Marine biopeptide for promoting skin wound repair and preparation method and application thereof

Abstract

The application discloses a marine organism peptide capable of effectively promoting skin wound repair, and a preparation method and application thereof. The marine organism peptide has good skin compatibility, can significantly promote cell proliferation, migration and vascular formation, effectively reduces inflammatory reaction, promotes high-quality repair of wounds, and reduces scar formation. The preparation method of the marine organism peptide comprises the following steps: taking a fresh and live soft body of a marine shellfish, performing protease enzymolysis, ultrafiltration and dextran gel separation, discarding an elution single peak in the last step, collecting or mixing other elution components, and freeze-drying to obtain the marine organism peptide. The method is simple, low in cost, green and environment-friendly, short in production period, and suitable for industrial production. The marine organism peptide can be applied to medicines, medical biomaterials and cosmetics, has a wide application range, and has a remarkable effect on promoting regeneration and repair of burn, scald, cutting, traffic, surgical, war injury, firearm injury combined with seawater immersion injury, diabetic ulcer, inflammatory ulcer, pressure ulcer and chronic refractory wounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a marine biological peptide that promotes skin wound repair, its preparation method, and its application. Background Technology

[0002] Skin, covering the body's surface, is one of the largest organs in the human body and serves as the body's first line of defense against external harm. Being in direct contact with the outside world, skin is highly susceptible to damage from trauma, burns, and other processes. According to the World Health Organization, approximately 9 million people suffered burns in 2017. Besides acute trauma, various common diseases (such as diabetic foot, aging, obesity, vascular disease, cancer, and infections) affect more than 1% of the world's population, causing slow or incomplete wound healing. Wound healing time is closely related to susceptibility to infection, duration of pain, length of hospital stay, and scarring rate.

[0003] Skin wound repair mechanisms are often influenced by numerous factors, such as age, nutritional status, endocrine changes, local blood circulation, infection, ionizing radiation, medications, and systemic diseases. Small-area injuries can heal within days without leaving scars. However, in wounds with significant tissue loss or large wounds, regenerated epidermis often fails to completely repair the wound. The body then fills the wound with connective tissue, frequently leaving scars after repair. Although the surface is covered with keratinized epidermis, these scars lack sweat glands, hair, and dermal papillae, losing their original tissue structure and function.

[0004] Furthermore, insufficient healing capacity for chronic wounds is particularly evident. In diabetes, the incidence of diabetic foot is as high as 25%, with abnormally high blood sugar leading to tissue cell damage and metabolic abnormalities, severely impairing the wound repair rate. In obese individuals, the thick layer of fat in the skin hinders microvascular angiogenesis, and wounds are prone to infection due to fat liquefaction, resulting in slower wound healing. In aging individuals, the decline in cellular compensatory function, weakened stem cell self-renewal function, and reduced ability to clear damaged cells significantly slows the wound healing rate.

[0005] Therefore, promoting rapid and high-quality healing of these acute and chronic wounds is an issue that cannot be ignored in clinical nursing. Clinical treatment primarily involves different approaches and medications for acute and chronic skin injuries. my country's Class I genetically engineered new drug, recombinant bovine basic fibroblast growth factor (rb-bFGF), promotes wound healing and is mainly used for burns, chronic superficial ulcers, and fresh wounds (including traumatic injuries, donor site wounds, and surgical wounds). However, due to the high cost of its preparation, this polypeptide and recombinant protein cannot be widely applied and promoted in fields such as medical biomaterials and cosmetics. Summary of the Invention

[0006] One objective of this invention is to provide a marine bioactive peptide that effectively promotes skin wound repair. This marine bioactive peptide exhibits good skin compatibility, significantly enhances cell vitality, promotes cell proliferation, migration, and angiogenesis, effectively reduces inflammatory responses, facilitates high-quality skin wound repair, and effectively reduces scar hyperplasia.

[0007] Another objective of this invention is to provide a method for preparing marine bioactive peptides via enzymatic hydrolysis, ultrafiltration, and separation using dextran gel G-15. This method is simple, low-cost, environmentally friendly, and has a short production cycle, making it suitable for large-scale industrial production.

[0008] Another objective of this invention is to provide an application of marine biopeptides, which can be used in pharmaceuticals, medical biomaterials, and cosmetics.

[0009] The present invention achieves the above objectives through the following technical solutions:

[0010] On the one hand, the present invention provides a marine biopeptide with good water solubility and skin compatibility.

[0011] The marine biopeptides of this invention are products obtained by enzymatic hydrolysis of the soft tissues of fresh marine shellfish using proteases.

[0012] These marine mollusks may include, but are not limited to, one or a combination of several of the following: Pinctada martensii, Bafi clam, and scallop.

[0013] The protease used can be an enzyme that can break down proteins, including but not limited to one or a combination of papain, neutral protease, and trypsin.

[0014] Previously, the inventors achieved good results using marine shellfish peptides in skincare and haircare research. However, due to the significant differences in physiological state and structure between traumatic skin tissue and normal skin tissue, there are currently no reports of substances used for general skincare being directly applicable to the care of traumatic 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 tissue formation; 3. Wound contraction and scar formation. These three stages overlap.

[0016] 1. Inflammatory Response: This begins immediately after injury and usually lasts 3-5 days. The main changes are blood coagulation and fibrinolysis, immune response, increased microvascular permeability, and the exudation of inflammatory cells (initially neutrophils, later monocytes). Its significance lies in clearing injury-causing factors (such as pathogens and other foreign substances) and necrotic tissue, preventing infection, and laying the foundation for tissue regeneration and repair. PDGF, IGF-1, EGF, and TGF-β released by platelets play important roles 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 to the wound.

