Protective effect of a novel antioxidative peptide purified from a marine Chlorella ellipsoidea protein against free radical-induced oxidative stress

Food and Chemical Toxicology 50 (2012) 2294–2302

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Protective effect of a novel antioxidative peptide purified from a marine Chlorella ellipsoidea protein against free radical-induced oxidative stress Seok-Chun Ko a,1, Daekyung Kim b,1, You-Jin Jeon a,⇑ a b

Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea Marine Bio Research Team, Korea Basic Science Institute (KBSI), Jeju 690-140, Republic of Korea

a r t i c l e

i n f o

Article history: Received 11 October 2011 Accepted 14 April 2012 Available online 21 April 2012 Keywords: Chlorella ellipsoidea Antioxidative peptide Free radical scavenging Oxidative stress

a b s t r a c t Protein derived the marine Chlorella ellipsoidea was hydrolyzed using different proteases (papain, trypsin, pepsin and a-chymotrypsin) for production of antioxidative peptide, and the antioxidant activities of their hydrolysates were investigated using free radical scavenging assay by electron spin resonance spin-trapping technique. Among the hydrolysates, the peptic hydrolysate exhibited the highest antioxidant activity compared to other hydrolysates. To identify antioxidant peptide, the peptic hydrolysate was purified using consecutive chromatographic methods, and the antioxidant peptide was identified to be Leu-Asn-Gly-Asp-Val-Trp (702.2 Da) by Q-TOF ESI mass spectroscopy. The antioxidant peptide scavenged peroxyl, DPPH and hydroxyl radicals at the IC50 values of 0.02, 0.92 and 1.42 mM, respectively. The purified peptide enhanced cell viability against AAPH-induced cytotoxicity on normal cells. Furthermore, the purified peptide reduced the proportion of apoptotic and necrotic cells induced by AAPH, as demonstrated by decreased sub-G1 hypodiploid cells and decreased apoptotic body formation by flow cytometry. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Reactive oxygen species (ROS) including free radicals such as peroxyl radicals (ROO), nitric oxide radicals (NO), hydroxyl radicals (OH), and superoxide radicals (O2) play an important role in many diseases (Heo and Jeon, 2008; Kim et al., 2007). ROS are physiological metabolites formed during aerobic life as a result of the metabolism of oxygen (Heo et al., 2005). Under normal conditions, ROS are effectively eliminated by antioxidant defense systems such as antioxidant enzymes and non-enzymatic factors (Qian et al., 2008). However, under pathological conditions, the balance between the generation and elimination of ROS is broken, as a result of these event, biomacromolecules are damaged by ROSinduced oxidative stress (Qian et al., 2008; Pryor, 1982; Butterfield et al., 2002). Therefore, antioxidants are important for bodily protection against oxidative stress. Generally, many synthetic antioxidants including butylated hydroxyanisol (BHA), butylated hydroxyltoluene (BHT), and tert-butylhydroquinone (TBHQ) have been used to reduce oxidative damage in the human body (Kim et al., 2008; Sherwin, 1990). However, the use of synthetic antioxidants in humans is associated with some side effects, such as carcinogenesis and liver damage (Lindenschmidt et al., 1986). In recent years, bio-resources ⇑ Corresponding author. Tel.: +82 64 754 3475; fax: +82 64 756 3493. 1

E-mail address: [email protected] (Y.-J. Jeon). These authors contributed equally to this study.

