Medicine for skin regeneration and repair and preparation method thereof

Abstract

The present application relates to a kind of skin regeneration repair medicine and preparation method thereof, belong to biological medicine technical field, and medicine includes oil palm extraction peptide, tiger palm silver ear extract, Gangdeng root-milletia nitida-Siberian milkvetch composite extract and pomegranate peel polyphenol etc.Oil palm extraction peptide is oil palm sequentially by cellulase, alpha-chymotrypsin and ficin enzymolysis, then by G-50 dextran gel column purification and obtain the product above 1kDa;Tiger palm silver ear extract is tiger palm silver ear fruit body by cellulase and bromelain common enzymolysis, and obtain the product below 3kDa;Gangdeng root-milletia nitida-Siberian milkvetch composite extract is Gangdeng root, milletia nitida and siberian milkvetch by gradient extraction of ethanol aqueous solution and obtain extract, sequentially by polyamide resin column adsorption and LH-60 dextran gel column purification and obtain the product.Multiple components construct good repair system, accelerate wound repair healing.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a medicine for skin regeneration and repair and a preparation method thereof. BACKGROUND

[0002] Skin is one of the organs most susceptible to damage, from minor daily abrasions and scalds to deep burns, surgical wounds, and chronic non-healing wounds such as diabetic foot and pressure sores, all of which can damage the integrity of the skin. Healthy skin has a high self-regeneration capacity, and its repair process exhibits precise timing: when the skin is damaged, the first response is to activate the hemostatic response by aggregating platelets to quickly seal the wound; then neutrophils and macrophages migrate to the injury site in succession to remove necrotic tissue and invading pathogens, clearing obstacles for subsequent repair; then fibroblasts proliferate and synthesize collagen to construct a granulation tissue scaffold, while endothelial cells form new blood vessels to supply nutrients to the wound; finally, epithelial cells migrate from the edge of the wound to the center to cover it, and collagen gradually remodels to restore the structure and function of the skin. The entire process usually takes 2-4 weeks to complete in healthy people. However, when the damage exceeds the skin's own repair threshold, or when the patient has underlying diseases or low immunity, the repair chain will be broken, inflammation will persist, fibroblast activity will be insufficient, and blood vessel formation will be blocked, ultimately leading to chronic non-healing wounds and chronic lesions.

[0003] Early interventions for skin damage mainly involve passive support: using vaseline gauze, ointment, and other dressings to isolate external stimuli and prevent secondary damage to the wound; for infected wounds, topical antibacterial drugs are used to control infection. Although this approach can provide a basic environment for wound healing, it cannot actively promote the regeneration process and has little effect on chronic wounds. With the development of cell biology research, drug development has entered the active regulation stage, and biological agents such as recombinant human epidermal growth factor and fibroblast growth factor have been introduced. By directly activating the proliferation and migration signals of local cells in the wound, these agents significantly shorten the healing time of small and medium-sized wounds and have become commonly used drugs in clinical practice. In recent years, the integration of material science and regenerative medicine has further driven the upgrade of drugs. Gels or patches using hyaluronic acid and collagen as carriers can achieve slow release of growth factors, extending the duration of action; new ingredients such as stem cell exosomes and bioactive peptides can improve the repair microenvironment from multiple dimensions such as inflammation control and blood vessel formation, providing a new direction for the treatment of chronic non-healing wounds.

[0004] Despite this, existing drugs still face many technical challenges: growth factor drugs have poor stability and are easily affected by temperature and enzymatic degradation; stem cell-related preparations have immune rejection and ethical issues; and for deep skin damage, the repair effect is slow. Therefore, it is necessary to continuously develop safer and more stable, efficient, and more skin-physiological-regeneration-mechanism-based drug ingredients to meet the needs of skin repair. SUMMARY

[0005] In view of the fact that the skin regeneration and repair drugs in the prior art still need to be continuously improved in terms of safety and high efficiency, the present application provides a skin regeneration and repair drug and a preparation method thereof, effective active ingredients such as Cyperus esculentus extraction peptide, Tremella fuciformis extraction, and composite extract of Rubus corchorifolius root, Spatholobus suberectus, and Cyathula officinalis are prepared, the physiological repair mechanisms of skin hemostasis, anti-inflammation, proliferation, vascularization, and collagen remodeling are precisely matched, a repair system with multi-component synergy and full-link support is constructed, the problems of persistent inflammation, insufficient cell activity, and blocked vascularization in the repair of chronic non-healing wounds in existing drugs are effectively solved, and the drug is safe and efficient. The specific technical scheme is as follows:

[0006] A skin regeneration and repair drug, the drug is composed of the following raw materials in parts by mass: Cyperus esculentus extraction peptide 6-8 parts, Tremella fuciformis extraction 4.5-5.5 parts, composite extract of Rubus corchorifolius root, Spatholobus suberectus, and Cyathula officinalis 8-10 parts, pomegranate peel polyphenol 1-3 parts, and modified hyaluronic acid-chitosan hydrogel 80-90 parts, and the pH is adjusted to 6.8-7.2.

[0007] The Cyperus esculentus extraction peptide is a product with a molecular weight of more than 1kDa obtained by sequentially subjecting Cyperus esculentus to cellulase, alpha-chymotrypsin, and ficin enzymolysis and then purifying the product through a G-50 dextran gel column; the Tremella fuciformis extraction is a product with a molecular weight of less than 3kDa obtained by subjecting Tremella fuciformis fruiting bodies to cellulase and bromelain enzymolysis; the composite extract of Rubus corchorifolius root, Spatholobus suberectus, and Cyathula officinalis is a product obtained by sequentially subjecting the extract obtained by gradient extraction with an ethanol aqueous solution in a mass ratio of Rubus corchorifolius root:Spatholobus suberectus:Cyathula officinalis=(3-4):(1-2):(2-3) to a polyamide resin column adsorption and an LH-60 dextran gel column purification; and the modified hyaluronic acid-chitosan hydrogel is a biocompatible hydrogel formed by carboxymethylated hyaluronic acid and chitosan crosslinked with glutaraldehyde.

