CN110522900A

CN110522900A - A kind of application of oyster active peptides in wound repair

Info

Publication number: CN110522900A
Application number: CN201910813981.4A
Authority: CN
Inventors: 秦小明; 林海生; 章超桦; 曹文红; 高加龙; 伍彬; 郑惠娜; 杨发明
Original assignee: Guangdong Ocean University
Current assignee: Guangdong Ocean University
Priority date: 2019-08-30
Filing date: 2019-08-30
Publication date: 2019-12-03

Abstract

The present invention provides a kind of application of oyster active peptides in wound repair, and the oyster active peptides have the new application for repairing skin trauma；The preparation method of the oyster active peptides is the following steps are included: oyster whole viscera is cleaned, mashing, adjust pH value to 7.0, add the animal protease of 800-1700U/g albumen, digest 3.5-5.5h, 100 DEG C of 10 min of enzyme deactivation after enzymatic hydrolysis, supernatant is taken after 8000--12000 r/min centrifugation, selecting aperture is after 0.05-0.5um ceramic membrane sufficiently filters, to be separated using the ultrafiltration membrane of different molecular weight cut off, obtains the enzymolysis liquid that molecular weight is less than 3000Da, concentration, -80 DEG C save for 24 hours, and freeze-drying obtains oyster active peptides.The present invention provides a kind of oyster active peptides in the new application for repairing skin burn, the skin traumas drug such as wound, burn, scald, and effect is substantially better than the drug of the prior art, and wound is repaired fast, and preventing from scar, rush wound repair effect become apparent from.

Description

A kind of application of oyster active peptides in wound repair

Technical field

The invention belongs to biomedicine technical fields, and in particular to a kind of application of oyster active peptides in wound repair.

Background technique

Hong Kong oyster (Crossostrea sikamea) is the main breed variety of China's South China Sea, delicious meat, nutrition It is abundant comprehensive, it is known as the title of " ocean milk ".Oyster is the food of first selected food and medicine consangunity, this research team and other Person, which studies discovery oyster peptide, has the function of that anti-oxidant, immune, antifatigue, antitumor, antibacterial, improvement memory, relieving alcoholism and protecting liver etc. are living Property, this is promotes wound healing to establish theoretical basis, but there has been no the relevant reports for go deep into it medical value, therefore The research of sufficiently development and utilization oyster functional value is very important.

Wound healing is a complicated process, and the good coordination for being related to biology and molecule is integrated, such as cell migration, cell Proliferation and extrtacellular matrix deposition, including three/tetra- stacking stages: hemostasis/inflammation, cell Proliferation and remodeling, and each Regulate and control link, the factor of its wound repair of interference effect is numerous.Skin is the maximum organ in vertebrate, complete human body Skin can be used as the powerful barrier for resisting mechanical attacker, and prevent pathogen from entering in vivo, and have self-regeneration and self The ability of update.Currently, common wound repair drug mainly has: the genetically engineered cell factor, antibacterials, enzyme debridement system Agent, Chinese materia medica preparation, antioxidant and corticosteroid etc..

Medical dressing has been widely used for promoting the healing of various injury types, however, being directed to the natural nothing of skin injury Scar preparation and trauma patient tailored version clinical nutrition product but lack very much, and being badly in need of exploitation facilitates the natural activity of wound healing Substance, the rush for developing convenience and high-efficiency are cured product.

Summary of the invention

In view of this, the present invention provides a kind of application of oyster active peptides in wound repair.The present invention provides one kind Oyster active peptides are substantially better than in the new application for repairing skin burn, the skin traumas drug such as wound, burn, scald, effect The drug of the prior art, wound is repaired fastly, and preventing from scar, and primary treatment effect includes hemostasis, antibacterial, anti-inflammatory, adjusting is thin Intracellular cytokine promotes wound healing and inhibits cicatrization isoreactivity, promotees wound repair effect and becomes apparent from.

The technical solution of the present invention is as follows:

The application of a kind of oyster active peptides in wound repair, which is characterized in that the oyster active peptides, which have, repairs skin wound The new application of wound；

The preparation methods of the oyster active peptides the following steps are included:

Oyster whole viscera is cleaned, is beaten, adjusts pH value to 7.0, adds the animal protease of 800-1700U/g albumen, digest 3.5- 5.5h takes supernatant after 100 DEG C of enzyme deactivation 10 min, 8000-12000 r/min centrifugations after enzymatic hydrolysis, and selections aperture is 0.05- It after 0.5um ceramic membrane sufficiently filters, is separated using the ultrafiltration membrane of different molecular weight cut off, obtains molecular weight less than 3000Da Enzymolysis liquid, concentration, -80 DEG C save for 24 hours, freeze-drying, obtain oyster active peptides (as EPO).

Further, application of the oyster active peptides in wound repair, comprising the following steps:

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

(3) efficacy assessments are carried out to wound repair effect, the efficacy assessments include level-one efficacy assessments and secondary efficacy evaluation； The level-one efficacy assessments include styptic activity evaluation, antibacterial activity evaluation it is any one or two kinds of；The secondary efficacy is commented Valence is skin wound healing evaluation, and the skin wound healing evaluation includes the measurement of wound healing rate, falls the measurement of scab time, epidermis The combination of any one or more of growth factor measurement, inflammatory factor measurement, collagen generation analysis.

