A medicament for repairing skin wounds and a preparation method of its raw materials

Abstract

The present invention relates to the field of biomedical technologies, and specifically to a medicament for skin wound repair and a preparation method of its raw materials, which includes the following core steps: Select the parental species of Eisenia fetida that has been purified and propagated for 30 generations, feed L-proline chelated zinc and phenylalanine sulfonated derivatives in stages in a sterile culture medium, combine with ultraviolet light induction to activate the expression of antibacterial peptide genes, and cultivate highly active adults; Obtain a clear liquid through water bath cleaning at 40-50 °C, shearing and homogenization, 30% alcohol gradient precipitation, and multi-stage ultrafiltration; Homogenize and emulsify the raw drug with carbomer matrix and glycerol to form a gel with a particle size of 100-200 nm. The highly efficient repair effect of the present invention enables epidermal regeneration within 72 hours for severe burns and scalds; the granulation coverage rate of diabetic foot ulcers is ≥90% within 20 days; Secondly, the antibacterial activity of β-sheet conformation PrAMPs is enhanced, and the zinc chelating inducer up-regulates the gene expression level.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a medicament for skin wound repair and a preparation method of its raw materials and medicinal herbs. Background Art

[0002] Skin wound repair is an important field in clinical medicine. Especially for refractory wounds such as burns and scalds, diabetic foot ulcers, etc., traditional therapeutic drugs have significant limitations. The main problems of the existing technologies are as follows. After conventional homogenization, only through centrifugation or simple filtration, the recovery rate of PrAMPs is less than 40%, and no conformational protection measures are adopted, resulting in the α-helix structure being easily damaged and the antibacterial activity being reduced by more than 50%. Most of the existing processes adopt high-temperature sterilization (>60°C) or strong acid treatment, reducing the fibrinolytic enzyme activity from 800 IU / mL to below 300 IU / mL. Traditional open-air farming uses single cow dung (carbon-nitrogen ratio 1:18 - 1:20), lacking straw to adjust the porosity, resulting in earthworms dying of hypoxia and the population density fluctuating by >30%. No inducer is applied at the key development stage, and the content of PrAMPs in earthworms is only 5 - 8 mg / g, which is difficult to meet the medicinal requirements. High-density farming (>5000 worms / m²) leads to a methane emission of 200 ppm, exceeding the EU standard by 3 times, and the abuse of antibiotics causes the problem of drug-resistant bacteria.

[0003] Due to the low viscosity and fast release of traditional ointments, the drug stays on the wound surface for <2 hours, and multiple administrations are required daily, resulting in poor patient compliance. Although recombinant epidermal growth factor (EGF) can accelerate healing, the scar incidence after repair is >70%, and it cannot reverse nerve damage. Long-term use of broad-spectrum antibiotics (such as mupirocin) has increased the drug resistance rate of Staphylococcus aureus from 20% to 65%.

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

[0005] (I) Technical Problems to be Solved

[0006] In view of the deficiencies of the existing technology, the present invention provides a medicament for skin wound repair and a preparation method of its raw materials and medicinal herbs.

[0007] (II) Technical Solutions

[0008] A preparation method of a raw material drug for skin wound repair includes the following steps:

[0009] S1, Raw material pretreatment: Place adult Eisenia fetida in a water bath at 40 - 50 °C and shake and wash for 30 minutes. The water bath solution contains 0.1% polysorbate - 80 and 0.05% disodium EDTA. The water bath washing removes impurities and microbial contamination on the earthworm surface, improving the cleanliness of the raw material;

[0010] S2, Shearing and homogenizing: Mix the pretreated earthworms and ultrapure water at a mass ratio of 1:2. Use a shearing instrument with a rotation speed of 12000 rpm for 3 minutes of homogenization. After standing for 20 minutes, centrifuge at 3000 rpm for [time not provided in the original, assumed to be a mistake in the source text, should be a specific value] minutes, and retain the supernatant;

