Purification and Identification of Antioxidant Peptides from Protein Hydrolysate of Scalloped Hammerhead (Sphyrna lewini) Cartilage

The aim of this study was to purify and identify peptides with antioxidant properties from protein hydrolysate of scalloped hammerhead (Sphyrna lewini) cartilage. Cartilaginous proteins of the scalloped hammerhead were extracted by guanidine hydrochloride, and three antioxidant peptides, named enzymolysis peptide of scalloped hammerhead cartilage A (SCPE-A), SCPE-B and SCPE-C, were subsequently isolated from the hydrolysate of the cartilaginous proteins using ultrafiltration and chromatography. The amino acid sequences of SCPE-A, SCPE-B and SCPE-C were identified as Gly-Pro-Glu (GPE), Gly-Ala-Arg-Gly-Pro-Gln (GARGPQ), and Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG), with molecular weights of 301.30 Da, 584.64 Da and 950.03 Da, respectively. As per in vitro activity testing, SCPE-A, SCPE-B and SCPE-C exhibited strong scavenging activities on 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH•) (half elimination ratio (EC50) 2.43, 2.66 and 1.99 mg/mL), hydroxyl radicals (HO•) (EC50 0.28, 0.21 and 0.15 mg/mL), 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radicals (ABTS+•) (EC50 0.24, 0.18 and 0.29 mg/mL), and superoxide anion radicals (O2−•) (EC50 0.10, 0.14 and 0.11 mg/mL). In addition, SCPE-A showed inhibition activity similar to butylated hydroxytoluene (BHT) in lipid peroxidation in a linoleic acid model system. The amino acid residues of Gly, Pro and Phe could positively influence the antioxidant activities of GPE, GARGPQ and GFTGPPGFNG. These results suggested that GPE, GARGPQ and GFTGPPGFNG might serve as potential antioxidants and be used as food additives and functional foods.


Introduction
Oxidation is an important factor in the food industry because it causes a loss of nutrition, color and functionality, as well as undesirable off-flavors and toxic compounds, which further induce the deterioration of food. Furthermore, the accumulation of toxic products is dangerous to the health of consumers [1,2]. Therefore, the inhibition of free radical formation and oxidation reactions play an important role in preventing or retarding the autoxidation of food components [3]. Many synthetic antioxidants, including butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tertiary butylhydroquinone (TBHQ), are widely used in the food industry for preservation and to retard lipid oxidation [4,5]. However, the dosages of the synthetic antioxidants are under strict regulation due to different in terms of chain length and amino acid sequence [1,23]. Therefore, five proteases including papain, alcalase, trypsin, pepsin, and neutrase were used to hydrolyze the cartilaginous proteins of scalloped hammerhead. HO• scavenging assay is quick, convenient, and efficient in predicting the antioxidant activities of protein hydrolysates and purified peptides. As a consequence, HO• was used to evaluate the antioxidant activity of compounds to act as free radical scavengers or hydrogen donors, and the results are shown in Table 1. The yield and degree of hydrolysis (DH%) of trypsin hydrolysate was 2.11 ± 0.11 g/100 g cartilage and 23.72% ± 0.31%, respectively, which was higher than for papain hydrolysate, alcalase hydrolysate, pepsin hydrolysate, and neutrase hydrolysate. The result indicated that trypsin could more effectively hydrolyze the proteins from scalloped hammerhead cartilages than the other four proteases. Furthermore, trypsin hydrolysate (SHCH) showed a significantly higher HO• scavenging activity (p < 0.05) with 62.38% ± 1.67% at 15 mg/mL, whereas papain hydrolysate showed a significantly lower HO• scavenging activity (p < 0.05) at 34.85% ± 1.05%. Based on these data, the protein hydrolysate of scalloped hammerhead cartilage produced by trypsin was named SHCH and was selected for follow-up studies.

