Composition for promoting wound healing and preparation method thereof

Abstract

The invention provides a composition for promoting wound healing and a preparation method thereof, and belongs to the technical field of medicines. Comprising the following raw materials in parts by weight: 3-5 parts of pseudo-ginseng, 1-3 parts of turmeric, 3-7 parts of astragalus membranaceus, 2-5 parts of herba epimedii, 3-5 parts of rhizoma bletillae, 45-60 parts of lecithin, 10-15 parts of cholesterol, 2-4 parts of ginseng, 0.5-1 part of a surfactant, 0.1-0.2 part of protein peptide, 10-15 g of agarose, 0.2-0.3 part of tetramethylpiperidine oxide, 1-1.5 parts of sodium bromide, 2-4 parts of mussel mucin and 0.1-0.2 part of zinc citrate. The composition is safe, free of side effects, free of chemical cross-linking agent addition, biodegradable, capable of effectively preventing wound reinfection, achieving intelligent drug delivery and promoting rapid healing of wounds, suitable for wound repair in various scenes and wide in application range.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a composition for promoting wound healing and its preparation method. Background Technology

[0002] Wound healing is a complex and dynamic process, generally divided into four stages: hemostasis, inflammation, tissue formation, and remodeling, with each stage highly coordinated. Under physiological conditions, during the hemostasis and inflammation phase, macrophages accumulate and engulf bacteria and damaged tissue, typically requiring 72 hours. This is followed by a proliferation phase with the accumulation of numerous cells and connective tissue, the formation of new granulation tissue, and angiogenesis. Angiogenesis occurs within hours to 4 days after tissue injury. Platelets, extracellular matrix, and macrophages produced after wound formation secrete growth factors that bind to their receptors, activating intracellular signaling cascades. This causes activated endothelial cells to secrete proteolytic enzymes, dissolving the basement membrane and stimulating the proliferation and migration of various endothelial cells to the wound site, constructing vascular lumens and forming new capillaries, a process known as "budding." The channels of these newly formed capillary buds interconnect, forming vascular rings. They then absorb pericytes and smooth muscle cells, differentiating into mature arteries and veins, providing the material basis for subsequent collagen fiber formation and tissue remodeling. This establishes the interrelationships between the stages, enabling dynamic repair and timely wound transition.

[0003] Under pathological conditions, tissue repair is hindered, and wound healing is delayed, primarily manifested as a persistent inflammatory response. The wound site contains a large number of neutrophils and macrophages, affecting the expression of endogenous growth factors, restricting endothelial cell proliferation, leading to insufficient angiogenesis, delaying subsequent tissue re-epithelialization, and hindering keratinocyte formation and fibroblast protein synthesis, migration, and proliferation. Simultaneously, the loss of the vascular network reduces the transport of oxygen and nutrients and the removal of metabolic waste, causing microcirculatory disturbances in chronic wounds. The large amount of missing collagen fibers cannot reconstruct the damaged area, delaying wound healing and leading to chronic skin ulcers. If these ulcers persist, there is a risk of cancerous transformation, increasing the socioeconomic burden.

[0004] Therefore, in the pathological context of chronic ulcers, timely completion of each stage of wound healing is crucial for repairing damaged tissues. Current research on biological agents often employs combination therapy for wound repair, such as alprostadil combined with recombinant human epidermal growth factor (rhGGF) for treating diabetic skin ulcers, nano-silver dressings combined with rhGGF gel for treating deep second-degree burns, compound polymyxin B ointment combined with rhGGF for treating refractory diabetic skin ulcers, or rhGGF combined with phototherapy M22 for treating common acne-related atrophic scars. However, research data shows a significant discrepancy between animal experimental results and clinical efficacy for biological agents containing growth factors such as vascular endothelial growth factor (VEGF), PDGF, and epidermal growth factor (EGF). In clinical practice, some patients experience adverse reactions such as local erythema, irritation, and itching when using topical rhGGF preparations. Furthermore, chronic wounds prolong the course of the disease, leading to drug resistance, and the prolonged course of the disease increases the psychological and economic burden on patients. Summary of the Invention

[0005] The purpose of this invention is to provide a composition for promoting wound healing and its preparation method. The composition is safe, has no side effects, contains no chemical cross-linking agents, and is biodegradable. It effectively prevents wound reinfection, achieves pH-responsive intelligent drug delivery, promotes rapid wound healing, and has good patient compliance.