Further, in the step (1), mouse holostrome skin lesion trauma model method are as follows: use 5 ~ 6 week old male mices It is raised by animal health and processing routine, 350 mg/kg chloral hydrate anesthesias, skin of back depilation disinfection is injected intraperitoneally Afterwards, a circular hole is made a call at mouse back with 8 mm punch of diameter, cuts full thickness skin, be deep to fascia, every animal sub-cage rearing.

Further, in the step (2), the mode of administration are as follows: take oyster active peptides 25-55mg total nitrogen content/ml water (paste) smear wound, until filling, daily smear be administered once, use sterile saline for negative control group (as NT), successive administration 28 days；Positive controls (as PC) use same method.

Oyster active peptides are smeared to trauma skin by external application mode, since oyster active peptides have good biology Characteristic, it is good with the compatibility of periwound tissue, and there is good antibacterial activity；It can promote skin by inductive factor Skin and nerve increase, and conducive to the proliferation for repairing of epithelial cell, promote the healing of the surface of a wound, reparing skin defect and tissue defect；Skin Skin also has good absorption effect to oyster active peptides, and Rapid Combination is at itself collagen, to form normal knot Tissue is formed, impaired skin is made to be filled and be repaired, reduces the formation of scar.

Further, in the step (2), another feasible pattern of administration are as follows: according to 0.15-0.45g/kgbw's Dosage carries out stomach-filling to mouse using oyster active peptides, and daily gastric infusion is primary, and stomach-filling amount is 0.1mL/10gbw, adopts It is negative control group with sterile saline, successive administration 28 days.

Oyster active peptides are provided for human body by mode for oral administration, since oyster active peptides molecular weight is low, has and digests and assimilates Property is fast, can reach corporal parts directly by intestinal absorption, into blood.After external source replenishing collagen peptide, skin The collagen of itself can be synthesized with fibroblast, fat cell and capillary etc. in connective tissue, to be formed just Normal connective tissue makes to be damaged, the skin of aging is filled and repaired.

Oyster active peptides of the invention have extremely strong bioactivity and diversity, can efficiently play health-care efficacy, entirely Face adjusts human body physiological function, is particularly helpful to accelerating wound healing and repairs superficial wound, can both take orally, can also be to wound It is smeared in face.

Further, the method for the styptic activity evaluation are as follows: fresh anticoagulation rabbit blood is centrifuged 10 points at 1000rpm Clock, blood plasma separation use；The blood plasma of 0.1 mL is added in every pipe, the 0.1 mL sample that concentration is 1mg/mL is added, is added 0.1mL, 2% citric acid solution are as negative control, and it is positive control that the drug that 0.1mL concentration is 1mg/mL, which is added, and at 37 °C It is incubated in water-bath；1 minute, the 0.025 mol/L calcium chloride solution of 0.1 mL is then added, mixes and is put into 37 °C of water-baths, Start stopwatch simultaneously, it is primary slowly to tilt test tube within every 15 seconds, and start to spread in the mixture；When fine using white particulate When fibrillarin, the stupid cured time is recorded.

Further, the method for the antibacterial activity evaluation are as follows: strain relevant to infection is selected, by activated bacterium solution It is diluted with sterile saline, 50uL physiological saline is added in 96 orifice plates and is blank control, 50ul, 25-55mg/mL sample is added Product, sample concentration is in terms of total nitrogen content, and bacterium solution after 50ulTSB culture medium and 50uL dilution is added mixes, in 550nm wavelength, It is recorded as the OD value of 0h, 37 DEG C of cultures measure 8h and OD value for 24 hours respectively, calculate separately bacteriostasis rate.

Specifically, the method for the antibacterial activity evaluation are as follows: wish escherichia coli, staphylococcus aureus, seaweed The kind such as watt Salmonella, listeria spp, pseudomonas aeruginosa, the thermophilic steam bacterium of loach, Streptococcusagalactiae is in 3%TSB fluid nutrient medium It is activated, vibrio alginolyticus, vibrio parahemolyticus is activated using 3%TSB-3%NaCl culture medium.By activated bacterium solution nothing Bacterium normal saline dilution is to OD₅₅₀Value is 0.1, and 50uL physiological saline is added in 96 orifice plates and is blank control, 50 ul is added, 20-70 mg/mL sample (being calculated with total nitrogen) is sample sets, and the bacterium solution after 50ulTSB culture medium and 50uL dilution is added in each group, It mixing, 550 nm measure absorbance, are recorded as 0 h OD value, and it is then placed in 37 DEG C of incubators and cultivates a 8 h OD value of measurement, 24 h measure an OD value.Bacteriostasis rate is calculated separately, shown in formula following (1):

。

Further, the wound healing rate measures and falls the method for measuring of scab time are as follows: smears and gives in the step (2) After medicine, since the 0th day, wound surface situation is photographed to record in same fixed height within every 2 days, calculate wound healing with software Rate, record fall the scab time；The formula (2) of wound healing rate measurement are as follows:

,

N is number of days after modeling, unit: day in formula.

Further, in the step (3), the factor involved in the human epidermal growth factor determination and inflammatory factor measurement Including in TNF-a, IL-1 β, TGF-β 1, COL1A1 (I-type collagen) mRNA, TGF-β 1, a-SMA, TGF-BRII, Smad7 Any two or more.

