A biocompatible skin wound healing drug and its preparation method

Abstract

This invention relates to a biocompatible skin wound healing drug and its preparation method, belonging to the field of biomedical technology. The drug includes liposome-encapsulated ovalbumin peptide, red okra seed extract, taro stem extract, liposome-encapsulated acetyl tetrapeptide-9, and vitamin E. The ovalbumin peptide is obtained by pre-cooling goose egg white followed by enzymatic hydrolysis with trypsin and ginger protease, and the hydrolysate is purified using an XAD16 non-ionic macroporous resin column. The red okra seed extract is obtained by ultrasonic hydrolysis of red okra seeds in an ethanol-water solution using hemicellulase and ginger protease, and the hydrolysate is purified using an AB-8 macroporous adsorption resin column. The taro stem extract is obtained by grinding taro stems into a pulp, ultrasonically extracting it with an ethanol-water solution, and purifying the extract using an HP-20 macroporous adsorption resin column. The synergistic use of these components constructs a "multifunctional integrated" repair system, solving the problems of insufficient biocompatibility, single function, and low repair efficiency of traditional drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biocompatible skin wound healing drug and its preparation method. Background Technology

[0002] As the largest barrier organ in the human body, the skin's wound healing is a complex physiological process involving multiple stages such as inflammatory response, cell proliferation, and matrix remodeling. When the trauma exceeds the body's self-repair capacity, intervention with drugs or biomaterials is necessary. Biocompatibility, as a core evaluation indicator, directly determines whether a drug will cause adverse reactions such as immune rejection or exacerbation of inflammation, and is a key prerequisite for the development of drugs for skin wound healing.

[0003] Currently, clinically used skin wound healing drugs and auxiliary materials are mainly divided into the following three categories: (1) Traditional chemical drugs: with the core functions of promoting local blood circulation and preventing infection, such as recombinant human epidermal growth factor (rhEGF) and silver sulfadiazine cream. rhEGF can accelerate the proliferation and migration of epidermal cells and shorten the healing time of superficial wounds; silver sulfadiazine cream has both antibacterial and astringent effects and is widely used for the prevention of burn wound infection, but the biocompatibility of these drugs is limited to "no direct toxicity" and the overall regulatory ability of the wound microenvironment is weak. (2) Natural biomaterials: relying on the low allergenicity and bioactivity of natural components, they have become a research hotspot in recent years, mainly including chitosan dressings, hyaluronic acid gel, etc. However, the degradation rate of some products does not match the wound healing process. (3) Synthetic polymer materials: represented by polylactic acid (PLA) and polycaprolactone (PCL), etc., performance customization can be achieved through controllable synthesis, which can simulate the physical barrier function of the skin, and the degradation products can be metabolized by the human body, and the biocompatibility is significantly improved compared with traditional synthetic materials. These materials are often made into hydrogels or porous scaffolds for the repair of chronic wounds (such as diabetic foot ulcers). However, the monomers remaining during the synthesis process may cause mild local inflammation and lack the bioactivity of natural materials. Additional active ingredients such as growth factors are required to improve the repair effect.

[0004] Although current technologies can meet the treatment needs of most superficial, acute wounds, there are still many shortcomings:

[0005] 1. Incomplete biocompatibility: Although some drugs or materials have no obvious toxicity, they may still activate the body's "foreign body recognition" mechanism, triggering a chronic inflammatory response, leading to redness, swelling and increased exudation in the wound. In addition, the degradation products of some synthetic materials are too acidic, which can disrupt the acid-base balance of the wound microenvironment and delay the healing process.

[0006] 2. Limitations of Single Function: Most products focus only on a single aspect such as "anti-infection" or "proliferation promotion." However, chronic wounds, due to insufficient angiogenesis and low cell activity, require materials that simultaneously possess multiple functions such as promoting angiogenesis, antibacterial properties, and immune regulation.

[0007] 3. Low repair efficiency: For example, traditional growth factor drugs are easily degraded by proteases in chronic wounds, resulting in low bioavailability.

[0008] Therefore, it is necessary to continuously develop new drug ingredients, design scientific formulations, and build a "multifunctional integrated" repair system to achieve safer and more efficient wound healing. Summary of the Invention

[0009] To address the problems of incomplete biocompatibility, limited single function, and low repair efficiency in existing skin wound healing drugs, this invention provides a biocompatible skin wound healing drug and its preparation method. The drug comprises ovalbumin peptides prepared by a special method, okra seed extract, and taro stem extract, which are used synergistically with acetyl tetrapeptide-9 and vitamin E to construct a "multifunctional integrated" repair system. This solves the core problems of insufficient biocompatibility, limited function, and low repair efficiency of traditional drugs. The specific technical solution is as follows:

[0010] A biocompatible skin wound healing drug comprises ovalbumin peptide, red okra seed extract, taro stem extract, acetyl tetrapeptide-9, and vitamin E in a mass ratio of (6-8):(2-3):(1-2):(3-5):(1-1.5); the ovalbumin peptide is encapsulated in 2.5-4 times its mass of liposomes, and the acetyl tetrapeptide-9 is encapsulated in 8-10 times its mass of liposomes; the ovalbumin peptide is obtained by pre-cooling goose egg white and then enzymatically hydrolyzing it with trypsin and ginger protease in sequence, and the hydrolysate is purified by XAD16 non-ionic macroporous resin column; the red okra seed extract is obtained by enzymatically hydrolyzing red okra seeds in an ethanol aqueous solution with hemicellulase and ginger protease by ultrasound, and the hydrolysate is purified by AB-8 macroporous adsorption resin column; the taro stem extract is obtained by grinding taro stems into a pulp, ultrasonically extracting it with an ethanol aqueous solution, and the extract is purified by HP-20 macroporous adsorption resin column.

[0011] The preparation of the ovalbumin peptide in the above-mentioned drug includes: taking ovalbumin from goose eggs, adding Tris-HCl buffer at pH 8.0-8.5, stirring and homogenizing to obtain crude ovalbumin solution, pre-cooling, adding trypsin, and enzymatically hydrolyzing at 36℃-39℃ for 1.5h-2h, adjusting the pH to 6.5-7.0, adding ginger protease, and enzymatically hydrolyzing at 52℃-58℃ for 1h-1.5h, centrifuging, taking the supernatant, inactivating the enzyme, loading the sample onto an XAD16 non-ionic macroporous resin column, first rinsing with 1.5BV-2BV deionized water to remove impurities, then eluting sequentially with 1.5BV-2BV 10%-15% volume concentration of ethanol aqueous solution and 1.5BV-2BV 20%-30% volume concentration of ethanol aqueous solution, collecting and combining the eluents, and freeze-drying to obtain ovalbumin peptide.

[0012] In the preparation of the above-mentioned ovalbumin peptide, the amount of Tris-HCl buffer used is 4 to 5 times the volume of ovalbumin; the pre-cooling is 3℃ to 5℃ for 1 to 2 hours; the amount of trypsin added is 0.5% to 1.0% of the ovalbumin mass; the amount of ginger protease added is 0.5% to 1.0% of the ovalbumin mass; the centrifugation is 8000 rpm to 8500 rpm for 10 to 15 minutes; the enzyme inactivation is 80℃ to 85℃ for 10 to 15 minutes; after enzyme inactivation, the solution is concentrated under reduced pressure at 45℃ to 50℃ to 25% to 35% of the volume; the eluent is concentrated under reduced pressure at 45℃ to 50℃ to remove ethanol.