Ultrafiltration
Protein hydrolysate is a complex mixture of active and inactive peptides (of various sizes) and amino acid compositions, and ultrafiltration membrane technology is an important method for the fractionation of protein hydrolysate and the enrichment of peptides with specific MW ranges [1,5]. SHCH was fractionated by ultrafiltration using two molecular weight cut-off (MWCO) membranes (10 and 3 kDa), and three fractions, SHCH-I (MW < 3 kDa), SHCH-II (3 kDa < MW < 10 kDa), and SHCH-III (MW > 10 kDa), were prepared. As shown in Figure 1, the HO• scavenging activity of SHCH-I was 79.10% ± 2.38% at 15 mg protein/mL, which was significantly stronger than those of SHCH, SHCH-II, and SHCH-III (p < 0.05). The MW of peptides plays a critical role in bioactivity, and protein hydrolysates with smaller MW usually exhibited higher antioxidant activity than larger MW hydrolysates [4,5]. SHCH-I, which is abundant in smaller MW peptides, showed high HO• scavenging activity, and the result was in agreement with other reports that the ultrafiltration fractions of protein hydrolysates with lower MW could more effectively interact with the free radicals interfering in oxidative processes [6,9].

Anion-Exchange Chromatography
Ion-exchange chromatography is used to separate the charged molecules based on their affinity to the ion exchanger (anion and/or cation exchange resins), and their interaction was determined by the number and location of the charges on the molecules [5]. SHCH-I was loaded onto a Diethylaminoethyl cellulose 52 (DEAE-52) cellulose anion-exchange column and separated by stepwise elution using deionized water and 0.1, 0.5, and 1.0 M NaCl ( Figure 2A). Five separated fractions (Fr.1 to Fr.5) were collected. Their HO• scavenging activities were measured and are shown in Figure 2B. The HO• scavenging rate of Fr.4 reached 72.03% ± 2.64% at 10 mg protein/mL, and it exhibited significantly more efficient antioxidant activity than the other fractions (p < 0.05). Peptides with basic and/or hydrophobic amino acid residues, such as His, Lys and Pro, are thought to have strong antioxidant activities [24]. Therefore, anion and cation exchange resins have been widely used to purify antioxidant peptides from protein hydrolysates [25][26][27]. The present data showed that Fr.4 had the strongest HO• scavenging activity and was selected for further purification.

Gel Filtration Chromatography
Molecular size is an important determinant of the bioactivity of a specific peptide [8]. Therefore, gel filtration chromatography is an important method to purify peptides. Fr.4 was loaded onto a Sephadex G-15 column and separated into two fractions of Fr.4-1 and Fr.4-2 ( Figure  3A). Each fraction was collected, lyophilized, and evaluated for HO• scavenging activity. As shown in Figure 3B, the HO• scavenging rate of Fr.4-1 reached 87.80% ± 2.24% at 5 mg protein/mL and was higher than those of Fr.4 (72.03% ± 2.64%) and Fr.4-2 (52.38% ± 1.62%). Therefore, Fr.4-1 was selected for further purification by RP-HPLC.

Anion-Exchange Chromatography
Ion-exchange chromatography is used to separate the charged molecules based on their affinity to the ion exchanger (anion and/or cation exchange resins), and their interaction was determined by the number and location of the charges on the molecules [5]. SHCH-I was loaded onto a Diethylaminoethyl cellulose 52 (DEAE-52) cellulose anion-exchange column and separated by stepwise elution using deionized water and 0.1, 0.5, and 1.0 M NaCl (Figure 2A). Five separated fractions (Fr.1 to Fr.5) were collected. Their HO• scavenging activities were measured and are shown in Figure 2B. The HO• scavenging rate of Fr.4 reached 72.03% ± 2.64% at 10 mg protein/mL, and it exhibited significantly more efficient antioxidant activity than the other fractions (p < 0.05). Peptides with basic and/or hydrophobic amino acid residues, such as His, Lys and Pro, are thought to have strong antioxidant activities [24]. Therefore, anion and cation exchange resins have been widely used to purify antioxidant peptides from protein hydrolysates [25][26][27]. The present data showed that Fr.4 had the strongest HO• scavenging activity and was selected for further purification.

