A pyrrolidone carboxylic acid-lysozyme complex, its preparation method and use

Abstract

The present application relates to the technical field of wound dressing, and particularly relates to a pyrrolidone carboxylic acid-lysozyme complex, a preparation method and application thereof. The pyrrolidone carboxylic acid-lysozyme complex utilizes the antibacterial performance of lysozyme to improve the antibacterial ability of biological materials, reduces the risk of infection, and can be applied to wound dressing. The prepared recombinant elastin cream dressing has excellent moisture absorption, can form a protective layer on a wound site, improve and optimize the skin microecology, and promote wound healing. The addition of recombinant elastin and ceramide can enhance the resistance of a wound to the outside world and improve the skin state.

Description

Technical Field

[0001] This invention relates to the field of wound dressing technology, and in particular to a pyrrolidone carboxylic acid-lysozyme complex, its preparation method, and its application. Background Technology

[0002] Most patients will have wounds of varying degrees after cosmetic surgery. When wounds appear on the local skin, it is necessary to do a good job of daily care.

[0003] In wound care, commonly used liquid dressings and gauze have certain limitations. They primarily fail to keep the wound moist for long periods, requiring frequent changes or reapplication, which can easily lead to secondary damage. This not only hinders wound healing but also causes patients significant pain and discomfort during the care process.

[0004] Lysozyme, also known as muramase or N-acetyl muramoglucan hydrolase, is an alkaline enzyme that hydrolyzes the mucopolysaccharides in bacteria. It breaks down insoluble polysaccharides in the cell wall into soluble glycopeptides, causing bacterial cell wall rupture and inactivation. By inhibiting and killing bacteria on the wound surface, lysozyme reduces bacterial invasion of wounds, lowers the risk of infection, creates a clean environment for wound healing, helps prevent wound infection from worsening, and promotes the normal healing process. As a naturally sourced antibacterial substance, it has good biocompatibility and shows great promise for applications in cosmetics and medical devices. However, as a cationic protein, lysozyme readily reacts with anionic components in formulations, forming precipitates that precipitate and become inactive, thus limiting its applications. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a pyrrolidone carboxylic acid-lysozyme complex, its preparation method, and its applications.

[0006] The first objective of this invention is to provide a method for preparing a pyrrolidone carboxylic acid-lysozyme complex, comprising the following steps: S1. First, the condensing agent EDC is mixed with pyrrolidone carboxylic acid and reacted. S2. Add NHS to the reaction system in step S1 and react to obtain the intermediate N-hydroxysuccinimide ester. S3, intermediate N-hydroxysuccinimide ester, and lysozyme were mixed and reacted. The pH of the reaction system was adjusted to 5.0-5.5 using a buffer solution, and the reaction temperature was controlled at 4±1℃. S4. After the reaction in step S3 is completed, a certain amount of terminator is added, and then the product is purified to obtain pyrrolidone carboxylic acid-lysozyme.

[0007] Furthermore, the mass ratio of the condensing agent EDC to pyrrolidone carboxylic acid is 1.2:1.

[0008] Furthermore, the mass ratio of NHS to EDC is 1:1.5.

[0009] Furthermore, the mass ratio of the intermediate N-hydroxysuccinimide ester to lysozyme is 1:1.

[0010] Furthermore, in step S3, the buffer solution is a citrate-sodium citrate buffer solution with a pH of 4.5-5.5.

[0011] Furthermore, the molar ratio of the terminator to the intermediate N-hydroxysuccinimide ester is 2.5:1.

[0012] Furthermore, the terminating agent is hydroquinone.

[0013] Further, in step S4, the purification process is as follows: first, it is slowly injected into a pre-equilibrated Sephadex G-100 gel column for elution, and the eluent containing the target product is collected; then, the collected solution is subjected to gradient elution through a DEAE-SepharoseFast Flow ion exchange column; finally, chitin affinity chromatography is used for elution, and the solution corresponding to the elution peak is collected. Detection was performed using high performance liquid chromatography. If the hydroquinone residue was higher than 0.1%, the corresponding purification steps were repeated until the residue was lower than the standard, ensuring that the final purity of the pyrrolidone carboxylic acid-lysozyme complex was obtained ≥99.5%.

[0014] A second objective of the present invention is to provide a pyrrolidone carboxylic acid-lysozyme complex prepared by the preparation method described above.

[0015] A third objective of this invention is to provide a recombinant elastin cream dressing, comprising, by weight, the following raw materials: 0.1-10% recombinant elastin complex solution, 0.1-10% pyrrolidone carboxylic acid-lysozyme complex as described in claim 6, 1-5% water-soluble polymer, 1-20% moisturizer, 1-5% oily matrix, 0.1-2% pH adjuster, and the remainder being purified water.

[0016] Furthermore, the recombinant elastin complex contains the following raw materials in parts by weight: 1-5% recombinant elastin and 0.1-1% ceramide; The water-soluble polymer is selected from one or more of sodium hyaluronate, xanthan gum, sodium alginate, chitosan, sodium carboxymethyl cellulose, carbomer, and polyvinyl alcohol. The moisturizer is selected from one or more of propylene glycol, glycerin, trehalose, urea, xylitol, and disodium EDTA; The oily matrix is ​​selected from one or more of olive oil, stearic acid, polyethylene glycol succinate, clove oil, soybean oil, corn oil, and white petrolatum. The pH adjuster is selected from one or more of potassium hydroxide, sodium hydroxide, arginine, triethanolamine, anhydrous disodium hydrogen phosphate, and anhydrous sodium dihydrogen phosphate.

[0017] The fourth objective of this invention is to provide a method for preparing the recombinant elastin cream dressing as described above, comprising the following steps: dissolving recombinant elastin and ceramide in purified water, stirring thoroughly until the solution is clear and transparent to obtain a first aqueous phase matrix; the pH value is 4.5-5.5; The second aqueous phase matrix is ​​obtained by uniformly mixing and dispersing the pyrrolidone carboxylic acid-lysozyme complex, water-soluble polymer and humectant; The pH adjuster was fully dissolved in purified water until it became clear and free of particles, thus obtaining the third aqueous phase matrix. The oily matrix is ​​mixed to obtain the first oil phase matrix; Pour the second aqueous phase matrix into the emulsification pot, stir and heat to 80-85℃ at 30-50r / min, keep warm and stir for 10-20min to ensure the material is completely dissolved; Add the first oil phase matrix to the oil phase pot, stir and heat at 30-50 r / min to 80-85℃, keep warm and stir for 10-15 min to ensure that the material is completely dissolved; Under homogenized conditions, the dissolved oil phase matrix is ​​extracted into the emulsification pot, vacuumed at -0.03 to -0.1 MPa, homogenized for 3-5 minutes, kept warm and stirred for 10-20 minutes, and then the cooling water is turned on to cool down. When the temperature drops to 60-70℃, turn off the cooling water, add the first aqueous phase matrix and the third aqueous phase matrix, continue to keep warm and stir for 10-20 minutes, then turn on the cooling water and stir at 30-40 r / min to continue cooling. When the temperature drops to 30-38℃, samples are taken for testing to obtain the cream dressing. The obtained cream dressing was irradiated with an electron beam at a dose of 20-30 kJ to obtain a sterile recombinant elastin cream dressing.

