken wounds, damaging newly formed granulation tissue, delaying wound healing, and have no long-term protective or repairing effects; existing antimicrobial peptide preparations are mostly single antimicrobial peptide components, possessing only a single bactericidal function, and cannot simultaneously address the four core issues of peristomal skin infection, inflammation, barrier damage, and continuous irritation from excrement, and are easily inactivated in the moist environment of the stoma, which is rich in proteases, resulting in poor stability; the film-forming matrix of existing preparations often has defects such as poor air permeability, affecting ostomy bag adhesion, and being easily washed away by excrement, resulting in extremely low clinical compliance. In addition, existing plant and animal-derived bioactive peptide preparations generally suffer from problems such as random enzymatic hydrolysis processes, low enrichment rate of bioactive peptides, insufficient purity, and excessive levels of endotoxins and impurities, which can easily trigger sensitization reactions and fail to meet the safety requirements for long-term administration to broken skin around stomas. Summary of the Invention

[0004] In view of this, the present invention proposes an antimicrobial peptide spray, its preparation method, and its application in the prevention and treatment of peristomal skin infections, thereby solving the above-mentioned problems.

[0005] The technical solution of the present invention is implemented as follows: an antimicrobial peptide spray, comprising the following components by mass percentage: 0.05%-0.5% composite antimicrobial peptide, 0.1%-5% moisturizing and repairing film-forming matrix, 0.05%-0.2% buffer, 0.8%-1.2% isotonic regulator, 0.01%-0.5% stabilizer, 0.005%-0.05% metal ion chelating agent, with the balance being water for injection;

[0006] The composite antimicrobial peptide is composed of bovine lactoferrin antimicrobial peptide, sericin oligopeptide, and buckwheat-derived anti-inflammatory active peptide in a mass ratio of (3-5):(2-3):1.

[0007] Furthermore, the bovine lactoferrin antimicrobial peptide is obtained from fresh bovine milk lactoferrin through targeted enzymatic hydrolysis and multi-stage membrane separation purification. It has a molecular weight of 3000-5000 Da, a purity ≥98%, and a minimum inhibitory concentration (MIC) ≤4 μg / mL against common pathogens causing stomach infections. It possesses both broad-spectrum bactericidal and bacterial biofilm inhibitory activities. Specific steps:

[0008] A1. Ultrasonic pretreatment: Dissolve lactoferrin in a buffer solution with a pH of 6.5-7.5 to prepare a solution with a mass-volume concentration of 3%-8%, and then perform ultrasonic pretreatment for 5-15 minutes at a frequency of 20-40kHz and a power density of 100-300W / L, with the treatment temperature controlled at 4-10℃.

[0009] A2. Targeted enzymatic hydrolysis: Adjust the temperature of the pretreated solution to 37-42°C, first add trypsin, and then perform enzymatic hydrolysis for 30-60 minutes with the assistance of 50-150W / L under the same ultrasonic frequency; then adjust the pH to 2.0-3.5, add pepsin, and continue enzymatic hydrolysis at 37°C for 60-120 minutes to obtain the enzymatic hydrolysate;

[0010] A3. Multistage membrane separation and purification:

[0011] Primary separation: The enzyme hydrolysate is heated to 85-90°C and held for 10 minutes to inactivate the enzyme. After cooling, it is then passed through a microfiltration membrane with a pore size of 1.0 μm to remove large particulate impurities.

[0012] Secondary separation: The primary permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the permeate is collected to remove undigested large protein molecules and large fragments.

[0013] Three-stage separation: The secondary permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 3kDa, and the retentate is collected to obtain the target antimicrobial peptide components with molecular weights concentrated in the range of 3-10kDa.

[0014] A4. Drying: The retentate obtained from the three-stage separation is subjected to vacuum freeze-drying to obtain the bovine lactoferrin antimicrobial peptide powder.

[0015] Furthermore, the sericin oligopeptides are obtained from silkworm cocoons or silk-making byproducts through water extraction, targeted enzymatic hydrolysis, and glycosylation modification. Their molecular weight is concentrated between 2-10 kDa, and they possess film-forming moisturizing, skin-repairing, and auxiliary antibacterial activities. The specific preparation steps are as follows:

[0016] B1. Water extraction and purification of sericin: Silkworm cocoons or silk processing waste are mixed with water at a mass ratio of 1:15-1:30 and subjected to high temperature and high pressure water extraction at 115-125°C and 0.1-0.2MPa for 30-60 minutes; after cooling, the extract is first coarsely filtered through a 100-mesh sieve, and then purified by fractionation through an ultrafiltration membrane with a molecular weight cutoff of 50kDa. The permeate is collected and concentrated to obtain a purified sericin solution;

[0017] B2. Stepwise controlled enzymatic hydrolysis with complex protease: Adjust the pH of the sericin solution obtained in step B1 to 8.5-9.5 and maintain the temperature at 50-55°C. First, add proteinase K at 1-3% of the sericin protein mass and hydrolyze for 1-2 hours. Then, without changing the temperature, adjust the pH to 10.0-11.0, add alkaline protease at 2-4% of the sericin protein mass, and continue hydrolysis for 2-3 hours to obtain the hydrolysate.

[0018] B3. Multistage membrane separation and purification:

[0019] Primary separation: The inactivated enzyme hydrolysate is cooled and then passed through a microfiltration membrane with a pore size of 0.1 μm to remove insoluble impurities.

[0020] Secondary separation: The primary permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the permeate is collected to remove large protein molecules and insufficiently degraded fragments.

[0021] Three-stage separation: The secondary permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 2kDa, and the retentate is collected to obtain a sericin oligopeptide solution with a molecular weight concentrated in the range of 2-10kDa.