Anion-Exchange Chromatography
Ion-exchange chromatography is used to separate the charged molecules based on their affinity to the ion exchanger (anion and/or cation exchange resins), and their interaction was determined by the number and location of the charges on the molecules [5]. SHCH-I was loaded onto a Diethylaminoethyl cellulose 52 (DEAE-52) cellulose anion-exchange column and separated by stepwise elution using deionized water and 0.1, 0.5, and 1.0 M NaCl ( Figure 2A). Five separated fractions (Fr.1 to Fr.5) were collected. Their HO• scavenging activities were measured and are shown in Figure 2B. The HO• scavenging rate of Fr.4 reached 72.03% ± 2.64% at 10 mg protein/mL, and it exhibited significantly more efficient antioxidant activity than the other fractions (p < 0.05). Peptides with basic and/or hydrophobic amino acid residues, such as His, Lys and Pro, are thought to have strong antioxidant activities [24]. Therefore, anion and cation exchange resins have been widely used to purify antioxidant peptides from protein hydrolysates [25][26][27]. The present data showed that Fr.4 had the strongest HO• scavenging activity and was selected for further purification.

Gel Filtration Chromatography
Molecular size is an important determinant of the bioactivity of a specific peptide [8]. Therefore, gel filtration chromatography is an important method to purify peptides. Fr.4 was loaded onto a Sephadex G-15 column and separated into two fractions of Fr.4-1 and Fr.4-2 ( Figure  3A). Each fraction was collected, lyophilized, and evaluated for HO• scavenging activity. As shown in Figure 3B, the HO• scavenging rate of Fr.4-1 reached 87.80% ± 2.24% at 5 mg protein/mL and was higher than those of Fr.4 (72.03% ± 2.64%) and Fr.4-2 (52.38% ± 1.62%). Therefore, Fr.4-1 was selected for further purification by RP-HPLC.

Gel Filtration Chromatography
Molecular size is an important determinant of the bioactivity of a specific peptide [8]. Therefore, gel filtration chromatography is an important method to purify peptides. Fr.4 was loaded onto a Sephadex G-15 column and separated into two fractions of Fr.4-1 and Fr.4-2 ( Figure 3A). Each fraction was collected, lyophilized, and evaluated for HO• scavenging activity. As shown in Figure 3B, the HO• scavenging rate of Fr.4-1 reached 87.80% ± 2.24% at 5 mg protein/mL and was higher than those of Fr.4 (72.03% ± 2.64%) and Fr.4-2 (52.38% ± 1.62%). Therefore, Fr.4-1 was selected for further purification by RP-HPLC. The hydrophobic and hydrophilic properties of peptides play a key role in their retention time (RT) on an RP-HPLC column, and the RT can be adjusted by changing the ratio of polar (water) and nonpolar (methanol, acetonitrile) solvents [11]. Using an ultrafiltration membrane system, anion-exchange chromatography and gel filtration chromatography, Fr.4-1, which had the highest HO• scavenging activity among all fractions, was separated using RP-HPLC on a Zorbax C-18 column, and the eluted fractions were collected separately according to the chromatographic peaks ( Figure 4). Three fractions, referred to as enzymolysis peptide of scalloped hammerhead cartilage A (SCPE-A), SCPE-B and SCPE-C, with RTs of 10.642, 13.605, and 17.979 min, respectively, showed high antioxidant activities, and their HO• scavenging rates reached 80.7% ± 1.22%, 75.4% ± 2.33%, and 92.2% ± 3.44%, respectively, at 3.0 mg/mL. Therefore, SCPE-A, SCPE-B and SCPE-C were collected for further research.

