CN107266530A - Anti-oxidant ultrashort peptide, preparation method and applications - Google Patents
Anti-oxidant ultrashort peptide, preparation method and applications Download PDFInfo
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- CN107266530A CN107266530A CN201710529190.XA CN201710529190A CN107266530A CN 107266530 A CN107266530 A CN 107266530A CN 201710529190 A CN201710529190 A CN 201710529190A CN 107266530 A CN107266530 A CN 107266530A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 230000003078 antioxidant effect Effects 0.000 title claims description 21
- 239000003963 antioxidant agent Substances 0.000 title claims description 17
- 235000006708 antioxidants Nutrition 0.000 title claims description 17
- 230000003064 anti-oxidating effect Effects 0.000 claims abstract description 22
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 8
- 239000003814 drug Substances 0.000 claims abstract description 7
- 239000002537 cosmetic Substances 0.000 claims abstract description 5
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 2
- 238000011033 desalting Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 7
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- 235000013373 food additive Nutrition 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 230000002000 scavenging effect Effects 0.000 abstract description 2
- 231100001083 no cytotoxicity Toxicity 0.000 abstract 1
- 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 8
- 210000004027 cell Anatomy 0.000 description 8
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 7
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 150000003254 radicals Chemical class 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 4
- 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
- 108010024636 Glutathione Proteins 0.000 description 4
- 239000000490 cosmetic additive Substances 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 150000002978 peroxides Chemical class 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
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- 238000000034 method Methods 0.000 description 3
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- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-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
- -1 Oxygen anion Chemical class 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
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- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
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- 235000016709 nutrition Nutrition 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
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- 150000005839 radical cations Chemical class 0.000 description 2
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- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 206010014561 Emphysema Diseases 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 229910002567 K2S2O8 Inorganic materials 0.000 description 1
- 208000003351 Melanosis Diseases 0.000 description 1
- ZSEMWHCVIJETNE-UHFFFAOYSA-N N1=CC=CC2=CC(=C3C=CC=NC3=C12)S(=O)(=O)O.C(C)N1CSC2=C1C=CC=C2 Chemical compound N1=CC=CC2=CC(=C3C=CC=NC3=C12)S(=O)(=O)O.C(C)N1CSC2=C1C=CC=C2 ZSEMWHCVIJETNE-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
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- 230000032683 aging Effects 0.000 description 1
- 230000003679 aging effect Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
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- 206010003246 arthritis Diseases 0.000 description 1
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- 230000036952 cancer formation Effects 0.000 description 1
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- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000010612 desalination reaction 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
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- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
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- 231100000350 mutagenesis Toxicity 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
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- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical class [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 235000019394 potassium persulphate Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000012599 radical scavenging assay Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Classifications
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- 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
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- 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
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Polymers & Plastics (AREA)
- Dermatology (AREA)
- Birds (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Nutrition Science (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Cosmetics (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to the ultrashort peptide of antioxidizing, preparation method and applications, belong to field of biomedicine technology.Described ultrashort peptide is the ultrashort peptide of straight chain, and the molecular weight of Ansin 3 is 971.16Da, and isoelectric point is 9.00, and containing 8 amino acid residues, complete sequence primary structure is：Thr‑Ser‑Arg‑Cys‑Phe‑Arg‑Val‑Cys；The molecular weight of Magin 3 is 1017.23Da, and isoelectric point is 9.02, and containing 8 amino acid residues, complete sequence primary structure is：Phe‑Phe‑Arg‑Ser‑Cys‑Arg‑Val‑Cys；After chemical synthesis, the two shows extremely strong antioxidation activity, extremely strong Scavenging ability, there is no cytotoxicity, and molecular weight is small, simple in construction, extremely low using the cost of chemical synthesis process, may be used as preparing anti-oxidation medicine, food additives, antioxidation cosmetic product new raw material.
Description
Technical field
The present invention relates to the ultrashort peptide of antioxidizing, preparation method and applications, belong to field of biomedicine technology.