[0017] 2. Tissue proliferation and granulation formation: 24-48 hours after injury, epithelial cells at the wound margin begin to proliferate. Some basal cells detach from the dermis and migrate towards the defect area, and mitosis is observed. Simultaneously, fibroblasts and myofibroblasts with abundant cytoplasm, appearing as spindle-shaped or star-shaped cells at the injury site. The latter are similar to the former but contain microfilament bundles parallel to the cell's long axis and attach to the membrane (facilitating cell contraction). Angiogenesis mainly involves the "sprouting" of existing blood vessels into new capillaries; existing vascular loops may also elongate. Fibroblasts secrete IGF-1, bFGF, TGF-β, PDGF, and KGF; endothelial cells synthesize bFGF and PDGF; keratinocytes synthesize TGF-β, TGF-α, and keratinocyte-derived autocrine factor (KAF). These growth factors stimulate cell proliferation, intercellular matrix protein synthesis, and angiogenesis.

[0018] 3. Wound Contraction and Scar Formation: 3-5 days after injury, the wound edges begin to move and contract towards the center to eliminate the wound surface and restore the continuity of body tissues. This often occurs before the wound surface is fully epithelialized, initially due to the contraction of microfibril bundles of epithelial cells at the wound edge, and eventually due to the contraction of myofibroblasts located in the center of the wound. PDGF and TGF-β play important roles in the transformation of granulation tissue into scar tissue. As the healing process progresses, collagen fibers continuously increase, while fibroblasts and capillaries gradually decrease, eventually transforming into scar tissue with fewer cells and blood vessels but more fibers.

[0019] During wound healing, as fibroblasts grow in, the content of fibronectin in the wound area increases and distributes along collagen within the granulation tissue. As new epithelium covers the wound and collagen matures, fibronectin gradually disappears. In the later stages of wound healing, a large number of fibroblasts are present at the wound site; these are the main repair cells, and their primary function is to synthesize collagen fibers. Collagen undergoes a dynamic process of intracellular synthesis, extracellular deposition, and reabsorption during wound healing. 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 for the first time used marine bioactive peptides obtained after removing the last eluted single peak component of dextran gel G-15 for the care of wounded skin.

[0021] As an alternative implementation method, the preparation method of marine bioactive peptides involves enzymatic hydrolysis of the soft body of marine shellfish by protease, ultrafiltration, separation by dextran gel G-15, discarding the last elution single peak component, collecting or combining each elution peak component separately, and freeze-drying to obtain marine bioactive peptides.

[0022] Preferably, in some embodiments, the papain hydrolysate of the soft body of marine shellfish can be treated with an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the resulting filtrate (less than 10 kDa) can be directly separated by dextran gel G-15.

[0023] Preferably, in some embodiments, the trypsin hydrolysate of the soft body of marine shellfish can be treated with an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the resulting filtrate (less than 3 kDa) can be separated by dextran gel G-15.

[0024] Preferably, in some embodiments, the neutral proteolytic enzyme hydrolysate from the soft tissue of marine shellfish can be sequentially treated with ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa, and the resulting retentate (3 kDa-10 kDa) can then be separated by dextran gel G-15.

[0025] The specific steps are as follows: Fresh marine shellfish are shelled, and the soft body is homogenized. This homogenate is diluted with 2.5-4 times its volume of water or PBS buffer. Protease (2000 U / g shellfish meat - 4000 U / g shellfish meat) is added, and the mixture is hydrolyzed at 40℃-50℃ and pH 6.5-7.5 for 4-6 hours. The enzyme is then inactivated by boiling in a water bath for 10 minutes. The mixture is centrifuged at 6000×g-9000×g for 10-15 minutes. The supernatant is ultrafiltered using a 10kDa molecular weight cutoff ultrafiltration membrane. The filtrate may be ultrafiltered again using a 3kDa molecular weight cutoff ultrafiltration membrane. The active peptides (less than 10kDa) from the papain hydrolysate, the active peptides (3kDa-10kDa) from the neutral protease hydrolysate, and the active peptides (less than 3kDa) from the trypsin hydrolysate are concentrated separately. These solutions are then separated using a dextran gel G-15. After discarding the last elution peak, the remaining elution peaks are collected or combined, and then freeze-dried to obtain marine bioactive peptides.

[0026] Preferably, the elution peak components are a first elution peak component, a second elution peak component, a third elution peak component, a fourth elution peak component, a fifth elution peak component, or any combination of these elution peak components.

[0027] Preferably, the collected elution peak components are active peptide components with less than 10 kDa from papain hydrolysate, which are collected after separation by dextran gel G-15 (e.g., active peptides from Pinctada martensii, Pterocarya serrata, and Scallop in Example 1), and have significant effects in promoting cell proliferation and skin wound repair.

[0028] Preferably, when the collected elution peak components are the active peptide components of neutral protease hydrolysate (3kDa-10kDa) collected after separation by dextran gel G-15 (e.g., N1 separation components F1-F6 in Example 2 and N1F1 and N1F2 in Example 3), they have significant effects in promoting cell proliferation and skin wound repair.

[0029] Preferably, when the collected elution peak components are active peptide components with less than 3kDa from trypsin hydrolysate separated by dextran gel G-15 (e.g., T2F1 in Example 4 and T2 separation components F1-F5 in Example 5), their effects on promoting cell proliferation and skin wound repair are significant.

