CN110538312A - skin wound repair ointment and preparation method thereof - Google Patents
skin wound repair ointment and preparation method thereof Download PDFInfo
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- CN110538312A CN110538312A CN201910813932.0A CN201910813932A CN110538312A CN 110538312 A CN110538312 A CN 110538312A CN 201910813932 A CN201910813932 A CN 201910813932A CN 110538312 A CN110538312 A CN 110538312A
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- marine shellfish
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- ointment
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
- A61K38/014—Hydrolysed proteins; Derivatives thereof from animals from connective tissue peptides, e.g. gelatin, collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
the invention provides an ointment for repairing skin wounds, which is characterized by comprising the following components in parts by weight: 20-25 parts of lanolin, 40-50 parts of white vaseline, 20-25 parts of glycerol, 2-6 parts of marine shellfish active peptide, 0.1-0.3 part of collagen peptide and 1-5 parts of water; the marine shellfish active peptide is a marine shellfish mantle enzymolysis product; the collagen peptide is Sipunculus nudus collagen enzymolysis product. The invention has the effects of resisting bacteria and diminishing inflammation, removing putrefaction and promoting tissue regeneration, converging and healing, and inhibiting scar hyperplasia, is particularly suitable for wound healing, and reduces scar residues.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to skin wound repair ointment and a preparation method thereof.
background
Human body injuries caused by various wounds, burns, operations and the like are common clinical phenomena. According to the statistics of the world health organization, the number of fatalities caused by various wounds is 510 million in 1990, and is expected to reach 840 million in 2020. Thus, trauma is a disease that is not negligible. Among them, skin wounds and skin repair disorders are not only susceptible to the possibility of causing related complications, but also seriously affect the quality of life of patients. Therefore, the development of a biological wound repair technique with rapid healing and no scar is one of the major points and hot spots of surgical research. The healing of skin soft tissue wound is a dynamic coordination tissue repair process, comprising three stages of inflammatory reaction, necrotic tissue removal, cell proliferation, granulation tissue generation and new tissue remodeling. At present, the main common wound repair drugs are: genetically engineered cytokines, antibacterial drugs, enzyme debridement (enzymic debridement) preparations, Chinese medicinal preparations, antioxidants, corticosteroids and the like.
Bioactive substances are a basic prerequisite for the study of pharmaceuticals, functional foods and biologics. The great difference between the growth environment of marine organisms and terrestrial organisms is a source for developing novel natural active substances with novel structures. In recent years, researchers have focused on marine active substances that contribute to wound healing, such as krill trypsin, collagenase, sea cucumber polysaccharides, fish polypeptides, marine collagen peptides, mussel decorin protein (decorin), and the like, but there are few marine shellfish-derived skin soft tissue wound healing active substances. Therefore, it is necessary to develop a product having a skin wound healing function by making full use of marine resources.
disclosure of Invention
In view of the above, the invention provides an ointment for repairing skin wounds, which has the effects of resisting bacteria and diminishing inflammation, removing putrefaction and promoting tissue regeneration, converging and healing, and inhibiting scar hyperplasia, is particularly suitable for healing wounds, and reduces scar residues.
the technical scheme of the invention is as follows:
The skin wound repair ointment is characterized by comprising the following components in parts by weight: 20-25 parts of lanolin, 40-50 parts of white vaseline, 20-25 parts of glycerol, 2-6 parts of marine shellfish active peptide, 0.1-0.3 part of collagen peptide and 1-5 parts of water;
the marine shellfish active peptide is a marine shellfish mantle enzymolysis product;
the collagen peptide is Sipunculus nudus collagen enzymolysis product.
further, the preparation raw material of the marine shellfish active peptide is any one of chlamys nobilis, pinna shell and Pinctada martensii.
further, the preparation method of the marine shellfish active peptide comprises the following steps:
A1. Preparing marine shellfish zymolyte;
Separating out the mantle of the marine shellfish through pretreatment; the protease selected by the enzymolysis of the marine shellfish mantle comprises: any one of neutral protease, animal protease, flavourzyme, alkaline protease and bromelain, wherein the addition amount of the protease is 3000-;
A2. and D, selecting the ceramic membrane with the aperture of 0.05-0.5um from the supernatant obtained in the step A1, fully filtering, separating by adopting ultrafiltration membranes with different molecular weight cut-off, collecting peptide sections with different molecular weights, and freeze-drying to obtain the marine shellfish active peptide.
further, the active peptide of the marine shellfish comprises any one or the combination of two of active peptide with the molecular weight of less than 3000Da, active peptide with the molecular weight of 3000Da-5000Da and active peptide with the molecular weight of 5000Da-10000 Da.