0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.04.022

have become the focus of a considerable amount of attention from researchers searching for protective agents against ROS-induced effects (Wang et al., 2010). As such, there is motivation to search for safe and natural antioxidants from various marine bio-resources (Byun et al., 2009). Several studies on the antioxidant effects of hydrolysates from marine bio-resource proteins, such as marine rotifer (Byun et al., 2009), tuna backbone (Je et al., 2007), round scad (Thiansilakul et al., 2007), and squid muscle, (Rajapakse et al., 2005) and grass carp (Ren et al., 2008) have been previously conducted. Recently, interest has emerged to identify and characterize bioactive peptides from bio-resources (Sarmadi and Ismail, 2010). Bioactive peptides can be derived by enzymatic proteolysis of bio-resource proteins and may act as potential physiological modulators of metabolism during gastrointestinal digestion of nutrients (Byun et al., 2009; Je et al., 2007). Bioactive peptides are in the size of 2–20 amino acids (Meisel and FitzGerald, 2003). Based on their structural properties their amino acid sequences may exhibit various bioactivities such as antihypertensive (Zhao et al., 2009), antioxidative (Mendis et al., 2005), immunomodulatory (Gauthier et al., 2006), and hypocholestrolemic (Zhong et al., 2007). Chlorella has been a popular foodstuff worldwide, and it contains protein, minerals, vitamins, chlorophyll, and bioactive substances. Several studies on these kinds of components have indicated a variety of biological benefits including antioxidant (Lee et al., 2010), anti-diabetes (Rodriguez-Lopez and LopezQuijada, 1971), anti-hypercholesterolemic (Okudo et al., 1975),

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anti-inflammatory and immunomodulatory (Guzman et al., 2003) effects. These potential benefits have been attributed to the effects of specific ingredients in Chlorella, such as dietary fiber, minerals, and chlorophylls (Lee et al., 2010). Chlorella ellipsoidea, used in this study, is a type of single-celled marine microalga. C. ellipsoidea proteins are usually used as live feed for rotifer and shrimp because of their poor functional properties. However, C. ellipsoidea proteins can be converted into value-added products by enzymatic hydrolysis, which may be widely applied to improve the functional and nutritional properties of C. ellipsoidea proteins. Therefore, the objective of this study was to purify antioxidant peptides from marine C. ellipsoidea protein by enzymatic hydrolysis and to evaluate their antioxidant properties using different in vitro systems. In addition, the protective effects of the purified peptides against free radical-induced cellular damage were also determined. 2. Materials and methods 2.1. Materials Marine C. ellipsoidea, was obtained from Marine Bioprocess Co., Korea, and lyophilized at 70 °C using a freeze dryer. Lyophilized C. ellipsoidea powder was stored at 80 °C until use. Proteases for enzymatic hydrolysis (pepsin, trypsin, and a-chymotrypsin), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2,2-azobis(2-amidinopropane) hydrochloride (AAPH) were purchased from Sigma Chemical Co. (St. Louis, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and streptomycin–penicillin was purchased from Gibco (Gaithersburg, MD). The other chemicals and reagents used were of analytical grade.

2.2. Preparation of marine C. ellipsoidea hydrolysates To obtain bioactive peptides from marine C. ellipsoidea, enzymatic hydrolysis was performed according to the method by Lee et al. (2009) and using various commercial proteases (papain, trypsin, pepsin and a-chymotrypsin) under optimal conditions. Enzyme and substrate were mixed at the ratio of enzyme-to-substrate (1:100, w/w). The mixture was incubated for 12 h at each optimal temperature with stirring and then heated in a boiling water bath for 10 min to inactivate the enzyme. All the gastrointestinal digests were kept 20 °C for further experiments.

2.3. Purification of antioxidant peptide The marine C. ellipsoidea hydrolysate showing antioxidant activity was utilized to purify potent radical scavenging peptide sequences and radical scavenging activity was tested at each purification step. The marine C. ellipsoidea hydrolysate showing free radical scavenging activity was dissolved in distilled water and loaded onto a Sephadex G-25 gel filtration column (2.5  75 cm), equilibrated with distilled water. The column was eluted with distilled water at a flow rate of 2.0 ml/min. The antioxidative fraction obtained were then applied to reverse-phase high-performance liquid chromatography (HPLC) on a YMC-Pack ODS-A column (5 lm, 4.6  250 mm, YMC Co., Kyoto, Japan) with a linear gradient of acetonitrile (0– 20% v/v, 30 min) containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 1.0 mL/min. Elution peaks were detected at 280 nm.