[0030] On the other hand, this invention provides an application of marine bioactive peptides that can effectively promote cell proliferation and skin wound repair. The minimum effective dose is 100 μg / mL.

[0031] As an alternative implementation, marine biopeptides can be used to promote the repair of skin wounds.

[0032] In a preferred embodiment of the present invention, marine bioactive peptides can be used in pharmaceuticals; preferably, these marine bioactive peptides are particularly suitable for pharmaceuticals that can effectively repair skin wounds, wherein the pharmaceutical dosage forms include lotions, ointments, tinctures, liniments, varnishes, powders, oils, pastes, plasters, films, aerosols, etc. When used in pharmaceuticals, the mass fraction of the marine bioactive peptides in the pharmaceuticals is 0.01% to 2.5%.

[0033] In a preferred embodiment of the present invention, marine biopeptides can also be used in medical biomaterials; preferably, the marine biopeptides are particularly suitable for medical biomaterials that can effectively repair skin wounds, wherein the dosage forms of the medical biomaterials 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 biopeptides in the medical biomaterials is 0.8% to 2.5%.

[0034] In a preferred embodiment of the present invention, marine bioactive peptides can be used in cosmetics; preferably, these marine bioactive peptides are particularly suitable for cosmetics that can effectively promote skin wound repair, wherein the cosmetic dosage forms include creams, lotions, gels, jelly, aqueous solutions, sprays, etc. When used in cosmetics, the mass fraction of the marine bioactive peptides in the cosmetic is 0.05% to 1.0%.

[0035] It is worth mentioning that the marine bioactive peptides provided by this invention, as functional ingredients, do not cause skin allergies and can significantly reduce inflammatory responses, promote cell proliferation, and repair skin wounds. Therefore, when the marine bioactive peptides of this invention are applied in pharmaceuticals, medical biomaterials, or cosmetics, the selection of the types, dosages, and preparation processes of pharmaceutical raw materials, medical biomaterial raw materials, and cosmetic raw materials is relatively broad. All components used in pharmaceuticals, medical biomaterials, and cosmetics should be skin-acceptable and should not affect the original performance of the marine bioactive peptides of this invention; that is, when in contact with human skin or in combination with other components, they should not cause inappropriate toxicity, incompatibility, instability, or allergic reactions.

[0036] The solution of this invention is based on the inventors' understanding of skin wound repair mechanisms and structure-activity relationships of marine biological peptides, combined with the research results of modern pharmacology, medical biomaterials and cosmetics. Through a large number of creative experiments, the inventors have searched for, explored, prepared and selected marine biological peptides that are compatible with the skin, non-allergenic and have significant effects on promoting skin wound repair.

[0037] The present invention has the following outstanding beneficial effects:

[0038] (1) Exploring the efficacy of marine bioactive peptides in promoting skin wound repair. From numerous marine shellfish, *Pinctada martensii*, *Bacillus undulatus*, and scallops were selected. Fresh soft tissues of these shellfish were used, and marine bioactive peptides were prepared using enzymatic hydrolysis, ultrafiltration, and dextran gel separation techniques. These active peptides can rapidly penetrate the skin surface, exerting multiple effects such as promoting cell proliferation and wound repair. Even at extremely low doses (50 μg / mL), the marine bioactive peptides of this invention can still significantly promote cell proliferation and wound repair.

[0039] (2) The preparation process of the marine biopeptides of the present invention is simple, low-cost, environmentally friendly, and has a short production cycle, making it suitable for large-scale industrial production and with great application prospects. Based on the characteristics of marine shellfish composition, protease specificity, and action sites, papain, neutral protease, and trypsin are selected from a large number of proteases. The soft parts of marine shellfish are subjected to targeted enzymatic hydrolysis. The hydrolysate is then treated with an ultrafiltration membrane with a molecular weight cutoff of 10kDa and / or 3kDa to remove components with poor water solubility, large molecular weight, and no significant cell proliferation-promoting activity (which may contain salts, free amino acids, fatty acids, etc.), resulting in better physiological efficacy of the obtained active peptides and minimal environmental pollutant emissions.

[0040] (3) The marine bioactive peptides of the present invention have good water solubility and skin compatibility, do not cause skin irritation or sensitization, and have significant efficacy, and can be widely used in biomedicine, medical biomaterials, and cosmetics. By separating with dextran gel G-15, smaller molecular weight and less active eluting components are removed, which can further enrich the active peptides. While improving physiological efficacy, the dosage is greatly reduced, thereby effectively reducing the requirements and operational difficulties of stability and compatibility of the marine bioactive peptides in the formulation of biomedicine, medical biomaterials, and cosmetics.

[0041] (4) The present invention uses a cell activity experimental model to evaluate the activity of each prepared active peptide component in a high-throughput manner. The marine biopeptides selected in the end showed excellent skin wound repair effects at both the cellular and animal levels, indicating that the cell experimental model can accurately guide the directional separation and high enrichment of the marine biopeptides of the present invention. Attached Figure Description

[0042] Figure 1 This is the separation pattern of dextran gel G-15 for the N1 (3kDa-10kDa) component in Example 2.

[0043] Figure 2 This is the separation pattern of dextran gel G-15 for the N2 (less than 3 kDa) component in Example 2.

[0044] Figure 3 This is the separation pattern of dextran gel G-15 for the N1 (3kDa-10kDa) component in Example 3.

