# Functional Characterization of Mung Bean Meal Protein-Derived Antioxidant Peptides
## 2 Results and Discussion  
### Antioxidant Activities of Peptide Fractions from the First Round of RP(C12)-HPLC  
Peptides present in the <1 kDa fraction of mung bean protein hydrolysate that contributed to a high antioxidant activity potential were identified using two consecutive rounds of RP(C12)HPLC as previously described [23]. In the first RP(C12)HPLC round, the mung bean peptides were pooled into twelve fractions (1-12) based on their retention time (Figure 1).  
Figure 1: Initial separation of mung bean protein hydrolysate by reversed-phase (C12) HPLC into 12 fractions (1–12). Reprinted from _Journal of Functional Foods_, Vol. 64, [PERSON], [PERSON], [PERSON], [PERSON], & [PERSON] Identification of antihypertensive peptides from mung bean protein hydrolysate and their effects in spontaneously hypertensive rats, Article 103635. Copyright (2020), with permission from Elsevier.  
The principle of the RP-HPLC involves separating peptides based on their differences in hydrophobicity (hydrophobic/hydrophilic characters). The hydrophilic peptides were eluted first while the more hydrophobic peptides become eluted later. The twelve peptide fractions were assayed for their antioxidant activities using five different methods including DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity, FRAP, and metal chelating activity (Figure 2).  
The results indicate that fraction P1 had the highest DPPH scavenging, hydroxyl, and superoxide radical scavenging activities at 34.92%, 41.15%, and 25.63% respectively. Fractions P5 and P9 had the strongest FRAP with 0.21 and 0.20 mM Fe\({}^{2+}\) reduced/mg peptide, respectively, while fraction 3 showed the highest metal chelation activity at 28.79%. The results suggest that most of the fractions contributed to at least one antioxidant activity, which reflect the presence of several antioxidant peptides with a wide range of hydrophobicity (directly related to elution time) in the mung bean protein hydrolysate. Most of the later-eluting fractions (P5-P12) showed a high potential for antioxidant activities, except for metal chelation. Fraction P1 had the best antioxidant activities for DPPH radical, hydroxyl radical and superoxide radical but poor activity for FRAP and metal ion chelation. Therefore, the results revealed that not only later-eluting (higher hydrophobic character)  
Figure 2: Antioxidant activities of crude mung bean meal protein hydrolysate (MMPH), <1 kDa peptides and peptide fractions derived from the 1 st round of RP(C12)HPLC (P1–P10): DPPH scavenging activity (**A**), hydroxyl radical scavenging activity (**B**), superoxide radical scavenging activity (**C**), FRAP (**D**), and metal ion chelation activity (**E**). Data are expressed as mean \(\pm\) standard deviation (\(n\) = 3). a,b,c,... Different letters indicate significantly different mean values (\(p\) < 0.05).  
peptide fractions have strong antioxidant activities. This might have been influenced by the high hydrophilic character of fraction P1, which indicate presence of excess electrons available for neutralizing free radicals. The results are consistent with an earlier report suggesting that the presence of aspartic acid residues enhance DPPH radical scavenging activity of food protein hydrolysates [27]. Fraction P1 was selected for further separation by a second round of RP(C12)HPLC and subsequent peptide sequence identification because of the strong free radical scavenging activities.