Molecular Mass and Amino Acid Sequences of the Purified Peptides
The properties of peptides are related to their composition, structure, MW, amino acid sequence and hydrophobicity. Considering the radical-scavenging ability, the amino acid sequences and molecular mass of the three isolated peptides were analyzed using a protein sequencer and quadrupole-time of flight mass spectrometry (Q-TOF MS), respectively. The mass spectra of the three isolated peptides were shown in Figure 5. The amino acid sequences of SCPE-A, SCPE-B and SCPE-C were identified as Gly-Pro-Glu (GPE), Gly-Ala-Arg-Gly-Pro-Gln (GARGPQ), and Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG), with molecular masses of 301.30 Da, The hydrophobic and hydrophilic properties of peptides play a key role in their retention time (RT) on an RP-HPLC column, and the RT can be adjusted by changing the ratio of polar (water) and nonpolar (methanol, acetonitrile) solvents [11]. Using an ultrafiltration membrane system, anion-exchange chromatography and gel filtration chromatography, Fr.4-1, which had the highest HO• scavenging activity among all fractions, was separated using RP-HPLC on a Zorbax C-18 column, and the eluted fractions were collected separately according to the chromatographic peaks ( Figure 4). Three fractions, referred to as enzymolysis peptide of scalloped hammerhead cartilage A (SCPE-A), SCPE-B and SCPE-C, with RTs of 10.642, 13.605, and 17.979 min, respectively, showed high antioxidant activities, and their HO• scavenging rates reached 80.7% ± 1.22%, 75.4% ± 2.33%, and 92.2% ± 3.44%, respectively, at 3.0 mg/mL. Therefore, SCPE-A, SCPE-B and SCPE-C were collected for further research. The hydrophobic and hydrophilic properties of peptides play a key role in their retention time (RT) on an RP-HPLC column, and the RT can be adjusted by changing the ratio of polar (water) and nonpolar (methanol, acetonitrile) solvents [11]. Using an ultrafiltration membrane system, anion-exchange chromatography and gel filtration chromatography, Fr.4-1, which had the highest HO• scavenging activity among all fractions, was separated using RP-HPLC on a Zorbax C-18 column, and the eluted fractions were collected separately according to the chromatographic peaks ( Figure 4). Three fractions, referred to as enzymolysis peptide of scalloped hammerhead cartilage A (SCPE-A), SCPE-B and SCPE-C, with RTs of 10.642, 13.605, and 17.979 min, respectively, showed high antioxidant activities, and their HO• scavenging rates reached 80.7% ± 1.22%, 75.4% ± 2.33%, and 92.2% ± 3.44%, respectively, at 3.0 mg/mL. Therefore, SCPE-A, SCPE-B and SCPE-C were collected for further research.

Molecular Mass and Amino Acid Sequences of the Purified Peptides
The properties of peptides are related to their composition, structure, MW, amino acid sequence and hydrophobicity. Considering the radical-scavenging ability, the amino acid sequences and molecular mass of the three isolated peptides were analyzed using a protein sequencer and quadrupole-time of flight mass spectrometry (Q-TOF MS), respectively. The mass spectra of the three isolated peptides were shown in Figure 5. The amino acid sequences of SCPE-A, SCPE-B and SCPE-C were identified as Gly-Pro-Glu (GPE), Gly-Ala-Arg-Gly-Pro-Gln (GARGPQ), and Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG), with molecular masses of 301.30 Da,

Molecular Mass and Amino Acid Sequences of the Purified Peptides
The properties of peptides are related to their composition, structure, MW, amino acid sequence and hydrophobicity. Considering the radical-scavenging ability, the amino acid sequences and molecular mass of the three isolated peptides were analyzed using a protein sequencer and quadrupole-time of flight mass spectrometry (Q-TOF MS), respectively. The mass spectra of the three isolated peptides were shown in Figure 5. The amino acid sequences of SCPE-A, SCPE-B and SCPE-C were identified as Gly-Pro-Glu (GPE), Gly-Ala-Arg-Gly-Pro-Gln (GARGPQ), and Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG), with molecular masses of 301. 30
the protein hydrolysates of Chinese leek, blue mussel, grass carp skin, mussel sauce and corn gluten meal. Therefore, the present results suggested that SCPE-A, SCPE-B and SCPE-C were DPPH• inhibitors and primary antioxidants that reacted with free radicals.