[0045] Figure 4 The effect of the N1 (3kDa-10kDa) isolated component in Example 3 on HaCaT cell activity (*p<0.05, **p<0.01 compared with the blank control).

[0046] Figure 5 This is the separation pattern of dextran gel G-15 for component T1 (3kDa-10kDa) in Example 4.

[0047] Figure 6 This is the separation pattern of dextran gel G-15 for the T2 (less than 3 kDa) component in Example 4.

[0048] Figure 7 This describes the effects of the T1 (3kDa-10kDa) and T2 (less than 3kDa) isolated components from Example 4 on HaCaT cell activity (*p<0.05, **p<0.01 compared to the blank control).

[0049] Figure 8 This is the RP-HPLC separation chromatogram of N1(3kDa-10kDa)F4 in Example 6.

[0050] Figure 9 The effect of the RP-HPLC separation fraction of N1(3kDa-10kDa)F4 in Example 6 on HaCaT cell activity (*p<0.05, **p<0.01 compared with the blank control).

[0051] Figure 10 These are images of the wound 10 days after administration of the marine biopeptide in Example 8 (Control: blank control; HG: active peptide of Pinctada martensii; HL: active peptide of Clam flounder; HM: active peptide of Scallop) (Compared with the blank control, *p<0.05, **p<0.01).

[0052] Figure 11 HE staining of skin tissue after administration of marine biological peptides in Example 8 (Control: blank control; HG: active peptide from Pinctada martensii; HL: active peptide from Clam of Sharon; HM: active peptide from Scallop).

[0053] Figure 12 This is the RT-qPCR analysis of skin tissue after administration of marine biological peptides in Example 8 (Control: blank control; HG: active peptide of Pinctada martensii; HL: active peptide of Clam flounder; HM: active peptide of scallop) (compared with blank control, *p<0.05, **p<0.01). Detailed Implementation

[0054] The present invention will be further described below with reference to the accompanying drawings. However, the embodiments of the present invention are not limited to the following examples. All equivalent changes or modifications made according to the method of the present invention should be considered within the scope of protection of the present invention. Unless otherwise specified, all raw materials used below are commercially available.

[0055] Example 1

[0056] Fresh Pinctada martensii oysters meeting relevant Chinese seafood usage standards were shelled, and the soft body was homogenized. 3.4 times the volume of distilled water and papain (4000 U / g of oyster meat) were added, and the mixture was enzymatically hydrolyzed at 50℃ and pH 6.5 for 6 hours. The hydrolysate was boiled for 10 minutes and centrifuged at 7000×g for 10 minutes. The supernatant was then passed sequentially through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The ultrafiltration filtrate (active peptide fractions less than 10 kDa) was concentrated to approximately 100 mg / mL and then separated using a dextran gel G-15 (100×4.0 cm): 15 mL was loaded, distilled water was used as the mobile phase, the flow rate was 10 mL / min, and the detection wavelengths were 220 nm and 280 nm. The eluent fractions, except for the last elution peak, were combined, concentrated, and lyophilized to obtain Pinctada martensii active peptides (HG). Following the same method, wavy clam active peptide (HL) and scallop active peptide (HM) were prepared respectively.

[0057] The efficacy of these bioactive peptides (HG, HL, HM) was evaluated using in vitro cell experiments (specific experimental methods are described in Example 7). The study found that at various dosages (50 μg / mL–1600 μg / mL), HG, HL, and HM significantly promoted HaCaT cell proliferation (significantly different from the control group). Specifically, at a lower concentration of 50 μg / mL, HG, HL, and HM significantly increased HaCaT cell activity by 10.93% (p<0.05), 11.02% (p<0.05), and 11.21% (p<0.05), respectively.

[0058] Applying active peptide solutions from *Pinctada martensii* (containing 2% HG, with physiological saline as the solvent, filtered and sterilized at 0.22μm), *Clam flounder* (containing 2% HL, with physiological saline as the solvent, filtered and sterilized at 0.22μm), and scallops (containing 2% HM, with physiological saline as the solvent, filtered and sterilized at 0.22μm) to the eschar removal wounds of deep second-degree burns and third-degree burns can reduce the infection of pathogens (*Pseudomonas aeruginosa*, *Acinetobacter baumannii*, *Staphylococcus aureus*, etc.), alleviate the inflammatory response, effectively prevent the wound from deepening, promote the formation of new tissue, and improve the wound healing speed and quality. These solutions have a significant effect on promoting the regeneration and repair of burn wounds.

[0059] Example 2

[0060] Fresh, live scallops meeting relevant Chinese seafood usage standards were shelled, and the soft body was homogenized. Three volumes of PBS buffer (0.01M, pH 7.5) and neutral protease (3000 U / g scallop meat) were added, and the mixture was enzymatically hydrolyzed at 45℃ for 5 hours. The hydrolysate was boiled for 10 minutes and centrifuged at 7000×g for 12 minutes. The supernatant was then ultrafiltered sequentially through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa. The 3 kDa-10 kDa active peptide fraction was designated N1, and the fraction less than 3 kDa was designated N2. The N1 and N2 fractions were concentrated to approximately 100 mg / mL and then separated using a dextran gel G-15 (100×4.0 cm): 15 mL was loaded, distilled water was used as the mobile phase, the flow rate was 10 mL / min, and the detection wavelengths were 220 nm and 280 nm.

[0061] The N1 (3kDa-10kDa) fraction was eluted using dextran gel G-15. Figure 1 Fractions F1, F2, F3, F4, F5, F6, and F7 were collected, concentrated, and lyophilized. Similarly, the N2 (less than 3 kDa) fraction was eluted using a dextran gel G-15. Figure 2 The separated components F1, F2, F3, F4, F5, and F6 were collected, concentrated, and freeze-dried.

