A marine shellfish active peptide that promotes skin wound repair

Abstract

The present invention discloses a marine shellfish active peptide that can effectively promote the repair of skin wounds. The marine shellfish active peptide has good skin compatibility, can significantly promote cell proliferation, migration and angiogenesis, effectively reduce inflammatory response, promote high-quality wound repair, and reduce scar formation. The preparation method of the marine shellfish active peptide is to take the soft part of fresh marine shellfish, perform protease hydrolysis, ultrafiltration, and dextran gel separation, discard the last elution single peak, collect or mix the other elution components respectively, and freeze-dry them. The method is simple, low-cost, green and environmentally friendly, has a short production cycle, and is suitable for industrial production. The marine shellfish active peptide of the present invention can be applied to medicines, medical biomaterials and cosmetics, has a wide range of uses, and has significant effects on promoting the regeneration and repair of burns, scalds, cuts, traffic injuries, surgical injuries, combat injuries, firearm injuries combined with seawater immersion injuries, diabetic ulcers, inflammatory ulcers, pressure ulcers and chronic difficult-to-heal wounds.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a marine shellfish active peptide for promoting skin wound repair, and a preparation method and application thereof. Background Art

[0002] The skin, covering the entire body, is one of the largest organs and the body's first defense against external damage. Due to its direct contact with the outside world, the skin is highly susceptible to damage from trauma, burns, and other processes. In addition to acute trauma, many common diseases (such as diabetic foot, aging, obesity, vascular disease, cancer, and infection) cause wounds to heal slowly or incompletely. Wound healing time is closely related to susceptibility to infection, duration of pain, length of hospital stay, and incidence of scarring.

[0003] Skin wound repair mechanisms are often influenced by numerous factors, including age, nutritional status, endocrine changes, local blood circulation, infection, ionizing radiation, medications, and systemic diseases. Small injuries heal within days without scarring. However, in wounds with significant tissue cell loss and excessive wound size, regenerated epidermis struggles to fully repair the wound. The body then fills the wound with connective tissue, often leaving a scar. Although covered by a keratinized epidermis, the scar lacks sweat glands, hair, and dermal papillae, losing its original tissue structure and function.

[0004] Furthermore, the inadequate healing capacity of chronic wounds is particularly pronounced. Among diabetics, the incidence of diabetic foot is as high as 25%. Abnormally high blood sugar levels lead to tissue cell damage and metabolic abnormalities, severely impairing wound repair. In obese individuals, the thick fat layer of the skin hinders microvascular regeneration, and wounds are more susceptible to fat liquefaction and infection, resulting in slower wound healing. In the aging population, wound healing is significantly slowed due to the decline in cellular compensatory function, diminished stem cell self-renewal, and reduced ability to clear damaged cells.

[0005] It can be seen that promoting the rapid and high-quality healing of these acute and chronic wounds is an issue that cannot be ignored in clinical care. In clinical treatment, different treatments and medications are mainly used for acute skin trauma and chronic skin trauma. my country's genetically engineered Class I new drug recombinant bovine basic fibroblast growth factor (rb-bFGF) promotes wound healing and is mainly used for burns, chronic ulcers on the body surface, and fresh wounds (including trauma, donor site wounds, surgical wounds, etc.). However, due to the high preparation cost, this polypeptide and recombinant protein cannot be widely used and promoted in the fields of medical biomaterials, cosmetics, etc. Summary of the Invention

[0006] One of the objectives of the present invention is to provide a marine shellfish active peptide that can effectively promote skin wound repair. The marine shellfish active peptide of the present invention has good skin compatibility, can significantly improve cell viability, promote cell proliferation, migration and angiogenesis, effectively reduce inflammatory reactions, promote high-quality skin wound repair, and effectively reduce scar hyperplasia.

[0007] Another object of the present invention is to provide a method for preparing marine shellfish active peptides by enzymatic hydrolysis, ultrafiltration, and Sephadex G-15 separation. The method is simple, low-cost, environmentally friendly, has a short production cycle, and is suitable for large-scale industrial production.

[0008] Another object of the present invention is to provide an application of marine shellfish active peptides, which can be used in medicines, medical biomaterials and cosmetics.

[0009] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0010] In one aspect, the present invention provides a marine shellfish active peptide having good water solubility and skin compatibility.

[0011] The marine shellfish active peptide of the present invention is a product obtained by enzymatic hydrolysis of the soft part of fresh marine shellfish by protease.

[0012] These marine shellfish may include, but are not limited to, one or a combination of Pinctada martensii, Paphia undulata, and scallops.

[0013] The protease used may be an enzyme that can enzymatically hydrolyze proteins, including but not limited to neutral protease.

[0014] The inventors previously used marine shellfish peptides in skin and hair care research with good results. However, due to the significant differences in physiological state and structure between damaged skin tissue and normal skin tissue, there are currently no reports that substances used in general skin care can be directly used to care for damaged skin tissue.

