CN115505618A - Marine shellfish active peptide for promoting skin wound repair - Google Patents

Abstract

The invention discloses a marine shellfish active peptide capable of effectively promoting skin wound repair. The active peptide of the marine shellfish has good skin compatibility, can obviously promote cell proliferation, migration and angiogenesis, effectively relieve inflammatory reaction, promote high-quality repair of wounds and reduce scar formation. The preparation method of the marine shellfish active peptide comprises the steps of taking a fresh marine shellfish soft body part, performing protease enzymolysis, ultrafiltration and sephadex separation, discarding the last elution single peak, respectively collecting or mixing other elution components, and freeze-drying to obtain the marine shellfish active peptide. The method is simple, low in cost, green, environment-friendly, short in production period and suitable for industrial production. The active peptide of the marine shellfish can be applied to medicines, medical biomaterials and cosmetics, has wide application range, and has obvious effects of promoting the regeneration and repair of burns, scalds, cuts, traffic injuries, surgical injuries, war injuries and firearm injuries which are combined with seawater soaking injuries, diabetic ulcers, inflammatory ulcers, pressure ulcers and chronic intractable wounds.

Description

Marine shellfish active peptide for promoting skin wound repair

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a marine shellfish active peptide for promoting skin wound repair, and a preparation method and application thereof.

Background

The skin is covered on the surface of the human body, is one of the organs with the largest area of the human body, and is also the first barrier for the human body to resist the external injury. The skin is directly contacted with the outside and is very easy to be damaged by the processes of trauma, burn and the like. In addition to acute trauma, many common diseases (such as diabetic foot, aging, obesity, vascular diseases, cancer, infection, etc.) cause slow or incomplete healing of wounds, and the healing time of the wounds is closely related to infection susceptibility, pain duration, hospitalization time and scar incidence.

The mechanisms of skin wound repair are often affected by a number of factors, such as age, nutritional status, endocrine changes, local blood circulation, infection, ionizing radiation, drugs, systemic diseases, and the like. The small-area injury can heal for several days without scars. However, in wounds with more tissue cells and overlarge wounds, the wounds are difficult to completely repair by regenerating epidermis, the body fills the wounds by connective tissues, scars are often left after the wounds are repaired, although the surfaces of the scars are covered by angular epidermis, sweat glands, hairs and dermal papilla are lacked, and the original tissue structures and functions are lost.

In addition, the insufficient healing capacity of chronic wounds is also obvious. In diabetes, the incidence of diabetes is as high as 25%, and abnormal hyperglycemia causes damage to tissue cells and metabolic abnormalities, severely impairing the rate of wound repair. In obese people, the skin is not beneficial to the angiogenesis due to the thick fat layer, and the wound is easy to cause fat liquefaction to cause infection, so that the wound recovery speed is slow. In aging people, the self-renewal function and the impaired cell clearance of stem cells are weakened due to the decline of the cell compensation function, and the wound healing rate is obviously slowed down.

It follows that promoting rapid, high quality healing of these acute and chronic wounds is a significant problem in clinical care. In clinical treatment, different treatments and medicines are mainly performed on acute skin wounds and chronic skin wounds. Chinese genetic engineering I-class new medicine recombinant bovine basic fibroblast growth factor (rb-bFGF) promotes wound healing, and is mainly used for burns, body surface chronic ulcers and fresh wounds (including wounds, donor skin area wounds, operation wounds and the like). However, the polypeptide and the recombinant protein cannot be applied and popularized in large scale in the fields of medical biomaterials, cosmetics and the like due to high preparation cost.

Disclosure of Invention

One of the purposes of the invention is to provide a marine shellfish active peptide which can effectively promote the repair of skin wounds. The marine shellfish active peptide has good skin compatibility, can obviously improve the cell activity, promotes cell proliferation, migration and angiogenesis, effectively relieves inflammatory reaction, promotes high-quality repair of skin wounds, and effectively reduces scar hyperplasia.

The invention also aims to provide a method for preparing the marine shellfish active peptide by enzymolysis, ultrafiltration and sephadex G-15 separation. The method is simple, low in cost, green, environment-friendly, short in production period and suitable for large-scale industrial production.

Still another object of the present invention is to provide the use of a marine shellfish active peptide, which can be applied in pharmaceuticals, as well as in medical biomaterials and cosmetics.

The invention realizes the purpose through the following technical scheme:

on one hand, the invention provides the marine shellfish active peptide which has good water solubility and skin compatibility.

The marine shellfish active peptide is a product obtained by carrying out protease enzymolysis on a soft body part of a fresh marine shellfish.

The marine shellfish can include, but are not limited to, one or a combination of several of pinctada martensii, paphia undulata and scallops.

The protease used may be an enzyme capable of enzymatically hydrolyzing a protein, including but not limited to neutral proteases.

The inventor uses the marine shellfish peptide for skin care and hair care research to obtain good effect. However, since there is a clear difference in physiological state and structure between skin wound tissue and normal skin tissue, there is no report that a substance for general skin care can be directly used for the care of skin wound tissue.

