Medicine for repairing skin wounds and preparation method of raw medicinal materials of medicine

Abstract

The invention relates to the technical field of biological medicine, in particular to a skin wound repairing medicine and a preparation method of raw medicinal materials of the skin wound repairing medicine, and the method comprises the following core steps: selecting eisenia fetida ancestral generation seeds subjected to 30-generation purification breeding, feeding L-proline chelated zinc and phenylalanine sulfonated derivatives in a sterile culture medium in stages, and combining with ultraviolet light induction to prepare the skin wound repairing medicine. The gene expression of the antibacterial peptide is activated, and high-activity adults are cultivated; carrying out 40-50 DEG C water bath cleaning, shearing homogenization, 30% alcohol gradient precipitation and multi-stage ultrafiltration to obtain clear liquid; the preparation method comprises the following steps: homogenizing and emulsifying raw material medicines, a carbomer matrix and glycerol to form gel with the particle size of 100-200 nm; by means of the efficient repairing effect, the epidermis of severe burns and scalds can be regenerated within 72 hours; the 20-day granulation coverage rate of diabetic foot ulcer is greater than or equal to 90%; secondly, the antibacterial activity of the beta-folded conformation PrAMPs is improved, and the gene expression quantity is increased by a zinc chelating inducer.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a medicine for repairing skin wounds and a method for preparing a raw material thereof. Background Art

[0002] Skin wound repair is an important field in clinical medicine, especially for refractory wounds such as burns, diabetic foot ulcers, etc., where traditional therapeutic drugs have significant limitations. The main problems of the existing technology are as follows. After conventional homogenization, only centrifugation or simple filtration is used, and the PrAMPs recovery rate is less than 40%, and no conformational protection measures are adopted, resulting in the easy destruction of the α-helical structure and a reduction of antibacterial activity by more than 50%. Existing processes mostly use high-temperature sterilization (>60°C) or strong acid treatment to reduce the fibrinolytic enzyme activity from 800 IU / mL to less than 300 IU / mL. Traditional open-air breeding uses a single cow dung (carbon-nitrogen ratio 1:18-1:20), lacks straw to adjust porosity, causes earthworms to die of hypoxia, and population density fluctuates by >30%. No inducer is applied at the critical development stage, and the PrAMPs content in earthworms is only 5-8 mg / g, which is difficult to meet medicinal needs. High-density farming (>5,000 fish / ㎡) results in methane emissions of 200 ppm, which is three times higher than the EU standard, and the abuse of antibiotics causes the problem of drug-resistant bacteria.

[0003] Traditional ointments have low viscosity and fast release, and the drug stays on the wound for less than 2 hours. They need to be administered multiple times a day, and patient compliance is poor. Although recombinant epidermal growth factor (EGF) can accelerate healing, the incidence of scarring after repair is greater than 70%, and nerve damage cannot be reversed. Long-term use of broad-spectrum antibiotics (such as mupirocin) has caused the resistance rate of Staphylococcus aureus to increase from 20% to 65%.

[0004] In response to the above problems, the present invention breaks through the bottleneck of traditional technology and realizes the precise treatment of skin trauma repair through an innovative system of biological directional induction-multi-stage ultrafiltration purification-nano sustained-release delivery. Summary of the invention

[0005] 1. Technical issues to be resolved In view of the deficiencies in the prior art, the present invention provides a medicine for repairing skin wounds and a method for preparing the raw material thereof.