[0015] It is understood that skin tissue repair and wound healing generally go through three basic stages: 1. Inflammatory response; 2. Tissue proliferation and granulation formation; 3. Wound contraction and scar formation. These three stages overlap with each other.

[0016] 1. Inflammatory response: This begins immediately after injury and usually lasts for 3-5 days. The main changes are blood coagulation and fibrinolysis, immune response, increased microvascular permeability, and infiltration of inflammatory cells (initially neutrophils, then monocytes). Its significance is to remove wound-causing factors (such as pathogens and other foreign bodies) and necrotic tissue, prevent infection, and lay the foundation for tissue regeneration and repair. PDGF, IGF-1, EGF, TGF-β, etc. released by platelets play an important role as chemotactic agents for inflammatory cells; macrophages synthesize and secrete TGF-β, TGF-α, bFGF, MDGF, and HB-EGF at the wound site. These factors can stimulate fibroblasts, epidermal cells, and vascular endothelial cells to migrate toward the wound.

[0017] 2. Tissue proliferation and granulation: 24-48 hours after injury, epithelial cells at the wound edge begin to proliferate. Some basal cells detach from the dermis and migrate toward the defect area, with mitosis observed. Simultaneously, fibroblasts and myofibroblasts with rich cytoplasm and spindle or stellate shapes appear at the wound site. The latter are similar to the former but contain microfilament bundles parallel to the long axis of the cell and are attached to the pellicle (facilitating cell contraction). Angiogenesis primarily occurs through the "sprouting" of new capillaries from existing vessels, and existing vascular loops may also be extended. Fibroblasts secrete IGF-1, bFGF, TGF-β, PDGF, and KGF; endothelial cells produce bFGF and PDGF; and keratinocytes produce TGF-β, TGF-α, and keratinocyte-derived autocrine factor (KAF). These growth factors stimulate cell proliferation, synthesis of intercellular matrix proteins, and angiogenesis.

[0018] 3. Wound Contraction and Scar Formation: 3-5 days after injury, the edges of the wound begin to move toward the center and contract, eliminating the wound and restoring tissue continuity. This often occurs before the wound has fully epithelialized, initially due to contraction of epithelial microfibril bundles at the wound edge and ultimately due to contraction of myofibroblasts located in the center of the wound. PDGF, TGF-β, and other proteins play a crucial role in the transformation of granulation tissue into scar. As the healing process progresses, collagen fibers increase, while fibroblasts and capillaries gradually decrease, ultimately transforming into scar tissue with fewer cells and blood vessels and more fibers.

[0019] During wound healing, as fibroblasts infiltrate, fibronectin levels increase within the wound area and are distributed along collagen in the granulation tissue. As new epithelium covers the wound and collagen matures, fibronectin gradually disappears. Later in wound healing, fibroblasts are abundant in the wound site. They are the primary repair cells, whose primary function is to synthesize collagen fibers. During wound healing, collagen undergoes a dynamic process of intracellular synthesis, extracellular deposition, and resorption. Growth factors play a crucial regulatory role in collagen synthesis and metabolism, primarily by influencing collagen gene expression.

[0020] Based on the above understanding of the skin wound repair process and mechanism, the inventors used for the first time the marine shellfish active peptides obtained after removing the last elution single peak component of polysaccharide gel G-15 for the care of wounded skin.

[0021] As an optional embodiment, the preparation method of marine shellfish active peptides is to hydrolyze the soft part of marine shellfish with protease, ultrafilter, and then separate it through polysaccharide gel G-15. After discarding the last elution single peak component, each elution peak component is collected or combined separately, and freeze-dried to obtain marine shellfish active peptides.

[0022] Preferably, in some embodiments, the neutral protease hydrolysate of the soft part of marine shellfish can be processed sequentially through ultrafiltration membranes with a molecular weight cutoff of 10 kDa and 3 kDa, and the obtained retentate (3 kDa-10 kDa) can be separated by dextran gel G-15.

[0023] The method is as follows: fresh marine shellfish are shelled, the soft part is homogenized, and 2.5-4 times the volume of water or PBS buffer is added for dilution, and protease (2000U / g shellfish meat to 4000U / g shellfish meat) is added. The mixture is enzymatically hydrolyzed at a temperature of 40°C to 50°C and a pH value of 6.5-7.5 for 4-6 hours, the enzyme is inactivated in a boiling water bath for 10 minutes, and the supernatant is ultrafiltered at 6000×g to 9000×g for 10-15 minutes. The supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10kDa, and the filtrate is ultrafiltered again through an ultrafiltration membrane with a molecular weight cutoff of 3kDa. The 3kDa to 10kDa active peptide solution of the neutral protease hydrolysis solution is concentrated and then separated through Sephadex G-15. After discarding the last eluted single peak fraction, the other eluted peak fractions are collected or combined, and freeze-dried to obtain marine shellfish active peptides.

