CN110606872B - Antioxidant polypeptide OA-GL17 for promoting skin wound repair and preparation method and application thereof - Google Patents
Antioxidant polypeptide OA-GL17 for promoting skin wound repair and preparation method and application thereof Download PDFInfo
- Publication number
- CN110606872B CN110606872B CN201910966274.9A CN201910966274A CN110606872B CN 110606872 B CN110606872 B CN 110606872B CN 201910966274 A CN201910966274 A CN 201910966274A CN 110606872 B CN110606872 B CN 110606872B
- Authority
- CN
- China
- Prior art keywords
- wound repair
- skin wound
- promoting skin
- antioxidant
- promoting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000037314 wound repair Effects 0.000 title claims abstract description 62
- 230000001737 promoting effect Effects 0.000 title claims abstract description 61
- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 53
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 50
- 206010072170 Skin wound Diseases 0.000 title claims abstract description 47
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 47
- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 44
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
- 235000006708 antioxidants Nutrition 0.000 claims description 38
- 239000003814 drug Substances 0.000 claims description 5
- 229940079593 drug Drugs 0.000 claims description 5
- 239000002537 cosmetic Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 15
- 238000000338 in vitro Methods 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 abstract description 6
- 238000000746 purification Methods 0.000 abstract description 6
- 238000010171 animal model Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 4
- 239000000463 material Substances 0.000 description 20
- 238000004128 high performance liquid chromatography Methods 0.000 description 13
- 230000028327 secretion Effects 0.000 description 10
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 7
- 230000008439 repair process Effects 0.000 description 7
- 230000029663 wound healing Effects 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 206010052428 Wound Diseases 0.000 description 4
- 208000027418 Wounds and injury Diseases 0.000 description 4
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 230000035876 healing Effects 0.000 description 4
- 241000149138 Nanorana yunnanensis Species 0.000 description 3
- 241000410394 Odorrana grahami Species 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 238000009777 vacuum freeze-drying Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 235000019154 vitamin C Nutrition 0.000 description 2
- 239000011718 vitamin C Substances 0.000 description 2
- -1 2, 2-diphenyl-1-picrylhydrazino Chemical group 0.000 description 1
- IIQFOVQMXGABDP-UHFFFAOYSA-N 3-ethyl-2-[(3-ethyl-6-sulfo-2h-1,3-benzothiazol-2-yl)diazenyl]-2h-1,3-benzothiazole-6-sulfonic acid Chemical compound S1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C1N=NC1N(CC)C2=CC=C(S(O)(=O)=O)C=C2S1 IIQFOVQMXGABDP-UHFFFAOYSA-N 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 101800000068 Antioxidant peptide Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- OKQLXOYFUPVEHI-CIUDSAMLSA-N Gln-Ser-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N OKQLXOYFUPVEHI-CIUDSAMLSA-N 0.000 description 1
- SOEATRRYCIPEHA-BQBZGAKWSA-N Gly-Glu-Glu Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SOEATRRYCIPEHA-BQBZGAKWSA-N 0.000 description 1
- TVUWMSBGMVAHSJ-KBPBESRZSA-N Gly-Leu-Phe Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 TVUWMSBGMVAHSJ-KBPBESRZSA-N 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- ZNAPAUSAUBHENO-IHPCNDPISA-N Lys-Trp-His Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)NC(=O)[C@H](CCCCN)N ZNAPAUSAUBHENO-IHPCNDPISA-N 0.000 description 1
- 241000517769 Odorrana Species 0.000 description 1
- 206010053159 Organ failure Diseases 0.000 description 1
- OLHDPZMYUSBGDE-GUBZILKMSA-N Pro-Arg-Cys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)O OLHDPZMYUSBGDE-GUBZILKMSA-N 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 108010066198 glycyl-leucyl-phenylalanine Proteins 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000003227 neuromodulating effect Effects 0.000 description 1
- 239000002581 neurotoxin Substances 0.000 description 1
- 231100000618 neurotoxin Toxicity 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 230000037390 scarring Effects 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000037070 skin defense Effects 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 230000002618 waking effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Dermatology (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- Birds (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Polymers & Plastics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses an antioxidant polypeptide OA-GL17 for promoting skin wound repair and a preparation method and application thereof. The amino acid sequence of the antioxidant polypeptide OA-GL17 for promoting skin wound repair is GLFKWHPRCGEEQSMWT. The antioxidant polypeptide OA-GL17 for promoting skin wound repair, which is obtained by purification, shows stronger activity in-vitro antioxidant activity detection, wound repair cells and animal model experiments, and the antioxidant polypeptide OA-GL17 for promoting skin wound repair disclosed by the invention has potential application prospects. The invention has the beneficial effects that: the antioxidant polypeptide OA-GL17 for promoting skin wound repair has the beneficial characteristics of simple structure and strong activity for promoting wound repair.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an antioxidant polypeptide OA-GL17 for promoting skin wound repair and a preparation method and application thereof.