[0013] The method for encapsulating the ovalbumin peptide in the aforementioned drug comprises: dissolving soybean lecithin in anhydrous ethanol at a mass ratio of (3.5–4.5):1, rotary evaporating to form a film, and vacuum drying to obtain liposomes; preparing a 5 wt%–8 wt% peptide solution using a pH 7.0–7.4 phosphate buffer; adding the peptide solution to the liposomes at a mass ratio of ovalbumin peptide:liposome = 1:(2.5–4), shaking in a water bath to obtain a proemulsion; sonicating in an ice bath to obtain a suspension; filtering with a microporous membrane, taking the permeate, and freeze-drying to obtain the ovalbumin peptide encapsulated in liposomes.

[0014] The preparation of the red okra seed extract in the above-mentioned drug includes: crushing red okra seeds into seed powder, adding 6 to 8 times the weight of the seed powder to a 20% to 30% volume concentration ethanol aqueous solution, adjusting the pH to 5.0 to 5.5, adding hemicellulase and ginger protease, ultrasonically hydrolyzing, inactivating the enzyme, centrifuging, taking the supernatant, loading the sample onto an AB-8 macroporous adsorption resin column, first rinsing with 2 BV to 2.5 BV of deionized water and 1.5 BV to 2 BV of 10% to 15% volume concentration ethanol aqueous solution to remove impurities, eluting with 70% to 75% volume concentration ethanol aqueous solution for 3 BV to 4 BV, collecting the eluent, freeze-drying, and obtaining the red okra seed extract.

[0015] In the preparation of the above-mentioned red okra seed extract, the particle size of the seed powder is sieved through an 80-100 mesh sieve; the amount of hemicellulase added is 0.5%-1% of the seed powder mass; the amount of ginger protease added is 1%-2% of the seed powder mass; the ultrasonic enzymatic hydrolysis is performed at 50℃-55℃ and 200W-250W for 1.5h-2h; the enzyme inactivation is performed by heating to 80℃-85℃ for 15min-20min; after enzyme inactivation, the solution is concentrated under reduced pressure at 45℃-50℃ to 25%-35% of its volume to remove ethanol; the centrifugation is performed at 8000rpm-8500rpm for 10min-15min; and the eluent is concentrated under reduced pressure at 45℃-50℃ to remove ethanol.

[0016] The preparation of the taro stem extract in the above-mentioned drug includes: grinding fresh taro stems into a slurry, ultrasonically extracting the slurry with 5 to 6 times its mass of 60% to 65% (v / v) ethanol aqueous solution, centrifuging, collecting the supernatant, loading it onto an HP-20 macroporous adsorption resin column, rinsing with 2 to 3 BV of 5% to 10% (v / v) ethanol aqueous solution to remove impurities, then eluting with 70% to 75% (v / v) ethanol aqueous solution for 3 to 5 BV, collecting the eluent, and freeze-drying to obtain the taro stem extract.

[0017] In the preparation of the above-mentioned taro stem extract, the ultrasonic extraction is performed at room temperature and 400W-500W for 40-60 minutes; the centrifugation is performed at 6000rpm-8000rpm for 15-20 minutes; the supernatant is concentrated under reduced pressure at 45℃-50℃ to 25%-35% of its volume to remove ethanol; and the eluent is concentrated under reduced pressure at 45℃-50℃ to remove ethanol.

[0018] The method for encapsulating the acetyl tetrapeptide-9 in liposomes in the above-mentioned drug includes: dissolving acetyl tetrapeptide-9 in anhydrous ethanol at a mass ratio of soybean lecithin to cholesterol of (3.5-4.5):1, rotary evaporating to form a film, and vacuum drying to obtain liposomes; preparing a 4wt%-6wt% peptide solution with phosphate buffer at pH 6.4-6.8; adding the peptide solution to the liposomes at a mass ratio of acetyl tetrapeptide-9:liposomes = 1:(6-8), shaking in a water bath to obtain a primary emulsion; sonicating in an ice bath to obtain a suspension; filtering with a microporous membrane, taking the permeate, and freeze-drying to obtain acetyl tetrapeptide-9 encapsulated in liposomes.

[0019] The preparation method of the above-mentioned biocompatible skin wound healing drug includes the following steps:

[0020] According to the formula mass ratio, ovalbumin peptide, red okra seed extract, taro stem extract, acetyl tetrapeptide-9 and vitamin E are mixed to obtain a pharmaceutical composition; the pharmaceutical composition is then combined with pharmaceutically available excipients to prepare a gel or spray.

[0021] This invention provides a biocompatible skin wound healing drug and its preparation method, the beneficial effects of which include:

[0022] I. This invention utilizes a synergistic design combining natural active ingredients, biomimetic peptides, and liposome inclusion technology to construct a multifunctional integrated repair system, addressing the core issues of insufficient biocompatibility, limited functionality, and low repair efficiency of traditional drugs. Liposome inclusion technology reduces the cytotoxicity of ovalbumin peptide and acetyl tetrapeptide-9, and minimizes enzymatic degradation, while the low allergenicity of each natural component further optimizes biocompatibility. Through multi-faceted intervention including anti-inflammatory, antibacterial, proliferative, and collagen-synthetic stimulation, it addresses both the infection and rapid healing needs of acute wounds and the low cell activity in chronic wounds, improving the repair efficiency of difficult-to-heal wounds. The antioxidant effects of taro stem extract, red okra seed extract, and vitamin E scavenge free radicals in the wound, reducing oxidative stress damage.

[0023] 2. Ovalbumin is enzymatically hydrolyzed stepwise by trypsin and ginger protease (avoiding insufficient or excessive hydrolysis caused by single enzymatic hydrolysis, which can produce allergenic peptides), generating low-allergenic small molecule peptides. These peptides provide amino acid raw materials for fibroblasts and keratinocytes, activating cell proliferation pathways. Liposome encapsulation effectively prevents peptides from being degraded by proteases, improving bioavailability, while reducing direct stimulation of erythrocyte membranes by peptide fragments, thus optimizing biocompatibility. The ratio of soybean lecithin to cholesterol (3.5–4.5:1) ensures a balance between the stability and permeability of the liposome membrane, avoiding a ratio that is too high, leading to an excessively thick membrane (affecting component release) or too low, leading to membrane rupture (reducing stability).

[0024] Third, red okra seeds are enzymatically hydrolyzed by hemicellulase and ginger protease to efficiently release flavonoids and polysaccharides. Flavonoids reduce inflammation by scavenging free radicals, while polysaccharides enhance antibacterial activity. AB-8 resin purification enriches highly active flavonoids, avoiding non-specific inflammation caused by impurities.

[0025] IV. Taro stems are extracted using ultrasound at room temperature to preserve the film-forming and moisturizing properties of polysaccharides. HP-20 resin purification removes small molecule impurities to prevent them from disrupting the acid-base balance of the wound. The membrane structure formed by the polysaccharides can isolate external pollution, maintain wound moisture, and exert antibacterial effects by altering the permeability of bacterial cell membranes.

[0026] V. Acetyl tetrapeptide-9 can activate collagen synthesis pathways (promoting collagen secretion by fibroblasts) and angiogenesis-related genes, but it has poor stability and is easily degraded by proteases. Liposome encapsulation effectively protects its active structure and promotes its transdermal absorption, allowing it to act precisely on dermal fibroblasts.