Further, in the step (3), human epidermal growth factor determination, inflammatory factor measurement use real time fluorescent quantitative PCR analyzes (RT-PCR).Method particularly includes: total serum IgE is extracted from wound tissue using RNAIso Plus and homogenizer.It uses Prime ScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) kit carries out inverse Transcription synthesis cDNA, is carried out glimmering with TB GreenTM Premix Ex TaqTM II (Tli RNaseH Plus) (* 2) kit Light is quantitative.Reaction condition are as follows: 95 DEG C of 30s, 95 DEG C of 5s, 60 DEG C of 30s, 40 circulations.Every to extend once, all progress real-time fluorescences are fixed Measurement.Use glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene, for correcting the expression for calculating target gene, It is calculated using CT method.The the 1, the 14th, 21 and 28 day measurement COL1A1 (I of 7th day measurement TNF-a, IL-1 β and TGF-β Collagen type) mRNA level in-site.

Further, in step (3), collagen generates analysis and uses collagenous fibres transmission electron microscope analysis (TEM).Specifically Are as follows: take the 21st day after modeling skin wound tissue to be put into 2.5% glutaraldehyde, in 4 DEG C it is fixed overnight.After glutaraldehyde is fixed Tissue immerses in PBS (0.1mol/L), rinses 3 times, and supernatant is removed in centrifugation, every time 20 min, then 1% osmic acid being pre-chilled with 4 DEG C, In Then 4 DEG C of fixed 2-3 h embathe 3 times with PBS(0.1mol/L), every time 20 min.By gradient 30%, 50%, 70%, 80%, 85%, 90%, 95%, 100% ethanol dehydration, every kind concentration ethanol 1 time, every time 15 min, then be thoroughly dehydrated 2 times with 100% ethyl alcohol, 10 min every time.Then acetone: epoxy resin (2:1), acetone: epoxy resin (1:1), epoxy resin is used, in 37 DEG C of insulating boxs It is successively permeated, every time 12 h.Then embed, solidify and repair block, after the double dyeing of lead and uranium, in 200 KV acceleration voltages and The observation of enlargement ratio 5000 is taken pictures.The diameter of each collagenous fibres in each image is measured using the analysis of ImageJ software.

In the present invention, the optional apparent drug of wound repair effect in the prior art of positive controls, such as Yunnan are white Medicine, jingwanhong soft plaster ointment, the antibacterial frost of silver-colored zinc etc..The Yunnan Baiyao is that the prior art is given birth to by Yunnan Paiyao Group Corp., Ltd The Yunnan Baiyao pulvis of production, national drug standard Z53020798；The jingwanhong soft plaster ointment is prior art Tianjin Da Rentang jingwanhong soft plaster medicine The jingwanhong soft plaster ointment of industry Co., Ltd production, national drug standard Z12020440；The antibacterial frost of silver-colored zinc is that the remittance of prior art Henan is rich The antibacterial frost of Bang Erkang silver-colored zinc of medical limited liability company's production, registration certificate number (Henan), which is defended to disappear, demonstrate,proves word (2004) the 0056th.

Further, the oyster active peptides can be made into including powdered, solution shape, emulsus, paste, gel, compound The membranaceous any product of fiber.Preferably, the oyster active peptides are the outer embranes that pearl shell is isolated by pre-processing, and are led to Protease hydrolyzed is crossed, ceramic membrane, ultrafiltration system separation, dry oyster active peptides obtained are passed sequentially through.

Oyster active peptides of the present invention have good biological characteristics, good with the compatibility of periwound tissue, can pass through Inductive factor promotes skin and nerve to increase, and conducive to the proliferation for repairing of epithelial cell, promotes the healing of the surface of a wound, repairs skin Skin defect and tissue defect；Skin also has good absorption effect to active peptide, Rapid Combination at itself collagen, from And normal connective tissue is formed, so that impaired skin is filled and is repaired, reduces the formation of scar.Due to oyster active peptides Molecular weight is low, has the property digested and assimilated fast, can reach corporal parts directly by intestinal absorption, into blood.Pass through external source After replenishing collagen peptide, fibroblast, fat cell and capillary etc. can synthesize itself in skin and connective tissue Collagen make to be damaged, the skin of aging is filled and repaired to form normal connective tissue, reach and delay skin The purpose of skin aging.Therefore, oyster active peptides may act on the post-hemorrhagic period of wound, the phase four inflammatory phase, proliferation period and remodeling wounds Recovery conjunction period can be used as the treatment that the regenerative healing active material that anti-abnormal scar is formed is used for dermal lesions.

The present invention provides a kind of oyster active peptides to repair skin burn, the skin traumas drug such as wound, burn, scald New application, effect is substantially better than the drug of the prior art, and wound repairs fast, and preventing from scar, primary treatment effect packet Hemostasis is included, antibacterial, anti-inflammatory, adjusting cell factor, promotes wound healing and inhibits cicatrization isoreactivity, promotees wound repair effect It becomes apparent from.

Further, the membranaceous product of the composite fibre is the electrospun composite fibers as made from electrostatic spinning technique Film, it is described the preparation method comprises the following steps:

A. oyster active peptides are dissolved in ultrapure water, are made it completely dissolved using prior arts such as ultrasounds, oyster activity is made Peptide solution；

B. carrier material is dissolved in solvent, carrier material solution is made；The carrier material includes base-material and auxiliary material, described The mass ratio of base-material and auxiliary material is 1:1-4:1；The base-material is polyvinyl alcohol or polycaprolactone, and the auxiliary material is sea cucumber collagen egg White or chitosan；The solvent is any one of hexafluoroisopropanol, trifluoroacetic acid, trifluoroethanol；The carrier material solution Concentration be 5%-10%；

C. the resulting solution sufficient vortex of step a and step b is mixed, dissolves it sufficiently, spinning solution is made；Spinning solution The concentration of middle oyster active peptides is 3-12%；

D. biological active dressing is prepared using the method for electrostatic spinning using step c obtained spinning solution；Static Spinning Silk solvent for use is any one of hexafluoroisopropanol, trifluoroacetic acid, trifluoroethanol.