Background technology
The free radical (free radical) that body oxidation reaction is produced is the atomic group containing a unpaired electron, such as super
Oxygen anion, hydroxy radical etc..Free radical has strong oxidizing property, with age or under pathological state, and free radical is produced
If can not excessively be removed in time, it will accumulate in the cell, with the large biological molecule in body, such as protein, nucleic acid, lipid
Deng interaction, excessive oxide and peroxide is generated, body metabolism is finally influenceed, and body is produced irreversible
Damage.Aging and many chronic diseases are such as cancer, angiocardiopathy, pulmonary emphysema, hepatic sclerosis, arthritis etc. all with free radical
Damage is relevant.Although body has Antioxidative Defense System, it completely effectively can not resist or repair oxidation band
The damage come.How appropriate removing free radical, it is maintained a relatively low level in vivo, so as to delay body to decline
Always, it is to work as previous important research topic.
Polyphenoils rises in the application of cosmetic field in recent years.Because the light aging effect of skin is due to ultraviolet
Line inducing skin cells produce substantial amounts of oxygen radical accumulation and caused.Antioxidant is added in present many skin care item, is had
Reduce freckle, radiation proof, anti-aging, immune and raising skin self-protection the effect of regulation.In addition nutritional ingredient in food
Oxidation reaction can produce peroxide.Peroxide influences nutritive value of food, or even can cause disease, finds peace
Full antioxidant with suppress peroxide produce be always biochemical nutrition study hotspot.
Now widely used antioxidant is generally synthetics, such as BHA, BHT, though antioxidant effect is good,
There is accumulative carcinogenesis to human liver, spleen, lung.Clinically common antioxidant such as vitamin C, vitamin E and gluathione
Peptide etc..Vitamin C is main to remove based on OH, but need it is heavy dose of using can be only achieved purpose, but simultaneously, megavitamin
C is using being easily caused acid poisoning；Vitamin E mainly reaches oxidation resistant mesh by suppressing the lipid peroxidation on cell membrane
, but be with then there is potential mutagenesis risk for a long time.And the industrialization barrier of clinical reduced glutathione is higher, by day
This Kyowa Hakkokogyo Co., Ltd almost monopolizes the supply of global glutathione, and per kilogram is up to 8000 U.S. dollars.Meanwhile, now
Glutathione pharmaceutical raw material used in domestic pharmacy corporation can't break away from import, and glutathione price remains high always.Can
See, activity is good, be easily-synthesized, the novel oxidation-resistant peptide that yield is high is urgently developed in the fields such as medical treatment, food, cosmetics.
Small molecule ultrashort peptide Ansin-3, Magin-3, with extremely strong antioxidation activity, and acellular poison, stability are high.
Ansin-3, Magin-3 primary sequence total length only have 8 amino acid residues, molecular weight it is small, simple in construction, without protein modified, lead to
Chemical synthesis process preparation is crossed, technique is easy, yield is high and purity is high.Therefore, the present invention in two kinds of ultrashort peptide Ansin-3,
Magin-3 has very prominent beneficial effect.
The content of the invention
It is an object of the invention to provide the ultrashort peptide of antioxidizing, preparation method and applications, including ultrashort peptide
Ansin-3 and ultrashort peptide Magin-3 and its preparing the application of anti-oxidation medicine, food, cosmetic additive agent.
Technical scheme：
The ultrashort peptide of antioxidizing, including ultrashort peptide Ansin-3 and ultrashort peptide Magin-3；
(1) ultrashort peptide Ansin-3：A kind of straight chain small peptide, molecular weight is 971.76Da, and isoelectric point is 9.00, contains 8 ammonia
Base acid residue, the primary structure of complete sequence is:Thr-Ser-Arg-Cys-Phe-Arg-Val-Cys；
(2) ultrashort peptide Magin-3：A kind of straight chain small peptide, molecular weight is 1017.23Da, and isoelectric point is 9.02, contains 8
Amino acid residue, the primary structure of complete sequence is:Phe-Phe-Arg-Ser-Cys-Arg-Val-Cys.
The preparation method of the ultrashort peptide of antioxidizing, first, according to the amino acid sequence of ultrashort peptide, uses automatic Peptide systhesis
Instrument synthesizes the complete sequence of ultrashort peptide；Again by HPLC reversed phase column chromatography desalting and purifyings, and determine that its purity is more than 95%；Finally,
The molecular weight of ultrashort peptide is determined with MALDI-TOF-MS.
