CN107226841A - The ultrashort peptide of antioxidizing and its application - Google Patents
The ultrashort peptide of antioxidizing and its application Download PDFInfo
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- CN107226841A CN107226841A CN201710533712.3A CN201710533712A CN107226841A CN 107226841 A CN107226841 A CN 107226841A CN 201710533712 A CN201710533712 A CN 201710533712A CN 107226841 A CN107226841 A CN 107226841A
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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
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- 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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Abstract
The invention provides the ultrashort peptide of antioxidizing and its application, belong to field of biomedicine technology.Described ultrashort peptide is the ultrashort peptide of straight chain, and Purin WH1 molecular weight is 544.71Da, and isoelectric point is 8.19, containing 5 amino acid residues, and the primary structure of complete sequence is valine valine cysteine proline lysine；Purin WH2 molecular weight is 641.83Da, and isoelectric point is 8.19, containing 6 amino acid residues, and the primary structure of complete sequence is valine valine proline proline cysteine lysine.After chemical synthesis, the two shows extremely strong antioxidation activity, compared to clinical antioxidant gluthathione, understands that free radical effect is more excellent, and without hemolytic and cytotoxicity.It may be used as preparing anti-oxidation medicine, food additives, antioxidation cosmetic product.
Description
Technical field
The present invention relates to the ultrashort peptide of antioxidizing and its application, belong to field of biomedicine technology.
Background technology
Free radical is one group of material with Strong oxdiative activity, is easily reacted.Active oxygen radical can not only make one
With protein chain type oxidation reaction, which occurs, for internal lipid causes cell membrane, tissue, enzyme and gene impairing, and damage is caused to human body
Evil and aging, and relevant gene mutation can be promoted, induce related disease, such as arteriosclerosis, hypertension, rheumatoid
Arthritis, cataract, senile dementia, Parkinson's disease and nerve retrograde affection etc..Increasing medical research and
Clinical test evidence shows that the interior free yl content more high life is shorter, and free radical is " source of ten thousand diseases ".
It is anti-oxidant in recent years very extensive in the application of cosmetic field.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 seriously can also result in disease,
The antioxidant of safety is found to suppress the study hotspot that peroxide produces always biochemical nutrition.
At present, chemical field is often generally synthetics with antioxidant, such as BHA, BHT, though antioxidant effect is good,
But there is accumulative carcinogenesis to human liver, spleen, lung.Clinically common antioxidant such as vitamin C, vitamin E and paddy Guang
Sweet 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, heavy dose dimension life
Plain 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 Purin-WH1, Purin-WH2, with extremely strong antioxidation activity, in vitro to it is a variety of from
Clinical anti-oxidation preparation --- glutathione is better than by the clearance rate of base, and acellular poison, without haemolysis, stability is high.Purin-
WH1, Purin-WH2 primary sequence total length only have 5-6 amino acid residue, 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, the clinical reduced glutathione of contrast, production cost is big
Big reduction.Therefore, two kinds of ultrashort peptide Purin-WH1, Purin-WH2 in this patent have very prominent beneficial effect.
The content of the invention
The present invention relates to the ultrashort peptide of antioxidizing, including two kinds of anti-oxidant ultrashort peptide Purin-WH1, Purin-WH2 and
It is preparing the application of anti-oxidation medicine, food, cosmetic additive agent.
The beneficial effects of the present invention are：Two kinds of anti-oxidant ultrashort peptide Purin-WH1, Purin-WH2 that the present invention is provided
The method of chemical synthesis is obtained.Wherein Purin-WH1 molecular weight is 544.71Da, and isoelectric point is 8.19, residual containing 5 amino acid
Base；Purin-WH2 molecular weight is 641.83Da, and isoelectric point is 8.19, contains 6 amino acid residues；Two kinds of ultrashort peptides are respectively provided with
Extremely strong antioxidation activity, concentration can reach 58.54%, 59.89% when being 3mM to DPPH free radical scavenging activities I%；It is right
ABTS·+Radical cation clearance rate I% is 96.02%, 72.16%.And two kinds of anti-oxidant ultrashort peptide Purin-WH1,
Purin-WH2 molecular weight is small, simple in construction, hemolytic activity is low, cytotoxicity is extremely low, preparation method is simple etc., therefore is answered
In anti-oxidation medicine, food or cosmetic additive agent, the anti-oxidant of medicine, food or cosmetic additive agent can be improved
Effect, with good application prospect.