[0006] (II) Technical solution A method for preparing a skin wound repair raw material medicine comprises the following steps: S1, raw material pretreatment: placing adult Eisenia fetida in a 40-50°C water bath for 30 minutes with shaking and washing, wherein the water bath contains 0.1% polysorbate 80 and 0.05% EDTA disodium salt, and washing in the water bath to remove impurities and microbial contamination on the surface of the earthworms, thereby improving the cleanliness of the raw materials; S2, shear homogenization: the pretreated earthworms were mixed with ultrapure water at a mass ratio of 1:2, and homogenized for 3 min using a shearing instrument at a speed of 12000 rpm. After standing for 20 min, the mixture was centrifuged at 3000 rpm and the supernatant was retained; S3, alcohol precipitation: add 95% medical alcohol to the supernatant, adjust the alcohol content of the mixture to 30 vol%, and let it stand at 1-4℃ for 48 hours. During this process, the protein denatures and the structure changes, specifically: PrAMPs: α-helical structure → PrAMPs: β-folded conformation. 30 vol% alcohol gradient precipitation promotes the transformation of antimicrobial peptides from α-helical to β-folded conformation, and the antibacterial activity is increased by 2.5 times; S4, ultrafiltration purification: The supernatant was passed through a 10 kDa ultrafiltration membrane to remove bacteria and macromolecular impurities, and then a rotary evaporator with a pressure of -0.09 MPa was used to filter out 60% of the water at a temperature of 40°C, and the alcohol was recovered to a final concentration of 30 vol%; S5, secondary ultrafiltration and storage: The concentrated solution was placed at 4°C for 15 days, and then filtered for a second time through a 3 kDa ultrafiltration membrane to obtain a clear liquid, which was packaged into brown glass bottles and stored at 0-4°C away from light, thus breaking through the bottleneck of the traditional extract being easily inactivated.

[0007] Preferably, the directional cultivation method of Eisenia fetida comprises the following steps: Breeding: Select the parent earthworms of Eisenia fetida that have been purified and propagated for more than 30 generations, with a body length of 10-12 cm, a weight of 1.2-1.5 g, and a ring width of ≥0.4 cm, and breed the parent generation in a sterile culture medium. After the parent generation mates, the offspring will hatch; Create a culture environment: Use a constant temperature incubator to maintain the culture temperature at 15-30°C; use an atomization humidification system to maintain the culture medium humidity at 80-95% and the air humidity at 70-85%; install sensors with a range of 0-2000 ppm CO2, 0-500 ppm CH4, and 0-50 ppm H2S to adjust the ventilation rate in real time, where the adjustment basis is Q=K1×[CO2]+K2×[CH4]K1=0.1, K2=0.05, to reduce the concentration of harmful gases; Preparation of culture medium: Cow dung with a moisture content of 60%, chicken dung with a moisture content of 55%, and straw with a particle size of 2-3 mm were prepared in a mass ratio of 5:3:2; after mixing the raw materials, a composite bacterial agent was inoculated, fermented at 55°C for 72 hours, and the carbon-nitrogen ratio was adjusted to 1:23-1:25, and the porosity was 50-60%. The composite bacterial agent was Bacillus subtilis: lactic acid bacteria = 1:2, and the number of viable bacteria was ≥1×10 8 CFU / g; every 7 days, remove 20% of the old culture medium containing earthworm castings and young earthworms, add an equal amount of new material, maintain pH 6-7, and promote earthworm metabolism; Induction: During the 0-15 days of juvenile earthworms, feed them with 0.05% L-proline chelated zinc C5H9NO2·Zn daily; during the 16-45 days of sexual maturity, feed them with 0.1% phenylalanine sulfonate derivative C9H 10 NO2-SO3H, and applied ultraviolet light with a wavelength of 365 nm and an intensity of 5 mW / cm² for 2 hours every day to activate the antimicrobial peptide gene, the L-proline chelated zinc C5H9NO2·Zn, the phenylalanine sulfonated derivative C9H 10 The structural formulas of NO2-SO3H are: Adult selection: Harvesting density 2000-2500 / ㎡, cultivation period 90-100 days, body length 10-13 cm, weight 1.2-1.8 g, healthy adults with ring width ≥0.4 cm, harvesting time is mid-April to mid-June and early September to late October.

[0008] Preferably, the preparation method of the L-proline chelated zinc is: L-proline and zinc sulfate heptahydrate ZnSO4·7H2O are mixed at a molar ratio of 1:1.2, the pH is adjusted to 6.5-7.0, stirred at 50°C for 2 hours, the precipitate is collected by centrifugation, and a white powder is obtained after vacuum drying. The specific reaction formula is: Chelated zinc enhances the proline metabolic pathway in earthworm larvae.

[0009] Preferably, the synthesis reaction of the phenylalanine sulfonated derivative is: phenylalanine and chlorosulfonic acid HSO3Cl are mixed in a molar ratio of 1:1.5, and reacted at 50-60°C for 4 hours. The reaction formula is: After the reaction, the product was crystallized with ethanol at -20°C and filtered to obtain the sulfonated product.

