Antioxidant activity of cyclolinopeptides

Abstract

Cyclolinopeptides (CLs) are hydrophobic cyclic peptides found in flaxseed. They show immunosuppressive activity, but the biological function of these compounds is largely unknown. This thesis presents the results of studies that were conducted to determine whether CLs could act as antioxidants. In the first study, flaxseed oil was passed over a silica adsorbent column to remove polar compounds. The polar compounds were then eluted from the silica absorbant using a series of increasingly polar solvents. Individual polar fractions were then added back to the silica-treated flaxseed oil and the oxidative stability index of these samples was determined at 100 °C. A polar fraction containing mainly CLA, β/γ- and δ-tocopherol increased the induction time of silica-treated flaxseed oil from 2.3 ± 0.28 h to 3.2 ± 0.41 h. A positive effect of the polar fraction containing a mixture of CLA and CLD-CLG on the oxidative stability of oil was also observed. The antioxidant mechanism of CLs was investigated in several model systems using electron spin resonance spectroscopy. The concentration of radicals in a DMPO (5,5-dimethyl-1-pyrroline-N-oxide) radical-CLs reaction mixture was monitored. All CLs exhibited dose dependent scavenging activities. CLA–CLC reactions with DMPO-OH at a concentration of 5 mM resulted in a 24–30% decrease in electron paramagnetic resonance (EPR) signal intensity. The reaction of CLs and the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•) revealed a more complex interaction than simple radical scavenging. Peptides (CLG and CLG") that contained both tryptophan and methionine showed stronger radical scavenging activity than did CLs containing methionine or methionine sulfoxide but not tryptophan (CLB and CLC). Irradiation of the reaction mixture of DPPH• and peptide with UV light also affected the radical scavenging behaviour. Scavenging activities of DPPH• by CLB, CLC and CLA were enhanced by light, whereas scavenging of DPPH• by the tryptophan containing peptides CLG and CLG″ was not affected. High-performance liquid chromatography with mass spectrometry (HPLC-MS) analysis of the reaction mixtures after a radical scavenging reaction was used to determine the impact of radical scavenging on the peptides. These reactions revealed new masses that were identified and characterized. It was established that DPPH• reacted with the methionine of CLB and with tryptophan in CLG and CLG, by formation of a new covalently-bonded species. Covalent linkages between these amino acids (alone or in peptides) and DPPH• have not been reported previously.