[0024] Preferably, the elution peak component is the first elution peak component, the second elution peak component, the third elution peak component, the fourth elution peak component, the fifth elution peak component, the sixth elution peak component or any combination of these elution peak components.

[0025] Preferably, when the elution peak fraction collected is the elution peak fraction collected after separation of the 3kDa-10kDa active peptide fraction of the neutral protease hydrolyzate by polysaccharide gel G-15 (for example, N1 separation fractions F1-F6 in Example 2 and N1F1 and N1F2 in Example 3), it has a significant effect in promoting cell proliferation and skin wound repair.

[0026] In another aspect, the present invention provides an application of marine shellfish active peptides that can effectively promote cell proliferation and skin wound repair, with a minimum effective dose of 100 μg / mL.

[0027] As an optional embodiment, marine shellfish active peptides can be used to promote skin wound repair.

[0028] In a preferred embodiment of the present invention, the marine shellfish active peptide can be used in medicines; preferably, the marine shellfish active peptide is particularly suitable for use in medicines that can effectively repair skin wounds. The pharmaceutical dosage forms include lotions, ointments, tinctures, liniments, spirits, powders, oils, pastes, plasters, film coatings, aerosols, etc. When used in medicines, the mass fraction of the marine shellfish active peptide in the medicine is 0.01% to 2.5%.

[0029] In a preferred embodiment of the present invention, the marine shellfish active peptide can also be used in medical biomaterials; preferably, the marine shellfish active peptide is particularly suitable for use in medical biomaterials that can effectively repair skin wounds. The medical biomaterial dosage forms include lotions, solutions, dressings, ointments, etc., and the dressing types include films, hydrocolloids, hydrogels, sponges, sprays, etc. When used in medical biomaterials, the mass fraction of the marine shellfish active peptide in the medical biomaterial is 0.8% to 2.5%.

[0030] In a preferred embodiment of the present invention, the marine shellfish active peptide can be used in cosmetics; preferably, the marine shellfish active peptide is particularly suitable for use in cosmetics that can effectively promote skin wound repair, and the cosmetic dosage forms include creams, lotions, gels, jelly, lotions, sprays, etc. When used in cosmetics, the mass fraction of the marine shellfish active peptide in the cosmetic is 0.05% to 1.0%.

[0031] It is worth mentioning that the marine shellfish active peptide provided by the present invention, as a functional ingredient, does not cause skin allergies, and can also significantly reduce inflammatory reactions, promote cell proliferation and skin wound repair. Therefore, when the marine shellfish active peptide of the present invention is used in medicines, medical biomaterials or cosmetics, the selection of the type, dosage, preparation process, etc. of pharmaceutical raw materials, medical biomaterial raw materials, and cosmetic raw materials is relatively wide. All ingredients used in medicines, medical biomaterials, and cosmetics should be acceptable to the skin and do not affect the performance of the original marine shellfish active peptide of the present invention, that is, when in contact with human skin or when combined with other components, they will not cause inappropriate toxicity, incompatibility, instability, and allergic reactions.

[0032] The solution of the present invention is based on the inventor's understanding of the skin wound repair mechanism, the structure-activity relationship of marine shellfish active peptides, etc., combined with the research results of modern pharmacology, medical biomaterials and cosmetics. Through a large number of creative experiments, the inventor has searched, explored, prepared and selected marine shellfish active peptides that have good skin compatibility, are non-allergenic, and have significant effects on promoting skin wound repair.

[0033] The present invention has the following outstanding beneficial effects:

[0034] (1) Exploiting the efficacy of marine shellfish active peptides in promoting skin wound repair. From a variety of marine shellfish, the pinctada martensii, undulata clam and scallop were selected, and the soft parts of fresh shellfish were selected. The marine shellfish active peptides were prepared by using protease hydrolysis, ultrafiltration and dextran gel separation technology. The active peptides can quickly penetrate into the surface layer of the skin and exert multiple effects such as promoting cell proliferation and wound repair. The marine shellfish active peptides of the present invention can still significantly promote cell proliferation and wound repair at an extremely low dose (50 μg / mL).

[0035] (2) The preparation process of the marine shellfish active peptide of the present invention is simple, low-cost, green and environmentally friendly, and has a short production cycle. It is suitable for large-scale industrial production and has great application prospects. According to the characteristics of the composition structure of marine shellfish, the specificity of proteases and the action sites, neutral proteases are preferably selected from a variety of proteases to carry out targeted enzymolysis of the soft part of marine shellfish. The enzymolysis solution is then treated with ultrafiltration membranes with a molecular weight cutoff of 10kDa and 3kDa, which can remove components with poor water solubility, large molecular weight, and no significant cell proliferation activity (possibly containing salts, free amino acids, fatty acids, etc.), so that the obtained active peptides have better physiological effects and emit very few environmental pollutants.