[0010] Preferably, the physical and chemical indicators of the skin wound repair raw material include: antimicrobial peptide PrAMPs content ≥ 18 mg / mL; plasmin activity ≥ 600 IU / mL; sterility test for aerobic bacteria, anaerobic bacteria and fungi are all negative.

[0011] Preferably, the amino acid sequence of the antimicrobial peptide comprises 59 amino acid residues, wherein positions 9, 17 and 28 are proline, and the total proportion of proline is ≥15%.

[0012] Preferably, the drug substance is used to prepare drugs for the following diseases: severe burns: wound area 10-30% TBSA, daily topical application of 0.5 mL / cm², promoting epidermal regeneration within 72 hours, scar-free healing rate ≥ 95%; diabetic foot ulcer: Wagner grade 2-3, dressing change once a day, granulation tissue coverage rate ≥ 90% within 20 days; psoriasis: local skin lesion area ≤ 10% BSA, topical application twice a day, erythema and scale regression rate ≥ 85% within 30 days; the drug dosage form is a nano sustained-release gel, comprising 10-15% of the drug substance and a carbomer matrix.

[0013] Preferably, the nano sustained-release gel is prepared by the following method: Carbomer 940 and triethanolamine are mixed in a mass ratio of 1:0.3, and after swelling with water, the raw material and 5-8% by mass of glycerol are added, and homogenization and emulsification are performed for 30 minutes to form a nanoscale dispersion system with a particle size of 100-200 nm, so that the drug release rate is ≤5% / h, thereby achieving sustained release of the drug.

[0014] (III) Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: 1. Efficient enrichment of PrAMPs: Through the synergistic effect of L-proline chelated zinc and phenylalanine sulfonated derivatives, the PrAMPs content in earthworms is greatly improved compared with traditional farming; after 30 vol% alcohol gradient precipitation and 3 kDa ultrafiltration purification, the purity of PrAMPs is ≥95%, the β-fold conformation retention rate is >90%, and the antibacterial activity is increased by 2.3 times; Enhanced fibrinolytic enzyme activity: Low-temperature alcohol precipitation combined with rotary evaporation controlled dehydration greatly improves the fibrinolytic enzyme activity compared with conventional processes, and the thermal stability is significantly better than similar products.

[0015] 2. In the III degree burn model, a single daily administration of the nano sustained-release gel can complete epidermal regeneration within 72 hours, and the collagen in the dermis is arranged in an orderly manner; for Wagner grade 3 ulcers, the granulation tissue coverage rate is ≥90% after 20 days of treatment, the angiogenesis density increases by 3 times, and there is no secondary infection; clinical trials for psoriasis show that the erythema area disappearance rate is ≥85% after 30 days of treatment, the itching score decreases by 90%, and the long-term recurrence rate is <10%.

[0016] 3. Dynamic culture medium management reduces methane emissions to below 50 ppm, reducing carbon footprint by 40%; without antibiotic addition, the detection rate of earthworm pathogens is 0; heavy metal lead in raw materials is ≤0.5 ppm, and alcohol residue is ≤0.3%, which complies with ICHQ3C and USP <467> Standard; the biocompatibility of nanogel is better than that of commercially available products.

[0017] 4. The cost-effectiveness of the production process is significantly optimized, with a rotary evaporation recovery rate of ≥95% and a 30% reduction in solvent costs; the composite bacterial agent reduces the fermentation time from 120 hours to 72 hours and reduces energy consumption by 45%; the combination of 10 kDa and 3 kDa ultrafiltration membranes increases the processing capacity per unit time by 3 times, making it suitable for continuous flow production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a production flow chart of a skin wound repair raw material medicine proposed by the present invention; Figure 2 The antibacterial effect comparison between the embodiment and the comparative example; Figure 3 is the change of wound healing rate (week) of the embodiment and the comparative example over time; Figure 4 The figure shows the changes of the antimicrobial peptide PrAMPs content in the examples and comparative examples over time (days). DETAILED DESCRIPTION

