Food and Chemical Toxicology 52 (2013) 113–120

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Food and Chemical Toxicology

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Purification and determination of two novel antioxidant peptides from flounderfish (Paralichthys olivaceus) using digestive proteases

Ju-Young Ko a, Ji-Hyeok Lee a, Kalpa Samarakoon a, Jin-Soo Kim b, You-Jin Jeon a,⇑a Department of Marine Life Science, Jeju National University, Jeju 690-756, Republic of Koreab Department of Seafood Science and Technology/Institute of Marine Industry, Gyeongsang National University, Tongyeong 650-160, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 2 August 2012Accepted 29 October 2012Available online 9 November 2012

Keywords:FlounderParalichthys olivaceusAntioxidative peptideEnzymatic hydrolysisRadical scavenginga-Chymotrypsin

0278-6915/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.fct.2012.10.058

Abbreviations: FFM, flounder fish muscle; ESR, ereactive oxygen species; FPH, fish protein hydrolysaAAPH, 2,2-azobis-(2-amidinopropane) dihydrochlorVCSV, Val-Cys-Ser-Val; DPPH, 1,1-diphenyl-2-picrylhdodecyl sulfate–polyacrylamide gel electrophoresis.⇑ Corresponding author. Tel.: +82 64 754 3475; fax

E-mail address: youjinj@jejunu.ac.kr (Y.-J. Jeon).

We investigated the effects of bioactive-peptides from hydrolysates of flounder fish muscle (FFM) onantioxidant activity. The hydrolysates were prepared by enzymatic reactions of FFM using eightcommercial proteases such as papain, pepsin, trypsin, neutrase, alcalase, kojizyme, protamex, anda-chymotrypsin. The a-chymotrypsin hydrolysate showed the strongest antioxidant activity among theeight enzymatic hydrolysates. Further separation of the a-chymotrypsin hydrolysate was performed byultrafiltration, gel filtration, and reverse-phase high performance liquid chromatography. Consequently,two novel peptides with high antioxidant activity were purified, and their amino acid sequences weredetermined (Val-Cys-Ser-Val [VCSV] and Cys-Ala-Ala-Pro [CAAP], respectively). The two peptides showedgood scavenging activity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical (IC50 values,111.32 and 26.89 lM, respectively) and high cytoprotective activities against 2,2-azobis-(2-amidino-propane) dihydrochloride (AAPH) without cytotoxicity and scavenged total reactive oxygen species inVero cells. In particular, apoptotic bodies produced by AAPH dose-dependently decreased followingtreatment with the CAAP peptide. These results revealed firstly the two peptides with strong antioxidativeeffects from FFM.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Free radicals are produced from oxygen by aerobic organismsduring the normal course of respiration. Reactive oxygen species(ROS) include a variety of free radicals, such as superoxide anion(O2

��), hydroxyl radical (HO�), nitric oxide radical (NO�), peroxylradical (RO��), and non-free radical species such as hydrogen per-oxide (H2O2). ROS in normal cells can be effectively eliminatedby an enzyme-mediated system such as superoxide dismutase,peroxidase, catalase, and glutathione peroxidase. However, if ROSare excessively produced by many factors such as exposure to radi-ation, major surgery, smoking, drinking, food additives, stress, pol-lution, infections, and excessive exercise, excess ROS may causediseases such as cancers (Leanderson et al., 1997), gastric ulcers(Debashis et al., 1997), Alzheimer’s, arthritis, and cause problemsduring ischemic reperfusion (Vajragupta et al., 2000). Moreover,

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lectron spin resonance; ROS,tes; MW, molecular weight;ide; CAAP, Cys-Ala-Ala-Pro;ydrazyl; SDS–PAGE, sodium

: +82 64 756 3493.

oxidation of fatty acids and lipids induced by free radicals alsoleads to deterioration in food quality (Liceaga-Gesualdo andLi-Chan, 1999; Kristinsson and Rasco, 2000). An antioxidant is amolecule capable of inhibiting the oxidation of other molecules.Commercial synthetic antioxidants with stronger antioxidantactivity are butylated hydroxyanisole, butylated hydroxytolueneand propyl gallate. But the amount used must be under strictregulation due to potential health hazards (Park et al., 2001).

