## 3 Results and Discussion
### Cellular Antioxidant Activity  
To further evaluate the antioxidant potential of BWSPHs, their ability to affect the endogenous antioxidant defence systems was assessed. The RAW264.7 cell line was selected due to its ability to generate intracellular oxidants as well as express enzymatic and nonenzymatic antioxidants [43]. Table 3 details the effects of BWSPHs and SGID BWSPHs on intracellular GSH concentration, CAT activity, and ROS production. Oxidative stress was induced via treatment with known oxidants \(t\)BOOH or H\({}_{2}\)O\({}_{2}\). The organic peroxide, \(t\)BOOH, was chosen as the oxidative stressor to reduce GSH levels as H\({}_{2}\)O\({}_{2}\) did not significantly reduce GSH concentration in RAW264.7 cells (data not shown). Unlike endogenous H\({}_{2}\)O\({}_{2}\), the xenobiotic \(t\)BOOH, related to industrial air pollution, has no specific detoxifying element in the cell. It has been proposed that the glutathione system catalyses the decomposition of \(t\)BOOH to tert-butyl alcohol and glutathione disulphide, resulting in depletion of GSH [44; 45; 46]. Although BW-SPH-B, -C, -D, -E, and -F did not protect against the suppression of GSH in \(t\)BOOH-challenged cells, treatment with BW-SPH-A did increase GSH concentration significantly compared with the control (\(p<0.05\)) (Table 3). Similarly, BW-SPH-A was the only hydrolysate to increase CAT activity significantly in H\({}_{2}\)O\({}_{2}\)-challenged RAW264.7 cells compared with the H\({}_{2}\)O\({}_{2}\) control (\(p<0.01\)) (Table 3). Similar to the BWSPHs tested in this study, which contained a high content of low MW peptides, large yellow croaker (_Pseudosciena crocca_) protein hydrolysate (MW \(<\) 3 kDa) exhibited O\({}_{2}\)\({}^{-}\) scavenging activity and DPPH scavenging activity in vitro and also effectively and dose-dependently (50-300 \(\mu\)g/mL) increased the activity of antioxidant enzymes GSH-Px, SOD, and CAT in H\({}_{2}\)O\({}_{2}\)-induced oxidative stress in HepG2 cells (\(p<0.05\)) [3]. In addition, numerous fish-derived peptides have demonstrated ability to modulate oxidative stress pathways in vitro [4; 47; 5; 9; 48]. The simulated GI digest of BW-SPH-A also significantly increased GSH concentration in oxidatively stressed RAW264.7 cells compared to treated controls (\(p<0.05\)), indicating that antioxidant peptides and/or antioxidant free amino acids were released from parent peptides during digestion (Table 3). SGID did not alter the ability of BW-SPH-A to increase GSH levels in oxidatively stressed RAW264.7 cells (Table 3) but did inhibit its ability to promote CAT activity (Table 3) indicating different components of the hydrolysate mediate GSH and CAT. In contrast, BW-SPH-E had no effect on CAT activity, whereas BW-SPH-E-GI did indeed enhance CAT activity compared with the H\({}_{2}\)O\({}_{2}\) control (\(p<0.05\)), possibly due to the release of encrypted antioxidant peptides/free amino acids during SGID. Interestingly, BW-SPH-A and BW-SPH-E were observed to have high DH values (43.19 \(\pm\) 2.16 and 42.97 \(\pm\) 3.30, respectively) and a high quantity of components \(<\)1 kDa (77.86 \(\pm\) 0.16 and 69.74 \(\pm\) 0.13, respectively) [26], therefore it is possible that the generation of short-chain peptides and free amino acids influenced subsequent antioxidant potential. Although BW-SPH-B and its simulated GI digest, BW-SPH-B-GI, did not alter GSH concentration or CAT activity significantly compared with controls, there was a significant difference between activities pre- and post-SGID (\(p<0.05\)). The fluorescence indicator DCFH-DA was used to detect H\({}_{2}\)O\({}_{2}\)-induced ROS production in RAW264.7 cells, which were pre-incubated with BWSPHs and SGID BWSPHs. RAW264.7 cells treated with H\({}_{2}\)O\({}_{2}\) for 60 min significantly increased ROS generation (\(p<0.001\)) (data not shown). Interestingly, cells pre-incubated with either BW-SPH-A or its digest BW-SPH-A-GI (0.5% (\(w/v\) dw)) significantly attenuated the production of ROS in H\({}_{2}\)O\({}_{2}\)-challenged RAW264.7 cells (\(p<0.05\)).