[0018] The present invention discloses a pyrrolidone carboxylic acid-lysozyme complex, which utilizes the antibacterial properties of lysozyme to improve the antibacterial ability of biomaterials and reduce the risk of infection, and can be applied to wound dressings.

[0019] This invention provides a recombinant elastin cream dressing and its preparation method. Combining advancements in modern chemical technology, the cream dressing is applied to the wound site to form a protective layer, isolating it from external contaminants and effectively preventing the invasion of dust or bacteria. It maintains the moisture of the wound skin for a prolonged period, promotes granulation tissue regeneration, and thus effectively accelerates skin healing, reducing the transition time for patients during the wound healing process.

[0020] Compared with the prior art, the beneficial effects of the present invention are: The components of this invention are scientifically proportioned and work synergistically to produce a recombinant elastin cream dressing with excellent moisture absorption. When applied to the wound site, it forms a protective layer, improving and optimizing the skin's microecology and promoting wound healing. The addition of recombinant elastin and ceramides enhances the wound's resistance to external factors and improves skin condition.

[0021] The addition of the pyrrolidone carboxylic acid-lysozyme complex alters the spatial structure of lysozyme, enhancing its binding ability with the bacterial cell wall and allowing for more effective substrate binding, thus synergistically strengthening its antibacterial efficacy. Furthermore, pyrrolidone carboxylic acid itself is a natural skin moisturizing factor; after condensation with lysozyme, it imparts new functional properties to the enzyme. For example, when the skin is dry, pyrrolidone carboxylic acid helps maintain a better active space, which can promote wound healing to some extent.

[0022] Electron beam irradiation sterilization is employed to prevent the inactivation of recombinant elastin, lysozyme, ceramides, and other components in this ointment dressing during high-temperature sterilization. Sterilized ointment dressings are more conducive to wound healing and have high safety. This product uses an emulsification process, resulting in good permeability and easy absorption of moisturizing ingredients, which can promote granulation tissue growth and effectively improve wound healing. Detailed Implementation

[0023] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0024] Example 1: Preparation of pyrrolidone carboxylic acid-lysozyme complex I. Experimental Materials 1. Condensing agent EDC (purity ≥ 99%) 2. Pyrrolidone carboxylic acid (purity ≥ 99%) 3. N-hydroxysuccinimide (NHS, purity ≥99%) 4. Lysozyme derived from egg white (purity ≥98%) 5. Hydroquinone (analytical grade) 6. Ethanol (analytical grade) 7. Ethyl acetate (analytical grade) 8. Citric acid (analytical grade) 9. Sodium citrate (analytical grade) 10. Phosphate (analytical grade) 11. Sodium chloride (analytical grade) 12. Glucosamine (analytical grade) 13. Acetic acid (analytical grade) 14. Sephadex G-100 Gel 15. DEAE-Sepharose Fast Flow Ion Exchange Resin 16. Chitin affinity chromatography column materials II. Experimental Instruments 1.250mL three-necked flask 2.100mL conical flask 3. High-precision low-temperature thermostat bath (DC-2006) 4. Precision pH meter (PHS-3C) 5. Mechanical mixer 6. Magnetic stirrer 7. Reflux condenser 8. Thermometer 9. Microsyringe (10mL) 10. Nitrogen gas introduction device 11. Rotary Evaporator 12. High-performance liquid chromatograph (Agilent 1260 Infinity II) 13. Thin-layer chromatograph 14. Electrophoresis apparatus (Mini-Protean Tetra vertical electrophoresis system) 15. Melting point apparatus (WRS-1B) 16. Online Infrared Spectrometer (Nicolet iS50) 17. Glass chromatography columns (60cm length, 2.6cm inner diameter; 30cm length, 1.6cm inner diameter; 20cm length, 1.0cm inner diameter) 18. Centrifuge III. Experimental Procedure (a) Screening experiment for early-stage terminators 1. Preliminary Experiment In twelve 50 mL Erlenmeyer flasks, 20 mL of a simulated reaction system (containing 10 mL of N-hydroxysuccinimide ester) was added to each flask. 3 (mol / L), place the conical flasks in a high-precision low-temperature thermostat bath, control the temperature at 4±1℃, and adjust the pH to 5.0 using a precision pH meter. Then, add 5 mL of a 10 mol / L solution to each conical flask. 2 A solution of hydroquinone, catechol, resorcinol, ethanol, propanol, ascorbic acid, sodium sulfite, sodium metabisulfite, sodium thiosulfate, dithiothreitol (DTT), β-mercaptoethanol, and oxalic acid (mol / L) was prepared and mechanically stirred for 15 minutes. After the reaction, thin-layer chromatography (TLC) was used for detection. A silica gel GF254 plate was used as the stationary phase, and chloroform-methanol (8:2, v / v) was used as the developing solvent. Spots were observed under UV light at 254 nm. Based on the degree of reduction in spot area, hydroquinone, catechol, and resorcinol were selected to proceed to the next stage. 2. Comparative Experiment In three 250 mL three-necked flasks, the same reaction system as the actual preparation (N-hydroxysuccinimide 10) was added. 3 mol / L, lysozyme 10 3 The concentration of hydroquinone, catechol, and resorcinol was controlled at 4±1℃ and pH 5.0. Equimolar amounts (1:1 molar ratio with the remaining active intermediate) of hydroquinone, catechol, and resorcinol were added to a three-necked flask, and the reaction was stopped after mechanical stirring for 20 minutes. After the reaction, high-performance liquid chromatography (HPLC) was used for analysis. An Agilent 1260 Infinity II HPLC system was used with a ZORBAX Eclipse XDB-C18 column (4.6×150mm, 5µm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. Gradient elution was used (0-5min, 5%B; 5-30min, 5-50%B; 30-35min, 50-95%B; 35-40min, 95%B), flow rate 1.0mL / min, detection wavelength 280nm, and injection volume 10µL. Based on the residual amount of the active intermediate and the purity of the product, hydroquinone was determined to be the better terminator. 3. Dosage optimization experiment Five 250 mL three-necked flasks were prepared, and a standard reaction system (under the same conditions as the control experiment) was added. Hydroquinone was prepared as a 10% (w / v) ethanol solution and added to the system according to the molar ratios of hydroquinone to the remaining active intermediate of 1:1, 1.5:1, 2:1, 2.5:1, and 3:1, respectively. The mixture was mechanically stirred for 20 minutes. After the reaction, the residual amount of the active intermediate and the purity of the product were determined by HPLC. The results showed that when the molar ratio was 2.5:1, the residual amount of the active intermediate was reduced to below the detection limit (<1×10⁻⁶). 5 (mol / L), with a product purity of 96.7%, this was determined to be the optimal dosage. 4. Compatibility Experiment The complete preparation reaction of the pyrrolidone carboxylic acid-lysozyme complex was carried out in a 250 mL three-necked flask. After the reaction was completed, hydroquinone was added at the optimal dosage (molar ratio 2.5:1) to terminate the reaction. The product was then purified sequentially by Sephadex G-100 gel column chromatography, DEAE-Sepharose Fast Flow ion exchange chromatography, and chitin affinity chromatography. The products at each stage were analyzed by HPLC, and the results showed that hydroquinone was compatible with the preparation process and did not affect the purification or product quality. (II) Preparation of the complex 1. Activation stage In a 250 mL three-necked flask, add 12 g (purity ≥99%) of condensing agent EDC, followed by 10 g (purity ≥99%) of pyrrolidone carboxylic acid. Stir mechanically at 180 rpm for 15 minutes. Then, slowly inject 8 g (purity ≥99%) of N-hydroxysuccinimide (NHS) using a 10 mL microsyringe within 5 minutes to form the activated intermediate. 2. Pro-nuclear attack phase The intermediate prepared above was transferred to a 100 mL Erlenmeyer flask, and 10 g (purity ≥98%) of egg white-derived lysozyme was added. The temperature was controlled at 4±1℃ in a high-precision cryostat bath, and the pH was adjusted to 4.5-5.5 by adding 0.1 mol / L citrate-sodium citrate buffer solution using a precision pH meter. Under nitrogen protection (nitrogen flow rate 100 mL / min), the reaction was stirred at 120 rpm for 2.5 hours using a magnetic stirrer.

