Potential Antioxidant Activity of Novel Antioxidant Peptides from Protein Hydrolysate of Peony Seed Dreg in Chemical and H 2 O 2 -Induced RAW264.7 Cells

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Introduction
Peroxyl radicals (ROO• − ), •OH, hydrogen peroxide (H 2 O 2 ), and superoxide anion (O 2 • − ) are typical ROS that play vital roles in the body, such as participating in intracellular signal transduction, energy supply, and anti-infammatory functions [1].Once the accumulation of ROS exceeds the capacity of the cellular free radical scavenging system, it leads to cellular oxidative stress and causes oxidative damage to various cellular components such as membrane lipids, proteins, and DNA [2], increasing the incidence of diferent diseases, including immune dysfunction [3], cancer [4], diabetes mellitus [5], neurodegenerative diseases [6,7], and cardiovascular diseases [8,9].Additionally, ROS-mediated lipid peroxidation contributes to food deterioration, unacceptable tastes, and the generation of potentially toxic products [10].Based on this, mitigating the ROS-induced oxidative damage via antioxidants is an efective treatment for free radical-related diseases, antiaging, and delaying food spoilage [11].Instead of synthetic antioxidants with their potential health hazards, natural antioxidants have received widespread attention [12].Antioxidant peptides show great advantages of low molecular weight, poor immunogenicity, excellent stability, and high absorption efciency [13,14].Antioxidant peptides are encrypted sequences in the amino acid sequence of precursor proteins, usually inactive, and can be released after hydrolysis by proteases.It was reported that antioxidant peptides can slow oxidative damage by scavenging or converting free radicals into stable or less active forms [15].
Peony (Paeonia sufruticosa Andr.) is a multifunctional traditional plant with ornamental, edible, and medicinal purposes [16], which has become an important cash crop in China [17].Many studies have shown that the application promising of peony seed oil as function-rich edible oil is very extensive [18]; however, the protein-rich cold-pressed peony seed dreg, a byproduct of oil processing, was only been used as organic fertilizer, animal fodder, or discarded as waste due to storage inconvenience [19,20].To explore the potential of peony seed dreg as a source of novel antioxidant peptides, we prepared antioxidant peptides from PSDP hydrolyzed by Pseudoalteromonas CSN423 extracellular proteases and characterized the antioxidant properties of the peptides via chemical-based antioxidant analysis and protective efect on H 2 O 2 -induced RAW264.7 cells.

Extraction of Peony Seed Dreg Protein (PSDP)
. PSDP was prepared from cold-pressed peony seed dreg by the traditional alkali dissolution and acid precipitation method [23].Defatted peony seed dreg was cracked and passed through an 80 mesh screen to obtain defatted peony seed dreg four.Te defatted peony seed dreg four was dissolved in distilled water (1 : 20, w/v), and the pH was adjusted to 8.5.Te slurry of peony seed dreg was stirred gently at 40 °C for 4 hours, and the supernatant was recovered by centrifugation (8000 × g, 10 min, 4 °C).Te supernatant was adjusted to pH 3.6 with 1M HCl and then centrifuged at 8000 × g for 10 minutes at 4 °C to collect the precipitate.Te precipitate was redissolved in distilled water, and the pH was adjusted to 7.0.Finally, the solution was lyophilized.

Preparation of PSDP Hydrolytic
Peptides.Hydrolysis of PSDP was performed with a substrate/enzyme ratio of 9 : 1 (v/v) at 40 °C for 30, 60, 90, 120, 150, and 180 min.After digestion, the enzyme was inactivated at 90 °C for 10 min.Te indene triketone colorimetric method was used to evaluate the free amino acid content at diferent treatment times [24].Te hydrolysates were centrifuged at 12, 000 × g for 5 min.After that, the supernatants were recovered and fltered through a 3 kDa ultrafltration membrane.
2.6.Amino Acid Analysis.Te ultrafltrated fraction with higher (>3 kDa) and lower molecular (<3 kDa) weights was designated as UF-1 and UF-2, respectively.UF-2 was further analyzed for its amino acid contents [25].Te amino acid analysis was evaluated after hydrolysis of UF-2 with 6 M HCl at 110 °C for 22 h, and its amino acid profle was detected using an HPLC system.Te methionine and cysteine contents were analyzed postperformic acid oxidation.