Many researchers have considered bioactive peptides from foodproteins as alternative materials to synthetic antioxidants. Bioac-tive peptides can be released from proteins by enzymatic proteol-ysis of proteins and may act as potential physiological modulatorsof metabolism during intestinal digestion. The possible regulatoryeffects of peptides are related to nutrient uptake, immune defense,opioid and antihypertensive activities, and antioxidant activities(Pihlanto-Leppälä, 2001). Fish protein hydrolysates (FPH) possessnutritional, antioxidative, antihypertensive, antimicrobial, andimmunomodulatory properties (Fujita and Yoshikawa, 1999). Inparticular, the antioxidant peptides of FPH hydrolyzed from vari-ous fish sources such as capelin (Amarowicz and Shahidi, 1997),tuna (Jao and Ko, 2002), mackerel (Wu et al., 2003), Alaska pollack(Je et al., 2005), and scad (Thiansilakul et al., 2007) have been re-ported. Angiotensin converting enzyme inhibitory peptides hydro-lyzed from tuna (Kohama et al., 1988), bonito (Matsumura et al.,

114 J.-Y. Ko et al. / Food and Chemical Toxicology 52 (2013) 113–120

1993), channel catfish (Theodore and Kristinsson, 2007), and chumsalmon (Ono et al., 2003) have also been reported.

Olive flounder, Paralichthys olivaceus, is one of the most impor-tant marketed fish on Jeju Island, and major cultivated fish speciesoccupy 98% of the domestic aquaculture market in South Korea.Annual production is about 25,000 tons and export is about4000 tons. The pathogens (Mizuki et al., 2006), functional feed(Vine et al., 2004), physical and chemical properties (Wassonet al., 1993; Wasson, 1993) and immunology (Carlson et al.,1993) of olive flounder during aquaculture have been reported.But, studies about bioactive peptides from flounder fish muscle(FFM) have not been reported until now.

Therefore, in this study, we evaluated the antioxidative activi-ties of hydrolysates from FFM prepared from eight commercialproteases such as papain, pepsin, trypsin, neutrase, alcalase, koji-zyme, protamex, and a-chymotrypsin, and identified the aminoacid sequences of the purified peptides with the strongest antiox-idative activity. We also investigated the antioxidative effects ofthe purified peptides against 2,2-azobis-(2-amidinopropane) dihy-drochloride (AAPH) induced cytotoxicity, total ROS, and apoptoticbody production.

2. Materials and methods

2.1. Materials

Flounder fish, P. olivaceus, were cultivated in Jeju island. Fish after capture werefilleted and muscle was collected. It was washed twice with freshwater, then imme-diately frozen and stored at �20 �C until used. The frozen sample was lyophilizedand homogenized with a grinder before hydrolysis.

2.2. Chemicals and reagent

1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2-azobis-(2-amidinopropane) dihy-drochloride (AAPH) were purchased from Sigma Chemical Co. (USA). Protein prote-ase such as papain, pepsin, trypsin, a-chymotrypsin were purchased from Sigmachemical Co. (USA) and neutrase, alcalase, kojizyme, protamex were purchasedfrom Novozyme Co. (Denmark). Dulbecco’s modified Eagle’s medium (DMEM), fetalbovine serum (FBS), penicillin–streptomycin, trypsine–EDTA, and Dulbecco’sPhosphate Buffered Saline (DPBS) were purchased from Gibco-BRL (Burlington,Ont, Canada). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT), 20 ,70-dichlorodihydrofluorescein diacetate (DCFH-DA), Hoechst 33342 wereobtained from Sigma (St. Louis, MO, USA). All the other chemicals used wereanalytical grade.

2.3. Approximate composition

The approximate composition of dried flounder fish muscle powder was deter-mined according to the AOAC methods. Moisture content was determined keepingin a dry oven at 105 �C for 24 h. Crude ash content was determined by calcinationsin furnace at 550 �C and crude protein content was determined by Kjeldahl method.Crude lipid was content was determined by Soxhlet method.

2.4. Preparation of enzymatic hydrolysate

2.4.1. Preparation of enzymatic hydrolysates by eight protein proteasesFlounder fish muscle enzymatic hydrolysis was performed according to the pre-

viously described procedures (Lee et al., 2009). Freeze dried flounder fish musclewas homogenized with a grinder. Five gram of the dried flounder fish powder putinto distilled water 100 ml and was adjusted to their optimal pH and temperatureconditions of the all proteases (Table 2). And then the enzymes were added to give a500:1 ratio of substrate to enzyme. Protein hydrolytic enzymes used are eight pro-teases such as papain, pepsin, trypsin, neutrase, alcalase, kojizyme, protamex anda-chymotrypsin. At the end of the reaction, the hydrolysates were adjusted to pH7.0 and heated at 97 �C for 10 min in order to inactivate the enzymes. The hydrol-ysates were centrifuged at 3500 rpm for 20 min to separate insoluble and solublefractions. The soluble phase was freeze dried using freeze dryer and stored at�20 �C for the further use.