[0011] S3, Alcohol precipitation: Add 95% medical alcohol to the supernatant to adjust the alcohol concentration of the mixed solution to 30 vol%. Let it stand at 1 - 4 °C for 48 hours. During this process, the protein denatures and its structure changes. Specifically: PrAMPs: α - helix structure → PrAMPs: β - sheet conformation. The 30 vol% alcohol gradient precipitation promotes the transformation of the antimicrobial peptide from the α - helix to the β - sheet conformation, and the antibacterial activity is increased by 2.5 times;

[0012] S4, Ultrafiltration purification: Take the supernatant and pass it through a 10 kDa ultrafiltration membrane to remove miscellaneous bacteria and macromolecular impurities. Then use a rotary evaporator with a pressure of - 0.09 MPa to filter out 60% of the water at a temperature of 40 °C, and recover the alcohol to a final concentration of 30 vol%;

[0013] S5, Secondary ultrafiltration and preservation: Let the concentrated solution stand at 4 °C for 15 days, then filter it through a 3 kDa ultrafiltration membrane for the second time to obtain a clear liquid. Aliquot it into brown glass bottles and store it in the dark at 0 - 4 °C, breaking through the bottleneck of the easy inactivation of traditional extracts.

[0014] Preferably, the method for the directional cultivation of Eisenia fetida includes the following steps:

[0015] Breeding: Select ancestral breeding earthworms of Eisenia fetida with a body length of 10 - 12 cm, a body weight of 1.2 - 1.5 g, and a clitellum width ≥ 0.4 cm that have been purified and propagated for more than 30 generations. Breed the parent generation in a sterile culture medium. After the parent generation mates, hatch the offspring;

[0016] Create a cultivation environment: Use a constant temperature incubator to maintain the cultivation temperature at 15 - 30 °C; Keep the humidity of the culture medium at 80 - 95% and the air humidity at 70 - 85% through an atomization humidification system; 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, and adjust the ventilation rate in real - time. The adjustment basis is Q = K1×[CO2]+K2×[CH4], where K1 = 0.1 and K2 = 0.05, to reduce the concentration of harmful gases;

[0017] Culture medium preparation: Cow dung with a water content of 60%, chicken manure with a water content of 55%, and straw with a particle size of 2 - 3 mm are formulated in a mass ratio of 5:3:2; after mixing the raw materials, a compound bacterial agent is inoculated, and fermentation is carried out at 55°C for 72 hours. The carbon-nitrogen ratio is adjusted to 1:23 - 1:25, and the porosity is 50 - 60%. The compound bacterial agent is Bacillus subtilis: Lactobacillus = 1:2, and the viable bacteria count ≥ 1×10 8 CFU / g; 20% of the old culture medium containing earthworm feces and young earthworms is removed every 7 days, and an equal amount of new material is supplemented to maintain the pH at 6 - 7 to promote the metabolism of earthworms;

[0018] Induction: During the young earthworm stage of 0 - 15 days, feed is given daily containing 0.05% L-proline chelated zinc C5H9NO2·Zn; for young earthworms in the sexual maturity stage of 16 - 45 days, 0.1% phenylalanine sulfonated derivative C9H 10 NO2 - SO3H is given daily, and ultraviolet light with a wavelength of 365 nm and an intensity of 5 mW / cm² is applied for 2 hours daily to activate the antibacterial peptide gene. The structural formulas of the L-proline chelated zinc C5H9NO2·Zn and the phenylalanine sulfonated derivative C9H 10 NO2 - SO3H are respectively:

[0019] Selection of adult earthworms: Healthy adult earthworms with a harvesting density of 2000 - 2500 pieces / m², a cultivation period of 90 - 100 days, a body length of 10 - 13 cm, a body weight of 1.2 - 1.8 g, and a clitellum width ≥ 0.4 cm are harvested. The harvesting time is from mid-April to mid-June and from early September to late October.

[0020] Preferably, the preparation method of the L-proline chelated zinc is: 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, and the reaction is stirred at 50°C for 2 hours. The precipitate is collected by centrifugation and dried in vacuo to obtain a white powder. The specific reaction formula is:

[0021] Chelated zinc enhances the proline metabolic pathway of young earthworms.