Purifcation of the Antioxidant Peptides.
Te fraction UF-2 (500 μg/ml, 1 ml) was loaded onto a Sephadex G-25 column (1.6 × 60 cm) and then washed with distilled water at 0.75 ml/min.Te absorbance of the eluates at 220 nm was monitored, and the antioxidant activities were estimated.

Chemical-Based Analysis of Antioxidant Capability
2.8.1.•OH Scavenging Capability.Te •OH scavenging capability was evaluated as previously described [17].Te FeSO 4 •7 H 2 O solution (60 μl, 6 mM), peptide solution (200 μl), and H 2 O 2 (60 μl, 6 mM) were mixed to form a homogenous mixture.After that, 60 μl of salicylic acid solution (6 mM) was added to form a colored reaction product, followed by the addition of 420 μl of distilled water.Te absorbance at 510 nm was detected after incubating at 37 °C for 30 min.500 μg/ml GSH was used as a standard reference.Te scavenging capability of •OH was calculated using the following equation: where A s is the absorbance of the sample group, B is the reaction system without salicylic acid, and A b is the absorbance of the blank group (the sample was replaced with distilled water).
After incubating the mixture at room temperature for 60 min in the dark, the absorbance at 517 nm was selected for the measurement of the scavenging capability.500 μg/ml GSH was used as a reference standard.Te DPPH• scavenging activity was calculated by the following equation: where A b is the absorbance of the blank group (the sample was replaced with distilled water) and A s is the absorbance of the sample.

Results and Discussion
3.1.Preparation of Proteases.Te protease E423 secreted by Pseudoalteromonas sp.CSN423 was isolated via anion exchange chromatography and gel fltration after precipitation with 60% ammonium sulfate.As shown in Table 1, the specifc protease activity of the crude enzyme was 10.5 U/mg and it was up to 595.2 U/mg after the purifcation treatment.By comparison, Wang et al. [17] used commercial alkaline protease (20 U/mg) to hydrolyze Paeonia ostii seed meal protein to obtain fve antioxidant peptides (FSAP, PVETVR, QEPLLR, EAAY, and VLRPPLS).Te antioxidant peptide SMRKPPG was obtained by hydrolysis of Paeonia seed protein with alcalase (62 U/mg) [20].Te cleavage site of proteases from diferent bacteria varies widely [30].Terefore, the sequence of antioxidant peptides prepared by E423 may also be diferent.

Te Hydrolysis of PSDP.
Te hydrolysis of PSDP was efective and reproducible.As shown in Figure 1(a), PSDP can be efciently digested by proteases produced by Pseudoalteromonas sp.CSN423 and the rate of hydrolysis reached close to a steady state after 120 minutes.Te DPPH• scavenging capacity of UF-2 increased with the hydrolysis time.UF-1 showed a lower DPPH• scavenging activity than UF-2 (Figure 1(b)).It is suggested that the disruption of protein's native structure by enzymatic hydrolysis might lead to unfolding, thereby enhancing the accessibility of their functional groups to ROS.However, over-hydrolysis can result in the conversion of peptides to amino acids or the destruction of their active groups, and the product activity may also be decreased.Terefore, the degree of enzymatic hydrolysis must be strictly controlled [31,32].2, the amino acid composition of UF-2 included seventeen kinds of amino acids.Hydrophobic amino acids, including leucine, alanine, glycine, valine, methionine, proline, phenylalanine, and isoleucine, accounted for 39.8% of the total amino acids.Te aromatic amino acids tyrosine and phenylalanine accounted for 9.44%.Hydrophobic amino acids such as Ile, Val, and Leu could enhance the solubility of peptides in lipids, thus enhancing their antioxidant activity.Aromatic amino acids, such as Phe and Tyr, can act as proton donors to maintain ROS stability during the free radical scavenging process [33,34].

Te Antioxidant Peptide Purifcation.
Figure 2 shows the column chromatographic profle of the UF-2, where eight major components were eluted.All the fractions displayed •OH and DPPH• scavenging activities with the highest activities observed in F7 (81.17 ± 3.69% and 73.0 ± 1.46%, respectively) (Figure 3).It is suggested that the fraction F7 may be the major contributor to the antioxidant activity of UF-2.

Detection of Antioxidant Activities
3.5.1.•OH Scavenging Activity.Hydroxyl radical is the most powerful oxidant that causes chronic diseases [35].Scavenging hydroxyl free radicals is an efective method for organisms to defend against various diseases.Te fraction F7 exhibited the highest •OH scavenging activity of 81.17 ± 3.69%, possibly due to the contribution of hydrogen atoms or blocking the generation of oxygen free radicals, as shown in Figure 3(a).