[0027] VI. Vitamin E can protect liposome membranes from oxidation and prevent inflammation caused by lipid peroxidation products. It can also directly eliminate free radicals on wound surfaces, while nourishing the stratum corneum and enhancing the skin barrier function.

[0028] VII. Targeted selection of different resins (XAD16, AB-8, HP-20) based on the polarity differences of target components (small molecule peptides, flavonoids, polysaccharides) to achieve efficient purification and avoid interference of impurities on drug performance.

[0029] VIII. The drug components achieve a synergistic effect of 1+1>2 through “functional complementarity-mechanism synergy”. (1) Biocompatibility synergy: Liposome inclusion technology reduces the cytotoxicity of ovalbumin peptide and acetyl tetrapeptide-9, the anti-inflammatory effect of red okra seed extract counteracts the mild inflammation that may be caused by the synthetic components, and the membrane stabilizing effect of taro stem extract further reduces the risk of hemolysis. The three together improve the overall biocompatibility of the drug. (2) Repair function synergy: Ovalbumin peptide provides energy for cell proliferation, and acetyl tetrapeptide-9 promotes collagen synthesis and angiogenesis. The two form a “raw material supply-functional activation” synergy. The antioxidant effect of red okra seed extract protects proliferating cells from free radical damage, and vitamin E enhances this effect, providing a good microenvironment for cell proliferation. (3) Antibacterial-anti-inflammatory synergy: Red okra seed extract and taro stem extract have a “broad-spectrum antibacterial” combination. At the same time, the anti-inflammatory effect of red okra seed extract inhibits the inflammatory response caused by bacterial infection. (4) Stability Synergy: Vitamin E protects the liposome membrane from oxidation, while the liposomes protect ovalbumin peptide and acetyl tetrapeptide-9 from rapid degradation by proteases, forming a dual guarantee of "component protection - dosage form stability". Detailed Implementation

[0030] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0031] Example 1

[0032] A biocompatible skin wound healing drug comprises ovalbumin peptide, red okra seed extract, taro stem extract, acetyl tetrapeptide-9, and vitamin E in a mass ratio of 7:2.5:1.5:4:1.2; wherein the ovalbumin peptide and the acetyl tetrapeptide-9 are encapsulated in liposomes.

[0033] The preparation of the ovalbumin peptides includes: taking ovalbumin from goose eggs, adding 4.5 volumes of pH 8.3 Tris-HCl buffer, stirring and homogenizing to obtain crude ovalbumin solution; pre-cooling the crude solution at 4℃ for 1.5 h (to inhibit endogenous protease activity); adding 0.8% trypsin by weight of ovalbumin, enzymatically hydrolyzing at 37℃ for 1.5 h, adjusting the pH to 6.7, adding 0.8% ginger protease by weight of ovalbumin, enzymatically hydrolyzing at 55℃ for 1.5 h; centrifuging at 8200 rpm for 12 min, taking the supernatant, inactivating the enzyme at 82℃ for 12 min, concentrating under reduced pressure at 48℃ to 30% of the volume, and loading the sample onto an XAD16 non-ionic macroporous resin column (pre-activated with 92% volume concentration ethanol aqueous solution and equilibrated with pH 8.3 Tris-HCl buffer), first rinsing with 1.5 BV deionized water at 1.8 BV / h to remove impurities, then sequentially rinsing with 2 BV 12% volume concentration ethanol aqueous solution and 1.5 BV... The eluent was eluted with a 25% (v / v) ethanol aqueous solution, the eluents were collected and combined, concentrated under reduced pressure at 48°C to remove ethanol, and then freeze-dried to obtain ovalbumin peptides.

[0034] The method for encapsulating the ovalbumin peptide via liposomes includes: dissolving soybean lecithin in anhydrous ethanol at a mass ratio of 4:1 (the amount of ethanol used is 5.5 times the total mass of soybean lecithin and cholesterol), rotary evaporating at 48°C to form a film, and vacuum drying for 1 hour to obtain liposomes; preparing a 6.5 wt% peptide solution using pH 7.2 phosphate buffer; adding the peptide solution to the liposomes at a mass ratio of ovalbumin peptide:liposome = 1:3.2, shaking in a water bath at 37°C for 40 minutes to obtain a proemulsion; sonicating at 300W for 3 seconds followed by a 5-second interval in an ice bath for 18 minutes to obtain a suspension; filtering through a 0.45 μm microporous membrane, collecting the permeate, and freeze-drying to obtain the ovalbumin peptide encapsulated in liposomes.

[0035] The preparation of the red okra seed extract includes: taking dried red okra seeds, pulverizing them and passing them through a 100-mesh sieve to obtain seed powder, adding 7 times the weight of the seed powder to a 25% volume concentration ethanol aqueous solution, adjusting the pH to 5.2, adding 0.8% hemicellulase and 1.5% ginger protease by weight of the seed powder, sonicating at 52℃ and 200W for 2 hours, raising the temperature to 82℃ to inactivate the enzyme for 18 minutes, concentrating under reduced pressure at 48℃ to 30% of the volume to remove ethanol, centrifuging at 8200rpm for 12 minutes, collecting the supernatant, and loading it onto an AB-8 macroporous adsorption resin column (activated with 5wt% hydrochloric acid aqueous solution and equilibrated to neutral with deionized water), first rinsing with 2BV of deionized water and 1.5BV of 12% volume concentration ethanol aqueous solution at 1.8BV / h to remove impurities, and finally eluting with 73% volume concentration ethanol aqueous solution for 3.5BV, collecting the eluent, concentrating under reduced pressure at 48℃ to remove ethanol, and freeze-drying to obtain the red okra seed extract.

[0036] The preparation of the taro stem extract includes: grinding fresh taro stems into a slurry, extracting the slurry with 5.5 times its mass of 63% (v / v) ethanol aqueous solution at room temperature and ultrasonically at 450W for 50 min, centrifuging at 7000 rpm for 18 min, collecting the supernatant, concentrating it under reduced pressure at 48℃ to 30% (v / v) to remove ethanol, loading the sample onto an HP-20 macroporous adsorption resin column (activated with 92% (v / v) ethanol aqueous solution and then equilibrated with deionized water), eluting with 2.5 BV of 8% (v / v) ethanol aqueous solution at 1.5 BV / h to remove impurities, then eluting with 73% (v / v) ethanol aqueous solution for 4 BV, collecting the eluent, concentrating it under reduced pressure at 48℃ to remove ethanol, and freeze-drying to obtain the taro stem extract.

[0037] The method for encapsulating acetyl tetrapeptide-9 with liposomes includes: dissolving soybean lecithin in anhydrous ethanol at a mass ratio of 4:1 (the amount of ethanol used is 5.5 times the total mass of soybean lecithin and cholesterol), rotary evaporating at 47°C to form a film, and vacuum drying for 1 hour to obtain liposomes; preparing a 5wt% peptide solution of acetyl tetrapeptide-9 with pH 6.5 phosphate buffer; adding the peptide solution to the liposomes at a mass ratio of acetyl tetrapeptide-9:liposomes = 1:7, shaking in a water bath at 37°C for 40 minutes to obtain a primary emulsion; sonicating at 300W for 3 seconds followed by a 5-second interval in an ice bath for 18 minutes to obtain a suspension; filtering through a 0.45μm microporous membrane, collecting the permeate, and freeze-drying to obtain acetyl tetrapeptide-9 encapsulated in liposomes.