2.4. Characterization of purified antioxidative peptide The molecular weight and amino acid sequence of the purified peptide from the marine C. ellipsoidea was determined using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled with electrospray ionisation (ESI) source. The purified peptide dissolved in methanol/water (1:1, v/v) was infused into the ESI source and the molecular weight was determined by doubly charged (M+2H)2+ state analysis in the mass spectrum. Following the molecular weight determination, the peptide was automatically selected for fragmentation and sequence information was obtained by tandem MS analysis.

2.5. Electron spin resonance (ESR) spectrometric assays 2.5.1. DPPH radical scavenging activity 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was measured using the method described by Nanjo et al. (1996). Sample solution (60 lL) was added to 60 lL of DPPH (60 lM/L) in methanol solution. After mixing vigorously for 10 s, the solutions were then transferred into a 100-lL Teflon capillary tube and fitted into the cavity of the ESR spectrometer (JES-FA machine, JOEL,

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Tokyo, Japan). The spin adduct was measured on an ESR spectrometer exactly 2 min later. Measurement conditions: central field 3475 G, modulation frequency 100 kHz, modulation amplitude 2 G, microwave power 5 mW, gain 6.3  105. 2.5.2. Hydroxyl radical scavenging activity Hydroxyl radicals were generated by Fenton reaction, and reacted rapidly with nitrone spin trap DMPO; the resultant DMPO-OH adduct was detectable with an ESR spectrometer (Rosen and Rauckman 1993). The ESR spectrum was recorded 2.5 min after mixing in a phosphate buffer solution (pH 7.4) with 0.3 M DMPO 0.2 mL, 10 mM FeSO4 0.2 mL and 10 mM H2O2 0.2 mL using JES-FA ESR spectrometer (JEOL, Tokyo, Japan) set at the following conditions: central field 3475 G, modulation frequency 100 kHz, modulation amplitude 2 G, microwave power 1 mW, gain 6.3  105. 2.5.3. Peroxyl radical scavenging activity Peroxyl radicals were generated by AAPH (Hiramoto et al., 1993). The phosphate buffered solution (pH 7.4) reaction mixtures containing 10 mM AAPH, 10 mM 4-POBN and indicated concentrations of tested samples, were incubated at 37 °C in a water bath for 30 min, and then transferred to a 100-lL Teflon capillary tube. The spin adduct was recorded on JES-FA ESR spectrometer (JEOL, Tokyo, Japan). Measurement conditions: central field 3475 G, modulation frequency 100 kHz, modulation amplitude 2 G, microwave power 10 mW, gain 6.3  105. 2.6. Cell culture study of the purified peptide 2.6.1. Cell culture Vero (Monkey kidney cell line) cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, streptomycin (100 mg/mL), and penicillin (100 U/mL). Cultures were maintained at 37 °C in 5% CO2 incubator. 2.6.2. Cytotoxic assessment using MTT assay Cells were seeded in a 96-well plate at a concentration of 1  105 cells/mL. Sixteen hours after seeding, the cells were treated with various concentrations of the purified peptide (25, 50 and 100 lM). The cells were then incubated for an additional 24 h at 37 °C. MTT stock solution (50 lL; 2 mg/mL in PBS) was then added to each well to a total reaction volume of 250 lL. After 4 h of incubation, the plates were centrifuged (800g, 5 min), and the supernatants were aspirated. The formazan crystals in each well were dissolved in 150 lL of dimethylsulfoxide (DMSO), and the absorbance was measured with an ELISA plate reader at 540 nm. 2.6.3. Intracellular radical measurement For the detection of intracellular AAPH, the Vero cells were seeded in 96-well plates at a concentration of 1  105 cells/mL. Sixteen hours after seeding, the cells were treated with various concentrations of the purified peptide (25, 50 and 100 lM). After 30 min, AAPH was added at a concentration of 10 mM, and then cells were incubated for an additional 30 min at 37 °C under a humidified atmosphere. Finally, 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA; 5 lg/mL) was introduced to the cells, and DCFH-DA was detected at an excitation wavelength of 485 nm and an emission wavelength of 535 nm, using a Perkin-Elmer LS-5B spectrofluorometer. 2.6.4. AAPH-induced cytotoxicity with the purified peptide Cells were seeded in a 96-well plate at a concentration of 1  105 cells/mL. Sixteen hours after seeding, the cells were treated with various concentrations of the purified peptide (25, 50 and 100 lM). After 1 h, AAPH was added at a concentration of 10 mM, and then cells were incubated for an additional 24 h at 37 °C under a humidified atmosphere. MTT stock solution (50 lL; 2 mg/mL in DPBS) was then added to each well to a total reaction volume of 250 lL. After 4 h of incubation, the plates were centrifuged (800g, 5 min), and the supernatants were aspirated. The formazan crystals in each well were dissolved in 150 lL of dimethylsulfoxide (DMSO), and the absorbance was measured with an ELISA plate reader at 540 nm. Relative cell viability was evaluated in accordance with the quantity of MTT converted to the insoluble formazan salt. The optical density of the formazan generated in the control cells was considered to represent 100% viability. The data are expressed as mean percentages of the viable cells versus the respective control. 2.6.5. Microscopic analysis for dead cells The type of cell death (apoptosis or necrosis) induced by AAPH was determined by fluorescent microscopy after staining with Hoechst 33342 (HO 342) or propidium iodide (PI), as described by Heo et al. (2009). Cells were seeded in a 24-well plate at a concentration of 1  105 cells/mL. Sixteen hours after seeding, the cells were treated with. various concentrations of the purified peptide (25, 50 and 100 lM).After 1 h, AAPH was added at a concentration of 10 mM, and then cells were incubated for an additional 24 h at 37 °C under a humidified atmosphere. Then, 1.5 lL of HO342 (stock 10 mg/mL) or PI were added to each well, followed by 10 min of incubation 37 °C. The stained cells were then observed under a fluorescence microscope equipped with a Moticam-Pro color digital camera, in order to examine the degree of nuclear condensation.