[0008] The preparation method of the Cyperus esculentus L. extract peptide comprises the following steps: crushing Cyperus esculentus L. cake into cake powder, dispersing the cake powder in a pH 4.5-5.5 acetic acid-sodium acetate buffer, adding cellulase, and carrying out enzymolysis at 50-55 ℃ for 2-3 h; adjusting the pH to 8.0-8.8, adding α-chymotrypsin, and carrying out enzymolysis at 35-40 ℃ for 1.5-2 h; inactivating the enzyme, adjusting the pH to 6.0-6.5 after cooling, adding ficin, and carrying out enzymolysis at 55-60 ℃ for 1-1.5 h; inactivating the enzyme, centrifuging, collecting the supernatant, filtering, taking the filtrate, and freeze-drying to obtain a crude extract; resuspending the crude extract in a pH 7.5-8.0 phosphate buffer, loading onto a G-50 dextran gel column, eluting with 2-3 BV of the pH 7.5-8.0 phosphate buffer, and then eluting with 3-4 BV of the pH 7.5-8.0 phosphate buffer containing 0.02-0.04 M NH4HCO3, and collecting the eluate; loading into a dialysis bag, dialyzing with deionized water, and freeze-drying to obtain the Cyperus esculentus L. extract peptide.

[0009] In the preparation method of the Cyperus esculentus L. extract peptide, the particle size of the cake powder is 60-80 mesh; the amount of the acetic acid-sodium acetate buffer is 10-12 times the mass of the cake powder; the amount of the added cellulase is 0.8%-1.5% of the mass of the cake powder; the amount of the added α-chymotrypsin is 0.8%-1.5% of the mass of the cake powder; the amount of the added ficin is 0.5%-1.0% of the mass of the cake powder; the inactivation of the enzyme is carried out at 80-85 ℃ for 10-15 min; the centrifugation is carried out at 6,000-8,000 r / min for 10-15 min; the filtration is carried out by using a filter membrane with a pore size of 0.45 μm; and the molecular weight cut-off of the dialysis bag is 1 kDa.

[0010] The preparation method of the tigerclaw wood ear extract comprises the following steps: crushing tigerclaw wood ear into powder, dispersing the powder in deionized water, and adjusting the pH to 4.5-5.0; adding cellulase, and carrying out enzymolysis at 50-55 ℃ for 1-2 h; adjusting the pH to 6.5-7.0, adding bromelain, and carrying out enzymolysis at 50-55 ℃ for 2-3 h; inactivating the enzyme, centrifuging, collecting the supernatant, ultrafiltering by using an ultrafilter membrane, collecting components with a molecular weight of less than 3 kDa, and freeze-drying to obtain the tigerclaw wood ear extract.

[0011] In the preparation method of the tiger-palm-tremella extract, the tiger-palm-tremella is dried tiger-palm-tremella fruiting body; the particle size of the powder is 120-150 mesh; the amount of the phosphate buffer is 10-12 times the mass of the powder; the amount of the cellulase added is 0.8-1.5% of the mass of the powder; the amount of the bromelain added is 0.8-1.5% of the mass of the powder; the enzyme inactivation is performed at 85-90°C for 10-15 min; and the centrifugation is performed at 6000-8000 r / min for 10-15 min.

[0012] The preparation method of the compound extract of gongreneng-jixueteng-chuanniuxie comprises the following steps: crushing gongreneng, jixueteng and chuanniuxie into a compound powder according to a mass ratio of (3-4):(1-2):(2-3), adding 70-75 vol% ethanol aqueous solution, ultrasonic extraction, centrifugation, to obtain extract A and precipitate residue A; adding 75-80 vol% ethanol aqueous solution to the precipitate residue A, reflux extraction, centrifugation, to obtain extract B; combining the extract A and the extract B, vacuum concentration to obtain a solid, dispersing the solid with deionized water, adjusting the pH to 5.0-6.0, loading the solid into a polyamide resin column, washing the column with 2-3 BV of deionized water to remove impurities, washing the column with 30-40 vol% ethanol aqueous solution containing 0.1-0.15 M NH4OH to remove impurities, eluting the column with 80-85 vol% ethanol aqueous solution containing 0.2-0.3 M NH4OH for 3-4 BV, collecting the eluate, vacuum concentration, dispersing the solid with 60-70 vol% ethanol aqueous solution, loading the solid into an LH-60 sephadex column, eluting the column with 80-90 vol% ethanol aqueous solution for 3-4 BV, collecting the eluate, vacuum concentration, vacuum drying, to obtain the compound extract of gongreneng-jixueteng-chuanniuxie.

[0013] In the preparation method of the compound extract of gongreneng-jixueteng-chuanniuxie, the particle size of the compound powder is 60-80 mesh; 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°C, 300-350 W and 30-40 kHz for 1-2 h; the amount of the 75-80 vol% ethanol aqueous solution is 10-12 times the mass of the precipitate residue A; the reflux extraction is performed at 75-80°C for 1-2 h; the centrifugation is performed at 6000-8000 r / min for 10-15 min; and the polyamide resin column is filled with 80-120 mesh polyamide resin.

[0014] The preparation method of the modified hyaluronic acid-chitosan hydrogel comprises the following steps: 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, freeze-drying to obtain carboxymethyl hyaluronic acid; dissolving carboxymethyl hyaluronic acid and chitosan in an acetic acid buffer according to a mass ratio of 1: (1-1.2) to a total concentration of 3wt%-5wt%, stirring until completely dissolved; adding glutaraldehyde, crosslinking to form a hydrogel, dialyzing with a dialysis bag, adjusting the viscosity to 2000mPa·s-3000mPa·s, and obtaining the modified hyaluronic acid-chitosan hydrogel.

[0015] In the preparation method of the modified hyaluronic acid-chitosan hydrogel, the concentration of the hyaluronic acid solution is 2wt%-3wt%; the mass ratio of the hyaluronic acid to chloroacetic acid is 1: (0.03-0.05); the stirring and reaction is carried out at 60°C-65°C and 200rpm-300rpm for 4h-5h; the amount of glutaraldehyde added is 0.1wt%-0.15wt% of the final concentration; the crosslinking reaction is carried out at 25°C-30°C and 200rpm-300rpm for 2h-3h; and the molecular weight cut-off of the dialysis bag is 10kDa.

[0016] The preparation method of the drug for skin regeneration and repair comprises the following steps:

[0017] The oil sedge peptide, the tiger palm stinkhorn extract, the compound extract of the root of the mountain vine, the millettia pinnata and the cyathula officinalis, and the pomegranate peel polyphenol are mixed according to mass parts, and then added into the modified hyaluronic acid-chitosan hydrogel, stirred uniformly, and adjusted to pH 6.8-7.2 to obtain a drug composition; and the drug composition is used for preparing a drug for skin wound repair.