[0038] The preparation method of the above-mentioned biocompatible skin wound healing drug includes the following steps:

[0039] According to the formula mass ratio, ovalbumin peptide, red okra seed extract, taro stem extract, acetyl tetrapeptide-9 and vitamin E are mixed to obtain a pharmaceutical composition; the pharmaceutical composition is then combined with pharmaceutically available excipients to prepare a gel or spray.

[0040] Example 2

[0041] A biocompatible skin wound healing drug comprises ovalbumin peptide, red okra seed extract, taro stem extract, acetyl tetrapeptide-9, and vitamin E in a mass ratio of 6:3:1:5:1; wherein the ovalbumin peptide and the acetyl tetrapeptide-9 are encapsulated in liposomes.

[0042] The preparation of the ovalbumin peptides includes: taking ovalbumin from goose eggs, adding 4 volumes of pH 8.5 Tris-HCl buffer, stirring and homogenizing to obtain crude ovalbumin solution; pre-cooling the crude solution at 3℃ for 2 hours (to inhibit endogenous protease activity); adding 0.5% trypsin by weight of ovalbumin, enzymatically hydrolyzing at 39℃ for 1.5 hours, adjusting the pH to 7.0, adding 0.5% ginger protease by weight of ovalbumin, enzymatically hydrolyzing at 58℃ for 1 hour; centrifuging at 8500 rpm for 10 minutes, taking the supernatant, inactivating the enzyme at 85℃ for 10 minutes, concentrating under reduced pressure at 50℃ to 25% of the volume, and loading the sample onto an XAD16 non-ionic macroporous resin column (pre-activated with 95% volume concentration ethanol aqueous solution and equilibrated with pH 8.0 Tris-HCl buffer), first rinsing with 2 BV deionized water at 1.5 BV / h to remove impurities, then sequentially rinsing with 2 BV 10% volume concentration ethanol aqueous solution and 2 BV... The eluent was eluted with a 20% (v / v) ethanol aqueous solution, the eluents were collected and combined, concentrated under reduced pressure at 50°C to remove ethanol, and then freeze-dried to obtain ovalbumin peptides.

[0043] The method for encapsulating the ovalbumin peptide via liposomes includes: dissolving soybean lecithin in anhydrous ethanol at a mass ratio of 3.5:1 (the amount of ethanol used is 6 times the total mass of soybean lecithin and cholesterol), rotary evaporating at 45°C to form a film, and vacuum drying for 1.5 h to obtain liposomes; preparing an 8 wt% peptide solution using pH 7.0 phosphate buffer; adding the peptide solution to the liposomes at a mass ratio of ovalbumin peptide:liposome = 1:2.5, shaking in a water bath at 40°C for 30 min to obtain a proemulsion; sonicating at 350 W for 3 s followed by a 5 s interval in an ice bath for 15 min to obtain a suspension; filtering through a 0.45 μm microporous membrane, collecting the permeate, and freeze-drying to obtain the ovalbumin peptide encapsulated in liposomes.

[0044] The preparation of the red okra seed extract includes: taking dried red okra seeds, pulverizing them and passing them through an 80-mesh sieve to obtain seed powder, adding 8 times the weight of the seed powder to a 20% volume concentration ethanol aqueous solution, adjusting the pH to 5.5, adding 0.5% hemicellulase and 2% ginger protease by weight of the seed powder, ultrasonically hydrolyzing at 50℃ and 250W for 1.5h, raising the temperature to 85℃ to inactivate the enzyme for 15min, concentrating under reduced pressure at 50℃ to 25% volume to remove ethanol, centrifuging at 8500rpm for 10min, taking the supernatant, and loading it onto an AB-8 macroporous adsorption resin column (activated with 6wt% hydrochloric acid aqueous solution and equilibrated to neutral with deionized water), first eluting with 2BV of deionized water and then 2BV of 10% volume concentration ethanol aqueous solution at 2BV / h to remove impurities, and finally eluting with 70% volume concentration ethanol aqueous solution for 4BV, collecting the eluent, concentrating under reduced pressure at 45℃ to remove ethanol, and freeze-drying to obtain the red okra seed extract.

[0045] The preparation of the taro stem extract includes: grinding fresh taro stems into a slurry, extracting the slurry with 6 times its mass of 60% (v / v) ethanol aqueous solution at room temperature and ultrasonically at 500W for 40 min, centrifuging at 8000 rpm for 15 min, collecting the supernatant, concentrating it under reduced pressure at 50℃ to 25% (v / v) to remove ethanol, loading the sample onto an HP-20 macroporous adsorption resin column (activated with 95% (v / v) ethanol aqueous solution and then equilibrated with deionized water), eluting with 2 BV of 10% (v / v) ethanol aqueous solution at 1.5 BV / h to remove impurities, then eluting with 75% (v / v) ethanol aqueous solution for 3 BV, collecting the eluent, concentrating it under reduced pressure at 50℃ to remove ethanol, and freeze-drying to obtain the taro stem extract.

[0046] The method for encapsulating acetyl tetrapeptide-9 with liposomes includes: dissolving soybean lecithin in anhydrous ethanol at a mass ratio of 4.5:1 (the amount of ethanol is 5 times the total mass of soybean lecithin and cholesterol), rotary evaporating at 50°C to form a film, and vacuum drying for 1 hour to obtain liposomes; preparing a 4wt% peptide solution of acetyl tetrapeptide-9 with pH 6.8 phosphate buffer; adding the peptide solution to the liposomes at a mass ratio of acetyl tetrapeptide-9:liposomes = 1:8, shaking in a water bath at 35°C for 50 minutes to obtain a primary emulsion; sonicating at 300W for 3 seconds followed by a 5-second interval in an ice bath for 20 minutes to obtain a suspension; filtering through a 0.45μm microporous membrane, collecting the permeate, and freeze-drying to obtain acetyl tetrapeptide-9 encapsulated in liposomes.

[0047] The preparation method of the above-mentioned biocompatible skin wound healing drug includes the following steps:

[0048] According to the formula mass ratio, ovalbumin peptide, red okra seed extract, taro stem extract, acetyl tetrapeptide-9 and vitamin E are mixed to obtain a pharmaceutical composition; the pharmaceutical composition is then combined with pharmaceutically available excipients to prepare a gel or spray.

[0049] Example 3

[0050] A biocompatible skin wound healing drug comprises ovalbumin peptide, red okra seed extract, taro stem extract, acetyl tetrapeptide-9, and vitamin E in a mass ratio of 8:2:2:3:1.5; wherein the ovalbumin peptide and the acetyl tetrapeptide-9 are encapsulated in liposomes.

[0051] The preparation of the ovalbumin peptides includes: taking ovalbumin from goose eggs, adding 5 volumes of pH 8.0 Tris-HCl buffer, stirring and homogenizing to obtain crude ovalbumin solution; pre-cooling the crude solution at 5℃ for 1 h (to inhibit endogenous protease activity); adding 1.0% trypsin by weight of ovalbumin, enzymatically hydrolyzing at 36℃ for 2 h, adjusting the pH to 6.5, adding 1.0% ginger protease by weight of ovalbumin, enzymatically hydrolyzing at 52℃ for 1.5 h; centrifuging at 8000 rpm for 15 min, taking the supernatant, inactivating the enzyme at 80℃ for 15 min, concentrating under reduced pressure at 45℃ to 35% of the volume, and loading the sample onto an XAD16 non-ionic macroporous resin column (pre-activated with 90% volume concentration ethanol aqueous solution and equilibrated with pH 8.5 Tris-HCl buffer), first rinsing with 1.5 BV deionized water at 2 BV / h to remove impurities, then sequentially rinsing with 1.5 BV 15% volume concentration ethanol aqueous solution and 1.5 BV... The eluent was eluted with a 30% (v / v) ethanol aqueous solution, the eluents were collected and combined, concentrated under reduced pressure at 45°C to remove ethanol, and then freeze-dried to obtain ovalbumin peptides.