[0025] 3. Amide bond formation and water formation stages During the reaction, infrared spectra were acquired every 15 minutes using an online infrared spectrometer (Nicolet iS50) to monitor the characteristic absorption peak of the amide bond (1650 cm⁻¹). -1 The characteristic absorption peaks of water (3400 cm⁻¹) and other components. -1 (Location). When the intensity of the characteristic absorption peak of the amide bond remains essentially unchanged within 30 minutes, and the intensity of the characteristic absorption peak of water no longer increases significantly, the reaction is considered to be essentially complete. 4. Termination and Purification Stage (1) Termination agent usage: After the reaction is complete, add 2.5 moles of hydroquinone for every mole of remaining active intermediate to prepare a 10% (w / v) ethanol solution of hydroquinone, and quickly add it to the reaction system. Use mechanical stirring and stir at 200 rpm for 20 minutes. Detect the reaction using TLC and HPLC. When the content of the active intermediate decreases below the detection limit, it is considered that the active intermediate has been basically completely deactivated. Transfer the reaction system to a rotary evaporator and distill under reduced pressure at 40℃ and -0.09 MPa to remove the ethanol solvent. (2) Purification treatment ① Gel column chromatography: Inject the concentrate into a pre-equilibrated Sephadex G-100 gel column (60cm long, 2.6cm inner diameter, equilibrated with 0.05mol / L phosphate buffer (pH 7.0)), elute at a flow rate of 0.8mL / min, and collect the eluent of the target product. Ion exchange chromatography: The collected solution was passed through a DEAE-Sepharose Fast Flow ion exchange column (30 cm long, 1.6 cm inner diameter) and eluted with a 0-0.5 M NaCl gradient to further remove charged impurities. Affinity chromatography: The treated solution was passed through a chitin affinity chromatography column (20 cm long, 1.0 cm inner diameter), equilibrated with 0.1 M acetate buffer (pH 4.5), eluted with acetate buffer containing 0.5 M glucosamine, and the solution corresponding to the elution peak was collected. After each purification step, HPLC was used to detect the residual amount of hydroquinone and the purity of the product. If the residual amount of hydroquinone was higher than 0.1%, the corresponding purification steps were repeated until the residual amount was lower than the standard, thus obtaining a high-purity (purity ≥99.5%) pyrrolidone carboxylic acid-lysozyme complex. V. Experimental Results The final product was a pyrrolidone carboxylic acid-lysozyme complex, with a purity of 99.6% as determined by HPLC and a hydroquinone residue of 0.08%. Electrophoresis analysis showed that the product had a single band, indicating that it was of high purity. VI. Experimental Conclusions Through the above experimental steps, a high-purity pyrrolidone carboxylic acid-lysozyme complex was successfully prepared. The operating parameters at each stage were reasonable and feasible, and the quality control was effective.

[0026] Example 2 A recombinant elastin cream dressing and its preparation method, wherein the cream dressing specifically comprises the following components, by weight: Recombinant elastin complex solution: 3.5%, comprising 3% recombinant elastin and 0.5% ceramide. Pyrrolidone carboxylic acid-lysozyme complex: 3% Water-soluble polymer: Sodium hyaluronate 1%, Carbomer 0.5% Moisturizers: 8% propylene glycol, 0.05% disodium ethylenediaminetetraacetate Oily base: 3% olive oil, 2% stearic acid, 0.5% vitamin E succinate (polyethylene glycol) pH adjuster: 0.5% potassium hydroxide Purified water: Balance The elastin used is the recombinant elastin disclosed in Chinese patent CN117551184B.

[0027] Preparation method: 1. Dissolve recombinant elastin and ceramide in purified water and stir thoroughly until the solution is clear and transparent to obtain the first aqueous phase matrix; 2. The pyrrolidone carboxylic acid-lysozyme complex, propylene glycol, sodium hyaluronate, carbomer, and disodium EDTA are stirred and dispersed evenly to avoid clumping, thus obtaining the second aqueous phase matrix; 3. Dissolve the pH adjuster thoroughly in purified water until it is clear and free of particles, to obtain the third aqueous phase matrix; 4. Mix olive oil, stearic acid, and vitamin E succinic acid polyethylene glycol ester to obtain the first oil phase matrix; 5. Pour the second aqueous phase matrix into the emulsification pot, stir and heat to 82°C at 40 rpm, keep warm and stir for 15 minutes to ensure the material is completely dissolved; 6. Add the first oil phase matrix to the oil phase pot, stir and heat to 82°C at 40 r / min, keep warm and stir for 12 min to ensure that the material is completely dissolved; 7. Under homogenized conditions, extract the dissolved oil phase matrix into the emulsification pot, vacuum -0.06MPa, homogenize for 4 min, keep warm and stir for 15 min, then turn on the cooling water to cool down; 8. When the temperature drops to 65℃, turn off the cooling water, add the first aqueous phase matrix and the third aqueous phase matrix, continue to keep warm and stir for 15 minutes, then turn on the cooling water and stir at 35 r / min to continue cooling. 9. When the temperature drops to 35℃, samples are taken for testing to obtain the cream dressing; 10. The obtained cream dressing is irradiated with an electron beam at a dose of 25 kJ to obtain a sterile recombinant elastin cream dressing.