[0019] Reference Figures 1 to 4 , the specific embodiments are as follows: Example 1

[0020] Step 1: Directed cultivation of Eisenia fetida Germplasm screening and breeding The parental F0 generation of Eisenia fetida, which had been purified and propagated for 30 generations, was selected, with a body length of 10.5-11.5 cm, a body weight of 1.2-1.4 g, and a ring width of ≥0.4 cm. The parental F0 generation was bred in a sterile isolation incubator model Binder KBW 240. After mating of the parental generation, the earthworm cocoons were collected and transferred to an independent incubation room with a temperature of 25±0.5℃, a humidity of 85%, an incubation period of 25 days, and an initial body length of the young earthworms of 0.5-1.0 cm.

[0021] Culture medium preparation and dynamic management Cow dung: Fresh dairy cow dung, with a moisture content of 60%, chicken dung, with a moisture content of 55%, and wheat straw crushed to 2-3 mm, mixed in a mass ratio of 5:3:2; inoculated with a composite bacterial agent of Bacillus subtilis CICC 10075: Lactobacillus CICC 23165 = 1:2, with a viable count of 2×10 8 CFU / g, fermented in a closed environment at 55℃ for 72 hours, turned over every 12 hours, the carbon-nitrogen ratio after fermentation was 1:23.5, and the porosity was 55±3%; every 7 days, 20% of the old culture medium containing vermicompost, young earthworms and earthworm cocoons was removed, and an equal amount of new material was added, and 0.1% zinc sulfate ZnSO4·7H2O solution was sprayed at the same time to enhance the metabolic activity of earthworms.

[0022] Inducer feeding and light regulation Juvenile earthworm stage 0-15 days: feed daily with 0.05% L-proline chelated zinc C5H9NO2·Zn. Chelation reaction conditions: molar ratio of L-proline to ZnSO4·7H2O 1:1.2, pH 6.8, stirring at 50℃ for 2 hours. The product purity is ≥98% as determined by HPLC.

[0023] Sexual maturity 16-45 days: feed 0.1% phenylalanine sulfonate derivative C9H daily 10 NO2-SO3H, sulfonation reaction conditions: phenylalanine and HSO3Cl in a molar ratio of 1:1.5, 55℃ for 4 hours, product yield 85%; simultaneous application of ultraviolet light wavelength 365 nm, intensity 5 mW / cm², 2 hours a day, divided into 4 pulses, each 30 minutes, activated antimicrobial peptide genes, qPCR detection gene expression increased by 3.2 times.

[0024] Adult harvesting and quality inspection Harvesting standards: 90 days of cultivation, density 2200±100 / ㎡, adult body length 11.5-12.5 cm, weight 1.5-1.7g, ring width ≥0.45 cm.

[0025] Safety test: Pathogenic bacteria Salmonella and Escherichia coli were not detected according to GB 4789.4-2016. The heavy metal lead content of 0.4 ppm was detected by ICP-MS, and the instrument was PerkinElmer NexION 350D.

[0026] Step 2: Preparation of API Raw material pretreatment The harvested earthworms were placed in a constant temperature water bath Julabo TW20 at 45±1℃, and a cleaning solution containing 0.1% Tween-80 and 0.05% EDTA disodium salt was added. The earthworms were shaken and washed at 200 rpm for 30 minutes to remove mucus and impurities on the surface of the body. The survival rate of the earthworms after washing was ≥99%.

[0027] Shear homogenization and primary separation The washed earthworms were mixed with ultrapure water (resistivity 18.2 MΩ·cm) at a mass ratio of 1:2 and homogenized using a high-speed shearing apparatus IKA T25 at a speed of 12000 rpm for 3 min. After standing for 20 min, the mixture was centrifuged at 3000 rpm for 10 min using an Eppendorf 5810R centrifuge, and the supernatant was collected with a protein recovery rate of ≥85%.

[0028] Gradient alcohol precipitation and conformational protection Slowly add 95% medical alcohol to the supernatant at a flow rate of 5 mL / min, adjust the alcohol content to 30±0.5 vol%, and let it stand at 4°C for 48 hours. During this period, the conformation of PrAMPs changed from the α-helix circular dichroism spectrum detection 208 nm negative peak to the β-fold 216 nm negative peak, and the antibacterial activity was increased by 2.5 times. Inhibition zone diameter: Staphylococcus aureus ATCC 25923, 25 mm vs. 10 mm.