Background
As the largest organ of the human body, the skin can regulate body temperature and sense the surrounding environment. It can protect internal tissues and organs from physical and chemical damage, as well as from pathogenic microorganisms. The process of wound repair of the skin is complex and time consuming, and any disturbance of the repair process can exacerbate the condition and delay wound healing. Refractory wounds can also lead to pathogen invasion, resulting in secondary infection, water and electrolyte disorders, infectious shock, organ failure and even death. To improve the healing process and reduce the complications that arise during wound healing, it is important to promote wound healing effectively and quickly. At present, several known wound repair medicines have certain defects, including poor stability, low activity, high storage cost, scar hyperplasia and the like. Therefore, the bioactive peptide with multiple functions, high activity, high stability and specific characteristics has high exploration value.
Amphibians, a species with naked skin and complex habitat, are vulnerable to external damage, and thus they form a protective skin defense system. Previous studies on secretions from amphibians have identified a wide variety of functionally diverse bioactive peptides, including antimicrobial peptides, antioxidant peptides, wound healing peptides, cytokines, neuromodulatory peptides, and neurotoxins, among others. It is noteworthy that the skin of amphibians heals rapidly after damage with little scarring, indicating the presence of certain process substances in the skin secretions of amphibians that may shorten wound healing.
A novel peptide, OA-GL17, is separated from skin secretion of Rana yunnanensis has high antioxidant activity, can effectively promote wound healing in vivo and in vitro, and has high stability and wound repairing activity.
Disclosure of Invention
The first aim of the invention is to provide an antioxidant polypeptide OA-GL17 for promoting skin wound repair; the second purpose is to provide a preparation method of the antioxidant polypeptide OA-GL17 for promoting skin wound repair; the third purpose is to provide the application of the antioxidant polypeptide OA-GL17 for promoting skin wound repair.
The first object of the invention is realized by that the amino acid sequence of the antioxidant polypeptide OA-GL17 for promoting skin wound repair is GLFKWHPRCGEEQSMWT.
The second object of the present invention is achieved by comprising the steps of:
A. dissolving the skin secretion of Rana grahami in PBS by electric stimulation, and vacuum freeze drying to obtain material a, and storing at-80 deg.C;
B. dissolving the material a in water to obtain a material b, eluting the material b by using a Tris-HCl buffer solution, and collecting an eluent to obtain a material c;
C. performing high performance liquid chromatography reverse phase chromatography on the material c for the first time to obtain a material d;
D. and performing high performance liquid chromatography reverse phase chromatography on the material d for the second time to obtain the target substance antioxidant polypeptide OA-GL17 for promoting skin wound repair.
The third purpose of the invention is realized by the application of the antioxidant polypeptide OA-GL17 for promoting skin wound repair in the preparation of health-care food, cosmetics or medicines for promoting skin wound repair.
The invention separates a novel peptide OA-GL17 from skin secretion of rana grahami, which has higher antioxidant activity, can effectively promote the healing of wounds in vivo and in vitro, and has higher stability and activity of promoting wound repair. The OA-GL17 has the characteristics of simple structure and activity of promoting wound repair, and can be obtained by separating and purifying the skin secretion of rana grahami. The antioxidant polypeptide OA-GL17 for promoting skin wound repair provided by the invention can be prepared from odorrana yunnanensis skin secretion by a biochemical separation and purification method. The antioxidant polypeptide OA-GL17 for promoting skin wound repair prepared by the method can be identified by mass spectrum and activity for promoting wound repair, and the antioxidant polypeptide OA-GL17 for promoting skin wound repair can be prepared by a conventional method in the field by adopting a chemical synthesis or gene expression mode. The antioxidant polypeptide OA-GL17 for promoting skin wound repair, which is obtained by purification, shows stronger activity in-vitro antioxidant activity detection, wound repair cells and animal model experiments, and the antioxidant polypeptide OA-GL17 for promoting skin wound repair disclosed by the invention has potential application prospects. The invention has the beneficial effects that: the antioxidant polypeptide OA-GL17 for promoting skin wound repair has the beneficial characteristics of simple structure and strong activity for promoting wound repair.