[0028] Example 3 This invention provides a recombinant elastin cream dressing, comprising the following raw materials by weight: Recombinant elastin complex solution: 3.5%, comprising 3% recombinant elastin and 0.5% ceramide; Pyrrolidone carboxylic acid-lysozyme complex: 3%; Water-soluble polymer: Sodium hyaluronate 1%, Carbomer 0.5%; Moisturizers: 8% propylene glycol, 0.05% disodium ethylenediaminetetraacetate; Oily base: 3% olive oil, 2% stearic acid, 0.5% polyethylene glycol succinate (vitamin E); pH adjuster: 0.5% potassium hydroxide; Purified water: Balance.

[0029] The preparation method is the same as in Example 2.

[0030] Example 4 This invention provides a recombinant elastin cream dressing, comprising the following raw materials by weight: Recombinant elastin complex solution: 1.3%, comprising 1% recombinant elastin and 0.3% ceramide; Pyrrolidone carboxylic acid-lysozyme complex: 2%; Water-soluble polymers: Sodium hyaluronate 0.3%, Carbomer 0.5%; Moisturizers: 5% propylene glycol, 0.03% disodium ethylenediaminetetraacetate; Oily base: 2% olive oil, 1% stearic acid, 0.5% polyethylene glycol succinate (vitamin E); pH adjuster: 0.3% potassium hydroxide; Purified water: Balance.

[0031] The preparation method is the same as in Example 2.

[0032] Example 5: This invention provides a recombinant elastin cream dressing, comprising the following raw materials by weight: Recombinant elastin complex solution: 3.5%, comprising 3% recombinant elastin and 0.5% ceramide; Pyrrolidone carboxylic acid-lysozyme complex: 2%; Water-soluble polymers: Sodium hyaluronate 0.3%, Carbomer 0.5%; Moisturizers: 5% propylene glycol, 0.03% disodium ethylenediaminetetraacetate; Oily base: 3% olive oil, 2% stearic acid, 0.5% polyethylene glycol succinate (vitamin E); pH adjuster: 0.5% potassium hydroxide; Purified water: Balance.

[0033] The preparation method is the same as in Example 2.

[0034] Example 6: This invention provides a recombinant elastin cream dressing, comprising the following raw materials by weight: Recombinant elastin complex solution: 5% recombinant elastin, 1% ceramide; Pyrrolidone carboxylic acid-lysozyme complex: 3%; Water-soluble polymers: Sodium hyaluronate 0.3%, Carbomer 0.5%; Moisturizers: 5% propylene glycol, 0.03% disodium ethylenediaminetetraacetate; Oily base: 3% olive oil, 2% stearic acid, 0.5% polyethylene glycol succinate (vitamin E); pH adjuster: 0.5% potassium hydroxide; Purified water: Balance.

[0035] The preparation method is the same as in Example 2.

[0036] Application Example 1: Lysozyme Activity Assay I. Experimental Objective The antibacterial activity of the pyrrolidone carboxylic acid-lysozyme complex was accurately determined and compared with that of ordinary lysozyme solution and other control solutions to verify its superior antibacterial ability.

[0037] II. Experimental Materials 1. Indicator bacteria: Staphylococcus aureus ATCC 25923, purchased from the American Center for Type Culture Collection.

[0038] 2. Culture medium: Tryptic soybean agar (TSA).

[0039] 3. Solution (1) Control group lysozyme solution: lysozyme (purity ≥98%, purchased from Sigma-Aldrich), with a concentration of 10 mg / mL.

[0040] (2) Pyrrolidone carboxylic acid-lysozyme complex solution: prepared according to the method of Patent Example 1, with a concentration of 10 mg / mL.

[0041] (3) Complex solution of lysozyme and inactive modified material (bovine serum albumin): lysozyme and bovine serum albumin (purchased from Sigma-Aldrich) were mixed at a ratio of 1:1 (g / g) to prepare a concentration of 10 mg / mL.

[0042] 4. Other materials: 6mm diameter sterile qualitative filter paper, micropipette (10-100μL), petri dishes, incubator, etc.

[0043] III. Experimental Procedure Culture medium preparation: Prepare the culture medium according to the instructions of tryptic soybean agar (TSA). After heating and dissolving, pour about 15-20 mL into sterile petri dishes while hot, and allow the plates to solidify before use.

[0044] Solution addition: Using a micropipette, accurately pipette 20 μL of the control group lysozyme solution, the pyrrolidone carboxylic acid-lysozyme complex solution, and the lysozyme-inactive modifier complex solution, and add them to a sterile filter paper, ensuring that the solution is evenly distributed on the filter paper.

[0045] Plate culture: Carefully place the filter paper discs with the added solutions onto TSA plates containing indicator bacteria. Place 5 filter paper discs on each plate (1 for each solution, repeated 5 times), and gently press to ensure full contact between the filter paper discs and the culture medium. Invert the plates and incubate at 37°C for 24 hours.

[0046] Results measurement: After the culture was completed, the plate was removed and the diameter of the inhibition zone around each filter paper was measured with a vernier caliper (accurate to 0.1 mm), and the data was recorded.

[0047] IV. Experimental Results and Analysis 1. Data Recording

[0048] 2. Data Analysis The data were statistically analyzed using a t-test to determine whether the differences in the diameter of the inhibition zone between different solution groups were statistically significant.

[0049] Compared with the control group of lysozyme solution, the pyrrolidone carboxylic acid-lysozyme complex solution group showed a highly significant difference (t=17.23, degrees of freedom df=8, P<0.001).

[0050] Compared with the pyrrolidone carboxylic acid-lysozyme complex solution group and the lysozyme-inactive modifier complex solution group: t=18.45, degrees of freedom df=8, P<0.001, the difference was extremely significant.

[0051] V. Experimental Conclusions The above experiments and data analysis show that the pyrrolidone carboxylic acid-lysozyme complex has significantly stronger antibacterial ability than ordinary lysozyme solution and the complex of lysozyme and inactive modifiers. This indicates that the complex can effectively enhance the antibacterial activity of lysozyme and has potential application value in fields such as medical aesthetics.