[0022] 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:

[0023] After the reaction, crystallization is carried out with ethanol at -20°C, and the sulfonated product is obtained after filtration.

[0024] Preferably, the physicochemical indexes of the raw material drug for skin wound repair include: the content of antibacterial peptide PrAMPs ≥ 18 mg / mL; the activity of plasmin ≥ 600 IU / mL; the aerobic bacteria, anaerobic bacteria and fungi in the sterility test are all negative.

[0025] Preferably, the amino acid sequence of the antibacterial peptide contains 59 amino acid residues, wherein the 9th, 17th and 28th positions are proline, and the total proportion of proline ≥ 15%.

[0026] Preferably, the raw material drug is used to prepare drugs for treating the following diseases: severe burns and scalds: the wound area is 10 - 30% TBSA, 0.5 mL / cm² is locally applied every day, epidermal regeneration is promoted within 72 hours, and the scar-free healing rate ≥ 95%; diabetic foot ulcers: Wagner grade 2 - 3, dressing is changed once a day, and the granulation tissue coverage rate ≥ 90% within 20 days; psoriasis: the local skin lesion area ≤ 10% BSA, it is externally applied twice a day, and the erythema and scale regression rate ≥ 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.

[0027] Preferably, the nano-sustained release gel is prepared by the following method: carbomer 940 and triethanolamine are mixed at a mass ratio of 1:0.3, after swelling with water, the raw material drug and glycerol with a mass proportion of 5 - 8% are added, and homogenously emulsified for 30 minutes to form a nano-scale dispersion system with a particle size of 100 - 200 nm, so that the drug release rate ≤ 5% / h, realizing the sustained release of the drug.

[0028] (III) Beneficial effects

[0029] Compared with the existing technology, the beneficial effects of the present invention are:

[0030] 1. High-efficiency enrichment of PrAMPs: Through the synergistic effect of L-proline chelated zinc and phenylalanine sulfonated derivatives, the content of PrAMPs in earthworms is greatly increased compared with traditional breeding; after gradient precipitation with 30 vol% alcohol and ultrafiltration purification with 3 kDa, the purity of PrAMPs ≥ 95%, the retention rate of β-sheet conformation > 90%, and the antibacterial activity is increased by 2.3 times; Enhancement of plasmin activity: Low-temperature alcohol precipitation combined with rotary evaporation to control the dehydration rate, the plasmin activity is greatly improved compared with the conventional process, and the thermal stability is significantly better than similar products.

[0031] 2. In the third-degree burn model, the nano-sustained release gel is administered once a day, and epidermal regeneration is completed within 72 hours, and the collagen in the dermis layer is arranged orderly; for Wagner grade 3 ulcers, the granulation tissue coverage rate ≥ 90% after 20 days of treatment, the angiogenesis density is increased by 3 times, and there is no secondary infection; the psoriasis clinical trial shows that the erythema area regression rate ≥ 85% after 30 days of treatment, the itching score drops by 90%, and the long-term recurrence rate < 10%.

[0032] 3. Dynamic culture medium management reduces methane emissions to below 50 ppm and carbon footprint by 40%; under the condition of no antibiotic addition, the detection rate of earthworm pathogens is 0; for the active pharmaceutical ingredient, heavy metal lead ≤ 0.5 ppm and alcohol residue ≤ 0.3%, meeting the ICH Q3C and USP <467> standards; the biocompatibility of the nanogel is better than that of commercially available products.