Drawings
FIG. 1 is HPLC reversed phase C of the antioxidant polypeptide OA-GL17 for promoting skin wound repair of the present invention18Column chromatography graph;
FIG. 2 is a graph showing the antioxidant activity of the antioxidant polypeptide OA-GL17 for promoting skin wound repair according to the present invention;
FIG. 3 is a graph of the cell model scratch repair promoting activity of the isolated and purified antioxidant polypeptide OA-GL17 for promoting skin wound repair according to the present invention;
FIG. 4 is a diagram of the wound repair promoting activity of an animal model for isolating and purifying the antioxidant polypeptide OA-GL17 for promoting skin wound repair.
Detailed Description
The present invention is further illustrated by the following examples and the accompanying drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
The amino acid sequence of the antioxidant polypeptide OA-GL17 for promoting skin wound repair is GLFKWHPRCGEEQSMWT.
The preparation method of the antioxidant polypeptide OA-GL17 for promoting skin wound repair, disclosed by the invention, comprises the following steps of:
A. dissolving the skin secretion of Rana yunnanensis with PBS by electric stimulation, and vacuum freeze drying to obtain material a, and storing at-80 deg.C;
B. dissolving the material a in water to obtain a material b, eluting the material b with a Tris-HCl buffer solution, and collecting an eluent to obtain a material c;
C. performing high performance liquid chromatography reverse phase chromatography on the material c for the first time to obtain a material d;
D. and (3) performing high performance liquid chromatography reverse phase chromatography on the material d for the second time to obtain the target antioxidant polypeptide OA-GL17 for promoting skin wound repair.
And the water in the step B is deionized water.
And the elution in the step B is to take the material B with the protein content of 80-150 mg to a Sephadex G75 column which is balanced by 20mM Tris-HCl buffer solution for 24h in advance, and then elute by using Tris-HCl buffer solution.
The flow rate of elution is 1.5ml/10 min.
The pH value of the Tris-HCl buffer solution is 7.8, and the Tris-HCl buffer solution contains 0.1M NaCl.
The first high performance liquid chromatography reverse phase chromatography is to load the material c into a Hypersil ODS 25 mm column which is pre-balanced by ultrapure water containing 0.1% trifluoroacetic acid, and elute the material c under the condition of linear gradient by acetonitrile containing 0.1% trifluoroacetic acid at the flow rate of 0.5-1.5 ml/min, wherein the detection wavelength is 215 nm.
And the second high performance liquid chromatography reverse phase chromatography is to dissolve the material d in deionized water after vacuum freeze drying, and then repeat the first high performance liquid chromatography reverse phase chromatography process.
The application of the antioxidant polypeptide OA-GL17 for promoting skin wound repair is the application of the antioxidant polypeptide OA-GL17 for promoting skin wound repair in preparation of health-care food, cosmetics or medicines for promoting skin wound repair.
The invention is further illustrated by the following specific examples:
example 1
Separation, purification and identification of antioxidant polypeptide OA-GL17 for promoting skin wound repair
1. Separating and purifying
The living Rana yunnanensis is collected from Yunnan Baoshan, and its skin secretion (dissolved in PBS) is obtained by electric stimulation, vacuum freeze-dried, and stored at-80 deg.C for use.
The first step is as follows: sephadex G75 molecular sieve: lyophilized powder of skin secretion obtained as described above was dissolved in deionized water, and 1ml (protein content: 100 mg) of Sephadex G75 (GE Healthcare, ultra-fine) column (length 40 cm, inner diameter width 1.5 cm) equilibrated in advance with 20mM Tris-HCl buffer (pH =7.8, containing 0.1M NaCl) for 24 hours was eluted with the same buffer at a flow rate of 1.5ml/10min and collected once every 10 min. And (3) measuring the wound repair promoting activity of the collected sample by using a HaCaT cell scratch experiment, and further purifying the sample with stronger activity by using HPLC (high performance liquid chromatography).
The second step is that: first high performance liquid chromatography reverse phase chromatography:
the sample obtained in the first step was applied to Hypersil ODS 25 mm column (Uliprit product, size 4.6 mm. times.300 mm) equilibrated in advance with ultrapure water (containing 0.1% trifluoroacetic acid), the experimental apparatus was a Waters 1525 high pressure liquid phase system, elution was carried out with acetonitrile (containing 0.1% trifluoroacetic acid) under a linear gradient (0-100%, 100 min) at a flow rate of 1 ml/min, the monitoring wavelength was 215nm, the separation and purification pattern obtained was as shown in FIG. 1, the arrow indicates a peak having a wound repair promoting activity, and OA-GL17 shows a peak.