[0052] Application Example 2 1. To investigate the difference in antibacterial activity between the pyrrolidone carboxylic acid-lysozyme complex and ordinary lysozyme in a recombinant elastin dressing system, the following experiment was conducted: 2. Experimental Methods The agar diffusion method was used. Nutrient agar medium was melted and poured into sterile petri dishes, approximately 15-20 mL per dish, and allowed to solidify. Small holes (6 mm in diameter) were punched in the plates using a sterile punch. 20 μL of the recombinant elastin dressings from Examples 2 and 3, prepared at a concentration of 5 mg / mL, was added to the corresponding wells. An equal volume of sterile physiological saline was used as a negative control, and a solution containing a commonly used antibacterial agent (e.g., gentamicin, at a concentration of 10 mg / mL) was used as a positive control. The plates were incubated at 37°C for 24 hours.

[0053] 3. Experimental Results

[0054] 4. Experimental Conclusions Based on the above experimental results, the following conclusions can be drawn: In the recombinant elastin dressing system, the dressing solution containing the pyrrolidone carboxylic acid-lysozyme complex exhibits significantly better antibacterial activity against Staphylococcus aureus and Escherichia coli than the dressing solution containing ordinary lysozyme. Specifically, the dressing solution containing the pyrrolidone carboxylic acid-lysozyme complex forms a larger average inhibition zone diameter in the culture experiments of both bacteria, indicating a stronger inhibitory effect on these two common bacteria. This demonstrates its more effective antibacterial efficacy, providing better antibacterial protection for wounds and contributing to wound healing and recovery.

[0055] Application Example 3 I. Experimental Objective Using a rat skin trauma model, the efficacy of the product of the present invention was evaluated by comparing the recombinant elastin cream dressing of Example 4 of the present invention with that of other brands of cream dressings in promoting wound healing.

[0056] II. Experimental Materials 1. Experimental animals: Forty healthy SPF-grade SD rats, weighing 200-220g, half male and half female, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Before the experiment, the rats were housed in an environment with a temperature of 22±2℃, humidity of 50-60%, and a 12-hour day-night cycle for 7 days for acclimatization, with free access to food and water.

[0057] 2. Experimental dressings (1) Recombinant elastin cream dressing of the present invention Example 4: prepared according to the above formula.

[0058] (2) Control dressing: Select two common brands of similar cream dressings on the market (labeled as control dressing A and control dressing B respectively).

[0059] (3) Other materials: sodium pentobarbital (manufacturer: Sigma-Aldrich, item number: ABC123) for anesthesia; iodine tincture, normal saline (Hualan Biological Engineering Co., Ltd., specification: 250ml / bag); scalpel blades (Shanghai Medical Instruments (Group) Co., Ltd. Surgical Instrument Factory), sterile gauze (Winner Medical Products Co., Ltd.), vernier calipers (accuracy 0.02mm, Guilin Guanglu Digital Measurement and Control Co., Ltd.), tissue fixative (4% paraformaldehyde), etc.

[0060] III. Experimental Methods 1. Trauma Model Establishment: Forty rats were randomly divided into four groups of ten each: experimental group (using the dressing of this invention), control dressing group A, control dressing group B, and blank control group (wound treated only with physiological saline). Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30 mg / kg). After anesthesia took effect, the hair on the rats' backs was shaved with an electric shaver. Then, a circular full-thickness skin defect, approximately 1.5 cm in diameter, was created on each side of the spine using a sterilized scalpel blade, reaching the subcutaneous fascia layer.

[0061] 2. Dressing treatment (1) Experimental group: After the wound hemostasis, the recombinant elastin cream dressing of the present invention was applied evenly immediately with a thickness of about 0.5 mm, and then covered with sterile gauze and fixed with medical tape.

[0062] (2) Control dressing group A and control dressing group B: respectively, the corresponding brand of cream dressing was used, and the treatment method was the same as that of the experimental group.

[0063] (3) Blank control group: Apply an equal amount of physiological saline to the wound and cover it with sterile gauze for fixation.

[0064] 3. Observation and Data Collection (1) Observation of wound healing: The wound healing of rats was observed and recorded at regular intervals every day, including wound redness and swelling, exudation, and signs of infection.

[0065] (2) Wound area measurement: On days 0, 3, 7, 10, and 14 after modeling, the longest diameter and the shortest diameter perpendicular to the longest diameter of the wound were measured using vernier calipers. The wound area was calculated according to the formula: Wound area = π × (longest diameter / 2) × (short diameter / 2), and the wound healing rate was calculated. Wound healing rate (%) = [(initial wound area - wound area at measurement) / initial wound area] × 100%.

[0066] (3) Histological analysis: On the 14th day of the experiment, five rats were randomly selected from each group, euthanized by overdose, and the wound and surrounding tissue were taken, fixed in 4% paraformaldehyde fixative, paraffin embedded, sectioned, stained with HE, and histological changes were observed under an optical microscope to assess the growth of new epithelial tissue, granulation tissue and inflammatory cell infiltration.

[0067] (4) Heme content determination: Skin tissue around the wounds of the remaining 5 rats was taken and the heme content was determined using a heme content determination kit (purchased from Nanjing Jiancheng Bioengineering Institute, catalog number: A012-2-1). The operation steps were strictly performed in accordance with the kit instructions.

[0068] (5) Allergy rate statistics: During the experiment, observe the rats for allergic reaction symptoms such as erythema, edema, and itching around the wound and on the whole body every day, count the number of allergic rats in each group, and calculate the allergy rate.

[0069] IV. Experimental Results 1. Wound healing status (1) Experimental group: On the 3rd day after surgery, the redness and swelling of the wound were significantly reduced and the exudate was less; on the 7th day, obvious new epithelial tissue could be seen at the edge of the wound and the granulation tissue grew well; on the 10th day, the wound area was significantly reduced; on the 14th day, the wound was basically healed and only a small amount of scar remained.

[0070] (2) Control dressing group A: On the 3rd day, the wound still had a lot of exudate and the redness and swelling subsided slowly; on the 7th day, the growth of new epithelial tissue was not obvious; on the 14th day, the wound was not completely healed and a large area of ​​wound remained.

[0071] (3) Control dressing group B: The wound healing was similar to that of control dressing group A, but the healing speed was slower, and some rats showed mild infection symptoms during the experiment.

[0072] (4) Blank control group: The wound healed slowly, and there was still a large wound on the 14th day, with obvious inflammatory response and a lot of inflammatory cell infiltration.

[0073] 2. Wound area and healing rate

[0074] 3. Histological analysis (1) Experimental group: A large number of newly formed epithelial cells covered the wound, collagen fibers in the granulation tissue were neatly arranged, and there were few inflammatory cells.

[0075] (2) Control dressing group A and group B: fewer new epithelial cells, more disordered collagen fiber arrangement in granulation tissue, and more inflammatory cell infiltration.

[0076] (3) Blank control group: The wound was mainly composed of inflammatory cells, and the growth of new epithelial and granulation tissue was not obvious.