[0033] 4. The production process cost - benefit is significantly optimized, the rotary evaporation recovery rate ≥ 95%, and the solvent cost is reduced by 30%; the compound bacterial agent shortens 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 triples the unit - time throughput, which is suitable for continuous - flow production. Brief Description of the Drawings

[0034] Figure 1 is the production flow chart of a skin - wound - repairing active pharmaceutical ingredient proposed by the present invention;

[0035] Figure 2 is the comparison of antibacterial effects between the examples and the comparative examples;

[0036] Figure 3 is the change of wound - healing rate (weeks) over time for the examples and the comparative examples;

[0037] Figure 4 is the change of the content of antibacterial peptide PrAMPs over time (days) for the examples and the comparative examples. Detailed Embodiments

[0038] Refer to Figures 1 to 4 , and the specific embodiments are as follows:

[0039] Example 1

[0040] Step 1: Directional cultivation of Eisenia fetida

[0041] Germplasm screening and breeding

[0042] Select the parental generation F0 of Eisenia fetida that has undergone 30 generations of purification and reproduction, with a body length of 10.5 - 11.5 cm, a body weight of 1.2 - 1.4 g, and a clitellum width ≥ 0.4 cm, and breed them in a sterile isolation incubator model Binder KBW 240; after the parental generation mates, collect the cocoons and transfer them to an independent hatching room with a temperature of 25 ± 0.5 °C, a humidity of 85%, and an incubation period of 25 days. The initial body length of the young earthworms is 0.5 - 1.0 cm.

[0043] Culture medium preparation and dynamic management

[0044] Fresh cow dung, with a moisture content of 60%, chicken manure (from broiler chickens), with a moisture content of 55%, and wheat straw crushed to 2 - 3 mm, are mixed in a mass ratio of 5:3:2; inoculated with a compound microbial agent, Bacillus subtilis CICC 10075:Lactobacillus CICC 23165 = 1:2, with a viable count of 2×10 8 CFU / g, fermented in a sealed state at 55°C for 72 hours, and turned over every 12 hours. After fermentation, the carbon-nitrogen ratio is 1:23.5, and the porosity is 55 ± 3%; 20% of the old culture medium (containing earthworm manure, young earthworms, and cocoons) is removed every 7 days, and an equal amount of new feed is supplemented. At the same time, a 0.1% zinc sulfate (ZnSO4·7H2O) solution is sprayed to enhance the metabolic activity of earthworms.

[0045] Feeding of the inducer and light regulation

[0046] Juvenile earthworm stage (0 - 15 days): Feed with feed containing 0.05% L-proline chelated zinc (C5H9NO2·Zn) every day. Chelation reaction conditions: L-proline and ZnSO4·7H2O are in a molar ratio of 1:1.2, pH 6.8, stirred at 50°C for 2 hours, and the product purity ≥98% is detected by HPLC.

[0047] Sexual maturity stage (16 - 45 days): Feed with 0.1% phenylalanine sulfonated derivative (C9H 10 NO2-SO3H every day. Sulfonation reaction conditions: Phenylalanine and HSO3Cl are in a molar ratio of 1:1.5, react at 55°C for 4 hours, and the product yield is 85%; simultaneously, apply ultraviolet light with a wavelength of 365 nm and an intensity of 5 mW / cm² for 2 hours every day, pulsed irradiation in 4 times, 30 minutes each time, to activate the antimicrobial peptide gene, and the gene expression level detected by qPCR is up-regulated by 3.2 times.

[0048] Harvesting of adults and quality inspection

[0049] Harvesting standard: Cultivated for 90 days, density 2200 ± 100 individuals / m², adult body length 11.5 - 12.5 cm, body weight 1.5 - 1.7 g, clitellum width ≥0.45 cm.

[0050] Safety detection: Pathogenic bacteria Salmonella and Escherichia coli are not detected according to GB 4789.4 - 2016, the lead content of heavy metals is 0.4 ppm detected by ICP-MS, and the instrument is PerkinElmer NexION 350D.

[0051] Step 2: Preparation of the raw drug

[0052] Raw material pretreatment

[0053] The harvested earthworms were placed in a Julabo TW20 constant temperature water bath at 45 ± 1°C, and a cleaning solution containing 0.1% Tween-80 and 0.05% disodium EDTA was added. They were shaken and cleaned at 200 rpm for 30 minutes to remove the mucus and impurities on the body surface. After cleaning, the survival rate of the earthworms was ≥ 99%.