The ultrashort peptide of antioxidizing is used to prepare anti-oxidation medicine, food additives, antioxidation cosmetic as main component
Product.
Beneficial effects of the present invention：Two kinds of anti-oxidant ultrashort peptide Ansin-3, Magin-3 that the present invention is provided are by chemistry
The method of synthesis is obtained.Two kinds of ultrashort peptides are respectively provided with extremely strong antioxidation activity, and concentration is that DPPH free radical scavenging activities I% is up to
97.35%, to ABTS·+Radical cation clearance rate I% can reach 84.41%；And two kinds of anti-oxidation peptide Ansin-3,
Magin-3 molecular weight is small, simple in construction, cytotoxicity is extremely low, preparation method is simple etc., therefore is applied in antioxidant drug
In thing, food or cosmetic additive agent, the antioxidant effect of medicine, food or cosmetic additive agent can be improved, with very
Good application prospect.
Brief description of the drawings
Fig. 1 is the toxicity figure of Ansin-3, Magin-3 to B16 cells.
In figure：
Embodiment
Below in conjunction with accompanying drawing and technical scheme, the embodiment of the present invention is further illustrated.
Embodiment 1
Two kinds of anti-oxidant ultrashort peptide Ansin-3, Magin-3 chemical synthesis：
(1) two kind of anti-oxidant ultrashort peptide Ansin-3, Magin-3 chemical synthesis process：According to ultrashort peptide ammino acid sequence
Row, the complete sequence of the two is synthesized with automatic Peptide synthesizer (433A, Applied Biosystems), passes through HPLC reversed-phase columns layer
Analyse desalination.
(2) molecular weight determination uses MALDI-TOF-MS (MALDI-TOF).
(3) two kinds of anti-oxidant ultrashort peptide Ansin-3, Magin-3 of purifying identify it with high-efficient liquid phase chromatogram HPLC method
Purity, molecular weight determination uses MALDI-TOF-MS (MALDI-TOF), and isoelectric focusing electrophoresis is surveyed
Determine isoelectric point, amino acid sequence structure is determined with automatic Protein Sequencer.
Two kinds of anti-oxidant ultrashort peptide Ansin-3, Magin-3 are the ultrashort peptides of straight chain, and wherein Ansin-3 molecular weight is
971.16Da, isoelectric point is 9.00, and containing 8 amino acid residues, the primary structure of complete sequence is：Thr-Ser-Arg-Cys-
Phe-Arg-Val-Cys；Magin-3 molecular weight is 1017.23Da, and isoelectric point is 9.02, contains 8 amino acid residues, total order
The primary structure of row is：Phe-Phe-Arg-Ser-Cys-Arg-Val-Cys；
Embodiment 2
Two kinds of anti-oxidant ultrashort peptide Ansin-3, Magin-3 Antioxidative Activity Determinations：
(1) DPPH free radical scavenging activities (DPPH radical scavenging assay)
A certain amount of DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate, Sigma, the U.S.) is weighed, is used
Methanol dissolves, and is made into 6 × 10-5M solution, it is now with the current.48 μ l DPPH solution and 2 μ l samples (2mg/ml) are mixed (final
Sample and DPPH mass ratio are 3:1), lucifuge stands 30min at room temperature, and light absorption value is determined at 517nm.Blank control group with
Sample dissolving medium replaces testing sample.Experiment do three it is parallel, ultraviolet specrophotometer return to zero when use methanol.
DPPH clearance rates (%)=(AB-AA)/A B × 100 (AB:Blank control group light absorption value；AA:Sample sets extinction
Value).
(2)ABTS·+Radical cation scavenging capacity
ABTS (3-ethylbezothiazoline-6-sulfonic acid) (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid)
2mM ABTS storing liquids are made into PBS (pH7.4).By ABTS storing liquids and 70mM potassium peroxydisulfates (K2S2O8) aqueous solution
By volume 250:1 mixing, in room temperature avoid light place 15-16h.Before on-test, by ABTS·+Release to the suction at 734nm wavelength
Light value is 0.80 ± 0.03.By 4 μ l not sample and the above-mentioned corrected ABTS of 96 μ l·+Solution is mixed, and room temperature is placed after 10min,
The light absorption value of reaction solution is detected at 734nm wavelength.Blank control group is sterile deionized water used in sample dissolution.Experiment does three
It is individual parallel.