It is understood that skin tissue repair and wound healing generally proceeds through three basic stages: 1. inflammatory reaction; 2. tissue proliferation and granulation; 3. wound contraction and scarring. The 3 stages overlap each other.

1. Inflammatory reaction: it begins immediately after injury and usually lasts for 3-5 days. The main changes of the method are blood coagulation and fibrinolysis, immune response, permeability increase of microvessels, exudation of inflammatory cells (neutrophiles at first and monocytes at later), and the significance of the method is to remove injury factors (such as pathogens and other foreign matters) and necrotic tissues and prevent infection so as to lay the foundation of tissue regeneration and repair. PDGF, IGF-1, EGF, TGF-. Beta.and the like released from platelets play an important role as chemotactic agents for inflammatory cells; macrophages synthesize and secrete TGF-. Beta.s, TGF-. Alpha.s, bFGF, MDGF, and HB-EGF at the wound, which stimulate fibroblasts, epidermal cells, and vascular endothelial cells to move toward the wound.

2. Tissue proliferation and granulation: after 24-48h, the epithelial cells begin to proliferate, and a part of the basal cells are detached from the dermis and move to the defect area, and mitosis is observed. Meanwhile, the wound has abundant cytoplasm and spindle or star-shaped fibroblasts and myofibroblasts, and the latter is similar to the former but contains micro-tows parallel to the long axis of the cell and is attached to the membrane (facilitating cell contraction). Angiogenesis is primarily the "sprouting" of existing blood vessels to grow new capillaries, and the existing vascular loops may also lengthen. Fibroblasts secrete IGF-1, bFGF, TGF-beta, PDGF and KGF; endothelial cells synthesize bFGF and PDGF; keratinocytes synthesize TGF-beta, TGF-alpha and keratinocyte derived autocrine factor (KAF). These growth factors stimulate cell proliferation, intercellular matrix protein synthesis and angiogenesis.

3. Wound contraction and scarring: 3-5 days after wound, the edge of wound begins to move and contract towards the center to eliminate the wound surface and restore the continuity of organism tissues. It often occurs when the wound is not yet completely epithelialized, initially due to contraction of the bundles of wound-limbal epithelial microfibrils, and finally contraction of myofibroblasts located in the center of the wound. PDGF, TGF-beta and the like play an important role in the process of converting granulation tissues into scars. As the healing process progresses, collagen fibers increase, fibroblasts and capillaries gradually decrease, and finally turn into scar tissue with fewer cells and blood vessels and more fibers.

During wound healing, as fibroblasts grow in, the fibronectin content in the wound area increases and is distributed in granulation tissue along the collagen. Fibronectin gradually disappears as the neoepithelium covers the wound and collagen matures. At a later stage of wound healing, the wound has a large number of fibroblasts, which are the main repair cells and the main function is to synthesize collagen fibers. Collagen generally undergoes a dynamic process of intracellular synthesis, extracellular deposition and resorption in wound healing. Growth factors play an important regulatory role in the anabolism of collagen, primarily by affecting collagen gene expression.

Based on the above knowledge of the process and mechanism of skin wound repair, the inventor firstly uses the marine shellfish active peptide obtained after removing the last single peak component eluted from the sephadex G-15 for nursing the wound skin.

As an optional implementation scheme, the preparation method of the marine shellfish active peptide comprises the steps of performing enzymolysis and ultrafiltration on marine shellfish soft parts through protease, separating through sephadex G-15, discarding the last elution single peak component, respectively collecting or combining the elution peak components, and freeze-drying to obtain the marine shellfish active peptide.

Preferably, in some embodiments, the marine shellfish soft body neutral protein enzymolysis liquid is sequentially treated by ultrafiltration membranes with the molecular weight cutoff of 10kDa and 3kDa, and the obtained retentate (3 kDa-10 kDa) is separated by sephadex G-15.

The method comprises the following specific steps: removing shell of fresh marine shellfish, homogenizing soft part, diluting with 2.5-4 times of water or PBS buffer solution, adding protease (2000U/g shellfish meat-4000U/g shellfish meat), performing enzymolysis at 40-50 deg.C and pH 6.5-7.5 for 4-6 h, inactivating enzyme in boiling water bath for 10min, centrifuging at 6000 Xg-9000 Xg for 10min-15min, ultrafiltering the supernatant with ultrafiltration membrane with molecular weight cutoff of 10kDa, and ultrafiltering the filtrate with ultrafiltration membrane with molecular weight cutoff of 3 kDa. Concentrating the active peptide solution of 3kDa-10kDa of neutral protease enzymolysis liquid, separating by Sephadex G-15, discarding the last eluted single peak component, respectively collecting or combining other eluted peak components, and lyophilizing to obtain active peptide of marine shellfish.

Preferably, the elution peak component is 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, a sixth elution peak component or any combination of the elution peak components.