[0077] 4. Heme content

[0078] 5. Allergy rate (1) Experimental group: allergy rate was 0%.

[0079] (2) Control dressing group A: allergy rate was 15%.

[0080] (3) Control dressing group B: allergy rate was 20%.

[0081] (4) Blank control group: No allergic reaction.

[0082] 6. Overall healing score A comprehensive scoring method was used, based on wound healing rate (40%) and histological appearance (40%). The assessment was based on four aspects: weight gain (30%), heme content (20%), allergies (10%), and a maximum score of 10. The assessment results are as follows:

[0083] V. Experimental Conclusions Experiments using a rat skin trauma model showed that the recombinant elastin cream dressing of this invention is significantly superior to other brand control dressings in promoting wound healing, reducing inflammatory response, lowering heme content, and preventing allergic reactions, and has good application prospects.

[0084] Application Example 4 I. Experimental Objective Through a human stimulation experiment using capsaicin, simulating a skin irritation and trauma scenario, the differences between the recombinant elastin cream dressing of Example 5 of this invention and other brands of dressings in promoting wound repair, alleviating inflammatory response, and safety were compared.

[0085] II. Experimental Materials 1. Subjects: Ten healthy subjects were recruited, with no history of skin diseases, drug allergies, or immune system diseases.

[0086] 2. Experimental dressings (1) Recombinant elastin cream dressing of the present invention Example 5: prepared according to the above formula.

[0087] (2) Comparison dressing: Select commercially available well-known brand dressings of the same type.

[0088] (3) Other materials: capsaicin solutions with concentrations of 0%, 1.0%, and 1.5%, sterile cotton swabs, sterile gauze, medical tape, and heme content detection kit.

[0089] III. Experimental Methods On the inner forearm of each subject, 0%, 1.0%, and 1.5% capsaicin solutions were applied symmetrically using sterile cotton swabs, with each application area approximately 2cm × 2cm. After 5 minutes of capsaicin application, the recombinant elastin cream dressing of this invention was applied to the left wound area to a thickness of approximately 0.3mm, and an equal amount of the control dressing was applied to the right wound area. Both were then covered with sterile gauze and secured with medical tape. The dressings were changed once daily, and the wound healing time, hemoglobin levels, and allergic reactions were continuously observed and recorded. A comprehensive score was calculated based on four dimensions: healing speed, hemoglobin changes, allergic reactions, and comfort, with a maximum score of 10 points.

[0090] IV. Experimental Results

[0091] V. Overall Conclusion Based on the results of the rat skin trauma model experiment in Application Example 3 and the human capsaicin stimulation experiment in this embodiment, it is evident that, in both animal and human experiments, the recombinant elastin cream dressing of this invention is significantly superior to the control dressing in promoting wound healing, reducing inflammatory response, and preventing allergies. This dressing provides nutrition to the wound, maintains a moist environment, effectively improves wound healing, shortens healing time, and is highly safe, thus enhancing the patient's recovery experience.

[0092] Application Example 5 Since the recombinant elastin cream dressing of the present invention is a medical device dressing and is applied to wounds, the recombinant elastin cream dressing of Example 6 needs to undergo routine five-item biocompatibility tests. The test results are as follows: 1. Cytotoxicity test The cytotoxicity test was conducted according to the GB / T16886.1-2022 standard, and the specific steps are as follows: 1.1 Experimental Preparation: All instruments that came into contact with the samples and cells were sterilized by moist heat to ensure sterility. L929 cells in the logarithmic growth phase and in good condition, along with the recombinant elastin cream dressing to be tested, were prepared using DMEM high-glucose medium to a concentration of 4 × 10⁻⁶. 4 Cell suspensions of 100 cells / ml were seeded into 96-well plates. DMEM high-glucose medium containing 10% fetal bovine serum was used as the extraction medium. The test sample was cut into strips of 0.5cm × 2cm with a thickness of 0.5mm and placed in glass containers at a ratio of 6cm² / ml. Extraction was carried out at 37±1℃ for 24h.

[0093] 1.2 Experimental Procedure: After cell seeding, the cell seeding density of each well was checked using a phase-contrast microscope. Once confirmed to be correct, the wells were incubated at 37±1℃ and 5%±1% CO2 for 24 hours. After 24 hours of incubation, the original culture medium was discarded, and cell morphology, quantity, and growth were observed under a microscope to qualitatively determine whether the sample extract had a toxic effect. On days 2, 4, and 7 after changing the culture medium, three vials from each group were taken to observe and count the cells for morphological analysis. Subsequently, MTT solution was added, and the cells were incubated in a CO2 incubator for another 2 hours. Isopropanol was then added for color development. After shaking the culture plate, the 96-well plate was placed on a microplate reader, and the absorbance of each well was measured at a wavelength of 490 nm. The average value was used to calculate the relative cell proliferation.

[0094] 1.3 Test Results 1.3.1 Cell morphology analysis: Under a phase-contrast microscope, the cells showed normal morphology, good adherence and growth, and formed uniform, irregular triangles. Based on the cell morphology analysis standards of the "Cytotoxicity Test Method" in the Pharmacopoeia of the People's Republic of China (2020 Edition, Part IV), the degree of reaction was determined to be non-toxic.

[0095] 1.3.2 Relative cell proliferation analysis (calculated based on cell concentration on day 7):

[0096] A relative cell proliferation rate of 90% or higher generally indicates that the material has good biocompatibility with cells and is suitable for medical device applications. According to the cell relative proliferation grading table in the "Cytotoxicity Test Method" of the Pharmacopoeia of the People's Republic of China (2020 Edition, Part IV), the cytotoxicity reaction of this dressing is Grade 1, indicating that the dressing has passed the cytotoxicity test and has a high level of safety.

[0097] 2. Sensitization test In accordance with GB / T16886.1-2022 standard, considering the characteristics of recombinant elastin cream dressings as surface application products, a closed-application test (Buehler test) was conducted, as follows: 2.1 Experimental Preparation: Fifteen albino guinea pigs weighing 300-500g and aged 2-3 months were selected as test animals for skin sensitization and housed in separate cages in a constant-temperature room. The housing environment followed the requirements of ISO10993-2, with the temperature controlled at 22±1℃, humidity at 40%-70%, and environmental noise below 60dB. The guinea pigs were housed for 3 days under stable dietary and environmental conditions. Before the experiment, the fur on the left back of the guinea pigs was removed.

[0098] 2.2 Experimental Procedure 2.2.1 Preliminary Experiment Eight-cm² patches were used, soaked in both the test material and medical saline solution (as an extract), and applied to the hair-removed areas of guinea pigs. The patches were secured with a closed bandage for 6 hours. At 24 and 48 hours after patch removal, the degree of skin erythema and edema at the test sites was evaluated according to the Magnusson and Kligman grading system.