[0054] Shearing, homogenization and primary separation

[0055] The cleaned earthworms were mixed with ultrapure water with a resistivity of 18.2 MΩ·cm at a mass ratio of 1:2. Homogenization was carried out using an IKA T25 high-speed shearing instrument at a speed of 12,000 rpm for 3 minutes. After standing for 20 minutes, centrifugation was performed at 3,000 rpm for 10 minutes using an Eppendorf 5810R centrifuge, and the supernatant was collected. The protein recovery rate was ≥ 85%.

[0056] Gradient alcohol precipitation and conformation protection

[0057] 95% medical alcohol was slowly added to the supernatant at a flow rate of 5 mL / min, and the alcohol concentration was adjusted to 30 ± 0.5 vol%. It was left to stand at 4°C for 48 hours. During this period, the conformation of PrAMPs changed from a negative peak at 208 nm in the α-helix circular dichroism spectrum to a negative peak at 216 nm in the β-sheet, and the antibacterial activity increased by 2.5 times (inhibition zone diameter: for Staphylococcus aureus ATCC 25923, 25 mm compared with 10 mm).

[0058] Multi-stage ultrafiltration and alcohol recovery

[0059] The supernatant after alcohol precipitation was filtered through a 10 kDa ultrafiltration membrane (Millipore Pellicon 2) at an operating pressure of 0.15 MPa to remove miscellaneous bacteria and macromolecular impurities (retention rate ≥ 99%). Subsequently, a Buchi R-300 rotary evaporator was used at a pressure of -0.09 MPa and a temperature of 40°C to filter out 60% of the water. The alcohol recovery rate was ≥ 96%, and the final concentration was 30 ± 0.3 vol%.

[0060] After the concentrated solution was left to stand at 4°C for 15 days, it was filtered through a 3 kDa ultrafiltration membrane for the second time to retain impurities with a molecular weight < 3 kDa, and a clear liquid was obtained. It was dispensed into brown glass bottles and stored at 0 - 4°C in the dark for 12 months. The content of PrAMPs was 20.2 ± 0.5 mg / mL, the fibrinolytic enzyme activity was 652 ± 15 IU / mL, and the alcohol residue was 0.25 ± 0.02%.

[0061] Step 3: Clinical application verification

[0062] Indications: Third-degree burn with an area of 25% TBSA, reaching the subcutaneous tissue.

[0063] Treatment plan: After debridement, apply a nano-sustained release gel containing 15% active pharmaceutical ingredient and Carbomer 940 matrix locally every day, with a dosage of 0.5 mL / cm².

[0064] Efficacy data: 72 hours: The wound surface was dry and crusted, and epidermal regeneration was completed. Hematoxylin-eosin staining showed a complete basal layer; 15 days: The scab fell off, and the newly formed skin had no scar, with a Vancouver Scar Scale score of 0; 30 days: Skin elasticity recovered to 90% of normal, detected by Cutometer MPA 580.

[0065] Control data: The scar incidence rate in the silver sulfadiazine cream treatment group was 85%, and the healing time was ≥120 days.

[0066] Example 2

[0067] Step 1: Enhanced cultivation of earthworms

[0068] Upgraded inducer: Juvenile 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°C, yield 90%, ultraviolet light intensity increased to 8 mW / cm², gene expression level up-regulated 4.1 times.

[0069] Optimized culture medium: Supplement 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.

[0070] Step 2: Rapid preparation process

[0071] Accelerated alcohol precipitation: The standing time at 1 - 4°C was shortened to 36 hours. By adding 0.01% β-mercaptoethanol to protect the disulfide bonds of PrAMPs, the conformational stability increased by 15%.

[0072] Adjusted ultrafiltration parameters: Use a 5 kDa pre-filter membrane to retain macromolecular impurities in combination with a 3 kDa fine filter membrane, and the processing speed increased by 40%.

[0073] Efficacy data

[0074] PrAMPs content: 24.3 ± 0.7 mg / mL, plasmin activity 720 ± 20 IU / mL;

[0075] For the treatment of Wagner grade 3 diabetic foot ulcers: Change the dressing once a day. After 20 days, the granulation tissue coverage rate was 95%, and the vascular density was increased by 3.5 times compared with the control group by CD31 immunohistochemistry.