[0022] B4. Targeted modification and drying: Adjust the pH of the sericin oligopeptide solution obtained in step B3 to 7.0-8.0, add 5-15% dextran by weight of the oligopeptide, and carry out Maillard reaction at 55-65°C for 30-60 minutes to glycosylate the oligopeptide; after nanofiltration desalting, the reaction solution is freeze-dried under vacuum to obtain the sericin oligopeptide.

[0023] Furthermore, the buckwheat-derived anti-inflammatory active peptide, obtained from sweet buckwheat seeds through low-temperature extraction and targeted enzymatic hydrolysis purification, has a molecular weight ≤2000 Da. It can significantly inhibit the release of pro-inflammatory factors such as IL-6 and TNF-α, and alleviate skin irritation and inflammation. The specific preparation steps are as follows:

[0024] C1. Raw material pretreatment and extraction: After crushing the sweet buckwheat seeds, pass them through a 60-80 mesh sieve. Use low-voltage pulsed electric field assisted treatment: Mix the powder with a citrate buffer solution of pH 3.5-4.5 at a material-to-liquid ratio of 1:8-1:12 (g / mL), and treat for 5-15 minutes under the conditions of electric field strength of 10-20kV / cm, pulse frequency of 100-200Hz, and temperature of 15-25°C. Then, let it stand at 4°C for 2-4 hours for extraction, centrifuge and collect the supernatant to obtain the crude extract.

[0025] C2. Two-enzyme stepwise directional enzymatic hydrolysis: Adjust the pH of the crude extract to 7.0-7.5, add 1%-3% of the substrate mass of proteinase K, and hydrolyze at 45-50°C for 1-2 hours; then adjust the pH to 6.0-6.5, add 2%-4% of the substrate mass of glutaminase, and continue enzymatic hydrolysis at 50-55°C for 2-3 hours;

[0026] C3. Combination, separation, and purification:

[0027] C31. Membrane separation and enrichment: After the enzyme is inactivated by the enzymatic hydrolysate, it is passed sequentially through an ultrafiltration membrane system with a molecular weight cutoff of 5 kDa and 1 kDa. The retentate from the 1 kDa ultrafiltration membrane is collected to obtain a peptide enrichment solution with a molecular weight ≤ 2 kDa.

[0028] C32. Decolorization and purification: The above enrichment solution is pumped into a column packed with macroporous adsorption resin, eluted with deionized water to remove pigments and small molecule impurities, and then eluted with a 20%-40% (v / v) ethanol aqueous solution to collect the target component.

[0029] C33. Reversed-phase chromatography purification: After concentrating the target component, preparative separation was performed using reversed-phase high-performance liquid chromatography. Gradient elution was performed using acetonitrile-water (containing 0.1% trifluoroacetic acid) as the mobile phase. The gradient elution program was as follows: 0-5 minutes, acetonitrile volume fraction 5%; 5-25 minutes, acetonitrile volume fraction linearly increased from 5% to 35%; 25-30 minutes, acetonitrile volume fraction maintained at 35%. The main peak fraction was collected and freeze-dried to obtain buckwheat-derived anti-inflammatory active peptides with a molecular weight ≤2000 Da.

[0030] Furthermore, the moisturizing and repairing film-forming matrix is ​​composed of recombinant type III human collagen, graded sodium hyaluronate, and ceramide NP in a mass ratio of (1-2):(2-4):1; the graded sodium hyaluronate has a molecular weight of (1.0-1.8)×10⁻⁶. 6 Da's high molecular weight sodium hyaluronate has a molecular weight of (1.0-3.0)×10 4 Da's low molecular weight sodium hyaluronate is compounded in a mass ratio of 1:1-3.

[0031] Furthermore, the buffer is a histidine-histidine hydrochloride buffer; the isotonic regulator is sodium chloride; the stabilizer is a mixture of trehalose and mannitol in a mass ratio of 1:(0.8-1.2); and the metal ion chelating agent is EDTA-2Na.

[0032] The present invention also provides a method for preparing the above-mentioned antimicrobial peptide spray, comprising the following steps:

[0033] S1. Preparation of aqueous phase: Heat 70-90% of the prescribed amount of water for injection to 40-50°C, and add buffer, isotonicity regulator, stabilizer and metal ion chelating agent in sequence while stirring. Stir until completely dissolved to obtain a clear aqueous phase, and cool to 25-30°C.

[0034] S2. Dispersion of active ingredients: The prescribed amounts of bovine lactoferrin antimicrobial peptide powder, sericin oligopeptide powder and buckwheat anti-inflammatory active peptide powder are slowly added to the aqueous phase obtained in step S1 and dispersed evenly to obtain an active ingredient dispersion.

[0035] S3. Matrix Dissolution and Mixing: Recombinant type III human collagen, graded sodium hyaluronate, and ceramide NP are mixed with the remaining water for injection in proportion and stirred at low speed at 4-10°C until completely swollen and dissolved to obtain a transparent gel-like matrix solution; under low speed stirring, the matrix solution is slowly added to the active ingredient dispersion in step S2 and mixed evenly.

[0036] S4. Volume adjustment and filtration: Adjust the volume to the total volume with water for injection, stir evenly, and then sterilely filter through 0.45μm and 0.22μm polyethersulfone membranes to obtain the final spray solution;

[0037] S5. Aseptic filling: Under aseptic conditions, the drug solution obtained in step S4 is filled into a light-proof, sterile pressurized or non-pressurized single-dose spray container to obtain the final product.

[0038] Furthermore, in step S1, the heating rate is 1-2°C / min, the stirring rate is 300-500 rpm, and the cooling rate is 0.5-1.0°C / min.

