Antioxidant Peptides from Collagen Hydrolysate of Redlip Croaker (Pseudosciaena polyactis) Scales: Preparation, Characterization, and Cytoprotective Effects on H2O2-Damaged HepG2 Cells

Bioactive peptides from fish collagens with antioxidant properties have become a topic of great interest for health, food, and processing/preservation industries. To explore the high-value utilized way of scales produced during the fish processing, collagen hydrolysates of redlip croaker (Pseudosciaena polyactis) scales were prepared using six different proteases, and the hydrolysate (RSCH) prepared using neutrase showed the highest degree of hydrolysis (21.36 ± 1.18%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH·) radical scavenging activity (30.97 ± 1.56%) among the six hydrolysates. Subsequently, six antioxidant peptides were purified from RSCH using membrane ultrafiltration and serial chromatography, and their amino acid sequences were identified as DGPEGR, GPEGPMGLE, EGPFGPEG, YGPDGPTG, GFIGPTE, and IGPLGA with molecular masses of 629.61, 885.95, 788.96, 762.75, 733.80, and 526.61 Da, respectively. Among six collagen peptides, GPEGPMGLE, EGPFGPEG, and GFIGPTE exhibited the strongest scavenging activities on DPPH· radical (EC50 0.59, 0.37, and 0.45 mg/mL), hydroxyl radical (EC50 0.45, 0.33, and 0.32 mg/mL), and superoxide anion radical (EC50 0.62, 0.47, and 0.74 mg/mL). GPEGPMGLE, EGPFGPEG, and GFIGPTE showed high inhibiting ability on lipid peroxidation in a linoleic acid model system and protective activities on oxidation-damaged DNA. More importantly, GPEGPMGLE, EGPFGPEG, and GFIGPTE could protect HepG2 cells from H2O2-induced oxidative damage through decreasing the levels of reactive oxygen species (ROS) and MDA and activating intracellular antioxidant enzymes of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). These results suggested that six collagen peptides (RCP1–RCP6), especially GPEGPMGLE, EGPFGPEG, and GFIGPTE, might serve as potential antioxidants applied in nutraceutical and pharmaceutical products.


Introduction
Collagen from mammal resources is widely employed in multifarious human applications, such as food, cosmetics, pharmaceutical/biomedical, and nutraceutical products [1][2][3]. However, the application of mammal collagen and its derivatives from mammalian species arouses extensive attention and anxiety of consumers due to disease transmission-connected reasons, allergy problems, Table 1. Degree of hydrolysis (DH, %) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) scavenging activity (%) of collagen hydrolysate of redlip croaker (P. polyactis) scale using six kinds of enzymes. All data are presented as the mean ± standard deviation (SD, n = 3). a-e Values with the same letters in each column indicate no significant difference (p > 0.05).

Fractionation of RSCH
Using ultrafiltration with molecular weight cut off (MWCO) membranes of 1, 3 and 5 kDa, RSCH was fractionated into four fractions, including RSCH-I (<1 kDa), RSCH-II (1-3 kDa), RSCH-III (3-5 kDa) and RSCH-IV (>5 kDa). As shown in Figure 1, the DPPH· scavenging activity of RSCH-I was 43.26 ± 2.76%, which was significantly stronger than those of RSCH (30.97 ± 1.53%), RSCH -II (35.63 ± 2.18%), RSCH-III (29.74 ± 1.39%), and RSCH-IV (19.34 ± 0.98%) at the concentration of 10 mg/mL (p < 0.05). Peptides in collagen hydrolysates showed different antioxidant activity due to their different amino acid compositions and chain lengths [19,35]. Previous reports indicated that hydrolysate fractions with smaller MW showed stronger antioxidant activity than those of larger MW hydrolysates because peptides with a short chain length were more accessible to free radicals and easy to trap the free radical [18,35]. The present result was in line with previous reports and RSCH-I accounting for 22.54 ± 1.73% of RSCH was selected for the subsequent chromatographic separation.
Mar. Drugs 2020, 18, x 4 of 19 and easy to trap the free radical [18,35]. The present result was in line with previous reports and RSCH-I accounting for 22.54 ± 1.73% of RSCH was selected for the subsequent chromatographic separation.

Anion-Exchange Chromatography
As shown in Figure 2A, five fractions (AEC-1 to AEC-5) were separated from RSCH-I using a DEAE-52 cellulose column. Amongst them, AEC-1 and AEC-2 were eluted using deionized water (DW); AEC-3 and AEC-4 were eluted using 0.1 mol/L NaCl; and AEC-5 was eluted using 0.5 mol/L NaCl. DPPH· radical scavenging activities of RSCH-I and its five fractions are presented in Figure  2B, and the results indicated that DPPH· radical scavenging activity of AEC-5 was 57.68 ± 3.21% at the concentration of 10 mg/mL, which was significantly stronger than those of RSCH (30.97 ± 1.53%), RSCH-I (43.26 ± 2.76%), AEC-1 (15.63 ± 0.95%), AEC-2 (19.74 ± 1.57%), AEC-3 (39.34 ± 2.55%), and AEC-4 (45.16 ± 2.38%), respectively (p < 0.05). Bioactive peptides usually contain basic and/or acidic amino acid residues, which can be adsorbed to the anion and/or cation exchange resins, and the interaction strength depends on the number and location of the charges on the peptide sequences [18,36]. Acidic and hydrophobic amino acid residues in peptide sequences can be absorbed by the anion-exchange resins on hydrogen bonds and/or van der Waals forces. The present results indicated that AEC-5 might contain some antioxidant peptides with acidic and hydrophobic amino acid residues. Therefore, AEC-5 accounting for 10.38 ± 0.68% of RSCH-I were selected for the following experiment.

Gel Filtration Chromatography (GFC)
As shown in Figure 3A, AEC-5 was further fractionated into three fractions (GFC-1 to GFC-3) using a Sephadex G-25 column, and their DPPH· radical scavenging activities were presented in

Anion-Exchange Chromatography
As shown in Figure 2A, five fractions (AEC-1 to AEC-5) were separated from RSCH-I using a DEAE-52 cellulose column. Amongst them, AEC-1 and AEC-2 were eluted using deionized water (DW); AEC-3 and AEC-4 were eluted using 0.1 mol/L NaCl; and AEC-5 was eluted using 0.5 mol/L NaCl. DPPH· radical scavenging activities of RSCH-I and its five fractions are presented in Figure 2B, and the results indicated that DPPH· radical scavenging activity of AEC-5 was 57.68 ± 3.21% at the concentration of 10 mg/mL, which was significantly stronger than those of RSCH (30.97 ± 1.53%), RSCH-I (43.26 ± 2.76%), AEC-1 (15.63 ± 0.95%), AEC-2 (19.74 ± 1.57%), AEC-3 (39.34 ± 2.55%), and AEC-4 (45.16 ± 2.38%), respectively (p < 0.05). Bioactive peptides usually contain basic and/or acidic amino acid residues, which can be adsorbed to the anion and/or cation exchange resins, and the interaction strength depends on the number and location of the charges on the peptide sequences [18,36]. Acidic and hydrophobic amino acid residues in peptide sequences can be absorbed by the anion-exchange resins on hydrogen bonds and/or van der Waals forces. The present results indicated that AEC-5 might contain some antioxidant peptides with acidic and hydrophobic amino acid residues. Therefore, AEC-5 accounting for 10.38 ± 0.68% of RSCH-I were selected for the following experiment.
Mar. Drugs 2020, 18, x 4 of 19 and easy to trap the free radical [18,35]. The present result was in line with previous reports and RSCH-I accounting for 22.54 ± 1.73% of RSCH was selected for the subsequent chromatographic separation.

Anion-Exchange Chromatography
As shown in Figure 2A, five fractions (AEC-1 to AEC-5) were separated from RSCH-I using a DEAE-52 cellulose column. Amongst them, AEC-1 and AEC-2 were eluted using deionized water (DW); AEC-3 and AEC-4 were eluted using 0.1 mol/L NaCl; and AEC-5 was eluted using 0.5 mol/L NaCl. DPPH· radical scavenging activities of RSCH-I and its five fractions are presented in Figure  2B, and the results indicated that DPPH· radical scavenging activity of AEC-5 was 57.68 ± 3.21% at the concentration of 10 mg/mL, which was significantly stronger than those of RSCH (30.97 ± 1.53%), RSCH-I (43.26 ± 2.76%), AEC-1 (15.63 ± 0.95%), AEC-2 (19.74 ± 1.57%), AEC-3 (39.34 ± 2.55%), and AEC-4 (45.16 ± 2.38%), respectively (p < 0.05). Bioactive peptides usually contain basic and/or acidic amino acid residues, which can be adsorbed to the anion and/or cation exchange resins, and the interaction strength depends on the number and location of the charges on the peptide sequences [18,36]. Acidic and hydrophobic amino acid residues in peptide sequences can be absorbed by the anion-exchange resins on hydrogen bonds and/or van der Waals forces. The present results indicated that AEC-5 might contain some antioxidant peptides with acidic and hydrophobic amino acid residues. Therefore, AEC-5 accounting for 10.38 ± 0.68% of RSCH-I were selected for the following experiment.

Gel Filtration Chromatography (GFC)
As shown in Figure 3A, AEC-5 was further fractionated into three fractions (GFC-1 to GFC-3) using a Sephadex G-25 column, and their DPPH· radical scavenging activities were presented in Figure 2. Elution profile of RSCH-I in DEAE-52 cellulose anion-exchange chromatography (A) and DPPH· radical scavenging activities (%) (B) of RSCH-I and its fractions at 10 mg/mL concentration. All data on DPPH· scavenging activity are presented as the mean ± SD (n = 3). (B) a-f Values with the same letters indicate no significant difference (p > 0.05).

Purification of Peptides from GFC-2 by RP-HPLC
Finally, GFC-2 was separated by the RP-HPLC system on an Agilent 1260 HPLC system with an Agilent Zorbax C-18 column, and the eluted peptides were collected separately on the chromatographic peaks ( Figure 4 Figure 3B. The data indicated that DPPH· scavenging activity of GFC-2 was 84.31 ± 2.24% at the concentration of 10 mg/mL, which was significantly stronger than those of RSCH (30.97 ± 1.53%), RSCH-I (43.26 ± 2.76%), AEC-5 (57.68 ± 3.21%), GFC-1 (49.39 ± 2.35%), and GFC-3 (25.25 ± 1.02%), respectively (p < 0.05) (p < 0.05). GFC is a popular method for the preparation of proteins or peptides on their molecular size used in food products and pharmaceutical industries [18]. The results further proved that molecular size is one of the key factors governing the antioxidative capacity of peptides. Therefore, GFC-2 accounting for 33.45 ± 2.41% of RSCH-I was suitable for the following separation process.

Antioxidant Activity
In the experiment, radical scavenging, lipid peroxidation inhibition, and oxidation-damaged DNA and HepG2 cells protective assays were used to evaluate the antioxidant activity of six CPs (RCP1-RCP6), and the results were presented in Table 3 and Figures 5 and 8. Table 3. Radical scavenging activity of six CPs (RCP1-RCP6) from collagen hydrolysate of redlip croaker (P. polyactis) scales.

No.
EC 50 All data are presented as the mean ± SD (N = 3). a-d Values with the same letters in each column indicate no significant difference of different samples at the same radicals (p > 0.05).

Radical Scavenging Activity
DPPH·Scavenging Activity As shown in Figure 5A, six CPs (RCP1-RCP6) could strong scavenge DPPH· in a concentration-dependent manner, but their scavenging activity was less than that of the positive control of GSH at the same concentration. Table 3 indicated that the EC 50 values of RCP2, RCP3, and RCP5 were 0.59 ± 0.06, 0.37 ± 0.05, and 0.45 ± 0.06 mg/mL, which were significantly lower than those of RCP1 (4.24 ± 0.18 mg/mL), RCP4 (1.76 ± 0.12 mg/mL), RCP6 (2.96 ± 0.17 mg/mL), respectively (p < 0.05). Furthermore, the EC 50 values of RCP2, RCP3 and RCP5 were lower than those of most antioxidant peptides from protein hydrolysates of hairtail (Trichiurus japonicas) muscle (QNDER: 4.95 mg/mL; TKA: 1.43 mg/mL) [37], salmon pectoral fin (TTANIEDRR: 2.503 mg/mL) [38], blue mussel (Mytilus edulis) (YPPAK: 2.62 mg/mL) [39], blood cockle (Tegillarca granosa) (EPLSD: 1.23 ± 0.09 mg/mL; WLDPDG: 1.82 ± 0.16 mg/mL; EPVV: 1.13 ± 0.14 mg/mL; CYIE: 1.30 ± 0.11 mg/mL) [40], and skipjack tuna (Katsuwonus pelamis) scales (His-Gly-Pro-Hyp-Gly-Glu: 1.34 mg/mL) [15]. Then, six CPs (RCP1-RCP6), especially RCP2, RCP3 and RCP5, had a strong radical scavenging capability to act as a hydrogen donor for preventing the DPPH· reaction.  Figure 5B showed that six CPs (RCP1-RCP6) could effectively clear HO· at their concentrations between 0 and 5.0 mg/mL. The EC50 values of RCP2 (0.45 ± 0.05 mg/mL), RCP3 (0.33 ± 0.04 mg/mL) and RCP5 (0.42 ± 0.05 mg/mL) were significantly lower than those of RCP1 (1.65 ± 0.11 mg/mL), RCP4 (1.95 ± 0.14 mg/mL), and RCP6 (4.47 ± 0.24 mg/mL), respectively (p < 0.05). However, there were no significantly differences among RCP2, RCP3 and RCP5 (p > 0.05). Moreover, the EC50 values of RCP2, RCP3 and RCP5 were lower than those of antioxidant peptides from protein hydrolysates of hairtail (T. japonicas) muscle (His-Gly-Pro-Hyp-Gly-Glu: 1.03 mg/mL; MLGPFGPS: 0.74 mg/mL) [37], loach (Misgurnus anguillicaudatus) (PSYV: 2.64 mg/mL) [41], blood cockle (T. granosa) (EPLSD: 2.18 ± 0.16 mg/mL; WLDPDG: 1.54 ± 0.11 mg/mL; EPVV: 1.09 ± 0.08 mg/mL; CYIE: 1.29 ± 0.13 mg/mL) [40], and conger eel (Conger myriaster) (LGLNGDDVN: 0.687 mg/mL) [42]. HO· could initiate the oxidative stress to damage biomacromolecules in organisms. The finding indicated that RCP2, RCP3, and RCP5 could reduce the HO· damage in biological systems through acting as a radical scavenger. HO· Scavenging Activity Figure 5B showed that six CPs (RCP1-RCP6) could effectively clear HO· at their concentrations between 0 and 5.0 mg/mL. The EC 50 values of RCP2 (0.45 ± 0.05 mg/mL), RCP3 (0.33 ± 0.04 mg/mL) and RCP5 (0.42 ± 0.05 mg/mL) were significantly lower than those of RCP1 (1.65 ± 0.11 mg/mL), RCP4 (1.95 ± 0.14 mg/mL), and RCP6 (4.47 ± 0.24 mg/mL), respectively (p < 0.05). However, there were no significantly differences among RCP2, RCP3 and RCP5 (p > 0.05). Moreover, the EC 50 values of RCP2, RCP3 and RCP5 were lower than those of antioxidant peptides from protein hydrolysates of hairtail (T. japonicas) muscle (His-Gly-Pro-Hyp-Gly-Glu: 1.03 mg/mL; MLGPFGPS: 0.74 mg/mL) [37], loach (Misgurnus anguillicaudatus) (PSYV: 2.64 mg/mL) [41], blood cockle (T. granosa) (EPLSD: 2.18 ± 0.16 mg/mL; WLDPDG: 1.54 ± 0.11 mg/mL; EPVV: 1.09 ± 0.08 mg/mL; CYIE: 1.29 ± 0.13 mg/mL) [40], and conger eel (Conger myriaster) (LGLNGDDVN: 0.687 mg/mL) [42]. HO· could initiate the oxidative stress to damage biomacromolecules in organisms. The finding indicated that RCP2, RCP3, and RCP5 could reduce the HO· damage in biological systems through acting as a radical scavenger.  Figure 5C indicates that six CPs (RCP1-RCP6) could strongly scavenge O − 2 · when their concentrations are between 0 and 5.0 mg/mL. The EC 50 values of RCP1-RCP6 were 7.98 ± 0.34, 0.62 ± 0.07, 0.47 ± 0.05, 0.99 ± 0.10, 0.74 ± 0.06, and 2.43 ± 0.13 mg/mL, respectively. RCP2 and RCP3 showed the highest O − 2 · scavenging ability among six CPs (RCP1-RCP6). In addition, the EC 50 values of RCP2 and RCP3 were lower than those of antioxidant peptides from protein hydrolysates of blood cockle (T. granosa) (EPLSD: 2.04 ± 0.23 mg/mL; WLDPDG: 2.49 ± 0.17 mg/mL; EPVV: 1.69 ± 0.14 mg/mL; CYIE: 2.31 ± 0.15 mg/mL) [40], bluefin leatherjacket (Navodon septentrionalis) heads (GVPLT: 2.8819 mg/mL) [36], skipjack tuna (K. pelamis) scales (His-Gly-Pro-Hyp-Gly-Glu: 1.19 mg/mL; DGPKGH: 0.71 mg/mL; MLGPFGPS: 1.59 mg/mL) [15], and hairtail (T. japonicas) muscle (AKG: 2.538 mg/mL; IYG: 1.355 mg/mL) [37]. O − 2 · is a main cause of oxidative stress because it can generate the highly reactive HO· and create toxic peroxy radicals. Therefore, RCP2 and RCP3 can play important roles in eliminating O − 2 · damage together with SOD in organisms. Figure 6 showed that the absorbance values of RCP2, RCP3, and RCP5 solutions at 500 nm were a little higher than that of the positive control of GSH and significantly lower than those of the blank control (without antioxidant) and the other three CPs (RCP1, RCP4, and RCP6). The data indicated that the abilities of RCP2 and RCP5 on lipid peroxidation inhibition were similar to that of GSH in the linoleic acid model system during 7 days incubation. Lipid oxidation involves in multifarious reactions of lipid radicals and produces various primary and secondary by-products, which significantly affect with food quality, nutritional, and health implications [18,36]. The present results indicated that RCP2 and RCP5 could serve as antioxidants used in food and healthy products because of their significant inhibitory effect on the lipid peroxidation.  Figure 5C indicates that six CPs (RCP1-RCP6) could strongly scavenge O -2 · when their concentrations are between 0 and 5.0 mg/mL. The EC50 values of RCP1-RCP6 were 7.98 ± 0.34, 0.62 ± 0.07, 0.47 ± 0.05, 0.99 ± 0.10, 0.74 ± 0.06, and 2.43 ± 0.13 mg/mL, respectively. RCP2 and RCP3 showed the highest O -2 · scavenging ability among six CPs (RCP1-RCP6). In addition, the EC50 values of RCP2 and RCP3 were lower than those of antioxidant peptides from protein hydrolysates of blood cockle (T. granosa) (EPLSD: 2.04 ± 0.23 mg/mL; WLDPDG: 2.49 ± 0.17 mg/mL; EPVV: 1.69 ± 0.14 mg/mL; CYIE: 2.31 ± 0.15 mg/mL) [40], bluefin leatherjacket (Navodon septentrionalis) heads (GVPLT: 2.8819 mg/mL) [36], skipjack tuna (K. pelamis) scales (His-Gly-Pro-Hyp-Gly-Glu: 1.19 mg/mL; DGPKGH: 0.71 mg/mL; MLGPFGPS: 1.59 mg/mL) [15], and hairtail (T. japonicas) muscle (AKG: 2.538 mg/mL; IYG: 1.355 mg/mL) [37]. O -2 · is a main cause of oxidative stress because it can generate the highly reactive HO· and create toxic peroxy radicals. Therefore, RCP2 and RCP3 can play important roles in eliminating O -2 · damage together with SOD in organisms. Figure 6 showed that the absorbance values of RCP2, RCP3, and RCP5 solutions at 500 nm were a little higher than that of the positive control of GSH and significantly lower than those of the blank control (without antioxidant) and the other three CPs (RCP1, RCP4, and RCP6). The data indicated that the abilities of RCP2 and RCP5 on lipid peroxidation inhibition were similar to that of GSH in the linoleic acid model system during 7 days incubation. Lipid oxidation involves in multifarious reactions of lipid radicals and produces various primary and secondary by-products, which significantly affect with food quality, nutritional, and health implications [18,36]. The present results indicated that RCP2 and RCP5 could serve as antioxidants used in food and healthy products because of their significant inhibitory effect on the lipid peroxidation.  Figure 7B). Figure 7C-F indicated the contents of OCF of plasmid DNA were significantly less than that of Figure 7B, which indicated that RCP2, RCP3, RCP5, and GSH had different protective function on plasmid DNA against HO· damage, and the protective abilities of RCP2 and RCP3 were stronger than that of RCP5. In addition, the images of RCP2 and  Figure 7B). Figure 7C-F indicated the contents of OCF of plasmid DNA were significantly less than that of Figure 7B, which indicated that RCP2, RCP3, RCP5, and GSH had different protective function on plasmid DNA against HO· damage, and the protective abilities of RCP2 and RCP3 were stronger than that of RCP5. In addition, the images of RCP2 and RCP3 were similar to that of the normal control (Figure 7). Oxidative DNA damage can cause toxicity and mutations, which further lead to cancer and premature aging [43,44]. Therefore, the present results indicated that RCP2 and RCP3 should have strong ability of protecting DNA from the oxidative damage through scavenging HO· generated from the reaction of Fe 2+ with H 2 O 2 and/or inhibiting the reaction. RCP3 were similar to that of the normal control (Figure 7). Oxidative DNA damage can cause toxicity and mutations, which further lead to cancer and premature aging [43,44]. Therefore, the present results indicated that RCP2 and RCP3 should have strong ability of protecting DNA from the oxidative damage through scavenging HO· generated from the reaction of Fe 2+ with H2O2 and/or inhibiting the reaction. As shown in Figure 8, RCP2, RCP3, and RCP5 exhibited no significantly effect on the viability of HepG2 Cells compared to the blank control group at the concentrations of 100.0 µM for 24 h treatment (P < 0.05). Cell viability is a determination of living or dead cell rate in a total cell sample, which is generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects [45]. The result indicated that RCP2, RCP3, and RCP5 showed no significant cytotoxic activity and could serve as the candidate biomolecules for the development of antioxidant food and drugs.  As shown in Figure 8, RCP2, RCP3, and RCP5 exhibited no significantly effect on the viability of HepG2 Cells compared to the blank control group at the concentrations of 100.0 µM for 24 h treatment (p < 0.05). Cell viability is a determination of living or dead cell rate in a total cell sample, which is generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects [45]. The result indicated that RCP2, RCP3, and RCP5 showed no significant cytotoxic activity and could serve as the candidate biomolecules for the development of antioxidant food and drugs. RCP3 were similar to that of the normal control ( Figure 7). Oxidative DNA damage can cause toxicity and mutations, which further lead to cancer and premature aging [43,44]. Therefore, the present results indicated that RCP2 and RCP3 should have strong ability of protecting DNA from the oxidative damage through scavenging HO· generated from the reaction of Fe 2+ with H2O2 and/or inhibiting the reaction. As shown in Figure 8, RCP2, RCP3, and RCP5 exhibited no significantly effect on the viability of HepG2 Cells compared to the blank control group at the concentrations of 100.0 µM for 24 h treatment (P < 0.05). Cell viability is a determination of living or dead cell rate in a total cell sample, which is generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects [45]. The result indicated that RCP2, RCP3, and RCP5 showed no significant cytotoxic activity and could serve as the candidate biomolecules for the development of antioxidant food and drugs.   Figure 9 showed the influences of RCP2, RCP3, and RCP5 on H 2 O 2 -damaged HepG2 cells, and the cell viability of RCP2, RCP3, and RCP5 treated groups were 86.79 ± 3.86%, 72.37 ± 3.35%, and 81.47 ± 2.45% at the concentrations of 100.0 µg/mL, respectively, which were significantly higher than that of model (treated with H 2 O 2 at the concentration of 300 µM) group (49.58 ± 3.91%) (p < 0.001). Oxidative stress reflects an imbalance between the systemic manifestation of ROS and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage [46]. Long-term oxidative stress can product excess peroxides and ROS, which damage functional molecules within cells and lead to aging, cancer, diabetes, atherosclerosis, inflammatory disorders, chronic fatigue syndrome, and Alzheimer's and Parkinson's diseases [47,48]. The finding suggested that RCP2, RCP3, and RCP5 could strongly protect HepG2 cells against H 2 O 2 -induced oxidative damage.  Figure 9 showed the influences of RCP2, RCP3, and RCP5 on H2O2-damaged HepG2 cells, and the cell viability of RCP2, RCP3, and RCP5 treated groups were 86.79 ± 3.86%, 72.37 ± 3.35%, and 81.47 ± 2.45% at the concentrations of 100.0 µg/mL, respectively, which were significantly higher than that of model (treated with H2O2 at the concentration of 300 µM) group (49.58 ± 3.91%) (P < 0.001). Oxidative stress reflects an imbalance between the systemic manifestation of ROS and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage [46]. Long-term oxidative stress can product excess peroxides and ROS, which damage functional molecules within cells and lead to aging, cancer, diabetes, atherosclerosis, inflammatory disorders, chronic fatigue syndrome, and Alzheimer's and Parkinson's diseases [47,48]. The finding suggested that RCP2, RCP3, and RCP5 could strongly protect HepG2 cells against H2O2-induced oxidative damage.

Effect of RCP2, RCP3, and RCP5 on the Levels of ROS in H2O2-Damaged HepG2 cells
The effects of RCP2, RCP3, and RCP5 on the levels of ROS in H2O2-damaged HepG2 cells were determined and presented in Figure 10. The level of ROS observed in the HepG2 exposed to H2O2 were 227.2 ± 14.8% of the blank control, which was significantly higher than those of the blank control group (P < 0.001). As expected, the intracellular ROS levels were significantly decreased by RCP2 (140.6 ± 10.8%), RCP3 (169.1 ± 8.6%), and RCP5 (182.1 ± 8.9%) at the concentrations of 100 µM. In addition, the intracellular ROS levels of RCP2 and RCP3 were significantly lower than that of model group (P < 0.001). ROS are generated by different physiological and biochemical oxidative processes in the organisms and associated with numerous physiological and pathophysiological processes [41,42]. High levels of intracellular ROS can oxidize and damage some key biological macromolecules and cytomembrane and play a major role in the pathogenesis of various human diseases [43,44]. The present data indicated that RCP2, RCP3, and RCP5 could protect HepG2 cells against the H2O2-induced oxidative damage through decreasing the level of ROS.

Effect of RCP2, RCP3, and RCP5 on the Levels of ROS in H 2 O 2 -Damaged HepG2 Cells
The effects of RCP2, RCP3, and RCP5 on the levels of ROS in H 2 O 2 -damaged HepG2 cells were determined and presented in Figure 10. The level of ROS observed in the HepG2 exposed to H 2 O 2 were 227.2 ± 14.8% of the blank control, which was significantly higher than those of the blank control group (p < 0.001). As expected, the intracellular ROS levels were significantly decreased by RCP2 (140.6 ± 10.8%), RCP3 (169.1 ± 8.6%), and RCP5 (182.1 ± 8.9%) at the concentrations of 100 µM. In addition, the intracellular ROS levels of RCP2 and RCP3 were significantly lower than that of model group (P < 0.001). ROS are generated by different physiological and biochemical oxidative processes in the organisms and associated with numerous physiological and pathophysiological processes [41,42]. High levels of intracellular ROS can oxidize and damage some key biological macromolecules and cytomembrane and play a major role in the pathogenesis of various human diseases [43,44]. The present data indicated that RCP2, RCP3, and RCP5 could protect HepG2 cells against the H 2 O 2 -induced oxidative damage through decreasing the level of ROS.  Figure 11A-C showed the effects of RCP2, RCP3, and RCP5 on the levels of intracellular antioxidant enzymes (SOD, CAT, and GSH-Px). Compared with the blank control group, the levels of SOD, CAT, and GSH-Px were significantly decreased by H2O2 in the HepG2 cells (P < 0.001), which indicated that the oxidative stress induced by H2O2 seriously damaged the enzymatic antioxidant defense system of HepG2 cells. However, the levels of SOD, CAT, and GSH-Px in HepG2 cells incubated by RCP2, RCP3, and RCP5 significantly higher than that of the H2O2 damaged group (P < 0.05). In addition, the effect of RCP2 on the enzymatic antioxidant defense system was higher than those of RCP3 and RCP5.  Figure 11D showed that the content of MDA (20.14 ± 1.58 nM/mg prot) in the H2O2-damaged HepG2 cells was significantly (P < 0.05) reduced compared with that of the blank control group (9.18 ± 0.54 nM/mg prot). At the concentration of 100 µM, the MDA content of RCP2 incubated Effects of RCP2, RCP3, and RCP5 on the Level of Intracellular Antioxidant Enzymes and MDA in H 2 O 2 -Damaged HepG2 Cells Figure 11A-C showed the effects of RCP2, RCP3, and RCP5 on the levels of intracellular antioxidant enzymes (SOD, CAT, and GSH-Px). Compared with the blank control group, the levels of SOD, CAT, and GSH-Px were significantly decreased by H 2 O 2 in the HepG2 cells (p < 0.001), which indicated that the oxidative stress induced by H 2 O 2 seriously damaged the enzymatic antioxidant defense system of HepG2 cells. However, the levels of SOD, CAT, and GSH-Px in HepG2 cells incubated by RCP2, RCP3, and RCP5 significantly higher than that of the H 2 O 2 damaged group (p < 0.05). In addition, the effect of RCP2 on the enzymatic antioxidant defense system was higher than those of RCP3 and RCP5.  Figure 11A-C showed the effects of RCP2, RCP3, and RCP5 on the levels of intracellular antioxidant enzymes (SOD, CAT, and GSH-Px). Compared with the blank control group, the levels of SOD, CAT, and GSH-Px were significantly decreased by H2O2 in the HepG2 cells (P < 0.001), which indicated that the oxidative stress induced by H2O2 seriously damaged the enzymatic antioxidant defense system of HepG2 cells. However, the levels of SOD, CAT, and GSH-Px in HepG2 cells incubated by RCP2, RCP3, and RCP5 significantly higher than that of the H2O2 damaged group (P < 0.05). In addition, the effect of RCP2 on the enzymatic antioxidant defense system was higher than those of RCP3 and RCP5. Figure 11. The effects of RCP2, RCP3, and RCP5 on the levels of intracellular antioxidant enzymes (SOD (A), CAT (B), and GSH-Px (C)) and content of malondialdehyde (MDA) (D) in H2O2-damaged HepG2 cells. All data are presented as the mean ± SD (n = 3). ### P < 0.001 versus the control group; *** P < 0.001, ** P < 0.01, and * P < 0.05 versus the model group (treated with H2O2 at the concentration of 300 µM). Figure 11D showed that the content of MDA (20.14 ± 1.58 nM/mg prot) in the H2O2-damaged HepG2 cells was significantly (P < 0.05) reduced compared with that of the blank control group (9.18 ± 0.54 nM/mg prot). At the concentration of 100 µM, the MDA content of RCP2 incubated  Figure 11D showed that the content of MDA (20.14 ± 1.58 nM/mg prot) in the H 2 O 2 -damaged HepG2 cells was significantly (p < 0.05) reduced compared with that of the blank control group (9.18 ± 0.54 nM/mg prot). At the concentration of 100 µM, the MDA content of RCP2 incubated group was 13.57 ± 0.96 nM/mg prot, which was lower than those of RCP3 (16.24 ± 1.37 nM/mg prot), and RCP5 (17.65 ± 1.28 nM/mg prot) and the H 2 O 2 treated groups. Moreover, the MDA content of RCP2, RCP3, and RCP5 were significantly lower than that of the model group (p < 0.05). Therefore, RCP2, RCP3, and RCP5 could significantly reduce the oxidative stress injury by restoration of endogenous antioxidation and the decrease of lipid peroxidation.
Cells contain enzymatic and non-enzymatic antioxidant defense systems for combatting and preventing oxidative stress [49]. The enzymatic antioxidant system is mainly comprised of SOD, CAT, and GSH-Px and can hold back the radicals attacking cellular membrane and components [50]. Many dietary antioxidants can help to increase the levels of intracellular antioxidant enzymes to maintain redox homeostasis in cells of the human body [50,51]. MDA is the oxidative metabolite of cell lipid oxidation, and the reduction of oxidative damage in cells was accompanied by the decrease of oxidative metabolites, which give an index to the degree of oxidative damage in cells [52,53]. Recently, antioxidant peptides have received widespread attention due to their nutritional function and activities, especially their ability on increasing the levels and activities of antioxidant enzymes [54]. Tonolo et al. reported that KVLPVPEK derived from milk could activate nuclear factor erythroid 2-related factor (Nrf2) pathway in Caco-2 cells to induce overexpression of phase II enzymes, including thioredoxin 1 (Trx1), thioredoxin reductase (TrxR1), gluthatione reductase (GR), NAD(P)H quinone dehydrogenase 1 (NQO1), and SOD1 [55]. Rahman et al. [56] confirmed that antioxidant peptide of YD1 from the strain Bacillus amyloliquefaciens CBSYD1 has strong antioxidant activity, hydrogen atom transfer ability, and the potential to prevent H 2 O 2 -induced oxidative stress in RAW 264.7 cells through the increase of cellular thiol content, GST activity, the expression of antioxidant enzymes and the expression of the redox-sensitive transcription factor Nrf2. Cai et al. [57] found that FPYLRH from the hydrolysate of miiuy croaker (M. miiuy) swim bladder could significantly increase the levels of SOD and GSH-Px and decrease the contents of ROS, MDA and nitric oxide (NO) in H 2 O 2 -induced oxidative damage human umbilical vein endothelial cells (HUVECs). In addition, FPYLRH could dose-dependently protect DNA in oxidative damage HUVECs model. The present results indicated that the antioxidant activities of RCP2, RCP3, and RCP5 were similar to KVLPVPEK, YD1, and FPYLRH, which can protect different cells from oxidative stress by enhancing endogenous antioxidant enzyme defense system.

Preparation of Protein Hydrolysate from ASC-RC
The dispersions of the ASC-RC (1%, w/v) was separately hydrolyzed by six different proteases according to the designed conditions ( Table 4). After that, the hydrolysates were kept in a 95 • C water bath for 15 min and centrifuged at 8000 g for 30 min. The resulting supernatants were freeze-dried and stored in a −20 • C refrigerator. The collagen hydrolysate prepared using neutrase was named as RSCH. The DH (%) of hydrolysates was measured using the previous method [30].
RSCH-I solution (5 mL, 40.0 mg/mL) was injected into a DEAE-52 cellulose column (1.6 cm × 80 cm) pre-equilibrated with DW, and stepwise eluted with 150 mL DW, 0.1 M NaCl, 0.5 M NaCl, and 1.0 M NaCl solution at a flow rate of 1.0 mL/min, respectively. Each eluate (5 mL) was monitored at 214 nm. Finally, five fractions (ACE-1 to ACE-5) were pooled and lyophilized on their chromatographic peaks. ACE-5 solution (5 mL, 20.0 mg/mL) was separated on a Sephadex G-25 column (2.6 cm × 160 cm) eluted with DW at a flow rate of 0.6 mL/min. Each eluate (3 mL) was collected and monitored at 214 nm, and the fraction of GFC-2 solution (25 µL, 10.0 mg/mL) was further purified on an Agilent 1260 HPLC system (Agilent Ltd., Santa Rosa, CA, USA) with an Agilent Zorbax, SB C-18 column (4.6 mm × 250 mm). The sample was eluated with a linear gradient of ACN (0%-50% in 0-40 min) in 0.1% TFA at a flow rate of 0.8 mL/min, and six CPs (RCP1 to RCP6) were isolated on the absorbance at 214 nm.
RSCH-I solution (5 mL, 40.0 mg/mL) was injected into a DEAE-52 cellulose column (1.6 cm × 80 cm) pre-equilibrated with DW, and stepwise eluted with 150 mL DW, 0.1 M NaCl, 0.5 M NaCl, and 1.0 M NaCl solution at a flow rate of 1.0 mL/min, respectively. Each eluate (5 mL) was monitored at 214 nm. Finally, five fractions (ACE-1 to ACE-5) were pooled and lyophilized on their chromatographic peaks. ACE-5 solution (5 mL, 20.0 mg/mL) was separated on a Sephadex G-25 column (2.6 cm × 160 cm) eluted with DW at a flow rate of 0.6 mL/min. Each eluate (3 mL) was collected and monitored at 214 nm, and the fraction of GFC-2 solution (25 µL, 10.0 mg/mL) was further purified on an Agilent 1260 HPLC system (Agilent Ltd., Santa Rosa, CA, USA) with an Agilent Zorbax, SB C-18 column (4.6 mm × 250 mm). The sample was eluated with a linear gradient of ACN (0%-50% in 0-40 min) in 0.1% TFA at a flow rate of 0.8 mL/min, and six CPs (RCP1 to RCP6) were isolated on the absorbance at 214 nm.

Amino Acid Sequence and Molecular Mass Analysis
The amino acid sequences and molecular masses of six CPs (RCP1 to RCP6) were measured on an Applied Biosystems 494 protein sequencer (Perkin Elmer/Applied Biosystems Inc, Foster City, CA, USA) and a Q-TOF mass spectrometer coupled with an electrospray ionization source, respectively.

Radical Scavenging Assays
The radical scavenging assays of six CPs (RCP1-RCP6) were determined by the previous method [5,30], and the results of the radical scavenging assays were expressed as a half elimination ratio (EC 50 ) defined as the concentration where a sample caused a 50% decrease of the initial radical concentration.
3.5.1. DPPH· Scavenging Activity 2.0 mL of samples consisting of distilled water and different concentrations of the analytes were placed in cuvettes, and 500 µL of an ethanolic solution of DPPH (0.02%) and 1.0 mL of ethanol were added. A control sample containing the DPPH solution without the sample was also prepared. In the blank, the DPPH solution was substituted with ethanol. The DPPH· scavenging activity was calculated using the following formula: where A s is the absorbance rate of the sample, A c is the control group absorbance, and A b is the blank absorbance.

HO· Scavenging Activity
First, 1.0 mL of a 1.865 mM 1,10-phenanthroline solution and 2.0 mL of the sample were added to a screw-capped tube and mixed. Then, 1.0 mL of a FeSO 4 ·7H 2 O solution (1.865 mM) was added to the mixture. The reaction was initiated by adding 1.0 mL of H 2 O 2 (0.03%, v/v). After incubating 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 a mixture without H 2 O 2 was used as the blank. The HO· scavenging activity was calculated using the following formula: where A s , A n , and A b are the absorbance values determined at 536 nm of the sample, the negative control, and the blank after the reaction, respectively.

O −
2 · Scavenging Activity In the experiment, superoxide anions were generated in 1.0 mL of nitrotetrazolium blue chloride (NBT) (2.52 mM), 1.0 mL of NADH (624 mM) and 1 mL of different sample concentrations. The reaction was initiated by adding 1.0 mL of phenazine methosulphate (PMS) solution (120 µM) to the reaction mixture. The absorbance was measured at 560 nm against the corresponding blank after 5-min incubation at 25 • C. The O − 2 · scavenging activity was calculated using the following equation: where A c is the absorbance without sample and A s is the absorbance with sample.

Lipid Peroxidation Inhibition Assay
The lipid peroxidation inhibition activity of the APs was measured in a linoleic acid model system using the method of Wang et al. [30]. Briefly, a sample (5.0 mg) was dissolved in 10 mL of 50 mM PBS (pH 7.0) and added to 0.13 mL of a solution 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 • C in a dark room, and the degree of oxidation was evaluated by measuring 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 at 500 nm following color development with FeCl 2 and thiocyanate at different intervals during the incubation period at 40 • C.

Cell Culture and Cell Viability Assay
MTT test was performed on the previous method to measure the effects of samples on the cell viability [59,60]. In brief, the HepG2 cells were cultured in DMEM medium contained 10% FBS supplemented with 2 mM l-Glu, and 1% penicillin-streptomycin solution at 37 • C and 5% CO 2 atmosphere. After 24 h incubation in a 96-well plate (7 × 10 3 cells/well), the HepG2 cells were cultured at the presence of designed concentrations of peptide solution for 12 h. After that, the wells were washed with phosphate-buffered saline (PBS) for twice and the MTT with the final concentration of 0.5 mg/mL was added into. After 4 h, the formazan crystals formed by active cells were dissolved in 150 µL of dimethyl sulfoxide (DMSO) and the absorbance at 570 nm of the solution was recorded. The cell viability was calculated by the following equation: Cell viability = (A sample /A control ) × 100%.

Cytoprotective Activity of RCP2, RCP3 and RCP5 on the Oxidative Damaged HepG2 Cells by H 2 O 2
The assay was performed on the previous method and the H 2 O 2 concentration of 300 µM was used to build the oxidative damage model of HepG2 cells [48]. In brief, the isolated CPs were dissolved in the Dulbecco's Modified Eagle's medium (DMEM) with the concentration of 100.0 µM. The HepG2 cells were grown (6 × 10 4 cells/well) in a 96-well plate for 24 h. Then the supernatant was aspirated and 100 µL of peptide samples were added into the protection groups respectively for incubating 8 h. After removing peptide samples, H 2 O 2 was respectively added into the damage and protection groups with the optimal concentration (300 µM) and sequentially incubated for 24 h. Finally, the cell viability was measured and calculated on the method of Section 3.8.

Determination of the Levels of ROS in H 2 O 2 -Induced HepG2 cells
Intracellular ROS accumulation in HepG2 cells was monitored according to the previous method described by Zheng et al. [13]. In brief, HepG2 cells were preincubated with samples at the concentrations of 10, 50, or 100 µM for 12.0 h, and then incubated with H 2 O 2 at the concentration of 300 µM for 2 h. After that, the cells were washed with PBS and incubated with 10 µM DCFH2-DA in fresh culture medium for 0.5 h. Intracellular ROS levels indicated by DCF fluorescence were quantified on a BD FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) using excitation and emission filters of 488 and 530 nm, respectively. The data were expressed as % of control values.

Determination of the Levels of Antioxidant Enzymes and MDA in H 2 O 2 -Induced HepG2 Cells
The assay was performed according to the previous methods [27,61]. The HepG2 cells were cultured in 6-well plates (1 × 10 6 cells/well). The isolated peptides (final concentration of 10.0, 50.0, and 100.0 µM, respectively) were added into the protection groups. After that, the damage and protection groups were induced by H 2 O 2 at the concentration of 300 µM. Finally, 500 mL of cell lysis buffer was added into each well on ice lysed for 0.5 h and centrifuged at 12,000 g, 4 • C for 10 min. The resulted liquid supernatant was followed with cold standby at 4 • C (the indicators should be measured in 6 h). The levels of superoxide dismutase (SOD), CAT, GSH-Px and MDA were measured using assay kits according to the protocols of manufacturer. The results were expressed as units of enzymatic activity per milligram of protein (U/mg prot).

Statistical Analysis
The data are reported as the mean ± standard deviation (SD, n = 3). A one-way analysis of variance (ANOVA) test for differences between the means of each group was applied to analyzed data using SPSS 19.0 (Statistical Program for Social Sciences, SPSS Corporation, Chicago, IL, USA).

Conclusions
In the experiment, scale collagen of redlip croaker (P. polyactis) was separately hydrolyzed using six different proteases and six CPs were purified from the collagen hydrolysate prepared using neutrase and identified as DGPEGR, GPEGPMGLE, EGPFGPEG, YGPDGPTG, GFIGPTE, and IGPLGA, respectively. Among them, GPEGPMGLE, EGPFGPEG, and GFIGPTE exhibited the strong radical scavenging activities, lipid peroxidation inhibiting ability, and oxidation-damaged DNA protective activities. Moreover, GPEGPMGLE, EGPFGPEG, and GFIGPTE could protect H 2 O 2 -damaged HepG2 cells from oxidative stress by decreasing ROS and MDA levels and enhancing endogenous antioxidant enzyme (SOD, CAT, and GSH-Px) defense system. These results suggested that the collagen hydrolysate and CPs from redlip croaker scales could serve as functional ingredients in nutraceuticals and pharmaceuticals.