[0052] The method for encapsulating the ovalbumin peptide via liposomes includes: dissolving soybean lecithin in anhydrous ethanol at a mass ratio of 4.5:1 (the amount of ethanol used is 5 times the total mass of soybean lecithin and cholesterol), rotary evaporating at 50°C to form a film, and vacuum drying for 1 hour to obtain liposomes; preparing a 5wt% peptide solution using pH 7.4 phosphate buffer; adding the peptide solution to the liposomes at a mass ratio of ovalbumin peptide:liposome = 1:4, shaking in a water bath at 35°C for 50 minutes to obtain a proemulsion; sonicating at 300W for 3 seconds followed by a 5-second interval in an ice bath for 20 minutes to obtain a suspension; filtering through a 0.45μm microporous membrane, collecting the permeate, and freeze-drying to obtain the liposome-encapsulated ovalbumin peptide.

[0053] The preparation of the red okra seed extract includes: taking dried red okra seeds, pulverizing them and passing them through a 100-mesh sieve to obtain seed powder, adding 6 times the weight of the seed powder to a 30% volume concentration ethanol aqueous solution, adjusting the pH to 5.0, adding 1% hemicellulase and 1% ginger protease of the seed powder, sonicating at 55℃ and 200W for 2 hours, raising the temperature to 80℃ to inactivate the enzyme for 20 minutes, concentrating under reduced pressure at 45℃ to 35% volume to remove ethanol, centrifuging at 8000rpm for 15 minutes, collecting the supernatant, loading it onto an AB-8 macroporous adsorption resin column (activated with 4wt% hydrochloric acid aqueous solution and equilibrated to neutral with deionized water), first rinsing with 2.5 BV of deionized water and then with 1.5 BV of 15% volume concentration ethanol aqueous solution at 1.5 BV / h to remove impurities, and finally eluting with 75% volume concentration ethanol aqueous solution for 3 BV, collecting the eluent, concentrating under reduced pressure at 50℃ to remove ethanol, and freeze-drying to obtain the red okra seed extract.

[0054] The preparation of the taro stem extract includes: grinding fresh taro stems into a slurry, extracting the slurry with 5 times its mass of 65% (v / v) ethanol aqueous solution at room temperature and 400W ultrasonic extraction for 60 min, centrifuging at 6000 rpm for 20 min, collecting the supernatant, concentrating it under reduced pressure at 45℃ to 35% (v / v) to remove ethanol, loading the sample onto an HP-20 macroporous adsorption resin column (activated with 90% (v / v) ethanol aqueous solution and then equilibrated with deionized water), rinsing with 3 BV of 5% (v / v) ethanol aqueous solution at 2 BV / h to remove impurities, then eluting with 70% (v / v) ethanol aqueous solution for 5 BV, collecting the eluent, concentrating it under reduced pressure at 45℃ to remove ethanol, and freeze-drying to obtain the taro stem extract.

[0055] The method for encapsulating acetyl tetrapeptide-9 with liposomes includes: dissolving soybean lecithin in anhydrous ethanol at a mass ratio of 3.5:1 (the amount of ethanol used is 6 times the total mass of soybean lecithin and cholesterol), rotary evaporating at 45°C to form a film, and vacuum drying for 1.5 h to obtain liposomes; preparing a 6 wt% peptide solution of acetyl tetrapeptide-9 with pH 6.4 phosphate buffer; adding the peptide solution to the liposomes at a mass ratio of acetyl tetrapeptide-9:liposomes = 1:6, shaking in a water bath at 40°C for 30 min to obtain a primary emulsion; sonicating at 350 W for 3 s followed by a 5 s interval in an ice bath for 15 min to obtain a suspension; filtering through a 0.45 μm microporous membrane, collecting the permeate, and freeze-drying to obtain acetyl tetrapeptide-9 encapsulated in liposomes.

[0056] The preparation method of the above-mentioned biocompatible skin wound healing drug includes the following steps:

[0057] According to the formula mass ratio, ovalbumin peptide, red okra seed extract, taro stem extract, acetyl tetrapeptide-9 and vitamin E are mixed to obtain a pharmaceutical composition; the pharmaceutical composition is then combined with pharmaceutically available excipients to prepare a gel or spray.

[0058] Comparative Example 1

[0059] The difference from Example 1 is that the mass ratio is ovalbumin peptide: red okra seed extract: taro stem extract: acetyl tetrapeptide-9: vitamin E = 7:3.8:0.2:4:1.2.

[0060] Comparative Example 2

[0061] The difference from Example 1 is that the mass ratio is ovalbumin peptide: red okra seed extract: taro stem extract: acetyl tetrapeptide-9: vitamin E = 3:2.5:1.5:8:1.2.

[0062] Comparative Example 3

[0063] The difference from Example 1 is that the mass ratio is ovalbumin peptide: red okra seed extract: taro stem extract: acetyl tetrapeptide-9: vitamin E = 3:3.8:0.2:8:1.2.

[0064] Comparative Example 4

[0065] The difference from Example 1 is that trypsin is not used for enzymatic hydrolysis in the preparation of ovalbumin peptides.

[0066] Comparative Example 5

[0067] The difference from Example 1 is that ginger protease is replaced with bromelain in the preparation of ovalbumin peptide.

[0068] Comparative Example 6

[0069] The difference from Example 1 is that in the preparation of ovalbumin peptide, XAD16 non-ionic macroporous resin is replaced with AB-8 macroporous adsorption resin.

[0070] Comparative Example 7

[0071] The difference from Example 1 is that ginger protease is replaced with bromelain in the preparation of red okra seed extract.

[0072] Comparative Example 8

[0073] The difference from Example 1 is that in the preparation of the red okra seed extract, ginger protease is replaced with papain.

[0074] Comparative Example 9

[0075] The difference from Example 1 is that in the preparation of red okra seed extract, AB-8 macroporous adsorption resin is replaced with XAD16 non-ionic macroporous resin.

[0076] Comparative Example 10

[0077] The difference from Example 1 is that in the preparation of taro stem extract, HP-20 macroporous adsorption resin is replaced with AB-8 macroporous adsorption resin.

[0078] The raw materials used in the above embodiments and comparative examples are as follows: Goose eggs are from ordinary farmed goose eggs. Taro stalks are from hairy taro stalks. Trypsin is from Xi'an Xinlu Biotechnology Co., Ltd., with an enzyme activity of 250,000 U / g. Ginger protease is from Shanghai Yimiao Chemical Technology Co., Ltd., with an enzyme activity of 400,000 U / g. XAD16 non-ionic macroporous resin is from Shanghai Yuanye Biotechnology Co., Ltd., model S51657. Soybean lecithin is from Peptide Biotechnology (Xi'an) Co., Ltd. Cholesterol is from Peptide Biotechnology (Xi'an) Co., Ltd. Hemicellulase is from Qingdao Haiweisen Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g. AB-8 macroporous adsorption resin is from Shanghai Yuanye Biotechnology Co., Ltd., model S30931. HP-20 macroporous adsorption resin is from Shanghai Yuanye Biotechnology Co., Ltd., model S27267. Acetyl tetrapeptide-9 is from Hubei Chenxin Pharmaceutical Co., Ltd., pharmaceutical grade. Vitamin E is from Peptide Biotechnology (Xi'an) Co., Ltd. Bromelain is from Peptide Biotechnology (Xi'an) Co., Ltd., with an enzyme activity of 100,000 U / g. Papain is derived from Peptide Biotechnology (Xi'an) Co., Ltd., with an enzyme activity of 100,000 U / g.