Further, the preparation method of the collagen peptide comprises the following steps:
B1. extracting Sipunculus nudus collagen; shearing fresh polypide, adding 0.2M NaOH 4-5 times the weight of the raw materials, stirring at 4 ℃ for 12-24 hours, and centrifuging to remove supernatant; washing the precipitate with distilled water, draining, adding 4-5 times of 0.5M acetic acid solution and 0.05-0.20% pepsin (w/v), extracting at 4 deg.C for 24-60 hr, centrifuging to obtain supernatant, slowly adding NaCl to 1M, stirring for 12-24 hr, centrifuging, collecting precipitate, dissolving with 0.5M acetic acid, dialyzing, and lyophilizing to obtain collagen;
B2. And (4) carrying out enzymolysis to prepare the sipunculus nudus collagen peptide.
Further, in B2, the specific method for enzymolysis includes: any two enzymes of neutral protease, animal protease, flavourzyme, alkaline protease and papain are selected for compounding, the addition amount of the two enzymes is 3000 plus 5000U/g, the pH value is 5.0-7.0, the enzymolysis temperature is 45-55 ℃, the enzymolysis time is 3-5 hours, after enzyme deactivation is carried out for 10 minutes at 100 ℃, the supernatant fluid is obtained by centrifugation, and freeze drying is carried out to obtain the Sipunculus nudus collagen peptide.
The invention also provides a preparation method of the skin wound repair ointment, which comprises the following steps:
(1) mixing vaseline and lanolin at a certain proportion, heating to dissolve, and adding glycerol to obtain ointment matrix;
(2) respectively adding active peptide of marine shellfish and collagen peptide before the matrix is not completely cooled, and stirring;
(3) And (3) after uniformly stirring until the color is not changed, sealing a preservative film, and standing for 1 hour to obtain the skin wound repair ointment.
In the invention, in order to avoid the influence of bacteria and impurities in the water on the quality of the ointment, the invention adopts pure water, including deionized water, distilled water, ultrapure water and the like.
The active peptide of the marine shellfish is from marine organisms and has the advantages of multiple functions, wide sources, strong specificity, small toxic and side effects and the like. The marine shellfish is one of important fishery resources, and the soft body part of the marine shellfish is a high-quality protein source with high protein and low fat, and is rich in a plurality of active substances such as polysaccharide, Zn, taurine, arginine and the like. The marine shellfish bioactive peptide is a marine shellfish mantle enzymolysis product, and the mantle tissue of the marine shellfish has the characteristics of tissue self-repairing capability and pearl layer secretion; the mantle is one of the by-products of the processing, the main components of which are protein (collagen) and polysaccharide substances, and simultaneously contains endogenous antibacterial protein, and is an important source for developing natural active substances for wound healing.
The marine shellfish comprises any one of chlamys nobilis, pinna shell and Pinctada martensii, the outer casing membrane of the marine shellfish is rich in collagen, and the collagen is one of important components of extracellular matrix, can induce the generation of various growth factors and is beneficial to wound healing. The enzymolysis product prepared by enzymolysis has effects of promoting wound healing and inhibiting scar hyperplasia. In order to better promote wound repair, preferably, the active peptide of the marine shellfish comprises any one of or the combination of two of active peptide with the molecular weight of less than 3000Da, active peptide with the molecular weight of 3000Da-5000Da and active peptide with the molecular weight of 5000Da-10000 Da.
the marine shellfish active peptide has good biological characteristics and good affinity with tissues around wounds, can promote the growth of skin and nerves by inducing growth factors, is beneficial to the proliferation and repair of epithelial cells, promotes the healing of wound surfaces, and repairs skin defects and tissue defects; the skin also has good absorption effect on the active peptide, and the active peptide is quickly combined into self collagen, so that normal connective tissue is formed, the damaged skin is filled and repaired, and the formation of scars is reduced. The active peptide of the marine shellfish has low molecular weight and quick digestion and absorption, and can be directly absorbed by intestinal tracts and enter blood to reach all parts of a body. After the collagen peptide is supplemented by external source, fibroblasts, fat cells, capillaries and the like in skin and connective tissue can synthesize collagen of the skin and connective tissue, so that normal connective tissue is formed, damaged and aged skin is filled and repaired, and the purpose of delaying skin aging is achieved. Therefore, the pearl oyster small molecular peptide can act on four wound healing periods of a wound in a hemostasis period, an inflammation period, a proliferation period and a remodeling period, and can be used as a regenerative healing active substance for resisting abnormal scar formation for treating dermal injury.