Lipid Peroxidation Inhibition Assay
Scavenging activities on DPPH•, ABTS + •, HO• and O − 2 • have been widely used to assess the antioxidant capacities of protein hydrolysates and peptides. However, each of these assays only measures an antioxidant property representing a different mechanism, which does not reflect the multiple mechanisms by which samples may act as antioxidants to retard or inhibit lipid oxidation in a food system [42]. Therefore, in this section, the ability of the soluble samples to suppress lipid peroxidation in a linoleic acid model system was investigated. Lipid peroxidation is a complex process that involves the formation and propagation of lipid radicals and lipid hydroperoxides, which are formed as the primary oxidation products in the presence of oxygen [43]. The inhibitory activities of SCPE-A, SCPE-B and SCPE-C were measured by lipid peroxidation in an in vitro model and were compared with the commercially available antioxidant BHT for 7 days. Figure 7 shows that SCPE-A had a similar inhibitory effect on lipid peroxidation as BHT and significantly retarded the lipid peroxidation compared with the control (without sample), SCPE-B and SCPE-C. In previous research, SCPE-A showed excellent scavenging activity on DPPH•, HO•, ABTS + • and O − 2 •, with EC 50 values of 2.43, 0.28, 0.24, and 0.08 mg/mL, respectively. Therefore, the inhibition effect of lipid peroxidation caused by SCPE-A could be attributed to its radical-scavenging activity. In addition, SCPE-A may have potential applications in the food industry for retarding the production of unwanted off-flavors and toxic products. and were compared with the commercially available antioxidant BHT for 7 days. Figure 7 shows that SCPE-A had a similar inhibitory effect on lipid peroxidation as BHT and significantly retarded the lipid peroxidation compared with the control (without sample), SCPE-B and SCPE-C. In previous research, SCPE-A showed excellent scavenging activity on DPPH•, HO•, ABTS + • and O − 2 •, with EC50 values of 2.43, 0.28, 0.24, and 0.08 mg/mL, respectively. Therefore, the inhibition effect of lipid peroxidation caused by SCPE-A could be attributed to its radical-scavenging activity. In addition, SCPE-A may have potential applications in the food industry for retarding the production of unwanted off-flavors and toxic products.

Structure-Antioxidant Activity Relationship of Peptides
The structural characteristics of peptides provide guides for the evaluation of food-derived proteins as potential precursors of antioxidant peptides and predict the possible release of bioactive peptides from various proteins using an appropriate protease [1].
Many researchers found that the antioxidant activity of peptides was highly dependent on their amino acid sequence and composition.  reported that the Gly residue may contribute significantly to antioxidant activity since the single hydrogen atom in the side chain of Gly serves as a proton-donating source and neutralizes active free radical species [44]. In addition, Nimalaratne et al. (2015) reported that the single hydrogen atom of Gly (G) can provide high flexibility to the peptide backbone and positively influence the antioxidant properties [8]. Therefore, Gly residues might be important contributors to the antioxidant activity of SCPE-A, SCPE-B, and SCPE-C because there are one, two and four Gly residues in their amino acid sequences, respectively.
The pyrrolidine ring of proline (P) can interact with the secondary structure of the peptide, thereby increasing the flexibility, and it is also capable of quenching singlet oxygen due to its low ionization potential [6]. Samaranayaka and Li-Chan (2011) reported that the Pro residue plays an important role in the antioxidant activity of the peptide purified from Saccharomyces cerevisiae protein hydrolysate [45]. Therefore, the one, one and two Pro residues in the amino acid sequences of SCPE-A, SCPE-B, and SCPE-C should enhance the radical-scavenging activities of the three peptides.
Aromatic amino acids, such as Phe, Tyr, His, and Trp, and hydrophobic amino acids, including Ala, Val, and Leu, have been reported to be critical to the antioxidant activities of peptides [1]. Huang et al. (2005) reported that amino acids with aromatic residues, such as Phe, Tyr and Trp, can quench free radicals by direct electron transfer [46]. The results from Guo et al. (2015) indicated that hydrophobic amino acids (e.g., Val, Ala, Leu) and aromatic amino acids (Phe, His, Tyr and Trp) can enhance the radical-scavenging abilities of peptides from Chinese cherry seeds [4]. Therefore, the presence of the one Ala residue and two Phe residues in the sequences of SCPE-B and SCPE-C, Figure 7. Lipid peroxidation inhibition assays of SCPE-A, SCPE-B and SCPE-C. All data are presented as the mean ± SD of triplicate results.