Preferably, when the collected elution peak components are collected after the neutral protease enzymolysis liquid 3kDa-10kDa active peptide component is separated by sephadex G-15 (such as N1 separation components F1-F6 in example 2 and N1F1 and N1F2 in example 3), the effects of promoting cell proliferation and repairing skin wounds are remarkable.

On the other hand, the invention provides the application of the marine shellfish active peptide capable of effectively promoting cell proliferation and skin wound repair. The lowest effective dose is 100. Mu.g/mL.

As an alternative embodiment, the marine shellfish active peptide may be used to promote skin wound repair.

In a preferred embodiment of the invention, the marine shellfish active peptide is useful in medicine; preferably, the marine shellfish active peptide is especially suitable for medicines capable of effectively repairing skin wounds, and the medicine dosage forms comprise lotion, ointment, tincture, liniment, spirit, powder, oil agent, cataplasm, plaster, plastics, aerosol and the like. When the marine shellfish bioactive peptide is used in the medicine, the mass fraction of the marine shellfish bioactive peptide in the medicine is 0.01-2.5%.

In a preferred embodiment of the invention, the marine shellfish active peptide may also be used in medical biomaterials; preferably, the marine shellfish active peptide is particularly suitable for medical biomaterials capable of effectively repairing skin wounds, the dosage forms of the medical biomaterials comprise lotions, solutions, dressings, ointments and the like, and the types of the dressings comprise films, hydrocolloids, hydrogels, sponges, sprays and the like. When the marine shellfish bioactive peptide is used in medical biomaterials, the mass fraction of the marine shellfish bioactive peptide in the medical biomaterials is 0.8-2.5%.

In a preferred embodiment of the invention, the marine shellfish active peptide may be used in cosmetics; preferably, the marine shellfish active peptide is particularly suitable for cosmetics which can effectively promote skin wound repair, and the cosmetic dosage forms comprise cream, lotion, gel, jelly, aqua, spray and the like. When the marine shellfish bioactive peptide is used in cosmetics, the mass fraction of the marine shellfish bioactive peptide in the cosmetics is 0.05-1.0%.

It is worth mentioning that the marine shellfish active peptide provided by the invention is used as a functional component, does not cause skin allergy, and can also obviously relieve inflammatory reaction, promote cell proliferation and repair skin wounds. Therefore, when the marine shellfish active peptide is applied to medicines, medical biological materials or cosmetics, the selection of the types, the dosage, the preparation process and the like of the medical raw materials, the medical biological materials and the cosmetics raw materials is wide. All ingredients used in medicine, medical biomaterials, cosmetics should be dermatologically acceptable and not affect the properties of the original marine shellfish active peptide of the present invention, i.e., not cause undue toxicity, incompatibility, instability, allergic response, etc., when in contact with human skin or when compatible with other components.

The solution of the invention is based on the understanding of the inventor on the skin wound repair mechanism, the structure-activity relationship of the marine shellfish active peptide and the like, and combined with the research results of modern pharmacology, medical biomaterials and cosmetics, the marine shellfish active peptide which has good compatibility with the skin, no sensitization and obvious skin wound repair promotion effect is searched, excavated, prepared and optimized through a large number of creative experiments.

The invention has the following outstanding advantages:

(1) Digging the active peptide of the marine shellfish to promote the repair of skin wound. The marine shellfish active peptide is prepared from a plurality of marine shellfish, preferably Pinctada martensii, paphia undulata and scallop, and by selecting a soft part of a fresh and alive shellfish, and utilizing protease enzymolysis, ultrafiltration and glucan gel separation technologies. The active peptide can rapidly penetrate into skin surface layer to promote cell proliferation and wound repair. The active peptide of the marine shellfish can still obviously promote cell proliferation and wound repair under the condition of extremely low dose (50 mu g/mL).

(2) The preparation process of the marine shellfish bioactive peptide is simple, low in cost, green and environment-friendly, short in production period, suitable for large-scale industrial production and has great application prospect. According to the characteristics of marine shellfish composition structure, protease specificity, action sites and the like, neutral protease is preferably selected from a plurality of proteases, the soft body part of the marine shellfish is subjected to directional enzymolysis, the enzymolysis liquid is treated by an ultrafiltration membrane with the molecular weight cut-off of 10kDa and 3kDa, and components (possibly containing salts, free amino acids, fatty acids and the like) with poor water solubility and large molecular weight and without significant cell proliferation activity promotion can be removed, so that the obtained active peptide has better physiological effect and less discharged environmental pollutants.

(3) The active peptide of the marine shellfish has good water solubility and skin compatibility, does not cause skin irritation, is not sensitized, has obvious effect, and can be widely applied to biological medicines, medical biomaterials and cosmetics. Through the separation of the glucan gel G-15, elution components with smaller molecular weight and poor activity are removed, so that the active peptide can be further highly enriched, the physiological effect is improved, the dosage is greatly reduced, and the requirements and the operation difficulty on the stability, the compatibility and the like of the marine shellfish active peptide in the preparation process of biological medicines, medical biomaterials and cosmetics are effectively reduced.