[0039] Furthermore, in step S3, the temperature of the low-speed stirring is 4-8°C, and the stirring speed is 100-200 rpm.

[0040] The present invention also provides the use of antimicrobial peptide spray in the preparation of a medicament for repairing peristomal skin infections.

[0041] Compared with the prior art, the beneficial effects of the present invention are:

[0042] This invention relates to an antimicrobial peptide spray, which utilizes a ternary composite system composed of bovine lactoferrin antimicrobial peptides, sericin oligopeptides, and buckwheat-derived anti-inflammatory active peptides in a specific ratio. These three components complement each other and synergistically enhance each other's effects. Through a physical membrane-breaking mechanism, it achieves broad-spectrum antibacterial activity against common pathogens causing ostomy infections and drug-resistant strains such as MRSA. It can inhibit and break down bacterial biofilms, making it less likely to induce bacterial resistance. Simultaneously, it possesses anti-inflammatory, soothing, and skin barrier repair effects, thus blocking the vicious cycle of infection-inflammation-barrier damage. The formulation is a weakly acidic isotonic system matched to the physiological environment of human skin, causing no irritation or sensitization risk to damaged skin. Combined with a moisturizing and repairing film-forming matrix of recombinant type III human collagen, graded sodium hyaluronate, and ceramide NP, it can form a thin, breathable, and water-resistant protective film on the skin surface, effectively isolating excrement irritation without affecting ostomy bag adhesion, while accelerating wound healing and meeting the needs of the entire ostomy care cycle.

[0043] The targeted enzymatic hydrolysis and process designed for the three active peptides can be used to selectively enrich highly active target peptides. The resulting products have high purity, stable activity, and low endotoxin content, meeting the quality control requirements for administration through broken skin. The overall preparation process is mild and controllable, with no high-temperature sterilization steps, high retention rate of active ingredients, good batch stability, and requires no special equipment. It is suitable for large-scale industrial production and has excellent clinical application value and industrialization prospects. Detailed Implementation

[0044] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0045] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0046] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0047] Skin irritation tests were conducted on the various embodiments and comparative examples of the present invention using a rabbit broken skin model. After single administration and multiple consecutive administrations, no abnormal reactions such as erythema or edema were observed at the test sites in each group, and the skin irritation score was 0, which was determined to be non-irritating, thus meeting the safety requirements for administration to broken skin around the stoma.

[0048] Example 1

[0049] Prescription composition (1000g, 100 bottles, 10g per bottle)

[0050] The composition includes 0.2% compound antimicrobial peptides, 2% moisturizing and repairing film-forming matrix, 0.1% histidine-histidine hydrochloride buffer, 0.9% sodium chloride, 0.2% stabilizer (trehalose:mannitol = 1:1), 0.02% EDTA-2Na@, and the remainder is water for injection.

[0051] Among them, the compound antimicrobial peptide is composed of bovine lactoferrin antimicrobial peptide, sericin oligopeptide, and buckwheat-derived anti-inflammatory active peptide in a mass ratio of 4:2:1.

[0052] The moisturizing and repairing film-forming matrix is ​​composed of recombinant type III human collagen, graded sodium hyaluronate, and ceramide NP in a mass ratio of 1.5:3:1; the graded sodium hyaluronate has a molecular weight of 1.5 × 10⁻⁶. 6 Da's high molecular weight sodium hyaluronate with a molecular weight of 2.0×10 4 Da's low molecular weight sodium hyaluronate is compounded in a mass ratio of 1:2.

[0053] Preparation of bioactive peptides

[0054] 1. Preparation of bovine lactoferrin antimicrobial peptides

[0055] A1. Ultrasonic pretreatment: Pure lactoferrin from fresh milk was dissolved in phosphate buffer at pH 7.0 to prepare a 5% (w / v) solution. The solution was then subjected to ultrasonic pretreatment for 10 minutes at a frequency of 30 kHz and a power density of 200 W / L, with the treatment temperature controlled at 6 ℃.

[0056] A2. Targeted enzymatic hydrolysis: Adjust the temperature of the pretreated solution to 37°C. First, add trypsin at 2% of the substrate protein mass and perform enzymatic hydrolysis for 45 minutes at a power density of 100 W / L under the same ultrasonic frequency. Then, adjust the pH of the system to 2.5 and add pepsin at 1.5% of the substrate protein mass. Continue enzymatic hydrolysis at 37°C for 90 minutes to obtain the enzymatic hydrolysate.

[0057] A3. Multistage membrane separation and purification: The enzyme hydrolysate is heated to 90℃ and kept at that temperature for 10 minutes to inactivate the enzyme. After cooling to room temperature, it is first passed through a 1.0μm microfiltration membrane to remove large particulate impurities; then the permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 10kDa and the permeate is collected; finally, the permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 3kDa and the retentate is collected to obtain the target antimicrobial peptide component.

[0058] A4. Drying: The retentate was freeze-dried under vacuum to obtain bovine lactoferrin antimicrobial peptide powder. The molecular weight was measured to be 3000-5000 Da, and the purity was 98.6%.

[0059] 2. Preparation of sericin oligopeptides

[0060] B1. Water extraction and purification of sericin: Impurity-free silk-making by-products were mixed with water for injection at a mass ratio of 1:20 and subjected to high-temperature and high-pressure water extraction at 121℃ and 0.12MPa for 45 minutes. After cooling, the extract was coarsely filtered through a 100-mesh sieve and then purified through an ultrafiltration membrane with a molecular weight cutoff of 50kDa. The permeate was collected and concentrated to obtain a purified sericin solution with a protein purity of 95.2%.

