Structure-activity Relationship And Directional Preparation Of Antioxidant Peptide

Antioxidant activities of peptides are widely accepted. In addition to their antioxidant activites, their nutritional values and ease of absorption also make them promising in developing anti-aging nutraceuticals. However, the research on antioxidant peptides is still in laboratory. There is little research on screening precursor protein for antioxidant peptides directionally. The structure-relationship of antioxidant peptides is still unclear. Therefore, it is hard for industrialization promotion of antioxidant peptides. In this paper, antioxidant activities of amino acids and dipeptides were studied systematically. The determining factor for the antioxidant activities of dipeptides was elucidated. Furthermore, the quantative structure-activity relationship of antioxidant dipeptides was studied. It was aimed to provide guidance on the directional screening of precursor protein for antioxidant peptides as well as developing an efficient approach of preparing antioxidant peptides. The main research contents and results are as follows:(1) Practical problems of DPPH and ABTS assays in assessing the antioxidant activities of amino acids and peptides were discussed and these two assays were further revised. Results showed that the deprotonation of DPPHH could occur in the presenc of Arg and further interfered with the spectrophotometric measurement at 515-525 nm. To avoid the interference, acetate buffer could be used to maintain the reaction mixture under mild acid condition, In ABTS assay, reaction p H value had varied effects on different types of amino acids and peptides in scavenging ABTS?+. The reaction rates of Tyr, Trp and their related peptides were strongly dependent on p H, while those of Cys and Cys-containing peptides were almost unaffected by p H. In addition, all these amino acids and dipeptides failed to reach equilibrium over the short incubation period of 6–10 min typically used in this assay at p H 7.4. At least 30 min was needed for most of the peptides to approach equilibrium.(2) Antioxidant activities of 20 protein-derived amino acids and 40 synthetic dipeptides were studied in ABTS, ORAC, reducing power and Fe(II) chelating ability assays. Effects of antioxidant amino acids on the antioxidant activities of dipeptides were discussed. In ABTS assay, Tyr and Trp as well as Tyr- and Trp-containing dipeptides showed very strong radical scavenging activities with TE values ranging from 0.74 to 5.90 μmol TE/μmol, followed by Cys and Met as well as Cys- and Met-containing dipeptides with TE values ranging from 0.14 to 2.00 μmol TE/μmol, while all other dipeptides did not show antioxidant activities in these two assays. In DPPH and reducing power assays, only Cys and Cys-containing dipeptides showed obvious activities with TE values ranging from 0.12 to 0.85 μmol TE/μmol. In addition, all tested amino acids and dipeptides did not show obvious Fe(II) chelating ability. It indicated that the presence of such antioxidant amino acid residues as Tyr, Trp, Cys and Met in dipeptides was the determining factor for their antioxidant activities. These findings were also validated by the antioxidant activites of nine polypeptides.(3) Protective effects of different types of dipeptides against AAPH-induced hemolysis in erythrocytes were studied. Additionally, Tyr-, Trp-, Cys- and Met-Gly were used to study the mechanism of their protective effects. Results showed that all Tyr-, Trp-, Cys- and Met-containing dipeptides could protect erythrocytes against AAPH-induced hemolysis, while other dipeptides absence of these amino acids did not show any protective effects. Additionally, Tyr-, Trp-, Cys- and Met-Gly protected erythrocytes against AAPH-induced hemolysis in a time- and dose-dependent manner. These dipeptides could also significantly(p < 0.05) retard the oxidation of hemoglobin and depletion of GSH in erythrocytes. It was suggested that these peptides protected erythrocytes against AAPH-induced oxidative damage by acting as the direct radical scavengers.(4) Antioxidant activities of 48 Tyr/Trp-containing dipeptides were determined. Quantitative structure–activity relationship(QSAR) modeling of these dipeptides was studied based on amino acid descriptors and density functional theory(DFT) calculations. Results showed that most Tyr- and Trp-containing dipeptides with Tyr or Trp at the N-terminus displayed stronger ABTS?+ and ROO? scavenging activities than those with Tyr or Trp at the C-terminus. In addition to the position of Tyr and Trp in the sequence, the neighboring residue also affected the radical scavenging activities of Tyr/Trp-containing dipeptides. The DPPS model indicated that the steric property, hydrophobicity, hydrogen-bond ability and electronic property of amino acid at the C-terminus had negative effects on the activities of Tyr-containing dipeptides, while the hydrophobicity and hydrogen-bond ability of amino acid at the N-terminus had positive effects on the activities of Tyr-containing dipeptides. DFT calculation indicated that the electronic cloud distribution of frontier molecular orbit as well as the BDE, IP, PA and ETE values of the functional group in Tyr and Trp was not affected by the neighboring residue. Their radical scavenging activities might be affected by filed and steric effects as well as hydrogen bonding.(5) Based on the amino acid composition characteristics of antioxidant peptides, precursor protein of antioxidant peptides were screened, and the corresponding antioxidant peptides were isolated and identified by consecutive Sephadex G-25 and UPLC-ESI-UHR-TOF-MS/MS. Results showed that, antioxidant activities of rice protein, casein, peanut protein isolate, soy protein isolate, wheat gluten and gelatin hydrolysates were highly related to their contents of antioxidant amino acids, i.e, Tyr, Trp, Cys and Met. Rice protein and casein which were rich in Tyr and Trp showed the highest antioxidant activities, while gelatin which was lack of Tyr, Trp, Cys and Met showed the weakest antioxidant activities. Rice protein was selcted as the protein source to prepare antioxidant peptides. Finally, 27 Tyr-, or Trp-containing peptides were identified from rice protein Protease M hydrolysate. All these peptides were synthesized and confirmed to show strong antioxidant activities. Among them, 20 peptides, including YR、WN、YY、AW、WV、YW、IW、YNPR、TYNPR、GYPR、NWR、WQSS、WQS、IGYPR、LGYPR、LQPY、TNPW、MYPIPR、MYPLPR and VNPW, showed stronger antioxidant activities than the standard antioxidant Trolox.