[0099] Magnusson and Kligman classification

[0100] Five concentration gradients were set up for the test samples: medical saline: recombinant elastin cream dressing = 4:1, 3:1, 2:1, 1:1, 1:2. The control group only applied a dressing soaked in medical saline.

[0101] The experimental results showed that no erythema reaction was observed on the skin of the test samples and control group guinea pigs at any concentration after 6 hours of application of the extract, and the Magnusson and Kligman grades were 0. Therefore, the main experimental concentration was determined to be medical saline: recombinant elastin cream dressing = 1:2.

[0102] 2.2.2 Main Experiment 2.2.2.1 Induction Phase Induction phase: According to the concentration selected in the preliminary experiment (medical saline: recombinant elastin cream dressing = 1:2), the extract was soaked in the dressing and applied to the upper left back of each guinea pig. After 6 hours, the closed bandage and dressing were removed. This operation was repeated for 3 consecutive days within 1 week, for a total of 3 weeks. The control group used saline dressing.

[0103] 2.2.2.2 Activation Phase Fourteen days after the final induction experiment, all experimental and control animals were challenged with the test sample and control sample, respectively. Similarly, the patch, soaked in the test sample at the selected concentration, was applied to the upper right back of the guinea pig. Six hours later, the fixation device, ties, and patch were removed. Scoring was performed according to the Magnusson and Kligman grading system at 24 and 48 hours after patch removal.

[0104] 2.2.2.3 Main Experiment Results Table 2

[0105] Note the calculation method: 1. During the observation, the number of times grade 0, grade 1, grade 2, and grade 3 skin reactions occurred were recorded as n0, n1, n2, and n3, respectively.

[0106] 2. Calculate the total score of the grade integrals: According to the formula S=n0×0+n1×1+n2×2+n3×3, calculate the total score S of the skin reaction grade integrals of all observed subjects.

[0107] 3. Calculate the average: Divide the sum of the rank integrals S by the total number of observed objects N (N=n0+n1+n2+n3) to obtain the average rank integral. The formula is =NS When the recombinant elastin cream dressing (concentration: medical saline: recombinant elastin cream dressing = 1:2) was used, the skin reaction on the back of the guinea pigs in the experimental group was grade 0. According to the evaluation criteria for the results of the closed dressing test in GB / T16886.1-2022, the recombinant elastin cream dressing obtained by this invention is non-sensitizing.

[0108] 3. Stimulus test The human skin irritation test was conducted according to the GB / T16886.23-2023 standard, and the steps are as follows: 3.1 Experimental Preparation: We are recruiting 30 volunteers (15 men and 15 women) aged 26-30, using 2.5cm diameter dressing patches.

[0109] 3.2 Experimental Procedure: The recombinant elastin cream dressing was moistened with 200 μl of purified water and applied to the inner side of the left upper arm of the volunteer, secured with a closed bandage with a gauze pad. Volunteers were allowed to leave the testing room after 15 and 30 minutes, continuing for 1, 2, 3, and 4 hours without experiencing skin irritation. If no irritation occurred after 48 hours, the dressing was applied to another untested site for extended periods to fully assess delayed irritation. 20% SDS was used as a positive control; its irritation properties were verified through characterization experiments.

[0110] 3.3.1 Clinical observation: Immediately after removing the patch, the skin reaction was graded, and re-graded at 1h, 2h, 24h, 48h, and 72h, with the observation period extended if necessary. Simultaneously, the skin condition (pigmentation and hydration level) before and after the test was accurately recorded. The skin reaction grading criteria are as follows: Table 3 Grading Table for Human Skin Irritation Tests

[0111] 3.4 Test Results Table 4

[0112] Note: The calculation method is the same as that for sensitization tests.

[0113] As shown in the table, compared with the positive control group, the average score of the irritation test for the recombinant elastin cream dressing of this invention is between 0 and 1. Based on the irritation test evaluation standard in GB / T 16886.23-2023 Medical Device Biological Evaluation, this dressing is determined to be non-irritating.

[0114] 4. Systemic toxicity In accordance with the requirements of GB / T 16886.11 standard, a systemic toxicity test was conducted, as follows: 4.1 Experimental Preparation: The experiment consisted of an experimental group and a control group, with five KM mice of the same origin and strain, weighing 18±2g in each group. All female mice were non-pregnant. A sterile environment was maintained for all experimental procedures. The recombinant elastin cream dressing was cut into strips of 0.5cm×3cm, 0.5mm thick, and immersed in 0.9% sodium chloride injection extraction medium at a ratio of 6cm² / ml. The control group used extraction medium without the dressing. Before injection, both the test and control solutions were vigorously shaken to ensure thorough mixing of the extracts.

[0115] 4.2 Experimental Procedure: The test solution and control solution were administered intravenously to mice in the experimental and control groups at a dose of 50 ml / kg, respectively, at an injection rate of 0.1 ml / s. Immediate responses were observed after injection, and the status, toxic reactions, and number of deaths of mice in both groups were recorded at 4 h, 24 h, 48 h, and 72 h. Mice were weighed at 72 h.

[0116] 4.3 Evaluation Grade – Observation Indicators of Mouse Response Table 5

[0117] 4.4 Evaluation of Test Results Table 6

[0118] Compared with the common systemic toxicity reactions of similar products, the recombinant elastin cream dressing of this invention did not induce any toxic symptoms in mice during the experiment, and the weight changes were normal. Based on the evaluation according to GB / T 16886.11 standard, the recombinant elastin cream dressing of this invention was determined to have no systemic toxicity reactions.

[0119] 5. Pyrogen test According to GB / T 14233.2-2022 standard, pyrogen testing was conducted based on the Chinese Pharmacopoeia, Part IV, "Pyrogen Testing Method". The specific steps are as follows: 5.1 Experimental Preparation Three healthy rabbits weighing 2.0 kg ± 0.2 kg were selected as experimental animals; the female rabbits were not pregnant. For the first 7 days, the rabbits underwent acclimatization rearing. The ambient temperature was maintained between 15-25℃, with a temperature difference not exceeding 3℃, and the ammonia content in the air below 20 ppm. The environment was kept quiet, avoiding strong light and noise interference. Body temperature was monitored before the experiment, measured every 30 minutes for a total of two measurements. Both measurements were required to be within the range of 38.0-39.6℃, with the highest and lowest temperature difference not exceeding 0.4℃, and the temperature difference within the same group not exceeding 1℃. After 48 hours of rest, pyrogen testing was performed on the rabbits to ensure that all containers that came into contact with the test samples were sterile and free of pyrogens.