DPPH• Scavenging
Ability.DPPH• exhibits a characteristic absorption at 517 nm.After exposure to free radical scavengers, the absorbance value of DPPH• decreased rapidly.We estimated the DPPH• scavenging ability of F7 to be 73.0 ± 1.46%, which was signifcantly higher than other isolated fractions (Figure 3(b)).It is suggested that F7 contained more efective antioxidant peptides that could convert DPPH• into a stable form and terminate the free radical chain reaction.

Inhibition of DNA Oxidative Damage.
Plasmid DNA exhibited diferent structures and electrophoretic mobility in agarose gel due to the varied degrees of damage.
•OH can cause oxidative stress-induced DNA strand breaks.Te formation of open-circle DNA indicates that one of the phosphodiester bonds was cleaved, whereas the formation of linear DNA indicates further cleavage near the frst break [36].As shown in Figure 4, H 2 O 2 decomposed to produce •OH, which damaged most of the supercoiled DNA in this reaction system (lane 3).However, the supercoiled DNA reappeared when the plasmid DNA was exposed to the fraction F7 (lanes 4 and 5).Te fraction F7 may have donated a hydrogen atom or an electron to scavenge •OH or directly prevent the reaction of Fe 2+ with H 2 O 2 to protect the pUC18 plasmid DNA from oxidative damage.

Te ORAC Assay of the Peptide Fraction F7.
Te ORAC assay was used to detect the oxygen radical scavenging capability of peptides, which is refected by the area under the fuorescence decay curve.From the results in Figure 5(a), the peptide fraction F7 exhibited a signifcant efect in decreasing fuorescence decay, in a dose-dependent manner.Te ORAC of the peptide fraction F7 was 4.04 ± 1.11 μmol TE/mg, as shown in Figure 5  Te results suggested that the component F7 is safe for RAW264.7 cells and, therefore, can evaluate the intracellular ROS scavenging ability of F7.

Intracellular Radical Scavenging Efects of the Isolated
Peptide Component F7.Compared with the in vitro chemical analysis, the evaluation of the efect of peptides on oxidative injured cells could more directly refect their ability to resist oxidative stress.To evaluate whether F7 has the capability of scavenging intracellular ROS, we labelled the RAW264.7 cells with DCFH-DA.Te ROS content in cells was refected by the fuorescence intensity.From the results in Figure 7, the ROS content signifcantly increased in cells treated with 500 μM H 2 2 , as compared with the untreated control cells.Te fuorescence intensity decreased when F7 was added.Hence, the protective efect of F7 on H 2 O 2 -induced RAW264.7 cells may be due to the efective removal of excessive intracellular ROS.

Assessment of Mitochondrial Membrane Potential
Changes.Te JC-1 is selectively taken up by the mitochondria.In apoptotic cells, JC-1 is monomeric and its color changes from red to green as the ΔΨm decreases.As shown in Figure 8, the number of C-gated cells refects the ΔΨm, with the greater number in the H 2 O 2 -induced group indicating a reduction in ΔΨm of the RAW 264.7 cells (p < 0.05).Cells treated with the peptide fraction F7 had higher ΔΨm than the H 2 O 2 -induced group (p < 0.05).

Inhibition of the Peptide Fraction F7 on Cell Apoptosis
Induced by H 2 O 2 .Cell apoptosis was evaluated through the Annexin V-FITC/PI staining via fow cytometry.As shown in Figure 9, the apoptosis rate of the H 2 O 2 -induced cells was higher than that of the control group (p < 0.05), which can reach about 35%.Te peptide fraction F7 signifcantly inhibited the cellular apoptosis (p < 0.05).In a state of oxidative stress, excess reactive oxygen species are produced, damaging a multitude of cellular functional molecules and further causing apoptosis due to reduced membrane potential, inactivation of antioxidant enzymes, and even transgeneration [37] Te peptide fraction F7 has a cytoprotective efect on H 2 O 2 -induced RAW 264.7 cells, and its mechanism may be related to the increase of reduced membrane potential.