The third step: and (3) performing reverse phase chromatography by high performance liquid chromatography for the second time:
the first peak having the activity of promoting wound repair was collected, lyophilized in vacuo and dissolved in deionized water, and the first HPLC procedure was repeated, and the resulting isolation and purification pattern was shown in FIG. 1, with the arrow indicating the peak of purified OA-GL 17.
2. Molecular identification
Determination of amino acid sequence:
the purified antioxidant polypeptide OA-GL17 for promoting skin wound repair is subjected to Edman degradation on a full-automatic protein sequencer (Shimadzu PPSQ-31A) to determine the complete amino acid sequence of the antioxidant polypeptide OA-GL17 for promoting skin wound repair, and the result shows that the antioxidant polypeptide OA-GL17 for promoting skin wound repair has the amino acid sequence shown in the invention: GLFKWHPRCGEEQSMWT are provided.
Example 2
Activity identification of antioxidant polypeptide OA-GL17 for promoting skin wound repair
1. In vitro antioxidant activity identification
The antioxidant activity of the antioxidant polypeptide OA-GL17 for promoting skin wound repair is detected by an in-vitro free radical scavenging activity detection experiment, and the result shows that the antioxidant polypeptide OA-GL17 for promoting skin wound repair has stronger antioxidant activity.
Antioxidant activity was measured by the 2, 2' -azobis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS) and 2, 2-diphenyl-1-picrylhydrazino (DPPH) scavenging activity of OA-GL 17. Incubating 2.8 mM potassium persulfate and 7 mM ABTS in water under darkroom conditions for at least 6 hr to prepare ABTS+Free radical stock solution, and used immediately. The stock solution was diluted 50-fold with double distilled water. Will ddH2The OA-GL17 dissolved in O was added to the diluted stock solution, and the same volume of solvent was used as a blank control and vitamin C (vc) was used as a positive control. The reaction was carried out in the dark for 30 minutes, and the absorbance at 415 nm was measured,
ABTS+radical clearance (%) = (a)blank-Asample)×100/AblankThe results are shown in FIG. 2.
At the same time, DPPH free radical is dissolved in methanol to 5X 10−5And (4) storing mother liquor of M at 4 ℃ in a dark place for later use. Mu.l of DPPH+The mother liquor was mixed with 10. mu.l of OA-GL17 solution and placed in a 96-well plate, incubated at room temperature for 30 minutes in the dark, and then the absorbance of the mixture at 517 nm was measured and recorded. Equal concentration of vitamin C as a positive control, equal volume of sample solvent (ddH)2O) as negative control and an equal volume of methanol as blank control, DPPH radical scavenging activity (%) = (A)blank-Asample)×100/AblankThe results are shown in FIG. 2.
2. In vitro cell scratch repair activity promoting identification
HaCaT cell scratch healing experiment is adopted to detect cell scratch repair promoting capability of OA-GL17. The cells were cultured in DMEM medium with 10% fetal bovine serum and 1% diabody (penicillin-streptomycin, 100U/ml) and incubated in a culture environment containing 5% carbon dioxide (37 ℃). HaCaT cells (2.5X 10) were cultured in 24-well plates5One/well), serum starved culture for 12-24 h, and mechanical scratching of the cells was made on the formed cell monolayer using the tip of a sterile pipette. The floating cells were removed by gentle washing twice with PBS, and then serum-free medium (500. mu.l/well) containing OA-GL17 at a certain concentration was added for further 24 hours. The same volume of serum-free medium without OA-GL17 was also used as a negative control. Images of cell scratch repair were taken at different time intervals using an inverted microscope. The repair of the cells was calculated using Image J software. The cell scratch repair promoting activity of the antioxidant polypeptide OA-GL17 for promoting skin wound repair is shown in FIG. 3.
