Corn silk (Stigma maydis) (CS) is the thread-like style at the top of an ear of corn. Although CS is discarded as an agricultural by-product worldwide, the value of CS as an herbal remedy has been recognized in the traditional medicines of some countries ^[1]. At present, CS health supplements and CS-based tea are also available to consumers. Phytochemically, CS is rich in flavonoids, which are responsible for some bioactivities of CS, such as antioxidant, anti-fatigue, and anti-hyperlipidemic ^[1]. In contrast to natural product exploration, the identification and characterization of CS-derived bioactive peptides are limited. CS consists of 17.6% of crude proteins by dry weight ^[1], hence it may be a potential source of bioactive peptides. To date, only antihypertensive ^[2] and anti-inflammatory ^[3] peptides were identified from CS, but there is no report of CS-derived antioxidant peptides. In our previous study, we found CS tryptic hydrolysate to be a more potent scavenger of hydrogen peroxide (H₂O₂) and superoxide than glutathione (GSH) and carnosine, two well-established peptidic antioxidants. Moreover, CS tryptic hydrolysate was more effective than the two aforementioned antioxidants in protecting human red blood cells from oxidative injury ^[4]. Nevertheless, the identity of the antioxidant peptides in the hydrolysate has not been unraveled.

Antioxidant peptides derived from food and agricultural waste/by-products are recognized for their potential applications as food additives, functional food, health-promoting supplements, and lead compounds for drug discovery. In the food industry, such peptidic antioxidants of natural origin could be substitutes for synthetic antioxidants, which have raised concerns about health risks. Besides attenuating food oxidation, antioxidant peptides could also serve as curative or preventive agents of reactive oxygen species (ROS)-mediated diseases, supporting the application of such peptides in functional food and health supplement formulation ^[5]. Antioxidant peptides could alleviate cellular oxidative damage by scavenging free radicals and/or by regulating the cellular production of antioxidants or oxidants. The Keap1/Nrf2 pathway is a major pathway that modulates cellular antioxidant responses, which can be activated by antioxidant peptides ^[6]. Under oxidative stress, nuclear factor erythroid 2-related factor 2 (Nrf2) bound to Kelch-like ECH-associated protein 1 (Keap1) will detach and migrate to the nucleus, where it activates the expression of antioxidant genes ^[7]. By contrast, myeloperoxidase (MPO) and xanthine oxidase (XO) are associated with cellular ROS production. Antioxidant peptides isolated from fish skin gelatin hydrolysate alleviated oxidative injury in mice by inhibiting MPO, besides activating the Nrf2 to upregulate the expression of antioxidant enzymes ^[8]. Antioxidant peptides derived from mackerel meat, egg white, and tuna backbone protein were also demonstrated as potent XO inhibitors ^[9].

2. Purification of 1-h Trypsin Hydrolysate by Ultrafiltration

1-h Trypsin hydrolysate (T1H) was separated by ultrafiltration (UF), yielding two UF fractions. The >3 kDa fraction exhibited stronger antioxidant activity than the <3 kDa fraction based on their EC₅₀ for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS^•+) (28.4 and 45.6 µg dry mass (DM)/mL, respectively) and H₂O₂ (174.5 and 461.7 µg DM/mL, respectively) scavenging activities (Figure 1C,D). Thiobarbituric acid reactive species (TBARS) value of the negative control increased from 0.7 to 1.6 µM malondialdehyde equivalents after incubation of the lecithin liposomes from 24 to 48 h, indicating an increase in lipid peroxidation. As revealed by the TBARS values, treatment with 0.5 mg DM/mL of >3 kDa and <3 kDa fractions inhibited lipid peroxidation by 22% and 16% after 24 h, and by 64% and 50% after 48 h, respectively (Figure 1E). Our results are concordant with previous observations on the antioxidant activities of the UF fractions of fennel seed hydrolysate ^[10]. In theirs and this study, the superiority of the >3 kDa fraction in scavenging radicals relative to the <3 kDa fraction was observed. This may be attributed, in part, to the presence of large compounds, such as long peptides, partially degraded proteins, or other components with antioxidant properties in the >3 kDa fraction. In fact, the protein content of the >3 kDa fraction was 8-fold higher than that of the <3 kDa fraction (Figure 1A). By contrast, the <3 kDa fraction was peptide-rich, with peptide content 5-fold greater than the >3 kDa fraction (Figure 1B). Notably, the <3 kDa fraction was comparable or superior to T1H in scavenging ABTS^•+ and repressing lipid peroxidation, based on activity data reported in our previous study ^[4]. This implies that at least part of the antioxidant activities exhibited by T1H could be attributed to the presence of antioxidant peptides in the hydrolysate. The <3 kDa fraction was weaker than antioxidant tripeptide GSH (EC₅₀ 6.7 µg DM/mL) as an ABTS^•+ scavenger but 3-fold stronger than GSH (EC₅₀ 1378.7 µg DM/mL) as H₂O₂ scavenger. H₂O₂ is an ROS molecule that can diffuse through biological membranes and convert to highly reactive hydroxyl radicals in the body cells ^[5]. Hence, the <3 kDa UF fraction likely contained antioxidant peptides with the ability to scavenge biologically relevant ROS. In this study, a time-dependent increase in the lipid peroxidation inhibitory activity of the UF fractions was observed (Figure 1E). This observation agrees with a previous study that compared the ability of corn gluten meal hydrolysates to inhibit lipid peroxidation in a ground pork system following 8 h and 16 h of treatment ^[11]. Our result showed that the lipid peroxidation inhibitory effects of the CS peptides could persist up to 48 h. Low molecular weight (MW) peptides are desirable because they could be more easily absorbed by the body compared to high MW peptides ^[5]. Thus, the peptide-enriched <3 kDa fraction was selected for further purification.