[0018] The drug for skin regeneration and repair and the preparation method thereof have the following beneficial effects:

[0019] I. The drug precisely matches the physiological repair mechanisms of skin hemostasis, anti-inflammation, proliferation, vascularization and collagen remodeling, constructs a repair system with multi-component synergy and full-link support, effectively solves the problems of persistent inflammation, insufficient cell activity and blocked neovascularization of existing drugs in chronic non-healing wound repair. The core advantage lies in the multi-dimensional effect from repair microenvironment regulation to functional cell activation, which can not only provide a repair carrier with good biocompatibility for the wound, but also actively regulate the key links of repair, avoid the repair chain breakage caused by single function loss, and finally realize efficient and safe skin regeneration and repair.

[0020] II. Cyrtosperma breviflora peptide: through step-by-step enzymolysis of cellulase, α-chymotrypsin and bromelain and G-50 dextran gel purification, it can precisely enrich high-activity functional small molecule peptides, directly activate fibroblast proliferation signals, and promote collagen synthesis, providing core power for granulation tissue construction and skin structure remodeling. G-50 dextran gel purification can precisely separate target active ingredients from impurities, improve ingredient purity, and avoid the inhibition of cell activity and drug efficacy interference by impurities.

[0021] III. Tiger palm stinkhorn extract: through cellulase and bromelain complex enzymolysis and 3 kDa ultrafiltration, it can enrich low molecular weight active polysaccharides and polypeptides, not only providing a moisturizing barrier for the wound to reduce water loss and external stimulation, but also assisting in activating fibroblast activity and enhancing anti-inflammatory effect, creating a stable microenvironment for the repair process.

[0022] IV. Compound extract of Gongdeng root, Millettia nitida and Cyathula officinalis: through gradient ethanol extraction, polyamide and LH-60 double column purification, it can precisely retain the effective components of Gongdeng root, Millettia nitida and Cyathula officinalis, and solve the problems of chronic wound inflammation, insufficient nutrition, and slow healing from the aspects of inhibiting pro-inflammatory factor expression, improving local blood supply, promoting vascular endothelial cell migration, and synergistically promoting cell proliferation and protein synthesis. Polyamide-LH-60 double column purification can remove harmful impurities, improve safety, and avoid the inhibition of cell activity and drug efficacy interference by impurities.

[0023] V. Pomegranate peel polyphenol: as a natural antibacterial ingredient, it can inhibit wound infection bacteria and reduce the interference of infection on the repair process, and its antioxidant properties can reduce oxidative stress damage to the wound and protect functional cell activity. Modified hyaluronic acid-chitosan hydrogel: prepared by carboxymethylation modification of hyaluronic acid and glutaraldehyde crosslinking of chitosan, it has good biocompatibility and viscosity stability, can act as a drug carrier to achieve slow release of active ingredients, prolong the action time, and form a physical barrier to isolate external pollution while providing a scaffold for cell migration.

[0024] VI. The strong anti-inflammatory effect of Gongdeng root removes inflammatory obstacles for vascular endothelial cell migration, Millettia nitida improves microcirculation to provide a nutritional basis for angiogenesis, and Cyathula officinalis further promotes vascular endothelial cell migration and new blood vessel formation, while the auxiliary anti-inflammatory effect of tiger palm stinkhorn extract enhances this synergistic effect, forming a double protection of strong anti-inflammatory and pro-vascular.

[0025] VII. Cyrtosperma breviflora peptide activates fibroblast proliferation and collagen synthesis, and tiger palm stinkhorn extract assists in enhancing cell activity, while modified hyaluronic acid-chitosan hydrogel not only provides a slow release carrier for these two ingredients, prolonging their action time, but also provides a physical scaffold for activated cells to migrate and proliferate.

[0026] In summary, the drug realizes good synergistic effect through functional complementation among components and precise control of step parameters, breaks through the limitation of single function of existing drugs, and provides a more physiological mechanism solution for skin regeneration and repair. DETAILED DESCRIPTION

[0027] The application will be further described below in combination with specific implementation cases, but the application is not limited to these examples.

[0028] Example 1

[0029] A drug for skin regeneration and repair, the drug comprising the following components in parts by mass: Cyperus esculentus L. extracted peptide 7 parts, Tigerlily-tremella extract 5 parts, compound extract of Potentilla kleiniana Wall, Spatholobus suberectus Dunn and Cyathula officinalis Duthie 9 parts, pomegranate peel polyphenol 2 parts, modified hyaluronic acid-chitosan hydrogel 85 parts, and the pH is adjusted to 7.0.

[0030] The preparation method of the Cyperus esculentus L. extracted peptide comprises the following steps: the oil-pressed Cyperus esculentus L. cake is crushed into cake powder passing through a 60-mesh sieve, and the cake powder is dispersed in a pH 5.0 acetic acid-sodium acetate buffer (0.1 M) according to a solid-liquid mass ratio of 1:11, 1.2% cellulase of the mass of the cake powder is added, and enzymolysis is carried out at 52°C for 2.5 h; the pH is adjusted to 8.5, 1.2% α-chymotrypsin of the mass of the cake powder is added, and enzymolysis is carried out at 38°C for 1.5 h; the enzyme is inactivated at 82°C for 12 min, after cooling, the pH is adjusted to 6.3, 0.8% ficin of the mass of the cake powder is added, and enzymolysis is carried out at 58°C for 1.5 h; the enzyme is inactivated at 82°C for 12 min, and centrifugation is carried out at 7000 r / min for 12 min, the supernatant is collected, filtered through a filter membrane with a pore size of 0.45 μm, the filtrate is taken, and freeze-drying is carried out to obtain a crude extract; the crude extract is resuspended in a pH 7.8 phosphate buffer (0.05 M) according to a solid-liquid mass ratio of 1:2.5, and loaded onto a G-50 dextran gel column, eluted with a pH 7.8 phosphate buffer (0.05 M) for 2.5 BV, and then eluted with a pH 7.8 phosphate buffer (0.05 M) containing 0.03 M NH4HCO3 for 3.5 BV, and the eluate is collected; the eluate is loaded into a dialysis bag with a molecular weight cut-off of 1 kDa, and dialyzed with deionized water for 50 h, and the deionized water is changed every 7 h, and freeze-drying is carried out to obtain the Cyperus esculentus L. extracted peptide.