[0006] The technical solution of this invention is implemented as follows: This invention provides a composition for promoting wound healing, which is prepared from the following raw materials in parts by weight: 3-5 parts of Panax notoginseng, 1-3 parts of turmeric, 3-7 parts of Astragalus membranaceus, 2-5 parts of Epimedium, 3-5 parts of Bletilla striata, 45-60 parts of lecithin, 10-15 parts of cholesterol, 2-4 parts of ginseng, 0.5-1 part of surfactant, 0.1-0.2 parts of protein peptide, 10-15 g of agarose, 0.2-0.3 parts of tetramethylpiperidine oxide, 1-1.5 parts of sodium bromide, 2-4 parts of mussel adhesive protein, and 0.1-0.2 parts of zinc citrate.

[0007] The present invention further protects a method for preparing the above-mentioned wound-healing composition, comprising the following steps: S1. Mix and pulverize Panax notoginseng, turmeric, Astragalus membranaceus, Epimedium, and Bletilla striata, add water, heat to boiling and extract, filter, dry the filtrate to obtain an aqueous extract, and keep the residue for use; S2. Add the filter residue to an organic solvent, heat and reflux to extract, filter, recover the organic solvent from the filtrate, add water to precipitate, filter, wash, dry, and obtain the organic extract; S3. Dissolve lecithin and cholesterol in a dichloromethane-ethanol mixture, add the organic extract, stir and mix well, remove the solvent by rotary evaporation, add PBS buffer solution, sonicate to hydrate, centrifuge, take the supernatant, filter, and obtain liposomes; S4. Wash the ginseng, add PBS buffer, homogenize, filter, add protease inhibitor, adjust the pH value, and obtain ginseng exosomes by gradient centrifugation. S5. Mix liposomes and ginseng exosomes evenly, sonicate at room temperature, and incubate by heating to obtain liposome-exosome complex; S6. Add surfactant to the liposome-exosome complex until the solution is clear, add protein peptide, shake at room temperature, and then remove surfactant using Bio-Bead SM-2 to form protein complex; S7. Dissolve agarose in water, heat and stir until homogeneous, cool, add tetramethylpiperidine oxide and sodium bromide, stir to dissolve, add sodium hypochlorite solution dropwise, adjust pH value, react at room temperature, add ethanol to terminate the reaction, adjust pH value, precipitate, filter, wash, and dry to obtain carboxylated agarose. S8. Carboxylated agarose and mussel adhesive protein are mixed and added to water, heated to dissolve, then water extract and protein complex are added and mixed evenly. Zinc citrate is added, and the mixture is stirred and mixed evenly. After cooling, a gel is formed to obtain a composition that promotes wound healing.

[0008] As a further improvement of the present invention, the heating and boiling extraction time in step S1 is 3-5 hours; the organic solvent in step S2 is 95 wt% ethanol, and the heating and reflux extraction time is 2-4 hours.

[0009] As a further improvement of the present invention, in step S3, the volume ratio of dichloromethane to ethanol in the dichloromethane-ethanol mixed solution is 1-3:1, the pH of the PBS buffer solution is 7.4, the ultrasonic hydration time is 40-60 min, the centrifugation speed is 3000-5000 r / min and the time is 20-30 min, and the filtration is performed using a 0.22 μm microporous filter membrane for granulation filtration.

[0010] As a further improvement of the present invention, the pH of the PBS buffer in step S4 is 7.4, the amount of protease inhibitor added is 2-3 wt%, and the gradient centrifugation method is specifically as follows: centrifuge at 400g, 800g, and 15000g for 15-25 min in sequence, take the supernatant, then centrifuge at 100000g for 50-70 min, take the precipitate, and resuspend the precipitate in 15-25 mmol / L Tris-HCl solution.