(4) According to the invention, through the established cell activity experimental model, high-throughput activity evaluation is carried out on each prepared active peptide component, and finally, the optimized marine shellfish active peptide shows excellent skin wound repair promotion effect at the cell and animal level, so that the cell activity experimental model can be used for accurately guiding the directional separation and high enrichment of the marine shellfish active peptide.

Drawings

FIG. 1 is the separation pattern of Sephadex G-15 of the N1 (3 kDa-10 kDa) component in example 2.

FIG. 2 is the separation profile of the N2 (less than 3 kDa) component sephadex G-15 of example 2.

FIG. 3 is the separation pattern of Sephadex G-15 of the N1 (3 kDa-10 kDa) component in example 3.

Figure 4 is the effect of the N1 (3 kDa-10 kDa) fraction on HaCaT cell activity in example 3 (p <0.05, p <0.01 compared to blank).

FIG. 5 is an RP-HPLC separation profile of N1 (3 kDa-10 kDa) F4 in example 4.

Figure 6 is the effect of RP-HPLC fractions of N1 (3 kDa-10 kDa) F4 on HaCaT cell activity in example 4 (p <0.05, p <0.01 compared to blank).

Detailed Description

The invention is further described with reference to the accompanying drawings, but the embodiments of the invention are not limited to the following examples, and equivalent variations or modifications of the method according to the invention are to be considered within the scope of the invention. The starting materials used below are all commercially available, unless otherwise indicated.

Example 1

Removing shell of fresh Pinctada martensii which meets the use standard of related marine products in China, homogenizing a soft part, adding 3.4 times of distilled water and neutral protease (4000U/g of shellfish meat) by volume, performing enzymolysis at 40 ℃ and pH6.5 for 6h. Boiling the enzymolysis solution for 10min, and centrifuging at 7000 Xg for 10min. The supernatant is ultrafiltered by ultrafiltration membrane with molecular weight cutoff of 10 kDa. The ultrafiltration filtrate (less than 10kDa active peptide fraction) was concentrated to a concentration of about 100mg/mL and separated by Sephadex G-15 (100X 4.0 cm): the sample loading amount is 15mL, the mobile phase is distilled water, the flow rate is 10mL/min, and the detection wavelength is 220nm and 280nm. Mixing the eluate components except the last eluate peak, concentrating, and lyophilizing to obtain active peptide (HG) of Pinctada martensii. According to the same method, paphia undulata active peptide (HL) and scallop active peptide (HM) are respectively prepared and obtained.

The efficacy of these active peptides (HG, HL, HM) was evaluated using in vitro cell experiments (see example 5 for specific experimental methods). It was found that HG, HL, HM significantly promoted HaCaT cell proliferation at each dose administered (50-1600 μ g/mL) (significant difference compared to placebo). Wherein, at a lower concentration of 50 mug/mL, the activity of the HaCaT cells can be obviously improved by HG, HL and HM by 9.85% (p is less than 0.05), 10.33% (p is less than 0.05) and 10.56% (p is less than 0.05).

The preparation method comprises the steps of respectively coating Pinctada martensii active peptide solution (containing HG 2%, solvent being physiological saline water and 0.22 mu m for filtration sterilization), pinctada martensii active peptide solution (containing HL2%, solvent being physiological saline water and 0.22 mu m for filtration sterilization) and scallop active peptide solution (containing HM 2%, solvent being physiological saline water and 0.22 mu m for filtration sterilization) on deep II-degree burn and III-degree burn cut scab wound surfaces, reducing infection of pathogenic bacteria (pseudomonas aeruginosa, acinetobacter baumannii, staphylococcus aureus and the like), relieving inflammatory reaction, effectively preventing the wound surfaces from deepening, promoting the formation of new tissues, improving the healing speed and the healing quality of the wound surfaces, and having remarkable efficacies of promoting the regeneration and the repair of the burn wound surfaces.

Example 2

Fresh chlamys farreri meeting the use standard of related marine products in China is taken and shelled, the soft part is homogenized, 3 times volume of PBS buffer solution (0.01M, pH7.5) and neutral protease (3000U/g shellfish meat) are added, the temperature is 45 ℃, and the enzymolysis is carried out for 5 hours. Boiling the enzymolysis liquid for 10min, centrifuging at 7000 Xg for 12min. The supernatant is ultrafiltered by ultrafiltration membranes with cut-off molecular weights of 10kDa and 3kDa in sequence. Wherein, the active peptide component with 3kDa-10kDa is marked as N1, and the active peptide component with less than 3kDa is marked as N2. The N1 and N2 fractions were concentrated to a concentration of about 100mg/mL, and separated by Sephadex G-15 (100X 4.0 cm): the sample loading amount is 15mL, the mobile phase is distilled water, the flow rate is 10mL/min, and the detection wavelength is 220nm and 280nm.