[0031] The preparation method of the tiger claw silver ear extract includes: crushing dried tiger claw silver ear fruiting bodies into powder passing through a 120-mesh sieve, dispersing in deionized water at a material-liquid mass ratio of 1:11, adjusting pH to 4.8, adding cellulase at 1.2% of the mass of the powder, and enzymolysis at 52 DEG C for 1.5 h, adjusting pH to 6.8, adding bromelain at 1.2% of the mass of the powder, and enzymolysis at 53 DEG C for 2.5 h; inactivating the enzyme at 88 DEG C for 12 min, centrifuging at 7000 r / min for 12 min, collecting the supernatant, ultrafiltrating through a 3-kDa ultrafiltration membrane, collecting components below 3 kDa, and freeze-drying to obtain the tiger claw silver ear extract.

[0032] The preparation method of the Gangdien root-Milletia reticulata root-Cyathula officinalis root composite extract includes: crushing the composite powder passing through an 80-mesh sieve at a Gangdien root:Milletia reticulata root:Cyathula officinalis root mass ratio of 3.5:1.5:2.5; adding 72vol% ethanol aqueous solution to the composite powder at a material-liquid mass ratio of 1:11, and ultrasonically extracting at 48 DEG C, 300 W and 30 kHz for 1.5 h, and centrifuging at 7000 r / min for 12 min to obtain extract A and precipitate dregs A; adding 78vol% ethanol aqueous solution to the precipitated dregs A at a material-liquid mass ratio of 1:11, and refluxing at 78 DEG C for 1.5 h, and centrifuging at 7000 r / min for 12 min to obtain extract B; combining the extract A and the extract B, and concentrating at 53 DEG C under reduced pressure to obtain a solid, dispersing the solid in 2.8 times the mass of deionized water, adjusting pH to 5.5, and loading onto a polyamide resin column with a mesh size of 80-120, washing with deionized water for 2.5 BV to remove impurities, washing with 35vol% ethanol aqueous solution containing 0.12 M NH4OH for 1.5 BV to remove impurities, eluting with 82vol% ethanol aqueous solution containing 0.25 M NH4OH for 3.5 BV, collecting the eluate, concentrating at 53 DEG C under reduced pressure, dispersing the solid in 2.8 times the mass of 65vol% ethanol aqueous solution, loading onto an LH-60 sephadex column, eluting with 85vol% ethanol aqueous solution for 3.5 BV, collecting the eluate, concentrating at 53 DEG C under reduced pressure, and vacuum drying at 48 DEG C to obtain the Gangdien root-Milletia reticulata root-Cyathula officinalis root composite extract.

[0033] 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.

[0034] The preparation method of the above-mentioned skin regeneration and repair drug includes the following steps:

[0035] 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.

[0036] Example 2

[0037] 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.

[0038] The preparation method of the Cyperus esculentus extracted peptide comprises the following steps: crushing the Cyperus esculentus cake after oil pressing into cake powder passing through a 60-mesh sieve, dispersing the cake powder in a pH 4.5 acetic acid-sodium acetate buffer (0.1 M) according to a material-liquid mass ratio of 1:12, adding 1.5% cellulase of the mass of the cake powder, and carrying out enzymolysis at 50 DEG C for 3 h; adjusting the pH to 8.0, adding 1.5% alpha-chymotrypsin of the mass of the cake powder, and carrying out enzymolysis at 35 DEG C for 2 h; inactivating the enzyme at 80 DEG C for 15 min, adjusting the pH to 6.0 after cooling, adding 1.0% ficin of the mass of the cake powder, and carrying out enzymolysis at 55 DEG C for 1.5 h; inactivating the enzyme at 80 DEG C for 15 min, centrifuging at 6000 r / min for 15 min, collecting the supernatant, filtering the supernatant through a filter membrane with a pore size of 0.45 mu m, taking the filtrate, and freeze-drying to obtain a crude extract; resuspending the crude extract in a pH 8.0 phosphate buffer (0.05 M) according to a material-liquid mass ratio of 1:2, loading the crude extract into a G-50 dextran gel column, eluting 3 BV of the crude extract with a pH 8.0 phosphate buffer (0.05 M), then eluting 4 BV of the crude extract with a pH 8.0 phosphate buffer (0.05 M) containing 0.02 M NH4HCO3, and collecting the eluate; loading the eluate into a dialysis bag with a molecular weight cut-off of 1 kDa, dialyzing the eluate in deionized water for 48 h, replacing the deionized water every 8 h, and freeze-drying to obtain the Cyperus esculentus extracted peptide.

[0039] The preparation method of the tiger claw white fungus extract comprises the following steps: crushing dried tiger claw white fungus fruiting bodies into powder passing through a 120-mesh sieve, dispersing the powder in deionized water according to a material-liquid mass ratio of 1:12, adjusting the pH to 4.5, adding 1.5% cellulase of the mass of the powder, and carrying out enzymolysis at 50 DEG C for 2 h, adjusting the pH to 6.5, adding 1.5% bromelain of the mass of the powder, and carrying out enzymolysis at 50 DEG C for 3 h; inactivating the enzyme at 85 DEG C for 15 min, centrifuging at 6000 r / min for 15 min, collecting the supernatant, ultrafiltering the supernatant through a 3-kDa ultrafiltration membrane, collecting components below 3 kDa, and freeze-drying to obtain the tiger claw white fungus 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 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*.

[0041] 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.

[0042] The preparation method of the above-mentioned skin regeneration and repair drug includes the following steps:

[0043] The Cyperus esculentus extracted peptide, the tiger palm silver ear extract, the compound extract of the root of the mountain vine, the millettia pinnata and the cyathula officinalis, and the pomegranate peel polyphenol are mixed according to the mass fraction, and then added into the modified hyaluronic acid-chitosan hydrogel (pre-mixed and dispersed in 10% hydrogel, and then added into the remaining hydrogel), stirred uniformly, and adjusted to pH 7.2 to obtain a drug composition; the drug composition is used for preparing a drug for repairing skin wounds.

[0044] Example 3

[0045] A drug for skin regeneration and repair, the drug comprising the following raw materials in mass fraction: Cyperus esculentus extracted peptide 8 parts, tiger palm silver ear extract 4.5 parts, compound extract of the root of the mountain vine, the millettia pinnata and the cyathula officinalis 10 parts, pomegranate peel polyphenol 1 part, modified hyaluronic acid-chitosan hydrogel 90 parts, and the pH is adjusted to 6.8.

