Antioxidant ultra short peptide and preparation method and application thereof
An anti-oxidation and ultra-short peptide technology, applied in the field of biomedicine, can solve the problems of high glutathione price, achieve extremely cytotoxicity, simple preparation method, and good application prospects
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Embodiment 1
[0018] Chemical synthesis of two antioxidant ultrashort peptides Ansin-3 and Magin-3:
[0019] (1) Chemical synthesis of two antioxidant ultrashort peptides Ansin-3 and Magin-3: According to the amino acid sequence of the ultrashort peptide, the full sequence of the two was synthesized with an automatic peptide synthesizer (433A, Applied Biosystems), and reversed by HPLC. Desalting by column chromatography.
[0020] (2) Molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).
[0021] (3) The purity of the purified two antioxidant ultrashort peptides Ansin-3 and Magin-3 was identified by high performance liquid chromatography (HPLC), and the molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), isoelectric focusing The isoelectric point was determined by electrophoresis, and the amino acid sequence structure was determined by an automatic ...
Embodiment 2
[0024] Determination of antioxidant activity of two antioxidant ultrashort peptides Ansin-3 and Magin-3:
[0025] (1) DPPH radical scavenging activity (DPPH radical scavenging assay)
[0026] Weigh a certain amount of DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate, Sigma, USA), dissolve it in methanol, and make 6×10 -5 The solution of M is prepared and used immediately. Mix 48 μl of DPPH solution with 2 μl of sample (2 mg / ml) (the mass ratio of final sample to DPPH is 3:1), let it stand in the dark for 30 minutes at room temperature, and measure the absorbance at 517 nm. In the blank control group, the sample to be tested was replaced by the sample dissolution medium. The experiment was done in triplicate, and methanol was used when the UV spectrophotometer was zeroed.
[0027] DPPH·clearance rate (%)=(AB-AA) / A B×100 (AB: absorbance value of blank control group; AA: absorbance value of sample group).
[0028] (2)ABTS ·+ Free radical cation scavenging activity
[0029] ABTS ...
Embodiment 3
[0037] Ansin-3, Magin-3 cytotoxicity assay:
[0038] The cytotoxicity of samples Ansin-3 and Magin-3 to mouse melanoma B16 was detected by MTT method.
[0039]The cells were first cultured in DMEM containing 10% fetal bovine serum and double antibodies (penicillin and streptomycin each 100 U / ml). After the cells were congested, they were digested with 0.25% trypsin and washed twice with the above medium. The second time, the cells were resuspended, and after cell counting, 100 μl of the cell suspension was added to a 96-well cell culture plate, so that the number of cells per well was 103. After the cells adhere to the wall overnight, aspirate the medium, add complete medium containing different concentrations of samples, add the same volume of complete medium for the control group, and store at 37°C, 5% CO 2 Cultivate in the incubator for 72 hours (change the medium once in the middle). After the culture was over, 20 μl of 5 mg / ml MTT solution (prepared in PBS buffer) was a...
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