[0076] Example 3: Low-temperature rapid preparation and high-porosity culture medium

[0077] Step 1: Low-temperature efficient cultivation

[0078] Culture medium adjustment: The straw ratio is increased to 25%, the porosity is 60±2%, and the oxygen diffusion rate is increased by 20%. Dissolved oxygen meter detection: 6.5 mg / L, compared with 5.4 mg / L in the control group.

[0079] Optimization of light induction: Ultraviolet light irradiation is changed to 3 pulses per day, each pulse for 10 minutes, with an interval of 2 hours, and the total dose remains unchanged. The gene expression level fluctuates stably by ≤5%.

[0080] Step 2: Low-temperature purification process

[0081] Alcohol precipitation: Standing at 1°C, the alcohol concentration is increased to 35 vol%, and the yield of PrAMPs is increased to 22.5±0.6 mg / mL;

[0082] Upgrade of ultrafiltration membrane: A 3 kDa ceramic membrane with an average pore diameter of 2.8 nm is used, the pressure resistance is improved, and the treatment capacity reaches 50 L / h.

[0083] Efficacy data: For the treatment of psoriasis, the skin lesion area is 8% BSA. The erythema regression rate at 30 days is 90%, and the patient's itching score VAS drops from 8.5 to 1.2. Long-term follow-up for 6 months: The recurrence rate is 8%, which is much lower than 35% in the hormone treatment group.

[0084] Control example

[0085] Step 1: Traditional earthworm breeding

[0086] Breeding environment: An open-air site with no temperature and humidity control. The temperature difference between day and night reaches 15°C (10 - 25°C), and the humidity fluctuates between 60 - 90%.

[0087] Culture medium: Unfermented cow dung with a carbon-nitrogen ratio of 1:18, a moisture content of 70%, no straw added, a porosity of <30%, and an ammonia concentration of >100 ppm, exceeding the standard by 3 times.

[0088] Management method: Replace the culture medium every 30 days, with a density of 5000 - 6000 earthworms per square meter. The growth of earthworms is slow. It takes 120 days for the adult body length to be 7±1 cm and the weight to be 0.8±0.2 g.

[0089] Step 2: Extensive preparation of raw materials for medicine

[0090] Homogenization and centrifugation: Earthworms are directly homogenized without pretreatment, and the supernatant after centrifugation is turbid, and the protein recovery rate is ≤40%.

[0091] One-step alcohol precipitation: 95% alcohol was directly added to 70 vol%, and it was left standing at room temperature of 25°C for 24 hours. The α-helix structure of PrAMPs was over-denatured and completely destroyed, and 80% of the activity was lost.

[0092] No ultrafiltration purification: Only rough filtration was carried out through filter paper. The alcohol residue was 1.8 ± 0.3%, and the content of impurity proteins was ≥ 30%.

[0093] Clinical application results: Treatment of third-degree burns: The healing time > 120 days, the scar incidence rate was 85%, and the secondary infection rate was 40% (Pseudomonas aeruginosa was detected); PrAMPs content: 5.2 ± 0.3 mg / mL, plasmin activity 248 ± 10 IU / mL; Safety issues: The content of heavy metal lead was 1.2 ppm, exceeding the standard by 2.4 times, and the incidence rate of irritant dermatitis caused by alcohol residue was 25%.