[0046] The preparation method of the Cyperus esculentus extracted peptide comprises the following steps: the oil-pressed Cyperus esculentus cake is crushed into cake powder passing through an 80-mesh sieve, and then dispersed in a pH 5.5 acetic acid-sodium acetate buffer (0.1M) according to a material-liquid mass ratio of 1:10; 0.8% cellulase of the mass of the cake powder is added, and enzymolysis is carried out at 55℃ for 2h; the pH is adjusted to 8.8, 0.8% alpha-chymotrypsin of the mass of the cake powder is added, and enzymolysis is carried out at 40℃ for 1.5h; enzyme inactivation is carried out at 85℃ for 10min; after cooling, the pH is adjusted to 6.5, 0.5% ficin of the mass of the cake powder is added, and enzymolysis is carried out at 60℃ for 1h; enzyme inactivation is carried out at 85℃ for 10min, and centrifugation is carried out at 8000r / min for 10min; the supernatant is collected, filtered through a filter membrane with a pore size of 0.45μm, and then the filtered liquid is obtained and freeze-dried to obtain a crude extract; the crude extract is resuspended in a pH 7.5 phosphate buffer (0.05M) according to a material-liquid mass ratio of 1:3, and then loaded into a G-50 dextran gel column; the column is eluted with a pH 7.5 phosphate buffer (0.05M) for 2BV, and then eluted with a pH 7.5 phosphate buffer (0.05M) containing 0.04M NH4HCO3 for 3BV; the eluate is collected; the eluate is loaded into a dialysis bag with a molecular weight cut-off of 1kDa, and then dialyzed with deionized water for 52h; the deionized water is replaced every 6h; and freeze-drying is carried out to obtain the Cyperus esculentus extracted peptide.

[0047] The preparation method of the tiger palm silver ear extract comprises the following steps: the dried tiger palm silver ear fruiting body is crushed into powder passing through a 150-mesh sieve, and then dispersed in deionized water according to a material-liquid mass ratio of 1:10; the pH is adjusted to 5.0; 0.8% cellulase of the mass of the powder is added, and enzymolysis is carried out at 55℃ for 1h; the pH is adjusted to 7.0; 0.8% bromelain of the mass of the powder is added, and enzymolysis is carried out at 55℃ for 2h; enzyme inactivation is carried out at 90℃ for 10min; centrifugation is carried out at 8000r / min for 10min; the supernatant is collected, ultrafiltered through a 3kDa ultrafiltration membrane, and then the components below 3kDa are collected; and freeze-drying is carried out to obtain the tiger palm silver ear extract.

[0048] The preparation method of the compound extract of Radix Rubus Chingii, Spatholobus suberectus and Cyathula officinalis includes: crushing the compound powder into a compound powder with a mesh size of 60 according to the mass ratio of Radix Rubus Chingii:Spatholobus suberectus:Cyathula officinalis = 4:1:3; adding 70vol% ethanol aqueous solution to the compound powder according to the mass ratio of material to liquid = 1:12, and extracting at 50°C, 300W and 40kHz for 1h, and centrifuging at 8000r / min for 10min to obtain extract A and precipitate residue A; adding 75vol% ethanol aqueous solution to the precipitate residue A according to the mass ratio of material to liquid = 1:12, and refluxing at 80°C for 1h, and centrifuging at 8000r / min for 10min to obtain extract B; combining the extract A and the extract B, and concentrating at 55°C under reduced pressure to obtain a solid, dispersing the solid with 2.5 times the mass of deionized water, adjusting the pH to 6.0, and loading onto a polyamide resin column with a mesh size of 80-120, washing with deionized water for 2BV to remove impurities, washing with 30vol% ethanol aqueous solution containing 0.15M NH4OH for 2BV to remove impurities, eluting with 85vol% ethanol aqueous solution containing 0.2M NH4OH for 3BV, collecting the eluate, concentrating at 55°C under reduced pressure to obtain a solid, dispersing the solid with 2.5 times the mass of 70vol% ethanol aqueous solution, and loading onto an LH-60 Sephadex gel column, eluting with 80vol% ethanol aqueous solution for 4BV, collecting the eluate, concentrating at 50°C under reduced pressure, and vacuum drying at 45°C to obtain the compound extract of Radix Rubus Chingii, Spatholobus suberectus and Cyathula officinalis.

[0049] The preparation method of the modified hyaluronic acid-chitosan hydrogel includes: preparing a 3wt% hyaluronic acid solution with deionized water, adjusting the pH to 11.0 with 0.8M NaOH aqueous solution, adding chloroacetic acid according to the mass ratio of hyaluronic acid to chloroacetic acid = 1:0.03, stirring at 200rpm for 5h at 65°C, loading into a dialysis bag with a molecular weight cutoff of 10kDa, dialyzing with deionized water for 48h, changing the deionized water every 8h, and freeze-drying to obtain carboxymethyl hyaluronic acid; dissolving the carboxymethyl hyaluronic acid and chitosan (degree of deacetylation 90%) in acetic acid buffer (0.1M, pH 5.5) according to the mass ratio of carboxymethyl hyaluronic acid to chitosan = 1:1, to a total concentration of 5wt%, and stirring until completely dissolved; adding glutaraldehyde with a final concentration of 0.1wt%, and crosslinking at 30°C and 200rpm for 3h to form a hydrogel, loading into a dialysis bag with a molecular weight cutoff of 10kDa, dialyzing with deionized water for 72h, changing the deionized water every 8h, and adjusting the viscosity to 2000mPa·s to obtain the modified hyaluronic acid-chitosan hydrogel.

[0050] The preparation method of the above-mentioned skin regeneration and repair drug includes the following steps:

[0051] The Cyperus esculentus extracted peptide, the tiger palm silver ear extract, the compound extract of the root of the Gangdian, the Millettia pinnata and the Cyathula officinalis, and the pomegranate peel polyphenol are mixed by mass fraction, added into the modified hyaluronic acid-chitosan hydrogel (pre-mixed and dispersed in 20% hydrogel, and then added into the remaining hydrogel), stirred uniformly, and adjusted to pH 6.8 to obtain a drug composition; the drug composition is used for preparing a drug for repairing a skin wound.