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2.6.6. Flow cytometry analysis Flow cytometry analyses were conducted to determine the proportion of apoptotic sub-G1 hypodiploid cells (Nicoletti et al., 1991). The Vero cells were placed in 6-well plates at a concentration of 1.0  105 cells/mL, and 16 h after plating, the cells were treated with various concentrations of the sample fraction (100 lg/ mL). After further 1 h incubation, H2O2 (1 mM) was added to the culture. After 24 h, the cells were harvested at the indicated times and fixed for 30 min in 1 mL of 70% ethanol at 4 °C. The cells were then washed twice with PBS and incubated for 30 min in the dark in 1 mL of PBS containing 100 lg PI and 100 lg RNase A at 37 °C. Flow cytometric analysis was conducted using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Effects on the cell cycle were determined by measuring changes in the percentage of cell distribution at each phase of the cell cycle and were assessed by histograms generated by the Cell Quest and Mod-Fit computer programs (Wang et al., 1999). 2.7. Statistical analysis All data were represented as the mean ± standard deviation of three determinations. Statistical comparisons of the mean values were performed by analysis of variance (ANOVA), followed by Duncan’s multiple range test using SPSS software. Statistical significance was considered at p < 0.05.

measured by dry weight, and observed to be 31.7%, 32.4%, 34.1%, and 30.8% for papain, pepsin, a-chymotrypsin, and trypsin, respectively (Table 1). Also, four proteolytic enzymes were selected to evaluate their effectiveness on degradation of marine C. ellipsoidea protein for antioxidant activities. Degrees of hydrolysis (DH) after proteolytic digestion were observed to be 40.3%, 72.3%, 70.1%, and 60.1% for papain, pepsin, a-chymotrypsin, and trypsin, respectively (Table 1). The antioxidative activity of a substance may be distinguished exactly by testing its scavenging activities on free radicals in oxidative systems. Consequently, the hydrolysates were evaluated for their free radical scavenging activities on DPPH, hydroxyl, and peroxyl radicals using the electron spin resonance technique. Among the hydrolysates, the pepsin-derived hydrolysate exhibited the highest hydroxyl and peroxyl radical scavenging activities compared to other hydrolysates (Table 1). Therefore, we selected peptic hydrolysate for the purification of the antioxidative peptide.