3. Purification of <3 kDa Fraction by Gel Filtration Chromatography

Purification by gel filtration chromatography (GFC) resulted in three pooled fractions: GF-I, GF-II, and GF-III (Figure 2A). Among the three, GF-I exhibited the strongest effects in ABTS^•+ scavenging activity (Figure 2C). GF-I possibly comprised more non-aromatic peptide residues with radical scavenging activity, such as Leu and Pro ^[12] than the other two fractions. Meanwhile, GF-III showed the highest H₂O₂ scavenging activity among the three pooled fractions (Figure 2D). All three pooled GFC fractions could dampen the time-dependent increase in lipid peroxidation in the liposome model at 0.1 mg peptide/mL, with 26–35% inhibition of TBARS formation after 48 h (Figure 2E), although their activities were significantly lower than that of GSH (57% inhibition after 48 h). The peptide content of GF-III (0.68 mg peptide/mg DM) was 3.5-fold greater than those of GF-I and GF-II (Figure 2B). When compared to GF-III, 3.5-fold higher DM of GF-I and GF-II was required to achieve the standardized peptide concentration used for evaluating the lipid peroxidation inhibitory activity depicted in Figure 2E. It can be anticipated that when expressed in terms of DM, the lipid peroxidation inhibitory activity of GF-III may exceed that of GF-I. Thus, we also analyzed the lipid peroxidation inhibitory activity of the three pooled fractions at 0.5 mg DM/mL. As expected, among the three fractions, GF-III showed the strongest inhibition of TBARS formation, with 26% and 51% inhibition after 24 and 48 h, respectively (Figure 2F). The antioxidant activity of GF-III could be owing to its relatively high absorbance at 280 nm (Figure 2A), which suggests an abundance of aromatic amino acid residues (e.g., Phe, Tyr, and Trp) in the pooled fraction. In keeping with this study, a previous study on Chinese chestnut also found that the GFC fraction with the most prominent absorbance at 280 nm had the highest antioxidant activity among all GFC fractions ^[13]. In our GFC experiment, GF-II (intermediate molecular size) had the lowest antioxidant activity, whereas GF-I (greatest molecular size) and GF-III (smallest molecular size) had relatively higher antioxidant activities. Hence, our results suggest that the antioxidant potential of the GFC fractions is not directly related to their molecular size. This is in agreement with the lack of explicit relationship between antioxidant activity and molecular size among 81 corn gluten meal peptide fractions collected in a GFC experiment ^[14]. On the other hand, peptide content data and the different trends in the lipid peroxidation inhibitory activities of the pooled fractions when tested based on DM and peptide mass pointed to the presence of non-peptide constituents in the GFC fractions. Thus, further purification was desirable. Considering that GF-III was the richest in peptide content and to discover peptides containing aromatic amino acid residues from T1H, we proceeded to perform purification on GF-III.