[0052] Comparative Example 1

[0053] The difference from Example 1 is that the Cyperus esculentus extracted peptide is modified to 2 parts, and the tiger palm silver ear extract is modified to 10 parts.

[0054] Comparative Example 2

[0055] The difference from Example 1 is that the tiger palm silver ear extract is modified to 11 parts, and the compound extract of the root of the Gangdian, the Millettia pinnata and the Cyathula officinalis is modified to 3 parts.

[0056] Comparative Example 3

[0057] The difference from Example 1 is that the Cyperus esculentus extracted peptide is modified to 12 parts, and the compound extract of the root of the Gangdian, the Millettia pinnata and the Cyathula officinalis is modified to 4 parts.

[0058] Comparative Example 4

[0059] The difference from Example 1 is that in the preparation of the Cyperus esculentus extracted peptide, the α-chymotrypsin is replaced by trypsin.

[0060] Comparative Example 5

[0061] The difference from Example 1 is that in the preparation of the Cyperus esculentus extracted peptide, the bromelain is replaced by bromelain.

[0062] Comparative Example 6

[0063] The difference from Example 1 is that in the preparation of the Cyperus esculentus extracted peptide, the G-50 dextran gel is replaced by G-75 dextran gel.

[0064] Comparative Example 7

[0065] The difference from Example 1 is that in the preparation of the tiger palm silver ear extract, the bromelain is replaced by papain.

[0066] Comparative Example 8

[0067] The difference from Example 1 is that in the preparation of the compound extract of the root of the Gangdian, the Millettia pinnata and the Cyathula officinalis, the mass ratio of the root of the Gangdian, the Millettia pinnata and the Cyathula officinalis is modified to 1:4:2.5 mass ratio.

[0068] Comparative Example 9

[0069] The difference from Example 1 is that the polyamide resin is replaced by AB-8 macroporous adsorption resin in the preparation of the Radix Rubus parvifolii-Acanthopanax gracilistylus-Radix Cyathulae composite extract.

[0070] The sources of raw materials involved in the above examples and comparative examples are as follows: the pomegranate peel polyphenol is from Shaanxi Jianhe Biotechnology Co., Ltd., and is of medical grade, mainly containing 40% ellagic acid and 50% polyphenol. The cellulase is from Jiangsu Changjing Bioengineering Co., Ltd. The α-chymotrypsin is from Guangzhou He Wei Pharmaceutical Technology Co., Ltd., model CD108568. The ficin is from Hubei Qi Fei Pharmaceutical Chemical Co., Ltd. The G-50 Sephadex gel is from Shanghai Yuan Ye Biotechnology Co., Ltd. The bromelain is from Shaanxi Yunqi Biotechnology Co., Ltd., model W301. The polyamide resin is from Shanghai Yuan Ye Biotechnology Co., Ltd., 80-120 mesh, model S14145. The LH-60 Sephadex gel is from Shanghai Yuan Ye Biotechnology Co., Ltd. The hyaluronic acid is from Shaanxi Angxi Biotechnology Co., Ltd., with a medium molecular weight of 110 kDa (Dalton). The chloroacetic acid is from Shandong Qiangsen Chemical Co., Ltd., a medical synthetic intermediate. The chitosan (degree of deacetylation 90%) is from Xi'an Jimei Biotechnology Co., Ltd. The trypsin is from Guangzhou He Wei Pharmaceutical Technology Co., Ltd., model CD101204. The papain is from Shanxi Ruisi Biotechnology Co., Ltd., model RS-0516-252. The G-75 Sephadex gel is from Shanghai Yuan Ye Biotechnology Co., Ltd. The AB-8 macroporous adsorption resin is from Shanghai Yuan Ye Biotechnology Co., Ltd.

[0071] The above pharmaceutical active ingredients are detected, and the active ingredients are Chikuzen root-Acanthopanax gracilistylus-Radix Cyathulae composite extract, pomegranate peel polyphenol, and oil palm extract peptide, according to the respective mass fraction ratio.

[0072] I. Fibroblast proliferation promotion detection:

[0073] Sample preparation: the active ingredient mixture is diluted to 250 μg / mL with DMEM culture medium containing 10% fetal bovine serum. A blank group containing only the culture medium (used for zero setting) and a negative control group containing cells and culture medium are set.

[0074] The detection method includes: taking human dermal fibroblasts (HDF) in the logarithmic growth phase, adjusting the cell concentration to 5×10 4cells / mL. Inoculate 96-well plates with 100 μL of the cell suspension (i.e. 5000 cells / well). Incubate at 37°C in a 5% CO2 incubator for 24 h to allow the cells to adhere completely. Carefully discard the original medium and add 100 μL of the test sample solution at 250 μg / mL to the experimental group, and add 100 μL of DMEM medium containing 10% fetal bovine serum to the negative control group, with 5 replicates in each group. Return the plates to the incubator and continue incubation for 48 h. Add 10 μL of CCK-8 solution directly to each well, mix gently, and continue incubation for 2 h. Measure the absorbance OD value at 450 nm using a microplate reader. Cell proliferation rate (%) = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] x 100%.

[0075] II. Detection of vascular endothelial cell migration

[0076] Sample preparation: Dilute the active ingredient mixture to 250 μg / mL using serum-free endothelial cell culture medium (ECM). Use serum-free medium as the blank control.

[0077] The detection method comprises: inoculate human umbilical vein endothelial cells (HUVEC) into a 6-well plate and culture until a dense monolayer is formed. Use a 200 μL sterile pipette tip (flat tip end to ensure consistent scratch width) to make a straight line scratch vertically on the bottom of each well. Gently rinse with PBS for 3 times to completely remove the scratched cells. Add 2 mL of the test sample solution at 250 μg / mL to the experimental group, and add 2 mL of serum-free ECM medium to the blank control group, with 3 independent replicates in each group. Incubate in a 37°C, 5% CO2 incubator. At 0 h and 24 h, randomly select 3 fixed fields of view for each scratch under a 100x inverted microscope, and measure the scratch area of each field of view using ImageJ software. Scratch healing rate (%) = (0 h scratch area - 24 h scratch area) / 0 h scratch area x 100%.

[0078] III. Detection of antibacterial activity

[0079] Sample preparation: Dilute the active ingredient mixture to 0.5 mg / mL of the test sample solution using sterile normal saline, and filter sterilize through a 0.22 μm filter membrane. Use normal saline as the blank control. Test strains: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853).

