Preparation of Antioxidant Peptide by Microwave- Assisted Hydrolysis of Collagen and Its Protective Effect Against H2O2-Induced Damage of RAW264.7 Cells

Antioxidant peptides have elicited interest for the versatility of their use in the food and pharmaceutical industry. In the current study, antioxidant peptides were prepared by microwave-assisted alkaline protease hydrolysis of collagen from sea cucumber (Acaudina molpadioides). The results showed that microwave irradiation significantly improved the degree of hydrolysis of collagen and the hydroxyl radical (OH⋅) scavenging activity of hydrolysate. The content and OH⋅ scavenging activity of collagen peptides with molecular weight ≤ 1 kDa (CPS) in the hydrolysate obtained at 250 W increased significantly compared with the non-microwave-assisted control. CPS could scavenge OH⋅ and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical in a dose-dependent manner. The scavenging activity of OH⋅ and DPPH radical was 93.1% and 41.2%, respectively, at CPS concentration of 1 mg/mL. CPS could significantly promote RAW264.7 cell proliferation and reduce the Reactive Oxygen Species (ROS) level of H2O2-induced damage in RAW264.7 cells in a dose-dependent manner. Furthermore, all CPS-treated groups exhibited an increase in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and a decrease in malondialdehyde (MDA) level compared with the control. These results showed that CPS could effectively protect RAW264.7 cells from H2O2-induced damage, implying the potential utilization of CPS as a natural antioxidant for food and pharmaceutical applications.


Introduction
Bioactive peptides are short peptides with some bioactive functions, such as antioxidant, antimicrobial, antitumor, or antihypertensive activity. Bioactive peptides have been widely used in health-food, nutraceuticals, and pharmaceutical preparations due to their significant biological functions and efficient absorption [1]. As one of several promising bioactive peptides, antioxidant peptides have become a topic of great interest because of the association between many human diseases and oxidative stress [2]. When humans are subjected to oxidative stress, large amounts of reactive oxygen species (ROS) are produced. Abundant quantities of ROS (superoxide, hydrogen peroxide, and hydroxyl) tend to react with biological molecules, such as protein and DNA, often leading to liver disease, heart disease, and cancer because of oxidative damage of cells [3,4]. ROS-induced oxidative cell damage is usually accompanied by an increase in lipid peroxides [5]. It was known that

Effect of Microwave Assisted on Alkaline Protease Hydrolysis of ASC-Am
The effect of microwave power on OH· scavenging activity of hydrolysate by alkaline protease digestion of ASC-Am was investigated ( Figure 1). The results demonstrated that OH· scavenging activities were significantly improved at all tested microwave powers (50-300 W) as compared to non-microwave assisted hydrolysis. The enhanced OH· scavenging activity when the microwave power increased from 50 to 250 W suggested that the higher microwave power enabled the higher probability of contact between ASC-Am and alkaline protease, resulting in more small peptides with antioxidant activity. However, excessive molecular collisions cause the denaturation of alkaline protease, which may make the OH· scavenging activity begin to decrease when the microwave power continues to increase to 300 W. The maximum OH· scavenging activity was observed at 250 W of microwave power, which increased from 70.3% in non-microwave assisted to 96.2% in this treatment. Therefore, 250 W was selected as the optimal power for microwave-assisted alkaline protease digestion of ASC-Am. In addition, we investigated the content and molecular weight distribution of the peptides in hydrolyzate under microwave assisted digestion with 250 W. As shown in Table 1, compared with 0 W, the total content of CP L at 250 W decreased, but the content of CP M and CP S increased by 48% and 30.2%, respectively. Furthermore, the proportions of CP M and CP S in hydrolyzate obtained at 250 W were both higher than those at 0 W. These results suggested that microwave assisted hydrolysis can significantly promote the breakdown of ASC-Am into smaller molecular weight peptides. The results of this study were similar to those of Uluko et al. (2015), which showed that the hydrolyzed product under microwave irradiation increased by 184.9% compared to the control [27]. The molecular weight of peptides has a significant effect on the antioxidant activity [28]. It was evident that the peptides with smaller molecular weight revealed a stronger antioxidant activity ( Figure 2), which was similar to the results of previous reports [29]. In addition, compared with those under 0 W of microwave power, the OH· scavenging activities of CP L , CP M , and CP S under 250 W power were significantly improved. The reasons for this result may be: (1) microwave radiation probably exposed more cleavage sites of collagen for alkaline protease hydrolysis, resulting in an improvement in antioxidant activity of the peptides due to altered amino acid sequences of peptides; (2) the microwaves probably altered the amino acid sequence of the alkaline protease, resulting in a change in the cleavage site of alkaline protease.

