Medicine for skin regeneration and repair and preparation method thereof

Abstract

The invention relates to a medicine for skin regeneration and repair and a preparation method thereof, and belongs to the technical field of biological medicine, the medicine comprises cyperus esculentus extract peptide, brooklet anemone root tremella extract, rhodomyrtus tomentosa root-suberect spatholobus stem-radix cyathulae composite extract, pomegranate peel polyphenol and the like. The cyperus esculentus extracted peptide is a product which is more than 1kDa and is obtained by performing enzymolysis on cyperus esculentus through cellulase, alpha-chymotrypsin and ficin protease in sequence and then performing purification through a G-50 sephadex column; the tremellodon gelatinosum tremella extract is a product which is less than 3kDa and is obtained by performing common enzymolysis on tremellodon gelatinosum tremella sporocarp through cellulase and bromelain; the rhodomyrtus tomentosa root-suberect spatholobus stem-radix cyathulae composite extract is a product obtained by performing gradient extraction on rhodomyrtus tomentosa root, suberect spatholobus stem and radix cyathulae through an ethanol water solution to obtain an extract, and performing polyamide resin column adsorption and LH-60 sephadex column purification in sequence. A good repairing system is constructed by multiple components, and wound repairing and healing are accelerated.

Description

Technical Field

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

[0002] Skin is one of the most vulnerable organs. From minor everyday abrasions and burns to deep burns, surgical wounds, and chronic, difficult-to-heal wounds such as diabetic foot ulcers and pressure sores, all can damage the integrity of the skin. Healthy skin possesses a highly efficient self-regenerative capacity, and its repair process exhibits a precise temporal sequence: When the skin is damaged, platelet aggregation first initiates a hemostatic response, quickly closing the wound; subsequently, neutrophils and macrophages migrate to the damaged site, clearing away necrotic tissue and invading pathogens, paving the way for subsequent repair; then, fibroblasts proliferate in large numbers and synthesize collagen, building a granulation tissue scaffold, while endothelial cells form new blood vessels to deliver nutrients to the wound; finally, epithelial cells migrate from the wound edge to the center to cover the wound, and collagen is gradually remodeled, completing the restoration of skin structure and function. The entire process typically takes 2-4 weeks in healthy individuals. However, when the damage exceeds the skin's own repair threshold, or when the patient has underlying diseases or low immunity, this repair chain will break down, the inflammatory response will persist, fibroblast activity will be insufficient, and angiogenesis will be hindered, ultimately leading to long-term non-healing of the wound and the formation of chronic lesions.

[0003] Early interventions for skin injuries primarily focused on passive support: using dressings such as petroleum jelly gauze and ointments to isolate external stimuli and prevent secondary damage to the wound; for infected wounds, topical antibiotics were used to control infection. While these methods provided a basic environment for wound healing, they could not actively promote the regeneration process and had little effect on chronic wounds. With the advancement of cell biology research, drug development entered a stage of active regulation. Biological agents such as recombinant human epidermal growth factor and fibroblast growth factor emerged, significantly shortening the healing time of small and medium-sized wounds by directly activating the proliferation and migration signals of local cells in the wound, and have become commonly used drugs in clinical practice. In recent years, the integration of materials science and regenerative medicine has further promoted drug upgrades. Gels or patches using hyaluronic acid and collagen as carriers can achieve slow release of growth factors, prolonging the duration of action; while novel ingredients such as stem cell exosomes and bioactive peptides can improve the repair microenvironment from multiple dimensions, such as regulating inflammation and promoting angiogenesis, providing new directions for the treatment of chronic and difficult-to-heal wounds.

[0004] Despite this, existing drugs still face numerous technical challenges: growth factor drugs have poor stability and are easily degraded by temperature and enzymatic degradation; stem cell-related preparations are subject to immune rejection and ethical controversies; and their repair effects are slow for deep skin damage. Therefore, there is a need to continuously develop safer, more stable, more efficient drug ingredients that are closer to the skin's physiological regeneration mechanisms to meet the needs of skin repair. Summary of the Invention

[0005] To address the need for continuous improvement in the safety and efficacy of existing skin regeneration and repair drugs, this invention provides a skin regeneration and repair drug and its preparation method. It prepares effective active ingredients such as tiger pea extract peptides, tiger paw thorn tremella extract, and a compound extract of *Rhizoma Cynodon dactylis* root, *Spatholobus suberectus*, and *Radix Achyranthis Bidentatae*. By precisely matching the physiological repair mechanisms of skin hemostasis, anti-inflammation, proliferation, vascularization, and collagen remodeling, a multi-component synergistic and comprehensive repair system is constructed. This effectively solves the problems of persistent inflammation, insufficient cell activity, and inhibited angiogenesis in the repair of chronic, difficult-to-heal wounds caused by existing drugs, ensuring safety and high efficacy. The specific technical solution is as follows:

[0006] A skin regeneration and repair drug, comprising the following raw materials in parts by weight: 6-8 parts tiger pea extract peptide, 4.5-5.5 parts tiger paw thorn tremella extract, 8-10 parts purslane root-chicken blood vine-Achyranthes bidentata compound extract, 1-3 parts pomegranate peel polyphenols, and 80-90 parts modified hyaluronic acid-chitosan hydrogel, with the pH adjusted to 6.8-7.2; The tiger nut extract peptide is a product with a value of over 1 kDa obtained by sequentially hydrolyzing tiger nut with cellulase, α-chymotrypsin, and fig protease, followed by purification using a G-50 dextran gel column. The tiger paw thorn tremella extract is a product with a value of less than 3 kDa obtained by co-hydrolyzing the fruiting body of tiger paw thorn tremella with cellulase and bromelain. The Rhododendron molle root-chrysanthemum vine-Achyranthes bidentata compound extract is obtained by gradient extraction with ethanol-water solution at a mass ratio of Rhododendron molle root:chrysanthemum vine:Achyranthes bidentata = (3-4):(1-2):(2-3), followed by sequential adsorption using a polyamide resin column and purification using an LH-60 dextran gel column. The modified hyaluronic acid-chitosan hydrogel is a biocompatible hydrogel formed by cross-linking carboxymethylated hyaluronic acid and chitosan with glutaraldehyde.

[0007] The preparation method of the above-mentioned tiger nut extract peptides includes: tiger nut meal being crushed into meal powder, dispersed in an acetate-sodium acetate buffer solution at pH 4.5–5.5, adding cellulase, and hydrolyzing at 50℃–55℃ for 2–3 hours; adjusting the pH to 8.0–8.8, adding α-chymotrypsin, and hydrolyzing at 35℃–40℃ for 1.5–2 hours; inactivating the enzyme, cooling, adjusting the pH to 6.0–6.5, adding fig protease, and hydrolyzing at 55℃–60℃ for 1–1.5 hours; inactivating the enzyme, centrifuging, collecting the supernatant, filtering, taking the filtrate, and freeze-drying to obtain the crude extract; resuspending the crude extract in a phosphate buffer solution at pH 7.5–8.0, loading it onto a G-50 dextran gel column, washing with a phosphate buffer solution at pH 7.5–8.0 for 2 BV–3 BV, and then washing with a phosphate buffer solution at pH 7.5–8.0 containing 0.02M–0.04M... Elute with phosphate buffer containing NH4HCO3 for 3-4 BV, collect the eluent, place it in a dialysis bag, dialyze with deionized water, and freeze-dry to obtain tiger nut extract peptides.

[0008] In the above method for preparing tiger nut extract peptides, the particle size of the meal powder is sieved through a 60-80 mesh sieve; the amount of the acetate-sodium acetate buffer solution is 10-12 times the mass of the meal powder; the amount of cellulase added is 0.8%-1.5% of the mass of the meal powder; the amount of α-chymotrypsin added is 0.8%-1.5% of the mass of the meal powder; the amount of fig protease added is 0.5%-1.0% of the mass of the meal powder; the enzyme inactivation is performed at 80-85℃ for 10-15 minutes; the centrifugation is performed at 6000-8000 r / min for 10-15 minutes; the filtration is performed using a 0.45 μm pore size filter membrane; and the molecular weight cutoff of the dialysis bag is 1 kDa.