The collagen peptide is Sipunculus nudus collagen enzymolysis product. Sipunculus nudus is rich in nutrients such as protein, polysaccharide and mineral elements and functional active substances, has crisp and tender meat and delicious taste, and has edible and medicinal values recorded in various pharmacopoeias of China. Modern nutritional studies indicate that the fresh Sipunculus nudus wall crude protein is a typical high-protein low-fat aquatic product and has 16.10 percent of crude protein content (80.86 percent on a dry basis).
the collagen is extracted from Sipunculus nudus, and the active peptide prepared by enzymolysis has good antibacterial activity and has good inhibition effect on staphylococcus aureus, listeria, streptococcus agalactiae, escherichia coli, pseudomonas aeruginosa, vibrio alginolyticus, vibrio parahaemolyticus, shiva alga aeromonas hydrophila and the like which are common in living environment and wound infection. Thus, the collagen peptide can effectively reduce bacterial infection in the wound healing process. Meanwhile, the collagen peptide participates in a series of related biological activities such as promotion of wound repair and the like through antioxidation, improvement of water content of stratum corneum, promotion of fibroblast proliferation, reduction of melanin formation, increase of hydroxyproline content in injured tissues and the like.
In the present invention, glycerin: on one hand, the water-absorbing and moisture-preserving function is exerted, and on the other hand, the water-absorbing and moisture-preserving agent is used as a surfactant, so that the water-absorbing and moisture-preserving agent is beneficial to being made into a paste and is convenient to use; white vaseline: ointment base, having lubricating and moisturizing effects, chemically inert so that it is not irritating to any type of skin; lanolin: the ointment matrix has viscosity, has moistening and softening effects, and can make the skin contact more comfortably.
The invention provides a preparation method of a skin wound repair cream, which comprises the following preparation steps:
(1) Mixing vaseline and lanolin at a certain proportion, heating to dissolve, and adding glycerol to obtain ointment matrix;
(2) Before the matrix is not completely cooled, adding the marine shellfish active peptide and the antibacterial peptide according to the proportion respectively, and uniformly stirring;
(3) and (3) after uniformly stirring until the color is not changed, sealing a preservative film, and standing for 1 hour to obtain the skin wound repair ointment.
the pasty skin wound repair paste prepared by the invention is a yellow semisolid ointment, has moderate viscosity, is fine and smooth, is easy to coat on skin, has a proper pH value, and has good stability.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The invention utilizes the wound healing promoting activity of the natural marine shellfish active peptide, compounds with the sipunculus nudus collagen peptide with antibacterial activity, plays a synergistic interaction role, and then is combined with the ointment matrix with moisturizing, moistening and non-irritating properties to prepare the fat-soluble matrix ointment; the invention has the effects of resisting bacteria and diminishing inflammation, removing putrefaction and promoting tissue regeneration, converging and healing, and inhibiting scar hyperplasia, is particularly suitable for wound healing, and reduces scar residues.
(2) The invention has the advantages of cheap and easily obtained raw materials, pure nature, safety, no toxicity, no need of adding antibiotics and preservatives, few components, simple preparation process and easy industrial production.
Drawings
FIG. 1 is a graph showing the effect of the skin wound repair cream of one embodiment of the present invention on promoting the healing of the skin wound of a mouse;
FIG. 2 is a graph showing the effect of the skin wound repair cream of one embodiment of the present invention on the rate of promoting the healing of the skin wound of a mouse;
FIG. 3 is a graph showing the effect of the skin wound repair cream of one embodiment of the present invention on EGF factors in the skin;
FIG. 4 is a graph showing the effect of a skin wound repair cream according to an embodiment of the present invention on FGF factors in skin;
FIG. 5 is a graph showing the effect of a skin wound repair cream according to an embodiment of the present invention on CD31 factor in skin;
FIG. 6 is a histological section of the effect of a skin wound repair cream on collagen production in skin according to an embodiment of the present invention;
FIG. 7 is a graph showing the effect of the skin wound healing cream on the rate of reduction of skin scar according to an embodiment of the present invention.
Detailed Description
the present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, materials, methods and equipment used in the present invention are conventional and commercially available in the art unless otherwise specified.
example 1
the embodiment provides a skin wound repair ointment which is characterized by comprising the following components in parts by weight: lanolin 22, white vaseline 44, glycerol 22, marine shellfish active peptide 4, collagen peptide 0.2, and water 3;
The marine shellfish active peptide is a marine shellfish mantle enzymolysis product;
The collagen peptide is Sipunculus nudus collagen enzymolysis product.
Furthermore, the raw material for preparing the marine shellfish active peptide is pinctada martensii.