3. Results

3.2. Purification and identification of antioxidative peptide

3.1. Preparation of marine C. ellipsoidea protein hydrolysates and their antioxidant properties

To identify the antioxidative peptide derived from the marine C. ellipsoidea hydrolysate that the antioxidant activity, the peptide was separated by Sephadex G-25 column chromatography in the four fractions (Fig. 1(A)). Fraction F4 was more potent at scavenging peroxyl and DPPH radicals (Fig. 1(B)). The potent fraction pool obtained from gel filtration chromatography was further separated

The marine C. ellipsoidea was sequentially hydrolyzed with various proteases such as papain, pepsin, trypsin, and a-chymotrypsin. The yields of the marine C. ellipsoidea hydrolysates were

Table 1 Yield and free radical scavenging activities from enzymatic hydrolysates of marine C. ellipsoidea. Enzyme

DHB (%)

Yield (%)

IC50 value (mg/ml) DPPH radical

Pepsin Trypsin a-Chymotrypsin Papain A B

A,d

34.1 ± 0.3 30.8 ± 0.2a 32.4 ± 0.1c 31.7 ± 0.3b

c

72.3 ± 1.4 60.1 ± 2.1b 70.1 ± 3.1c 40.3 ± 1.8a

Hydroxyl radical b

2.321 ± 0.037 2.698 ± 0.027c 1.841 ± 0.054a 2.598 ± 0.061c

a

0.494 ± 0.077 2.155 ± 0.053d 1.232 ± 0.102b 1.562 ± 0.058c

Peroxyl radical 0.754 ± 0.076a 0.888 ± 0.077ab 1.029 ± 0.081b 0.854 ± 0.051a

Means with different lowercase letters are significantly different by Duncan’s multiple range test (p < 0.05). DH, degree of hydrolysis.

Fig. 1. Sephadex G-25 gel filtration chromatogram of peptic hydrolysate. (A) Separation was performed with 2 mL/min and collected at a fraction volume (10 mL). The fractions isolated by Sephadex G-25. Gel column were separated into four fractions (F1–F4). (B) Free radical scavenging activities of each fraction.

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Fig. 2. RP-HPLC chromatogram of the potent free radical scavenging activity fraction F4 isolated from Sephadex G-25. (A) Separation into sub-fractions (A and B) was performed with linear gradient of acetonitrile from 0% to 20% at a flow rate of 1.0 mL/min and an YMC-Pack ODS-A column (5 lm, 4.6  250 mm). The elution was monitored at 280 nm. (B) Free radical scavenging activities of each fraction.

Fig. 3. RP-HPLC profile of the purified the antioxidant peptide.

with RP-HPLC on a YMC-Pack ODS-A column (5 lm, 4.6 (250 mm, YMC Co., Kyoto, Japan) with a linear gradient of acetonitrile (0– 20%) containing 0.1% trifluoroacetic acid (TFA), and two main fractions were obtained (Fig. 2(A)). Fraction A exhibited the most potent free radical scavenging activity (Fig. 2(B)). The purity of fraction A was confirmed by RP-HPLC analysis (Fig. 3) and its amino acid sequence was determined by Q-TOF ESI mass spectroscopy. The purified peptide A was identified as hexapeptide Leu-Asn-GlyAsp-Val-Trp (702.2 Da) (Fig. 4). In order to validate the antioxidant activities of the purified peptide, a synthetic peptide with the same sequence was synthesized and tested. The synthetic peptide exhibited the same antioxidant activities as the purified peptide from marine C. ellipsoidea protein hydrolysate (Table 2). The result suggests that the purified peptide actually possesses antioxidant activities. 3.3. Effect of the antioxidative peptide on cell viability in normal cells In this study, normal cells were treated with different concentrations of the antioxidative peptide to determine non-cytotoxic effects for further experiments. The cell viability data confirmed