Identifcation and Verifcation of the Peptide Components of F7.
Te peptide fraction F7 was sequenced by nano-RPLC-MS/MS.We identifed four novel antioxidant peptides YFPF, ECCASLAPL, YVSLK, and YFEM and considered them as the major contributors to the antioxidant activity of the fraction F7.We synthesized the peptides by the solid-phase method and assessed their antioxidant activities.As shown in Table 3, the YFPF peptide sequence showed 75.3 ± 1.08% hydroxyl radical scavenging at 500 μg/ ml, as compared with a powerful antioxidant like GSH (77.56 ± 1.40%).Te hydroxyl radical scavenging ability of YFPF was higher than that of EAAY (61.9% ± 1.3%) and SMRKPPG (16.48%) at 1 mg/ml, which were isolated from peony seed dreg [17,20].Te peptide ECCASLAPL exhibited a higher DPPH• scavenging ability than GSH at 200 μg/ml, which was 87.2 ± 1.03%.Among all the synthetic peptides, YVSLK exhibited the strongest ORAC value at 2.52 ± 1.20 μmol TE/mg.Te antioxidant activities of these peptides may be related to their molecular weight.Te molecular weight of the peptides identifed in this study is all less than 1000 Da, and they are more likely to react with reactive oxygen species due to their low steric resistance [38].Moreover, the efect of the amino acid composition of the peptides on antioxidant activity has been stressed in the literature.A high proportion of hydrophobic amino acids was  considered as the key factor in the peptide ability to scavenge radicals [33].Te hydrophobic amino acids in YFEM and YFPF accounted for 50% and 75%, respectively.Hydrophobic amino acids can promote the presence of peptides at the water-lipid interface, facilitate the scavenging of radicals in the lipid phase, and promote their accessibility to hydrophobic targets, making it easier for the peptides to pass through the cell membranes [39,40].Since the aromatic amino acids can provide electrons for free radicals to convert them into stable molecules, we hypothesized that such a high •OH scavenging capacity of YFPF requires a high proportion of aromatic amino acids up to 75%.In addition to the presence of hydrophobic amino acids and aromatic residues, some peptides containing sulfur (Cys or Met) have been found to possess strong antioxidant properties.Te thiol group in Cys and the thioether in Met are relatively easily oxidized.Cys is a hydrogen donor through the SH group or can lose an electron from its atom of sulfur [41].Terefore, it is suggested that the higher DPPH• scavenging ability of ECCASLAPL may be related to the SH group of Cys.

Conclusions
In summary, peony seed dreg, the byproduct remaining after oil extractions with a low added value, has proven to be an ideal protein resource for preparing antioxidant peptides.Te antioxidant peptide fraction F7 efectively protected the H 2 O 2induced RAW264.7 cells and is considered as a potential alternative peptide ingredient for synthetic antioxidants, which can be utilized by the nutraceutical, functional food, and cosmetics industries.Te novel antioxidant peptides YFPF, ECCASLAPL, YVSLK, and YFEM were considered to be major contributors to the antioxidant activity of F7.However, further research on gastrointestinal stability, bioavailability, and allergenicity assessment is needed. (b).

Figure 1 :
Figure 1: Te free amino acid content of PSDP hydrolyzed for diferent times (a); analysis of the DPPH• scavenging activities of the UF-1 and the UF-2 (b).Changes were analyzed for statistical signifcance, which is indicated with asterisks; * p < 0.05.

Figure 3 :
Figure 3: •OH and DPPH• scavenging activities of the F7 fraction.Te concentration of both the isolated fractions F1-F8 and GSH was 500 μg/ml.Changes compared to F7 were analyzed for statistical signifcance, which is indicated with asterisks; * p < 0.05, while nonsignifcant diferences are marked as ns.

Figure 6 :
Figure 6: Te efects of the peptide fraction F7 (a) and H 2 O 2 (b) on RAW264.7 cell viabilities at diferent concentrations and treatment times.
RAW 264.7 cells were incubated with the peptide fraction F7 for 24 h and then exposed to H 2 O 2 (500 µM) for 8 h.After all treatments, the supernatant was removed and 100 μl of 10 μM DCFH-DA (prepared with DMEM) was added and incubated for 20 min.Excess DCFH-DA was rinsed away by PBS (0.01 M).Statistical Analysis.SPSS 26 software was used to organize and analyze the data.Te quantitative data conforming to normal distribution were represented by mean ± standard deviation (x ± s).Comparison between the two groups was statistically analyzed by t-test.Comparison between multiple groups was statistically analyzed by ANOVA, and multiple experimental groups were compared with one control group using Dunnett's t-test.p < 0.05 indicated statistically signifcant.

Table 1 :
Protease activity in the purifcation process of E423.

Table 2 :
Amino acid composition of the fraction UF-2.
Results are expressed as the mean ± standard deviation (n � 3).