Further, the preparation method of the marine shellfish active peptide comprises the following steps:
A1. Preparing marine shellfish zymolyte;
separating out the mantle of the marine shellfish through pretreatment; the protease selected for the enzymolysis of the marine shellfish mantle is animal protease, the addition amount of the protease is 4000U/g, the pH value is 7.5, the enzymolysis temperature is 50 ℃, the enzymolysis time is 4 hours, and after enzyme deactivation is carried out for 10 minutes at 100 ℃, the supernatant is obtained by centrifugation;
A2. And D, selecting the ceramic membrane with the aperture of 0.05-0.5um from the supernatant obtained in the step A1, fully filtering, separating by adopting ultrafiltration membranes with different molecular weight cut-off, collecting peptide sections with different molecular weights, and freeze-drying to obtain the marine shellfish active peptide.
Furthermore, the marine shellfish active peptide is an active peptide with the molecular weight of less than 3000 Da.
Further, the preparation method of the collagen peptide comprises the following steps:
B1. extracting Sipunculus nudus collagen; shearing fresh polypide, adding 0.2M NaOH 4.5 times the weight of the raw materials, stirring at 4 ℃ for 16 hours, and centrifuging to remove supernatant; washing the precipitate with distilled water, draining, adding 4.5 times of 0.5M acetic acid solution and 0.10% pepsin (w/v), extracting at 4 deg.C for 30 hr, centrifuging to obtain supernatant, slowly adding NaCl to 1M, stirring for 16 hr, centrifuging, collecting precipitate, dissolving with 0.5M acetic acid, dialyzing, and lyophilizing to obtain collagen;
B2. and (4) carrying out enzymolysis to prepare the sipunculus nudus collagen peptide.
Further, in B2, the specific method for enzymolysis includes: selecting neutral protease and animal protease to compound, wherein the addition amount of the two enzymes is 3500U/g, the pH value is 7, the enzymolysis temperature is 50 ℃, the enzymolysis time is 3.5 hours, after enzyme deactivation is carried out for 10 minutes at 100 ℃, centrifuging, taking supernate, and freeze-drying to obtain the sipunculus nudus collagen peptide.
the present embodiment provides a preparation method of the above ointment for repairing skin wounds, which includes the following steps:
(1) Mixing vaseline and lanolin at a certain proportion, heating to dissolve, and adding glycerol to obtain ointment matrix;
(2) Respectively adding active peptide of marine shellfish and collagen peptide before the matrix is not completely cooled, and stirring;
(3) and (3) after uniformly stirring until the color is not changed, sealing a preservative film, and standing for 1 hour to obtain the skin wound repair ointment.
Example 2
the embodiment provides a skin wound repair ointment which is characterized by comprising the following components in parts by weight: lanolin 20, white vaseline 40, glycerol 20, marine shellfish active peptide 2, collagen peptide 0.1, and water 1;
The marine shellfish active peptide is a marine shellfish mantle enzymolysis product;
the collagen peptide is Sipunculus nudus collagen enzymolysis product.
Furthermore, the preparation raw material of the marine shellfish active peptide is chlamys nobilis.
Further, the preparation method of the marine shellfish active peptide comprises the following steps:
A1. preparing marine shellfish zymolyte;
Separating out the mantle of the marine shellfish through pretreatment; the protease selected for the marine shellfish mantle enzymolysis is neutral protease, the addition amount of the protease is 3000U/g, the pH value is 6.5, the enzymolysis temperature is 45 ℃, the enzymolysis time is 3 hours, and after enzyme deactivation is carried out for 10 minutes at 100 ℃, the supernatant is obtained by centrifugation;
A2. and D, selecting the ceramic membrane with the aperture of 0.05-0.5um from the supernatant obtained in the step A1, fully filtering, separating by adopting ultrafiltration membranes with different molecular weight cut-off, collecting peptide sections with different molecular weights, and freeze-drying to obtain the marine shellfish active peptide.
further, the active peptide of the marine shellfish comprises two combinations of active peptide with the molecular weight of less than 3000Da and active peptide with the molecular weight of 3000Da-5000 Da.
Further, the preparation method of the collagen peptide comprises the following steps:
B1. extracting Sipunculus nudus collagen; shearing fresh polypide, adding 0.2M NaOH 4 times the weight of the raw materials, stirring at 4 ℃ for 24 hours, and centrifuging to remove supernatant; washing the precipitate with distilled water, draining, adding 4 times of 0.5M acetic acid solution and 0.05% pepsin (w/v), extracting at 4 deg.C for 60 hr, centrifuging to obtain supernatant, slowly adding NaCl to 1M, stirring for 24 hr, centrifuging, collecting precipitate, dissolving with 0.5M acetic acid, dialyzing, and lyophilizing to obtain collagen;
B2. and (4) carrying out enzymolysis to prepare the sipunculus nudus collagen peptide.