[0079] I. Hemolysis rate test:

[0080] Test solution preparation: Prepare a 10 mg / mL drug stock solution with physiological saline, filter to remove bacteria, and then dilute with physiological saline to prepare test solutions with concentrations of 1000 μg / mL and 500 μg / mL.

[0081] Experimental Method: Human anticoagulated whole blood was centrifuged at 3000 rpm for 10 minutes with physiological saline, and the supernatant and leukocyte layer were carefully removed. Red blood cells were washed three times until the supernatant was clear. Finally, the hematocrit red blood cells were prepared into a 2% (v / v) suspension with physiological saline (0.5 mL hematocrit red blood cells to 24.5 mL physiological saline). Test group: 1.0 mL drug test solution + 0.5 mL 2% red blood cell suspension, with 3 replicates per group. Negative control group: 1.0 mL physiological saline + 0.5 mL 2% red blood cell suspension. Positive control group: 1.0 mL deionized water + 0.5 mL 2% red blood cell suspension. The tubes were gently vortexed to mix. All tubes were incubated in a 37°C water bath for 60 minutes. After incubation, all tubes were centrifuged at 3000 rpm for 5 minutes to precipitate unruptured red blood cells. 200 μL of the supernatant from each tube was carefully aspirated and added to a 96-well plate. The absorbance (OD value) was measured at a wavelength of 540 nm using an ELISA reader.

[0082] Formula for calculating hemolysis rate (%): Hemolysis rate (%) = [(OD test group - OD negative control) / (OD positive control - OD negative control)] × 100%.

[0083] II. Cell proliferation rate detection (CCK-8 assay):

[0084] Test solution preparation: Prepare a 10 mg / mL drug stock solution using DMEM high-glucose complete medium (containing 10% FBS and 1% penicillin-streptomycin), filter to sterilize, and then dilute with DMEM high-glucose complete medium (containing 10% FBS and 1% penicillin-streptomycin) to prepare a test solution with a concentration of 150 μg / mL.

[0085] Negative control: DMEM high-glucose complete medium (for cells with wells, without drugs). Blank control: DMEM high-glucose complete medium (for cells without wells, zeroed).

[0086] Positive control: DMEM high glucose complete medium containing 10 ng / mL EGF.

[0087] Cell lines: HaCaT (human keratinocytes), HDF (human dermal fibroblasts).

[0088] Experimental method: Cells in the logarithmic growth phase were digested and counted, with 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates, with 100 μL of complete culture medium added to each well. The plates were pre-cultured at 37°C with 5% CO2 for 24 hours to ensure cell adhesion >85%. The old culture medium was discarded, and the experimental groups were replaced with fresh complete culture medium containing the drug (100 μL per well). A negative control group was also established: the medium was replaced with fresh complete culture medium (without the drug). A blank control group was added only with fresh complete culture medium (without cells). A positive control group was added with fresh complete culture medium containing 10 ng / mL EGF. Six replicates were set up for each group. Cells were returned to the incubator and cultured for another 48 hours, with 10 μL of CCK-8 solution added to each well. The plates were then returned to the incubator and incubated in the dark for 2 hours. The absorbance (OD value) of each well was measured using a microplate reader at 450 nm.

[0089] Calculate cell proliferation rate (%): Cell proliferation rate (%) = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] × 100%.

[0090] III. Antibacterial properties test:

[0091] Test solution preparation: Prepare a 100 mg / mL stock solution of the drug using sterile DMSO, filter to remove bacteria, and then dilute the stock solution with sterile physiological saline to a concentration of 5 mg / mL (ensure the final DMSO concentration is less than 1%).

[0092] Strains selected: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853). All strains were pre-cultured in appropriate media to the logarithmic growth phase, and the bacterial suspension concentration was adjusted to 1.5 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL.

[0093] Detection method: Use standard BHI agar medium (pH 7.2). Pour 20 mL of medium onto each plate and allow it to solidify at room temperature for 30 minutes. Take 100 μL of a 1.5 × 10⁻⁶ agar solution. 8 Spread a CFU / mL bacterial suspension evenly onto an agar plate. Take a sterile filter paper disc (6 mm in diameter, 1 mm thick), immerse it in 10 μL of sample solution, and let it drain at room temperature for 2 minutes; then attach the disc to the agar surface. Incubate at 37°C for 24 hours in a 5% CO2, 95% air environment. Perform three replicates for each bacterial strain.

[0094] Evaluation metric: Diameter of the inhibition zone (mm), including the diameter of the paper.

[0095] IV. Antioxidant Test (DPPH Free Radical Scavenging Method):

[0096] Test solution preparation: Prepare a 10 mg / mL drug stock solution with anhydrous ethanol, filter to remove bacteria, and then dilute with anhydrous ethanol to a 100 μg / mL test solution.

[0097] Blank control: 100 μL DPPH solution + 100 μL anhydrous ethanol (i.e., a 1:1 mixture). Experimental method: Dissolve DPPH in anhydrous ethanol to prepare a 0.1 mM DPPH ethanol solution. Add 100 μL of DPPH solution to each well of a 96-well plate, followed by 100 μL of drug solutions of different concentrations. Each group has 3 replicates. Incubate at room temperature in the dark for 30 minutes. Measure the absorbance (OD value) at 517 nm using a microplate reader.

[0098] Calculate DPPH clearance rate (%): DPPH clearance rate (%) = (1 - OD experimental group / OD blank group) × 100%.

[0099] V. Detection of inflammatory factor expression (ELISA method):

[0100] Test solution preparation: Prepare a 10 mg / mL stock solution of the drug in DMEM medium containing 10% FBS, filter to sterilize, and dilute with DMEM medium containing 10% FBS to prepare a 100 μg / mL test solution.

[0101] Control group: DMEM medium containing 10% FBS (no LPS, no drugs).

[0102] LPS group: DMEM medium containing 1 μg / mL LPS (containing 10% FBS).

[0103] Cell line: RAW264.7 macrophages.

[0104] The experimental method included: seeding cells in 24-well plates at a density of 5 × 10⁶ cells per well. 4 Cells were cultured in DMEM medium containing 10% FBS for 24 hours. The old medium was discarded, and cells were divided into groups: control group (DMEM medium containing 10% FBS, no LPS, no drug); LPS group (medium containing 1 μg / mL LPS, no drug); and drug group (medium containing 1 μg / mL LPS and the drug). Each group had 3 replicates. Cells were cultured at 37°C and 5% CO2 for 24 hours. The cells were centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected.

[0105] After following the instructions for the ELISA kit, measure the absorbance at 450 nm. Calculate the concentrations of TNF-α and IL-6 based on the standard curve.