[0062] The efficacy of the 13 isolated components was evaluated using in vitro cell experiments (specific experimental methods are described in Example 7). The study found that, compared to the blank control, the cell activity of N1 isolated component F7 and N2 isolated component F6 was significantly reduced after administration (significant cytotoxicity); the cell activity of the N2 isolated components F1-F5 treatment groups showed no significant change (no cell proliferation promotion effect). However, N1 (3kDa-10kDa) isolated components F1-F6, even at a low concentration (50 μg / mL), significantly enhanced HaCaT cell activity (significantly different from the blank control).

[0063] Therefore, it can be seen that the N1 (3kDa-10kDa) fractions F1-F6 have a significant effect on promoting the proliferation of HaCaT cells. These six fractions can be used individually, or in combination or merging, to promote cell proliferation and skin wound repair.

[0064] The N1 (3kDa-10kDa) fractions F1-F6 were separately prepared into solutions (the effective dose of peptide was 1.5%, the solvent was physiological saline, and the solution was sterilized by 0.22μm filtration). These solutions were then applied to cut wounds and surgical suture wounds, respectively. This effectively reduced the inflammatory response, promoted the formation of new tissue, improved the wound healing speed and quality, and reduced scar formation. It has a significant effect on promoting the regeneration and repair of cut wounds.

[0065] Example 3

[0066] Fresh, live scallops meeting relevant Chinese seafood usage standards were shelled, and the soft body was homogenized. 2.5 times the volume of distilled water was added, the pH adjusted to 7.0, and neutral protease (2000 U / g scallop meat) was added. Enzymatic hydrolysis was performed at 50℃ for 5 hours. The hydrolysate was boiled for 10 minutes and centrifuged at 6000×g for 15 minutes. The supernatant was then ultrafiltered sequentially through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa. The 3 kDa-10 kDa active peptide fraction was designated N1. The N1 fraction was concentrated to approximately 200 mg / mL and then separated using a dextran gel G-15 (100×4.0 cm): 7 mL was loaded, distilled water was used as the mobile phase, the flow rate was 10 mL / min, and the detection wavelengths were 220 nm and 280 nm. Elution fractions N1F1, N1F2, and N1F3 were collected separately. Figure 3 ), and concentrated and freeze-dried.

[0067] The efficacy of the three isolated components was evaluated using in vitro cell experiments (see Example 7 for specific experimental methods). Figure 4 The study found that N1F3 administration significantly reduced cell viability (significant cytotoxicity). However, administration of the other two isolated components (N1F1 and N1F2) significantly enhanced cell viability. Specifically, N1F1 at a low concentration of 50 μg / mL increased HaCaT cell viability by 12.31% (p<0.05); and N1F2 at a concentration of 100 μg / mL increased HaCaT cell viability by 20.13% (p<0.01).

[0068] Therefore, it can be seen that the N1 (3kDa-10kDa) isolated components N1F1 and N1F2 have a significant effect on promoting HaCaT cell proliferation. N1F1 and N1F2 can be used alone or in combination to promote cell proliferation and skin wound repair.

[0069] Marine biomedical materials (N1F1@calcium alginate microspheres / collagen / chitosan scaffold and N1F2@calcium alginate microspheres / collagen / chitosan scaffold) were prepared from N1F1 and N1F2 respectively using the following methods, and their efficacy was evaluated.

[0070] N1F1@Calcium Alginate Microspheres: Sodium alginate (purchased from Shanghai Maclean 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 Maclean Biochemical Technology Co., Ltd.) was measured, and Tween 80 (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) was added at a 1% v / v ratio. The mixture was stirred at 600 rpm for 2 hours to prepare an organic phase. 1 00 mL of organic phase was added to 20 mL of aqueous phase under stirring at 650 rpm, and stirred at 750 rpm for 1 h. Then, 40 mL of 1% w / v calcium chloride solution (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) was added, and stirred at 500 rpm for 1 h. Then, 10 mL of isopropyl ketone (Tianjin Fuyu Chemical Reagent Factory, AR grade) was added, and stirred at 400 rpm for 30 min. The mixture was centrifuged at 6000 rpm for 5 min. The resulting precipitate was washed three times alternately with isopropyl ketone and distilled water, and then freeze-dried.

[0071] N1F1@Calcium Alginate Microspheres / Collagen / Chitosan Scaffold: Collagen was dissolved in a 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 at a 1:1 volume ratio, and N1F1@calcium alginate microspheres were added to achieve a final concentration of 0.01 g / mL. The mixture was magnetically stirred at 700 rpm for 1 h. The mixture was then added to a 48-well plate. After the lyophilized scaffold was demolded, it was soaked in EDC / NHS solution (EDC concentration 0.5 mol / L, NHS concentration 0.5 mol / L) for 2 h, washed three times with distilled water, and lyophilized to obtain the N1F1@calcium alginate microspheres / collagen / chitosan scaffold.

[0072] Following the same method, N1F2@calcium alginate microspheres / collagen / chitosan scaffolds were prepared.

[0073] Applying N1F1@calcium alginate microspheres / collagen / chitosan scaffolds and N1F2@calcium alginate microspheres / collagen / chitosan scaffolds to the wound surface after debridement of gunshot wounds (a type of combat wound) can rapidly absorb and drain wound exudate, providing a dry, slightly acidic, and low-oxygen environment for the wound, reducing pathogen infection (Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, etc.), alleviating inflammatory response, effectively preventing wound deepening, promoting the formation of new tissue, and improving the wound healing speed and quality. It has a significant effect on promoting the regeneration and repair of gunshot wounds.