In the present invention, any prior art realization, such as Chinese patent is can be used in the holothurian collagen The holothurian collagen of the extracting method of sea cucumber collagen, collagen " 200710159245.9, " preparation.Holothurian collagen (PSC) amino acid contained A wide selection of colours and designs has the composition of typical collagen.Total amino acid content reaches the 33.07% of PSC.Wherein, Total amount of essential amino acids reach the 5.29% of PSC, account for the 16% of total amino acid content；Fresh ear field total amount reaches the 19.04% of PSC;Drug effect Total amino acid content reaches the 17.69% of PSC, in these ingredients, has the function factor for promoting wound reparation, can be with oyster active peptides Synergistic compounding enhances the quick healing of its wound in composite cellulosic membrane, prevents the effect of cicatrization.

Further, the concentration of the oyster active peptides solution is 10-20mg/ml.

Further, electrostatic spinning voltage is 15-30kV in step d, and the distance between syringe needle and receiver board can For 10-15cm, the fltting speed of syringe pump can be 0.01-0.50mL/min, and spinning environment temperature can be 35-39 DEG C.

The present invention will join together with electrostatic spinning technique, be aided with carrier material and prepare a kind of adjustable immune micro-loop Border has anti-inflammatory effect, is conducive to the quick healing of wound, prevents cicatrization, and the new bio that can play dauer effect applies Material.

Particularly, the present invention provides a kind of oyster active peptides composite cellulosic membrane for the purposes in wound repair drug.

The tunica fibrosa of electrostatic spinning technique preparation has very high porosity and good gas permeability, wound can be made to keep Preferably wet degree, and be conducive to the respiration of cell.Drug ingedient is added in spinning solution during electrostatic spinning, The available nanofiber for being compounded with some drug ingedients can be by medicine after nanofiber degrades or encounters water-swellable Object ingredient slowly releases, and the drug effect of drug not only can be improved in this mode, reduces the toxic side effect of drug.And it is coaxial Electrostatic spinning technique can carry out functional modification in fiber surface, while functional material can also be wrapped in fibrous inside, To play the role of persistently playing effect.

The present invention prepares biological active dressing using electrostatic spinning technique by addition oyster active peptides, as wound Face dressing has biggish specific surface area and pore structure, sticks sertoli cell growth well, can provide natural bionical Class extracellular matrix has anti-inflammatory effect, and protecting it from being degraded by wound microenvironment inactivates, and promotes collagen and cell factor Synthesis, secretion, greatly improvement collagen deposition, form orderly basketry weave pattern arrangement, be allowed to have normal dermal Feature.Method provided by the invention is easy to operate, and repeatability is strong, there is huge application prospect in wound repair drug.

Oyster active peptides composite cellulosic membrane of the invention, using holothurian collagen or chitosan as carrier, due to sea Ginseng collagen or chitosan itself have good functional activity, are capable of the anti-of synergistic amplification oyster active peptides composite cellulosic membrane Inflammation effect and the effect for promoting quick healing of cut, so that its effect in wound repair is better than exclusive use oyster activity The effect of peptide directly smears wound.The electrostatic spinning raw material can be realized by any prior art.

Dressing preparation method of the present invention is simple and easy, and obtained biological dressing wound sticks well, safe and non-toxic, application It is convenient.Biologically active oyster active peptides dressing has hemostasis, antibacterial, moisturizing while the isolation surface of a wound, good permeability again, There is anti-inflammatory effect simultaneously, be conducive to the quick healing of wound, prevent cicatrization.

Detailed description of the invention

Fig. 1 is influence diagram of the EPO to wounds in mice of one embodiment of the invention preparation；

Fig. 2 is influence diagram of the EPO to inflammatory factor IL-10 in trauma skin tissue of one embodiment of the invention preparation；

Fig. 3 is influence diagram of the EPO to inflammatory factor IL-6 in trauma skin tissue of one embodiment of the invention preparation；

Fig. 4 is influence diagram of the EPO to inflammatory factor IL-10 in trauma skin tissue of another embodiment of the present invention preparation.

Specific embodiment

Technical solution of the present invention is clearly and completely described below in conjunction with the embodiment of the present invention, it is clear that described Embodiment be only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ability Domain those of ordinary skill every other embodiment obtained without making creative work, belongs to guarantor of the present invention The range of shield.

Embodiment 1

Oyster whole viscera is cleaned, is beaten, adjusts pH value to 7.0, adds the animal protease of 1400U/g albumen, digest 4.5h, enzymatic hydrolysis After 100 DEG C of enzyme deactivation 10 min, take supernatant after 10000 r/min centrifugation, selecting aperture is that 0.05-0.5um ceramic membrane fills It after dividing filtering, is separated using the ultrafiltration membrane of different molecular weight cut off, obtains the enzymolysis liquid that molecular weight is less than 3000Da, it is dense Contracting, -80 DEG C save for 24 hours, and freeze-drying obtains oyster active peptides (as EPO).

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

Further, in the step (2), the mode of administration are as follows: take oyster active peptides 35mg total nitrogen content/ml water (cream Shape) wound is smeared, it until filling, smears be administered once daily, use sterile saline for negative control group (as NT), even Continuous administration 28 days；Positive controls (as PC) use same method.

。

,

N is number of days after modeling, unit: day in formula.