[0080] The detection method comprises: prepare Mueller-Hinton (MH) agar plates with a thickness of 4 mm. Prepare 1.5 x 10 8CFU / mL of bacterial suspension, using a sterile cotton swab to dip the bacterial suspension, along the edge of the plate to the center of the uniform 3 times, each time after the rotation of the plate 60 °, and finally along the edge of the plate 1 circle, to ensure that the bacteria liquid evenly distributed. After standing for 5 min, with a sterile forceps 6 mm in diameter sterile Oxford cup gently placed on the agar surface (each plate placed 3 Oxford cup parallel sample, spacing 25 mm or more, to avoid overlapping of the inhibition zone). With a micropipette to each Oxford cup slowly add 200 μL of 0.5 mg / mL sample solution, blank control group added 200 μL sterile saline, to avoid liquid overflow. The plate upright placed in a 37 °C incubator, incubation for 24 h. Measure the diameter of the inhibition zone (accurate to 0.1 mm, when the longest diameter and the shortest diameter of the average value of the inhibition zone is measured).

[0081] Four, inflammation factor expression inhibition detection:

[0082] Sample preparation: the active ingredient mixture was diluted to 250 μg / mL with RPMI-1640 medium containing 10% fetal bovine serum. Set the blank control group (complete medium), model group (containing 1 μg / mL LPS complete medium).

[0083] The detection method includes: human monocyte macrophage (THP-1) adjusted concentration to 2×10 5 cells / mL, inoculated in 24 well plate, 1 mL per well. Add 100 nM PMA induced 48 h, so that it is differentiated into macrophages. Discard the old culture medium, gently washed with PBS once, to avoid residual PMA affect the subsequent experiment. Except for the blank control group, the rest of the groups were added with 1 μg / mL LPS complete medium 500 μL, stimulated for 4 h to establish an inflammation model. Discard the LPS containing medium, the experimental group was replaced with 1 mL of complete medium containing 250 μg / mL sample solution and 1 μg / mL LPS, the model group was added with 1 mL of complete medium containing 1 μg / mL LPS, and the blank control group was replaced with 1 mL of complete medium. Continue to culture for 24 h. Collect the cell supernatant, centrifuge at 1000 ×g for 10 min, and take the supernatant. Each group of 3 parallel samples. According to the TNF-α, IL-6 ELISA kit instructions for detection, according to the standard curve to get the concentration of inflammatory factors in the sample. Inflammatory factor inhibition rate (%) = [(inflammatory model group concentration-test group concentration) / inflammatory model group concentration] × 100%.

[0084] Five, collagen synthesis detection:

[0085] Sample preparation: the active ingredient mixture was diluted to 250 μg / mL with DMEM medium containing 10% fetal bovine serum. Set the blank control group containing cells and medium.

[0086] The detection methods include: HDF cells at 2.5 × 10⁻⁶ 4 Cells 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%.

[0087] Table 1. Test Results (Average Values)

[0088]

[0089] In the vascular endothelial cell migration assay, the scratch healing rate in the blank control group was 21.8%.

[0090] 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.

[0091] Comparative Example 1 to Comparative Example 3 (imbalanced component ratio): Insufficient dosage of individual components leads to poor synergistic effect, and excessive dosage of individual components leads to competition for binding sites, disrupting the balance of absorption of individual components, reducing the synergistic effect, and excessive components reaching saturation absorption, the remaining drugs cannot improve efficacy, and excessive impurities are introduced, affecting cell activity. The indicators change and decrease.

[0092] Comparative Example 4 to Comparative Example 5 (α-chymotrypsin is replaced by trypsin; or ficin is replaced by bromelain): The specific hydrolysis cleavage sites of trypsin and α-chymotrypsin are different, resulting in differences in peptide chain structure, and the compatibility of ficin is also different. The final small molecule peptides are different, the efficacy direction and effect are different, leading to changes in indicators, and non-optimal efficacy. Similarly, the specific hydrolysis cleavage sites of bromelain and ficin are different, and the compatibility with trypsin is also different. The final small molecule peptides are different, the efficacy direction and effect are different, leading to changes in indicators. This indicates that for the hydrolysis of cyperus esculentus protein, the combination of α-chymotrypsin and ficin is the optimal scheme, which can obtain more effective components and improve efficacy.

[0093] Comparative Example 6 (G-50 dextran gel is replaced by G-75 dextran gel): The separation range of G-75 gel is different from that of G-50 dextran gel, and for the purification of cyperus esculentus peptide, G-50 dextran gel can better enrich effective components; G-75 gel separation will lose part of the effective components, and retain different impurity peptides, which affect cell activity and reduce antibacterial properties, and the inflammation inhibitory property is also poor, leading to a decrease in performance effect.

[0094] Comparative Example 7 (in the preparation of tiger palm silver ear extract, bromelain is replaced by papain): The hydrolysis cleavage sites of papain and bromelain are different, and bromelain has a better effect of releasing polysaccharides and functional peptides, and produces fewer impurity peptides. After replacing bromelain with papain, the composition of the final extraction product changes, leading to a decrease in efficacy.

[0095] Comparative Example 8 (insufficient proportion of gangdeng root, excessive proportion of kudzu vine): Gangdeng root is the most active anti-inflammatory component in the complex extract, and its flavonoid components directly inhibit the transcription of TNF-α and IL-6. When the dosage is reduced, the anti-inflammatory core component is insufficient, and although kudzu vine contains saponin components, its anti-inflammatory efficiency is insufficient, and its main function is to improve microcirculation, and it cannot replace the strong anti-inflammatory effect. At the same time, the reduction of gangdeng root destroys the synergy with chuan niuxi, and the migration of vascular endothelial cells is blocked, leading to a decrease in the rate of wound healing and inflammation inhibition, and a decrease in antibacterial synergy, resulting in a decrease in comprehensive efficacy.

[0096] Comparative Example 9 (polyamide resin is replaced by AB-8 macroporous adsorption resin, and LH-60 dextran gel is replaced by G-50 dextran gel): AB-8 macroporous adsorption resin and polyamide resin have different purified components, and AB-8 macroporous adsorption resin is used for purifying components of Radix Acanthopanacis Senticosi-Caulis Spatholobi- Radix Cyathulae composite extract, which is difficult to remove harmful components, and the separation rate of effective components is low. LH-60 gel is a high-resolution gel, which can separate components with similar molecular weights and purify active monomers accurately; G-50 gel has low separation precision and cannot effectively separate target components and impurities with similar structures, resulting in a decrease in the purity of active components of the composite extract. At the same time, G-50 gel has a large elution volume, the concentration of target components is diluted, part of the active components are non-specifically combined with the gel matrix, the recovery rate is reduced, and finally the indicators are reduced.