2.4.2. Optimum conditions for the active enzymatic hydrolysateOptimum hydrolytic conditions assay was carried out for producing antioxida-

tive hydrolysate from flounder fish. Five grams of the dried flounder fish powderwere put into distilled water 100 ml and was adjusted to its optimal pH value.

And then various ratios of substrate to enzyme (1000:1, 500:1 and 100:1) and var-ious times (6, 12, 18 and 24 h) were examined for the optimal hydrolytic conditions.The next processing were the same as the previous.

Yields of the hydrolysates obtained by enzymatic hydrolysis of flounder fishmuscle were calculated by dry weight of hydrolyzed filtrate over dry weight ofthe flounder fish muscle sample used (Heo et al., 2005).

2.5. Separation and purification of antioxidative peptides

2.5.1. Molecular weight fractionation of active enzymatic hydrolysateThe enzymatic hydrolysate having the highest antioxidant activity was fraction-

ated by using ultrafiltration membranes (MWCO 5 and 10 kDa) with Millipore’s Labscale TFF system (Millipore Corporation, Bedford, Massachusetts, USA) at 4 �C andthree fractions including (>10 kDa, 5 � 10 kDa, and <5 kDa) were separated accord-ing to their molecular weights. The fractions lyophilized, were stored at �20 �C forthe next use.

2.5.2. Size exclusion chromatographyThe superior active fraction (100 mg) for the three fractions were separated by

ultrafiltration membranes and dissolved with 1 ml of distilled water. The fractionwas loaded onto a Sephadex G-25 column (2.5 � 100 cm). Previously the columnequilibrated with distilled water. And then elution was carried out with distilledwater at a flow rate of 1.5 ml/min. The fractions absorbance was read at 220 nmand collected peaks. This was lyophilized and stored at �20 �C until used.

2.5.3. Purification of antioxidant peptide on HPLCThe antioxidative peaks obtained on the size exclusion chromatography were

applied to a reverse-phase high performance liquid chromatography (RP-HPLC)having a YMC-Pack ODS-A column (5 lm, 4.6 � 250 mm, YMC Co., Kyoto, Japan)with a linear gradient of acetonitrile (0–100% v/v, 30 min) at a flow rate of1.0 ml/min. Elution peaks were detected at a 220 nm.

2.6. SDS–PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) wasperformed on the protein hydrolysates using a 15% Tris/HCl gel to characterizethe hydrolysates based on their molecular weights (MW). The MW of the hydroly-sates was calculated with reference to the migration of SDS–PAGE wide rangemolecular weight standard. Samples were heated at 100 �C for 5 min prior to theelectrophoresis run. After electrophoresis, the gels were stained with Bio-Rad Coo-massie Blue R-250. The bands in the samples were compared with known bands ofprotein standards.

2.7. Amino acid sequences

Amino acid sequences of the purified peptides from flounder fish were deter-mined using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled withelectrospray ionization (ESI) source. The purified peptide dissolved in distilledwater was infused into the ESI source and the molecular weight was determinedby doubly charged (M + 2H)2+ state analysis in the mass spectrum.

2.8. Free radical scavenging capacities using ESR spectrometer

The different radicals tested here were generated according to the previouslydescribed procedures (Heo et al., 2008), and the spin adducts were recorded usingJES-FA electron spin resonance (ESR) spectrometer (JES-FA ESR, JEOL, Tokyo, Japan).

2.8.1. DPPH radical scavenging activityDPPH radical scavenging activity was measured using an electron spin reso-

nance (ESR) spectrometer in accordance with the method described by Nanjoet al. (1996). A distilled water solution of 60 ll of each sample (or distilled wateras a control) was added to 60 ll DPPH (60 lM) in methanol solvent, and the samplewas mixed vigorously. After 2 min, the solution was transferred to a capillary tube,and the spectrum was recorded with an ESR spectrometer (JES-FA machine, JEOL,Tokyo, Japan). The experimental conditions were as follows: magnetic field,336.5 ± 5 mT; power, 1 mW, modulation frequency, 100 kHz; amplitude,10 � 100; modulation width, 0.8 mT; sweep width, 10 mT; sweep time, 30 s; andtime constant, 0.03 s. The extent of scavenging activity was calculated as follows.