[0120] 5.2 Test Procedure Cut the recombinant elastin cream dressing into strips of 0.5 cm × 3 cm with a thickness of 0.5 mm, immerse it in the extraction medium of 0.9% sodium chloride injection, and extract it充分浸提 according to the ratio of 6 cm² / ml. Select 3 rabbits. Within 15 minutes after measuring the body temperature, slowly inject the test solution warmed to 38°C into the ear vein. Subsequently, measure the body temperature once every 30 minutes. Subtract the normal body temperature value from the highest value among the 6 body temperature measurements, and the result is the body temperature increase value of the rabbit.

[0121] 5.3 Test evaluation method Evaluate according to the standard of "Pyrogen Test" in the fourth part of Chinese Pharmacopoeia. The judgment criteria are as follows: Table 7

[0122] 5.4 Test results Table 8

[0123] During the initial test of 3 rabbits, the body temperature increase was less than 0.6°C, and the total temperature increase at any time period was less than 1.3°C, which met the requirements of the "Pyrogen Test" in the fourth part of Chinese Pharmacopoeia. There was no need for a retest, and it was determined that the recombinant elastin cream dressing passed the pyrogen test.

[0124] Comprehensive conclusion: Through the five biocompatibility tests of cytotoxicity, sensitization, irritation, systemic toxicity reaction and pyrogen test, it can be seen that the recombinant elastin cream dressing prepared by the present invention has low cytotoxicity, no sensitization and irritation, no systemic toxicity reaction and passes the pyrogen test. It shows good performance in terms of biosafety and meets the safety requirements of medical device management.

[0125] For those not covered above, the prior art shall apply.

[0126] Although some specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are only for illustration and not for limiting the scope of the present invention. Those skilled in the art of the present invention can make various modifications or supplements to the described specific embodiments or use similar methods for substitution, but will not deviate from the direction of the present invention or exceed the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made based on the technical essence of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a pyrrolidone carboxylic acid-lysozyme complex, characterized in that: Includes the following steps: S1. First, the condensing agent EDC is mixed with pyrrolidone carboxylic acid and reacted. S2. Add NHS to the reaction system in step S1 and react to obtain the intermediate N-hydroxysuccinimide ester. S3, intermediate N-hydroxysuccinimide ester, and lysozyme were mixed and reacted. The pH of the reaction system was adjusted to 5.0-5.5 using a buffer solution, and the reaction temperature was controlled at 4±1℃. S4. After the reaction in step S3 is completed, a certain amount of terminator is added, and then the mixture is purified to obtain the pyrrolidone carboxylic acid-lysozyme complex. The mass ratio of condensing agent EDC to pyrrolidone carboxylic acid is 1.2:1; the mass ratio of NHS to EDC is 1:1.5; and the mass ratio of intermediate N-hydroxysuccinimide ester to lysozyme is 1:

1. The molar ratio of the terminator to the intermediate N-hydroxysuccinimide ester is 2.5:1; the terminator is hydroquinone.

2. The preparation method according to claim 1, characterized in that: In step S3, the buffer solution is a citric acid-sodium citrate buffer solution with a pH of 4.5-5.

5.

3. The preparation method according to claim 1, characterized in that: In step S4, the purification process is as follows: slowly inject the pre-equilibrated Sephadex G-100 gel column for elution, and collect the eluent containing the target product; then pass the collected solution through a DEAE-Sepharose Fast Flow ion exchange column for gradient elution; finally, elute with a chitin affinity chromatography column and collect the solution corresponding to the elution peak. Detection was performed using high performance liquid chromatography. If the hydroquinone residue was higher than 0.1%, the corresponding purification steps were repeated until the residue was lower than 0.1%, ensuring that the final pyrrolidone carboxylic acid-lysozyme complex with a purity ≥99.5% was obtained.

4. A pyrrolidone carboxylic acid-lysozyme complex prepared by the preparation method according to any one of claims 1-3.

5. The use of the pyrrolidone carboxylic acid-lysozyme complex as described in claim 4 in the preparation of wound dressings.

6. A recombinant elastin cream dressing, characterized in that: The product comprises, by weight parts, the following raw materials: 0.1-10% recombinant elastin complex solution, 0.1-10% pyrrolidone carboxylic acid-lysozyme complex as described in claim 4, 1-5% water-soluble polymer, 1-20% humectant, 1-5% oily matrix, 0.1-2% pH adjuster, and the remainder being purified water; the recombinant elastin complex solution comprises the following raw materials by weight parts: 1-5% recombinant elastin and 0.1-1% ceramide.

7. The recombinant elastin cream dressing as described in claim 6, characterized in that: The water-soluble polymer is selected from one or more of sodium hyaluronate, xanthan gum, sodium alginate, chitosan, sodium carboxymethyl cellulose, carbomer, and polyvinyl alcohol. The moisturizer is selected from one or more of propylene glycol, glycerin, trehalose, urea, and xylitol; The oily matrix is ​​selected from one or more of olive oil, stearic acid, polyethylene glycol succinate, clove oil, soybean oil, corn oil, and white petrolatum. The pH adjuster is selected from one or more of potassium hydroxide, sodium hydroxide, arginine, triethanolamine, anhydrous disodium hydrogen phosphate, and anhydrous sodium dihydrogen phosphate.

8. A method for preparing a recombinant elastin cream dressing as described in claim 6 or 7, characterized in that: Includes the following steps: Recombinant elastin and ceramide were dissolved in purified water and stirred thoroughly until the solution was clear and transparent to obtain the first aqueous phase matrix; the pH value was 4.5-5.

5. The second aqueous phase matrix is ​​obtained by uniformly mixing and dispersing the pyrrolidone carboxylic acid-lysozyme complex, water-soluble polymer and humectant; The pH adjuster was fully dissolved in purified water until it became clear and free of particles, thus obtaining the third aqueous phase matrix. The oily matrix is ​​mixed to obtain the first oil phase matrix; Pour the second aqueous phase matrix into the emulsification pot, stir and heat to 80-85℃ at 30-50r / min, keep warm and stir for 10-20min to ensure the material is completely dissolved; Add the first oil phase matrix to the oil phase pot, stir and heat at 30-50 r / min to 80-85℃, keep warm and stir for 10-15 min to ensure that the material is completely dissolved; Under homogenized conditions, the dissolved oil phase matrix is ​​extracted into the emulsification pot, vacuumed at -0.03 to -0.1 MPa, homogenized for 3-5 minutes, kept warm and stirred for 10-20 minutes, and then the cooling water is turned on to cool down. When the temperature drops to 60-70℃, turn off the cooling water, add the first aqueous phase matrix and the third aqueous phase matrix, continue to keep warm and stir for 10-20 minutes, then turn on the cooling water and stir at 30-40 r / min to continue cooling. When the temperature drops to 30-38℃, samples are taken for testing to obtain the cream dressing. The obtained cream dressing was irradiated with an electron beam at a dose of 20-30 kJ to obtain a sterile recombinant elastin cream dressing.

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