Eluting the N1 (3 kDa-10 kDa) fraction with Sephadex G-15 (FIG. 1), collecting fractions F1, F2, F3, F4, F5, F6, and F7, respectively, concentrating, and lyophilizing. Similarly, the N2 (less than 3 kDa) fraction was eluted through Sephadex G-15 (FIG. 2), and fractions F1, F2, F3, F4, F5, and F6 were collected, concentrated, and lyophilized, respectively.

The 13 fractions obtained were evaluated for efficacy using in vitro cell experiments (see example 5 for specific experimental methods). The research finds that compared with a blank control, after the N1 separation component F7 and the N2 separation component F6 are administrated, the cell activity is obviously reduced (obvious cytotoxicity is generated); the activity of the cells of the N2 separation component F1-F5 treatment group is not obviously changed (the cell proliferation promoting effect is not achieved). And the activity of the HaCaT cells can be obviously improved (compared with a blank control, the obvious difference is obtained) when the N1 (3 kDa-10 kDa) separation components F1-F6 are at a lower concentration (50 mu g/mL).

Therefore, N1 (3 kDa-10 kDa) separation components F1-F6 have the effect of remarkably promoting HaCaT cell proliferation, and the 6 separation components can be used independently, and can also be combined or combined for promoting cell proliferation and skin wound repair.

The N1 (3 kDa-10 kDa) separation components F1-F6 are respectively prepared into solutions (the effective dose of peptide is 1.5%, the solvent is physiological saline, and the filtration sterilization is carried out at 0.22 mu m), and the solutions are respectively coated on the surfaces of the diabetic ulcer wounds, so that the ulcer can be effectively protected from injury and secondary infection, the conditions of ischemia and edema can be improved, the formation of new tissues can be promoted, the healing speed and the healing quality of the wounds can be improved, and the effects of better promoting the regeneration and the repair of the surfaces of the diabetic ulcer wounds can be achieved.

Example 3

Taking fresh chlamys farreri meeting the use standard of related marine products in China, removing shells, homogenizing a soft body part, adding 2.5 times of distilled water by volume, adjusting the pH value to 7.0, adding neutral protease (2000U/g shell meat), and carrying out enzymolysis for 5 hours at the temperature of 50 ℃. Boiling the enzymolysis solution for 10min, and centrifuging at 6000 Xg for 15min. The supernatant is ultrafiltered by ultrafiltration membranes with cut-off molecular weights of 10kDa and 3kDa in sequence. Wherein, the active peptide component of 3kDa-10kDa is marked as N1. The N1 fraction was concentrated to a concentration of about 200mg/mL and separated by Sephadex G-15 (100X 4.0 cm): the sample loading amount is 7mL, the mobile phase is distilled water, the flow rate is 10mL/min, and the detection wavelength is 220nm and 280nm. The eluted fractions N1F1, N1F2, N1F3 (FIG. 3) were collected separately, concentrated and lyophilized.

The 3 fractions obtained were evaluated for efficacy using in vitro cell experiments (see example 5 for specific experimental methods) (FIG. 4). The research shows that after N1F3 is administrated, the cell activity is obviously reduced (obvious cytotoxicity). After the other 2 separated components (N1F 1 and N1F 2) are administrated, the cell activity is obviously improved. Wherein, when the N1F1 is at a lower concentration of 50 mug/mL, the HaCaT cell activity can be improved by 12.31% (p is less than 0.05); N1F2 increased HaCaT cell activity by 20.13% (p < 0.01) when administered at a concentration of 100. Mu.g/mL.

Therefore, N1 (3 kDa-10 kDa) separation components N1F1 and N1F2 have the effect of remarkably promoting HaCaT cell proliferation, and the N1F1 and the N1F2 can be used independently or combined for promoting cell proliferation and skin wound repair.

According to the following method, N1F1 and N1F2 are respectively prepared to obtain marine biomedical materials (N1F1 @ calcium alginate microsphere/collagen/chitosan scaffold, N1F2@ calcium alginate microsphere/collagen/chitosan scaffold), and efficacy evaluation is carried out.

N1F1@ calcium alginate microspheres: dissolving sodium alginate (purchased from Shanghai Michelin Biochemical technology Co., ltd., purity 98%) in distilled water to prepare a sodium alginate solution with a concentration of 2% w/v, adding N1F1 according to a mass ratio of 1.05, and stirring to prepare an aqueous phase; measuring a certain volume of oleum Olivarum (purchased from Shanghai Michelin Biochemical technology Co., ltd.), adding Tween 80 (purchased from Shanghai Michelin Biochemical technology Co., ltd.) according to a v/v ratio of 1%, stirring at 600rpm for 2h, and preparing into organic phase; measuring 100 mL of organic phase, adding 20mL of aqueous phase under the stirring condition of 650rpm, stirring for 1h at 750rpm, adding 40mL of 1% w/v calcium chloride (purchased from Shanghai Michelin Biochemical technology Co., ltd.), stirring for 1h at 500rpm, adding 10mL of isopropyl ketone (AR grade, fuyu chemical reagent factory, tianjin) and stirring for 30min at 400rpm, centrifuging the mixed solution at 6000rpm for 5min, washing the obtained precipitate with isopropyl ketone and distilled water alternately for 3 times, and freeze-drying to obtain the final product.