4. Purification of GF-III by Strong-Cation-Exchange Solid-Phase Extraction

GF-III was further purified by strong-cation-exchange solid-phase extraction (SCX-SPE), producing six SPE fractions. As shown in Figure 3A, most of the peptide constituents of GF-III were found in 50 mM KCl fraction (0.9 mg peptide/mL), which was 22–118 times greater than the other five SPE fractions. The 50 mM KCl fraction showed relatively low or no ABTS^•+ and H₂O₂ scavenging activities at the peptide concentrations tested (Figure 3B,C). Our result suggests that SCX-SPE has partitioned most of the non-antioxidant peptides and/or peptides with weak antioxidant activity into the 50 mM KCl fraction. The effectiveness of SCX-SPE in concentrating the antioxidant peptides into single fractions were also evident, as affirmed by the enhancement in antioxidant activity after the SPE step. Briefly, 30 µg peptide/mL of GF-III scavenged 57% of ABTS^•+ (Figure 2C). After the purification of GF-III by SCX-SPE, the resultant 0 and 20 mM KCl fractions scavenged 35% and 22% ABTS^•+ at a 43-fold lower concentration (0.7 µg peptide/mL), respectively (Figure 3B). By estimation, the 0 and 20 mM KCl fractions may be 26-fold and 17-fold stronger than GF-III as ABTS^•+ scavengers, respectively. Other studies also showed 33% ^[15] and 44% ^[10] improvement in the ABTS^•+ scavenging activity of a peptide fraction purified by the SCX-SPE. The potency of the 0 and 20 mM KCl fractions over the other SPE fractions as ABTS^•+ scavenger may be attributed to the presence of negatively charged amino acids (e.g., Glu) ^[16] or proton-donating amino acids (e.g., Trp and Gln) ^[14]. The presence of such residues may impart antioxidant activity to peptides by transferring electrons or protons to free radicals ^[12]. For instance, rapeseed peptides predominantly made up of Glu (19.5%) were reported to have potent radical scavenging activity ^[16]. Corn gluten meal-derived peptides made up of 67% of Trp and Gln displayed high ABTS^•+ scavenging activity ^[14]. A similar improvement in the antioxidant activity of SPE fractions following SCX-SPE was revealed by the H₂O₂ scavenging assay. For example, 150 µg peptide/mL of GF-III scavenged 65% H₂O₂ (Figure 2D). In contrast, at a 15-fold lower concentration (10 µg peptide/mL), the 0 and 200 mM KCl fractions scavenged more than 80% of H₂O₂ (Figure 3C). The 20 mM KCl fraction also scavenged 56% H₂O₂ when tested at 10 µg peptide/mL. Thus, based on theoretical calculations, our results imply a 13–21-fold improvement in the H₂O₂ scavenging activity of the 0, 20, and 200 mM KCl fractions resulting from the purification of GF-III by using SCX-SPE. Altogether, the 0, 20, and 200 mM KCl fractions potentially contained potent antioxidant peptides; thus, they were taken to peptide sequencing.