Further, in B2, the specific method for enzymolysis includes: selecting two enzymes of flavourzyme and papain for compounding, wherein the addition amount of the two enzymes is 3000U/g, the pH value is 5.0, the enzymolysis temperature is 45 ℃, the enzymolysis time is 3 hours, after enzyme deactivation is carried out for 10 minutes at 100 ℃, centrifuging, taking supernate, and freeze-drying to obtain the sipunculus nudus collagen peptide.
The present embodiment provides a preparation method of the above ointment for repairing skin wounds, which includes the following steps:
(1) mixing vaseline and lanolin at a certain proportion, heating to dissolve, and adding glycerol to obtain ointment matrix;
(2) respectively adding active peptide of marine shellfish and collagen peptide before the matrix is not completely cooled, and stirring;
(3) And (3) after uniformly stirring until the color is not changed, sealing a preservative film, and standing for 1 hour to obtain the skin wound repair ointment.
Example 3
The embodiment provides a skin wound repair ointment which is characterized by comprising the following components in parts by weight: lanolin 25, white vaseline 50, glycerol 25, marine shellfish active peptide 6, collagen peptide 0.3, and water 5;
The marine shellfish active peptide is a marine shellfish mantle enzymolysis product;
the collagen peptide is Sipunculus nudus collagen enzymolysis product.
further, the preparation raw material of the marine shellfish active peptide is atrina pectinata.
Further, the preparation method of the marine shellfish active peptide comprises the following steps:
A1. preparing marine shellfish zymolyte;
separating out the mantle of the marine shellfish through pretreatment; the protease selected for the ocean shellfish mantle enzymolysis is alkaline protease, the addition amount of the protease is 5000U/g, the pH value is 10, the enzymolysis temperature is 55 ℃, the enzymolysis time is 5 hours, the enzyme is inactivated at 100 ℃ for 10 minutes, and then the supernatant is obtained by centrifugation;
A2. and D, selecting the ceramic membrane with the aperture of 0.05-0.5um from the supernatant obtained in the step A1, fully filtering, separating by adopting ultrafiltration membranes with different molecular weight cut-off, collecting peptide sections with different molecular weights, and freeze-drying to obtain the marine shellfish active peptide.
further, the marine shellfish active peptide is an active peptide with the molecular weight of 3000Da-5000 Da.
Further, the preparation method of the collagen peptide comprises the following steps:
B1. Extracting Sipunculus nudus collagen; shearing fresh polypide, adding 0.2M NaOH 5 times the weight of the raw materials, stirring at 4 ℃ for 12 hours, and centrifuging to remove supernatant; washing the precipitate with distilled water, draining, adding 5 times of 0.5M acetic acid solution and 0.20% pepsin (w/v), extracting at 4 deg.C for 24 hr, centrifuging to obtain supernatant, slowly adding NaCl to 1M, stirring for 12 hr, centrifuging, collecting precipitate, dissolving with 0.5M acetic acid, dialyzing, and lyophilizing to obtain collagen;
B2. And (4) carrying out enzymolysis to prepare the sipunculus nudus collagen peptide.
Further, in B2, the specific method for enzymolysis includes: the Sipunculus nudus collagen peptide is prepared by compounding two enzymes of alkaline protease and papain, wherein the addition amount of the two enzymes is 5000U/g, the pH value is 7.0, the enzymolysis temperature is 55 ℃, the enzymolysis time is 5 hours, and after enzyme deactivation is carried out for 10 minutes at 100 ℃, the supernatant is obtained by centrifugation, and the Sipunculus nudus collagen peptide is obtained by freeze drying.
The present embodiment provides a preparation method of the above ointment for repairing skin wounds, which includes the following steps:
(1) Mixing vaseline and lanolin at a certain proportion, heating to dissolve, and adding glycerol to obtain ointment matrix;
(2) respectively adding active peptide of marine shellfish and collagen peptide before the matrix is not completely cooled, and stirring;
(3) and (3) after uniformly stirring until the color is not changed, sealing a preservative film, and standing for 1 hour to obtain the skin wound repair ointment.
example 4
The embodiment provides a skin wound repair ointment which is characterized by comprising the following components in parts by weight: lanolin 21, white vaseline 45, glycerol 23, marine shellfish active peptide 3, collagen peptide 0.15, and water 2;
the marine shellfish active peptide is a marine shellfish mantle enzymolysis product;
The collagen peptide is Sipunculus nudus collagen enzymolysis product.
Furthermore, the raw material for preparing the marine shellfish active peptide is pinctada martensii.
