*3.2. Antioxidant Properties*

Free radicals are short-lived and highly reactive chemical species that contain unpaired electrons [76]. Although reactive oxygen species (ROS) are formed during normal metabolic processes, they may also be formed due to exposure to exogenous factors such as ionizing radiation and UV light [77]. Free radicals can oxidatively modify nucleic acids, proteins, lipids, and sugars. An increased presence of these modified forms has been associated with an increased risk of various human diseases (e.g., cancer, Alzheimer's disease) [78]. Antioxidant mechanisms are present in most organisms and act to reduce or eliminate the levels of common ROS [77]. While many of the most effective mechanisms are enzymebased (e.g., superoxide dismutase and catalase). Many small molecules (e.g., vitamin C, glutathione) also play a role in maintaining the overall redox balance in the cell [79]. While

both natural and synthetic antioxidants have been added to foodstuffs, the potential toxicity of synthetic antioxidants, e.g., GHT has prompted the exploration of the use of peptides as antioxidant agents [77,79,80]. It is generally accepted that large peptides have less radical scavenging or antioxidant potential than small peptides [81]. Peptides have been reported to exert their antioxidant activity through direct metal chelation, ROS scavenging and inhibition of lipid peroxidation cascades [82].

A peptide from the microalgae *Isochrysis zhanjiangensis* has shown to have potent antioxidant capabilities towards alcohol-induced injury in cultured liver hepatoma cells (HepG2) [83]. This peptide was produced by in vitro gastro digestion using pepsin, trypsin and chymotrypsin resulting in the active sequence of NDAEYGICGF [83]. The treatment of cells with this peptide resulted in increased levels of the enzymes superoxide dismutase and glutathione. The antioxidant capabilities of the peptide appear to be related to a combination of the following factors: its molecular weight, hydrophobic amino acids (A, G, I) and aromatic amino acids (F, Y) in the sequence [83]. Other antioxidant peptides identified from *C. vulgaris* include VECYGPRPQF, which showed antioxidant capacity 26-fold higher (197 ng/mL Trolox equivalent) than trolox when tested by ORAC [84]. This peptide was found to slow oxidation by up to 10-fold compared to the control (PBS). The authors hypothesized that the Cu2+ chelating properties of this peptide were likely the main mechanism of action of antioxidant activity [84].