[0061] B2. Stepwise controllable enzymatic hydrolysis with complex protease: Adjust the pH of the sericin solution to 9.0 and maintain the temperature at 52℃. First, add 2% of the sericin protein mass of protein ...

[0062] B3. Multistage membrane separation and purification: The enzyme hydrolysate was incubated at 90℃ for 10 minutes to inactivate the enzyme, and after cooling, it was filtered through a 0.1μm microfiltration membrane to remove impurities; the permeate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 10kDa and the permeate was collected; it was then filtered through an ultrafiltration membrane with a molecular weight cutoff of 2kDa and the retentate was collected to obtain a sericin oligopeptide solution with a molecular weight of 2-10kDa.

[0063] B4. Targeted modification and drying: The pH of the sericin oligopeptide solution was adjusted to 7.5, and 10% of the dry weight of the oligopeptide was added with dextran. The Maillard reaction was carried out at 60°C for 45 minutes. After nanofiltration desalting, the reaction solution was freeze-dried under vacuum to obtain the sericin oligopeptide product.

[0064] 3. Preparation of Buckwheat-Derived Anti-inflammatory Active Peptides

[0065] C1. Raw material pretreatment and extraction: High-quality sweet buckwheat seeds were crushed and passed through an 80-mesh sieve. They were mixed with citrate buffer solution at pH 4.0 at a material-to-liquid ratio of 1:10 (g / mL). The mixture was treated with a low-voltage pulsed electric field at an electric field strength of 15kV / cm, a pulse frequency of 150Hz, and a temperature of 20℃ for 10 minutes. Then, the mixture was extracted by standing at 4℃ for 3 hours. The supernatant was collected by centrifugation at 8000r / min for 15 minutes to obtain the crude extract.

[0066] C2. Stepwise directional enzymatic hydrolysis with two enzymes: Adjust the pH of the crude extract to 7.2, add 2% proteinase K by substrate mass, and hydrolyze at 48℃ for 1.5 hours; then adjust the pH to 6.2, add 3% glutaminase by substrate mass, and continue enzymatic hydrolysis at 52℃ for 2.5 hours, and inactivate the enzyme at 90℃ for 10 minutes.

[0067] C3. Combined Separation and Purification: The enzymatic hydrolysate was sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 5 kDa and 1 kDa. The retentate from the 1 kDa membrane was collected to obtain a peptide enrichment solution. The enrichment solution was loaded onto a D101 macroporous adsorption resin column, and impurities were first eluted with deionized water, followed by elution with 30% ethanol aqueous solution. The target components were collected. After concentration, preparative separation was performed using reversed-phase high-performance liquid chromatography, eluting according to a preset gradient. The main peak fraction was collected and freeze-dried to obtain buckwheat-derived anti-inflammatory active peptides with a molecular weight ≤2000 Da.

[0068] Preparation of spray

[0069] S1. Preparation of aqueous phase: Take 800g of water for injection, heat it to 45℃ at a rate of 1.5℃ / min, and add 1g of histidine-histidine hydrochloride buffer, 9g of sodium chloride, 1g of trehalose, 1g of mannitol, and 0.2g of EDTA-2Na in sequence while stirring at 400rpm. Stir until completely dissolved to obtain a clear aqueous phase, and cool it to 25℃ at a rate of 0.8℃ / min.

[0070] S2. Dispersion of active ingredients: 1.14g of bovine lactoferrin antimicrobial peptide, 0.57g of sericin oligopeptide, and 0.29g of buckwheat anti-inflammatory active peptide were slowly added to the aqueous phase in sequence and stirred to disperse evenly to obtain a dispersion of active ingredients;

[0071] S3. Matrix Dissolution and Mixing: 4.62g of recombinant type III human collagen, 6.15g of high molecular weight sodium hyaluronate, 12.3g of low molecular weight sodium hyaluronate, and 3.08g of ceramide NP were mixed with the remaining 170g of water for injection. The mixture was stirred at 6°C and 150rpm until completely swollen and dissolved to obtain a transparent gel-like matrix solution. While stirring at 150rpm, the matrix solution was slowly added to the active ingredient dispersion and mixed evenly.

[0072] S4. Volume Adjustment and Filtration: Add water for injection to a total volume of 1000g, stir evenly, and then aseptically filter through 0.45μm and 0.22μm polyethersulfone membranes to obtain the spray solution; S5. Aseptic Filling: In a Class A clean environment, aseptically fill the solution into 10mL light-proof aseptic spray bottles, 10g per bottle, seal and cap to obtain the final product.

[0073] Testing showed that the pH value of the spray in this embodiment was 6.0 and the osmotic pressure was 298 mOsm / L, which met the design requirements.

[0074] Example 2

[0075] Prescription composition (1000g)

[0076] The composition includes 0.05% compound antimicrobial peptide, 0.1% moisturizing and repairing film-forming matrix, 0.05% histidine-histidine hydrochloride buffer, 0.8% sodium chloride, 0.01% stabilizer (trehalose:mannitol = 1:0.8), 0.005% EDTA-2Na@, and the balance is water for injection.

[0077] Among them, the compound antimicrobial peptide is composed of bovine lactoferrin antimicrobial peptide, sericin oligopeptide, and buckwheat-derived anti-inflammatory active peptide in a mass ratio of 3:2:1.

[0078] The moisturizing and repairing film-forming matrix is ​​composed of recombinant type III human collagen, graded sodium hyaluronate, and ceramide NP in a mass ratio of 1:2:1; the graded sodium hyaluronate has a molecular weight of 1.0 × 10⁻⁶. 6 Da's high molecular weight sodium hyaluronate and its molecular weight of 1.0×104 Da's low molecular weight sodium hyaluronate is compounded in a 1:1 mass ratio.