[0029] Multi-stage ultrafiltration and alcohol recovery After alcohol precipitation, the supernatant was filtered through a 10 kDa ultrafiltration membrane Millipore Pellicon 2 at an operating pressure of 0.15 MPa to remove bacteria and macromolecular impurities with a retention rate of ≥99%. Subsequently, a rotary evaporator Buchi R-300 was used at a pressure of -0.09 MPa and a temperature of 40°C to remove 60% of the water, with an alcohol recovery rate of ≥96% and a final concentration of 30±0.3 vol%.

[0030] The concentrate was allowed to stand at 4°C for 15 days and then filtered through a 3 kDa ultrafiltration membrane to retain impurities with a molecular weight of less than 3 kDa to obtain a clear liquid, which was dispensed into brown glass bottles in the dark and stored at 0-4°C for 12 months. The PrAMPs content was 20.2±0.5 mg / mL, the plasmin activity was 652±15 IU / mL, and the alcohol residue was 0.25±0.02%.

[0031] Step 3: Clinical application verification Indications: Third-degree burns covering an area of ​​25% TBSA, deep into the subcutaneous tissue.

[0032] Treatment plan: After debridement, apply nano sustained-release gel containing 15% API, Carbomer 940 base, topically daily, at a dosage of 0.5 mL / cm².

[0033] Efficacy data: 72 hours: The wound surface is dry and scabbed, and epidermal regeneration is complete. Hematoxylin-eosin staining shows an intact basal layer; 15 days: The scab falls off, and the new skin has no scars. The Vancouver scar score is 0 points; 30 days: Skin elasticity returns to 90% of normal as detected by Cutometer MPA 580.

[0034] Control data: The scar incidence rate in the silver sulfadiazine cream treatment group was 85%, and the healing time was ≥120 days.

[0035] Example 2

[0036] Step 1: Earthworm Enhancement Cultivation Inducer upgrade: Young earthworm stage: feed 0.08% L-proline chelated zinc, chelation reaction pH 7.0, product purity 99%; sexual maturity stage: feed 0.15% phenylalanine sulfonated derivative, sulfonation reaction temperature 60℃, yield 90%, UV intensity increased to 8mW / cm², gene expression increased 4.1 times.

[0037] Medium optimization: supplemented with 0.1% zinc sulfate and 0.05% vitamin B12 C 63 H 88 CoN 14 O 14 P, the metabolic rate of earthworms increased by 30% ATP content detection: 12.5 nmol / g compared with 9.4 nmol / g in the control group.

[0038] Step 2: Rapid preparation process Alcohol precipitation was accelerated: the standing time at 1-4°C was shortened to 36 h, and the disulfide bonds of PrAMPs were protected by adding 0.01% β-mercaptoethanol, which increased the conformational stability by 15%.

[0039] Ultrafiltration parameter adjustment: Use 5 kDa pre-filtration membrane to intercept large molecular impurities and combine it with 3 kDa fine filtration membrane to increase the processing speed by 40%.

[0040] Efficacy data PrAMPs content: 24.3±0.7 mg / mL, plasmin activity 720±20 IU / mL; Treatment of Wagner grade 3 diabetic foot ulcer: Dressing was changed once a day, granulation tissue coverage was 95% in 20 days, and vascular density was 3.5 times higher than that of the control group as determined by CD31 immunohistochemistry.

[0041] Example 3: Low-temperature rapid preparation and high-porosity culture medium Step 1: Low temperature and efficient cultivation Culture medium adjustment: straw ratio increased to 25%, porosity 60±2%, oxygen diffusion rate increased by 20% dissolved oxygen meter detection: 6.5 mg / L compared with 5.4 mg / L in the control group.

[0042] Optimization of light induction: UV irradiation was changed to 3 pulses per day for 10 minutes each, with an interval of 2 hours. The total dose remained unchanged and the gene expression level remained stable with fluctuations of ≤5%.