that the antioxidative peptide was non-cytotoxic in normal cells (Fig. 5). Therefore, it was determined that the antioxidative peptide could be used in further experiments. 3.4. Inhibitory effect of the antioxidative peptide on intracellular radicals generated by AAPH and cytotoxicity Intracellular radical scavenging activity could be detected via measurements of the level of DCF, the results of which are illustrated in Fig. 6. The fluorescence intensity of the control cells was recorded as 1274.9, whereas that of the AAPH-treated cells was recorded as 7824.2. However, the addition of the purified peptide to the cells mixed with AAPH reduced intracellular radical accumulation to levels of 6595.5, 3738.1, and 2502.4 at 25, 50, and 100 lM, respectively. Moreover, as shown in the Fig. 6 the scavenging activity of the purified peptide on intracellular radicals increased in a dose dependent manner. The results of this study suggest that this antioxidative peptide could be developed into a potential bio-molecular candidate to inhibit radical formation. Since this antioxidative peptide was found to exert radical scavenging effects, it was further evaluated with regard to the protec-

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Fig. 4. Identification of the molecular weight and amino acid sequence of the purified peptide using Q-TOF mass spectrometer.

Table 2 Comparison with antioxidant activities by purified and synthetic peptide. IC50 value DPPH radical Purified peptide Synthetic peptide

Hydroxyl radical A,B,a

a

Peroxyl radical

0.654 ± 0.048 0.649 ± 0.086a

0.986 ± 0.138 0.997 ± 0.059a

0.014 ± 0.003a 0.012 ± 0.012a

0.92 ± 0.12C

1.42 ± 0.08

0.02 ± 0.01

A

Means with different lowercase letters are significantly different by Duncan’s multiple range test (p < 0.05). B mg/mL of purified and synthetic peptide. C lM of synthetic peptide.

Fig. 6. The intracellular radical generated was detected by DCFH-DA assay. Bar graph exhibited fluorescence intensity and line graph showed the intracellular radical scavenging activity. Values are expressed as means ± SD in triplicate experiments. Statistical evaluation was performed to compare the experimental groups and AAPH-treated cells. ⁄p < 0.05 and ⁄⁄p < 0.01.

tive effect against AAPH-induced cell damage. The effect of the antioxidative peptide on cell viability in AAPH-induced Vero cells was measured via MTT assay. As shown in Fig. 7, AAPH treatment without the antioxidative peptide decreased cell viability to 48.6%, while the antioxidative peptide prevented cells from AAPH-induced cell damage, restoring cell survival to 56.3%, 72.3%, and 79.4% at the concentrations of 25, 50, and 100 lM, respectively. 3.5. Protective effect of the antioxidative peptide against AAPHinduced cell damage Fig. 5. The cytotoxic effect of the purified peptide on viability in normal cells. Cells were treated with the purified peptide at the indicated concentrations (25, 50 and 100 lM). After 24 h to treat the purified peptide cell viability was assessed by MTT assay. Values are expressed as means ± SD in triplicate experiments.

The micrograph in Fig. 8 shows that the control cells had intact nuclei (Fig. 8A), and the AAPH-treated cells exhibited significant nuclear fragmentation and destruction, which are characteristics

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Fig. 7. Protective effect of the purified peptide on AAPH-induced oxidative damage in normal cells. The viability of cells on AAPH treatment was assessed by MTT assay. Values are expressed as means ± SD in triplicate experiments. Statistical evaluation was performed to compare the experimental groups and AAPH-treated cells. ⁄ p < 0.05 and ⁄⁄p < 0.01.