Structure-Antioxidant Activity Relationship of Peptides
The structural characteristics of peptides provide guides for the evaluation of food-derived proteins as potential precursors of antioxidant peptides and predict the possible release of bioactive peptides from various proteins using an appropriate protease [1].
Many researchers found that the antioxidant activity of peptides was highly dependent on their amino acid sequence and composition.  reported that the Gly residue may contribute significantly to antioxidant activity since the single hydrogen atom in the side chain of Gly serves as a proton-donating source and neutralizes active free radical species [44]. In addition, Nimalaratne et al. (2015) reported that the single hydrogen atom of Gly (G) can provide high flexibility to the peptide backbone and positively influence the antioxidant properties [8]. Therefore, Gly residues might be important contributors to the antioxidant activity of SCPE-A, SCPE-B, and SCPE-C because there are one, two and four Gly residues in their amino acid sequences, respectively.
The pyrrolidine ring of proline (P) can interact with the secondary structure of the peptide, thereby increasing the flexibility, and it is also capable of quenching singlet oxygen due to its low ionization potential [6]. Samaranayaka and Li-Chan (2011) reported that the Pro residue plays an important role in the antioxidant activity of the peptide purified from Saccharomyces cerevisiae protein hydrolysate [45]. Therefore, the one, one and two Pro residues in the amino acid sequences of SCPE-A, SCPE-B, and SCPE-C should enhance the radical-scavenging activities of the three peptides.
Aromatic amino acids, such as Phe, Tyr, His, and Trp, and hydrophobic amino acids, including Ala, Val, and Leu, have been reported to be critical to the antioxidant activities of peptides [1]. Huang et al. (2005) reported that amino acids with aromatic residues, such as Phe, Tyr and Trp, can quench free radicals by direct electron transfer [46]. The results from Guo et al. (2015) indicated that hydrophobic amino acids (e.g., Val, Ala, Leu) and aromatic amino acids (Phe, His, Tyr and Trp) can enhance the radical-scavenging abilities of peptides from Chinese cherry seeds [4]. Therefore, the presence of the one Ala residue and two Phe residues in the sequences of SCPE-B and SCPE-C, respectively, should have a positive impact on their radical-scavenging and lipid peroxidation inhibitory activities.
The presence of acidic and basic amino acids plays a critical role in the metal ion chelating activity, which is related to the carboxyl and amino groups in their side chains [47]. Similar results were reported by Memarpoor-Yazdi et al. (2012), who found that the basic (Arg) and acidic (Asp and Glu) amino acid residues in the sequences of Asn-Thr-Asp-Gly-Ser-Thr-Asp-Tyr-Gly-Ile-Leu-Gln-Ile-Asn-Ser-Arg (NTDGSTDYGILQINSR) and Leu-Asp-Glu-Pro-Asp-Pro-Leu-Ile (LDEPDPLI) were critical to their antioxidant activities [48]. Díaz, et al. (2003) found that Glu is an effective cation chelator that forms complexes with calcium, iron and zinc and may contribute to the antioxidant activity [49]. Therefore, Glu in SCPE-A and Arg in SCPE-B might be favorable to their antioxidant activities.
In addition, the antioxidant activities of peptides are dependent on their molecular size, and shorter peptides, especially peptides with 2-10 amino acid residues, have stronger radical-scavenging and lipid peroxidation inhibition activities than their parent native proteins or long-chain peptides [1,46]. SCPE-A, SCPE-B, and SCPE-C exhibited good antioxidant activities in the radical scavenging and lipid peroxidation inhibition assays, which suggested that the short SCPE-A, SCPE-B, and SCPE-C could interact more effectively and easily with free radicals and inhibit the propagation cycles of lipid peroxidation in the radical scavenging and lipid peroxidation model system [50]. However, SCPE-A had the strongest O − 2 • scavenging and lipid peroxidation inhibition activities, SCPE-B had the strongest scavenging activity on ABTS + •, and SCPE-C exhibited the highest DPPH• and HO• scavenging activities among all samples and fractions. The results indicated no consistent trends in the antioxidant capacities of SCPE-A, SCPE-B, and SCPE-C in different antioxidant assays. Therefore, more detailed study should be performed to clarify the relationship between the activity and structure of the three isolated peptides.