3. Identification of wound healing promoting activity of in vivo animal model
SPF-grade Kunming mice weighing 22-25g were selected for the experiment and randomly divided into two groups of 10 mice each. Mice were first anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.1 ml/20 g), then the mice were shaved off of their back hairs and sterilized with 75% alcohol, and finally two holes of symmetrical size, about 8mm x 8mm, were punched on both sides of the mice back with a hole-drilling machine. After operation, the mice are placed beside the heater and placed back to the breeding room for normal breeding after waking up. Twice daily, about 20. mu.l per well, first group of mice were coated with saline on the left back and OA-GL17 on the right; the second group of mice was coated with a new healing patch, OA-GL17 on the left side of their backs. The back wounds of the mice were photographed every 2 days and finally the wound repair rate was calculated by Image J software. The promoter model wound repair activity of the antioxidant polypeptide OA-GL17 for promoting skin wound repair is shown in FIG. 4.
SEQUENCE LISTING
<110> university of Kunming medical science
<120> antioxidant polypeptide OA-GL17 for promoting skin wound repair and preparation method and application thereof
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> PRT
<213> antioxidant polypeptide OA-GL17 for promoting skin wound repair and preparation method and application thereof
<400> 1
Gly Leu Phe Lys Trp His Pro Arg Cys Gly Glu Glu Gln Ser Met Trp
1 5 10 15
Thr
Claims (2)
1. An antioxidant polypeptide OA-GL17 for promoting skin wound repair, which is characterized in that the amino acid sequence of the antioxidant polypeptide OA-GL17 for promoting skin wound repair is GLFKWHPRCGEEQSMWT.
2. The application of the anti-oxidant polypeptide OA-GL17 for promoting skin wound repair of claim 1, wherein the anti-oxidant polypeptide OA-GL17 for promoting skin wound repair is used for preparing cosmetics or medicines for promoting skin wound repair.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910966274.9A CN110606872B (en) | 2019-10-12 | 2019-10-12 | Antioxidant polypeptide OA-GL17 for promoting skin wound repair and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910966274.9A CN110606872B (en) | 2019-10-12 | 2019-10-12 | Antioxidant polypeptide OA-GL17 for promoting skin wound repair and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110606872A CN110606872A (en) | 2019-12-24 |
| CN110606872B true CN110606872B (en) | 2022-06-21 |
Family
ID=68894545
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910966274.9A Active CN110606872B (en) | 2019-10-12 | 2019-10-12 | Antioxidant polypeptide OA-GL17 for promoting skin wound repair and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110606872B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111606973B (en) * | 2020-05-21 | 2022-03-18 | 昆明医科大学 | Anti-gout active polypeptide RDP3, and preparation method and application thereof |
| CN118221777A (en) * | 2020-08-26 | 2024-06-21 | 四川好医生攀西药业有限责任公司 | Polypeptide for repairing skin wound or mucous membrane injury and application thereof |
| CN112316110B (en) * | 2020-11-12 | 2023-06-23 | 温州大学 | Pharmaceutical preparation for promoting skin wound repair and preparation method thereof |
| CN112574282B (en) * | 2020-11-16 | 2022-03-04 | 昆明学院 | Antioxidant damage skin protection active polypeptide RL-PP10 and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107344959A (en) * | 2017-06-29 | 2017-11-14 | 常州市千康生物科技有限公司 | One kind promotees skin repair ultrashort peptide Purin WH, its preparation method and application |
| CN108484747A (en) * | 2018-03-22 | 2018-09-04 | 昆明医科大学 | Japan frog skin repair peptide cathelicidin-NV and its gene and application |
| CN113698452A (en) * | 2021-08-23 | 2021-11-26 | 四川丽妍工坊生物科技有限公司 | Skin repair promoting peptide, preparation method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AUPQ645700A0 (en) * | 2000-03-24 | 2000-04-20 | University Of Queensland, The | Novel polypeptides and polynucleotides and methods of using them |
| US20040038230A1 (en) * | 2001-11-05 | 2004-02-26 | Alsobrook John P. | Therapeutic polypeptides, nucleic acids encoding same, and methods of use |
| PL2289535T3 (en) * | 2004-12-21 | 2018-12-31 | Musc Foundation For Research Development | Compositions and methods for promoting wound healing and tissue regeneration |
-
2019
- 2019-10-12 CN CN201910966274.9A patent/CN110606872B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107344959A (en) * | 2017-06-29 | 2017-11-14 | 常州市千康生物科技有限公司 | One kind promotees skin repair ultrashort peptide Purin WH, its preparation method and application |