[0094] Comparison table of preparation process parameters:

[0095] Comparison items Example 1 Example 2 Example 3 Control example Alcohol concentration (vol%) 30 30 35 70 Precipitation temperature (°C) 1-4 1-4 1 25 (room temperature) Precipitation time (hours) 48 36 36 24 Molecular weight cut-off of ultrafiltration membrane (kDa) 10→3 5→3 5→3 Without ultrafiltration Alcohol recovery rate (%) ≥95 ≥95 ≥96 ≤80 Yield of PrAMPs (%) ≥90 ≥92 ≥93 ≤40

[0096] Conclusion: In Examples 1 - 3, through the processes of gradient alcohol precipitation (30 - 35 vol%), low-temperature standing (1 - 4°C) and multi-stage ultrafiltration (10 kDa → 3 kDa), the yield of PrAMPs ≥ 90% and the alcohol recovery rate ≥ 95% were achieved, and the alcohol residue ≤ 0.3%, which was far superior to the one-step alcohol precipitation process of the comparative example (PrAMPs yield ≤ 40%, alcohol residue 1.8%). The data showed that through precise temperature control and hierarchical purification, the present invention effectively retained the active conformation (β-sheet) of antibacterial peptides, and solved the core problems of large component loss and high impurity residue in the traditional process.

[0097] Comparison table of earthworm cultivation conditions:

[0098] Comparison items Example 1 Example 2 Example 3 Control example Carbon-nitrogen ratio of culture medium 1:23-1:25 1:23-1:25 1:22-1:24 1:18-1:20 Concentration of inducer 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 Ultraviolet light intensity (mW / cm²) 5 8 5 (pulse mode) None Stocking density (fish / m²) 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

[0099] Conclusion: In Examples 1 - 3, by adopting dynamic medium management (carbon-nitrogen ratio 1:22 - 1:25), staged inducer feeding (Zn-proline, sulfonated phenylalanine) and ultraviolet gene activation, the weight of adult earthworms reached 1.5 - 1.9 g (only 0.6 - 0.8 g in the comparative example), and the PrAMPs content increased by 3 - 4 times. Due to open-air mixed cultivation and no induction measures in the comparative example, the development of earthworms was retarded and the active ingredients were poor. Through the coordinated regulation of environment and metabolism, the present invention provides a high-active substance basis for raw pharmaceutical materials.

[0100] Comparison table of clinical efficacy and safety:

[0101] Comparison items Example 1 Example 2 Example 3 Control example Burn healing time (days) 70 65 68 >120 Healing rate of diabetic foot ulcer (%) 97 95 93 45 Regression rate of psoriasis (%) 85 88 90 30 Incidence rate of scar (%) 0 0 0 85 Side effect of alcohol residue None None None Irritant dermatitis Heavy metal lead content (ppm) 0.4 0.3 0.35 1.2 (exceeding the standard)

[0102] Conclusion: In the treatment of severe burns, diabetic foot ulcers, and psoriasis, the scar-free healing rate of Examples 1-3 is ≥93%, the erythema regression rate is ≥85%, and there are no side effects of alcohol residue (the scar rate of the comparative example is 85%, and the incidence of dermatitis is 25%). In terms of safety, the heavy metal lead content in the examples is ≤0.4 ppm (2.4 times higher than the standard in the comparative example), and the alcohol residue is ≤0.3% (1.8% in the comparative example). The data verifies the dual advantages of the present invention in efficient repair and low toxicity and safety, filling the gap that traditional drugs cannot balance efficacy and risk.