Further, the preparation method of the marine shellfish active peptide comprises the following steps:
A1. Preparing marine shellfish zymolyte;
separating out the mantle of the marine shellfish through pretreatment; the protease selected for the ocean shellfish mantle enzymolysis is flavourzyme, the addition amount of the protease is 3500/g, the pH value is 7, the enzymolysis temperature is 48 ℃, the enzymolysis time is 3.8 hours, after enzyme deactivation at 100 ℃ is 10 minutes, the supernatant is obtained by centrifugation;
A2. and D, selecting the ceramic membrane with the aperture of 0.05-0.5um from the supernatant obtained in the step A1, fully filtering, separating by adopting ultrafiltration membranes with different molecular weight cut-off, collecting peptide sections with different molecular weights, and freeze-drying to obtain the marine shellfish active peptide.
Further, the active peptide of the marine shellfish comprises two combinations of active peptide with the molecular weight of less than 3000Da and active peptide with the molecular weight of 5000Da-10000 Da.
Further, the preparation method of the collagen peptide comprises the following steps:
B1. extracting Sipunculus nudus collagen; shearing fresh polypide, adding 0.2M NaOH 4.5 times the weight of the raw materials, stirring at 4 ℃ for 18 hours, and centrifuging to remove supernatant; washing the precipitate with distilled water, draining, adding 4.5 times of 0.5M acetic acid solution and 0.15% pepsin (w/v), extracting at 4 deg.C for 40 hr, centrifuging to obtain supernatant, slowly adding NaCl to 1M, stirring for 18 hr, centrifuging, collecting precipitate, dissolving with 0.5M acetic acid, dialyzing, and lyophilizing to obtain collagen;
B2. and (4) carrying out enzymolysis to prepare the sipunculus nudus collagen peptide.
Further, in B2, the specific method for enzymolysis includes: selecting neutral protease and papain to compound, adding 4500U/g of both enzymes, pH 6.5, enzymolysis temperature 52 ℃, enzymolysis time 4.5 hours, inactivating enzyme at 100 ℃ for 10 minutes, centrifuging, taking supernatant, and freeze-drying to obtain the Sipunculus nudus collagen peptide.
the present embodiment provides a preparation method of the above ointment for repairing skin wounds, which includes the following steps:
(1) mixing vaseline and lanolin at a certain proportion, heating to dissolve, and adding glycerol to obtain ointment matrix;
(2) Respectively adding active peptide of marine shellfish and collagen peptide before the matrix is not completely cooled, and stirring;
(3) And (3) after uniformly stirring until the color is not changed, sealing a preservative film, and standing for 1 hour to obtain the skin wound repair ointment.
example 5
The embodiment provides a skin wound repair ointment which is characterized by comprising the following components in parts by weight: lanolin 24, white vaseline 47, glycerol 21, marine shellfish active peptide 5, collagen peptide 0.25, and water 4;
the marine shellfish active peptide is a marine shellfish mantle enzymolysis product;
the collagen peptide is Sipunculus nudus collagen enzymolysis product.
furthermore, the raw material for preparing the marine shellfish active peptide is pinctada martensii.
Further, the preparation method of the marine shellfish active peptide comprises the following steps:
A1. Preparing marine shellfish zymolyte;
separating out the mantle of the marine shellfish through pretreatment; the protease selected for the enzymolysis of the marine shellfish mantle is bromelain, the addition amount of the protease is 4200U/g, the pH value is 7.5, the enzymolysis temperature is 46 ℃, the enzymolysis time is 4.2 hours, after enzyme deactivation is carried out for 10 minutes at 100 ℃, the supernatant is obtained by centrifugation;
A2. and D, selecting the ceramic membrane with the aperture of 0.05-0.5um from the supernatant obtained in the step A1, fully filtering, separating by adopting ultrafiltration membranes with different molecular weight cut-off, collecting peptide sections with different molecular weights, and freeze-drying to obtain the marine shellfish active peptide.
Further, the active peptide of the marine shellfish comprises a combination of two active peptides with the molecular weight of 3000Da-5000Da and active peptides with the molecular weight of 5000Da-10000 Da.
further, the preparation method of the collagen peptide comprises the following steps:
B1. extracting Sipunculus nudus collagen; shearing fresh polypide, adding 0.2M NaOH 4.5 times the weight of the raw materials, stirring at 4 ℃ for 20 hours, and centrifuging to remove supernatant; washing the precipitate with distilled water, draining, adding 4.5 times of 0.5M acetic acid solution and 0.13% pepsin (w/v), extracting at 4 deg.C for 35 hr, centrifuging to obtain supernatant, slowly adding NaCl to 1M, stirring for 20 hr, centrifuging, collecting precipitate, dissolving with 0.5M acetic acid, dialyzing, and lyophilizing to obtain collagen;
B2. And (4) carrying out enzymolysis to prepare the sipunculus nudus collagen peptide.
further, in B2, the specific method for enzymolysis includes: selecting neutral protease and flavourzyme, compounding the neutral protease and flavourzyme, adding the two enzymes into the mixture, wherein the adding amount of the two enzymes is 3700U/g, the pH value is 6.2, the enzymolysis temperature is 49 ℃, the enzymolysis time is 3.6 hours, and after enzyme deactivation is carried out for 10 minutes at 100 ℃, centrifuging, taking supernate, and carrying out freeze drying to obtain the sipunculus nudus collagen peptide.