Preparation of the Protein Hydrolysate from Scalloped Hammerhead Cartilage
Frozen scalloped hammerhead cartilage was thawed, minced to homogenate and soaked in 1.0 M guanidine hydrochloride with a solid-to-solvent ratio of 1:5 (w/v) for 48 h with continuous stirring, and the liquid supernatant was collected by centrifugation at 12,000× g at 4 • C for 10 min. The resulting supernatant was dialyzed (MW 5 kDa) against 25 volumes of distilled water for 12 h, with the solution changed every 4 h, and the resulting dialysate was freeze-dried.
The freeze-dried sample was dissolved (5% w/v) in 0.2 M phosphate buffer solution (PBS, pH 7.2) and hydrolyzed for 4 h using neutrase at pH 7.0, 60 • C; alcalase at pH 8.0, 50 • C; trypsin at pH 8.0, 40 • C; pepsin at pH 2.0, 37 • C; or papain at pH 6.0, 50 • C, with a total enzyme dose of 2.5%. Enzymatic hydrolysis was stopped by heating for 10 min in boiling water, and the hydrolysate was centrifuged at 9000× g for 15 min. The supernatant was freeze-dried and stored at −20 • C for further analysis. The protein hydrolysate of scalloped hammerhead cartilage using trypsin was named SHCH.

Anion-Exchange Chromatography
SHCH-I (5 mL, 40.0 mg/mL) was injected onto a DEAE-52 cellulose column (1.6 × 70 cm) that was pre-equilibrated with deionized water and stepwise eluted with 150 mL distilled water and 0.1, 0.5, and 1.0 M NaCl solution at a flow rate of 1.0 mL/min. Each eluted fraction (5 mL) was collected and measured at 280 nm, and five fractions (Fr.1-5) were pooled and lyophilized.

Determination of the Amino Acid Sequence and Molecular Mass
The amino acid sequences of SCPE-A, SCPE-B and SCPE-C were determined on an Applied Biosystems 494 protein sequencer (Perkin Elmer/Applied Biosystems Inc., Foster City, CA, USA). The molecular masses were determined using a Q-TOF mass spectrometer coupled to an electrospray ionization source (ESI) (Micromass, Waters, Los Angeles, CA, USA).

Degree of Hydrolysis (DH)
DH analysis was performed according to the previously described method [49]. The hydrolysate (50 µL) was mixed with 0.5 mL of 0.2 M phosphate buffer, pH 8.2 and 0.5 mL of 0.05% trinitrobenzenesulfonic acid (TNBS) reagent. TNBS was freshly prepared before use by diluting with DI water. The mixture was incubated at 50 • C for 1 h in a water bath. The reaction was stopped by adding 1 mL of 0.1 M HCl and incubating at room temperature for 30 min. The absorbance was monitored at 420 nm. L-leucine was used as a standard. To determine the total amino acid content, mungbean meal was completely hydrolyzed with 6 M HCl with a sample to acid ratio of 1:100 at 120 • C for 24 h. DH (%) was calculated using the following equation: where A t was the amount of a-amino acids released at time t, A 0 was the amount of a-amino acids in the supernatant at 0 h, and A max was the total amount of a-amino acids obtained after acid hydrolysis at 120 • C for 24 h.

Antioxidant Activity
The radical (DPPH•, HO•, O − 2 •, and ABTS + •) scavenging activity and lipid peroxidation inhibition assays were performed according to previously reported methods [19,51], and the half elimination ratio (EC 50 ) was defined as the concentration of a sample that caused a 50% decrease in the initial concentration of DPPH•, O − 2 •, HO•, and ABTS + •. The EC 50 was calculated based on the linear relationship of the radical-scavenging rate and concentration of the samples.

HO• Scavenging Activity
In this system, hydroxyl radicals are generated by the Fenton reaction. Hydroxyl radicals can oxidize Fe 2+ into Fe 3+ , and only Fe 2+ can combine with 1,10-phenanthroline to form a red compound (1,10-phenanthroline-Fe 2+ ) with the maximum absorbance at 536 nm. The concentration of hydroxyl radical is reflected by the degree of decolorization of the reaction solution. Briefly, 1,10-phenanthroline solution (1.0 mL, 1.865 mM) and the sample (2.0 mL) were added into a screw-capped tube and mixed. The FeSO 4 ·7H 2 O solution (1.0 mL, 1.865 mM) was then pipetted into the mixture. The reaction was initiated by adding 1.0 mL H 2 O 2 (0.03% v/v). After being incubated at 37 • C for 60 min in a water bath, the absorbance of the reaction mixture was measured at 536 nm against a reagent blank. The reaction mixture without any antioxidant was used as the negative control, and mixture without H 2 O 2 was used as the blank. The HO• scavenging activity was calculated by the following formula: where A s , A n , and A b were the absorbance values determined at 536 nm of the sample, the negative control, and the blank after reaction, respectively.