Claims

1. A medicament for skin regeneration and repair, characterized by, The medicine is composed of the following raw materials in parts by mass: Cyperus esculentus L. extracted peptide 6-8 parts, Tremella fuciformis Berk. extract 4.5-5.5 parts, Saurauia apiona root-Acanthopanax gracilistylus-Taliniella sinensis complex extract 8-10 parts, Punica granatum L. pericarp polyphenol 1-3 parts and modified hyaluronic acid-chitosan hydrogel 80-90 parts, and the pH is adjusted to 6.8-7.2; The modified hyaluronic acid-chitosan hydrogel is a biocompatible hydrogel formed by carboxymethylated hyaluronic acid and chitosan under cross-linking of glutaraldehyde; The preparation method of the Cyperus esculentus L. extracted peptide comprises the following steps: crushing Cyperus esculentus L. cake into cake powder, dispersing the cake powder in a pH 4.5-5.5 acetic acid-sodium acetate buffer, adding cellulase, and carrying out enzymolysis at 50-55 DEG C for 2-3 h; adjusting the pH to 8.0-8.8, adding alpha-chymotrypsin, and carrying out enzymolysis at 35-40 DEG C for 1.5-2 h; inactivating the enzyme, adjusting the pH to 6.0-6.5 after cooling, adding ficin, and carrying out enzymolysis at 55-60 DEG C for 1-1.5 h; inactivating the enzyme, centrifuging, collecting the supernatant, filtering, taking the filtrate, and freeze-drying to obtain a crude extract; resuspending the crude extract in a pH 7.5-8.0 phosphate buffer, loading the resuspension onto a G-50 dextran gel column, washing the column with a pH 7.5-8.0 phosphate buffer for 2-3 BV, then eluting the column with a pH 7.5-8.0 phosphate buffer containing 0.02-0.04 M NH4HCO3 for 3-4 BV, and collecting the eluate; loading the eluate into a dialysis bag, dialyzing with deionized water, and freeze-drying to obtain the Cyperus esculentus L. extracted peptide; The preparation method of the Tremella fuciformis Berk. extract comprises the following steps: crushing Tremella fuciformis Berk. into powder, dispersing the powder in deionized water, and adjusting the pH to 4.5-5.0; adding cellulase, and carrying out enzymolysis at 50-55 DEG C for 1-2 h; adjusting the pH to 6.5-7.0, adding bromelain, and carrying out enzymolysis at 50-55 DEG C for 2-3 h; inactivating the enzyme, centrifuging, collecting the supernatant, ultrafiltering the supernatant with an ultrafiltration membrane, collecting components below 3 kDa, and freeze-drying to obtain the Tremella fuciformis Berk. extract; The preparation method of the compound extract of Radix Rubus parvifolii-Milletia reticulata-Cyathostemon fortunei comprises the following steps: crushing Radix Rubus parvifolii-Milletia reticulata-Cyathostemon fortunei into a compound powder according to a mass ratio of (3-4):(1-2):(2-3), adding 70vol%-75vol% ethanol water solution, ultrasonic extraction, centrifugation, to obtain extract A and precipitate dregs A; adding 75vol%-80vol% ethanol water solution to the precipitated dregs A, reflux extraction, centrifugation, to obtain extract B; combining the extract A and the extract B, reducing pressure concentration to obtain a solid, dispersing the solid with deionized water, adjusting the pH to 5.0-6.0, loading onto a polyamide resin column, washing with 2BV-3BV deionized water to remove impurities, washing with 1.5BV-2BV 30vol%-40vol% ethanol water solution containing 0.1M-0.15M NH4OH to remove impurities, eluting with 3BV-4BV 80vol%-85vol% ethanol water solution containing 0.2M-0.3M NH4OH, collecting the eluate, reducing pressure concentration, dispersing the solid with 60vol%-70vol% ethanol water solution, loading onto an LH-60 sephadex column, eluting with 3BV-4BV 80vol%-90vol% ethanol water solution, collecting the eluate, reducing pressure concentration, vacuum drying, to obtain the compound extract of Radix Rubus parvifolii-Milletia reticulata-Cyathostemon fortunei.

2. The drug for skin regeneration and repair according to claim 1, characterized in that, In the preparation method of the Elaeagnus umbellata extract peptide, the particle size of the meal powder is 60-80 mesh; the amount of the acetic acid-sodium acetate buffer solution is 10-12 times the mass of the meal powder; the amount of the cellulase added is 0.8-1.5% of the mass of the meal powder; the amount of the α-chymotrypsin added is 0.8-1.5% of the mass of the meal powder; the amount of the ficin added is 0.5-1.0% of the mass of the meal powder; the enzyme inactivation is performed at 80-85°C for 10-15 min; the centrifugation is performed at 6000-8000 r / min for 10-15 min; the filtration is performed by using a filter membrane with a pore size of 0.45 μm; and the molecular weight cut-off of the dialysis bag is 1 kDa.

3. The drug for skin regeneration and repair according to claim 1, characterized in that, In the preparation method of the Morinda officinalis-Tremella fuciformis extract, the Morinda officinalis-Tremella fuciformis is dried Morinda officinalis-Tremella fuciformis fruiting body; the particle size of the powder is 120-150 mesh; the amount of the cellulase added is 0.8-1.5% of the mass of the powder; the amount of the bromelain added is 0.8-1.5% of the mass of the powder; the enzyme inactivation is performed at 85-90°C for 10-15 min; and the centrifugation is performed at 6000-8000 r / min for 10-15 min.

4. The drug for skin regeneration and repair according to claim 1, characterized in that, In the preparation method of the compound extract of *Rhizoma Cynanchi* root, *Spatholobus suberectus*, and *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.

5. The drug for skin regeneration and repair 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.

6. The drug for skin regeneration and repair according to claim 5, 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.

7. The method of claim 1, wherein the skin regenerative medicine is prepared by the steps of: a) mixing the skin regenerative medicine of claim 1 with a pharmaceutically acceptable carrier; b) sterilizing the mixture; and c) filling the mixture into a container. 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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