The present embodiment provides a preparation method of the above ointment for repairing skin wounds, which includes the following steps:
(1) mixing vaseline and lanolin at a certain proportion, heating to dissolve, and adding glycerol to obtain ointment matrix;
(2) respectively adding active peptide of marine shellfish and collagen peptide before the matrix is not completely cooled, and stirring;
(3) And (3) after uniformly stirring until the color is not changed, sealing a preservative film, and standing for 1 hour to obtain the skin wound repair ointment.
test for wound repair test
experimental tests related to wound repair were performed on the skin wound-repairing ointment prepared in example 1.
1.1 wound healing assay: taking 57 healthy mice, wherein the mice are all male, and depilating and disinfecting the backs of the mice; the mouse is anesthetized by intraperitoneal injection of 10% chloral hydrate, and a wound surface is prepared on the back of the mouse by using laboratory scissors, wherein the wound surface area is about 1 cm 2. After the molding was completed, the area of the wound of the mouse was photographed and recorded as day 0. The remaining 57 model mice were randomly divided into 3 groups, blank control (group 1), positive control (group 1) and sample (group 1), 19/group. The blank control group is not administrated after the model building, and the positive control group and the sample group are administrated on the day of the model building, are smeared once a day and are smeared for 18 days continuously. The ointment (Tianjin Da Rentang Jingwanhong pharmaceutical Co., Ltd.) was used as a positive control drug, and the sample group and the positive control group were applied with a plaster dose of 0.5g per wound surface. Recording the wound healing area by adopting a photographing recording method on the 2 nd, 4 th, 6 th, 8 th, 10 th, 12 th, 14 th, 16 th and 18 th days of modeling respectively, encircling the wound area on the back of the mouse along the edge of the wound by ImageJ software, recording the area numerical value, and calculating the healing rate according to the following formula:
Wound healing rate (%) = [ (day 0 wound area-day n wound area)/day 0 wound area ]. 100%
scar area was recorded by taking a photograph recording on day 18 of the experiment, scar: different from the normal skin, rough and pigmented. And enclosing the scar area of the back of the mouse along the scar edge by ImageJ software, recording the area numerical value, and calculating the scar reduction rate according to the following formula:
scar reduction (%) = [ (wound area on day 0-scar area on day n)/wound area on day 0 ]. 100%.
1.2 histological examination: skin tissues were taken on days 7 and 18 of treatment, fixed with 4% neutral formaldehyde, paraffin sectioned, immunohistochemically stained for CD31, EGF and FGF, and observed under a microscope for quantitative analysis using ImageJ software treatment of the immunohistochemical images (100 ×) of the tissues on days 7 and 18.
skin tissues were collected at day 18 of the treatment group, and the tissue sections at day 18 were stained by sirius red-picric acid staining method, and the collagen type change was observed under a microscope, and the tissue section image (200 ×) at day 18 of the sample treatment group, the quantitative results after treatment with ImageJ software, and the scar reduction rate results.
As can be seen from the wound healing test data in figures 1-2, the healing rate is steadily increased with the increase of days, and the ointment of the invention has obvious healing promotion effect on the wound surface of the skin wound of the mouse.
From the immunohistochemical experimental data of fig. 3-5, it can be seen that the ointment of the present invention can effectively promote the repair of the vascular system and the proliferation of EGF and FGF cells, thereby facilitating the repair of the epidermal layer and dermal layer of the wound surface, and thus, the ointment of the present invention has the efficacy of promoting wound healing.
As can be seen from the experimental data of sirius red-picric acid staining and scar reduction rate of FIGS. 6-7, the ointment treatment group significantly reduced the type I/II collagen ratio and the scar reduction rate (P < 0.05); compared with a negative control group, the skin wound repair ointment has the effect of inhibiting scar hyperplasia. Therefore, the skin wound repair ointment disclosed by the invention not only promotes wound healing, but also has the effect of inhibiting scar hyperplasia.
it is noted that the ointments prepared in examples 2-5 of the present invention achieved comparable levels to example 1 in the evaluation of the wound repair test efficacy test, which resulted in comparable levels of both wound healing promotion and scar formation inhibition.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. The skin wound repair ointment is characterized by comprising the following components in parts by weight: 20-25 parts of lanolin, 40-50 parts of white vaseline, 20-25 parts of glycerol, 2-6 parts of marine shellfish active peptide, 0.1-0.3 part of collagen peptide and 1-5 parts of water;
the marine shellfish active peptide is a marine shellfish mantle enzymolysis product;
The collagen peptide is Sipunculus nudus collagen enzymolysis product.