N1F1@ calcium alginate microspheres/collagen/chitosan scaffold: dissolving collagen in acetic acid solution with concentration of 2% w/v to obtain collagen solution with concentration of 0.015 g/mL; dissolving carboxymethyl chitosan in distilled water to prepare a carboxymethyl chitosan solution with the concentration of 0.04 g/mL; mixing a collagen solution and a carboxymethyl chitosan solution according to a volume ratio of 1.

Preparing the N1F2@ calcium alginate microsphere/collagen/chitosan scaffold by the same method.

Respectively applying N1F1@ calcium alginate microspheres/collagen/chitosan scaffold and N1F2@ calcium alginate microspheres/collagen/chitosan scaffold to a wound surface of a firearm wound (one type of war wound) after debridement, can quickly absorb drainage wound surface seepage liquid, provide a dry, slightly acidic and hypoxic environment for the wound surface, reduce infection of pathogenic bacteria (pseudomonas aeruginosa, acinetobacter baumannii, staphylococcus aureus and the like), relieve inflammatory reaction, effectively prevent the wound surface from deepening, promote the formation of new tissues, improve the healing speed and quality of the wound surface, and have the effects of remarkably promoting the regeneration and repair of the wound surface of the firearm wound.

Example 4

The bioactive peptide of marine shellfish can be further separated and refined by RP-HPLC technology.

Separation and purification of active peptides were carried out by using Agilent 1260, a YMC Triart C18 column (250X 10mm, S-5 μm,12 nm). Mobile phase A: water (0.1% formic acid); and (3) mobile phase B: acetonitrile (0.1% formic acid), temperature 40 ℃. The sample concentration is 150mg/mL, the sample loading amount is 50 muL, the flow rate is 2.5mL/min, and the detection wavelengths are 200nm, 220nm, 254nm and 280nm.

Since the elution conditions of the individual components of the active peptide of marine shellfish of the present invention are slightly different, the N1F4 component in example 2 will now be exemplified. The elution conditions were: 0-7min,100% A;7-37min,100% -80% A;37-44min,80% -65% A; 44-50min,65% -30% A;50-51min,30% -10% A;51-56min,10% by weight A;56-58min,10% -100% A. Under this elution condition, 6 fractions can be collected separately: 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 fractions were concentrated and lyophilized to obtain N1 (3 kDa-10 kDa) F4 fractions F1, F2, F3, F4, F5 and F6 (FIG. 5).

The 6 fractions prepared (F1, F2, F3, F4, F5, F6) were evaluated for efficacy using in vitro cell experiments (see example 5 for specific experimental methods) (fig. 6). It was found that although F1, F5, and F6 significantly increased the activity of HaCaT cells at each concentration (37.5 μ g/mL-600 μ g/mL) (significantly different from the blank control), their cellular activity was still lower than that of the N1F4 fraction before isolation.

It can be seen that the N1F4 fraction can be used directly to promote cell proliferation and skin wound repair without further isolation and refinement by RP-HPLC techniques.

According to the preparation method of the biomaterial for traditional Chinese medicine in the embodiment 3, the N1F4@ calcium alginate microspheres/collagen/chitosan scaffold is prepared, is applied to the debrided wound surface of a firearm wound combined with seawater soaking injury, can quickly absorb wound exudate, provides a dry, clean, slightly acidic and low-oxygen environment for the wound surface, protects the wound surface, reduces infection of pathogenic bacteria (vibrio vulnificus, pseudomonas aeruginosa, acinetobacter baumannii, staphylococcus aureus and the like), relieves inflammatory reaction, effectively prevents the wound surface from further deepening, improves blood circulation, promotes formation of new tissues, improves the healing speed and quality of the wound surface, and has the effects of promoting regeneration and repair of the firearm wound surface combined with seawater soaking injury wound surface.

Example 5

The marine shellfish active peptide prepared in each example was evaluated for cell proliferation promoting activity by using an in vitro cell assay.

The cell activity was measured by the CCK-8 method: will have a density of 6X 10 ⁴ The suspension of HaCaT cells was inoculated into 96-well plates (100. Mu.L/well) and cultured in a 5-vol CO2 incubator at 37 ℃ for 24 hours. mu.L of the sample solution was aspirated from each well, and 50. Mu.L/well of the sample solution was added to the sample group at the corresponding concentration ( final concentration 50, 100, 200, 400, 800, 1600. Mu.g/mL, sterilized by a 0.22 μm pore size syringe filter). The blank control group was prepared in DMEM complete medium at 50. Mu.L/well. After each group was further cultured in a CO2 incubator for 24 hours, the cell morphology was observed using a fluorescence inverted phase contrast microscope CKX41 (Olympus, japan). Subsequently, 10. Mu.L of CCK-8 solution was added to each well, and the incubation was continued for 4 hours, and absorbance was measured for each well at a measurement wavelength of 450nm and a reference wavelength of 650nm for each set of 6 parallel wells using a microplate reader Multiskan GO (Thermo Fisher Scientific Co.).