[0106] VI. Collagen Synthesis Detection:

[0107] Test solution preparation: Prepare a 10 mg / mL stock solution of the drug in DMEM medium containing 10% FBS, filter to sterilize, and dilute with DMEM medium containing 10% FBS to prepare a test solution with a concentration of 100 μg / mL.

[0108] Negative control: DMEM medium containing 10% FBS (without drugs).

[0109] Cell line: HDF (human dermal fibroblasts).

[0110] Experimental method: Cells were seeded in 6-well plates at a density of 1 × 10⁶ cells per well. 5 Cells were cultured in DMEM medium containing 10% FBS for 24 hours. The old medium was discarded and replaced with medium containing the drug. Three replicates were set up for each group; a negative control was also included. Cells were cultured for another 48 hours. Cells were washed three times with PBS, and 1 mL of 6M HCl was added to each well. The cells were hydrolyzed at 110°C for 24 hours. Following the instructions of the hydroxyproline assay kit, the hydrolysate was neutralized and brought to a final volume, then reacted with the assay reagent, and the absorbance was measured at 560 nm. Simultaneously, the total protein content was determined using the BCA method: another portion of the cell lysate was taken, and the absorbance was measured at 562 nm according to the BCA kit. The total protein content was calculated based on the standard curve. Three replicates were set up for each group.

[0111] Calculate hydroxyproline content (μg / mg protein): Hydroxyproline content / Total protein content.

[0112] Table 1. Test Results (Average)

[0113]

[0114] The results above show that the pharmaceutical compositions of Examples 1 to 3 have the optimal component ratios, preparation and purification processes, which can comprehensively improve the various indicators of the products and make them safer and more reliable.

[0115] Comparative Example 1 (extremely low levels of taro stem extract and excessive levels of red okra seed extract): When red okra seed extract is in excess, its polyphenols act non-specifically on the erythrocyte membrane, increasing permeability. However, the membrane-stabilizing components in taro stem extract are insufficient, failing to counteract the stimulation and reducing biocompatibility. The polysaccharides and flavonoids in taro stems can promote cell adhesion and proliferation, but their low proportion directly weakens this effect. Combined with the inhibition of metabolic enzymes by red okra seed polyphenols, cell proliferation is weakened. Antibacterial activity depends on the synergistic effect of multiple components. The deficiency of taro stem reduces the number of antibacterial targets, resulting in limited antibacterial effect of red okra seed alone. The antioxidant synergy of "flavonoids-vitamin E-polyphenols" is disrupted due to the absence of taro stem, leading to a decrease in free radical scavenging efficiency. Taro stem extract weakens the inhibition of inflammatory factor secretion. Fibroblasts have a weak collagen synthesis capacity.

[0116] Comparative Example 2 (Very Low Ovalbumin Peptide, Excessively High Acetyl Tetrapeptide-9): Excessive acetyl tetrapeptide-9 alters the drug's osmotic pressure and charge, and its electrostatic interaction with erythrocyte membrane phospholipids disrupts the membrane structure, resulting in insufficient membrane stabilization of ovalbumin peptide and decreased biocompatibility; ovalbumin peptide provides essential amino acids for cell proliferation, and its deficiency leads to a lack of metabolic raw materials, while excessive acetyl tetrapeptide-9 competes with integrin receptors to inhibit adhesion, resulting in a decrease in cell proliferation rate; ovalbumin peptide contains antimicrobial peptide components, and its deficiency leads to a loss of antimicrobial activity; ovalbumin peptide contains components that scavenge free radicals, have anti-inflammatory properties, and are precursors to collagen synthesis, and its deficiency weakens the synergistic effects of antioxidation, anti-inflammation, and collagen synthesis.

[0117] Comparative Example 3 (Very low levels of ovalbumin peptide and taro stem, and excessively high levels of red okra seed and acetyl tetrapeptide-9): The synergistic effects of all factors were broken, and all indicators were worse than those of Comparative Example 1 and Comparative Example 2.

[0118] Comparative Example 4 (without trypsin hydrolysis): Trypsin hydrolyzes ovalbumin to generate low-sensitivity small peptides. Ginger protease alone cannot fully hydrolyze it, and the remaining large protein molecules are recognized as foreign substances, damaging the erythrocyte membrane and reducing biocompatibility. Specific small peptides are absorbed by cells and activate proliferation pathways, while large fragments are difficult to absorb and compete for nutrient transporters, resulting in decreased cell proliferation. The small peptides hydrolyzed by trypsin have a high cation density, making them easy to penetrate bacterial membranes, while the large fragments have a low cation density, weakening antibacterial activity. Trypsin releases antioxidant active sites, while the active sites of large fragments are encapsulated, reducing antioxidant activity and decreasing the inhibition of inflammatory factor secretion. Small peptides provide collagen synthesis precursors, while large fragments require further hydrolysis, resulting in insufficient precursor supply and reduced collagen synthesis.

[0119] Comparative Example 5 (ginger protease replaced with bromelain): Bromelain and ginger protease have different cleavage sites. Ginger protease, by hydrolyzing specific substrates of ovalbumin, produces peptides with better effects, effectively reducing sensitizing fragments, promoting cell proliferation, and enhancing antibacterial, antioxidant, and anti-inflammatory properties. Ginger protease hydrolysates peptides that activate collagen synthesis pathways, promoting collagen synthesis. Bromelain hydrolysates have different characteristics, resulting in decreased efficacy.

[0120] Comparative Example 6 (XAD16 resin replaced with AB-8 resin): The adsorption and purification of substances by AB-8 resin and XAD16 resin differed. AB-8 elution of residual ions disrupted the ion balance of erythrocyte membranes, leading to decreased biocompatibility; AB-8 had a low impurity removal rate, and residual impurities inhibited metabolic enzymes, resulting in decreased cell proliferation; AB-8 exhibited low selectivity for bioactive peptides, and residual impurities competed for bacterial binding sites, weakening antibacterial activity; the antioxidant and inflammatory factor secretion inhibitory effects of the purified product were reduced; the proportion of bioactive peptides in the AB-8 product was low, leading to decreased collagen synthesis.

[0121] Comparative Example 7 (ginger protease replaced with bromelain): Bromelain and ginger protease have different cleavage sites. Ginger protease, by hydrolyzing specific substrates from red okra seeds, produces peptides with better effects, effectively reducing sensitizing fragments, promoting cell proliferation, and enhancing antibacterial, antioxidant, and anti-inflammatory properties. The hydrolyzed peptides of ginger protease activate collagen synthesis pathways, promoting collagen synthesis. Bromelain hydrolysates showed poorer effects.

[0122] Comparative Example 8 (ginger protease replaced with papain): Papain produces hemolytic active peptides at the extraction pH, resulting in decreased biocompatibility; the different cleavage sites of papain disrupt the active peptide sequence, and the residual fragments inhibit key steps of proliferation signaling, leading to decreased cell proliferation; papain has a low extraction rate of effective active peptides, and the residual fragments compete for bacterial targets, resulting in weakened antibacterial activity; furthermore, the product's antioxidant and anti-inflammatory effects are reduced; ginger protease hydrolyzes polysaccharides, promoting collagen synthesis gene expression, while the product of papain has a poorer effect on promoting collagen synthesis.

[0123] Comparative Example 9 (AB-8 resin replaced with XAD16 resin): For okra seed extract, AB-8 resin specifically adsorbs acidic antibacterial and antioxidant components, resulting in high product efficacy. The XAD16 purified product showed relatively poor efficacy.