Scavenging activity % ¼ ðHC�HSÞ=HC� 100

where HC is the relative peak heights of the radical signals without sample, and HS isthe relative peak heights of the radical signals with sample.

2.8.2. Hydroxyl radical scavenging activityHydroxyl radicals were generated by the iron-catalyzed Haber–Weiss reaction

(Fenton-driven Haber–Weiss reaction; Fe2+ + H2O2 ?�OH + �OH) and the hydroxyl

radicals rapidly reacted with nitrone spin trap DMPO (Rosen and Rauckman,1980). The resultant DMPO-OH adducts were detected using an ESR spectrometer.

Table 2Optimum conditions and radicals scavenging activities of enzymatic hydrolysatesprepared from flounder fish with eight proteinase.

Enzymatic digests Optimum conditions Radicals (IC50 mg/ml)

J.-Y. Ko et al. / Food and Chemical Toxicology 52 (2013) 113–120 115

2.8.3. Peroxyl radical scavenging activityPeroxyl radicals were generated by AAPH and their scavenging effects were

investigated by the method described by Hiramoto et al. (1993). The reaction mix-ture containing 40 mM AAPH and 40 mM 4-POBN were mixed with PBS. The solu-tion was incubated for 30 min at 37 �C in a water bath, and then transferred tocapillary tube.

2.9. Cell experiments for antioxidant activity assay

2.9.1. Cell cultureVero (Monkey kidney fibroblast line) cell was grown on DMEM medium supple-

mented with 10% (v/v) heat-inactivated FBS, 1% (v/v) antibiotic. Cultures weremaintained at 37 �C in a 5% CO2 incubator.

2.9.2. Measuring of cytoprotective effect by MTT assayThe cytoprotective effect of peptides against Vero cell was determined by a

colorimetric MTT assay. Vero cells were seeded in a 96-well culture plates at1 � 105 cells/ml. After 16 h, cells were treated with various concentrations of pep-tides (12.5, 25, 50 and 100 lg/ml) and 2 h later 10 mM of AAPH was added to theculture. Cells were incubated for an additional 24 h at 37 �C. After incubation,50 ll (stock concentration 2 mg/ml in DPBS) of MTT solution was added into eachwell and cells were incubated at 37 �C for 4 h. The plates were centrifuged for10 min at 2000 rpm and the supernatants were aspirated. The formazan crystalsin each well were dissolved in DMSO. The amount of purple formazan was assessedby measuring the absorbance at 540 nm. The optical density of the formazanformed in the control cells was taken as 100% viability. Data are mean percentagesof viable cells versus respective controls.

2.9.3. Intracellular ROS measurementThe DCFH-DA method was used to detect the levels of intracellular ROS

(Rosenkranz et al., 1992). Vero cells were seeded on a 96-well culture plates at1 � 105 cells/ml. After 16 h, cells were treated with various concentrations of pep-tides (12.5, 25, 50, and 100 lg/ml) and 2 h later 10 mM of AAPH was added to theculture. Cells were incubated for an additional 30 min at 37 �C. Finally, cell wastreated with DCFH-DA (5 lg/ml). Intracellular production of ROS was measuredafter 30 min incubation at 37 �C by fluorometric detection of DCF oxidation on aspectrofluorometer (Perkin-Elmer LS-5B) with an excitation wavelength of485 nm and emission wavelength of 535 nm. The DCF fluorescence intensity is pro-portional to the amount of ROS formed intracellularly. Results are expressed as thepercentage of ROS generation as compared to the control.

2.9.4. Apoptotic body produced by AAPH in Vero cell lineHoechst 33342 staining assay Vero cells were seeded in a 24-well culture plates

at 1 � 105 cells/ml. After 16 h, cells were treated with various concentrations ofpeptides (12.5, 25, 50, and 100 lg/ml) and 2 h later 10 mM of AAPH was added tothe culture. Cells were incubated for an additional 24 h at 37 �C. After 24 h, the celltreated with final concentration 10 lg/ml of Hoechst 33342 for 10 min at 37 �C indark. The stained cells were observed under a fluorescence microscope equippedwith a CoolSNAP-Pro color digital camera in order to determine the degree of nucle-ar condensation.