According to the above experimental procedures, each component prepared in the examples of the present invention was subjected to efficacy evaluation. The study found that the last eluted monomodal component obtained by separation on sephadex G-15: as in the case of the fraction F7 isolated from N1 in example 2 and the fraction F3 isolated from N1 in example 3, the cell activity was significantly reduced (significant cytotoxicity) after administration; and the other separated components: the cell activity was significantly increased after administration (compared to the blank control, with a significant difference) as in the N1 fraction F1-F6 of example 2 and N1F1, N1F2 of example 3.

Therefore, after the final elution unimodal component of the sephadex G-15 is discarded, the prepared marine shellfish active peptide can effectively promote cell proliferation and skin wound repair.

Example 6

The active peptide of marine shellfish (taking the active peptide HG of Pinctada martensii, the active peptide HL of Paphia undulata and the active peptide HM of scallop in the embodiment 1 as examples) prepared by the invention is subjected to skin wound repair efficacy evaluation (taking a full-layer skin defect model as an example).

Ointment base: PEG400 (400 mL), PEG4000 (50 g), span 40 (1 mL) and distilled water (9 mL) are dissolved and homogenized in a hot water bath at 60 ℃.

Nacre martensii active peptide (HG) ointment: contains 2% of Pinctada martensii active peptide, and the rest is ointment matrix.

Paxta paphia bioactive peptide (HL) ointment: contains 2% Paphia undulata active peptide and the rest is ointment matrix.

Scallop active peptide (HM) ointment: contains 2% scallop active peptide, and the rest is ointment matrix.

(1) Full-layer skin defect molding: SPF grade 6-8 week old male BALB/c mice 72 were purchased from Guangdong province medical laboratory animal center. Animals were randomized into 4 groups, placebo, HG, HL, HM, 18 mice each. The mice were anesthetized and dehaired and the back was scratched across the full skin wound by approximately 1.5X 1.5cm.

(2) Administration: the blank control group and the seashell bioactive peptide treatment group (HG, HL, HM) were administered 1 time each day (about 0.1g ointment/mouse) for 14 days. Wherein the blank control group is administered with ointment matrix, and the active peptide treatment group of marine shellfish is administered with corresponding ointment sample.

(3) Photographing the wound surface (calculating the healing rate): and observing and recording the wound healing condition every day, taking a picture of the back wound 2 times a week, keeping the visual field, the light and the picture magnification consistent, measuring and calculating the wound area by using IPP6.0 software, and calculating the wound healing rate. On days 5, 10 and 14, 6 mice were euthanized and skin at the wound margin (skin at the same position as much as possible) was cut off and examined as follows.

(4) And (3) histopathological detection: taking a paraffin section of the skin tissue, carrying out HE staining, carrying out microscopic examination and collecting images, and measuring and calculating the thickness of the epidermal layer and the dermal layer of the skin tissue and the inflammatory score by using Image J software.

(5) And (3) RT-qPCR detection: after skin tissues are taken and mRNA is extracted (operated according to the corresponding extraction kit specification), RT-qPCR technology is adopted respectively, microscopic examination and image acquisition are combined, and the mRNA expression conditions of transforming growth factor TGF-beta 1, fibronectin, vascular endothelial growth factor VEGF, collagen-I and epidermal growth factor EGF in the skin tissues are detected.

Researches find that the active peptide of Pinctada martensii, the active peptide of Paphia undulata and the active peptide of scallop can effectively promote cell proliferation and migration, collagen and elastic fiber generation, promote capillary vessel regeneration, greatly reduce inflammatory factor generation, accelerate wound surface contraction, promote re-epithelization and hair follicle accessory regeneration, reduce scar formation and effectively improve healing speed and quality.

Compared with a blank control group, the expression levels of mRNA of TGF-beta 1, fibrinectin, VEGF, collagen-I and EGF in skin tissues are obviously increased at the 5 th day in a Pinctada martensii active peptide (HG), paphia undulata active peptide (HL) and scallop active peptide (HM) treatment group; on day 10, the wound healing rate of each treatment group was significantly improved by 23.54% (p < 0.01), 21.22% (p < 0.01), and 20.34% (p < 0.01).

Therefore, the marine shellfish active peptide prepared by the invention has the obvious effect of promoting skin wound repair.

It is noted that the active peptides of marine shellfish prepared in examples 2-4 of the present invention, in the evaluation of the efficacy of skin wound repair, could reach a level comparable to example 1, which is comparable to the level of promoting wound healing and reducing scar hyperplasia.

