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Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster, Crassostrea gigas against free radical induced DNA damage
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Bioresource Technology 99 (2008) 3365–3371
Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster, Crassostrea gigas against free radical induced DNA damage Zhong-Ji Qian a, Won-Kyo Jung c, Hee-Guk Byun d, Se-Kwon Kim
a,b,*
a Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea Department of NOAA Sea Grant Development and Food Science, Louisiana State University, Baton Rouge, LA 70803, USA d Faculty of Marine Bioscience and Technology, Kangnung National University, Kangnung 210-702, Republic of Korea b
c
Received 3 July 2007; received in revised form 10 August 2007; accepted 12 August 2007 Available online 27 September 2007
Abstract In this study, in vitro gastrointestinal digestion was employed to obtain potent antioxidative peptide from protein of oyster, Crassostrea gias. The protein was subjected to hydrolysate using consecutive chromatographic methods, on a Hiprep 16/10 diethylaminoethyl fast flow (DEAE FF) anion exchange column and octadecylsilane (ODS) C18 reversed phase column. Finally, the amino acid sequence of the peptide was determined. The peptide, having the amino acid sequence Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-Gln-Glu (1.60 kDa), exhibited the higher activity against polyunsaturated fatty acid (PUFA) peroxidation than that of native antioxidant, atocopherol. The free radical scavenging assay conducted using electron spin resonance (ESR) spectroscopy, clearly exhibited that it scavenged hydroxyl radical and superoxide radical at IC50 values of 28.76 lM and 78.97 lM, respectively. Further, we investigated its antioxidant activities on cellular system, and the results showed that purified peptide significantly scavenged cellular radicals and protective effect on DNA damage caused by hydroxyl radicals generated. Furthermore (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) MTT assay showed no cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5) and mouse macrophages cell (RAW264.7), respectively. These results indicate that this peptide shows potent antioxidant. 2007 Elsevier Ltd. All rights reserved. Keywords: Antioxidant peptide; Oyster Crassostrea gias; In vitro gastrointestinal; Lipid peroxidation; Radical scavenging activity
1. Introduction Lipid oxidation by reactive oxygen species (ROSs) such as superoxide anion, hydroxyl radicals, and hydrogen peroxide causes a decrease in nutritional value of lipids, their safety and appearance. In addition, it is the predominant cause of qualitative decay of foods, which leads to rancidity, toxicity, and destruction of biochemical components that are important in physiologic metabolisms. These rad* Corresponding author. Address: Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea. Tel.: +82 51 620 6375; fax: +82 51 628 8147. E-mail address: [email protected] (S.-K. Kim).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.08.018
icals are very unstable and react rapidly with other groups or substances in the body, leading to cell or tissue injury. Moreover free radicals-mediated modification of DNA, proteins, lipids, and small cellular molecules is associated with a number of pathological processes, including atherosclerosis, arthritis, diabetes, cataractogenesis, muscular dystrophy, pulmonary dysfunction, inflammatory disorders, ischemiareperfusion tissue damage, and neurological disorders such as Alzheimer’s disease (Frlich and Riederer, 1995). Specially, lipid peroxidation in foods affects the nutritive value and may cause disease conditions following consumption of potentially toxic reaction products. Therefore, during last few decade human nutrition and biochemistry research focused on an antioxidant derived from food
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ingredient that could retard lipid peroxidation. Free radical scavenger is a preventive antioxidant. Therefore, antioxidants are suggested to play an important role for bodily protection against oxidative stress. Further antioxidants are used to preserve food products to retard discoloration and deterioration that occur as a result of oxidation. This illustrates one of the many mechanisms which oxidative stress cause damage to lipids by stimulating the free radical chain reaction. In this context lipid peroxidation is of great concern to the food industry and consumers because it leads to the development of undesirable off-flavors and potentially toxic reaction products (Maillard et al., 1996). Presently, natural antioxidants such as vitamin C and atocopherol are continuously used as a means of enhancing biological functions and improving the stability of lipid and lipid-containing products. However, the use of synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), and propyl gallate (PG) is under strict regulation because of the potential health hazards (Hettiarachchy et al., 1996). Therefore, the search for natural antioxidants as alternatives to synthetic ones is of great interest among researchers. Recently a great interest is been developing to study about the structural, compositional and sequential properties of concerning bioactive peptides. They have shown the different kinds of bioactivities such as antioxidative (Jung et