[0074] Example 4

[0075] Fresh, live scallops meeting relevant Chinese seafood usage standards were shelled, and the soft body was homogenized. Three times the volume of deionized water was added, and the pH was adjusted to 7.5. Trypsin (3000 U / g scallop meat) was added, and the mixture was enzymatically hydrolyzed at 40℃ for 4 hours. The hydrolysate was boiled for 10 minutes and centrifuged at 8000×g for 12 minutes. The supernatant was then passed sequentially through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa. The 3 kDa-10 kDa active peptide fraction was designated T1, and the fraction less than 3 kDa was designated T2. The T1 and T2 fractions were concentrated to approximately 100 mg / mL and then separated using a dextran gel G-15 (100×4.0 cm) spectrophotometer: 10 mL sample was loaded, distilled water was used as the mobile phase, the flow rate was 10 mL / min, and the detection wavelengths were 220 nm and 280 nm. The T1 (3kDa-10kDa) fraction was separated using a dextran gel G-15, and T1F1, T1F2, and T1F3 were collected separately. Figure 5 The T2 fraction (less than 3 kDa) was concentrated and lyophilized. Similarly, the T2 fraction was separated using a dextran gel G-15, and T2F1 and T2F2 were collected separately. Figure 6 ), and concentrated and freeze-dried.

[0076] The efficacy of the five isolated components was evaluated using in vitro cell experiments (specific experimental methods are described in Example 7). The study found that T1F3 and T2F2 exhibited significant cytotoxicity, resulting in a significant decrease in cell viability after administration (significantly different from the blank control); T1F1 and T1F2 showed little change in cell viability after administration (no significant difference compared to the blank control). However, T2F1, at a low concentration of 100 μg / mL, significantly increased HaCaT cell viability by 10.12% (p<0.01).

[0077] Therefore, it can be seen that the T2 (less than 3kDa) isolated component T2F1 has a significant effect on promoting the proliferation of HaCaT cells and can be used to promote skin wound repair.

[0078] Following the preparation method of TCM biomaterials in Example 3, T2F1@calcium alginate microspheres / collagen / chitosan scaffolds were prepared and applied to diabetic ulcer wounds. This provided a clean, slightly acidic, and low-oxygen environment for the wound, effectively protecting the ulcer from damage and secondary infection, improving local ischemia and edema, promoting the formation of new tissue, and improving the wound healing speed and quality. It has a good effect on promoting the regeneration and repair of diabetic ulcer wounds.

[0079] Example 5

[0080] Following the preparation method of Example 4, the T2 (less than 3 kDa) fraction was prepared and concentrated to a concentration of approximately 150 mg / mL. It was then separated using a dextran gel G-15 (100 × 4.0 cm): 8 mL of sample was loaded, distilled water was used as the mobile phase, the flow rate was 10 mL / min, and the detection wavelengths were 220 nm and 280 nm. The first to sixth elution peaks were collected sequentially, concentrated, and lyophilized to obtain the T2 (less than 3 kDa) fraction. This fraction was then concentrated to a concentration of approximately 150 mg / mL and separated using a dextran gel G-15 (100 × 4.0 cm): 8 mL of sample was loaded, and the T2 (less than 3 kDa) fractions F1, F2, F3, F4, F5, and F6 were separated.

[0081] The efficacy of the six isolated components was evaluated using in vitro cell experiments (specific experimental methods are described in Example 7). The study found that the T2 (less than 3 kDa) isolated component F6 exhibited significant cytotoxicity, resulting in a significant decrease in cell activity after administration (significantly different from the blank control); while the T2 (less than 3 kDa) isolated components F1-F5, at a low concentration (50 μg / mL), significantly enhanced HaCaT cell activity (significantly different from the blank control).

[0082] Therefore, it can be seen that the T2 (less than 3kDa) isolated components F1-F5 have a significant effect on promoting the proliferation of HaCaT cells. These five isolated components can be used individually, or in combination or merging, to promote cell proliferation and skin wound repair.

[0083] The T2 (less than 3 kDa) fractions F1-F5 were prepared into solutions (effective dose 0.8%, solvent is physiological saline, 0.22 μm filtration sterilization) and applied to deep second-degree and third-degree burn wounds. This can reduce the infection of pathogens (Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, etc.), alleviate the inflammatory response, effectively prevent the wound from deepening, promote the formation of new tissue, improve the wound healing speed and healing quality, and has a significant effect on promoting the regeneration and repair of burn wounds.

[0084] Example 6

[0085] RP-HPLC technology can be used to further separate and purify marine biopeptides.

[0086] High-performance liquid chromatography (HPLC) using an Agilent 1260 system with a YMC Triart C18 column (250 × 10 mm, S-5 μm, 12 nm) was employed for the separation and purification of bioactive peptides. Mobile phase A consisted of water (containing 0.1% formic acid); mobile phase B consisted of acetonitrile (containing 0.1% formic acid); the temperature was 40 °C. The sample concentration was 150 mg / mL, the loading volume was 50 μL, the flow rate was 2.5 mL / min, and the detection wavelengths were 200 nm, 220 nm, 254 nm, and 280 nm.