[0103] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation method of a raw material drug for skin wound repair, characterized in that: It includes the following steps: S1, raw material pretreatment: Place the adult Eisenia fetida in a 40 - 50 °C water bath and oscillate and wash for 30 minutes. The aqueous solution in the water bath contains 0.1% polysorbate - 80 and 0.05% disodium EDTA; S2, shearing and homogenizing: Mix the pretreated earthworms and ultrapure water at a mass ratio of 1:2, use a shearing instrument with a rotation speed of 12000 rpm for 3 minutes of homogenization, let it stand for 20 minutes, and then centrifuge at 3000 rpm for min, and retain the supernatant; S3, alcohol precipitation: Add 95% medical alcohol to the supernatant, adjust the alcohol content of the mixed solution to 30 vol%, and let it stand at 1 - 4 °C for 48 hours. During this process, the protein denatures and its structure changes. Specifically: PrAMPs: α - helix structure → PrAMPs: β - sheet conformation; S4, ultrafiltration purification: Take the supernatant and remove miscellaneous bacteria and macromolecular impurities through a 10 kDa ultrafiltration membrane, and then use a rotary evaporator with a pressure of - 0.09 MPa to filter out 60% of the water at a temperature of 40 °C, and recover the alcohol to a final concentration of 30 vol%; S5, secondary ultrafiltration and preservation: Let the concentrated solution stand at 4 °C for 15 days, then filter it twice through a 3 kDa ultrafiltration membrane to obtain a clear liquid, which is dispensed into brown glass bottles and stored in the dark at 0 - 4 °C; The method for the directional cultivation of the Eisenia fetida includes the following steps: Induction: During the juvenile earthworm stage of 0 - 15 days, feed the earthworms with feed containing 0.05% L - proline chelated zinc every day; for the juvenile earthworms in the sexual maturity stage of 16 - 45 days, feed them with 0.1% phenylalanine sulfonated derivative every day, and apply ultraviolet light with a wavelength of 365 nm and an intensity of 5 mW / cm² for 2 hours every day to activate the antibacterial peptide gene. The structural formulas of the L - proline chelated zinc and phenylalanine sulfonated derivative are respectively: Selection of adult individuals: Healthy adult individuals with a harvesting density of 2000 - 2500 individuals per square meter, a cultivation period of 90 - 100 days, a body length of 10 - 13 cm, a body weight of 1.2 - 1.8 g, and a clitellum width of ≥ 0.4 cm are selected. The harvesting time is from mid-April to mid-June and from early September to late October.

2. The preparation method of a raw material drug for skin wound repair according to claim 1, wherein: The preparation method of the L - proline chelated zinc is: Mix L - proline and zinc sulfate heptahydrate at a molar ratio of 1:1.2, adjust the pH to 6.5 - 7.0, stir and react at 50 °C for 2 hours, centrifuge to collect the precipitate, and obtain a white powder after vacuum drying. The specific reaction formula is: 。 3. The preparation method of a raw material drug for skin wound repair according to claim 1, characterized in that: The synthesis reaction of the phenylalanine sulfonated derivative is: Mix phenylalanine and chlorosulfonic acid at a molar ratio of 1:1.5, and react at 50 - 60 °C for 4 hours. The reaction formula is: After the reaction, crystallization is carried out with ethanol at -20°C, and the sulfonated product is obtained after filtration.

4. The raw material drug for skin wound repair prepared by the method according to claim 1, characterized in that: Its physical and chemical indexes include: the content of antibacterial peptide PrAMPs ≥ 18 mg / mL; the fibrinolytic enzyme activity ≥ 600 IU / mL; for the sterility test, aerobic bacteria, anaerobic bacteria and fungi are all negative.

5. The active pharmaceutical ingredient according to claim 4, wherein: The amino acid sequence of the antibacterial peptide contains 59 amino acid residues, among which the 9th, 17th and 28th positions are proline, and the total proportion of proline ≥ 15%.

6. The application of the raw material drug for skin wound repair according to claim 4, characterized in that: The raw drug substance is used to prepare drugs for treating the following diseases: severe burn and scald, diabetic foot ulcer, psoriasis; wherein, for severe burn and scald: the wound area is 10 - 30% TBSA, 0.5 mL / cm² is topically applied daily, promoting epidermal regeneration within 72 hours, and the scar-free healing rate is ≥95%; for diabetic foot ulcer: Wagner grade 2 - 3, dressing is changed once a day, and the granulation tissue coverage rate is ≥90% within 20 days; for psoriasis: the local skin lesion area is ≤10% BSA, it is topically applied twice a day, and the erythema and scale regression rate is ≥85% within 30 days; the drug dosage form is a nano-sustained release gel, which contains 10 - 15% of the raw drug substance and a carbomer matrix.

7. The application according to claim 6, wherein: The nano-sustained release gel is prepared by the following method: Carbomer 940 and triethanolamine are mixed at a mass ratio of 1:0.3, after swelling with water, the raw drug substance and glycerol with a mass ratio of 5 - 8% are added, and homogenously emulsified 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, realizing the sustained release of the drug.

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