[0011] As a further improvement of the present invention, the room temperature ultrasonic treatment in step S5 for 2-3 minutes is performed by ultrasonic treatment for 30 seconds and stopping for 30 seconds, and the heating incubation temperature is 37°C and the incubation time is 50-70 minutes.

[0012] As a further improvement of the present invention, the surfactant in step S6 is Brij-35, the sequence of the protein peptide is IKFQFHFD, as shown in SEQ ID NO.1, and the room temperature shaking time is 30-50 min.

[0013] As a further improvement of the present invention, the room temperature reaction time in step S7 is 5-7 hours, and the heating and stirring temperature is 90-95°C.

[0014] As a further improvement of the present invention, the heating temperature in step S8 is 35-45°C.

[0015] The present invention further protects the use of the above-described wound-healing composition in the preparation of a medicament for treating wound healing in chronic ulcer conditions.

[0016] The inventive concept and mechanism of this invention: The aqueous and organic extracts of Panax notoginseng, turmeric, Astragalus membranaceus, Epimedium, and Bletilla striata, obtained through water and ethanol extraction, contain abundant active components. These include water-soluble polysaccharides such as Bletilla striata polysaccharides, Panax notoginseng saponins, ginsenosides, Panax notoginseng polysaccharides, Astragalus membranaceus polysaccharides, Astragalus membranaceus saponins, and Epimedium polysaccharides, as well as fat-soluble icariin, isoflavone glycosides, curcumin, and Panax notoginseng flavonoids. These substances possess antibacterial, analgesic, anti-inflammatory, and tissue-forming properties, which are beneficial for repairing wound tissue and shortening wound healing time. Water-soluble substances can be directly added to the hydrogel system, subsequently releasing the drug and exerting its activity. However, fat-soluble drugs are often insoluble or poorly soluble in hydrogel systems, making them prone to aggregation and precipitation, resulting in poor efficacy and difficulty in achieving the desired effect.

[0017] This invention utilizes liposomes to encapsulate lipid-soluble organic extracts, which, while offering high drug loading capacity and improved water solubility, suffers from limitations such as inability to intelligently release the drug and poor efficacy, thus restricting its application. This invention combines the prepared liposomes with ginseng exosomes to obtain a complex. On one hand, the complex, incorporating ginseng exosomes, exhibits highly effective wound healing, anti-inflammatory, and antioxidant effects. Ginseng exosomes also possess a lipid bilayer membrane structure, facilitating fusion, and contain components such as mRNA, miRNA, and proteins. Plant-derived exosomes also offer advantages such as wide availability, safety, non-toxicity, and low immunogenicity. On the other hand, the complex possesses excellent transdermal delivery capabilities, facilitating the delivery of the loaded drug to the wound for maximum efficacy. The next challenge is controlling the drug release from the complex.

[0018] This invention utilizes surfactant-mediated induction to fuse a pH-responsive octapeptide into the membrane layer of a complex. This octapeptide, IKFQFHFD, is a mature protein peptide that is unstable under acidic conditions, causing membrane rupture and drug release. Furthermore, this octapeptide also exhibits antibacterial activity under acidic conditions, thereby enhancing the wound's anti-infection efficacy. Normal skin is neutral, while the pH of wound skin decreases, creating an acidic wound environment. This allows the protein complex to intelligently release the drug only at the wound site, achieving highly efficient drug utilization.

[0019] Furthermore, the carboxylated agarose and mussel adhesive protein prepared in this invention, after being combined, can form a gel through physical cross-linking at a medium temperature of around 45°C after dissolving through the interaction of carboxyl groups, polysaccharides, and proteins, without requiring high temperatures such as above 70°C. At the same time, zinc ions can also cross-link the agarose and protein chains. Under acidic conditions, the ionic cross-linking is destroyed, and the hydrogel network becomes loose, thereby releasing the water extract and protein complex. In addition, mussel adhesive protein is rich in dopa, which has good adhesion, helping the hydrogel to adhere to the skin surface, making it less likely to fall off, and prolonging the drug administration time.

[0020] The present invention has the following beneficial effects: 1. The entire formulation of this invention is safe, has no side effects, contains no chemical cross-linking agents, and is biodegradable, making the resulting wound-healing composition more convenient, safe, and effective, with better patient compliance.