[0087] Because the elution conditions for each component of the marine bioactive peptides in this invention are slightly different, 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 respectively: 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). Each component was concentrated and freeze-dried separately to obtain N1(3kDa-10kDa)F4 separated components F1, F2, F3, F4, F5, and F6. Figure 8 ).

[0088] The efficacy of the six prepared components (F1, F2, F3, F4, F5, and F6) was evaluated using in vitro cell experiments (specific experimental methods are described in Example 7). Figure 9 The study found that although F1, F5, and F6 at various concentrations (37.5 μg / mL-600 μg / mL) could significantly enhance the activity of HaCaT cells (significantly different from the blank control), their cell activity was still lower than that of the N1F4 component before separation.

[0089] Therefore, the N1F4 component can be directly used to promote cell proliferation and skin wound repair without further separation and purification by RP-HPLC technology.

[0090] Following the preparation method of traditional Chinese medicine biomaterials in Example 3, N1F4@calcium alginate microspheres / collagen / chitosan scaffolds were prepared. When applied to the wound surface after debridement of gunshot wounds combined with seawater immersion injuries, it can quickly absorb wound exudate, provide a dry, clean, slightly acidic and low-oxygen environment for the wound surface, protect the wound surface from pathogen infection (Vibrio vulnificus, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, etc.), reduce inflammatory response, effectively prevent the wound from deepening further, improve blood circulation and promote the formation of new tissue, improve the wound healing speed and healing quality, and has the effect of promoting the regeneration and repair of gunshot wounds combined with seawater immersion injuries.

[0091] Example 7

[0092] The cell proliferation-promoting activity of the marine biopeptides prepared in each example was evaluated using in vitro cell experiments.

[0093] Cell viability was determined using the CCK-8 assay: cells with a density of 6 × 10⁶ cells / year were tested. 4 HaCaT cell suspension of 100 μL / well was seeded into 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. 50 μL was aspirated from each well, and 50 μL of the corresponding concentration sample solution (final concentrations of 50, 100, 200, 400, 800, and 1600 μg / mL, sterilized using a 0.22 μm needle filter) was added to each well for the sample groups. The blank control group received 50 μL of complete DMEM culture medium. After culturing in a CO2 incubator for another 24 h, cell morphology was observed using a fluorescence inverted phase-contrast microscope (CKX41, Olympus Corporation, Japan). Subsequently, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 4 h. The absorbance of each well was measured using a Multiskan GO microplate reader (Thermo Fisher Scientific) at a wavelength of 450 nm (reference wavelength 650 nm), with 6 parallel wells per group.

[0094] Following the experimental steps described above, the efficacy of each component prepared in the embodiments of this invention was evaluated. The study found that the last eluted single-peak component obtained by dextran gel G-15 separation—such as component F7 separated by N1 in Example 2, N1F3 in Example 3, T2F2 in Example 4, and component F6 separated by T2 in Example 5—significantly reduced cell activity after administration (exhibiting obvious cytotoxicity); while other separated components—such as components F1-F6 separated by N1 in Example 2, N1F1 and N1F2 in Example 3, T2F1 in Example 4, and components F1-F5 separated by T2 in Example 5—significantly increased cell activity after administration (significantly different from the blank control).

[0095] Therefore, it can be seen that the marine biopeptides prepared after discarding the last elution single peak component of dextran gel G-15 can effectively promote cell proliferation and skin wound repair.

[0096] Example 8

[0097] The marine bioactive peptides prepared in this invention (taking the active peptides of Pinctada martensii HG, active peptide of Baccarat chrysophagus HL, and active peptide of scallop HM in Example 1 as examples) were evaluated for their skin wound repair efficacy (taking a full-thickness skin defect model as an example).

[0098] Ointment base: PEG400 (400mL), PEG4000 (50g), Span 40 (1mL), distilled water (9mL), dissolved and homogenized in a 60℃ hot water bath.

[0099] Pinctada martensii active peptide (HG) ointment: contains 2% Pinctada martensii active peptide, the remainder being ointment base.

[0100] Rippling barnacle active peptide (HL) ointment: contains 2% rippling barnacle active peptide, the remainder being ointment base.

[0101] Scallop Active Peptide (HM) Ointment: Contains 2% scallop active peptides, with the remainder being an ointment base.

[0102] (1) Full-thickness skin defect model: Seventy-two male BALB / c mice aged 6-8 weeks of age (SPF grade) were purchased from Guangdong Provincial Medical Experimental Animal Center. The animals were randomly divided into four groups: blank control group, HG, HL, and HM, with 18 mice in each group. After anesthetizing the mice, the hair was removed, and a full-thickness skin wound of about 1.5 × 1.5 cm was made on the back.

[0103] (2) Administration: The blank control group and the marine biopeptide treatment groups (HG, HL, HM) were administered the ointment once a day (approximately 0.1 g ointment / mouse) for 14 consecutive days. The blank control group was given the ointment base, while the marine biopeptide treatment groups were given the corresponding ointment samples.

[0104] (3) Wound photography (for healing rate calculation): Wound healing was observed and recorded daily. Photos of the back wound were taken twice a week, maintaining consistent field of view, lighting, and image magnification. The wound area was measured using IPP6.0 software to calculate the wound healing rate. Six mice were euthanized on days 5, 10, and 14, and skin samples were taken from the wound edges (preferably from the same location) for the following tests.

[0105] (4) Histopathological examination: Paraffin sections of skin tissue were taken, stained with HE, examined under a microscope and images were acquired. Image J software was used to calculate the thickness of the epidermis and dermis of the skin tissue and the inflammation score.