2.10. Statistical analysis

All experiments were conducted in triplicate (n = 3) and an ANOVA test (usingSPSS 11.5 statistical software) was used to analyze the data. Significant differencesbetween the means of parameters were determined by using the Duncan’s test(p < 0.05).

3. Results and discussion

3.1. Approximate composition

Approximate chemical compositions of the raw flounder fishmuscle are shown in Table 1. Moisture, ash, crude protein, and lipidare indicated on a dry weight basis, in which crude protein content

Table 1Chemical composition of the dried flounder fish.

Composition Sources (%)

Moisture 5.00 ± 0.01Ash 1.63 ± 0.14Crude protein 87.82 ± 1.37Crude lipid 5.55 ± 1.29

Mean ± SD from triplicate determinations.

was 87.82%. FFM protein content was similar to the protein contentof flounder muscle reported by Liu et al. (2008).

3.2. Preparation of enzymatic hydrolysates from FFM and theirantioxidant activities

The types of enzymes used for enzymatic hydrolyzes are veryimportant because of different peptide bond cleavage patternsaccording to the enzymes (Shahidi and Ying, 2008). Therefore,FFM was hydrolyzed with proteolytic enzymes, including papain,pepsin, trypsin, neutrase, alcalase, kojizyme, protamex, anda-chymotrypsin in a batch reactor, and a distilled water extractwas used as the control. The antioxidant activities of the hydroly-sates against DPPH and hydroxyl and peroxyl radicals wereevaluated using an ESR spectrometer and their activities are shownin Table 2. Among the FFM hydrolysates prepared with differentenzymes, the a-chymotrypsin hydrolysate showed the highestantioxidative activities on DPPH and peroxyl radicals. (IC50 values,2.775 and 0.156 mg/ml, respectively). Although the pepsinhydrolysate showed the lowest IC50 value for hydroxyl radicalscavenging activities, the a-chymotrypsin hydrolysate indicatedthe second lowest IC50 value. Therefore, we chose the a-chymo-trypsin hydrolysate. Antioxidative peptides are obtained by enzy-matic hydrolysis of various proteins derived from marineorganisms (Rajapakse et al., 2005; Himaya et al., 2011). Jun et al.(2004) reported that yellow fin sole protein is hydrolyzed bya-chymotrypsin to produce an antioxidative peptide and that thepeptide had good antioxidant activity.

3.3. Optimum FFM hydrolysis conditions using a-chymotrypsin

Enzymatic hydrolysis is influenced by several factors such aspH, time, enzyme to substrate ratio, and temperature, which coop-eratively influence enzyme activity (Viera et al., 1995; Liaset et al.,2000). Mullaly et al. (1995) reported that the choice of substrates,proteases added, and degrees of hydrolysis generally affect thephysicochemical properties of the resulting hydrolysates.a-Chymotrypsin is a non-specific protease with an optimum pHof 7.8 and an optimum temperature of 37 �C. Optimal hydrolysisconditions for producing peptides with maximal activity werestudied by manipulating the E/S ratio and reaction time. ROSscavenging activity of the a-chymotrypsin hydrolysates obtainedwith different reaction times (6, 12, 18, and 24 h) at E/S ratios of1:1000, 1:500, and 1:100, respectively, are shown in Table 3. Asthe E/S ratio increased, antioxidative activities of the hydrolysateagainst DPPH and the hydroxyl and peroxyl radicals decreased.An 18 h reaction time resulted in the highest antioxidativeactivities (IC50 values, 1.968 mg/ml for DPPH, 0.347 mg/ml for thehydroxyl radical, and 0.148 mg/ml for the peroxyl radical). Wuet al. (2003) reported that the higher hydrolysis of minced

pH Temp. (�C) DPPH Hydroxyl Peroxyl

Distilled water 7.0 37 10< 1.653 1.423Papain 6.2 37 5.850 0.666 0.244Pepsin 2.0 37 10< 0.124 0.248Trypsin 7.6 37 8.685 1.459 0.286Neutrase 6.0 50 10< 0.594 0.308Alcalase 8.0 50 10< 0.446 0.339Kojizyme 6.0 40 3.227 0.461 0.184Protamex 6.0 40 6.642 0.580 0.181a-Chymotrypsin 7.8 37 2.775 0.403 0.156

Table 3Radicals scavenging activities of a-chymotrypsin hydrolysate from flounder fish, according to hydrolysis times and substrate to enzyme ratios.