[0036] (3) The marine shellfish active peptides of the present invention have good water solubility and skin compatibility, do not cause skin irritation, are non-sensitizing, and have significant efficacy, and can be widely used in biopharmaceuticals, medical biomaterials, and cosmetics. By separating with polysaccharide gel G-15, the eluted components with smaller molecular weight and poor activity are eliminated, which can further enrich the active peptides. While improving the physiological efficacy, the dosage is greatly reduced, thereby effectively reducing the requirements for stability, compatibility, etc. of the marine shellfish active peptides and the difficulty of operation in the preparation of biopharmaceuticals, medical biomaterials, and cosmetics.

[0037] (4) The present invention established a cell activity experimental model to conduct high-throughput activity evaluation on the prepared active peptide components. The marine shellfish active peptides finally selected showed excellent efficacy in promoting skin wound repair at both cell and animal levels, indicating that the use of this cell experimental model can accurately guide the directional separation and high enrichment of the marine shellfish active peptides of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the Sephadex G-15 separation pattern of the N1 (3kDa-10kDa) component in Example 2.

[0039] Figure 2 This is the Sephadex G-15 separation pattern of the N2 (less than 3 kDa) component in Example 2.

[0040] Figure 3 This is the Sephadex G-15 separation pattern of the N1 (3kDa-10kDa) component in Example 3.

[0041] Figure 4 The figure shows the effect of the N1 (3 kDa-10 kDa) fraction in Example 3 on the activity of HaCaT cells (compared with the blank control, *p<0.05, **p<0.01).

[0042] Figure 5 This is the RP-HPLC separation pattern of N1(3kDa-10kDa)F4 in Example 4.

[0043] Figure 6 The figure shows the effects of the RP-HPLC fractions of N1 (3 kDa-10 kDa) F4 in Example 4 on the activity of HaCaT cells (compared with the blank control, *p<0.05, **p<0.01). DETAILED DESCRIPTION

[0044] The present invention is further described below with reference to the accompanying drawings. However, the embodiments of the present invention are not limited to the following examples. Any equivalent changes or modifications made according to the method of the present invention should be considered as the scope of protection of the present invention. Unless otherwise specified, the raw materials used below are commercially available.

[0045] Example 1

[0046] Fresh Pinctada martensii oysters that meet relevant Chinese seafood standards were shelled and the soft parts homogenized. The mixture was then added with 3.4 volumes of distilled water and a neutral protease (4000 U / g of flesh) at 40°C and pH 6.5 for 6 hours. The hydrolyzate was boiled for 10 minutes and centrifuged at 7000 × g for 10 minutes. The supernatant was then ultrafiltered through a 10 kDa molecular weight cutoff ultrafiltration membrane. The filtrate (active peptide fractions less than 10 kDa) was concentrated to a concentration of approximately 100 mg / mL and then separated on a Sephadex G-15 (100 × 4.0 cm) filter. The sample volume was 15 mL, the mobile phase was distilled water, the flow rate was 10 mL / min, and the detection wavelengths were 220 nm and 280 nm. The eluate fractions, except the last eluting peak, were combined, concentrated, and lyophilized to obtain Pinctada martensii active peptides (HG). According to the same method, the active peptides from Paphia undulata (HL) and Scallop (HM) were prepared respectively.

[0047] The efficacy of these active peptides (HG, HL, and HM) was evaluated using in vitro cell experiments (see Example 5 for specific experimental methods). The study found that at various doses (50 μg / mL-1600 μg / mL), HG, HL, and HM significantly promoted HaCaT cell proliferation (significantly different from the blank control). Among them, at a lower concentration of 50 μg / mL, HG, HL, and HM significantly increased HaCaT cell activity by 9.85% (p < 0.05), 10.33% (p < 0.05), and 10.56% (p < 0.05), respectively.

[0048] Applying a solution of active peptides from Pinctada martensii (containing 2% HG, in saline solution, sterilized by 0.22 μm filtration), an active peptide solution from Paphia undulata (containing 2% HL, in saline solution, sterilized by 0.22 μm filtration), and an active peptide solution from Scallop (containing 2% HM, in saline solution, sterilized by 0.22 μm filtration) to deep second-degree burns and third-degree burn scab wounds, respectively, can reduce infection by pathogens (Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, etc.), alleviate inflammatory reactions, effectively prevent wound deepening, promote new tissue formation, improve wound healing speed and quality, and have a significant effect in promoting the regeneration and repair of burn wounds.

[0049] Example 2

[0050] Fresh, live scallops that meet the relevant Chinese seafood standards were shelled, the soft parts homogenized, and then lysed with 3 volumes of PBS buffer (0.01 M, pH 7.5) and neutral protease (3000 U / g of scallop flesh) at 45°C for 5 hours. The hydrolyzate was boiled for 10 minutes and centrifuged at 7000 × g for 12 minutes. The supernatant was then ultrafiltered through 10 kDa and 3 kDa ultrafiltration membranes, respectively. The active peptide fraction between 3 kDa and 10 kDa was designated N1, and the active peptide fraction less than 3 kDa was designated N2. The N1 and N2 fractions were each concentrated to a concentration of approximately 100 mg / mL and then separated on Sephadex G-15 (100 × 4.0 cm). The sample volume was 15 mL, the mobile phase was distilled water, the flow rate was 10 mL / min, and the detection wavelengths were 220 nm and 280 nm.