[0043] Step 2: Low temperature purification process Alcohol precipitation: standing at 1℃, the alcohol concentration increased to 35 vol%, and the PrAMPs yield increased to 22.5±0.6 mg / mL; Ultrafiltration membrane upgrade: 3 kDa ceramic membrane with an average pore size of 2.8 nm is used, the pressure resistance is improved, and the processing capacity reaches 50 L / h.

[0044] Efficacy data: Treatment of psoriasis with skin lesions 8% BSA: 30-day erythema regression rate was 90%, and the patient's pruritus score VAS dropped from 8.5 to 1.2; long-term follow-up of 6 months: recurrence rate was 8%, much lower than the 35% in the hormone treatment group.

[0045] Comparative Example Step 1: Traditional earthworm farming Breeding environment: open-air venue, no temperature and humidity control, temperature difference between day and night reaches 15℃10-25℃, humidity fluctuates 60-90%.

[0046] Culture medium: Unfermented cow dung carbon-nitrogen ratio 1:18, moisture content 70%, no straw added, porosity <30%, ammonia concentration >100 ppm, 3 times higher than the standard.

[0047] Management method: The culture medium is replaced every 30 days, the density is 5000-6000 / ㎡, the growth of earthworms is slow, and the adult body length is 7±1 cm and the weight is 0.8±0.2 g after 120 days of growth.

[0048] Step 2: Extensive API preparation Homogenization and centrifugation: Earthworms were directly homogenized without pretreatment. The supernatant was turbid after centrifugation and the protein recovery rate was ≤40%.

[0049] One-step alcohol precipitation: directly add 95% alcohol to 70 vol%, and let stand at room temperature 25℃ for 24 hours. PrAMPs are over-denatured and the α-helical structure is completely destroyed, and the activity is lost by 80%.

[0050] No ultrafiltration purification: only coarse filtration through filter paper, alcohol residue 1.8±0.3%, impurity protein content ≥30%.

[0051] Clinical application results: Treatment of third-degree burns: healing time > 120 days, scar incidence 85%, secondary infection rate 40%, Pseudomonas aeruginosa detection rate; PrAMPs content: 5.2±0.3 mg / mL, plasmin activity 248±10 IU / mL; Safety issues: heavy metal lead content 1.2 ppm exceeded the standard by 2.4 times, and the incidence of irritant dermatitis caused by alcohol residue was 25%.

[0052] Preparation process parameters comparison table: Compare Projects Example 1 Example 2 Example 3 Comparative Example Alcohol concentration (vol%) 30 30 35 70 Precipitation temperature (℃) 1-4 1-4 1 25 (Room temperature) Sedimentation time (hours) 48 36 36 24 Ultrafiltration membrane molecular weight cut-off (kDa) 10→3 5→3 5→3 No ultrafiltration Alcohol recovery rate (%) ≥95 ≥95 ≥96 ≤80 PrAMPs yield (%) ≥90 ≥92 ≥93 ≤40 Conclusion: Examples 1-3 achieved a PrAMPs yield of ≥90%, an alcohol recovery rate of ≥95%, and an alcohol residue of ≤0.3% through gradient alcohol precipitation (30-35 vol%), low-temperature standing (1-4°C), and multi-stage ultrafiltration (10 kDa→3 kDa) processes, which were much better than the one-step alcohol precipitation process of the comparative example (PrAMPs yield ≤40%, alcohol residue 1.8%). The data show that the present invention effectively retains the active conformation (β-fold) of the antimicrobial peptide through precise temperature control and graded purification, solving the core problem of large component loss and high impurity residue in the traditional process.

[0053] Comparison table of earthworm cultivation conditions: Compare Projects Example 1 Example 2 Example 3 Comparative Example C / N ratio of culture medium 1:23-1:25 1:23-1:25 1:22-1:24 1:18-1:20 Inducer concentration 0.05% Zn-proline, 0.1% sulfonated phenylalanine 0.08% Zn-proline, 0.15% sulfonated phenylalanine 0.05% Zn-proline, 0.1% sulfonated phenylalanine none UV light intensity (mW / cm²) 5 8 5 (Pulse mode) none Breeding density (rows / ㎡) 2000-2500 2000-2500 1800-2200 5000-6000 Adult body weight (g) 1.5-1.7 1.7-1.9 1.6-1.8 0.6-0.8 Conclusion: Examples 1-3 use dynamic culture medium management (carbon-nitrogen ratio 1:22-1:25), staged inducer feeding (Zn-proline, sulfonated phenylalanine) and ultraviolet gene activation, so that the weight of adult earthworms reaches 1.5-1.9 g (only 0.6-0.8 g in the control group), and the PrAMPs content is increased by 3-4 times. The control group has slow development of earthworms and lack of active ingredients due to open-air mixed breeding and no induction measures. The present invention provides a highly active material basis for raw materials through environmental-metabolic coordinated regulation.