Radicals Substrate:enzyme (S:E) Hydrolysis times, (IC50 mg/ml)

6 h 12 h 18 h 24 h

DPPH 1000:1 1.963 2.890 1.968 3.803500:1 3.760 3.778 2.548 2.775100:1 3.379 7.945 7.668 2.919

Hydroxyl 1000:1 0.212 0.301 0.347 0.313500:1 0.366 0.324 0.353 0.403100:1 0.326 0.416 0.672 0.793

Peroxyl 1000:1 0.172 0.228 0.148 0.200500:1 0.152 0.208 0.158 0.156100:1 0.177 0.326 0.156 0.209

116 J.-Y. Ko et al. / Food and Chemical Toxicology 52 (2013) 113–120

mackerel fish fillet may result in a decrease in antioxidative activ-ity, suggesting that increasing hydrolysis time may decrease anti-oxidative activity of fish protein hydrolysates, which may be dueto breakdown of antioxidative peptide sequences formed duringthe early stages of hydrolysis.

The size of peptides in the hydrolysates was determined by so-dium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with stacking and resolving gels. Proteins were visualizedby Coomassie Brilliant Blue R-250 staining. The molecular size ofthe hydrolysates decreased with increasing hydrolysis times, andbands with molecular sizes <6 kDa increased (Fig. 1).

3.4. Separation and purification of antioxidant peptides from thea-chymotrypsin hydrolysate (substrate to enzyme ratio of 1000:1for 18 h)

The hydrolysates were ultrafiltered using 5 and 10 kDa cut-offmembranes to analyze the molecular weight (MW) distributionof the hydrolysates. Ultrafiltration is a fast and easy techniquefor separating peptides, based on their molecular weight as it re-quires numerous washings and prolonged separation times tocompletely eliminate low MW fractions (i.e. <5 kDa fractions)(Sivakumar and Hordur, 2009). The yields and total protein con-tents of the three fractions separated using ultrafiltration mem-branes are shown in Table 4. The >10 kDa fraction possessed thehighest yield and total protein content, compared with those of

209KDa117KDa97KDa

55KDa

37KDa

29KDa

6KDa

19KDa

B C D EA

Fig. 1. SDS–PAGE pattern of molecular weight fractions from a-chymotrypsinhydrolysate. A: Marker, B: a-chymotrypsin hydrolysate (1000:1, 18 h), C: >10 kDafraction, D: 5 � 10 kDa fraction. E: <5 kDa fraction.

the other fractions with lower MW compounds. The MW patternsof the fractions were confirmed by SDS–PAGE (Fig. 1). The free rad-icals scavenging activities of the fractions are shown in Table 4. Allfractions exhibited dose-dependent scavenging activities of vary-ing capacities. The <5 kDa fraction had the highest peroxyl radicalscavenging activity (IC50 value, 0.130 mg/ml) than that of the otherfractions.

The hydrolysates were partially purified using a size-exclusioncolumn to clarify the active fractions in the FMHs. The <5 kDa frac-tion from the a-chymotrypsin hydrolysate was loaded on a size-exclusion column containing Sephadex G-25. Sephadex G-25 hasan optimal MW range of 1000–5000 Da and separated the antiox-idative active fractions, as shown in Fig. 2A. We obtained four frac-tions, which showed peroxyl radical scavenging activity at 1 mg/ml, 38.29%, 66.82%, 60.04% and 90.54% for fractions 1, 2, 3, and 4,respectively (Table 5).

Fraction 4 obtained from size-exclusion chromatography wasfurther separated by reverse-phase-high performance liquid chro-matography on an YMC-Pack ODS-A column (5 lm, 4.6 � 250 mm,YMC Co., Kyoto, Japan) with a linear gradient of acetonitrile (0–100%), and three fractions were obtained (Fig. 2B). Fractions 4–2and 4–3 exhibited the highest radical scavenging activity (Table 5).Thus, the amino acid sequences of fractions 4–2 and 4–3 weredetermined by quantitative time of flight (Q-TOF) ESI mass spec-troscopy (Fig. 3). The two purified peptides were identified asVal-Cys-Ser-Val (406.1 Da) in fraction 4–2 and Cys-Ala-Ala-Pro(360.1 Da) in fraction 4–3. We named the two peptides VCSV andCAAP. In particular, the two purified peptides exhibited higherscavenging effects against peroxyl radical (IC50 values, 15.26 and29.86 lM), compared with those of the other radicals. It is com-monly believed that His, Met, and Cys are very important aminoacids for peptide radical scavenging activity due to their specialstructural characteristics. Cys is hydrophobic and can interact di-rectly with free radicals by donating a sulfur hydrogen (Hernan-dez-Ledesma et al., 2005). Je et al. (2008) reported that peptidescontaining Pro, Leu, Ala, and Tyr play an important role in radicalscavenging effects. Two non-polar aliphatic amino acids, Val andAla highly react with hydrophobic polyunsaturated fatty acids,which is one of the mechanisms to inhibit the radical mediatedperoxidizing chain reaction. In the present study, both purifiedpeptides obtained from the FFM hydrolysate consisted of highlyhydrophobic amino acids such as valine and alanine. Therefore,these peptides were suggested to possess the ability to interactwith lipids and scavenge lipid-derived radicals by donatingprotons.