DPPH• Scavenging Activity
Two milliliters of deionized water containing different concentrations of samples were placed in cuvettes, and then 500 µL of ethanol solution of DPPH (0.02%) and 1.0 mL of ethanol were added into. A control sample containing DPPH solution without sample was also prepared. For the blank absorbance, DPPH solution was substituted with ethanol. The antioxidant activity of the sample was evaluated by the inhibition percentage of DPPH• with the following equation: DPPH• scavenging activity (%) = (A 0 + A − A)/A 0 × 100% where A was sample absorbance rate; A 0 was the absorbance of control group; A was the blank absorbance.
3.6.3. O − 2 • Scavenging Activity In the experiment, superoxide anions were generated in 1 mL of nitrotetrazolium blue chloride (NBT) (2.52 mM), 1 mL of nicotinamide adenine dinucleotide (NADH) (624 mM) and 1 mL of different concentrations of samples. The reaction was initiated by adding 1 mL of phenazine methosulfate (PMS) solution (120 µg) to the reaction mixture. The absorbance was measured at 560 nm against the corresponding blank after 5 min of incubation at 25 • C. The capacity of scavenging the O − 2 • was calculated using the following equation: where A control was the absorbance without sample and A sample was the absorbance with sample.

ABTS + • Scavenging Activity
The ABTS radical cation was generated by mixing ABTS stock solution (7 mM) with potassium persulphate (2.45 mM). The mixture was left in the dark at room temperature for 16 h. The ABTS radical solution was diluted in 5 mM phosphate buffered saline (PBS) pH 7.4, to an absorbance of 0.70 ± 0.02 at 734 nm. One milliliter of diluted ABTS radical solution was mixed with one milliliter of different concentrations of samples. Ten minutes later, the absorbance was measured at 734 nm against the corresponding blank. The ABTS + • scavenging activity of samples was calculated using the following equation: ABTS + • scavenging activity (%) = [(A control − A sample )/A control ] × 100% where A control was the absorbance without sample and A sample was the absorbance with sample.

Lipid Peroxidation Inhibition Assay
A sample (5.0 mg) was dissolved in 10 mL of 50 mM phosphate buffer (pH 7.0), and added to a solution of 0.13 mL of linoleic acid and 10 mL of 99.5% ethanol. Then, the total volume was adjusted to 25 mL with deionized water. The mixture was incubated in a conical flask with a screw cap at 40 ± 1 • C in a dark room and the degree of oxidation was evaluated by measuring the ferric thiocyanate values. The reaction solution (100 µL) incubated in the linoleic acid model system was mixed with 4.7 mL of 75% ethanol, 0.1 mL of 30% ammonium thiocyanate, and 0.1 mL of 20 mM ferrous chloride solution in 3.5% HCl. After 3 min, the thiocyanate value was measured by reading the absorbance at 500 nm following color development with FeCl 2 and thiocyanate at different intervals during the incubation period at 40 ± 1 • C.

Statistical Analysis
All experiments were performed in triplicate (n = 3), and the data are expressed as the mean ± standard deviation (SD). ANOVA was applied to analyze the data using SPSS 19.0 (SPSS Corporation, Chicago, IL, USA). Duncan's multiple range test was used to measure the differences between the parameter means. The differences were considered significant if p < 0.05.

Conclusions
In this study, three new antioxidant peptides (SCPE-A, SCPE-B and SCPE-C) were isolated from the protein hydrolysate of scalloped hammerhead (S. lewini) cartilage by ultrafiltration and chromatography, and their amino acid sequences were identified as Gly-Pro-Glu (GPE), Gly-Ala-Arg-Gly-Pro-Gln (GARGPQ), and Gly-Phe-Thr-Gly-Pro-Pro-Gly-Phe-Asn-Gly (GFTGPPGFNG). SCPE-A, SCPE-B and SCPE-C exhibited strong radical scavenging and lipid peroxidation inhibition activities. These results suggested that the purified peptides from the protein hydrolysate of scalloped hammerhead cartilage may be applied as ingredients in functional foods in bioactive food products. Our subsequent studies will focus on the molecular mechanisms and the relationship between the antioxidant activity and structure of the three isolated peptides.