Antioxidant Activity of CP S
In order to better investigate its antioxidant properties, the DPPH and OH· scavenging assays at different concentration of CP S were studied and compared with the positive controls containing ascorbic acid (AA). The CP S The OH· scavenging activity of CP S increased from 24.9% at 0.1 mg/mL to 93.1% at 1.0 mg/mL ( Figure 3A), indicating it scavenged OH· in a concentration-dependent manner. The EC 50 value of CP S was 0.4 mg/mL for OH·, which was lower than that of the peptide BSH-III (Mw ≤ 1 kDa) from protein hydrolysate obtained from bluefin leatherjacket skin (IC 50 of 0.746 mg/mL) [30]. It is known that OH· can have a destructive effect on many biological macromolecules, such as proteins and nucleic acids; therefore, the high OH· scavenging activity of CP S suggested the potential utilization of CP S as a natural antioxidant for reducing or eliminating damage induced by OH· in food and pharmaceutical applications. DPPH radicals are often used in antioxidant experiments because of their high stability [31]. The mechanism of DPPH assay is based on the reduction of DPPH solution in the presence of a hydrogen donor, leading to the formation of the non-radical form DPPH-H [32]. The scavenging of DPPH radicals by CP S was well correlated with the concentration of CP S ( Figure 3B). The DPPH scavenging activity of CP S was found to be 20.5% at a concentration of 0.1 mg/mL, which increased to 41.2% at 1.0 mg/mL, still much lower than that of AA. However, the DPPH scavenging activity of CP S was significantly higher than that of the protein hydrolysate fraction (Mw < 1 kDa) from bluefin leatherjacket skin and skate cartilage [30,33].  Figure 4). When the concentration of H 2 O 2 was greater than 300 µM and the treatment time was longer than 8 h, the cell viability was significantly inhibited. Cell viability exposed to 500 µmol/L H 2 O 2 for 8 h was 46.2% of the control value. In the subsequent experiments, the RAW264.7 cells were treated with 500 µmol/L H 2 O 2 for 8 h to study the effect of CP S on H 2 O 2 -induced injury. The effect of CP S -treated on the proliferation of RAW264.7 cells was shown in Figure 5. The viability of RAW264.7 cells in CP S treatment group was significantly improved compared to the control group when the concentration of CP S increased from 20 µg/mL to 150 µg/mL ( Figure 5). This demonstrated that CP S could effectively promote the proliferation of RAW264.7 cells in a dose-dependent manner. However, a significant increase in cell viability was not observed when cells were treated with 200 µg/mL. Therefore, 100 µg/mL, 150 µg/mL and 200 µg/mL were selected as the low-, middle-and high-dose groups for subsequent experiments, respectively.