[0009] The preparation method of the above-mentioned Tiger Paw Thorn Tremella extract includes: pulverizing Tiger Paw Thorn Tremella into powder, dispersing it in deionized water, and adjusting the pH to 4.5-5.0; adding cellulase and hydrolyzing it at 50℃-55℃ for 1-2 hours, adjusting the pH to 6.5-7.0, adding bromelain and hydrolyzing it at 50℃-55℃ for 2-3 hours; inactivating the enzyme, centrifuging, collecting the supernatant, ultrafiltration membrane ultrafiltration, collecting the fraction below 3kDa, and freeze-drying to obtain Tiger Paw Thorn Tremella extract.

[0010] In the above-mentioned method for preparing the extract of Tremella fuciformis, the Tremella fuciformis is a dried fruiting body; the particle size of the powder is sieved through a 120-150 mesh sieve; the amount of phosphate buffer is 10-12 times the mass of the powder; the amount of cellulase added is 0.8%-1.5% of the mass of the powder; the amount of bromelain added is 0.8%-1.5% of the mass of the powder; the enzyme inactivation is performed at 85-90℃ for 10-15 minutes; and the centrifugation is performed at 6000-8000 r / min for 10-15 minutes.

[0011] The preparation method of the above-mentioned compound extract of *Rhizoma Rhizoma Barkti* root, *Spatholobus suberectus*, and *Radix Achyranthis Bidentatae* includes: pulverizing the compound powder according to the mass ratio of *Rhizoma Rhizoma Barkti* root: *Spatholobus suberectus*: *Radix Achyranthis Bidentatae* = (3-4):(1-2):(2-3), adding it to a 70 vol%-75 vol% ethanol aqueous solution, ultrasonically extracting, centrifuging to obtain extract A and precipitated residue A; adding precipitated residue A to a 75 vol%-80 vol% ethanol aqueous solution, reflux extracting, centrifuging to obtain extract B; combining extract A and extract B, concentrating under reduced pressure to obtain a solid, dispersing with deionized water, adjusting the pH to 5.0-6.0, loading the sample onto a polyamide resin column, rinsing with deionized water for 2-3 BV to remove impurities, rinsing with a 30 vol%-40 vol% ethanol aqueous solution containing 0.1 M-0.15 M NH4OH for 1.5 BV-2 BV to remove impurities, and rinsing with a 0.2 M-0.3 M NH4OH solution... Elute 3-4 BV of NH4OH with 80-85 vol% ethanol aqueous solution, collect the eluent, concentrate under reduced pressure to obtain a solid, disperse with 60-70 vol% ethanol aqueous solution, load the sample onto an LH-60 dextran gel column, elute 3-4 BV with 80-90 vol% ethanol aqueous solution, collect the eluent, concentrate under reduced pressure, and vacuum dry to obtain a compound extract of *Rhizoma Melilotifolia* root, *Spatholobus suberectus*, and *Achyranthes bidentata*.

[0012] In the preparation method of the above-mentioned compound extract of *Rhizoma Cynanchi* root-*Spatholobus suberectus*-*Radix Achyranthis Bidentatae*, the particle size of the compound powder is sieved through a 60-80 mesh sieve; the amount of the 70-75 vol% ethanol aqueous solution is 10-12 times the mass of the compound powder; the ultrasonic extraction is performed at 45-50℃, 300-350W, and 30-40kHz for 1-2 hours; the amount of the 75-80 vol% ethanol aqueous solution is 10-12 times the mass of the precipitated residue A; the reflux extraction is performed at 75-80℃ for 1-2 hours; the centrifugation is performed at 6000-8000 r / min for 10-15 minutes; and the polyamide resin column is packed with 80-120 mesh polyamide resin.

[0013] The preparation method of the above-mentioned modified hyaluronic acid-chitosan hydrogel includes: preparing a hyaluronic acid solution with deionized water, adjusting the pH to 10.0-11.0, adding chloroacetic acid, stirring and reacting, dialyzing with a dialysis bag, and freeze-drying to obtain carboxymethyl hyaluronic acid; dissolving carboxymethyl hyaluronic acid in an acetate buffer solution at a mass ratio of carboxymethyl hyaluronic acid: chitosan = 1:(1-1.2) to a total concentration of 3wt%-5wt%, stirring until completely dissolved; adding glutaraldehyde, performing a cross-linking reaction to form a hydrogel, dialyzing with a dialysis bag, and adjusting the viscosity to 2000 mPa·s-3000 mPa·s to obtain the modified hyaluronic acid-chitosan hydrogel.

[0014] In the above-mentioned method for preparing modified hyaluronic acid-chitosan hydrogel, the concentration of the hyaluronic acid solution is 2wt% to 3wt%; the mass ratio of hyaluronic acid to chloroacetic acid is 1:(0.03 to 0.05); the stirring reaction is carried out at 60℃ to 65℃ and 200rpm to 300rpm for 4h to 5h; the amount of glutaraldehyde added is 0.1wt% to 0.15wt% at a final concentration; the crosslinking reaction is carried out at 25℃ to 30℃ and 200rpm to 300rpm for 2h to 3h; and the molecular weight cutoff of the dialysis bags is 10kDa.

[0015] The preparation method of the above-mentioned skin regeneration and repair drug includes the following steps: According to the mass fractions, tiger pea extract peptide, tiger paw thorn tremella extract, jujube root-chicken blood vine-achyranthes root compound extract and pomegranate peel polyphenol are mixed and added to modified hyaluronic acid-chitosan hydrogel, stirred evenly, and the pH is adjusted to 6.8-7.2 to obtain a pharmaceutical composition; the pharmaceutical composition is used to prepare a drug for skin wound repair.

[0016] This invention provides a skin regeneration and repair drug and its preparation method, the beneficial effects of which include: I. This medication precisely matches the physiological repair mechanisms of skin hemostasis, anti-inflammation, proliferation, vascularization, and collagen remodeling, constructing a multi-component synergistic and comprehensive repair system. This effectively addresses the problems of persistent inflammation, insufficient cell activity, and hindered angiogenesis encountered by existing medications in the repair of chronic, difficult-to-heal wounds. Its core advantage lies in its multi-dimensional effects, from regulating the repair microenvironment to activating functional cells. It provides a biocompatible repair carrier for the wound and actively regulates key repair processes, avoiding the breakage of the repair chain due to the loss of a single function, ultimately achieving efficient and safe skin regeneration and repair.

[0017] II. Tiger Pea Extract Peptides: Through stepwise enzymatic hydrolysis with cellulase, α-chymotrypsin, and fig protease, followed by G-50 dextran gel purification, highly active small molecule peptides can be precisely enriched. These peptides can directly activate fibroblast proliferation signals and promote collagen synthesis, providing core impetus for granulation tissue construction and skin structure remodeling. G-50 dextran gel purification can precisely separate the target active ingredients from impurities, improving ingredient purity and avoiding the inhibition of cell activity and interference with drug efficacy caused by impurities.

[0018] 3. Tiger Paw Thorn Tremella Extract: Through complex enzymatic hydrolysis with cellulase and bromelain and 3kDa ultrafiltration, low molecular weight active polysaccharides and peptides are enriched. This can not only provide a moisturizing barrier for the wound, reducing water loss and external irritation, but also help activate fibroblast activity, enhance anti-inflammatory effects, and create a stable microenvironment for the repair process.