of apoptosis (left; bright blue color2) and necrosis (right; red color2), respectively (Fig. 8B). However, the amount of fragmentation and destruction of the AAPH-treated cells were reduced when the cells were treated with the antioxidative peptide (Fig. 8C–F). The protective effect of the purified peptide was confirmed by flow cytometry. DNA content analyses conducted following the AAPH treatment of Vero cells revealed an increase in the proportion of cells with subG1 DNA content to 32.95% (Fig. 9). This result indicated that apoptosis was induced by the added AAPH. However, the purified peptide pretreatment dose-dependently reduced the sub-G1 DNA contents in the cells induced by the treatment with AAPH. These data indicate that the purified peptide may have notable apoptosis inhibition activity against Vero cells. 4. Discussion Recently, due to the relatively limited natural products of land, many researchers have focused on marine natural products with various biological effects. Chlorella have become good candidates for sources of natural antioxidants, as revealed by a number of recent studies (Lee et al., 2009, 2010). In the present study, we used the ESR technique spin trapping is the most direct method to detect highly reactive free radicals. With this ESR technique, a higher steady-state concentration of free radicals is achieved, which can overcome the sensitivity problem inherent in the detection of endogenous radicals in biological systems (Janzen et al., 1987; Auddy et al., 2003). DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. Therefore, DPPH is used as a substrate to evaluate the antioxidant activity of an antioxidant. Hydroxyl radicals were generated in a Fenton reaction and were visualized by an ESR spectrometer. AAPH can decompose to form peroxyl radicals that can react swiftly with O2 to yield peroxyl radicals to stimulate lipid peroxidation (Qian et al., 2008). The aim of this study was to purify and identify antioxidant peptides from marine C. ellipsoidea protein by enzymatic hydrolysis and to evaluate their antioxidant properties using reactive oxygen species (ROS) scavenging assays, and the protective effects of the purified peptides against free radical-induced cell damage. To obtain the novel active antioxidant peptide, four proteases were used under optimal conditions hydrolyze marine C. ellipsoi2 For interpretation of the references to color in this figure, the reader is referred to the web version of this article.

Fig. 8. Protective effect of the purified peptide against AAPH-induced apoptosis and necrosis in normal cells. The cellular morphological changes were observed under a fluorescence microscope after HO342 (left) and PI (right) staining. (A) Control (untreated cells); (B) 10 mM AAPH; (C) 25 lM of the purified peptide + 10 mM AAPH; (D) 50 lM of the purified peptide + 10 mM AAPH; (E) 100 lM of the purified peptide + 10 mM AAPH.

dea protein. Peptic hydrolysate showed the highest free radical scavenging activities and was selected for further isolation using HPLC. Compared to previous reports, the free radical scavenging activities of pepsin-derived hydrolysate was more effective than that of food grade protease and cabohydrase hydrolysates from C. ellipsoidea (Lee et al., 2009). The hydrolytic reaction depends on the availability of susceptible peptide bonds on which the

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Fig. 9. Effects of the purified peptide on cell cycle pattern and apoptotic cell proportion in Vero cells with AAPH (10 mM) via flow cytometric analysis. (A) Control (nontreated); (B) 10 mM AAPH-treated; (C) cells treated with 25 lM of purified peptide + 10 mM AAPH; (D) cells treated with 50 lM of purified peptide + 10 mM AAPH; (E) cells treated with 100 lM of purified peptide + 10 mM AAPH; (F) bar graph of sub-G1 content of Vero cells. Values are expressed as means ± SD in triplicate experiments. Statistical evaluation was performed to compare the experimental groups and AAPH-treated cells. ⁄p < 0.05 and ⁄⁄p < 0.01.