Effects of CP S on the ROS Levels in RAW264.7 Cells
In this section, the effect of CP S on the ROS levels in RAW264.7 cells was investigated. The fluorescence intensity in RAW264.7 following H 2 O 2 treatment was significantly larger than that of the control group ( Figure 6). However, the addition of CP S effectively reduced the ROS level relative to the model group, and the ROS level gradually decreased with the increase in CP S concentration. The results suggested that the protective effects of CP S against H 2 O 2 -induced injury of RAW264.7 cells may have resulted from inhibition of intracellular ROS production.  Figure 7B,C. The results showed that H 2 O 2 caused a significant decrease in GSH-Px and SOD activity in RAW264.7 cells. Fortunately, the levels of GSH-Px and SOD were both markedly promoted in CP S -treated groups as compared to the control group. The group with the higher dose exhibited the highest level of GSH-Px, but the results between the low-dose and middle-dose groups were not statistically significant. The highest levels of SOD were observed in the high-dose and middle-dose groups, but the difference between them was not statistically significant. Qiu et al. had reported that collagen peptides could up-regulate the levels of SOD and GSH-Px, and down-regulate the contents of MDA, playing a protective role in antioxidant effects on Drosophila [34]. The collagen peptides from cod skin protected liver tissue against oxidative injure by increasing the activity of SOD and decreasing MDA [35]. Therefore, these results in this study demonstrated that collagen peptides from sea cucumber Acaudina molpadioides could protect RAW264.7 cells against H 2 O 2 -induced injury by inhibition of lipid peroxides and enhancement of antioxidant enzyme activity.

Preparation of Collagen Peptides
ASC-Am was prepared from the body wall of Acaudina molpadioides according to the method reported previously [26]. Two grams of ASC-Am were added into 200 mL buffer solution (pH 10.0) and pretreated with irradiation (0, 50, 150, 200, 250, and 300 W) for 30 minutes each in a microwave (XH-300A; Beijing Xianghu, China). Alkaline protease (≥200 U/mg, Shanghai Ryon Biological Technology Co., Ltd., Shanghai, China) was added to the sample for hydrolysis at 45 • C. After 1 h, the alkaline protease was inactivated in boiling water for 10 min. After centrifuging the hydrolysates at 10,000 rpm for 10 min, the supernatants were freeze-dried, and the scavenging activity of hydroxyl radical (OH·) was measured at the concentration of 0.2 mg/mL.
The hydrolysates obtained under the optimal microwave power were separated by ultrafiltration membranes with size exclusion of 5 kDa and 1 kDa. Three fractions (CP S , collagen peptides with Mw ≤ 1 kDa; CP M , collagen peptides with 1 kDa < Mw ≤ 5 kDa; and CP L , collagen peptides with Mw > 5 kDa) were collected and freeze-dried separately to measure the peptide content and scavenging activity of OH· radical at the concentration of 0.2 mg/mL.

Scavenging Activity of Hydroxyl Radical
The scavenging activity of OH· of peptide was determined using the Fenton method [36]. Two mL peptide samples were mixed with 2 mL of phosphate buffer (0.2 M, pH 7.4), 1 mL of 1,10-phenanthroline (0.75 mM), 1 mL of FeSO 4 (0.75 mM), and 1 mL of 0.3% H 2 O 2 (v/v). The mixture was heated at 37 • C for 30 min, and the absorbance (A 1 ) measured at 510 nm. The scavenging activity of OH· was calculated using the following formula, where A 0 is the absorbance of the blank with 2 mL of water instead of the peptide solution and A 2 the absorbance of mixture with 1 mL of deionized water instead of the FeSO 4 solution.