[0019] IV. *Rhizoma Cynanchi Root*-*Spatholobus Suberectus*-*Radix Achyranthis Bidentatae* Compound Extract: This extract, obtained through gradient ethanol extraction and dual-column purification with polyamide and LH-60, precisely preserves the effective components of *Rhizoma Cynanchi Root*, *Spatholobus Suberectus*, and *Radix Achyranthis Bidentatae*. It addresses the problems of persistent inflammation, insufficient nutrition, and slow healing in chronic wounds by inhibiting the expression of pro-inflammatory factors, improving local blood supply, promoting vascular endothelial cell migration, and synergistically promoting cell proliferation and protein synthesis. The polyamide-LH-60 dual-column purification effectively removes harmful impurities, improves safety, and avoids the inhibition of cell activity and interference with drug efficacy caused by impurities.

[0020] V. Pomegranate peel polyphenols: As a natural antibacterial component, they inhibit wound infection bacteria, reduce the interference of infection on the repair process, and their antioxidant properties reduce oxidative stress damage to the wound, protecting the activity of functional cells. Modified hyaluronic acid-chitosan hydrogel: Prepared by carboxymethylating hyaluronic acid and cross-linking chitosan with glutaraldehyde, it possesses both good biocompatibility and viscosity stability. It can serve as a drug carrier to achieve slow release of active ingredients, prolonging the duration of action, and can also adhere to the wound surface to form a physical barrier, isolating external contamination, while providing a scaffold for cell migration.

[0021] VI. The strong anti-inflammatory effect of the root of *Rhizoma Cynanchi* clears inflammatory obstacles for the migration of vascular endothelial cells, *Spatholobus suberectus* improves microcirculation and provides a nutritional basis for angiogenesis, *Radix Achyranthis Bidentatae* further promotes the migration of vascular endothelial cells and the formation of new blood vessels, and the auxiliary anti-inflammatory effect of *Fructus Tremellae* extract enhances this synergistic effect, forming a dual guarantee of strong anti-inflammatory and angiogenesis promotion.

[0022] VII. Tiger pea extract peptides activate fibroblast proliferation and collagen synthesis, while tiger paw thorn tremella extract helps enhance cell activity. Modified hyaluronic acid-chitosan hydrogel not only provides a slow-release carrier for these two components, prolonging their action time, but also provides a physical scaffold for the migration and proliferation of activated cells.

[0023] In summary, this drug achieves excellent synergistic effects through the functional complementarity between its components and the precise control of the step parameters, breaking through the limitations of existing drugs with single functions and providing a solution for skin regeneration and repair that is more in line with physiological mechanisms. Detailed Implementation

[0024] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0025] Example 1 A skin regeneration and repair drug comprising the following raw materials in parts by weight: 7 parts tiger pea extract peptide, 5 parts tiger paw thorn tremella extract, 9 parts purslane root-chicken blood vine-Achyranthes bidentata compound extract, 2 parts pomegranate peel polyphenol, 85 parts modified hyaluronic acid-chitosan hydrogel, with pH adjusted to 7.0.

[0026] The preparation method of tiger nut extract peptides includes: taking tiger nut cake after oil pressing, crushing it into meal powder that passes through a 60-mesh sieve, dispersing it in a pH 5.0 acetate-sodium acetate buffer (0.1M) at a material-to-liquid mass ratio of 1:11, adding 1.2% (by weight of the meal powder) of cellulase, and enzymatically hydrolyzing at 52℃ for 2.5h; adjusting the pH to 8.5, adding 1.2% (by weight of the meal powder) of α-chymotrypsin, and enzymatically hydrolyzing at 38℃ for 1.5h; inactivating the enzyme at 82℃ for 12min, cooling, adjusting the pH to 6.3, and adding 0.8% (by weight of the meal powder) of... Fig protease was hydrolyzed at 58℃ for 1.5 h; the enzyme was inactivated at 82℃ for 12 min, centrifuged at 7000 r / min for 12 min, and the supernatant was collected. The supernatant was filtered through a 0.45 μm pore size filter membrane, and the filtrate was freeze-dried to obtain a crude extract. The crude extract was resuspended in pH 7.8 phosphate buffer (0.05 M) at a material-to-liquid mass ratio of 1:2.5 and loaded onto a G-50 dextran gel column. The column was washed with pH 7.8 phosphate buffer (0.05 M) for 2.5 BV, followed by elution with pH 7.8 phosphate buffer containing 0.03 M NH4HCO3 (0.05 M) for 3.5 BV. The eluent was collected, placed in a dialysis bag with a molecular weight cutoff of 1 kDa, and dialyzed with deionized water for 50 h, with the deionized water changed every 7 h. The extract was then freeze-dried to obtain tiger nut extract peptides.

[0027] The preparation method of the tiger paw thorn tremella extract includes: pulverizing the dried tiger paw thorn tremella fruiting body into powder that passes through a 120-mesh sieve, dispersing it in deionized water at a material-to-liquid mass ratio of 1:11, adjusting the pH to 4.8, adding 1.2% (by weight) of cellulase from the powder, enzymatically hydrolyzing at 52℃ for 1.5 h, adjusting the pH to 6.8, adding 1.2% (by weight) of bromelain from the powder, enzymatically hydrolyzing at 53℃ for 2.5 h, inactivating the enzyme at 88℃ for 12 min, centrifuging at 7000 r / min for 12 min, collecting the supernatant, ultrafiltration through a 3 kDa ultrafiltration membrane, collecting the fraction below 3 kDa, and freeze-drying to obtain the tiger paw thorn tremella extract.

[0028] The preparation method of the compound extract of *Rhizoma Rhizoma Barkti* root, *Spatholobus suberectus*, and *Radix Achyranthis Bidentatae* includes: pulverizing the compound powder into a sieve passing through an 80-mesh sieve according to a mass ratio of *Rhizoma Rhizoma Barkti*: *Spatholobus suberectus*: *Radix Achyranthis Bidentatae* = 3.5:1.5:2.5; adding the compound powder to a 72 vol% ethanol aqueous solution at a mass ratio of 1:11; ultrasonically extracting at 48℃, 300W, and 30kHz for 1.5 h; centrifuging at 7000 r / min for 12 min to obtain extract A and precipitated residue A; the precipitate... Residue A was added to a 78 vol% ethanol aqueous solution at a material-to-liquid mass ratio of 1:11, and extracted by reflux at 78℃ for 1.5 h. The extract was then centrifuged at 7000 r / min for 12 min to obtain extract B. Extracts A and B were combined and concentrated under reduced pressure at 53℃ to obtain a solid. The solid was dispersed in 2.8 times its mass of deionized water, and the pH was adjusted to 5.5. The solid was loaded onto an 80-120 mesh polyamide resin column, washed with 2.5 BV of deionized water to remove impurities, and then rinsed with a solution containing 0.12 M... The sample was washed with 35 vol% ethanol aqueous solution of NH4OH for 1.5 BV to remove impurities, and then eluted with 82 vol% ethanol aqueous solution containing 0.25 M NH4OH for 3.5 BV. The eluent was collected and concentrated under reduced pressure at 53 °C to obtain a solid. The solid was dispersed with 2.8 times its mass of 65 vol% ethanol aqueous solution and loaded onto an LH-60 dextran gel column. The solid was then eluted with 85 vol% ethanol aqueous solution for 3.5 BV. The eluent was collected and concentrated under reduced pressure at 53 °C and dried under vacuum at 48 °C to obtain a compound extract of *Rhizoma Melilotifolia* root, *Spatholobus suberectus*, and *Achyranthes bidentata*.