In the present invention, positive controls select the Yunnan Baiyao of the prior art.The Yunnan Baiyao is the prior art by cloud The Yunnan Baiyao pulvis of southern baiyao Group Plc production, national drug standard Z53020798.

Embodiment 2

Oyster whole viscera is cleaned, is beaten, adjusts pH value to 7.0, adds the animal protease of 1000U/g albumen, digest 3.8h, enzymatic hydrolysis After 100 DEG C of enzyme deactivation 10 min, take supernatant after 9000 r/min centrifugation, selecting aperture is that 0.05-0.5um ceramic membrane is abundant It after filtering, is separated using the ultrafiltration membrane of different molecular weight cut off, obtains the enzymolysis liquid that molecular weight is less than 3000Da, concentration ,- 80 DEG C save for 24 hours, and freeze-drying obtains oyster active peptides (as EPO).

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

Further, in the step (1), mouse holostrome skin lesion trauma model method and embodiment 1 are consistent.

Further, the method and embodiment 1 of the styptic activity evaluation are consistent.

Further, the method and embodiment 1 of the antibacterial activity evaluation are consistent.

Further, the wound healing rate measures and falls the method for measuring of scab time and embodiment 1 is consistent.

Further, in the step (3), human epidermal growth factor determination, inflammatory factor measurement use real time fluorescent quantitative PCR analyzes (RT-PCR), and specific method and embodiment 1 are consistent.

Further, in step (3), collagen generates analysis and uses collagenous fibres transmission electron microscope analysis (TEM), specifically Method and embodiment 1 are consistent.

Embodiment 3

Oyster whole viscera is cleaned, is beaten, adjusts pH value to 7.0, adds the animal protease of 1200U/g albumen, digest 4h, enzymatic hydrolysis knot Supernatant is taken after 100 DEG C of enzyme deactivation 10 min, 11000 r/min centrifugations after beam, selecting aperture is that 0.05-0.5um ceramic membrane is abundant It after filtering, is separated using the ultrafiltration membrane of different molecular weight cut off, obtains the enzymolysis liquid that molecular weight is less than 3000Da, concentration ,- 80 DEG C save for 24 hours, and freeze-drying obtains oyster active peptides (as EPO).

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

Further, in the step (2), the mode of administration are as follows: take oyster active peptides 40mg total nitrogen content/ml water (cream Shape) wound is smeared, it until filling, smears be administered once daily, use sterile saline for negative control group (as NT), even Continuous administration 28 days；Positive controls (as PC) use same method.

Embodiment 4

Oyster whole viscera is cleaned, is beaten, adjusts pH value to 7.0, adds the animal protease of 1600U/g albumen, digest 4.8h, enzymatic hydrolysis After 100 DEG C of enzyme deactivation 10 min, take supernatant after 10000 r/min centrifugation, selecting aperture is that 0.05-0.5um ceramic membrane fills It after dividing filtering, is separated using the ultrafiltration membrane of different molecular weight cut off, obtains the enzymolysis liquid that molecular weight is less than 3000Da, it is dense Contracting, -80 DEG C save for 24 hours, and freeze-drying obtains oyster active peptides (as EPO).

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

Further, in the step (2), the mode of administration are as follows: take oyster active peptides 50mg total nitrogen content/ml water (cream Shape) wound is smeared, it until filling, smears be administered once daily, use sterile saline for negative control group (as NT), even Continuous administration 28 days；Positive controls (as PC) use same method.

In the present invention, the antibacterial frost of silver-colored zinc in the prior art is can be selected in positive controls, and the antibacterial frost of silver-colored zinc is existing skill Converge the antibacterial frost of Bang Erkang silver-colored zinc of rich medical limited liability company's production in art Henan, and registration certificate number (Henan), which is defended to disappear, demonstrate,proves word (2004) the No. 0056.

Embodiment 5

Oyster whole viscera is cleaned, is beaten, adjusts pH value to 7.0, adds the animal protease of 800U/g albumen, digest 5.2h, enzymatic hydrolysis After 100 DEG C of enzyme deactivation 10 min, take supernatant after 12000 r/min centrifugation, selecting aperture is that 0.05-0.5um ceramic membrane fills It after dividing filtering, is separated using the ultrafiltration membrane of different molecular weight cut off, obtains the enzymolysis liquid that molecular weight is less than 3000Da, it is dense Contracting, -80 DEG C save for 24 hours, and freeze-drying obtains oyster active peptides (as EPO).

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

Further, in the step (2), the mode of administration are as follows: take oyster active peptides 38mg total nitrogen content/ml water (cream Shape) wound is smeared, it until filling, smears be administered once daily, use sterile saline for negative control group (as NT), even Continuous administration 28 days；Positive controls (as PC) use same method.

In the present invention, jingwanhong soft plaster ointment in the prior art is can be selected in positive controls.The jingwanhong soft plaster ointment is existing skill The jingwanhong soft plaster ointment of art Tianjin Darentang Wanhong Pharmaceutical Co., Ltd production, national drug standard Z12020440.

Embodiment 6

The preparation method and embodiment 1 of the oyster active peptides are consistent.

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

Further, in the step (2), the mode of administration are as follows: according to the dosage of 0.25g/kgbw, using oyster Active Peptide in Mice carries out stomach-filling, and daily gastric infusion is primary, and stomach-filling amount is 0.1mL/10gbw, using sterile saline For negative control group, successive administration 28 days.