[0079] Preparation method

[0080] The preparation method of the active peptide is the same as that in Example 1. The preparation method of the spray is the same as that in Example 1. Only the dosage of the components is adjusted according to the prescription. The final spray has a pH of 5.5 and an osmotic pressure of 282 mOsm / L.

[0081] Example 3

[0082] Prescription composition (1000g)

[0083] The composition includes 0.5% compound antimicrobial peptides, 5% moisturizing and repairing film-forming matrix, 0.2% histidine-histidine hydrochloride buffer, 1.2% sodium chloride, 0.5% stabilizer (trehalose:mannitol = 1:1.2), 0.05% EDTA-2Na@, and the remainder is water for injection.

[0084] Among them, the compound antimicrobial peptide is composed of bovine lactoferrin antimicrobial peptide, sericin oligopeptide, and buckwheat-derived anti-inflammatory active peptide in a mass ratio of 5:3:1.

[0085] The moisturizing and repairing film-forming matrix is ​​composed of recombinant type III human collagen, graded sodium hyaluronate, and ceramide NP in a mass ratio of 2:4:1; the graded sodium hyaluronate has a molecular weight of 1.8 × 10⁻⁶. 6 Da's high molecular weight sodium hyaluronate with a molecular weight of 3.0×10 4 Da's low molecular weight sodium hyaluronate is compounded in a mass ratio of 1:3.

[0086] Preparation method

[0087] The preparation method of the active peptide is the same as that in Example 1. The preparation method of the spray is the same as that in Example 1. Only the dosage of the components is adjusted according to the prescription. The final spray has a pH of 6.5 and an osmotic pressure of 318 mOsm / L.

[0088] Comparative Example 1

[0089] The only difference between this comparative example and Example 1 is that bovine lactoferrin antimicrobial peptides are removed from the composite antimicrobial peptides, and only sericin oligopeptides and buckwheat-derived anti-inflammatory active peptides are retained, with a mass ratio of 2:1 and a total addition amount of 0.2%. The rest of the formulation and preparation method are completely consistent with Example 1.

[0090] Comparative Example 2

[0091] The only difference between this comparative example and Example 1 is that the sericin oligopeptide is removed from the composite antimicrobial peptide, and only bovine lactoferrin antimicrobial peptide and buckwheat anti-inflammatory active peptide are retained, with a mass ratio of 4:1 and a total addition amount of 0.2%. The rest of the formulation and preparation method are completely consistent with Example 1.

[0092] Comparative Example 3

[0093] The only difference between this comparative example and Example 1 is that the buckwheat-derived anti-inflammatory active peptides are removed from the compound antimicrobial peptides, and only bovine lactoferrin antimicrobial peptides and sericin oligopeptides are retained, with a mass ratio of 4:2 and a total addition amount of 0.2%. The rest of the formulation and preparation method are completely consistent with Example 1.

[0094] Comparative Example 4

[0095] The comparative example used was commercially available 2% mupirocin ointment, which served as a positive control.

[0096] Comparative Example 5

[0097] This comparative example uses commercially available 0.2%@LL-37 antimicrobial peptide spray as a control for similar products.

[0098] Test Example 1 @ In vitro antibacterial activity test

[0099] 1. Test materials

[0100] Test samples: Antimicrobial peptide spray stock solutions prepared in Examples 1, 2, and 3; Comparative Examples 1, 2, and 3; Comparative Example 4 (2% mupirocin ointment, prepared into a solution of the corresponding concentration using Mueller-Hinton broth); Comparative Example 5 (0.2% LL-37 antimicrobial peptide spray stock solution).

[0101] Standard strains: Staphylococcus aureus (ATCC@25923), methicillin-resistant Staphylococcus aureus (MRSA, ATCC@43300), Escherichia coli (ATCC@25922), and Pseudomonas aeruginosa (ATCC@27853), all of which are the most common pathogens causing peristomal skin infections in clinical practice.

[0102] Culture medium: Mueller-Hinton broth, trypsin-soybean broth, crystal violet staining solution, acetic acid.

[0103] Main instruments: microplate reader, constant temperature incubator, biosafety cabinet, 96-well cell culture plate.

[0104] 2. Testing Methods

[0105] 2.1 Determination of minimum inhibitory concentration and minimum bactericidal concentration

[0106] The procedure was performed according to the CLSIM07-A10 micro-broth dilution method.

[0107] Preparation of bacterial culture: After the test strain was revived and cultured, it was diluted 1:100 with Mueller-Hinton broth to obtain 1.5 × 10⁻⁶ bacterial culture. 6 CFU / mL inoculated bacterial solution.

[0108] Sample dilution: The samples were serially diluted 2-fold in 96-well plates with Mueller-Hinton broth, with a volume of 100 μL per well, to a concentration range of 128 μg / mL to 0.25 μg / mL (based on total peptides or active ingredients).

[0109] Inoculation and culture: Add an equal volume of bacterial solution to each well to make a final bacterial count of approximately 5 × 10⁻⁶. 5 CFU / mL, incubated at 37℃ for 18 hours.

[0110] Simultaneously set:

[0111] Growth control: bacterial solution + MH broth.

[0112] Solvent control: Bacterial culture + MH broth containing the highest concentration of the sample solvent.

[0113] Blank control: MH broth only.

[0114] MIC determination: The lowest sample concentration that is completely clear to the naked eye and shows no visible bacterial growth is the minimum inhibitory concentration.

[0115] MBC determination: Take 10 μL of liquid from the well with a concentration of MIC or higher, spread it on an MH agar plate, and incubate at 37°C for 24 hours. The lowest sample concentration with a kill rate ≥99.9% is taken as the MBC concentration.