[0029] The preparation method of the modified hyaluronic acid-chitosan hydrogel includes: preparing a 2.5wt% hyaluronic acid solution with deionized water, and using 0.9M... Adjust the pH to 10.5 with NaOH aqueous solution. Add chloroacetic acid at a mass ratio of 1:0.04 (hyaluronic acid to chloroacetic acid). Stir at 63℃ and 250rpm for 4.5h. Transfer the mixture to a dialysis bag with a molecular weight cutoff of 10kDa and dialyze with deionized water for 54h, changing the deionized water every 7h. Freeze-dry to obtain carboxymethyl hyaluronic acid. Dissolve carboxymethyl hyaluronic acid in 0.1M acetate buffer (pH 5.5) at a mass ratio of 1:1.1 to chitosan (90% degree of deacetylation) to a total concentration of 4wt%. Stir until completely dissolved. Add 0.12wt% glutaraldehyde and crosslink at 28℃ and 250rpm for 2.5h to form a hydrogel. Transfer the hydrogel to a dialysis bag with a molecular weight cutoff of 10kDa and dialyze with deionized water for 76h, changing the deionized water every 7h. Adjust the viscosity to 2500mPa·s to obtain modified hyaluronic acid-chitosan hydrogel.

[0030] The preparation method of the above-mentioned skin regeneration and repair drug includes the following steps: According to the mass fractions, tiger pea extract peptides, tiger paw thorn tremella extract, jujube root-chicken blood vine-achyranthes root compound extract and pomegranate peel polyphenols were mixed and added to modified hyaluronic acid-chitosan hydrogel (pre-mixed and dispersed with 15% hydrogel first, then added to the remaining hydrogel), stirred evenly, and the pH was adjusted to 7.0 to obtain the pharmaceutical composition; the pharmaceutical composition is used to prepare a drug for skin wound repair.

[0031] Example 2 A skin regeneration and repair drug comprising the following raw materials in parts by weight: 6 parts tiger pea extract peptide, 5.5 parts tiger paw thorn tremella extract, 8 parts jujube root-chicken blood vine-Achyranthes bidentata compound extract, 3 parts pomegranate peel polyphenol, 80 parts modified hyaluronic acid-chitosan hydrogel, with pH adjusted to 7.2.

[0032] The preparation method of tiger nut extract peptides includes: crushing tiger nut cake after oil pressing into meal powder that passes through a 60-mesh sieve, dispersing it in a pH 4.5 acetate-sodium acetate buffer (0.1M) at a material-to-liquid mass ratio of 1:12, adding 1.5% (by weight of the meal powder) of cellulase, and enzymatically hydrolyzing at 50℃ for 3 hours; adjusting the pH to 8.0, adding 1.5% (by weight of the meal powder) of α-chymotrypsin, and enzymatically hydrolyzing at 35℃ for 2 hours; inactivating the enzyme at 80℃ for 15 minutes, cooling, adjusting the pH to 6.0, and adding 1.0% (by weight of the meal powder) of... Fig protease was hydrolyzed at 55℃ for 1.5 h; the enzyme was inactivated at 80℃ for 15 min, centrifuged at 6000 r / min for 15 min, and the supernatant was collected. The supernatant was filtered through a 0.45 μm pore size filter membrane, and the filtrate was freeze-dried to obtain the crude extract. The crude extract was resuspended in pH 8.0 phosphate buffer (0.05 M) at a material-to-liquid mass ratio of 1:2 and loaded onto a G-50 dextran gel column. The column was washed with pH 8.0 phosphate buffer (0.05 M) for 3 BV, followed by elution with pH 8.0 phosphate buffer containing 0.02 M NH4HCO3 (0.05 M) for 4 BV. The eluent was collected and placed in a dialysis bag with a molecular weight cutoff of 1 kDa. The column was dialyzed with deionized water for 48 h, with the deionized water changed every 8 h. The column was then freeze-dried to obtain tiger nut extract peptides.

[0033] The preparation method of the tiger paw thorn tremella extract includes: pulverizing the dried tiger paw thorn tremella fruiting body into powder that passes through a 120-mesh sieve, dispersing it in deionized water at a material-to-liquid mass ratio of 1:12, adjusting the pH to 4.5, adding 1.5% (by weight) of cellulase from the powder, enzymatically hydrolyzing at 50℃ for 2 hours, adjusting the pH to 6.5, adding 1.5% (by weight) of bromelain from the powder, enzymatically hydrolyzing at 50℃ for 3 hours, inactivating the enzyme at 85℃ for 15 minutes, centrifuging at 6000 r / min for 15 minutes, collecting the supernatant, ultrafiltration through a 3 kDa ultrafiltration membrane, collecting the fraction below 3 kDa, and freeze-drying to obtain the tiger paw thorn tremella extract.

[0034] The preparation method of the compound extract of *Rhizoma Rhizoma Barkti* root, *Spatholobus suberectus*, and *Radix Achyranthis Bidentatae* includes: *Rhizoma Rhizoma Barkti* root: *Spatholobus suberectus*: *Radix Achyranthis Bidentatae* are pulverized into a compound powder that passes through an 80-mesh sieve at a mass ratio of 3:2:2; the compound powder is added to a 75 vol% ethanol aqueous solution at a mass ratio of 1:10, and ultrasonically extracted at 45℃, 350W, and 30kHz for 2 hours, followed by centrifugation at 6000 r / min for 15 minutes to obtain extract A and precipitated residue A; precipitated residue A is added to an 80 vol% ethanol aqueous solution at a mass ratio of 1:10, and refluxed at 75℃ for 2 hours, followed by centrifugation at 6000 r / min for 15 minutes to obtain extract B; extract A and extract B are combined, concentrated under reduced pressure at 50℃ to obtain a solid, dispersed with 3 times its mass of deionized water, adjusted to pH 5.0, loaded onto an 80-120 mesh polyamide resin column, and washed with deionized water for 3 BV to remove impurities, followed by washing with a solution containing 0.1 M... The sample was washed with 40 vol% ethanol aqueous solution of NH4OH for 1.5 BV to remove impurities, and then eluted with 80 vol% ethanol aqueous solution containing 0.3 M NH4OH for 4 BV. The eluent was collected and concentrated under reduced pressure at 50 °C to obtain a solid. The solid was dispersed with 3 times its mass of 60 vol% ethanol aqueous solution and loaded onto an LH-60 dextran gel column. The solid was eluted with 90 vol% ethanol aqueous solution for 3 BV. The eluent was collected and concentrated under reduced pressure at 55 °C and dried under vacuum at 50 °C to obtain the compound extract of *Rhizoma Melilotifolia* root, *Spatholobus suberectus*, and *Radix Achyranthis Bidentatae*.

[0035] The preparation method of the modified hyaluronic acid-chitosan hydrogel includes: preparing a 2wt% hyaluronic acid solution with deionized water, and using 1M... Adjust the pH to 10.0 with NaOH aqueous solution. Add chloroacetic acid at a mass ratio of 1:0.05 (hyaluronic acid to chloroacetic acid). Stir at 60℃ and 300rpm for 4 hours. Transfer the mixture to a dialysis bag with a molecular weight cutoff of 10kDa. Dialyze with deionized water for 60 hours, changing the deionized water every 6 hours. Freeze-dry to obtain carboxymethyl hyaluronic acid. Dissolve carboxymethyl hyaluronic acid in 0.1M acetate buffer (pH 5.5) at a mass ratio of 1:1.2 to a total concentration of 3wt%. Stir until completely dissolved. Add 0.15wt% glutaraldehyde. Crosslink the mixture at 25℃ and 300rpm for 2 hours to form a hydrogel. Transfer the hydrogel to a dialysis bag with a molecular weight cutoff of 10kDa. Dialyze with deionized water for 80 hours, changing the deionized water every 6 hours. Adjust the viscosity to 3000mPa·s to obtain modified hyaluronic acid-chitosan hydrogel.