In the present invention, the Yunnan Baiyao of the prior art is can be selected in positive controls.The Yunnan Baiyao be the prior art by The Yunnan Baiyao pulvis of Yunnan Paiyao Group Corp., Ltd's production, national drug standard Z53020798.

Embodiment 7

The preparation method and embodiment 2 of the oyster active peptides are consistent.

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

Further, in the step (2), the mode of administration are as follows: according to the dosage of 0.2g/kgbw, using oyster Active Peptide in Mice carries out stomach-filling, and daily gastric infusion is primary, and stomach-filling amount is 0.1mL/10gbw, using sterile saline For negative control group, successive administration 28 days.

Embodiment 8

The preparation method and embodiment 3 of the oyster active peptides are consistent.

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

Further, in the step (2), the mode of administration are as follows: according to the dosage of 0.3g/kgbw, using oyster Active Peptide in Mice carries out stomach-filling, and daily gastric infusion is primary, and stomach-filling amount is 0.1mL/10gbw, using sterile saline For negative control group, successive administration 28 days.

Embodiment 9

The preparation method and embodiment 4 of the oyster active peptides are consistent.

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

Further, in the step (2), the mode of administration are as follows: according to the dosage of 0.35g/kgbw, using oyster Active Peptide in Mice carries out stomach-filling, and daily gastric infusion is primary, and stomach-filling amount is 0.1mL/10gbw, using sterile saline For negative control group, successive administration 28 days.

Embodiment 10

The preparation method and embodiment 5 of the oyster active peptides are consistent.

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

Further, in the step (2), the mode of administration are as follows: according to the dosage of 0.4g/kgbw, using oyster Active Peptide in Mice carries out stomach-filling, and daily gastric infusion is primary, and stomach-filling amount is 0.1mL/10gbw, using sterile saline For negative control group, successive administration 28 days.

Embodiment 11

In the present embodiment, the oyster active peptides of any preparation of embodiment 1-10 be can be made into the membranaceous product of composite fibre.Preferably, The oyster active peptides are to isolate the outer embrane of pearl shell by pre-processing, by protease hydrolyzed, pass sequentially through ceramic membrane, Ultrafiltration system separation, dry oyster active peptides obtained.

B. carrier material is dissolved in solvent, carrier material solution is made；The carrier material includes base-material and auxiliary material, described The mass ratio of base-material and auxiliary material is 1:1；The base-material is polyvinyl alcohol, and the auxiliary material is holothurian collagen；The solvent is In trifluoroethanol；The concentration of the carrier material solution is 7%；

C. the resulting solution sufficient vortex of step a and step b is mixed, dissolves it sufficiently, spinning solution is made；Spinning solution The concentration of middle oyster active peptides is 9%；

D. biological active dressing is prepared using the method for electrostatic spinning using step c obtained spinning solution；Static Spinning Silk solvent for use is hexafluoroisopropanol.

Further, the concentration of the oyster active peptides solution is 15mg/ml.

Further, electrostatic spinning voltage is 26kV in step d, and the distance between syringe needle and receiver board can be 12cm, the fltting speed of syringe pump can be 0.4mL/min, and spinning environment temperature can be 37 DEG C.

Embodiment 12

B. carrier material is dissolved in solvent, carrier material solution is made；The carrier material includes base-material and auxiliary material, described The mass ratio of base-material and auxiliary material is 4:1；The base-material is polycaprolactone, and the auxiliary material is chitosan；The solvent is trifluoro second Acid；The concentration of the carrier material solution is 8%；

C. the resulting solution sufficient vortex of step a and step b is mixed, dissolves it sufficiently, spinning solution is made；Spinning solution The concentration of middle oyster active peptides is 10%；

D. biological active dressing is prepared using the method for electrostatic spinning using step c obtained spinning solution；Static Spinning Silk solvent for use is trifluoroacetic acid.

Further, the concentration of the oyster active peptides solution is 20mg/ml.

Further, electrostatic spinning voltage is 18kV in step d, and the distance between syringe needle and receiver board can be 11cm, the fltting speed of syringe pump can be 0.1mL/min, and spinning environment temperature can be 36 DEG C.

Embodiment 13

B. carrier material is dissolved in solvent, carrier material solution is made；The carrier material includes base-material and auxiliary material, described The mass ratio of base-material and auxiliary material is 3:1；The base-material is polycaprolactone, and the auxiliary material is holothurian collagen；The solvent is Hexafluoroisopropanol；The concentration of the carrier material solution is 6%；

C. the resulting solution sufficient vortex of step a and step b is mixed, dissolves it sufficiently, spinning solution is made；Spinning solution The concentration of middle oyster active peptides is 5%；

D. biological active dressing is prepared using the method for electrostatic spinning using step c obtained spinning solution；Static Spinning Silk solvent for use is trifluoroethanol.

Further, the concentration of the oyster active peptides solution is 10mg/ml.

Further, electrostatic spinning voltage is 28kV in step d, and the distance between syringe needle and receiver board can be 14cm, the fltting speed of syringe pump can be 0.4mL/min, and spinning environment temperature can be 38 DEG C.

Embodiment 14

B. carrier material is dissolved in solvent, carrier material solution is made；The carrier material includes base-material and auxiliary material, described The mass ratio of base-material and auxiliary material is 2:1；The base-material is polyvinyl alcohol, and the auxiliary material is chitosan；The solvent is trifluoro second Acid；The concentration of the carrier material solution is 8%；

Further, the concentration of the oyster active peptides solution is 13mg/ml.