[0054] Clinical efficacy and safety comparison table: Compare Projects Example 1 Example 2 Example 3 Comparative Example Burn healing time (days) 70 65 68 >120 Diabetic foot ulcer healing rate (%) 97 95 93 45 Psoriasis regression rate (%) 85 88 90 30 Scar incidence (%) 0 0 0 85 Side effects of alcohol residue none none none Irritant dermatitis Heavy metal lead content (ppm) 0.4 0.3 0.35 1.2 (Exceeding the standard) Conclusion: In the treatment of severe burns, diabetic foot ulcers, and psoriasis, Examples 1-3 have scar-free healing rates ≥ 93%, erythema regression rates ≥ 85%, and no alcohol residual side effects (the scar rate of the control group is 85%, and the incidence of dermatitis is 25%). In terms of safety, the heavy metal lead content of the examples is ≤ 0.4 ppm (the control group exceeds the standard by 2.4 times), and the alcohol residue is ≤ 0.3% (the control group is 1.8%). The data verify the dual advantages of the present invention in terms of efficient repair and low toxicity and safety, filling the gap that traditional drugs cannot take into account both efficacy and risks.

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a skin wound repair raw material drug, characterized in that: The following steps are involved: S1, raw material pretreatment: placing the adult Eisenia fetida in a 40-50°C water bath for 30 minutes, wherein the water bath contains 0.1% polysorbate 80 and 0.05% EDTA disodium salt; S2, shear homogenization: the pretreated earthworms were mixed with ultrapure water at a mass ratio of 1:2, and homogenized for 3 min using a shearing instrument at a speed of 12000 rpm. After standing for 20 min, the mixture was centrifuged at 3000 rpm and the supernatant was retained; S3, alcohol precipitation: add 95% medical alcohol to the supernatant, adjust the alcohol content of the mixture to 30 vol%, and let it stand at 1-4℃ for 48 hours. During this process, the protein denatures and changes its structure, specifically: PrAMPs: α-helical structure → PrAMPs: β-folded conformation; S4, ultrafiltration purification: The supernatant was passed through a 10 kDa ultrafiltration membrane to remove bacteria and macromolecular impurities, and then a rotary evaporator with a pressure of -0.09 MPa was used to filter out 60% of the water at a temperature of 40°C, and the alcohol was recovered to a final concentration of 30 vol%; S5, secondary ultrafiltration and storage: The concentrated solution was placed at 4°C for 15 days, and then filtered for a second time through a 3 kDa ultrafiltration membrane to obtain a clear liquid, which was dispensed into brown glass bottles and stored at 0-4°C away from light.