[0036] The preparation method of the above-mentioned skin regeneration and repair drug includes the following steps: According to the mass fractions, tiger pea extract peptides, tiger paw thorn tremella extract, jujube root-chicken blood vine-achyranthes root compound extract and pomegranate peel polyphenols were mixed and added to modified hyaluronic acid-chitosan hydrogel (pre-mixed and dispersed with 10% hydrogel first, then added to the remaining hydrogel), stirred evenly, and the pH was adjusted to 7.2 to obtain the drug composition; the drug composition is used to prepare a drug for skin wound repair.

[0037] Example 3 A skin regeneration and repair drug comprising the following raw materials in parts by weight: 8 parts tiger pea extract peptide, 4.5 parts tiger paw thorn tremella extract, 10 parts purslane root-chicken blood vine-Achyranthes bidentata compound extract, 1 part pomegranate peel polyphenol, 90 parts modified hyaluronic acid-chitosan hydrogel, with pH adjusted to 6.8.

[0038] The preparation method of tiger nut extract peptides includes: crushing tiger nut cake after oil pressing into meal powder that passes through an 80-mesh sieve, dispersing it in a pH 5.5 acetate-sodium acetate buffer (0.1M) at a material-to-liquid mass ratio of 1:10, adding 0.8% (by weight of the meal powder) of cellulase, and enzymatically hydrolyzing at 55℃ for 2 hours; adjusting the pH to 8.8, adding 0.8% (by weight of the meal powder) of α-chymotrypsin, and enzymatically hydrolyzing at 40℃ for 1.5 hours; inactivating the enzyme at 85℃ for 10 minutes, cooling, adjusting the pH to 6.5, and adding 0.5% (by weight of the meal powder) of... The ficus protease was hydrolyzed at 60℃ for 1 h; the enzyme was inactivated at 85℃ for 10 min, centrifuged at 8000 r / min for 10 min, and the supernatant was collected. The supernatant was filtered through a 0.45 μm pore size filter membrane, and the filtrate was freeze-dried to obtain the crude extract. The crude extract was resuspended in pH 7.5 phosphate buffer (0.05 M) at a material-to-liquid mass ratio of 1:3, loaded onto a G-50 dextran gel column, and washed with pH 7.5 phosphate buffer (0.05 M) for 2 BV, followed by elution with pH 7.5 phosphate buffer containing 0.04 M NH4HCO3 (0.05 M) for 3 BV. The eluent was collected, placed in a dialysis bag with a molecular weight cutoff of 1 kDa, and dialyzed with deionized water for 52 h, with the deionized water changed every 6 h. The extract was then freeze-dried to obtain tiger nut extract peptides.

[0039] The preparation method of the tiger paw thorn tremella extract includes: pulverizing the dried tiger paw thorn tremella fruiting body into powder that passes through a 150-mesh sieve, dispersing it in deionized water at a material-to-liquid mass ratio of 1:10, adjusting the pH to 5.0, adding 0.8% (by weight) of cellulase from the powder, enzymatically hydrolyzing at 55℃ for 1 hour, adjusting the pH to 7.0, adding 0.8% (by weight) of bromelain from the powder, enzymatically hydrolyzing at 55℃ for 2 hours, inactivating the enzyme at 90℃ for 10 minutes, centrifuging at 8000 r / min for 10 minutes, collecting the supernatant, ultrafiltration through a 3 kDa ultrafiltration membrane, collecting the fraction below 3 kDa, and freeze-drying to obtain the tiger paw thorn tremella extract.

[0040] The preparation method of the compound extract of *Rhizoma Rhizoma Barkti* root, *Spatholobus suberectus*, and *Radix Achyranthis Bidentatae* includes: *Rhizoma Rhizoma Barkti* root: *Spatholobus suberectus*: *Radix Achyranthis Bidentatae* are pulverized into a compound powder that passes through a 60-mesh sieve at a mass ratio of 4:1:3; the compound powder is added to a 70 vol% ethanol aqueous solution at a mass ratio of 1:12, and ultrasonically extracted at 50℃, 300W, and 40kHz for 1 h, followed by centrifugation at 8000 r / min for 10 min to obtain extract A and precipitated residue A; precipitated residue A is added to a 75 vol% ethanol aqueous solution at a mass ratio of 1:12, and refluxed at 80℃ for 1 h, followed by centrifugation at 8000 r / min for 10 min to obtain extract B; extract A and extract B are combined, concentrated under reduced pressure at 55℃ to obtain a solid, dispersed with 2.5 times the mass of deionized water, the pH is adjusted to 6.0, and the sample is loaded onto an 80-120 mesh polyamide resin column. The column is washed with deionized water for 2 BV to remove impurities, and then treated with a solution containing 0.15 M... The sample was eluted with 30 vol% ethanol aqueous solution of NH4OH for 2 BV to remove impurities, and then eluted with 85 vol% ethanol aqueous solution containing 0.2 M NH4OH for 3 BV. The eluent was collected and concentrated under reduced pressure at 55 °C to obtain a solid. The solid was dispersed with 2.5 times its mass of 70 vol% ethanol aqueous solution and loaded onto an LH-60 dextran gel column. The solid was eluted with 80 vol% ethanol aqueous solution for 4 BV. The eluent was collected and concentrated under reduced pressure at 50 °C and dried under vacuum at 45 °C to obtain the compound extract of *Rhizoma Melilotifolia* root, *Spatholobus suberectus*, and *Radix Achyranthis Bidentatae*.

[0041] The preparation method of the modified hyaluronic acid-chitosan hydrogel includes: preparing a 3wt% hyaluronic acid solution with deionized water, and using 0.8M... Adjust the pH to 11.0 with NaOH aqueous solution. Add chloroacetic acid at a mass ratio of 1:0.03 (hyaluronic acid to chloroacetic acid). Stir at 65℃ and 200 rpm for 5 hours. Transfer the mixture to a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze with deionized water for 48 hours, changing the deionized water every 8 hours. Freeze-dry to obtain carboxymethyl hyaluronic acid. Dissolve carboxymethyl hyaluronic acid and chitosan (90% degree of deacetylation) in acetate buffer (0.1 M, pH 5.5) at a mass ratio of 1:1 to a total concentration of 5 wt%. Stir until completely dissolved. Add 0.1 wt% glutaraldehyde. Crosslink the mixture at 30℃ and 200 rpm for 3 hours to form a hydrogel. Transfer the hydrogel to a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze with deionized water for 72 hours, changing the deionized water every 8 hours. Adjust the viscosity to 2000 mPa·s to obtain modified hyaluronic acid-chitosan hydrogel.

[0042] The preparation method of the above-mentioned skin regeneration and repair drug includes the following steps: According to the mass fractions, tiger pea extract peptides, tiger paw thorn tremella extract, jujube root-chicken blood vine-achyranthes root compound extract and pomegranate peel polyphenols were mixed and added to modified hyaluronic acid-chitosan hydrogel (pre-mixed and dispersed with 20% hydrogel first, then added to the remaining hydrogel), stirred evenly, and the pH was adjusted to 6.8 to obtain the drug composition; the drug composition is used to prepare a drug for skin wound repair.

[0043] Comparative Example 1 The difference from Example 1 is that the tiger pea extract peptide was modified to 2 parts, and the tiger paw thorn tremella extract was modified to 10 parts.

[0044] Comparative Example 2 The difference from Example 1 is that the extract of Tremella fuciformis was modified to 11 parts, and the compound extract of Rhizoma Cynanchi var. sarcodactylis, Rhizoma Spatholobi, and Rhizoma Achyranthis Bidentatae was modified to 3 parts.

[0045] Comparative Example 3 The difference from Example 1 is that the tiger nut extract peptide was modified to 12 parts, and the compound extract of *Rhizoma Cynanchi* root, *Spatholobus suberectus*, and *Radix Achyranthis Bidentatae* was modified to 4 parts. Comparative Example 4 The difference from Example 1 is that in the preparation of tiger nut extract peptides, α-chymotrypsin is replaced with trypsin.

[0046] Comparative Example 5 The difference from Example 1 is that in the preparation of tiger nut extract peptides, fig protease is replaced with bromelain.