Further, electrostatic spinning voltage is 20kV in step d, and the distance between syringe needle and receiver board can be 13cm, the fltting speed of syringe pump can be 0.25mL/min, and spinning environment temperature can be 37 DEG C.

Functional evaluation result

Fig. 1-2, table 1-3 are the functional evaluation result correlation graph of embodiment 1.

By Fig. 1, each group healing state as shown, the 2nd day after modeling, all formed a scab by each test group animal, wound week Existing redness is crossed, no liquid is oozed out, and contraction of wounds is unobvious.2nd, 4 day, each group surface of a wound incrustation part was constantly hardened, wound Circumferential surface out-of-flatness, edge of wound skin contraction are more apparent.6th, 8,10 day, the incrustation of negative control group wound constantly thickend, at black Red, but scar area change is small.Positive controls wound contraction, and beginning decrustation, contracting scab are obvious.Administration group is the 6th It when fallen scab, contracting scab is substantially better than two control groups.12nd day, each group all fell scab, started to grow hair at healing.Wherein give The further contracting scab of medicine group.14th day, most wound diameters of control group were less than 2mm, and each administration group wound diameter is respectively less than 1 mm, Flesh pink neoplastic skin is presented, newborn yellowish pink tissue is complete.Administration group is good according to group healing state compared with to feminine gender, without Hypertrophic Scar and keloid generate.

In conjunction with Tables 1 and 2,6 days after modeling, the wound healing rate of administration group is 35% or more, with negative control group phase Than being higher by 21%(respectivelyP<0.05)；10d after modeling, administration group and negative control group have significant difference (P<0.05)；14th It when, administration group it is high compared with negative control group by 14% or more (P<0.05), healing rate is up to 94% or more；As shown in Table 2, administration group scar Minification be significantly higher than negative control group (P<0.05).

As shown in Table 1, administration group is in wound healing period, and wound healing rate is above negative control group, administration group with Negative control group is compared to no significant difference.Wound healing later period, each test group wound healing rate gradually tend to balance, administration group Effect is prominent.Positive controls and administration group wound healing rate are without significant difference, and administration group effect is more preferable, and scar is smaller, Almost seamless state.Composition detection is the results show that EPO main component is protein, and wherein peptide content is higher, and has Film permeability is good, active cell is active, repairs the bioactivity such as human body degeneration of cells, has correlation function active peptide and rises main Effect.

Influence of 1 EPO of table to mouse skin wound healing

The influence that 2 EPO of table reduces mouse skin scar

As shown in Figure 2, the 3rd day, administration group IL-10 content be significantly higher than negative control group (P<0.05)；5th day, positive control Group IL-10 content is extremely significant be higher than other groups (P<0.01)；7th day, compared with negative control group, administration group IL-10 content without Statistical significance (P<0.05).EPO has the albumen and peptide of high level, this, which is conducive to EPO, has promotion IL-10 secretion to rise To the effect for inhibiting inflammatory reaction.

As shown in Table 3, administration group FGF-2 content is compared with other groups, have significant difference (P<0.05)；It is right with feminine gender Compared according to a group TGF-β content, administration group all present significant difference (P<0.05)；Administration group CCND1 content be significantly higher than two it is right According to group (P<0.05)；Administration group EGF content is significantly higher than negative control group, but not statistically significant (P>0.05).Thus it can push away Pass through during surveying EPO wound healing and the secretion of cell factor TGF-β is accelerated to promote fibroblasts to secrete collagen, increase Add extracellular matrix deposition, neovascularization, promote epidermal growth.Simultaneously also by acceleration cell cycle regulating protein It is same to realize to accelerate the mitosis of cell that the expression of CCND1 promotes the conversion of cell cycles to the division stages such as fibroblast The consistent wound healing of healing rate result.The result shows that smearing EPO can pass through point of promotion TGF-β and CCND1 in proliferation period It secretes, to accelerate mouse skin wound healing.

Influence of 3 EPO of table to growth factor in trauma skin tissue

Fig. 3-4, table 4-5 are the functional evaluation result correlation graph of embodiment 6,7,10.

Functional evaluation is carried out by embodiment 6, show that each test group animal was all formed a scab with the 2nd day after modeling of drawing a conclusion, There is red and swollen, no liquid exudation in wound circumference, and contraction of wounds is unobvious.4th, 6,8 day, the incrustation of each group surface of a wound was constantly hardened, Periwound surface irregularity, edge of wound skin contraction are more apparent, wherein each administration group incrustation thickness is better than control group, and part is created Dark red is presented in face.10th day, control group wound incrustation partial exfoliation, but scar area change is small.Each administration group: wound is bright It is aobvious to reduce, and beginning decrustation, contracting scab are obvious.12nd day, each group all fell scab, started to grow hair at healing.Each administration The further contracting scab of group, the secondary incrustation of middle dose group.14th day, most wound diameters of control group were less than 4 mm, each administration group wound Mouth diameter is respectively less than 2 mm, and flesh pink neoplastic skin is presented in middle and high dosage group, and newborn yellowish pink tissue is completely.Each administration group is more right Good according to group healing state, no hyperplastic scar and keloid generate.

Influence of 4 EPO of table to mouse skin wound healing

As can be seen from Table 4,6 days after modeling, embodiment 6,7 groups of embodiment of wound healing rate are right with feminine gender 25% or more It is compared according to group, is higher by 21%, 12%(respectivelyP<0.05)；10 days after modeling, embodiment 6,7 groups of embodiment have with negative control group it is aobvious Work sex differernce (P<0.05)；At the 14th day, 7 groups of embodiment 6, embodiment dosage groups it is high compared with negative control group by 17% or more (P< 0.05), healing rate is up to 97% or more；By Fig. 3, Fig. 4 it is found that oyster active peptides prepared by the present invention are to mouse trauma skin tissue Middle inflammatory factor IL-6 has certain inhibitory effect, while having and promoting IL-10 secretion to play the work for inhibiting inflammatory reaction With.