[0051] The N1 (3kDa-10kDa) fraction was eluted by Sephadex G-15 ( Figure 1 ), and the separated fractions F1, F2, F3, F4, F5, F6, and F7 were collected, concentrated, and freeze-dried. Similarly, the N2 (less than 3 kDa) fraction was eluted through Sephadex G-15 ( Figure 2 ), and the separated fractions F1, F2, F3, F4, F5, and F6 were collected, concentrated, and freeze-dried.

[0052] The efficacy of the 13 isolated components obtained was evaluated using in vitro cell experiments (see Example 5 for specific experimental methods). The study found that compared with the blank control, the cell activity of the N1 isolated component F7 and the N2 isolated component F6 was significantly reduced after administration (with obvious cytotoxicity); the cell activity of the N2 isolated component F1-F5 treatment group did not change significantly (no effect of promoting cell proliferation). However, the N1 (3kDa-10kDa) isolated components F1-F6 at a lower concentration (50μg / mL) can significantly increase the activity of HaCaT cells (compared with the blank control, there is a significant difference).

[0053] It can be seen that the N1 (3kDa-10kDa) separation components F1-F6 have the effect of significantly promoting the proliferation of HaCaT cells. These six separation components can be used alone, or they can be combined or merged to promote cell proliferation and skin wound repair.

[0054] The N1 (3kDa-10kDa) separated components F1-F6 were respectively prepared into solutions (the effective peptide dose was 1.5%, the solvent was normal saline, and the solution was sterilized by 0.22μm filtration) and applied to the diabetic ulcer wound. This can effectively protect the ulcer from damage and secondary infection, improve local ischemia and edema, promote the formation of new tissue, improve the speed and quality of wound healing, and have a good effect in promoting the regeneration and repair of diabetic ulcer wounds.

[0055] Example 3

[0056] Fresh scallops that meet the relevant seafood use standards of my country were shelled, the soft part was homogenized, 2.5 times the volume of distilled water was added, the pH value was adjusted to 7.0, neutral protease (2000U / g scallop meat) was added, and the temperature was 50°C for enzymatic hydrolysis for 5 hours. The enzymatic hydrolyzate was boiled for 10 minutes and centrifuged at 6000×g for 15 minutes. The supernatant was ultrafiltered through ultrafiltration membranes with a molecular weight cutoff of 10kDa and 3kDa in sequence. Among them, the 3kDa-10kDa active peptide component was recorded as N1. The N1 component was concentrated to a concentration of about 200mg / mL and then separated by dextran gel G-15 (100×4.0cm): the sample volume was 7mL, the mobile phase was distilled water, the flow rate was 10mL / min, and the detection wavelengths were 220nm and 280nm. The eluted components N1F1, N1F2, N1F3 ( Figure 3 ), concentrated and freeze-dried.

[0057] The efficacy of the three isolated components was evaluated using in vitro cell experiments (see Example 5 for specific experimental methods). Figure 4The study found that after administration of N1F3, cell viability was significantly reduced (showing significant cytotoxicity). However, after administration of the other two isolated components (N1F1 and N1F2), cell viability was significantly increased. N1F1, at a low concentration of 50 μg / mL, increased HaCaT cell viability by 12.31% (p < 0.05); N1F2, at a concentration of 100 μg / mL, increased HaCaT cell viability by 20.13% (p < 0.01).

[0058] It can be seen that the N1 (3kDa-10kDa) separated components N1F1 and N1F2 have the effect of significantly promoting the proliferation of HaCaT cells. N1F1 and N1F2 can be used alone or combined to promote cell proliferation and skin wound repair.

[0059] According to the following method, N1F1 and N1F2 were prepared to obtain marine biomedical materials (N1F1@calcium alginate microspheres / collagen / chitosan scaffold and N1F2@calcium alginate microspheres / collagen / chitosan scaffold), and their efficacy was evaluated.

[0060] N1F1@calcium alginate microspheres: Sodium alginate (purchased from Shanghai McLean Biochemical Technology Co., Ltd., purity 98%) was dissolved in distilled water to prepare a 2% w / v sodium alginate solution, N1F1 was added at a mass ratio of 1:0.05, and stirred to prepare an aqueous phase; a certain volume of olive oil (purchased from Shanghai McLean Biochemical Technology Co., Ltd.) was measured, Tween 80 (purchased from Shanghai McLean Biochemical Technology Co., Ltd.) was added at a ratio of 1% v / v, and stirred at 600 rpm for 2 h to prepare an organic phase; 100 mL of the organic phase was measured, 20 mL of the aqueous phase was added under stirring at 650 rpm, stirred at 750 rpm for 1 h, 40 mL of a 1% w / v calcium chloride solution (purchased from Shanghai McLean Biochemical Technology Co., Ltd.) was added, stirred at 500 rpm for 1 h, and then 10 mL Isopropyl alcohol (Tianjin Fuyu Chemical Reagent Factory, AR grade) was stirred at 400 rpm for 30 min, and the mixture was centrifuged at 6000 rpm for 5 min. The obtained precipitate was washed alternately with isopropyl alcohol and distilled water for 3 times and freeze-dried.