2. the skin wound repair ointment of claim 1, wherein the marine shellfish active peptide is prepared from any one of chlamys nobilis, pinna shell and Pinctada martensii.
3. The ointment for skin wound healing according to claim 2, wherein the marine shellfish active peptide is prepared by:
A1. Preparing marine shellfish zymolyte;
separating out the mantle of the marine shellfish through pretreatment; the protease selected by the enzymolysis of the marine shellfish mantle comprises: any one of neutral protease, animal protease, flavourzyme, alkaline protease and bromelain, wherein the addition amount of the protease is 3000-;
A2. and D, selecting the ceramic membrane with the aperture of 0.05-0.5um from the supernatant obtained in the step A1, fully filtering, separating by adopting ultrafiltration membranes with different molecular weight cut-off, collecting peptide sections with different molecular weights, and freeze-drying to obtain the marine shellfish active peptide.
4. The skin wound repair ointment of claim 3, wherein the marine shellfish active peptides comprise any one of or a combination of two of active peptides having a molecular weight of less than 3000Da, active peptides having a molecular weight of 3000Da to 5000Da, active peptides having a molecular weight of 5000Da to 10000 Da.
5. the ointment for skin wound healing according to claim 1, wherein the collagen peptide is prepared by:
B1. Extracting Sipunculus nudus collagen; shearing fresh polypide, adding 0.2M NaOH 4-5 times the weight of the raw materials, stirring at 4 ℃ for 12-24 hours, and centrifuging to remove supernatant; washing the precipitate with distilled water, draining, adding 4-5 times of 0.5M acetic acid solution and 0.05-0.20% pepsin (w/v), extracting at 4 deg.C for 24-60 hr, centrifuging to obtain supernatant, slowly adding NaCl to 1M, stirring for 12-24 hr, centrifuging, collecting precipitate, dissolving with 0.5M acetic acid, dialyzing, and lyophilizing to obtain collagen;
B2. and (4) carrying out enzymolysis to prepare the sipunculus nudus collagen peptide.
6. The skin wound repair ointment according to claim 5, wherein in B2, the specific method for enzymatic hydrolysis comprises: any two enzymes of neutral protease, animal protease, flavourzyme, alkaline protease and papain are selected for compounding, the addition amount of the two enzymes is 3000 plus 5000U/g, the pH value is 5.0-7.0, the enzymolysis temperature is 45-55 ℃, the enzymolysis time is 3-5 hours, after enzyme deactivation is carried out for 10 minutes at 100 ℃, the supernatant fluid is obtained by centrifugation, and freeze drying is carried out to obtain the Sipunculus nudus collagen peptide.
7. A process for the preparation of a skin wound healing ointment according to any one of claims 1 to 6, characterized in that it comprises the following steps:
(1) Mixing vaseline and lanolin at a certain proportion, heating to dissolve, and adding glycerol to obtain ointment matrix;
(2) Respectively adding active peptide of marine shellfish and collagen peptide before the matrix is not completely cooled, and stirring;
(3) and (3) after uniformly stirring until the color is not changed, sealing a preservative film, and standing for 1 hour to obtain the skin wound repair ointment.
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| CN112190687A (en) * | 2020-11-19 | 2021-01-08 | 温州医科大学附属第一医院 | Medicine for reducing skin wound scar and application thereof |
| CN113481271A (en) * | 2021-06-22 | 2021-10-08 | 中国科学院南海海洋研究所 | Marine biological active peptide capable of effectively relieving skin sunburn and preparation method and application thereof |
| CN113633818A (en) * | 2021-07-20 | 2021-11-12 | 南方医科大学 | Development of antibiotic artificial dressing of polypeptide |
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| CN111000974A (en) * | 2020-02-24 | 2020-04-14 | 重庆大学 | Rabbit milk active small peptide solution and extraction method and application thereof |
| CN112190687A (en) * | 2020-11-19 | 2021-01-08 | 温州医科大学附属第一医院 | Medicine for reducing skin wound scar and application thereof |
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| CN113633818A (en) * | 2021-07-20 | 2021-11-12 | 南方医科大学 | Development of antibiotic artificial dressing of polypeptide |
| CN115444924A (en) * | 2022-09-29 | 2022-12-09 | 广东海洋大学 | Pearl oyster protein peptide composition and preparation and application thereof |
| CN115444924B (en) * | 2022-09-29 | 2023-03-10 | 广东海洋大学 | Pearl oyster protein peptide composition and preparation and application thereof |
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Application publication date: 20191206 |