Influence of 5 EPO of table to growth factor in trauma skin tissue

As shown in Table 5, by accelerating the secretion of cell factor TGF-β, EGF to promote into fibre during EPO wound healing Cell secretion collagen is tieed up, increases extracellular matrix deposition, neovascularization, promote epidermal growth.It is thin simultaneously also by accelerating The expression of born of the same parents' cycle regulatory protein CCND1 promotes the conversion of cell cycles to the division stages such as fibroblast to accelerate having for cell Silk division is to realize the same consistent wound healing of healing rate result.

Composite fibre membrane biological dressing prepared by embodiment 11-14, takes a small amount of target fibers, in its surface metal spraying, in Fiber morphology is observed under scanning electron microscope.The results show that the composite fiber membrane material of oyster active peptides is carried, electrospinning fibre more light Sliding, irregular arrangement, also without apparent droplet formation, the tunica fibrosa substance eventually formed is that cellular is presented.

It is worth noting that, the embodiment of the present invention 2-5, is cured in styptic activity evaluation, antibacterial activity evaluation, skin trauma Closing, which can reach in evaluation, is on close level with the comparable level of embodiment 1, promotion wound healing with inhibition cicatrization；This hair Bright embodiment 7-10 can reach and embodiment 6 in styptic activity evaluation, antibacterial activity evaluation, skin wound healing evaluation Similar level has the function of promoting wound healing and inhibits cicatrization；The embodiment of the present invention 11-14 is prepared compound Fibre Membrane Bio dressing is better than the water of embodiment 1 in styptic activity evaluation, antibacterial activity evaluation, skin wound healing evaluation It is flat, promote wound healing and inhibits the better effect of cicatrization.

It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims Variation is included within the present invention.

In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art The other embodiments being understood that.It is noted that the technical characteristic being not described in detail in the present invention, it can be by appointing One prior art is realized.

Claims

1. a kind of application of oyster active peptides in wound repair, which is characterized in that the oyster active peptides, which have, repairs skin The new application of wound；

Oyster whole viscera is cleaned, is beaten, adjusts pH value to 7.0, adds the animal protease of 800-1700U/g albumen, digest 3.5- 5.5h takes supernatant after 100 DEG C of enzyme deactivation 10 min, 8000-12000 r/min centrifugations after enzymatic hydrolysis, and selections aperture is 0.05- It after 0.5um ceramic membrane sufficiently filters, is separated using the ultrafiltration membrane of different molecular weight cut off, obtains molecular weight less than 3000Da Enzymolysis liquid, concentration, -80 DEG C save for 24 hours, freeze-drying, obtain oyster active peptides.

2. application of the oyster active peptides according to claim 1 in wound repair, which is characterized in that including following step It is rapid:

(1) mouse holostrome skin lesion trauma model is established；

(2) mouse of trauma model is administered；

3. application of the oyster active peptides according to claim 2 in wound repair, which is characterized in that the step (2) In, the mode of administration are as follows: take oyster active peptides 25-55mg total nitrogen content/ml water to smear wound, until filling, smear give daily Medicine is primary, uses sterile saline for negative control group, and successive administration 28 days.

4. application of the oyster active peptides according to claim 2 in wound repair, which is characterized in that the step (2) In, the mode of administration are as follows: according to the dosage of 0.15-0.45g/kgbw, stomach-filling is carried out to mouse using oyster active peptides, often Its gastric infusion is primary, and stomach-filling amount is 0.1mL/10gbw, uses sterile saline for negative control group, successive administration 28 It.

5. application of the oyster active peptides according to claim 3 or 4 in wound repair, which is characterized in that the wound Healing rate measures and falls the method for measuring of scab time are as follows: after smearing administration in the step (2), since the 0th day, every 2 days Same fixed height photographs to record wound surface situation, calculates wound healing rate with software, record falls the scab time；Wound healing rate The formula (2) of measurement are as follows:

,

N is number of days after modeling, unit: day in formula.

6. application of the oyster active peptides according to claim 3 or 4 in wound repair, which is characterized in that the step (3) in, the human epidermal growth factor determination and inflammatory factor measurement involved in the factor include TNF-a, IL-1 β, TGF-β 1, COL1A1 mRNA, TGF-β 1, in a-SMA, TGF-BRII, Smad7 any two or more.

7. application of the oyster active peptides according to claim 1 in wound repair, which is characterized in that the oyster activity Peptide can be made into any product membranaceous including powdered, solution shape, emulsus, paste, gel, composite fibre.

8. application of the oyster active peptides according to claim 7 in wound repair, which is characterized in that the composite fibre Film-shaped products be the electrospun composite fibers film as made from electrostatic spinning technique, it is described the preparation method comprises the following steps:

A. oyster active peptides are dissolved in ultrapure water, are made it completely dissolved, oyster active peptides solution is made；

9. application of the oyster active peptides according to claim 8 in wound repair, which is characterized in that electrostatic in step d Spinning voltage is 15-30kV, and the distance between syringe needle and receiver board can be 10-15cm, and the fltting speed of syringe pump can For 0.01-0.50mL/min, spinning environment temperature can be 35-39 DEG C.