[0047] Comparative Example 6 The difference from Example 1 is that in the preparation of tiger nut extract peptides, G-50 dextran gel is replaced with G-75 dextran gel.

[0048] Comparative Example 7 The difference from Example 1 is that in the preparation of the tiger paw thorn tremella extract, bromelain is replaced with papain.

[0049] Comparative Example 8 The difference from Example 1 is that in the preparation of the compound extract of Rhizoma Amomum villosum root-Spatholobus suberectus-Achyranthes bidentata, the mass ratio of Rhizoma Amomum villosum root, Spatholobus suberectus and Achyranthes bidentata is modified to 1:4:2.5.

[0050] Comparative Example 9 The difference from Example 1 is that in the preparation of the compound extract of Rhizoma Cynanchi Aristolochiae, Rhizoma Spatholobi, and Rhizoma Achyranthis Bidentatae, the polyamide resin is replaced with AB-8 macroporous adsorption resin; and the LH-60 dextran gel is replaced with G-50 dextran gel.

[0051] The raw materials used in the above embodiments and comparative examples are as follows: Pomegranate peel polyphenols are from Shaanxi Jianhe Biotechnology Co., Ltd., pharmaceutical grade, with main components of 40% ellagic acid and 50% polyphenols. Cellulase is from Jiangsu Changjing Bioengineering Co., Ltd. α-chymotrypsin is from Guangzhou Hewei Pharmaceutical Technology Co., Ltd., model CD108568. Fig protease is from Hubei Qifei Pharmaceutical Chemical Co., Ltd. G-50 dextran gel is from Shanghai Yuanye Biotechnology Co., Ltd. Bromelain is from Shaanxi Yunqi Biotechnology Co., Ltd., model W301. Polyamide resin is from Shanghai Yuanye Biotechnology Co., Ltd., 80-120 mesh, model S14145. LH-60 dextran gel is from Shanghai Yuanye Biotechnology Co., Ltd. Hyaluronic acid is from Shaanxi Angxu Biotechnology Co., Ltd., medium molecular weight 110W (Daltons). Chloroacetic acid is from Shandong Qiangsen Chemical Co., Ltd., a pharmaceutical synthesis intermediate. Chitosan (90% deacetylation) is from Xi'an Jingmei Biotechnology Co., Ltd. Trypsin is from Guangzhou Hewei Pharmaceutical Technology Co., Ltd., model CD101204. Papain was obtained from Shanxi Ruisi Biotechnology Co., Ltd., model RS-0516-252. G-75 dextran gel was obtained from Shanghai Yuanye Biotechnology Co., Ltd. AB-8 macroporous adsorption resin was obtained from Shanghai Yuanye Biotechnology Co., Ltd.

[0052] The active ingredients of the above-mentioned drugs were tested and found to be tiger pea extract peptides, tiger paw thorn tremella extract, compound extract of jujube root-chicken blood vine-achyranthes root, and pomegranate peel polyphenols, according to their respective mass fractions.

[0053] I. Detection of fibroblast proliferation promotion: Sample preparation: The active ingredient mixture was diluted to 250 μg / mL using DMEM medium containing 10% fetal bovine serum. A blank control group containing only the medium (for zeroing) and a negative control group containing both cells and the medium were established.

[0054] The detection method includes: taking human dermal fibroblasts (HDF) in the logarithmic growth phase and adjusting the cell concentration to 5 × 10⁻⁶ cells with culture medium. 4Cells / mL. 100 μL of cell suspension (i.e., 5000 cells / well) was added to each well of a 96-well plate. The plates were incubated at 37°C with 5% CO2 for 24 h to allow complete cell adhesion. The original culture medium was carefully discarded. 100 μL of 250 μg / mL sample solution was added to the experimental group, and 100 μL of DMEM medium containing 10% fetal bovine serum was added to the negative control group. Five replicates were made for each group. The plates were returned to the incubator and incubated for another 48 h. 10 μL of CCK-8 solution was added directly to each well, gently shaken to mix, and incubated for another 2 h. The absorbance (OD) at 450 nm was measured using a microplate reader. Cell proliferation rate (%) = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] × 100%.

[0055] II. Detection of vascular endothelial cell migration: Sample preparation: The active ingredient mixture was diluted to 250 μg / mL with serum-free endothelial cell culture medium (ECM). Serum-free culture medium was used as a blank control group.

[0056] The detection method included: Human umbilical vein endothelial cells (HUVECs) were seeded into 6-well plates and cultured until they fused to form a dense monolayer. Using a 200 μL sterile pipette tip (with a flush tip to ensure consistent scratch width), a straight scratch was made perpendicular to the bottom of the plate, centered on each well. The plates were gently rinsed three times with PBS to thoroughly remove the scratched cells. 2 mL of 250 μg / mL sample solution was added to the experimental group, and 2 mL of serum-free ECM medium was added to the blank control group. Each group had three independent replicates. The plates were incubated at 37°C in a 5% CO2 incubator. At 0 h and 24 h, three fixed fields of view were randomly selected for each scratch under a 100x inverted microscope. The scratch area in each field was measured using ImageJ software. Scratch healing rate (%) = (0 h scratch area - 24 h scratch area) / 0 h scratch area × 100%.

[0057] III. Antibacterial activity test: Sample preparation: The active ingredient mixture was diluted with sterile physiological saline to a sample solution of 0.5 mg / mL, and then filtered through a 0.22 μm filter membrane for sterilization. Physiological saline was used as a blank control. Tested bacterial species: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853).

[0058] The detection method includes: preparing Mueller-Hinton (MH) agar plates with a thickness controlled at 4 mm. Prepare 1.5 × 10⁻⁶ agar plates. 8Prepare a bacterial suspension of CFU / mL. Using a sterile cotton swab, spread the suspension evenly three times along the edge of the agar plate towards the center, rotating the plate 60° after each spread. Finally, spread the suspension around the edge of the plate once to ensure even distribution of the bacterial suspension. After standing for 5 minutes, gently place a 6mm diameter sterile Oxford cup onto the agar surface using sterile forceps (place three parallel Oxford cups on each plate, spaced at least 25mm apart to avoid overlapping inhibition zones). Slowly add 200μL of 0.5mg / mL sample solution to each Oxford cup using a micropipette. For the blank control group, add 200μL of sterile physiological saline, avoiding spillage. Incubate the plates upright in a 37℃ incubator for 24 hours. Measure the diameter of the inhibition zone (accurate to 0.1mm, using the average of the longest and shortest diameters of the inhibition zone).

[0059] IV. Detection of Inhibition of Inflammatory Factor Expression: Sample preparation: The active ingredient mixture was diluted to 250 μg / mL using RPMI-1640 medium containing 10% fetal bovine serum. A blank control group (complete medium) and a model group (complete medium containing 1 μg / mL LPS) were established.

[0060] The detection method included adjusting the concentration of human mononuclear macrophages (THP-1) to 2 × 10⁻⁶. 5 Cells / mL were seeded into 24-well plates, 1 mL per well. 100 nM PMA was added for 48 h to induce differentiation into macrophages. The old culture medium was discarded, and the cells were gently washed once with PBS to avoid residual PMA affecting subsequent experiments. Except for the blank control group, all other groups were added 500 μL of complete culture medium containing 1 μg / mL LPS and stimulated for 4 h to establish an inflammation model. The LPS-containing culture medium was discarded. The experimental groups were replaced with 1 mL of complete culture medium containing 250 μg / mL sample solution and 1 μg / mL LPS, the model group was added only with 1 mL of complete culture medium containing 1 μg / mL LPS, and the blank control group was replaced with 1 mL of complete culture medium. Cells were cultured for another 24 h. The cell supernatant was collected and centrifuged at 1000 × g for 10 min. Three replicates were performed for each group. TNF-α and IL-6 ELISA kits were used for detection, and the concentrations of inflammatory factors in the samples were obtained based on the standard curve. Inflammatory factor inhibition rate (%) = [(concentration of inflammation model group - concentration of experimental group) / concentration of inflammation model group] × 100%.