3.5. Scavenging and cytoprotective effects of antioxidant peptideson AAPH-induced intracellular ROS

The cytotoxicity of the isolated antioxidative peptides wasdetermined by the MTT assay, which showed no cytotoxicity after24 h (Fig. 4A). Hence, the two peptides were used to determine the

Table 4Yields, total protein contents and radicals scavenging activities of different molecular weight fractions from a-chymotrypsin hydrolysate of flounder fish.

Molecular sizes Yields (%) Protein contents (mg/g) Radicals (IC50 mg/ml)

Hydroxyl Peroxyl

Unfractionated 43.83 ± 0.00c 306.86 ± 4.21c 0.347 0.148>10 kDa 54.07 ± 0.05c 418.30 ± 5.52c 0.165 0.2965 � 10 kDa 15.07 ± 0.09a 98.93 ± 8.95a 0.413 0.250<5 kDa 22.13 ± 0.14b 111.08 ± 27.13b 0.332 0.130

Mean ± SD from triplicate determinations.Significant differences at p < 0.05 indicated with different letters.

)B()A(

0

0.5

1

1.5

2

2.5

3

3.5

4

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

Abs

orba

nce

at 2

20 n

m

Fraction unmber

Fr.1

Fr.3 Fr.4

Fr.2

Fr.4-1

Fr.4-2

Fr.4-3

Fig. 2. Sephadex G-25 gel filtration chromatogram of <5 kDa fraction from a-chymotrypsin hydrolysate of flounder fish and RP-HPLC chromatogram of the isolated Fr. 4isolated on Sephadex G-25. (A) Separation on Sephadex G-25 was performed with a elution rate of 1.5 ml/min and a fraction volume (7.5 ml), and the four fractions wereseparation. (B) Separation on RP-HPLC with an YMC-Pack ODS-A column (5 lm, 4.6 � 250 mm) was performed with a linear gradient of acetonitrile from 0% to 100% at a flowrate of 1.0 ml/min. The elution was monitored at 220 nm. Three fractions were separated on RP-HPLC (Fr.4–1 � Fr.4–3).

Table 5Separation and purification of the <5 kDa fraction from a-chymotrypsin hydrolysateof flounder fish.

Purification step Radicals (IC50 mg/ml)

DPPH Hydroxyl Peroxyl

Gel filtration chromatographyFr. 1 0.5< 2< 0.837Fr. 2 0.5< 0.768 0.232Fr. 3 0.5< 0.975 0.361Fr. 4 0.239 0.301 0.094

RP-HPLCFr. 4–1 0.5< 0.25< 0.25<Fr. 4–2 0.193 0.300 0.071Fr. 4–3 0.086 0.282 0.110

Purified peptidesVCSV 0.045 0.253 0.006CAAP 0.009 0.252 0.010

J.-Y. Ko et al. / Food and Chemical Toxicology 52 (2013) 113–120 117

protective effect of 2,2-azobis-(2-amidinopropane) dihydrochlo-ride (AAPH)-induced cell cytotoxicity in Vero cells. Fig. 4B showsthat the purified FFM peptides increased viability in AAPH-damaged Vero cells in a dose-dependent manner. However,significant cell damage was observed in the control after treatmentwith AAPH. Total ROS were measured by fluorometric assay with20,70-dichlorofluorescent (DCFH-DA), which is used extensively tomonitor oxidation and to detect and quantify intracellular ROSproduced in biological systems (Chen and Wong, 2009). Therefore,the DCFH-DA assay has been utilized as a probe to analyze