protease attack is concentrated, as well as the physical structure of the protein (Ngo et al., 2010). Previous reports also indicate that pepsin is capable of producing antioxidative peptides from other proteins (Byun et al., 2009; Je et al., 2007; Sheih et al., 2009). After two-step isolation, the novel antioxidative peptide which was composed of six amino acids was obtained. The antioxidant properties of the peptides were highly influenced by molecular weight and structure (Sheih et al., 2009). Most of the reported peptides showing antioxidant effects were those with low molecular

weights, increasing their chance of crossing the intestinal barrier and exerting biological effects (Rajapakse et al., 2005; Sheih et al., 2009; Roberts et al., 1999). The purified peptide in this study had a low molecular weight of 702.2 Da, and it also was intestinal enzyme tolerant. In addition, the characteristic amino acid sequence of the peptide was important for its antioxidant effects. The oxidative susceptibility of a given amino acid in a peptide to free radical attack is dictated in a large part by its R groups (Sheih et al., 2009; Elis et al., 2008). In general, antioxidative peptides

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containing aromatic amino acid residues (Trp and Tyr) at the C-terminus have strong radical scavenging activities (Samaranyaka and Li-Chan, 2011). The aromatic amino acid residues (Trp and Tyr) can make active oxygen stable through direct electron transfer (Qian et al., 2008). Furthermore, positioning of hydrophobic amino acids, such as leucine and valine, at the N-terminus of the peptide sequence is thought to be important for antioxidative activity, since it is assumed that leucine and valine can increase the presence of peptides at the water–lipid interface and therefore facilitate access to scavenge free radicals generated at the lipid phase (Ranathung et al., 2006; Chen et al., 1995). As a whole, the presence of specific amino acids and their specific positioning in the sequence could have attributed to the antioxidative activity of the purified peptide. ROS form as natural products from normal oxygen metabolism and perform important functions in cell signaling. Cells damages are normally able to defend themselves against ROS damage by the use of antioxidant enzymes, including catalases and superoxide dismutases (Heo and Jeon, 2008). However, an imbalance between ROS and antioxidant defense mechanisms can result in oxidative modification of the cellular membrane or intracellular molecules (Halliwell and Aruoma, 1991). The direct scavenging effects of the antioxidative peptide on cellular radicals were studied in order to confirm its ability to scavenge free radicals in a cellular environment. The intracellular radical scavenging effect of the antioxidative peptide was assessed in AAPH-induced cells using oxidantsensitive fluorescent probes (DCFH-DA). DCFH-DA exhibited no fluorescence without reactive oxygen species (ROS), and became fluorescent upon interaction with ROS (Handa et al., 2006). During labeling, non-fluorescent DCFH-DA dye that freely penetrates into the cells becomes hydrolyzed by intracellular esterase to DCFH, and is trapped inside the cells (Veerman et al., 2004). In the presence of ROS, DCFH was oxidized to highly fluorescent DCF (Heo and Jeon, 2009). The intracellular AAPH scavenging activity of purified peptide was expressed as 68% at the concentration of 100 lM, and the scavenging activities increased when increasing the purified peptide concentration. As the purified peptide generated in this study evidenced such good AAPH radical scavenging activity, this purified peptide was evaluated further with regard to its protective effects against AAPH-induced cellular damage. Active mitochondria of living cells can cleave MTT to produce formazan, the amount of which is directly correlated to the living cell number (Heo et al., 2010). The results of the MTT assay exhibited that formazan content was reduced due to AAPH treatment; however, it increased with the addition of the antioxidative peptide. Oxidative stress may be induced by increasing generation of ROS and other radicals. ROS induced by oxidative stress can ultimately lead to apoptotic or necrotic cell death (Fiers et al., 1999; Heo et al., 2009). These results suggest that the antioxidative peptide has the ability to protect normal cells from oxidative stress-related cellular injuries. In conclusion, we investigated the antioxidative potency of a peptide from marine C. ellipsoidea protein in different mechanisms of oxidation in vitro. In addition, the protective abilities of the purified peptide in free radical-induced cell damage were also tested. We have shown that this peptide of marine C. ellipsoidea protein has great efficiency in scavenging various free radicals. The peptide therefore has the potential to be a good dietary supplement for the prevention of oxidative stress. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgment This research was supported by a Grant (K31092) from the Korea Basic Science Institute (KBSI) to D. Kim.

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