[0061] V. Collagen Synthesis Detection: Sample preparation: The active ingredient mixture was diluted to 250 μg / mL using DMEM medium containing 10% fetal bovine serum. A blank control group containing cells and culture medium was set up.

[0062] The detection methods include: HDF cells at 2.5 × 10⁻⁶ 4Cells were seeded at a density of 1 / well in 24-well plates and cultured for 24 h to allow for cell adhesion and growth. The original culture medium was discarded. 1 mL of 250 μg / mL sample solution was added to the experimental groups, and 1 mL of DMEM medium containing 10% fetal bovine serum was added to the blank control group. Cultures were continued for 72 h. The supernatant was discarded, and each well was gently washed twice with PBS to remove residual culture medium. 1 mL of 4% paraformaldehyde was added to each well, and the plates were fixed at room temperature for 30 min. The fixative was discarded, and the plates were washed three times with PBS for 5 min each time to completely remove the fixative. 1 mL of 0.1% Sirius Red staining solution (dissolved in saturated picric acid) was added to each well, and the plates were stained at room temperature in the dark for 1 h. The staining solution was discarded, and the plates were washed repeatedly with 0.5% glacial acetic acid until the eluent was colorless. After drying, 1 mL of 0.1M NaOH-anhydrous ethanol solution (1:1, v / v) was added to each well, and the plates were shaken slowly at 100 rpm for 30 min on a shaker to ensure complete dissolution of the dye. Pipette 200 μL of solution into a 96-well plate, with 3 replicates per group. Measure the OD value using a microplate reader at 540 nm. Collagen synthesis increase rate (%) = [(OD experimental group - OD blank group) / OD blank group] × 100%.

[0063] Table 1. Test Results (Average Values) In the vascular endothelial cell migration assay, the scratch healing rate in the blank control group was 21.8%.

[0064] The technical solutions in Examples 1 to 3 precisely match the physiological mechanisms of skin regeneration and repair from raw material ratios and preparation processes to functional synergy, forming a multi-component synergistic and comprehensive repair system. The core functional ingredients are tiger nut extract peptides and a compound extract of *Rhizoma Cynogum* root, *Spatholobus suberectus*, and *Radix Achyranthis Bidentatae*, combined with *Fructus Tremellae* extract and pomegranate peel polyphenols, with the proportions falling within the optimal range. Among these, tiger nut extract peptides activate fibroblast proliferation and collagen synthesis; the compound extracts regulate inflammation and promote angiogenesis; *Fructus Tremellae* extract assists in moisturizing and anti-inflammatory effects, and aids in cell proliferation and collagen synthesis; and pomegranate peel polyphenols enhance antibacterial capabilities. These four ingredients synergistically cover the entire skin repair process, including hemostasis, anti-inflammation, proliferation, vascularization, and collagen remodeling, avoiding the breakage of the repair chain due to the loss of a single function. Tiger nut peptides are obtained through stepwise enzymatic hydrolysis with three enzymes and G-50 gel purification to ensure the acquisition of highly active small molecule peptides; tiger paw thorn tremella extract is enriched with low molecular weight active polysaccharides and peptides through synergistic enzymatic hydrolysis with two enzymes and 3kDa ultrafiltration; the compound extract is precisely preserved by gradient ethanol extraction, polyamide and LH-60 dual-column purification.

[0065] Comparative Examples 1 to 3 (component imbalance): Insufficient dosage of individual components led to a decrease in synergistic efficacy, while excessive dosage of individual components resulted in competition for binding sites, disrupting the balance of absorption among components, reducing the synergistic effect. Furthermore, once the excessive component reached saturation absorption, the remaining drug could not improve efficacy and introduced excessive impurities, affecting cell activity. This led to a decline in various indicators.

[0066] Comparative Examples 4 and 5 (α-chymotrypsin replaced with trypsin; or figoprotein protease replaced with bromelain): Trypsin and α-chymotrypsin have different specific hydrolytic cleavage sites, resulting in different peptide chain structures and different compatibility with figoprotein protease. The resulting small peptides have different effects and directions of action, leading to variations in indicators and suboptimal efficacy. Similarly, bromelain and figoprotein protease have different specific hydrolytic cleavage sites and different compatibility with trypsin, resulting in different small peptides with different effects and directions of action, leading to variations in indicators. This indicates that for the protein hydrolysis of tiger nut, using a combination of α-chymotrypsin and figoprotein protease is the optimal approach, yielding more effective components and improving efficacy.

[0067] Comparative Example 6 (G-50 dextran gel replaced with G-75 dextran gel): The separation range of G-75 gel is different from that of G-50 dextran gel. For the purification of tiger nut extract peptides, G-50 dextran gel can better enrich the active ingredients; while G-75 gel separation will lose some active ingredients and retain different impurity peptides. Impurities affect cell activity and reduce antibacterial activity, and the anti-inflammatory effect is also poor, resulting in a decline in performance.

[0068] Comparative Example 7 (In the preparation of Tiger Paw Tremella extract, bromelain was replaced with papain): Papain and bromelain have different hydrolytic cleavage sites. Bromelain has a better ability to release functional polysaccharides and functional peptides, and produces fewer impurity peptides. After being replaced with papain, the composition of the final extract changed, resulting in a decrease in efficacy.

[0069] Comparative Example 8 (insufficient Rhizoma Rhizoma et Radix et Rhizoma, excessive Rhizoma Spatholobiae): Rhizoma Rhizoma et ...

[0070] Comparative Example 9 (polyamide resin replaced with AB-8 macroporous adsorption resin, LH-60 dextran gel replaced with G-50 dextran gel): AB-8 macroporous adsorption resin and polyamide resin have different purification components. Furthermore, using AB-8 macroporous adsorption resin to purify the compound extract of *Rhizoma Cynanchi* root, *Spatholobus suberectus*, and *Radix Achyranthis Bidentatae* is insufficient to remove harmful components, and the separation rate of effective components is low. LH-60 gel is a high-resolution gel that can separate components with similar molecular weights, accurately purifying active monomers. G-50 gel has low separation precision and cannot effectively separate the target components and structurally similar impurities, leading to a decrease in the purity of the active components in the compound extract. Simultaneously, the large elution volume of G-50 gel dilutes the concentration of the target components, causing some active components to non-specifically bind to the gel matrix, resulting in a decreased recovery rate and ultimately affecting the reduction of various indicators.

Claims

1. A skin regeneration and repair drug, characterized in that, The drug is composed of the following raw materials in parts by weight: 6-8 parts tiger pea extract peptide, 4.5-5.5 parts tiger paw thorn tremella extract, 8-10 parts purslane root-chicken blood vine-Achyranthes bidentata compound extract, 1-3 parts pomegranate peel polyphenols, and 80-90 parts modified hyaluronic acid-chitosan hydrogel, with the pH adjusted to 6.8-7.2; The tiger nut extract peptide is a product with a value of over 1 kDa obtained by sequentially hydrolyzing tiger nut with cellulase, α-chymotrypsin, and fig protease, followed by purification using a G-50 dextran gel column. The tiger paw thorn tremella extract is a product with a value of less than 3 kDa obtained by co-hydrolyzing the fruiting body of tiger paw thorn tremella with cellulase and bromelain. The Rhododendron molle root-chrysanthemum vine-Achyranthes bidentata compound extract is obtained by gradient extraction with ethanol-water solution at a mass ratio of Rhododendron molle root:chrysanthemum vine:Achyranthes bidentata = (3-4):(1-2):(2-3), followed by sequential adsorption using a polyamide resin column and purification using an LH-60 dextran gel column. The modified hyaluronic acid-chitosan hydrogel is a biocompatible hydrogel formed by cross-linking carboxymethylated hyaluronic acid and chitosan with glutaraldehyde.