| CN108484747A (en) * | 2018-03-22 | 2018-09-04 | 昆明医科大学 | Japan frog skin repair peptide cathelicidin-NV and its gene and application |
| CN113698452A (en) * | 2021-08-23 | 2021-11-26 | 四川丽妍工坊生物科技有限公司 | Skin repair promoting peptide, preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| A small peptide with potential ability to promote wound healing;Jing Tang等;《PLOS ONE》;20140319;第9卷(第3期);1-10 * |
| 抗氧化肽AOP1对小鼠皮肤烫伤创面愈合修复的影响;秦迪等;《中国药理学通报》;20181231;第34卷(第2期);225-231 * |
| 林蛙皮肤多肽对大鼠皮肤激光损失的修复作用;吴琼等;《吉林农业大学学报》;20151231;第5卷(第5期);617-621 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110606872A (en) | 2019-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110606869B (en) | Peptide OA-GP11 for promoting skin healing and preparation method and application thereof | |
| CN110606872B (en) | Antioxidant polypeptide OA-GL17 for promoting skin wound repair and preparation method and application thereof | |
| CN110559476B (en) | Liquid band-aid containing peptide anti-inflammatory active ingredients and preparation method thereof | |
| CN107619437B (en) | A kind of skin ultrastructure peptide WHPP-OA1 and its method of purification and application | |
| AU2014278708B2 (en) | Tetrapeptides derived from human C-X-C chemokines useful for treatment of various skin conditions | |
| Guo et al. | Identification of multiple peptides with antioxidant and antimicrobial activities from skin and its secretions of Hylarana taipehensis, Amolops lifanensis, and Amolops granulosus | |
| CN116970071A (en) | Recombinant elastin with anti-aging activity and preparation method and application thereof | |
| CN111423495B (en) | Rapana venosa polypeptide with oxidative stress damage resistance and preparation method and application thereof | |
| CN110590913A (en) | Antioxidant damage skin protection active polypeptide AOP-P1 and preparation method and application thereof | |
| EP2125878B1 (en) | Short bio-active peptides for cellular and immunological modulation | |
| CN110590912A (en) | Novel antioxidant active polypeptide OA-VI12 as well as preparation method and application thereof | |
| KR101625502B1 (en) | A New Scolopendrasin-4 peptide for promoting growth of large intestine epithelial cell and its synthetic composition | |
| CN114989248B (en) | Clinopodium polycephalum polypeptide with anti-inflammatory and antioxidant activities, and preparation method and application thereof | |
| US20230112511A1 (en) | Liquid bandage containing peptide anti-inflammatory active ingredients and preparation method thereof | |
| CN104945490B (en) | Plant alexin polypeptide of separation and preparation method thereof and the purposes in lung cancer is treated | |
| CN114957394B (en) | Polypeptide PM-7 for promoting skin repair and application thereof | |
| CN114716515A (en) | Polypeptide analogue and preparation method and application thereof | |
| TWI602569B (en) | Polysaccharide extract of microalgae and extraction method thereof | |
| CN115724907A (en) | Dictyophora rubrovalvata polypeptide and application thereof | |
| CN117338856B (en) | Preparation method of lycium ruthenicum anthocyanin composition, product and application of lycium ruthenicum anthocyanin composition | |
| CN112457375B (en) | Polypeptide compound and preparation method and application thereof | |
| CN114053165B (en) | Use of biologically active substances for producing a composition for improving the condition of the skin | |
| CN110183528A (en) | Antibacterial peptide and its purposes for pharmacy and beauty | |
| TW200825100A (en) | Protein and nucleic acid for glutathione-dependent formaldehyde dehydrogenase (GFD), alcohol dehydrogenase and S-nitrosoglutathione reductase from Antrodia camphorata, manufacturing method and uses therefor | |
| CN118027152A (en) | Polypeptide with anti-inflammatory biological activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220531 Address after: 519000 floor 2, plant 1, No. 756, xiangtian Road, Jinwan District, Zhuhai City, Guangdong Province Applicant after: ZHUHAI YASHA MEDICAL DEVICES Co.,Ltd. Address before: 710016 B 1616, Tiandi Times Plaza, Fengcheng two road, Weiyang District, Xi'an, Shaanxi. Applicant before: Liu Jiaojiao Effective date of registration: 20220531 Address after: 710016 B 1616, Tiandi Times Plaza, Fengcheng two road, Weiyang District, Xi'an, Shaanxi. Applicant after: Liu Jiaojiao Address before: Kunming Medical University, 1168 Chunrong West Road, Chenggong District, Kunming, Yunnan 650500 Applicant before: KUNMING MEDICAL University |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |