Eight Collagen Peptides from Hydrolysate Fraction of Spanish Mackerel Skins: Isolation, Identification, and In Vitro Antioxidant Activity Evaluation

A previous report indicated that collagen hydrolysate fraction (F7) from Spanish mackerel (Scomberomorous niphonius) skins showed high reducing power and radical scavenging activities on 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (EC50 value of 1.57 mg/mL) and hydroxyl (EC50 value of 1.20 mg/mL). In this work, eight peptides were isolated from F7 and identified as Gly-Pro-Tyr (GPY, 335.31 Da), Gly-Pro-Thr-Gly-Glu (GPTGE, 459.47 Da), Pro-Phe-Gly-Pro-Asp (PFGPD, 531.52 Da), Gly-Pro-Thr-Gly-Ala-Lys (GPTGAKG, 586.65 Da), Pro-Tyr-Gly-Ala-Lys-Gly (PYGAKG, 591.69 Da), Gly-Ala-Thr-Gly-Pro-Gln-Gly (GATGPQG, 586.61 Da), Gly-Pro-Phe-Gly-Pro-Met (GPFGPM, 604.73 Da), and Tyr-Gly-Pro-Met (YGPM, 466.50 Da), respectively. Among them, PFGPD, PYGAKG, and YGPM exhibited strong radical scavenging activities on DPPH (EC50 values of 0.80, 3.02, and 0.72 mg/mL for PFGPD, PYGAKG, and YGPM, respectively), hydroxyl (EC50 values of 0.81, 0.66, and 0.88 mg/mL for PFGPD, PYGAKG, and YGPM, respectively), superoxide anion (EC50 values of 0.91, 0.80, and 0.73 mg/mL for PFGPD, PYGAKG, and YGPM, respectively), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) cation (EC50 values of 0.86, 1.07, and 0.82 mg/mL for PFGPD, PYGAKG, and YGPM, respectively) in a positive concentration–activity relationship. Furthermore, PFGPD, PYGAKG, and YGPM could effectively reduce Fe3+ to Fe2+ and inhibit lipid peroxidation. Hence, eight collagen peptides from hydrolysate of Spanish mackerel skins might be served as antioxidant candidates for various industrial applications.


Introduction
Reactive oxygen species (ROS) derived from molecular oxygen are indispensable cellular components in organisms that take part in cell signaling and homeostasis during physiological processes [1]. However, excess ROS during oxidative stress can result in oxidative damage to cell membranes, proteins, and DNA. Those damage will cause increased issues in a series of chronic diseases including hepatopathy, atherosclerosis, and diabetes [1,2]. In addition, radical mediated oxidation can cause deterioration in food products, which is a main concern during food processing and storage. This concern is because the toxic and smelly secondary metabolites produced by lipid peroxidation

Isolation of APs from FSP-III by RP-HPLC
FSP-III with high radical scavenging activities was further purified on a Kromasil C18 column (10 × 250 mm) in RP-HPLC system and the result is presented in Figure 2 I to FSP-IV) from F7 by ÄKTA avant 25 with a Superdex ® Peptide 10/300 GL column. All data are presented as the mean ± SD of triplicate results. a-e/A-E Values with same superscripts of this type indicate no significant difference (p > 0.05).

Isolation of APs from FSP-III by RP-HPLC
FSP-III with high radical scavenging activities was further purified on a Kromasil C18 column (10 × 250 mm) in RP-HPLC system and the result is presented in Figure 2

Antioxidant Activity
Radical scavenging assay, reducing power assay, and lipid peroxidation inhibition assay were employed to evaluate the activities of eight collagen peptides (F7-P1 to F7-P8), and their EC50 values on DPPH/hydroxyl/superoxide anion/ABTS radicals are presented in Table 2.

Reducing Power
Reducing power is an important index applied to evaluate the antioxidant activities of bioactive peptides through the decrease of Fe 3+ to Fe 2+ [4]. As shown in Figure 4, eight isolated collagen peptides (F7-P1 to F7-P8) showed dose-dependent reducing power when the concentration of tested peptides increased from 0 to 2.5 mg/mL. The present data indicated that F7-P3 had the highest ability to decrease ferric ions (Fe 3+ ) to ferrous ions (Fe 2+ ) when compared with the other seven isolated collagen peptides, but still lower than GSH at the same tested concentrations.  [5], and bluefin leatherjacket heads (GVPLT: 3.124 mg/mL; GPP: 2.472 mg/mL; WEGPK: 5.407 mg/mL) [12]. These findings indicate that eight isolated collagen peptides (F7-P1 to F7-P8), especially F7-P3 and F7-P8 possess a high capacity to turn ABTS + • into its neutral form and inhibit free radical chain reactions.

Reducing Power
Reducing power is an important index applied to evaluate the antioxidant activities of bioactive peptides through the decrease of Fe 3+ to Fe 2+ [4]. As shown in Figure 4, eight isolated collagen peptides (F7-P1 to F7-P8) showed dose-dependent reducing power when the concentration of tested peptides increased from 0 to 2.5 mg/mL. The present data indicated that F7-P3 had the highest ability to decrease ferric ions (Fe 3+ ) to ferrous ions (Fe 2+ ) when compared with the other seven isolated collagen peptides, but still lower than GSH at the same tested concentrations.

Lipid Peroxidation Inhibition Assay
Polyunsaturated lipids in biological membranes are easily oxidized by ROS to produce alkanes, MDA, and 4-hydroxyl 2-nonenal. The process will lead to the lipid peroxidation [28]. Furthermore, secondary metabolites of lipid peroxidation can react with cell macromolecules to create deleterious adducts, which will cause serious irreversible consequences on the biological function of cells, such as membrane permeability and gene mutation [4,29]. As a consequence, capabilities of eight isolated collagen peptides (F7-P1 to F7-P8) on lipid peroxidation inhibition were investigated using the linoleic acid system [16,30]. In the system, detected sample solution with a high absorbance value at 500 nm indicates high oxidation degree of linoleic acid, which highlights that the sample has low capability on inhibiting the oxidation of unsaturated fatty acid. In comparison with the negative control group (without antioxidant), eight isolated collagen peptides (F7-P1 to F7-P8) could effectively inhibit the lipid peroxidation during 7 days at 40 °C ( Figure 5). More importantly, the inhibiting ability of F7-P8 was close to that of the positive control of GSH and noticeably better than those of the other seven isolated collagen peptides. These data suggested that eight isolated collagen peptides (F7-P1 to F7-P8) could react with peroxyl radicals and inhibit the diffusion process of lipid peroxidation.

Lipid Peroxidation Inhibition Assay
Polyunsaturated lipids in biological membranes are easily oxidized by ROS to produce alkanes, MDA, and 4-hydroxyl 2-nonenal. The process will lead to the lipid peroxidation [28]. Furthermore, secondary metabolites of lipid peroxidation can react with cell macromolecules to create deleterious adducts, which will cause serious irreversible consequences on the biological function of cells, such as membrane permeability and gene mutation [4,29]. As a consequence, capabilities of eight isolated collagen peptides (F7-P1 to F7-P8) on lipid peroxidation inhibition were investigated using the linoleic acid system [16,30]. In the system, detected sample solution with a high absorbance value at 500 nm indicates high oxidation degree of linoleic acid, which highlights that the sample has low capability on inhibiting the oxidation of unsaturated fatty acid. In comparison with the negative control group (without antioxidant), eight isolated collagen peptides (F7-P1 to F7-P8) could effectively inhibit the lipid peroxidation during 7 days at 40 • C ( Figure 5). More importantly, the inhibiting ability of F7-P8 was close to that of the positive control of GSH and noticeably better than those of the other seven isolated collagen peptides. These data suggested that eight isolated collagen peptides (F7-P1 to F7-P8) could react with peroxyl radicals and inhibit the diffusion process of lipid peroxidation.

Discussion on Structure-Activity Relationship of APs
APs derived from dietary foods have given rise to more attention because they show enormous potential to be applied for both food and medical purposes due to their capability to promote human health by reducing oxidative stress [2,4]. Clarifying the structure-function relationship will contribute to predicting the bioactivities of peptides and screening the high-quality protein materials for the preparation of APs [7,22]. However, the antioxidant mechanism of food-derived peptides have not been fully worked out. Yet, investigations by Sila and Bougatef [2] and Pan et al. [5] have indicated that the biological functions of peptides are distinctly affected by their amino acid composition, amino acid sequence, and molecular size. In addition, spatial conformation is thought to play a key role in their biological activities [31,32].
APs can act as lipid peroxyl radical trap and hydrogen donor to play their functions [1]. Short peptides containing 2 to 10 amino acids usually show better activity than their parent native proteins do because they can easily react with active radicals, which allows them to display their potent actions in reaction system [2,33]. In the experiment, F7-P3, F7-P5 and F7-P8 showed high antioxidant effect in the assays of radical scavenging, reducing power, and lipid peroxidation inhibition. These findings demonstrated that F7-P3 with five amino acid residues, F7-P5 with six amino acid residues, and F7-P8 with four amino acid residues could more easily and effectively react with free radicals and inhibit the propagating systems of lipid peroxidation in the tested model. Nevertheless, the antioxidant activity of F7-P1 (tripeptide) with the smallest molecular size among eight isolated peptides, was lower than those of F7-P3, F7-P5, and F7-P8. In addition, the reducing power of F7-P8 was weaker than those of F7-P3 and F7-P5. These findings imply that the bioactivities of APs were not only independent of molecular size, but also relied on other structural properties, such as their amino acid composition and amino acid sequences.
It has been proven that the amino acid composition and sequence distinctively influence the bioactivities of APs. Previous investigations showed that Glycine (Gly) residue could supply high backbone flexibility of peptides and neutralize active radical species through donating the single hydrogen atom of its side chain [1,34]. Gly is the richest amino acid in the sequences of collagens, and all members belonging to the collagen family are manifested by repetition domains of the tripeptides (Gly-X-Y) and drawn into the structure of the triple helix [18]. As showed in Table 1, eight isolated collagen peptides (F7-P1 to F7-P8) from hydrolysate of Spanish mackerel skins had the number of Gly residues ranging from one to three, which could be contributed to their antioxidant activities.
Zhao et al. [1] and Gimenez et al. [8] reported that polar amino acids, such as acidic (Asp and Glu) and basic (Lys, Arg, and His) amino acid residues in peptide sequences have a critical function

Discussion on Structure-Activity Relationship of APs
APs derived from dietary foods have given rise to more attention because they show enormous potential to be applied for both food and medical purposes due to their capability to promote human health by reducing oxidative stress [2,4]. Clarifying the structure-function relationship will contribute to predicting the bioactivities of peptides and screening the high-quality protein materials for the preparation of APs [7,22]. However, the antioxidant mechanism of food-derived peptides have not been fully worked out. Yet, investigations by Sila and Bougatef [2] and Pan et al. [5] have indicated that the biological functions of peptides are distinctly affected by their amino acid composition, amino acid sequence, and molecular size. In addition, spatial conformation is thought to play a key role in their biological activities [31,32].
APs can act as lipid peroxyl radical trap and hydrogen donor to play their functions [1]. Short peptides containing 2 to 10 amino acids usually show better activity than their parent native proteins do because they can easily react with active radicals, which allows them to display their potent actions in reaction system [2,33]. In the experiment, F7-P3, F7-P5 and F7-P8 showed high antioxidant effect in the assays of radical scavenging, reducing power, and lipid peroxidation inhibition. These findings demonstrated that F7-P3 with five amino acid residues, F7-P5 with six amino acid residues, and F7-P8 with four amino acid residues could more easily and effectively react with free radicals and inhibit the propagating systems of lipid peroxidation in the tested model. Nevertheless, the antioxidant activity of F7-P1 (tripeptide) with the smallest molecular size among eight isolated peptides, was lower than those of F7-P3, F7-P5, and F7-P8. In addition, the reducing power of F7-P8 was weaker than those of F7-P3 and F7-P5. These findings imply that the bioactivities of APs were not only independent of molecular size, but also relied on other structural properties, such as their amino acid composition and amino acid sequences.
It has been proven that the amino acid composition and sequence distinctively influence the bioactivities of APs. Previous investigations showed that Glycine (Gly) residue could supply high backbone flexibility of peptides and neutralize active radical species through donating the single hydrogen atom of its side chain [1,34]. Gly is the richest amino acid in the sequences of collagens, and all members belonging to the collagen family are manifested by repetition domains of the tripeptides (Gly-X-Y) and drawn into the structure of the triple helix [18]. As showed in Table 1, eight isolated collagen peptides (F7-P1 to F7-P8) from hydrolysate of Spanish mackerel skins had the number of Gly residues ranging from one to three, which could be contributed to their antioxidant activities.
Zhao et al. [1] and Gimenez et al. [8] reported that polar amino acids, such as acidic (Asp and Glu) and basic (Lys, Arg, and His) amino acid residues in peptide sequences have a critical function in the HO• scavenging activities through metal ion chelating, which is positively related to the amino and carboxyl groups of their side chains. Arg and His residues in FPYLRH, and Glu residue in GIEWA show a significant effect on their oxidation resistance [1]. Glu, Asp, and Arg residues in LDEPDPLI and NTDGSTDYGILQINSR showed significant impact on their radical scavenging capacity [35]. Therefore, Asp residues may have a positive influence on the antioxidant capacity of F7-P3.
Hydrophobic amino acids in APs have their significant radical scavenging ability because their non-polar aliphatic groups have been shown to have strong reaction activity to hydrophobic PUFAs [1][2][3]. Aromatic amino acids (Phe and Tyr residues) can maintain ROS at a stable state by donating protons to free radicals that are deficient in electrons [6,32]. Pyrrolidine ring of Pro residue can strengthen the backbone flexibility of bioactive peptides and remove singlet oxygen because of its property of low ionization potential [5]. Zhao et al. [1] proved that Tyr residues could interdict the chain reaction of peroxidation mediated by free radicals because it could shift and change free radicals into steady phenoxy radicals. As a consequence, the hydrophobic and aromatic amino acid residues in the sequences of F7-P3 (Pro and Phe residues), F7-P5 (Pro, Tyr, and Lys residues), and F7-P8 (Tyr, Pro, and Met residues) should have an active influence on their activities of lipid peroxidation inhibitory and radical scavenging. Hydroxylproline (Hyp) is an important component in collagens, but no Hyp was found in the amino acid sequences of eight collagen peptides (F7-P1 to F7-P8). The reason for this phenomenon needs careful study in the future.
Therefore, the composition of amino acids and molecular size of eight collagen peptides (F7-P1 to F7-P8) plays a key role in their antioxidant activities. Small molecular size and hydrophobic amino acids help them access free radicals in an oxidant/antioxidant system with increased ease and efficiency. Moreover, antioxidant amino acids further change free radicals into more stable structures or inhibit the propagation of the peroxidizing chain reaction mediated by free radicals.

Isolation of APs from Hydrolysate Fraction of F7
The preparation processes of collagens, collagen hydrolysate, and hydrolysate fraction (F1 to F7) from Spanish mackerel skins were performed following a previously established protocol [18,19]. F7 solution (500 µL, 25.0 mg/mL) was further purified by ÄKTA avant 25 (GE Healthcare Life Sciences, Chicago, IL, USA) in a Superdex ® Peptide 10/300 GL column. The flow rate of mobile phase (Deionized water (DW)) was 0.75 mL/min and monitored at 214 nm. According to the chromatographic peaks, four fractions, defined as FSP-I, FSP-II, FSP-III, and FSP-IV, were collected and freeze-dried. Finally, FSP-III (100 µL, 25.0 mg/mL) with higher HO• scavenging activity than the other three fractions was purified on a Kromasil C18 column (10 × 250 mm) in RP-HPLC system. The sample was eluted at a flow rate of 0.8 mL/min by a linear gradient of ACN (0% to 40% for 0 to 25 min) in 0.05% TFA. The absorbance of the eluate was monitored at 214 nm. According to the chromatographic peaks, eight peptides (F7-P1 to F7-P8) were collected and freeze-dried.

Radical Scavenging Activity
The scavenging activities of eight isolated peptides (F7-P1 to F7-P8) on HO•, DPPH•, O − 2 •, and ABTS + • were measured according to previous methods [20,36]. The results 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. The counting method was based on the linear relationship between radical scavenging rates and peptide concentrations [1].

DPPH• Scavenging Activity
Two millilitres of samples consisting of distilled water (DW) 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 antioxidant activity of the sample was evaluated using the inhibition percentage of the DPPH• with the following equation: where A is the absorbance rate of the sample, A 0 is the control group absorbance, and A' is the blank absorbance.
HO• Scavenging Activity 1.0 mL of a 1.87 mM 1,10-phenanthroline solution and 2.0 mL of the sample were added to a screw-capped tube and mixed. Following this, 1.0 mL of a FeSO 4 ·7H 2 O solution (1.87 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 1.0 h 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 (HRSA) 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 Superoxide anions were generated in 1 mL of nitrotetrazolium blue chloride (NBT) (2.52 mM), 1 mL of NADH (624 mM), and 1 mL of different sample concentrations. The reaction was initiated by adding 1 mL of phenazine methosulphate 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 scavenging capacity of the O − 2 • was calculated using the following equation: where A control is the absorbance without sample and A sample is the absorbance with sample.
ABTS + • Scavenging Activity ABTS + • was generated by mixing an ABTS stock solution (7 mM) with K 2 S 2 O 8 (2.45 mM). The mixture was left in the dark for 16 h at room temperature. The ABTS + • 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 millilitre of diluted ABTS + • solution was mixed with one millilitre of the different sample concentrations. After 10 min, the absorbances at 734 nm were measured against the corresponding blank. The ABTS + • scavenging activities of the samples were calculated using the same equation as indicated in O − 2 • scavenging activity (%).

Reducing Power Assay
The reducing power assay of eight isolated peptides (F7-P1 to F7-P8) was carried out according to previously reported methods [21,37]. We blended 2.5 mL of 1% K 3 Fe(CN) 6 solution with 2.0 mL of peptide solution and incubated at 50 • C for 30 min. After that, 1.5 mL of 10% trichloroacetic acid was added into the mixed solution. Finally, 2.0 mL of the upper layer, 0.5 mL of 0.1% aqueous FeCl 3 , and 2.0 mL of DW were mixed, and the absorbance at 700 nm was applied to record the reaction mixture.

Lipid Peroxidation Inhibition Assay
This assay was performed according to previously reported methods [20,38]. In brief, 5.0 mg of a sample dissolved in 10 mL of phosphate buffer saline (50 mM, pH 7.0) was mixed with 0.13 mL of linoleic acid solution and 10 mL of absolute ethyl alcohol. The total volume was added to 25 mL with DW in a conical flask. In a dark room, the mixed solution was kept warm at 40 • C. Subsequently, 100 µL of the solution was blended with 0.1 mL of 30% NH 4 CNS, 4.7 mL of 75% ethanol, and 0.1 mL of 20 mM FeCl 2 in 3.5% HCl. After 3 min, the Fe(SCN) 3 value was recorded at 500 nm following color development with thiocyanate and FeCl 2 every 24 h during the incubation period at 40 • C.

Determination of Molecular Mass and AA Sequences
Molecular masses and AA sequences of F7-P1 to F7-P8 were measured according to previously reported methods [19]. A Q-TOF mass spectrometer (Micromass, Waters, Milford, MA, USA) combined with an electrospray ionization (ESI) were used to determine the molecular masses of F7-P1 to F7-P8. N-terminal AA sequencing of F7-P1 to F7-P8 were measured on an Applied Biosystems 494 protein sequencer (Perkin Elmer/Applied Biosystems Inc., Foster City, CA, USA).

Statistical Analysis
Data are presented as means ± standard deviation (SD) (n = 3). ANOVA test (SPSS 19.0 software) was used to comparative analysis the mean value of each treatment. Duncan s multiple range test was carried out to analyze the significant differences of samples (p < 0.05).

Conclusions
In this study, eight collagen peptides (F7-P1 to F7-P8) from protein hydrolysate fraction (F7) of Spanish mackerel skins were isolated and identified as GPY, GPTGE, PFGPD, GPTGAKG, PYGAKG, GATGPQG, GPFGPM, and YGPM. Among them, PFGPD, PYGAKG, and YGPM could effectively inhibit lipid peroxidation and scavenge free radicals in a dose-effect relationship. The present results indicate that eight isolated collagen peptides (F7-P1 to F7-P8) are good candidates as antioxidant additives for various industrial applications. Nonetheless, it is essential to carry out further investigations on the antioxidant mechanism and relationship between the structure and activity of the eight isolated collagen peptides using cell and animal models.