Brief description of the drawings
Fig. 1 is the toxicity figure of ultrashort peptide Purin-WH1, Purin-WH2 to HFF-1 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 Purin-WH1, Purin-WH2 chemical synthesis：
(1) two kind of anti-oxidant ultrashort peptide Purin-WH1, Purin-WH2 chemical synthesis process：According to ultrashort peptide ammino acid
Sequence, synthesizes the complete sequence of the two with automatic Peptide synthesizer (433A, Applied Biosystems), passes through HPLC reversed-phase columns
Chromatograph desalination.
(2) molecular weight determination uses MALDI-TOF-MS (MALDI-TOF).
(3) two kinds of anti-oxidant ultrashort peptide Purin-WH1, Purin-WH2 of purifying are reflected with high-efficient liquid phase chromatogram HPLC method
Its fixed purity, molecular weight determination uses MALDI-TOF-MS (MALDI-TOF), Isoelectric Focusing
Swimming determines isoelectric point, and amino acid sequence structure is determined with automatic Protein Sequencer.
Two kinds of anti-oxidant ultrashort peptide Purin-WH1, Purin-WH2 are straight chain small peptides, and wherein Purin-WH1 molecular weight is
544.71Da, isoelectric point is 8.19, containing 5 amino acid residues, and the primary structure of complete sequence is valine-Guang of valine-half
Propylhomoserin-proline-lysine；Purin-WH2 molecular weight is 641.83Da, and isoelectric point is 8.19, containing 6 amino acid residues,
The primary structure of complete sequence is valine-VAL-PRO-PRO-cysteine-lysine；
Embodiment 2
Two kinds of anti-oxidant ultrashort peptide Purin-WH1, Purin-WH2 anti-oxidant experiment：
(1) two kind of ultrashort peptide Purin-WH1, Purin-WH2 Antioxidative Activity Determination
(1.1) 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 are mixed (final sample with
DPPH mass ratio is 3:1), lucifuge stands 30min at room temperature, and light absorption value is determined at 517nm.Blank control group is molten with sample
Solution medium replaces testing sample.Experiment do three it is parallel, ultraviolet specrophotometer return to zero when use methanol.
DPPH clearance rates (%)=(AB-AA)/AB × 100 (AB:Blank control group light absorption value；AA:Sample sets extinction
Value).
(1.2)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 samples and the above-mentioned corrected ABTS of 96 μ l·+Solution is mixed, and room temperature is placed after 10min, in
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 parallel.
ABTS·+Clearance rate I (%)=(AB-AA)/AB × 100 (AB:Blank control group light absorption value；AA:Sample sets extinction
Value).
Ultrashort peptide Purin-WH1, Purin-WH2 antioxidation activity of table 1.
As a result as shown in table 1, ultrashort peptide Purin-WH1, Purin-WH2 have extremely strong antioxidation activity.It is dense in sample
When spending for 3mM, ultrashort peptide Purin-WH1, Purin-WH2 can reach 58.54% to DPPH free radical scavenging activities I%,
59.89%；To ABTS·+Radical cation clearance rate I% is 96.02%, 72.16%.Possess with clinical medicine glutathione
Suitable antioxidation activity.Therefore ultrashort peptide Purin-WH1, Purin-WH2 will have in fields such as antioxidant drug, food it is outstanding
Potentiality to be exploited.
(2) ultrashort peptide Purin-WH1, Purin-WH2 haemolysis and cytotoxic assay
(2.1) hemolytic is detected
The healthy human blood of collection is mixed into anti-freezing with Alsever's Solution, brine 2 times is simultaneously resuspended into 107-108cell/ml
Suspension.The good red cell suspension of above-mentioned dilution is mixed with being dissolved in the ultrashort peptide Mapin-WH2 samples of physiological saline, 37 DEG C
30min is incubated, 5min is centrifuged then at 1000rpm, supernatant surveys absorption value in 540nm.Negative control uses physiological saline, positive
Control uses Triton X-100, and percent hemolysis is calculated as follows：Percent hemolysis H%=A sample-A negative controls/
A positive control × 100%.