[0116] 2.2 Bacterial Biofilm Inhibition Experiment

[0117] The inhibitory effect on mature biofilms of Pseudomonas aeruginosa was evaluated using crystal violet staining.

[0118] Biofilm formation: Pseudomonas aeruginosa was cultured in 96-well plates for 24 hours to form a mature biofilm. Airborne bacteria were removed by gentle washing with PBS (200 μL / well, incubated for 30 seconds and then discarded, repeated 3 times).

[0119] Sample preparation: Add MH broth containing samples at concentrations of 1×MIC, 2×MIC, and 4×MIC, 200 μL per well. Include a biofilm positive control (MH broth only) and a negative control (sterile broth). Incubate at 37°C for 24 hours.

[0120] Staining and quantification: After washing with PBS, the sample was fixed with methanol, stained with 0.1% crystal violet, and dissolved in 33% glacial acetic acid. The absorbance at 590 nm was measured using a microplate reader.

[0121] Calculate the inhibition rate:

[0122] Biofilm inhibition rate (%) = [1 - (OD sample - OD negative) / (OD positive - OD negative)] × 100%

[0123] 3. Data Processing

[0124] All experiments were independently repeated 3 times, and the result was the average of the 3 experiments. The final results are shown in Tables 1 and 2.

[0125]

[0126] The MIC / MBC values ​​of Examples 1-3 were much lower than those of Comparative Examples 1-3, which lacked a single active peptide, and the biofilm inhibition rate was also significantly higher. This proves that the ternary combination of bovine lactoferrin antimicrobial peptide, sericin oligopeptide, and buckwheat anti-inflammatory active peptide is not a simple superposition, but rather forms a functionally complementary and synergistic complex system that can comprehensively cover the core pathogens of ostomy infection and effectively break through the defensive barrier of bacterial biofilm.

[0127] Experimental Example 2 @ In vitro anti-inflammatory activity test

[0128] 1. Cell Culture and Seeding: RAW264.7 mouse macrophages were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2 using standard methods; cells in the logarithmic growth phase were harvested and the density was adjusted to 1×10⁻⁶ cells / year. 6 Cells were seeded at a density of 100 μL / mL in 96-well cell culture plates and cultured for 24 h until the cells were fully adhered.

[0129] 2. Inflammation induction and drug administration: Discard the original culture medium and set up a blank control group (culture medium only), a model control group (1 μg / mL LPS), and a test sample group (1 μg / mL LPS + 100 μg / mL test sample), with 3 replicates for each group; incubate at 37℃ and 5% CO2 for 24 h.

[0130] 3. Sample collection and testing: Cell supernatant from each group was collected, and the absorbance (OD value) of IL-6 and TNF-α was measured using a commercial ELISA kit, strictly following the instructions. The content of each inflammatory factor was calculated.

[0131] 4. Calculation of results: Anti-inflammatory inhibition rate (%) = (Inflammatory factor content in model group - Inflammatory factor content in sample group) / (Inflammatory factor content in model group - Inflammatory factor content in blank group) × 100%.

[0132]

[0133] As shown in Table 2, Example 1 of the present invention has extremely strong anti-inflammatory activity and can significantly inhibit the release of pro-inflammatory factors. Its effect is far superior to that of Comparative Examples 2 and 3, which lack buckwheat-derived anti-inflammatory active peptides. This proves that buckwheat-derived anti-inflammatory active peptides are the core component of the anti-inflammatory function of the present invention. At the same time, the ternary composite system can synergistically enhance the anti-inflammatory effect.

[0134] Experimental Example 3: Efficacy Test of a Rat Peristostomy Skin Infection Model

[0135] 1. Test materials

[0136] Experimental animals: SPF-grade male SD rats, weighing 180-220g, acclimatized for one week.

[0137] Test strain: Methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300), consistent with the strain used in the in vitro antibacterial test.

[0138] Test samples: antimicrobial peptide spray prepared in Example 1, spray prepared in Comparative Examples 1-3, commercially available 2% mupirocin ointment in Comparative Example 4, and sterile physiological saline for the model control group.

[0139] 2. Test Methods

[0140] 2.1 Preparation of bacterial suspension

[0141] After activation, the MRSA strain was inoculated into MH broth and cultured at 37°C with shaking at 180 rpm until the logarithmic growth phase. The bacterial concentration was then adjusted to 1×10⁻⁶ with sterile physiological saline. 8 CFU / mL, prepare and use immediately.

[0142] 2.2 Construction of a peristomal skin infection model

[0143] After intraperitoneal anesthesia, the abdomen of rats was prepared and disinfected, and a colostomy was performed and the intestinal tract was fixed. A standardized lesion was constructed by abrading the skin around the stoma up to a point where the dermis bled. A 50 μL LMRSA bacterial suspension was evenly applied to the wound and covered with a sterile dressing. Twenty-four hours after inoculation, redness, swelling, and discharge appeared at the wound site, with a bacterial load ≥1×10⁻⁶. 6 CFU / g indicates successful model establishment.

[0144] 3. Experimental grouping and drug administration

[0145] Sixty rats that successfully developed the model were randomly divided into 6 groups of 10 rats each using a random number table. The grouping and drug administration regimens are as follows:

[0146] Model control group: Sterile saline was sprayed onto the broken skin around the stoma, 0.2 mL each time;

[0147] Example 1 group: Spray the antimicrobial peptide spray prepared in Example 1, 0.2 mL each time;

[0148] Comparative Example 1: Spray the spray prepared in Comparative Example 1, 0.2 mL each time;

[0149] Comparative Example 2: Spray the spray prepared in Comparative Example 2, 0.2 mL each time;

[0150] Comparative Example 3: Spray the spray prepared in Comparative Example 3, 0.2 mL each time;

[0151] Positive control group (comparative example 4): 0.2g of 2% mupirocin ointment was applied each time;

[0152] All groups were given the medication twice daily for 14 consecutive days.