[0021] 2. The wound healing composition prepared by this invention can effectively prevent wound reinfection, has good antibacterial properties, good skin adhesion, long duration of action, and can achieve intelligent drug delivery, promote rapid wound healing, and is suitable for wound repair in various scenarios with a wide range of applications.

[0022] 3. The raw materials of this invention are widely available, the preparation method is simple, it is easy to realize industrial application, the storage conditions are simple, and it is easy to transport and use, thus having broad application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1Images of the protein complex obtained in Example 1 are shown, where A is an appearance image of the protein complex, B is a photograph showing the Tyndall effect after blue laser irradiation, and C is a TEM image.

[0025] Figure 2 This is a comparison chart of the cumulative release rates of each group in Test Example 1. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1 This embodiment provides a method for preparing the above-mentioned wound-healing composition, comprising the following steps: S1. Mix and pulverize 3g Panax notoginseng, 1g Curcuma longa, 3g Astragalus membranaceus, 2g Epimedium brevicornu, and 3g Bletilla striata, add to 500mL of water, heat to boiling and extract for 3 hours, filter, dry the filtrate to obtain the water extract, and keep the filter residue for use; S2. Add the filter residue from step S1 to 500 mL of 95 wt% ethanol and heat under reflux for 2 h. Filter, recover the ethanol from the filtrate, add water to precipitate, filter, wash, and dry to obtain the organic extract. S3. Dissolve 45g of egg yolk lecithin and 10g of cholesterol in a dichloromethane-ethanol mixture (volume ratio 1:1), add the organic extract obtained in step S2, stir and mix evenly, remove the solvent by rotary evaporation, add 100mL of PBS buffer solution with pH=7.4, sonicate for 40min, centrifuge at 3000r / min for 20min, take the supernatant, and filter it through a 0.22μm microporous membrane to obtain liposomes; S4. Wash 2g of ginseng, add 50mL of PBS buffer (pH=7.4), homogenize, filter, add 2wt% protease inhibitor, adjust the pH to 7 with 1mol / L Tris-HCl solution, centrifuge at 400g, 800g, and 15000g for 15min, collect the supernatant, centrifuge at 100000g for 50min, collect the precipitate, resuspend the precipitate in 100mL of 15mmol / L Tris-HCl solution to obtain ginseng exosomes; S5. Mix the liposomes obtained in step S3 and the ginseng exosomes obtained in step S4 evenly, sonicate at room temperature for 2 min (sonicate for 30 s, stop for 30 s), and incubate at 37℃ for 50 min to obtain the liposome-exosome complex. S6. Add 0.5g of surfactant Brij-35 to the liposome-exosome complex obtained in step S5 until the solution is clear, add 0.1g of protein peptide IKFQFHFD, shake at room temperature for 30min, then remove the surfactant using Bio-Bead SM-2, centrifuge at 300000g, 4℃ for 2h, collect the precipitate, and resuspend it in a buffer solution at pH=7.4 to obtain the protein complex; Figure 1 Image A shows the appearance of the protein complex, image B shows the Tyndall effect after blue laser irradiation, and image C is a TEM image. As can be seen from the images, the particle size is between 200-400 nm.

[0028] S7. Dissolve 10g agarose in 500mL of water, heat to 90℃, stir and mix evenly, cool to 5℃, add 0.2g tetramethylpiperidine oxide and 1g sodium bromide, stir to dissolve, add 25mL of sodium hypochlorite solution containing 15wt% dropwise, adjust the pH to 10-11, react at room temperature for 5h, add 1g sodium borohydride to terminate the reaction, adjust the pH to 8, add sodium chloride and ethanol to precipitate, filter, wash, dry, and obtain carboxylated agarose; S8. The carboxylated agarose obtained in step S7 and 2g of mussel adhesive protein are mixed and added to 200mL of water, heated to 45℃ to dissolve, the water extract obtained in step S1 and the protein complex obtained in step S6 are added and mixed evenly, 0.1g of zinc citrate is added, and the mixture is stirred and mixed evenly. After cooling to room temperature, a gel is formed to obtain a composition that promotes wound healing.

[0029] Example 2 This embodiment provides a method for preparing the above-mentioned wound-healing composition, comprising the following steps: S1. Mix and pulverize 5g Panax notoginseng, 3g Curcuma longa, 7g Astragalus membranaceus, 5g Epimedium brevicornu, and 5g Bletilla striata, add to 500mL of water, heat to boiling and extract for 5 hours, filter, dry the filtrate to obtain the water extract, and keep the filter residue. S2. Add the filter residue from step S1 to 500 mL of 95 wt% ethanol and heat under reflux for 4 h. Filter, recover the ethanol from the filtrate, add water to precipitate, filter, wash, and dry to obtain the organic extract. S3. Dissolve 60g of egg yolk lecithin and 15g of cholesterol in a dichloromethane-ethanol mixture (volume ratio 3:1), add the organic extract obtained in step S2, stir and mix evenly, remove the solvent by rotary evaporation, add 100mL of PBS buffer solution with pH=7.4, sonicate for 60min, centrifuge at 5000r / min for 30min, take the supernatant, and filter it through a 0.22μm microporous membrane to obtain liposomes; S4. Wash 4g of ginseng, add 50mL of PBS buffer (pH=7.4), homogenize, filter, add 3wt% protease inhibitor, adjust the pH to 7 with 1mol / L Tris-HCl solution, centrifuge at 400g, 800g, and 15000g for 25min, collect the supernatant, centrifuge at 100000g for 70min, collect the precipitate, resuspend the precipitate in 100mL of 25mmol / L Tris-HCl solution to obtain ginseng exosomes; S5. Mix the liposomes obtained in step S3 and the ginseng exosomes obtained in step S4 evenly, sonicate at room temperature for 3 min (sonicate for 30 s, stop for 30 s), and incubate at 37℃ for 70 min to obtain the liposome-exosome complex; S6. Add 1g of surfactant Brij-35 to the liposome-exosome complex obtained in step S5 until the solution is clear, add 0.02g of protein peptide IKFQFHFD, shake at room temperature for 50min, then remove the surfactant using Bio-Bead SM-2, centrifuge at 300000g, 4℃ for 2h, collect the precipitate, and resuspend it in a buffer solution at pH=7.4 to obtain the protein complex; S7. Dissolve 15g agarose in 500mL of water, heat to 95℃, stir and mix evenly, cool to 5℃, add 0.3g tetramethylpiperidine oxide and 1.5g sodium bromide, stir to dissolve, add 30mL of sodium hypochlorite solution containing 15wt% dropwise, adjust the pH to 10-11, react at room temperature for 7h, add 1g sodium borohydride to terminate the reaction, adjust the pH to 8, add sodium chloride and ethanol to precipitate, filter, wash, dry, and obtain carboxylated agarose; S8. The carboxylated agarose obtained in step S7 and 4g of mussel adhesive protein are mixed and added to 200mL of water, heated to 45℃ to dissolve, the water extract obtained in step S1 and the protein complex obtained in step S6 are added and mixed evenly, 0.2g of zinc citrate is added, and the mixture is stirred and mixed evenly. After cooling to room temperature, a gel is formed to obtain a composition that promotes wound healing.

[0030] Example 3 This embodiment provides a method for preparing the above-mentioned wound-healing composition, comprising the following steps: S1. Mix and pulverize 4g Panax notoginseng, 2g Curcuma longa, 5g Astragalus membranaceus, 3g Epimedium brevicornu, and 4g Bletilla striata, add to 500mL of water, heat to boiling and extract for 4 hours, filter, dry the filtrate to obtain the water extract, and keep the filter residue. S2. Add the filter residue from step S1 to 500 mL of 95 wt% ethanol and heat under reflux for 3 h. Filter, recover the ethanol from the filtrate, add water to precipitate, filter, wash, and dry to obtain the organic extract. S3. Dissolve 52g of egg yolk lecithin and 12g of cholesterol in a dichloromethane-ethanol mixture (volume ratio 2:1), add the organic extract obtained in step S2, stir and mix evenly, remove the solvent by rotary evaporation, add 100mL of PBS buffer solution with pH=7.4, sonicate for 50min, centrifuge at 4500r / min for 25min, take the supernatant, and filter it through a 0.22μm microporous membrane to obtain liposomes; S4. Wash 3g of ginseng, add 50mL of PBS buffer (pH=7.4), homogenize, filter, add 2.5wt% protease inhibitor, adjust the pH to 7 with 1mol / L Tris-HCl solution, centrifuge at 400g, 800g, and 15000g for 20min, collect the supernatant, centrifuge at 100000g for 60min, collect the precipitate, resuspend the precipitate in 100mL of 20mmol / L Tris-HCl solution to obtain ginseng exosomes; S5. Mix the liposomes obtained in step S3 and the ginseng exosomes obtained in step S4 evenly, sonicate at room temperature for 2.5 min (sonicate for 30 s, stop for 30 s), and incubate at 37℃ for 60 min to obtain the liposome-exosome complex. S6. Add 0.7g of surfactant Brij-35 to the liposome-exosome complex obtained in step S5 until the solution is clear, add 0.15g of protein peptide IKFQFHFD, shake at room temperature for 40min, then remove the surfactant using Bio-Bead SM-2, centrifuge at 300000g, 4℃ for 2h, collect the precipitate, and resuspend it in a buffer solution at pH=7.4 to obtain the protein complex; S7. Dissolve 13g agarose in 500mL of water, heat to 95℃, stir and mix evenly, cool to 5℃, add 0.25g tetramethylpiperidine oxide and 1.2g sodium bromide, stir to dissolve, add 27mL of sodium hypochlorite solution containing 15wt%, adjust the pH to 10-11, react at room temperature for 6h, add 1g sodium borohydride to terminate the reaction, adjust the pH to 8, add sodium chloride and ethanol to precipitate, filter, wash, dry, and obtain carboxylated agarose; S8. The carboxylated agarose obtained in step S7 and 3g of mussel adhesive protein are mixed and added to 200mL of water, heated to 45℃ to dissolve, the water extract obtained in step S1 and the protein complex obtained in step S6 are added and mixed evenly, 0.15g of zinc citrate is added, and the mixture is stirred and mixed evenly. After cooling to room temperature, a gel is formed to obtain a composition that promotes wound healing.

[0031] Comparative Example 1 The only difference from Example 3 is that steps S4 and S5 were not performed, and in step S6 the liposome-exosome complex was replaced by the liposomes obtained in step S3.

[0032] Comparative Example 2 The only difference from Example 3 is that step S6 was not performed, and in step S8 the protein complex was replaced by the liposome-exosome complex obtained in step S5.

[0033] Comparative Example 3 The only difference from Example 3 is that zinc citrate was not added in step S8.

[0034] Comparative Example 4 The only difference from Example 3 is that in step S8, the protein complex is replaced by an equal mass of organic extract.

[0035] Comparative Example 5 The only difference from Example 3 is that no water extract was added in step S8.

[0036] Test Example 1 Equal amounts of the protein complex prepared in step S6 of Example 3 and the liposome-exosome complex prepared in step S5 were used to determine the in vitro release rate of baicalin under different pH conditions. The samples were placed in dialysis bags with a molecular weight cutoff of 4000 Da and then in 15 mL of PBS buffer solution at pH 5.5 and 7, respectively, and incubated at 37°C with shaking at 100 rpm. At 0.5, 1, 2, 4, 6, 8, and 12 h, 3 mL of the release medium was taken out, and 3 mL of the corresponding fresh release medium was added simultaneously. The taken-out release medium was filtered through a 0.45 μm microporous membrane, and the baicalin content was measured. The cumulative release rate was calculated, and release curves were plotted. The results are shown in [Figure number missing]. Figure 2 As shown in the figure, with the help of octapeptide IKFQFHFD, the protein complex can rapidly release the drug under acidic pH=5.5 conditions.

[0037] Test Example 2 Balb / c mice weighing 20-22g were randomly divided into four groups: a blank control group, a model group, groups 1-3 (Examples 1-3), and comparative groups 1-5, with 12 mice in each group. Except for the blank control group, the other mice were fed a high-fat diet (mouse feed mixed with lard) and high-sugar water (50% glucose solution) for two weeks, and were intraperitoneally injected with streptozotocin-citrate buffer (streptozotocin, 45mg / kg) for two consecutive days. One week later, blood glucose was measured; a fasting blood glucose level greater than 11.1mmol / L was considered a successful model of diabetes. After successful modeling, mice in each group were anesthetized, their backs were shaved and disinfected, and a full-skin wound of approximately 8mm in diameter was created on the back of each mouse using a stoma device. During the experiment, the mouse wounds were cleaned and disinfected twice daily. In the treatment groups, the wounds were treated with a prepared wound-healing composition (1mL / mouse) twice daily after disinfection. The blank control group and the model group were given the same amount of deionized water.

[0038] Wound healing status: On day 14 of the experiment, wound healing was observed, wound diameter was measured, and wound healing rate was calculated. Wound healing rate (%) = (original wound area - current wound area) / original wound area × 100%. The results are shown in Table 1.

[0039] Table 1

[0040] Note: * indicates P < 0.05 compared to the control group; # indicates P < 0.05 compared to the model group.

[0041] As can be seen from the table above, the wound-healing compositions prepared in Examples 1-3 of the present invention can significantly promote the healing of chronic ulcers caused by hyperglycemia.

[0042] Inflammatory cell count in wound tissue: On day 14 of the experiment, skin samples (0.5cm × 0.5cm) from the wound were fixed in paraformaldehyde, dehydrated, paraffin-embedded, and embedded, then sectioned at 5μm. The sections were rehydrated with ethanol, stained with hematoxylin and eosin, mounted, dried, and examined under a light microscope. Inflammatory cells were counted using the eyepiece grid counting method. Inflammatory cell count (%) = (Inflammatory cell count / Total cells in the field of view) × 100%. Results are shown in Table 2.

[0043] Table 2

[0044] Note: * indicates P < 0.05 compared to the control group; # indicates P < 0.05 compared to the model group.

[0045] As shown in the table above, the wound-healing compositions prepared in Examples 1-3 of this invention can significantly reduce the proportion of inflammatory cells in wound tissue and have a good anti-inflammatory effect.

[0046] The effects of Comparative Examples 1-5 all decreased. Comparative Example 1: The ginseng exosome complex exhibits highly effective wound healing, anti-inflammatory, and antioxidant effects, while also possessing excellent transdermal delivery capabilities, facilitating the delivery of the loaded drug to the wound for maximum efficacy. Therefore, its effect is superior to simple liposomes. Comparative Example 2: The protein complex incorporates a pH-responsive octapeptide into its membrane layer. Under acidic wound conditions, the membrane ruptures, releasing the drug. Its drug release effect is superior to the liposome-exosome complex. Furthermore, the octapeptide also possesses antibacterial activity under acidic conditions, enhancing the wound's anti-infection efficacy. Comparative Example 3: Zinc ions can also crosslink agarose chains and protein chains. Under acidic conditions, the ionic crosslinking breaks down, loosening the hydrogel network and releasing the water extract and protein complex, resulting in a better pH-responsive drug release effect. Comparative Examples 4 and 5: The protein complex can release organic extracts. Both the organic and aqueous extracts contain abundant active components of traditional Chinese medicine, exhibiting antibacterial, analgesic, anti-inflammatory, and tissue-promoting effects, which are beneficial for repairing wound tissue and shortening wound healing time. However, directly adding organic extracts to the hydrogel system results in insolubility or poor solubility, making them prone to aggregation and precipitation, leading to poor efficacy and difficulty in achieving the intended effect.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition for promoting wound healing, characterized in that, It is prepared from the following raw materials in parts by weight: Panax notoginseng 3-5 parts, turmeric 1-3 parts, Astragalus membranaceus 3-7 parts, Epimedium 2-5 parts, Bletilla striata 3-5 parts, lecithin 45-60 parts, cholesterol 10-15 parts, ginseng 2-4 parts, surfactant 0.5-1 part, protein peptide 0.1-0.2 parts, agarose 10-15g, tetramethylpiperidine oxide 0.2-0.3 parts, sodium bromide 1-1.5 parts, mussel adhesive protein 2-4 parts, and zinc citrate 0.1-0.2 parts.

2. A method for preparing the composition for promoting wound healing as described in claim 1, characterized in that, Includes the following steps: S1. Mix and pulverize Panax notoginseng, turmeric, Astragalus membranaceus, Epimedium, and Bletilla striata, add water, heat to boiling and extract, filter, dry the filtrate to obtain an aqueous extract, and keep the residue for use; S2. Add the filter residue to an organic solvent, heat and reflux to extract, filter, recover the organic solvent from the filtrate, add water to precipitate, filter, wash, dry, and obtain the organic extract; S3. Dissolve lecithin and cholesterol in a dichloromethane-ethanol mixture, add the organic extract, stir and mix well, remove the solvent by rotary evaporation, add PBS buffer solution, sonicate to hydrate, centrifuge, take the supernatant, filter, and obtain liposomes; S4. Wash the ginseng, add PBS buffer, homogenize, filter, add protease inhibitor, adjust the pH value, and obtain ginseng exosomes by gradient centrifugation. S5. Mix liposomes and ginseng exosomes evenly, sonicate at room temperature, and incubate by heating to obtain liposome-exosome complex; S6. Add surfactant to the liposome-exosome complex until the solution is clear, add protein peptide, shake at room temperature, and then remove surfactant using Bio-Bead SM-2 to form protein complex; S7. Dissolve agarose in water, heat and stir until homogeneous, cool, add tetramethylpiperidine oxide and sodium bromide, stir to dissolve, add sodium hypochlorite solution dropwise, adjust pH value, react at room temperature, add ethanol to terminate the reaction, adjust pH value, precipitate, filter, wash, and dry to obtain carboxylated agarose. S8. Carboxylated agarose and mussel adhesive protein are mixed and added to water, heated to dissolve, then water extract and protein complex are added and mixed evenly. Zinc citrate is added, and the mixture is stirred and mixed evenly. After cooling, a gel is formed to obtain a composition that promotes wound healing.

3. The preparation method according to claim 2, characterized in that, The heating and boiling extraction time in step S1 is 3-5 hours; the organic solvent in step S2 is 95 wt% ethanol, and the heating and reflux extraction time is 2-4 hours.

4. The preparation method according to claim 2, characterized in that, In step S3, the volume ratio of dichloromethane to ethanol in the dichloromethane-ethanol mixed solution is 1-3:1, the pH of the PBS buffer solution is 7.4, the ultrasonic hydration time is 40-60 min, the centrifugation speed is 3000-5000 r / min, and the time is 20-30 min. The filtration is performed using a 0.22 μm microporous filter membrane for granulation filtration.

5. The preparation method according to claim 2, characterized in that, In step S4, the pH of the PBS buffer is 7.4, the amount of protease inhibitor added is 2-3 wt%, and the gradient centrifugation method is as follows: centrifuge at 400g, 800g, and 15000g for 15-25 min in sequence, take the supernatant, centrifuge at 100000g for 50-70 min, take the precipitate, and resuspend the precipitate in 15-25 mmol / L Tris-HCl solution.

6. The preparation method according to claim 2, characterized in that, The room temperature ultrasonic treatment in step S5 is performed for 2-3 minutes, with the method being ultrasonic treatment for 30 seconds and stopping for 30 seconds. The heating incubation temperature is 37°C, and the incubation time is 50-70 minutes.

7. The preparation method according to claim 2, characterized in that, The surfactant mentioned in step S6 is Brij-35, the sequence of the protein peptide is IKFQFHFD, as shown in SEQ ID NO.1, and the room temperature shaking time is 30-50 min.

8. The preparation method according to claim 2, characterized in that, The room temperature reaction time in step S7 is 5-7 hours, and the heating and stirring temperature is 90-95°C.

9. The preparation method according to claim 2, characterized in that, The heating temperature in step S8 is 35-45℃.

10. Use of the wound-healing composition as described in claim 1 in the preparation of a medicament for treating wound healing in chronic ulcer conditions.

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