[0106] (5) RT-qPCR detection: Skin tissue was taken, mRNA was extracted (according to the instructions of the corresponding extraction kit), and RT-qPCR technology was used to detect the expression of transforming growth factor TGF-β1, fibronectin, vascular endothelial growth factor VEGF, collagen-I and epidermal growth factor EGF mRNA in skin tissue, combined with microscopic examination and image acquisition.

[0107] Skin wound healing rate on day 10 Figure 10 Histopathological examination results are shown in Figure 11 The RT-qPCR test results are shown in [link to results]. Figure 12 .

[0108] Studies have found that active peptides from Pinctada martensii, Baccarat clam, 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 healing speed and quality.

[0109] Compared with the blank control group, the expression levels of TGF-β1, fibronectin, VEGF, collagen-I, and EGF mRNA in the skin tissue of the treatment groups treated with Pinctada martensii active peptide (HG), Baccarat clams active peptide (HL), and scallop active peptide (HM) were significantly increased on day 5; and on day 10, the wound healing rates of each treatment group were significantly increased by 26.07% (p<0.01), 20.70% (p<0.01), and 22.32% (p<0.01), respectively.

[0110] Therefore, the marine biopeptides prepared by this invention have significant effects in promoting skin wound repair.

[0111] It is worth noting that the marine biopeptides prepared in Examples 2-5 of the present invention can achieve a level comparable to that of Example 1 in the evaluation of skin wound repair efficacy, with comparable levels of promoting wound healing and reducing scar hyperplasia.

[0112] Comparative Example 1

[0113] Compared with Example 3, except that the soft part of the live shellfish is replaced with the mantle, everything else is the same as Example 3.

[0114] The efficacy of the mantle active peptides prepared in this embodiment was evaluated using in vitro cell experiments (specific experimental methods are described in Example 7). The study found that at concentrations of 50 μg / mL to 400 μg / mL, the cell activity in the mantle active peptide treatment group showed no significant change (no significant difference compared to the blank control). However, the N1F1 prepared in this invention increased HaCaT cell activity by 12.31% at 50 μg / mL (p < 0.05 compared to the blank control); and the N1F2 prepared in this invention increased HaCaT cell activity by 20.13% at 100 μg / mL (p < 0.01 compared to the blank control).

[0115] Following the method in Example 3, the mantle active peptides were prepared into a medical biomaterial and applied to the wound surface after debridement of gunshot wounds (a type of war wound). Although it had a certain effect in promoting the formation of new tissue, its effect in improving the wound healing speed and healing quality was not very significant. Therefore, it can be seen that the mantle active peptides prepared in this embodiment are significantly less effective than the marine biological peptides prepared in Example 3 in promoting skin wound repair.

[0116] Comparative Example 2

[0117] Compared with Example 4, except that boiling for 10 minutes before enzymatic hydrolysis and adding ultrasonic treatment during enzymatic hydrolysis, everything else is the same as Example 4.

[0118] The efficacy of the bioactive peptides prepared in this embodiment was evaluated using in vitro cell experiments (specific experimental methods are described in Example 7). The study found that at concentrations of 50 μg / mL to 800 μg / mL, the cell activity in the bioactive peptide-treated groups showed no significant change (no significant difference compared to the blank control). However, the T2F1 prepared in this invention, at a lower concentration of 100 μg / mL, significantly increased the activity of HaCaT cells by 10.12% (p < 0.01 compared to the blank control).

[0119] Following the method in Example 4, the active peptides were used to prepare marine medical biomaterials, which were then applied to diabetic ulcer wounds. However, their effects on improving local ischemia and edema, promoting new tissue formation, increasing wound healing speed, and improving healing quality were not significant. Therefore, it is evident that the active peptides prepared in this embodiment are significantly less effective than the marine biopeptides prepared in Example 4 in promoting skin wound repair.

Claims

1. The application of a marine biopeptide in the preparation of pharmaceuticals, medical biomaterials, or cosmetics that promote the repair of gunshot wounds; characterized in that, The marine bioactive peptides are obtained through the following steps: Fresh, live scallops meeting relevant seafood usage standards are shelled, the soft body is homogenized, 2.5 times its volume of distilled water is added, the pH is adjusted to 7.0, 2000 U / g of neutral protease from the scallop meat is added, and the mixture is enzymatically hydrolyzed at 50°C for 5 hours. The hydrolysate is then boiled for 10 minutes and centrifuged at 6000×g for 15 minutes. The supernatant is then ultrafiltered sequentially through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa. The 3 kDa-10 kDa active peptide fraction is designated as N1. The N1 fraction is concentrated to approximately 200 mg / mL and then separated using a 100×4.0 cm dextran gel G-15: 7 mL is loaded, distilled water is used as the mobile phase, the flow rate is 10 mL / min, and the detection wavelengths are 220 nm and 280 nm. The elution fraction from N1F2 for 50-110 minutes is collected to obtain the marine bioactive peptides. The dosage forms of the medicine include lotions, ointments, tinctures, liniments, powders, oils, pastes, plasters, film-forming agents, or aerosols; the marine biological peptides in the medicine have a mass fraction of 0.01%-2.5%; The dosage forms of the medical biomaterials include lotions, solutions, dressings, ointments, or injections, and the marine biopeptides in the medical biomaterials have a mass fraction of 0.8%-2.5%. The dosage form of the cosmetic includes creams, lotions, gels, aqueous solutions, or sprays; the mass fraction of the marine bioactive peptides in the cosmetic is 0.05%-1.0%.

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