intracellular ROS. In the presence of cellular ROS, peroxides in par-ticular convert non-fluorescent (DCFH-DA) into highly fluorescentdichlorofluorescein (DCFH) through oxidation. The purified VCSVand CAAP peptide from FFM dose-dependently decreased totalROS (Fig. 4C). The fluorescence intensity of the control group wasrecorded as 5515 and 4106, and intracellular ROS accumulation in-creased following treatment with AAPH. However, adding the twopurified peptides reduced intracellular ROS accumulation. Further-more, the scavenging effect of the two purified peptides on intra-cellular ROS increased significantly in a dose-dependent manner(34.05, 36.26, 42.68 and 47.71% for the VCSV peptide, and 22.94,37.30, 46.67 and 47.09% for the CAAP peptide), respectively.

AAPH generates free radicals by reacting with oxygen mole-cules, resulting in the rapid formation of peroxyl radicals. These li-pid peroxyl radicals attack other lipid molecules to form lipidhydroperoxide and new lipid radicals. This chain reaction occursrepeatedly, resulting in attacks on various biological moleculesand the production of physiochemical alterations and cellulardamage (Yokozawa et al., 2000). Therefore, elevated peroxyl radi-cal levels must be minimized. In this study, the antioxidant activityof the two purified peptides could be utilized as a natural antioxi-dant to potentially reduce the levels of pro-oxidants such as AAPH.

3.6. Protective effects of the peptides on AAPH-induced cell damage

The protective effects of the two purified peptides onAAPH-induced cell damage were observed by double staining

Fig. 3. Molecular mass identification and amino acid sequence of the purified peptides from a-chymotrypsin hydrolysate of flounder fish by Q-TOF mass spectrometer.

118 J.-Y. Ko et al. / Food and Chemical Toxicology 52 (2013) 113–120

nuclei Vero cells with Hoechst 33342. The microscopic photographin Fig. 4D shows that the control group had intact nuclei, whereasthe AAPH-treated group exhibited significant nuclear fragmenta-tion and destruction, which characterizes apoptosis (bright bluecolor). However, the amount of fragmentation and destruction ofthe AAPH-treated group decreased dramatically following treat-ment with the purified VCSV and CAAP peptides. Numerous studieshave reported that internucleosomal DNA fragmentation is not

essential for apoptotic cell death, but that DNA fragmentationaccompanies some necrotic cell death. However, it may not be asufficient indicator of apoptosis. It is clear that the central mecha-nism of apoptosis is evolutionarily conserved and that caspase acti-vation is an essential step in this complex apoptotic pathway(Cohen et al., 1992). Therefore, our result showed that the twoantioxidant peptides provided protection against AAPH-inducedapoptosis in Vero cells.

(B)(A)

(C)

(D)

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VCSV CAAP VCSV CAAP

AAPH 10 mM + Peptide µg/ml

Fig. 4. The protective effects of VCSV and CAAP peptide against AAPH-induced oxidative damage in Vero cells. Cells were treated with VCSV and CAAP at the indicatedconcentrations (12.5, 25, 50, and 100 lg/ml). (A) The cytotoxic effect of VCSV and CAAP on viability. After 24 h of treatment of VCSV and CAAP the cell viabilities wereassessed by MTT assay. (B) Cytoprotective effects of VCSV and CAAP on AAPH-induced oxidative damage. Cell viabilities were assessed by MTT assay. (C) The intracellular ROSgenerated was detected by DCFH-DA assay. (h,j) Fluorescence intensity; (s,d) intracellular ROS scavenging activity. (D) Protective effects of VCSV and CAAP against AAPH-induced apoptosis in the cells. The apoptotic body formation was observed under a fluorescence microscope after Hoechst 33342 staining. ⁄p < 0.05 and ⁄⁄p < 0.01.

J.-Y. Ko et al. / Food and Chemical Toxicology 52 (2013) 113–120 119

120 J.-Y. Ko et al. / Food and Chemical Toxicology 52 (2013) 113–120

4. Conclusion

We demonstrated that two purified peptides (VCSV and CAAP)derived from flounder muscle protein possessed strong antioxida-tive activity. In particular, the purified peptides, consisting of ami-no acid residues in the sequence Pro, Ala, Val, and Cys may havecontributed to antioxidative activity. Taken together, our resultssuggest that the two antioxidative peptides could be useful as ther-apeutic agents.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgement

This research was supported by the National Research Founda-tion of Korea (NRF) grant funded by the Korea government (MEST)(No. 2012H1B8A2025863).

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