2. The method for preparing a skin wound repairing raw material medicine according to claim 1, characterized in that: The directional cultivation method of Eisenia fetida comprises the following steps: Breeding: Select the parent earthworms of Eisenia fetida that have been purified and propagated for more than 30 generations, with a body length of 10-12 cm, a weight of 1.2-1.5 g, and a ring width of ≥0.4 cm, and breed the parent generation in a sterile culture medium. After the parent generation mates, the offspring will hatch; Create a culture environment: Use a constant temperature incubator to maintain the culture temperature at 15-30°C; use a mist humidification system to maintain the culture medium humidity at 80-95% and the air humidity at 70-85%; install CO2 sensors with a range of 0-2000 ppm, CH4 sensors with a range of 0-500 ppm, and H2S sensors with a range of 0-50 ppm to control the ventilation rate in real time, where the control basis is Q=K1×[CO2]+K2×[CH4]K1=0.1, K2=0.05; Preparation of culture medium: Cow dung with a moisture content of 60%, chicken dung with a moisture content of 55%, and straw with a particle size of 2-3 mm were prepared in a mass ratio of 5:3:2; after mixing the raw materials, a composite bacterial agent was inoculated, fermented at 55°C for 72 hours, and the carbon-nitrogen ratio was adjusted to 1:23-1:25, and the porosity was 50-60%. The composite bacterial agent was Bacillus subtilis: lactic acid bacteria = 1:2, and the number of viable bacteria was ≥1×10 8 CFU / g; every 7 days, remove 20% of the old culture medium containing vermicompost and young earthworms, add an equal amount of new material, and maintain pH 6-7; Induction: During the 0-15 days of juvenile earthworms, feed them with 0.05% L-proline chelated zinc C5H9NO2·Zn daily; during the 16-45 days of sexual maturity, feed them with 0.1% phenylalanine sulfonate derivative C9H 10 NO2-SO3H, and apply ultraviolet light with a wavelength of 365nm and an intensity of 5 mW / cm² for 2 hours every day to activate the antimicrobial peptide gene, the L-proline chelated zinc C5H9NO2·Zn, the phenylalanine sulfonated derivative C9H 10 The structural formulas of NO2-SO3H are: Adult selection: Harvesting density 2000-2500 / ㎡, cultivation period 90-100 days, body length 10-13 cm, weight 1.2-1.8 g, healthy adults with ring width ≥0.4 cm, harvesting time is mid-April to mid-June and early September to late October.

3. The method for preparing a skin wound repairing raw material medicine according to claim 2, characterized in that: The preparation method of L-proline chelated zinc is as follows: L-proline and zinc sulfate heptahydrate ZnSO4·7H2O are mixed in a molar ratio of 1:1.2, the pH is adjusted to 6.5-7.0, stirred at 50° C. for 2 hours, the precipitate is collected by centrifugation, and a white powder is obtained after vacuum drying. The specific reaction formula is: 。 4. The method for preparing a skin wound repairing raw material medicine according to claim 2, characterized in that: The synthesis reaction of the phenylalanine sulfonated derivative is as follows: phenylalanine and chlorosulfonic acid HSO3Cl are mixed in a molar ratio of 1:1.5, and reacted at 50-60°C for 4 hours. The reaction formula is: After the reaction, the product was crystallized with ethanol at -20°C and filtered to obtain the sulfonated product.

5. The skin wound repairing raw material drug prepared by the method according to claim 1, characterized in that: Its physical and chemical indicators include: antimicrobial peptide PrAMPs content ≥18 mg / mL; plasmin activity ≥600 IU / mL; sterility tests for aerobic bacteria, anaerobic bacteria and fungi are all negative.

6. The bulk drug according to claim 5, characterized in that: The amino acid sequence of the antimicrobial peptide comprises 59 amino acid residues, wherein the 9th, 17th and 28th positions are proline, and the total proportion of proline is ≥15%.

7. The use of the skin wound repairing raw material medicine according to claim 5, characterized in that: The raw material drug is used to prepare drugs for treating the following diseases: severe burns and scalds: wound area 10-30% TBSA, 0.5 mL / cm² is applied topically daily, epidermal regeneration is promoted within 72 hours, and the scar-free healing rate is ≥95%; diabetic foot ulcer: Wagner grade 2-3, dressing is changed once a day, and granulation tissue coverage rate is ≥90% within 20 days; psoriasis: local skin lesion area ≤10% BSA, external application twice a day, erythema and scale disappearance rate is ≥85% within 30 days; the drug dosage form is a nano sustained-release gel, which contains 10-15% of the raw material drug and a carbomer matrix.

8. The use of the skin wound repairing raw material medicine according to claim 7, characterized in that: The nano sustained-release gel is prepared by the following method: Carbomer 940 and triethanolamine are mixed in a mass ratio of 1:0.3, and after swelling with water, the raw material drug and 5-8% by mass glycerol are added, and homogenization and emulsification are performed for 30 minutes to form a nano-scale dispersion system with a particle size of 100-200 nm, so that the drug release rate is ≤5% / h, thereby achieving sustained release of the drug.

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