Ultrashort peptide Purin-WH1, Purin-WH2 hemolytic of table 2.
Hemolysis rate when as a result showing ultrashort peptide Purin-WH1, Purin-WH2 at concentrations up to 160 μ g/ml only has
1.66%th, 1.27%.Illustrate that ultrashort peptide Purin-WH1, Purin-WH2 do not have hemolytic, be difficult to cause human erythrocyte to break
Split dissolving and produce human body injury, therefore be very beneficial to it to enter in anti-oxidation medicine, food or cosmetic additive agent field
The development and application of one step.
(2.2) ultrashort peptide Purin-WH1, Purin-WH2 cytotoxicity detection
Detect the ultrashort peptide of the group to human skin fibroblasts HFF-1 cells (being purchased from Shanghai cell bank) toxicity with mtt assay.
First fibroblast is trained in the DMEM containing 15% 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 every hole cell number is reached 105It is individual.Add sample
Product, control group adds the sterilizing ultra-pure water of same volume, puts 37 DEG C, 5%CO2Culture 24h in incubator.After culture terminates, 96
Porocyte culture plates add 20 μ l 5mg/ml MTT solution (being prepared with cell culture PBS) per hole, continue to cultivate 5h,
Liquid in hole is suctioned out with syringe, 100 μ l DMSO are added per hole, purple crystal is completely dissolved several times with liquid-transfering gun piping and druming.Enzyme
Instrument detection light absorbs are marked, wavelength 490nm, reference wavelength 630nm is determined.
As a result as shown in figure 1, cytotoxicity when ultrashort peptide Purin-WH1, Purin-WH2 concentration is 160 μ g/ml only has
15.48% and 2.84%, illustrate that ultrashort peptide Purin-WH1, Purin-WH2 are extremely low to the toxicity of human skin fibroblasts, it is right
Normal cell survival rate influences without conspicuousness.Will not produce injury to human normal Skin Cell, thus be very beneficial to its
Anti-oxidation medicine, the further development and application of food or cosmetic additive agent field.
Claims (2)
1. the ultrashort peptide of antioxidizing, including two kinds of anti-oxidant ultrashort peptides：Purin-WH1 and Purin-WH2；It is characterized in that：
Described anti-oxidant ultrashort peptide Purin-WH1：A kind of straight chain small peptide, molecular weight is 544.71Da, and isoelectric point is 8.19, is contained
There are 5 amino acid residues, the primary structure of complete sequence is Val-Val-Cys-proline-lysine；
Described anti-oxidant ultrashort peptide Purin-WH2：A kind of straight chain small peptide, molecular weight is 641.83Da, and isoelectric point is 8.19, is contained
There are 6 amino acid residues, the primary structure of complete sequence is valine-VAL-PRO-PRO-cysteine-bad ammonia
Acid.
2. the ultrashort peptide of antioxidizing described in claim 1 is used for preparing anti-oxidation medicine, food as main active
Additive and antioxidation cosmetic product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710533712.3A CN107226841B (en) | 2017-07-03 | 2017-07-03 | Antioxidant ultrashort peptide and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710533712.3A CN107226841B (en) | 2017-07-03 | 2017-07-03 | Antioxidant ultrashort peptide and application thereof |
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| Publication Number | Publication Date |
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| CN107226841A true CN107226841A (en) | 2017-10-03 |
| CN107226841B CN107226841B (en) | 2019-12-31 |
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Non-Patent Citations (3)
| Title |
|---|
| HAINING YUA ET AL.,: ""Host defense peptides in skin secretions of Odorrana tiannanensis: Proof for other survival strategy of the frog than merely anti-microbial"", 《BIOCHIMIE》 * |
| 王嘉榕等: ""功能性抗氧化肽制备与机制研究进展"", 《天然产物研究与开发》 * |
| 陈洁、胡晓赟: ""蛋白水解物的抗氧化性研究与展望"", 《中国食品学报》 * |
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