[0124] Comparative Example 10 (HP-20 resin replaced with AB-8 resin): The adsorption and purification substances of AB-8 resin and HP-20 resin differ. For taro stem extract, AB-8 showed poor selectivity for the active ingredient, resulting in a decrease in various indicators.

Claims

1. A biocompatible skin wound healing medicament, characterized in that, The drug comprises egg white peptide, red okra seed extract, taro stem extract, acetyl tetrapeptide-9 and vitamin E in a mass ratio of (6-8):(2-3):(1-2):(3-5):(1-1.5); the egg white peptide is encapsulated by 2.5-4 times mass of liposome, and the acetyl tetrapeptide-9 is encapsulated by 8-10 times mass of liposome; The egg white peptide is obtained by sequentially enzymolysis of goose egg white pre-cooled by trypsin and ginger protease, and purification of the enzymolysis solution by XAD16 non-ionic macroporous resin column; the red okra seed extract is obtained by ultrasonic enzymolysis of red okra seeds in an ethanol aqueous solution by hemicellulase and ginger protease, and purification of the enzymolysis solution by AB-8 macroporous adsorption resin column; and the taro stem extract is obtained by ultrasonic extraction of taro stem slurry in an ethanol aqueous solution, and purification of the extraction solution by HP-20 macroporous adsorption resin column.

2. A biocompatible skin wound healing drug as claimed in claim 1, wherein, The preparation of the egg white peptide comprises: taking goose egg white, adding pH 8.0-8.5 Tris-HCl buffer solution, stirring and homogenizing to obtain egg white crude solution, pre-cooling, adding trypsin, and enzymolysis at 36-39 DEG C for 1.5-2 h, adjusting pH to 6.5-7.0, adding ginger protease, and enzymolysis at 52-58 DEG C for 1-1.5 h, centrifuging, taking supernatant, inactivating enzyme, and loading into XAD16 non-ionic macroporous resin column, first washing and removing impurities by 1.5-2 BV of deionized water, and then eluting by 1.5-2 BV of 10-15% volume concentration ethanol aqueous solution and 1.5-2 BV of 20-30% volume concentration ethanol aqueous solution in sequence, collecting and freeze-drying the eluate to obtain egg white peptide.

3. A biocompatible skin wound healing medicament as claimed in claim 2, wherein, The Tris-HCl buffer solution is used in an amount of 4-5 times the volume of egg white; the pre-cooling is pre-cooling at 3-5 DEG C for 1-2 h; the trypsin is added in an amount of 0.5-1.0% of the mass of egg white; the ginger protease is added in an amount of 0.5-1.0% of the mass of egg white; the centrifuging is centrifuging at 8000-8500 rpm for 10-15 min; the enzyme inactivation is inactivation at 80-85 DEG C for 10-15 min; after the enzyme inactivation, the eluate is concentrated at 45-50 DEG C under reduced pressure to 25-35% of the volume; and the eluate is concentrated under reduced pressure at 45-50 DEG C to remove ethanol.

4. A biocompatible skin wound healing medicament as claimed in claim 1 or 2, wherein, The method for encapsulating the egg white peptide by liposome comprises: dissolving soybean lecithin and cholesterol in anhydrous ethanol according to a mass ratio of (3.5-4.5):1, rotary evaporation to form a film, and vacuum drying to obtain liposome; preparing a 5-8 wt% peptide solution by using pH 7.0-7.4 phosphate buffer solution to dissolve the egg white peptide; adding the peptide solution into the liposome according to a mass ratio of egg white peptide:liposome=1:(2.5-4), water bath oscillation to obtain a primary emulsion; ultrasonic treatment in an ice bath to obtain a suspension; filtering through a microporous filter membrane, taking the permeate, and freeze-drying to obtain the egg white peptide encapsulated by liposome.

5. The biocompatible skin wound healing drug as claimed in claim 1, wherein, The preparation of the red eggplant seed extract includes: crushing red eggplant seeds into seed powder, adding 6-8 times the mass of the seed powder of 20-30% volume concentration ethanol aqueous solution, adjusting pH to 5.0-5.5, adding hemicellulase and ginger protease, ultrasonic enzymolysis, enzyme inactivation, centrifugation, taking supernatant, loading to AB-8 macroporous adsorption resin column, first eluting impurities with 2BV-2.5BV deionized water and 1.5BV-2BV 10%-15% volume concentration ethanol aqueous solution in sequence, eluting with 70%-75% volume concentration ethanol aqueous solution for 3BV-4BV, collecting eluate, freeze-drying to obtain red eggplant seed extract.

6. A biocompatible skin wound healing medicament as claimed in claim 5, wherein, The particle size of the seed powder is 80-100 mesh; the hemicellulase is added in an amount of 0.5%-1% of the mass of the seed powder; the ginger protease is added in an amount of 1%-2% of the mass of the seed powder; the ultrasonic enzymolysis is carried out at 50-55°C, 200-250W for 1.5-2h; the enzyme inactivation is carried out by heating to 80-85°C for 15-20min; after the enzyme inactivation, the ethanol is removed by concentration under reduced pressure at 45-50°C to 25%-35% of the volume; the centrifugation is carried out at 8000-8500rpm for 10-15min; the eluate is concentrated under reduced pressure at 45-50°C to remove ethanol.

7. The biocompatible skin wound healing drug as claimed in claim 1, wherein, The preparation of the taro stem extract includes: grinding fresh taro stems into pulp, ultrasonic extraction of the pulp with 5-6 times the mass of 60%-65% volume concentration ethanol aqueous solution, centrifugation, taking supernatant, loading to HP-20 macroporous adsorption resin column, eluting impurities with 2BV-3BV 5%-10% volume concentration ethanol aqueous solution, further eluting with 70%-75% volume concentration ethanol aqueous solution for 3-5BV, collecting eluate, freeze-drying to obtain taro stem extract.

8. A biocompatible skin wound healing medicament as claimed in claim 7, wherein, The ultrasonic extraction is carried out at room temperature, 400-500W for 40-60min; the centrifugation is carried out at 6000-8000rpm for 15-20min; the supernatant is concentrated under reduced pressure at 45-50°C to 25%-35% of the volume to remove ethanol; the eluate is concentrated under reduced pressure at 45-50°C to remove ethanol.

9. The biocompatible skin wound healing drug as claimed in claim 1, wherein, The method for liposome encapsulation of acetyl tetrapeptide-9 includes: dissolving soybean lecithin and cholesterol in anhydrous ethanol according to a mass ratio of (3.5-4.5):1, rotary evaporation to form a film, vacuum drying to obtain liposomes; preparing a 4wt%-6wt% peptide solution of acetyl tetrapeptide-9 with pH 6.4-6.8 phosphate buffer; adding the peptide solution into the liposomes according to a mass ratio of acetyl tetrapeptide-9:liposomes=1:(6-8), water bath oscillation to obtain a primary emulsion; ultrasonic treatment in an ice bath to obtain a suspension; filtering with a microporous membrane, taking the permeate, freeze-drying to obtain liposome-encapsulated acetyl tetrapeptide-9.

10. The process for the preparation of a biocompatible skin wound healing drug as claimed in claim 1, wherein, The method comprises the following steps: According to a mass ratio of a formula, egg white peptide, red okra seed extract, taro stem extract, acetyl tetrapeptide-9 and vitamin E are mixed to obtain a pharmaceutical composition; the pharmaceutical composition is prepared into a gel or a spray with a pharmaceutically acceptable excipient.

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