2. The skin regeneration and repair drug according to claim 1, characterized in that, The preparation method of the tiger nut extract peptide includes: tiger nut meal being crushed into meal powder, dispersed in an acetate-sodium acetate buffer solution at pH 4.5–5.5, adding cellulase, and hydrolyzing at 50–55°C for 2–3 hours; adjusting the pH to 8.0–8.8, adding α-chymotrypsin, and hydrolyzing at 35–40°C for 1.5–2 hours; inactivating the enzyme, cooling, adjusting the pH to 6.0–6.5, adding fig protease, and hydrolyzing at 55–60°C for 1–1.5 hours; inactivating the enzyme, centrifuging, collecting the supernatant, filtering, taking the filtrate, and freeze-drying to obtain the crude extract; resuspending the crude extract in a phosphate buffer solution at pH 7.5–8.0, loading it onto a G-50 dextran gel column, washing with phosphate buffer solution at pH 7.5–8.0 for 2 BV–3 BV, and then washing with phosphate buffer solution at pH 7.5–8.0 containing 0.02 M–0.04 M... Elute with phosphate buffer containing NH4HCO3 for 3-4 BV, collect the eluent, place it in a dialysis bag, dialyze with deionized water, and freeze-dry to obtain tiger nut extract peptides.

3. The skin regeneration and repair drug according to claim 2, characterized in that, The meal powder has a particle size that passes through a 60-80 mesh sieve; the amount of the acetate-sodium acetate buffer solution is 10-12 times the mass of the meal powder; the amount of cellulase added is 0.8%-1.5% of the mass of the meal powder; the amount of α-chymotrypsin added is 0.8%-1.5% of the mass of the meal powder; the amount of fig protease added is 0.5%-1.0% of the mass of the meal powder; the enzyme inactivation is performed at 80-85℃ for 10-15 minutes; the centrifugation is performed at 6000-8000 r / min for 10-15 minutes; the filtration is performed using a 0.45 μm pore size filter membrane; the molecular weight cutoff of the dialysis bag is 1 kDa.

4. The skin regeneration and repair drug according to claim 1, characterized in that, The preparation method of the tiger paw thorn tremella extract includes: pulverizing tiger paw thorn tremella into powder, dispersing it in deionized water, and adjusting the pH to 4.5-5.0; adding cellulase and hydrolyzing it at 50℃-55℃ for 1-2 hours, adjusting the pH to 6.5-7.0, adding bromelain and hydrolyzing it at 50℃-55℃ for 2-3 hours; inactivating the enzyme, centrifuging, collecting the supernatant, ultrafiltration membrane ultrafiltration, collecting the fraction below 3kDa, and freeze-drying to obtain tiger paw thorn tremella extract.

5. The skin regeneration and repair drug according to claim 4, characterized in that, The tiger paw thorn tremella is the dried fruiting body of tiger paw thorn tremella; the particle size of the powder is sieved through a 120-150 mesh sieve; the amount of phosphate buffer is 10-12 times the mass of the powder; the amount of cellulase added is 0.8%-1.5% of the mass of the powder; the amount of bromelain added is 0.8%-1.5% of the mass of the powder; the enzyme inactivation is performed at 85-90℃ for 10-15 minutes; the centrifugation is performed at 6000-8000 r / min for 10-15 minutes.

6. The skin regeneration and repair drug according to claim 1, characterized in that, The preparation method of the compound extract of *Rhizoma Cynanchi* root, *Spatholobus suberectus*, and *Radix Achyranthis Bidentatae* includes: pulverizing the compound powder according to the mass ratio of *Rhizoma Cynanchi* root: *Spatholobus suberectus*: *Radix Achyranthis Bidentatae* = (3-4):(1-2):(2-3), adding it to a 70 vol%-75 vol% ethanol aqueous solution, ultrasonically extracting, centrifuging to obtain extract A and precipitated residue A; adding precipitated residue A to a 75 vol%-80 vol% ethanol aqueous solution, reflux extracting, centrifuging to obtain extract B; combining extract A and extract B, concentrating under reduced pressure to obtain a solid, dispersing with deionized water, adjusting the pH to 5.0-6.0, loading the sample onto a polyamide resin column, rinsing with deionized water for 2-3 BV to remove impurities, rinsing with a 30 vol%-40 vol% ethanol aqueous solution containing 0.1 M-0.15 M NH4OH for 1.5 BV-2 BV to remove impurities, and rinsing with a 0.2 M-0.3 M NH4OH solution... Elute 3-4 BV of NH4OH with 80-85 vol% ethanol aqueous solution, collect the eluent, concentrate under reduced pressure to obtain a solid, disperse with 60-70 vol% ethanol aqueous solution, load the sample onto an LH-60 dextran gel column, elute 3-4 BV with 80-90 vol% ethanol aqueous solution, collect the eluent, concentrate under reduced pressure, and vacuum dry to obtain a compound extract of *Rhizoma Melilotifolia* root, *Spatholobus suberectus*, and *Achyranthes bidentata*.

7. The skin regeneration and repair medicine according to claim 6, characterized in that, The particle size of the composite powder is such that it passes through a 60-80 mesh sieve; the amount of the 70-75 vol% ethanol aqueous solution is 10-12 times the mass of the composite powder; the ultrasonic extraction is performed at 45-50℃, 300-350W, and 30-40kHz for 1-2 hours; the amount of the 75-80 vol% ethanol aqueous solution is 10-12 times the mass of the precipitated residue A; the reflux extraction is performed at 75-80℃ for 1-2 hours; the centrifugation is performed at 6000-8000 r / min for 10-15 minutes; and the polyamide resin column is packed with 80-120 mesh polyamide resin.

8. The skin regeneration and repair medicine according to claim 1, characterized in that, The preparation method of the modified hyaluronic acid-chitosan hydrogel includes: preparing a hyaluronic acid solution with deionized water, adjusting the pH to 10.0-11.0, adding chloroacetic acid, stirring and reacting, dialyzing with a dialysis bag, and freeze-drying to obtain carboxymethyl hyaluronic acid; dissolving carboxymethyl hyaluronic acid in an acetate buffer solution at a mass ratio of carboxymethyl hyaluronic acid: chitosan = 1:(1-1.2) to a total concentration of 3wt%-5wt%, stirring until completely dissolved; adding glutaraldehyde, performing a cross-linking reaction to form a hydrogel, dialyzing with a dialysis bag, and adjusting the viscosity to 2000 mPa·s-3000 mPa·s to obtain the modified hyaluronic acid-chitosan hydrogel.

9. The skin regeneration and repair drug according to claim 1, characterized in that, The concentration of the hyaluronic acid solution is 2wt%–3wt%; the mass ratio of hyaluronic acid to chloroacetic acid is 1:(0.03–0.05); the stirring reaction is carried out at 60℃–65℃ and 200rpm–300rpm for 4–5 hours; the amount of glutaraldehyde added is 0.1wt%–0.15wt% to a final concentration; the crosslinking reaction is carried out at 25℃–30℃ and 200rpm–300rpm for 2–3 hours; the molecular weight cutoff of the dialysis bags is 10kDa.

10. The method for preparing a skin regeneration and repair drug according to claim 1, characterized in that, Includes the following steps: According to the mass fractions, tiger pea extract peptide, tiger paw thorn tremella extract, jujube root-chicken blood vine-achyranthes root compound extract and pomegranate peel polyphenol are mixed and added to modified hyaluronic acid-chitosan hydrogel, stirred evenly, and the pH is adjusted to 6.8-7.2 to obtain a pharmaceutical composition; the pharmaceutical composition is used to prepare a drug for skin wound repair.

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