## 2 Results and Discussion
### Purification of Antioxidant Peptides from MGCH
#### 2.2.3 Gel Filtration Chromatography of Frac.4  
Gel filtration chromatography is an efficient technique for preparing bioactive components on the basis of molecule size [30]. Therefore, it has been widely used to analyze MW, prepare samples with particular molecule size range, and remove salt from prepared macromolecules [3; 19]. In the experiment, Frac.4 was separated into two fractions of Frac.4-1 and Frac.4-2 using a Sephadex G-15 column (Figure 2A), and each fraction was collected, lyophilized, and then evaluated for DPPH\(\bullet\) and HO\(\bullet\) scavenging activity. As shown in Figure 2B, DPPH\(\bullet\) and HO\(\bullet\) scavenging activities of Frac.4-1 were 73.3% \(\pm\) 1.98% and 85.4% \(\pm\) 2.66%, respectively, at the concentration of 10 mg protein/mL. The radical scavenging activities of Frac.4-1 were higher than those of Frac.4 (DPPH\(\bullet\) 48.2% \(\pm\) 1.25%; HO\(\bullet\) 56.7% \(\pm\) 1.37%) and Frac.4-2 (DPPH\(\bullet\) 33.1% \(\pm\) 0.96%; HO\(\bullet\) 43.7% \(\pm\) 1.05%). In the gel filtration chromatography column, smaller molecules diffuse further into the pores of the resins and therefore move through the bed more slowly, while larger molecules enter less or not at all and thus move through the bed more quickly. Therefore, the average molecular size of Frac.4-1 was larger than that of Frac.4-2, and this result suggested that bioactivities of peptides were influenced by hydrophobicity, amino acid composition, and sequence apart from the MW [3; 24].  
Figure 1: Elution profile of MGCH-I through DEAE-52 cellulose chromatography (**A**); and 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH\(\bullet\)) and hydroxyl radicals (HO\(\bullet\)) scavenging activities of MGCH-I and its subfractions at a concentration of 15 mg protein/mL (**B**). All data are presented as the mean \(\pm\) SD of triplicate results.  
2.4 Isolation of Peptides from Frac.4-1 by Reverse-Phase High Performance Liquid Chromatography (RP-HPLC)  
RP-HPLC separates peptides on the basis of differences in their hydrophobicity, and the strength of the interaction between the peptides and the stationary phase depends on both hydrophobic interactions and polar interactions [5; 24]. The retention time of peptides can be adjusted by increasing or decreasing organic solvent (methanol, acetonitrile) into the flowing mobile phase, and the chromatographic peak can be applied to confirm the identity and quantity of isolated peptide. Therefore, RP-HPLC has become the widespread technique for the separation of different sized peptides due to its high speed, sensitivity, and good reproducibility [24; 31]. As shown in Figure 3, Frac.4-1 with the highest DPPH\(\bullet\) and HO\(\bullet\) scavenging activities among all hydrolysate fractions was finally measured and purified using RP-HPLC system on a Zorbax C-18 column, and the eluted fractions were collected separately according to chromatographic peaks. Among all fractions, three peptides named as MCPE-A, MCPE-B, and MCPE-C with retention times of 12.068 min, 14.078 min, and 15.714 min, respectively, showed high antioxidant activities. Therefore, MCPE-A, MCPE-B, and MCPE-C were collected and lyophilized for further research.