[0153] 4. Detection Indicators and Methods

[0154] 4.1 Detection of bacterial load in wound: On day 3 and day 7 of drug administration, 5 rats were randomly selected from each group. Under aseptic conditions, full-thickness skin tissue of the wound around the stoma was taken, weighed, and then mixed with sterile physiological saline at a ratio of 1:9 (g:mL) to prepare tissue homogenate. After dilution, the homogenate was spread on MH agar plates and incubated at 37℃ for 24h. The number of colonies was counted and the number of colonies per gram of tissue was calculated. The results are expressed as 1g CFU / g.

[0155] 4.2 Wound healing rate determination: After successful modeling (day 0 of drug administration) and on day 14 of drug administration, the major axis (a) and minor axis (b) of the ruptured wound around the stoma of each rat were measured with vernier calipers. The wound area was calculated using the formula: Wound area (mm²) = π × a / 2 × b / 2; then the wound healing rate was calculated using the following formula:

[0156] Wound healing rate (%) = (Initial wound area on day 0 of drug administration - Remaining wound area on day 14 of drug administration) / Initial wound area on day 0 of drug administration × 100%

[0157]

[0158] As shown in Table 3, Example 1 of the present invention can rapidly eliminate pathogens in infected wounds around stomas and significantly promote wound healing. After 14 days of administration, the wound healing rate reached 96.8%, which is far superior to Comparative Examples 1-4. Pathological observation showed that the skin structure of rats in Example 1 group was intact, inflammatory cell infiltration was significantly reduced, and new granulation tissue and collagen fibers were abundant, proving that the present invention can simultaneously achieve the dual effects of anti-infection and promoting healing, perfectly meeting the treatment needs of peristomal skin infections.

[0159] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antimicrobial peptide spray, characterized in that, The product comprises the following components by mass percentage: 0.05%-0.5% complex antimicrobial peptide, 0.1%-5% moisturizing and repairing film-forming matrix, 0.05%-0.2% buffer, 0.8%-1.2% isotonic regulator, 0.01%-0.5% stabilizer, 0.005%-0.05% metal ion chelating agent, with the balance being water for injection; the complex antimicrobial peptide is composed of bovine lactoferrin antimicrobial peptide, sericin oligopeptide, and buckwheat-derived anti-inflammatory active peptide in a mass ratio of (3-5):(2-3):

1.

2. The antimicrobial peptide spray as described in claim 1, characterized in that, The bovine lactoferrin antimicrobial peptides were obtained from fresh bovine milk lactoferrin through targeted enzymatic hydrolysis and multi-stage membrane separation purification, with a molecular weight of 3000-5000 Da; specific steps: A1. Ultrasonic pretreatment: Dissolve lactoferrin in a buffer solution with a pH of 6.5-7.5 to prepare a solution with a mass-volume concentration of 3%-8%, and then perform ultrasonic pretreatment for 5-15 minutes at a frequency of 20-40kHz and a power density of 100-300W / L, with the treatment temperature controlled at 4-10℃. A2. Targeted enzymatic hydrolysis: Adjust the temperature of the pretreated solution to 37-42°C, first add trypsin, and then perform enzymatic hydrolysis for 30-60 minutes with the assistance of 50-150W / L under the same ultrasonic frequency; then adjust the pH to 2.0-3.5, add pepsin, and continue enzymatic hydrolysis at 37°C for 60-120 minutes to obtain the enzymatic hydrolysate; A3. Multistage membrane separation and purification: Primary separation: The enzyme hydrolysate is heated to 85-90°C and held for 10 minutes to inactivate the enzyme. After cooling, it is then passed through a microfiltration membrane with a pore size of 1.0 μm to remove large particulate impurities. Secondary separation: The primary permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the permeate is collected to remove undigested large protein molecules and large fragments. Three-stage separation: The secondary permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 3kDa, and the retentate is collected to obtain the target antimicrobial peptide components with molecular weights concentrated in the range of 3-10kDa. A4. Drying: The retentate obtained from the three-stage separation is subjected to vacuum freeze-drying to obtain the bovine lactoferrin antimicrobial peptide powder.

3. The antimicrobial peptide spray as described in claim 1, characterized in that, The extraction method of the sericin oligopeptides includes the following steps: B1. Water extraction and purification of sericin: Silkworm cocoons or silk processing waste are mixed with water at a mass ratio of 1:15-1:30 and subjected to high temperature and high pressure water extraction at 115-125°C and 0.1-0.2MPa for 30-60 minutes; after cooling, the extract is first coarsely filtered through a 100-mesh sieve, and then purified by fractionation through an ultrafiltration membrane with a molecular weight cutoff of 50kDa. The permeate is collected and concentrated to obtain a purified sericin solution; B2. Stepwise controlled enzymatic hydrolysis with complex protease: Adjust the pH of the sericin solution obtained in step B1 to 8.5-9.5 and maintain the temperature at 50-55°C. First, add proteinase K at 1-3% of the sericin protein mass and hydrolyze for 1-2 hours. Then, without changing the temperature, adjust the pH to 10.0-11.0, add alkaline protease at 2-4% of the sericin protein mass, and continue hydrolysis for 2-3 hours to obtain the hydrolysate. B3. Multistage membrane separation and purification: Primary separation: The inactivated enzyme hydrolysate is cooled and then passed through a microfiltration membrane with a pore size of 0.1 μm to remove insoluble impurities. Secondary separation: The primary permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the permeate is collected to remove large protein molecules and insufficiently degraded fragments. Three-stage separation: The secondary permeate is passed through an ultrafiltration membrane with a molecular weight cutoff of 2kDa, and the retentate is collected to obtain a sericin oligopeptide solution with a molecular weight concentrated in the range of 2-10kDa. B4. Targeted modification and drying: Adjust the pH of the sericin oligopeptide solution obtained in step B3 to 7.0-8.0, add 5-15% dextran by weight of the oligopeptide, and carry out Maillard reaction at 55-65°C for 30-60 minutes to glycosylate the oligopeptide; after nanofiltration desalting, the reaction solution is freeze-dried under vacuum to obtain the sericin oligopeptide.