Scavenging Activity of DPPH Radical
The scavenging activity of DPPH radical was tested according to previously reported methods [37]. The samples of peptides were dissolved in water with different concentrations 0.1, 0.2, 0.5, 0.8, and 1.0 mg/mL. Two mL of peptide samples were mixed with 2 mL of DPPH solution (0.2 mM) and 1 mL of ethanol. The mixture was incubated for 30 min at room temperature and then centrifuged at 5000 rpm for 5 min. The absorbance of the sample (A s ) was measured at 517 nm using an ultraviolet-visible spectrophotometer. The scavenging activity of DPPH was calculated as shown in the formula: where A c is the absorbance of the control group with 2 mL of water instead of the peptide sample; A b is the absorbance of blank with 2 mL of ethanol in place of the DPPH solution.
3.4. The Effect of H 2 O 2 and Peptides on the Proliferation of RAW264.7 Cells RAW264.7 cells (purchased from Chinese Academy of Sciences) were grown in DMEM medium supplemented with 10% fetal bovine serum and incubated at 37 • C in a humidified atmosphere with 5% CO 2 . The peptides were dissolved in DMEM medium and diluted to different concentrations (20-200 µg/mL). Cell viability was determined using MTT assay. RAW264.7 cells were seeded in 96-well plates at 1 × 10 5 cells/mL and incubated at 37 • C with 5% CO 2 for 24 h. The cells were treated with different concentrations of H 2 O 2 (100, 200, 300, 400, 500, 600, 700, 800, and 900 µmol/L) or peptides. A volume of 20 µL of MTT solution (2 mg/mL) was added to each well and incubated for 4 h. The supernatant was removed and 100 µL of Dimethyl sulfoxide (DMSO) was added into each well to dissolve the formazan crystals. The plates were shaken for 5 min to dissolve the crystals completely, and absorbance was measured at 490 nm. The cell viability with H 2 O 2 or peptide treatment was evaluated as follows: Cell viability ( where A S and A N were the absorbance of treatment and control wells, respectively.

Reactive Oxygen Species (ROS) Assay
Production of ROS was monitored using a ROS Assay Kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). RAW 264.7 cells were cultured in 96-well plates with a density of 1 × 10 5 cells/mL for 24 h. Cells were treated with various concentrations of peptides for 24 h, and then exposed to H 2 O 2 (500 µmol/L) for 8 h. The experiments were implemented in the control group (not treated with peptides and H 2 O 2 ), the model group (treated with 500 µM H 2 O 2 ), the low-dose group (treated with 100 µg/mL peptides + 500 µM H 2 O 2 ), the middle-dose group (treated with 150 µg/mL peptides + 500 µM H 2 O 2 ), and the high-dose group (treated with 200 µg/mL peptides + 500 µM H 2 O 2 ). RAW264.7 were incubated for 20 min in a 37 • C cell incubator with 5 µL 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA). Cells were washed three times with serum-free cell culture medium and then observed under a fluorescence microscope.

Assays for Antioxidant Enzyme Activity
RAW264.7 cells (1 × 10 5 cells/mL) were seeded in 96-well plates, and exposed to 500 µmol/L H 2 O 2 for 8 h with or without various concentrations of peptide pre-treatment for 24 h. RAW264.7 cells were washed with Phosphate-buffered saline (PBS), and then lysed with cell lysate. The supernatants were collected following centrifugation at 2000 × g for 5 min at 4 • C. The activity of SOD, GSH-Px, and MDA content was determined by using corresponding diagnostic kits according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Statistical Analysis
Each experiment was carried out in triplicate. Data were presented as means and standard deviations. Results were analyzed using Microsoft Excel 2010 (Redmond, WA, USA), and significant differences (p < 0.05) between data were identified by Duncan's multiple range test in the software SPSS (SPSS Inc., Chicago, IL, USA).

Conclusions
Microwave-assisted hydrolysis of collagen is a promising method for preparation of antioxidant peptides. Microwave radiation can significantly increase the content and antioxidant activity of small molecular weight peptides. The hydrolyzate fragment CP S (Mw ≤ 1 kDa) obtained by microwave-assisted alkaline protease hydrolysis of collagen from sea cucumber Acaudina molpadioides exhibited the good scavenging activities of DPPH and OH•. CP S also exhibited a significant protective effect on H 2 O 2 -injured RAW264.7 cells by promoting cell proliferation, reducing the levels of ROS and MDA, and enhancing antioxidant enzyme (SOD and GSH-Px) activity. Therefore, it is recommended, as a result of the current study, to explore CP S as a potential natural antioxidant and utilize microwave-assisted hydrolysis as a method of obtaining high levels of antioxidant peptides from naturally occurring high-molecular weight protein molecules.