ABTS·+Clearance rate I (%)=(AB-AA)/AB × 100 (AB:Blank control group light absorption value；AA:Sample sets extinction
Value).
(3) reducing power is determined
10 μ l samples (2mg/ml) and 50 μ l sodium phosphate buffers and 50 μ l 1%K3Fe(CN)6Mixing, 50 DEG C of water-baths
20min；Then 50 μ l 10% trichloroacetic acid, 3000rpm centrifugations 10min are added；The μ l of supernatant 50 are taken, 50 μ l deionizations are added
Water and 10 μ l 1%FeCl3.Absorbed in 700nm light-meterings.Light absorbs more strong representation reducing power is stronger.
Table 1.Ansin-3, Magin-3 antioxidation activity in vitro
As a result as shown in table 1, Ansin-3, Magin-3 have extremely strong antioxidation activity.
Embodiment 3
Ansin-3, Magin-3 CTA：
The cytotoxicity of sample Ansin-3, Magin-3 to mouse melanoma B16 is detected with mtt assay.
First cell is trained in the DMEM containing 10% hyclone and dual anti-(penicillin and each 100U/ml of streptomysin)
Support, after cell is covered with, got off with 0.25% Trypsin Induced, washed twice with above-mentioned culture medium, again suspension cell, carefully
The μ l of cell suspending liquid 100 are added in 96 porocyte culture plates by born of the same parents after counting, and it is 103 to make every hole cell number.Treat thin overnight
After born of the same parents are adherent, culture medium is suctioned out, the complete medium of the sample containing various concentrations is added, control group adds the complete training of same volume
Base is supported, 37 DEG C, 5%CO is put2Culture 72h (changing liquid halfway once) in incubator.After culture terminates, 96 porocyte culture plates are per hole
20 μ l 5mg/ml MTT solution (being prepared with PBS) are added, continues to cultivate after 4h, suctions out liquid in hole, added per hole
150 μ l DMSO, shaking table vibration 10min.ELIASA detects light absorbs, and measure wavelength is 490nm, reference wavelength 630nm.
As a result as shown in figure 1, as a result showing, cell survival when sample Ansin-3, Magin-3 concentration are 160 μ g/ml
Rate still may be up to 90%, illustrate that sample Ansin-3, Magin-3 do not have cytotoxicity, therefore be very beneficial to it in antioxidant drug
Thing or food, cosmetic additive agent field are further developed.
Claims (3)
1. the ultrashort peptide of antioxidizing, including two kinds of anti-oxidant ultrashort peptide Ansin-3 and Magin-3, it is characterised in that：
Described anti-oxidant ultrashort peptide Ansin-3：A kind of straight chain small peptide, molecular weight is 971.76Da, and isoelectric point is 9.00, is contained
8 amino acid residues, the primary structure of complete sequence is:Thr-Ser-Arg-Cys-Phe-Arg-Val-Cys；
Described anti-oxidant ultrashort peptide Magin-3：A kind of straight chain small peptide, molecular weight is 1017.23Da, and isoelectric point is 9.02, is contained
There are 8 amino acid residues, the primary structure of complete sequence is:Phe-Phe-Arg-Ser-Cys-Arg-Val-Cys.
2. the preparation method of the ultrashort peptide of antioxidizing described in claim 1, it is characterised in that first, according to ultrashort peptide
Amino acid sequence, the complete sequence of ultrashort peptide is synthesized with automatic Peptide synthesizer；Again by HPLC reversed phase column chromatography desalting and purifyings, and
Determine that its purity is more than 95%；Finally, the molecular weight of ultrashort peptide is determined with MALDI-TOF-MS.
3. the ultrashort peptide of antioxidizing described in claim 1 is used to prepare anti-oxidation medicine, food addition as main component
Agent, antioxidation cosmetic product.
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