[0061] N1F1@calcium alginate microspheres / collagen / chitosan scaffold: Collagen was dissolved in 2% w / v acetic acid solution to prepare a collagen solution with a concentration of 0.015 g / mL; carboxymethyl chitosan was dissolved in distilled water to prepare a carboxymethyl chitosan solution with a concentration of 0.04 g / mL; the collagen solution and carboxymethyl chitosan solution were mixed in a 1:1 volume ratio, and N1F1@calcium alginate microspheres were added to make a final concentration of 0.01 g / mL. The mixture was magnetically stirred at 700 rpm for 1 hour, and the mixture was added to a 48-well plate. After the freeze-dried scaffold was demolded, it was soaked in EDC / NHS solution (EDC concentration 0.5 mol / L, NHS concentration 0.5 mol / L) for 2 hours, washed three times with distilled water, and freeze-dried to prepare the N1F1@calcium alginate microspheres / collagen / chitosan scaffold.

[0062] The N1F2@calcium alginate microspheres / collagen / chitosan scaffold was prepared by the same method.

[0063] The N1F1@calcium alginate microspheres / collagen / chitosan scaffold and the N1F2@calcium alginate microspheres / collagen / chitosan scaffold were applied to the wound surface of firearm wounds (a type of combat wound) after debridement. They can quickly absorb and drain wound exudate, provide a dry, slightly acidic and hypoxic environment for the wound surface, reduce infection by pathogens (Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, etc.), alleviate inflammatory response, effectively prevent wound deepening, promote the formation of new tissue, improve the speed and quality of wound healing, and significantly promote the regeneration and repair of firearm wounds.

[0064] Example 4

[0065] Using RP-HPLC technology, marine shellfish active peptides can be further separated and refined.

[0066] Active peptides were separated and purified using an Agilent 1260 high-performance liquid chromatograph coupled with a YMC Triart C18 column (250×10 mm, S-5 μm, 12 nm). Mobile phase A: water (containing 0.1% formic acid); mobile phase B: acetonitrile (containing 0.1% formic acid), at 40°C. The sample concentration was 150 mg / mL, the loading volume was 50 μL, the flow rate was 2.5 mL / min, and detection wavelengths were 200 nm, 220 nm, 254 nm, and 280 nm.

[0067] Because the elution conditions for the various components of the marine shellfish active peptide of the present invention vary slightly, the N1F4 component in Example 2 will be used as an example for illustration. The elution conditions are: 0-7 min, 100% A; 7-37 min, 100%-80% A; 37-44 min, 80%-65% A; 44-50 min, 65%-30% A; 50-51 min, 30%-10% A; 51-56 min, 10% A; 56-58 min, 10%-100% A. Under these elution conditions, six components can be collected: F1 (4-7 min), F2 (7-22 min), F3 (22-37 min), F4 (37-44 min), F5 (44-50 min), and F6 (50-56 min). The components were concentrated and lyophilized to obtain N1 (3kDa-10kDa)F4 separated components F1, F2, F3, F4, F5, F6 ( Figure 5 ).

[0068] The efficacy of the six components (F1, F2, F3, F4, F5, F6) prepared was evaluated using in vitro cell experiments (see Example 5 for specific experimental methods). Figure 6 The study found that although F1, F5, and F6 could significantly enhance the activity of HaCaT cells at various concentrations (37.5 μg / mL-600 μg / mL) (there was a significant difference compared with the blank control), their cell activity was still lower than that of the N1F4 component before separation.

[0069] It can be seen that the N1F4 component can be directly used to promote cell proliferation and skin wound repair without further separation and purification by RP-HPLC technology.

[0070] According to the preparation method of the traditional Chinese medicine biomaterial in Example 3, an N1F4@calcium alginate microsphere / collagen / chitosan scaffold was prepared. When applied to the wound surface of a firearm injury combined with seawater immersion injury after debridement, the scaffold can quickly absorb wound exudate, provide a dry, clean, slightly acidic and hypoxic environment for the wound surface, protect the wound surface, reduce infection by pathogens (such as Vibrio vulnificus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus), alleviate the inflammatory response, effectively prevent further deepening of the wound surface, improve blood circulation, promote the formation of new tissue, and improve the speed and quality of wound healing. It has the effect of promoting the regeneration and repair of the wound surface of the firearm injury combined with seawater immersion injury.

[0071] Example 5

[0072] The cell proliferation promoting activity of the marine shellfish active peptides prepared in each example was evaluated using an in vitro cell experiment.

[0073] The cell viability was determined by CCK-8 assay: the cell density was 6×10 4A suspension of HaCaT cells at a concentration of 1 μg / mL was seeded into a 96-well culture plate (100 μL / well) and incubated in a 37°C, 5% CO2 incubator for 24 hours. 50 μL of each well was aspirated and discarded. The sample groups were then treated with 50 μL / well of the corresponding sample solution (final concentrations of 50, 100, 200, 400, 800, and 1600 μg / mL, sterilized through a 0.22 μm syringe filter). The blank control group was treated with 50 μL / well of complete DMEM. After incubation for 24 hours in a CO2 incubator, cell morphology was observed using an inverted fluorescence microscope (CKX41, Olympus, Japan). Subsequently, 10 μL of CCK-8 solution was added to each well. Incubation continued for 4 hours, and the absorbance of each well was measured using a Multiskan GO microplate reader (Thermo Fisher Scientific) at a wavelength of 450 nm and a reference wavelength of 650 nm. Six replicate wells were included in each group.

[0074] Following the above experimental steps, the efficacy of each component prepared in the examples of the present invention was evaluated. The study found that the last eluted single peak fraction obtained by Sephadex G-15 separation, such as N1 fraction F7 in Example 2 and N1F3 in Example 3, significantly reduced cell viability (showed significant cytotoxicity) after administration; while the other fractions, such as N1 fraction F1-F6 in Example 2 and N1F1 and N1F2 in Example 3, significantly increased cell viability after administration (significantly different from the blank control).

[0075] It can be seen that after discarding the last eluted single peak component of the polysaccharide gel G-15, the marine shellfish active peptides prepared can effectively promote cell proliferation and skin wound repair.

[0076] Example 6

[0077] The marine shellfish active peptides prepared by the present invention (taking the Pinctada martensii active peptide HG, the Paphia undulata active peptide HL, and the scallop active peptide HM in Example 1 as examples) were evaluated for their skin wound repair efficacy (taking the full-thickness skin defect model as an example).

[0078] Ointment base: PEG400 (400 mL), PEG4000 (50 g), Span 40 (1 mL), and distilled water (9 mL), dissolved and homogenized in a 60°C hot water bath.

[0079] Pinctada martensii active peptide (HG) ointment: contains 2% Pinctada martensii active peptide, and the rest is ointment base.

[0080] Paphia undulata active peptide (HL) ointment: contains 2% Paphia undulata active peptide, and the rest is ointment base.

[0081] Scallop active peptide (HM) ointment: contains 2% scallop active peptide, and the rest is ointment base.

[0082] (1) Full-thickness skin defect model: 72 SPF-grade male BALB / c mice, aged 6-8 weeks, were purchased from the Guangdong Medical Laboratory Animal Center. The animals were randomly divided into four groups: blank control, HG, HL, and HM, with 18 mice in each group. After anesthesia, the mice were depilated and a full-thickness skin wound of approximately 1.5 × 1.5 cm was made on the back.

[0083] (2) Administration: The blank control group and the marine shellfish active peptide treatment groups (HG, HL, and HM) were administered once a day (approximately 0.1 g of ointment per mouse) for 14 consecutive days. The blank control group was administered with the ointment base, and the marine shellfish active peptide treatment groups were administered with the corresponding ointment samples.

[0084] (3) Wound photography (calculation of healing rate): Wound healing was observed and recorded daily. The back wound was photographed twice a week. The field of view, lighting, and image magnification were kept consistent. The wound area was measured using IPP6.0 software to calculate the wound healing rate. On the 5th, 10th, and 14th day, six mice were euthanized and the skin at the wound edge was cut (try to select the same location of skin) for the following tests.

[0085] (4) Histopathological examination: Paraffin sections of skin tissue were obtained and stained with HE. Microscopic examination and image collection were performed. The thickness of the epidermis and dermis of the skin tissue and the inflammatory score were calculated using Image J software.

[0086] (5) RT-qPCR detection: Skin tissue was collected and mRNA was extracted (according to the instructions of the corresponding extraction kit). Then, RT-qPCR technology was used, combined with microscopy and image acquisition, to detect the mRNA expression of transforming growth factor TGF-β1, fibronectin, vascular endothelial growth factor VEGF, collagen-I and epidermal growth factor EGF in the skin tissue.

[0087] Studies have found that active peptides from Pinctada martensii, Paphia undulata, and Scallop can effectively promote cell proliferation and migration, collagen and elastic fiber production, and capillary regeneration. They can also significantly reduce the production of inflammatory factors, accelerate wound contraction, promote re-epithelialization and regeneration of hair follicle appendages, reduce scar formation, and effectively improve the healing speed and quality.

[0088] Compared with the blank control group, the mRNA expression levels of TGF-β1, fibronectin, VEGF, collagen-I, and EGF in the skin tissue of the Pinctada martensii active peptide (HG), Paphia undulata active peptide (HL), and Scallop active peptide (HM) treatment groups were significantly increased on the 5th day; on the 10th day, the wound healing rates of each treatment group were significantly increased by 23.54% (p<0.01), 21.22% (p<0.01), and 20.34% (p<0.01).

[0089] It can be seen that the marine shellfish active peptide prepared by the present invention has a significant effect of promoting skin wound repair.

[0090] It is noteworthy that the marine shellfish active peptides prepared in Examples 2-4 of the present invention can achieve a level comparable to that of Example 1 in the evaluation of skin wound repair efficacy, and their levels of promoting wound healing and reducing scar hyperplasia are comparable.

[0091] Comparative Example 1

[0092] Compared with Example 3, except that the soft part of the fresh shellfish is replaced by the mantle, the rest is the same as Example 3.

[0093] The efficacy of the mantle active peptide prepared in this embodiment was evaluated using an in vitro cell experiment (see Example 5 for the specific experimental method). The study found that at concentrations of 50 μg / mL-400 μg / mL, there was no significant change in cell activity in the mantle active peptide-treated group (no significant difference compared to the blank control). However, N1F1 prepared by the present invention increased HaCaT cell activity by 12.31% at 50 μg / mL (p < 0.05 compared to the blank control); N1F2 prepared by the present invention increased HaCaT cell activity by 20.13% at 100 μg / mL (p < 0.01 compared to the blank control).

[0094] According to the method in Example 3, the mantle active peptide was prepared into a medical biomaterial and applied to deep second-degree burns and third-degree burns. Although it had a certain effect of promoting new tissue formation, it did not significantly improve the speed of wound healing and the quality of healing. Therefore, it can be seen that the mantle active peptide prepared in this embodiment is significantly inferior to the marine shellfish active peptide prepared in Example 3 in promoting the repair of skin wounds.

[0095] Comparative Example 2

[0096] Compared with Example 3, except that the solution was boiled for 10 minutes before enzymatic hydrolysis and ultrasonic treatment was added during the enzymatic hydrolysis, the other steps were the same as those in Example 3.

[0097] The efficacy of the active peptide prepared in this embodiment was evaluated using an in vitro cell experiment (see Example 5 for the specific experimental method). The study found that at a concentration of 50 μg / mL-800 μg / mL, there was no significant change in cell activity in the active peptide-treated group (no significant difference compared to the blank control). However, the N1F1 prepared by the present invention increased the activity of HaCaT cells by 12.31% at 50 μg / mL (p < 0.05 compared to the blank control); the N1F2 prepared by the present invention increased the activity of HaCaT cells by 20.13% at 100 μg / mL (p < 0.01 compared to the blank control).

[0098] Using the method described in Example 3, the active peptide was prepared into a marine medical biomaterial and applied to diabetic ulcer wounds. The material showed no significant improvement in ischemia and edema, in promoting new tissue formation, or in increasing the speed or quality of wound healing. This suggests that the active peptide prepared in this embodiment is significantly inferior to the marine shellfish active peptide prepared in Example 3 in promoting skin wound repair.

Claims

1. An application of a marine shellfish active peptide in the preparation of a skin repair product for diabetic ulcer wounds, characterized in that: The preparation of the marine shellfish active peptide comprises the following steps: Fresh scallops that meet the relevant seafood use standards of my country are shelled, the soft part is homogenized, 2.5 times the volume of distilled water is added, the pH value is adjusted to 7.0, neutral protease is added according to 2000U / g scallop meat, the temperature is 50℃, and enzymatic hydrolysis is carried out for 5h; the enzymatic hydrolysis solution is boiled for 10min and centrifuged at 6000×g for 15min; the supernatant is ultrafiltered through ultrafiltration membranes with a molecular weight cutoff of 10kDa and 3kDa in sequence; among them, the 3kDa-10kDa active peptide component is recorded as N1; the N1 component is concentrated to a concentration of about 200mg / mL, and then separated by polysaccharide gel G-15: the sample volume is 7mL, the mobile phase is distilled water, the flow rate is 10mL / min, and the detection wavelengths are 220nm and 280nm; the elution peak components within 50-110 minutes in the elution component N1F2 are collected, concentrated and freeze-dried to obtain the marine shellfish active peptide.

2. The use according to claim 1, characterized in that The dosage forms of the product include lotion, ointment, powder, oil, paste, plaster, film or aerosol; the mass fraction of the marine shellfish active peptide in the product is 0.01%-2.5%.

3. The use according to claim 1, characterized in that The product is a medical biomaterial, and its dosage forms include lotion, dressing, ointment or injection. The mass fraction of the marine shellfish active peptide in the medical biomaterial is 0.8%-2.5%.

4. The use according to claim 1, wherein The product is a cosmetic, and the dosage forms include cream, lotion, gel, aqueous solution or spray; the mass fraction of the marine shellfish active peptide in the cosmetic is 0.05%-1.0%.

Images