5. Identification and Characterization of Antioxidant Peptides

Liquid chromatography-tandem mass spectrometry analysis identified 29 peptide sequences comprising 6–14 residues (633.33 to 1517.81 Da) from the 0, 20, and 200 mM KCl fractions (Table 1). This range of peptide masses agrees with the observation that the molecular masses of food-derived antioxidant peptides commonly range between 500–1800 Da ^[12]. Twenty-three of the 29 peptides contain 11–56% aliphatic amino acid residues (Table 1). Such residues are responsible for the thermal stability of proteins ^[17]. Two thermal-stable antioxidant peptides WAFAPA and MYPGLA that were identified from the blue-spotted stingray, for instance, are composed of 50% and 33% of aliphatic residues, respectively ^[18]. Based on the comparison of the aliphatic index, 10 of the 29 peptides were likely superior to both WAFAPA and MYPGLA in terms of thermal stability (Table 1). The discovery of such peptides also supports our previous observation of the thermal stability of T1H, the protein hydrolysate from which the 29 peptides were purified. T1H retained its radical scavenging and ferric reducing activity at temperatures up to 100 °C ^[4]. These CS peptides that are likely to be thermal-stable can thus be utilized as alternatives for food additives to address the concerns regarding the food processing heat treatment. ^•+ more stably than could MYPGLA (Table 3) is also consistent with their relative in vitro antioxidant activity ^[18]. Altogether, our results suggest that the stronger ABTS^•+ scavenging activities of the 0 and 20 mM KCl fractions, relative to that of the 200 mM KCl fraction, could be accounted for, at least in part, by the affinity of their seven peptides to ABTS^•+. ^[5]. Hydrophobic residues made up 33–86% of the compositions of the 29 CS peptides (Table 1). The presence of hydrophobic residue-containing peptides may account for the lipid peroxidation inhibitory effect of T1H ^[4], as well as that of <3 kDa UF (Figure 1E) and GF-III (Figure 2E,F), the peptide fractions which the 29 peptides were purified from. Hydrophobic residues may enhance the interaction of antioxidant peptides with lipid-soluble free radicals, thus attenuating the progression of lipid peroxidation ^[5]. Lipid oxidation is one of the major contributors to the deterioration of food quality during food processing and storage ^[5]. Thus, these CS peptides, as well the CS hydrolysate and partially purified fractions containing them, may be useful in the preservation of lipid-rich foods. On the other hand, it has been reported that peptides with C-terminal Lys could act as potent H₂O₂ scavengers ^[19]. Thus, MAPRTPRK and THAVKGVVHK in the 0 mM KCl fraction, FMFFVYK and MCFHHHFHK in the 20 mM KCl fraction, as well as DFPGAK, AGFPLGK, and AMQQDK in the 200 mM KCl fraction may have contributed to the H₂O₂ scavenging activity of the three SPE fractions. The 0 mM KCl fraction had the highest proportion of peptides containing aromatic residues (9 peptides), followed by the 200 mM KCl (4 peptides) and the 20 mM KCl fractions (3 peptides). Aromatic residues may promote the antioxidant activity of peptides by donating protons to the electron-deficient radicals ^[12]. Notably, the 0 mM KCl fraction had the highest number of basic residue-containing peptides and the highest percentage of basic residues in peptides (Table 1). Thus, the strongest ABTS^•+ scavenging activity of the 0 mM KCl fraction may be attributed to its richness in peptides comprising aromatic and basic amino acids. Our result agrees with the finding that abundance in basic amino acids may account for the strong ABTS^•+ scavenging activity of a Chinese chestnut peptide fraction ^[13]. As revealed by the AnOxPePred analysis, 10 CS peptides (MCFHHHFHK, NLEGYR, AGFPLGK, FMFFVYK, NMVPGR, PVVWAAKR, DFPGAK, FSCPLVMKGPNGLR, RHGSGR, and VHFNKGKKR) had comparable or higher free radical scavenger (FRS) scores relative to the four reference peptides VGPWQK, MYPGLA, FPLPSF and WAFAPA (Table 2). The four reference peptides were empirically proven as ABTS^•+ scavengers ^[18][15][20]. Our results are in accordance with the finding that His, Trp, Tyr, and Pro are common in free-radical-scavenging peptides ^[21]. In this study, the four amino acids account for 11–44% of the residues making up the 10 CS peptides and the four reference peptides (Table 2). Notably, His-containing peptides were only found in the 0 mM KCl (THAVKGVVHK and VHFNKGKKR) and 20 mM KCl (RHGSGR and MCFHHHFHK) fractions, but none in the 200 mM KCl fraction (Table 1). This is in keeping with our observation that the 0 and 20 mM KCl fractions had at least 4-fold greater ABTS^•+ scavenging activity than the 200 mM KCl (Figure 3B). Our results, therefore, support the role of His residues in imparting radical scavenging activity to peptides ^[12]. ^•+ 12–15% less stable relative to VHFNKGKKR and PVVWAAKR. Similarly, both WAFAPA and MYPGLA were predicted to form comparable numbers of interactions with ABTS^•+. However, the lack of participation of aromatic residues in MYPGLA-ABTS^•+ interaction may explain the reported weaker ABTS^•+ scavenging activity of MYPGLA when compared with WAFAPA ^[18]. Moreover, we also observed the significant participation of basic residues in peptide-ABTS^•+ interactions. Briefly, basic residues in 90% of basic residue-containing CS peptides were involved in the interactions with ABTS^•+. Such interactions account for 33–80% of the total number of interactions between individual basic residue-containing peptides and ABTS^•+. To further verify the role of the basic residues of peptides in binding to ABTS^•+, in silico alanine substitution was performed on those that were involved in ABTS^•+-peptide interactions, followed by docking of the alanine-substituted peptides to ABTS^•+. The binding affinities of all CS peptides, except FSCPLVMKGPNGLR, were diminished upon alanine mutagenesis of selected basic residues in the peptides (Table 3 and Table 4). For instance, alanine substitution of His6 in MCFHHHFHK has resulted in a 15% reduction in the binding affinity of peptides towards ABTS^•+. Besides, a decline of 24% in binding affinity of NMVPGR towards ABTS^•+ was observed upon alanine substitution of Arg6. Our results suggest that the basic residues are likely to be critical in binding and stabilizing ABTS^•+. This observation further reinforces our finding that the 0 mM KCl fraction with the highest number of basic residue-containing peptides displayed the strongest ABTS^•+ scavenging activity (Table 1 and Figure 3B). Besides, Leu-ABTS^•+ interactions were observed in 67% of Leu-containing CS peptides (Table 3). Our observation agrees with a previous report of the participation of Leu in the binding between antioxidant peptides and ABTS^•+ [20].