4. The antimicrobial peptide spray as described in claim 1, characterized in that, The buckwheat-derived anti-inflammatory active peptides were obtained from sweet buckwheat seeds through low-temperature extraction and targeted enzymatic hydrolysis purification, with a molecular weight ≤2000Da. Specific steps included: C1. Raw material pretreatment and extraction: After crushing the sweet buckwheat seeds, pass them through a 60-80 mesh sieve. Use low-voltage pulsed electric field assisted treatment: Mix the powder with a citrate buffer solution of pH 3.5-4.5 at a material-to-liquid ratio of 1:8-1:12 (g / mL), and treat for 5-15 minutes under the conditions of electric field strength of 10-20kV / cm, pulse frequency of 100-200Hz, and temperature of 15-25°C. Then, let it stand at 4°C for 2-4 hours for extraction, centrifuge and collect the supernatant to obtain the crude extract. C2. Two-enzyme stepwise directional enzymatic hydrolysis: Adjust the pH of the crude extract to 7.0-7.5, add 1%-3% of the substrate mass of proteinase K, and hydrolyze at 45-50°C for 1-2 hours; then adjust the pH to 6.0-6.5, add 2%-4% of the substrate mass of glutaminase, and continue enzymatic hydrolysis at 50-55°C for 2-3 hours; C3. Combination, separation, and purification: C31. Membrane separation and enrichment: After the enzyme is inactivated by the enzymatic hydrolysate, it is passed sequentially through an ultrafiltration membrane system with a molecular weight cutoff of 5 kDa and 1 kDa. The retentate from the 1 kDa ultrafiltration membrane is collected to obtain a peptide enrichment solution with a molecular weight ≤ 2 kDa. C32. Decolorization and purification: The above enrichment solution is pumped into a column packed with macroporous adsorption resin, eluted with deionized water to remove pigments and small molecule impurities, and then eluted with a 20-40% (v / v) ethanol aqueous solution to collect the target component. C33. Reversed-phase chromatography purification: After concentrating the target component, preparative separation is performed by reversed-phase high-performance liquid chromatography with gradient elution. The main peak fraction is collected and freeze-dried to obtain buckwheat-derived anti-inflammatory active peptides with a molecular weight ≤2000 Da.

5. The antimicrobial peptide spray as described in claim 1, characterized in that, The moisturizing and repairing film-forming matrix is ​​composed of recombinant type III human collagen, graded sodium hyaluronate, and ceramide NP in a mass ratio of (1-2):(2-4):1; the graded sodium hyaluronate has a molecular weight of (1.0-1.8)×10 6 Da's high molecular weight sodium hyaluronate has a molecular weight of (1.0-3.0)×10 4 Da's low molecular weight sodium hyaluronate is compounded in a mass ratio of 1:1-3.

6. The antimicrobial peptide spray as described in claim 1, characterized in that, The buffer is histidine-histidine hydrochloride buffer; the isotonic regulator is sodium chloride; the stabilizer is a mixture of trehalose and mannitol in a mass ratio of 1:(0.8-1.2); and the metal ion chelating agent is EDTA-2Na.

7. A method for preparing an antimicrobial peptide spray according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Preparation of aqueous phase: Heat 70-90% of the prescribed amount of water for injection to 40-50°C, and add buffer, isotonicity regulator, stabilizer and metal ion chelating agent in sequence while stirring. Stir until completely dissolved to obtain a clear aqueous phase, and cool to 25-30°C. S2. Dispersion of active ingredients: The prescribed amounts of bovine lactoferrin antimicrobial peptide powder, sericin oligopeptide powder and buckwheat anti-inflammatory active peptide powder are slowly added to the aqueous phase obtained in step S1 and dispersed evenly to obtain an active ingredient dispersion. S3. Matrix Dissolution and Mixing: Recombinant type III human collagen, graded sodium hyaluronate, and ceramide NP are mixed with the remaining water for injection in proportion and stirred at low speed at 4-10°C until completely swollen and dissolved to obtain a transparent gel-like matrix solution; under low speed stirring, the matrix solution is slowly added to the active ingredient dispersion in step S2 and mixed evenly. S4. Volume adjustment and filtration: Adjust the volume to the total volume with water for injection, stir evenly, and then sterilely filter through 0.45μm and 0.22μm polyethersulfone membranes to obtain the final spray solution; S5. Aseptic filling: Under aseptic conditions, the drug solution obtained in step S4 is filled into a light-proof, sterile pressurized or non-pressurized single-dose spray container to obtain the final product.

8. The preparation method according to claim 7, characterized in that, In step S1, the heating rate is 1-2°C / min, the stirring rate is 300-500 rpm, and the cooling rate is 0.5-1.0°C / min.

9. The preparation method according to claim 7, characterized in that, In step S3, the temperature of low-speed stirring is 4-8°C, and the stirring speed is 100-200 rpm.

10. The use of the antimicrobial peptide spray according to any one of claims 1-6 in the preparation of a medicament for repairing peristomal skin infections.

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