Comparative example 1

Compared with the embodiment 3, the method is the same as the embodiment 3 except that the soft body part of the fresh and alive shellfish is replaced by the mantle.

The in vitro cell experiments (see example 5 for a specific experimental method) are utilized to evaluate the efficacy of the mantle active peptide prepared by the embodiment. The activity of the cells of the mantle active peptide-treated group was found to have no significant change (no significant difference compared with the blank control) at concentrations of 50-400. Mu.g/mL. The activity of HaCaT cells can be improved by 12.31 percent when the N1F1 prepared by the invention is 50 mu g/mL (compared with a blank control, p is less than 0.05); the N1F2 prepared by the invention can improve the activity of HaCaT cells by 20.13% when the concentration is 100 mu g/mL (compared with a blank control, p is less than 0.01).

According to the method in the embodiment 3, the mantle bioactive peptide is prepared into a medical biomaterial, and is applied to the deep II-degree and III-degree scald wounds, although the medical biomaterial has a certain effect of promoting the formation of new tissues, the improvement of the healing speed and the improvement of the healing quality of the wounds are not obvious. Therefore, the mantle active peptide prepared by the embodiment is obviously inferior to the marine shellfish active peptide prepared in example 3 in the aspect of promoting the skin wound repair effect.

Comparative example 2

Compared with the embodiment 3, the method is the same as the embodiment 3 except that the boiling is carried out for 10min before the enzymolysis, and the ultrasonic treatment is added in the enzymolysis process.

The active peptides prepared in this embodiment were evaluated for efficacy using in vitro cell experiments (see example 5 for a specific experimental procedure). The activity of the cells of the active peptide treatment group is not obviously changed (compared with a blank control, no significant difference is generated) when the concentration is between 50 and 800 mu g/mL. The activity of the HaCaT cell can be improved by 12.31 percent when the N1F1 prepared by the invention is 50 mug/mL (compared with a blank control, p is less than 0.05); the N1F2 prepared by the invention can improve the HaCaT cell activity by 20.13% when the concentration is 100 mu g/mL (compared with a blank control, p is less than 0.01).

According to the method in the embodiment 3, the active peptide is prepared into the marine medical biomaterial, and the marine medical biomaterial is applied to the diabetic ulcer wound, so that the effects of improving the conditions of ischemia and edema, promoting the formation of new tissues, improving the wound healing speed and improving the healing quality are not obvious. Therefore, the active peptide prepared by the embodiment is obviously inferior to the active peptide of the marine shellfish prepared in the example 3 in the aspect of promoting the skin wound repair effect.

Claims (10)

1. A preparation method of active peptide of marine shellfish is characterized by comprising the following steps: removing shells of fresh and live shellfish, taking a soft part homogenate, adding distilled water, adding protease according to the addition amount of 2000U/g shellfish meat to 4000U/g shellfish meat, boiling and inactivating at the temperature of 40-50 ℃, the pH value of 6.5-7.5, performing enzymolysis for 4-6 h, centrifuging to obtain a supernatant, performing ultrafiltration on the supernatant through an ultrafiltration membrane, separating by sephadex, collecting components except the last elution peak component, concentrating, and freeze-drying to obtain the marine shellfish active peptide.

2. The method for preparing a marine shellfish active peptide of claim 1, wherein said protease is a neutral protease; the molecular weight cut-off of the ultrafiltration membrane is 3kDa and 10kDa; the fraction separated by sephadex is a 3kDa-10kDa ultrafiltration fraction.

3. The method for preparing a marine shellfish active peptide according to claim 1 or 2, wherein said marine shellfish is Paphia undulata, pinctada martensii or scallop.

4. Marine shellfish active peptide produced by the process for producing marine shellfish active peptide according to any of claims 1-3.

5. Use of a marine shellfish active peptide as defined in claim 4 for the preparation of a medicament, medical biomaterial or cosmetic for promoting skin wound repair.

6. The use of claim 5, wherein the pharmaceutical product is in the form of a lotion, ointment, tincture, spirit, powder, oil, cataplasm, plaster, film coating agent or aerosol; the mass fraction of the marine shellfish active peptide in the medicine is 0.01-2.5%.

7. The use of claim 5, wherein the dosage form of the medical biomaterial comprises lotion, solution, dressing, ointment or injection, and the mass fraction of the marine shellfish active peptide in the medical biomaterial is 0.8% -2.5%.

8. The use of claim 5, wherein the cosmetic formulation comprises a cream, lotion, gel, lotion, or spray; the mass fraction of the marine shellfish active peptide in the cosmetics is 0.05-1.0%.

9. The skin wound of claim 5, wherein the skin wound comprises a burn, scald, cut, traffic wound, surgical wound, war wound, firearm wound combined with seawater soak wound or chronic intractable wound.

10. The chronic refractory wound of claim 9, wherein the chronic refractory wound comprises a diabetic ulcer, an inflammatory ulcer or a pressure ulcer.

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