al., 2005; Rajapakse et al., 2005), antihypertensive (Jung et al., 2006; Je et al., 2005; Suetsuna et al., 2004) and immunomodulatory effects (Chen et al., 1995; Tsuruki et al., 2003). These reports have confirmed that bioactive peptides released by enzymatic proteolysis of food proteins may act as potential physiological modulators of metabolism during intestinal digestion. Bioactive peptides usually contain 3–20 amino acid residues, and their activities are based on their amino acid composition and sequence (PihlantoLeppala, 2001). Some recent studies have reported the in vitro formation of antioxidant peptides from marine food sources and their possibilities to be used as alternative antioxidants. The digestion by gastrointestinal proteases can be used as a production process for antioxidant peptide, with the advantage that the formed peptides will resist the physiological digestion after oral intake. The scope of this paper is to further investigate the conditions of in vitro gastrointestinal digestion leading to the formation and/or degradation of antioxidant peptides and to elucidate their antioxidative effects. Shellfish and fish sauces are widely used in East and Southeast Asian countries. In Korea, the production of oyster was estimated to be 252,000 ton in 2005, and only a few shellfish and fish sauces have survived in local areas in Korea. However, shellfish and fish sauces have recently been rediscovered because of increased consumer interest in their taste and flavor. From this point of view, the present study intended to an antioxidant peptide derived from oyster Crassostrea gigas by gastrointestinal digestion and their antioxidative
effects on linoleic acid peroxidation and radical scavenging were assessed. 2. Methods 2.1. Materials Oyster (C. gigas) was donated by Chamson Food Co. (Busan, Korea). Proteases for enzymatic hydrolysis (pepsin, trypsin and a-chymotrypsin), Linoleic acid, ammonium thiocyanate, a-tocopherol, and radical-testing chemicals, including 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), FeSO4, and H2O2 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). MRC-5 (human lung fibroblast) and RAW264.7 (mouse macrophage) cell lines were obtained from American Type of Culture Collection (Manassas, VA, USA). Cell culture media and all the other materials required for culturing were obtained from Gibco BRL, Life Technologies (USA). Other chemicals and reagents were of analytical grade commercially available. 2.2. In vitro gastrointestinal digestion The digestion process was carried out using the method described by Kapsokefalou and Miller (1991). A 100 ml of 4% (w/v) oyster protein isolating solution was brought to the desired pH to represent the stomach digestion using 1 and 10 M HCl and NaOH under rigorous mixing. Pepsin (EC 3.4.23.1) was added at the enzyme to substrate ratio of 1/100 (w/w), then incubated at 37 C on a shaker. After 2 h the pH was set to 2.5 to obtain the conditions of small intestine digestion. Similarly trypsin (EC 3.4.21.4) and a-chymotrypsin (EC 3.4.21.1) were supplemented both at the enzyme to substrate ratio of 1/100 (w/w). Then the solution was further incubated at 37 C for 2.5 h. When samples were taken at the start and end of digestion, the pH was adjusted to 6.5. Samples were centrifuged at 10,000g for 15 min at 4 C and the supernatant was frozen and stored at 80 C. The frozen samples were subsequently lyophilized to obtain the dry powder. 2.3. Measurement of the antioxidative activity in linoleic acid model system The antioxidative activity was measured in a linoleic acid model system according to the methods of Osawa and Namiki (1985). Briefly, a sample (1.3 mg) was dissolved in 10 ml of 50 mM phosphate buffer (pH 7.0), and added to a solution containing 0.13 ml of linoleic acid and 10 ml of 99.5% ethanol. Then the total volume was adjusted to 25 ml with distilled water. The mixture was incubated in a conical flask with a screw cap at 40 ± 1 C in a dark room and the degree of oxidation was evaluated by measuring the ferric thiocyanate values. The ferric thiocyanate value was measured according to the method of Mitsuta et al. (1996). The reaction solution (100 ll) incubated in the linoleic acid model system was mixed with
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4.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate, and 0.1 ml of 2 · 102 M ferrous chloride solution in 3.5% HCl. After 3 min, the thiocyanate value was measured by reading the absorbance at 500 nm following color development with FeCl2 and thiocyanate at different intervals during the incubation period at 40 ± 1 C. 2.4. Assays conducted using electron spin resonance (ESR) spectroscopy 2.4.1. Hydroxyl radicals scavenging activity Hydroxyl radicals were generated by iron-catalyzed Fenton Haber–Weiss reaction and the generated hydroxyl radicals were rapidly reacted with nitrone spin trap DMPO (Rosen and Rauckman, 1984). The resultant DMPO-OH adducts was detectable with an ESR spectrometer. The peptide solution (0.2 ml) was mixed with DMPO (0.3 M, 20 ll), FeSO4 (10 mM, 20 ll) and H2O2 (10 mM, 20 ll) in a phosphate buffer solution (pH 7.4), and then transferred into a 100 ll quartz capillary tube. After 2.5 min, the ESR spectrum was recorded using an ESR spectrometer. The experimental conditions employed were as follows: magnetic field, 336.5 ± 5 mT; power, 1 mW; modulation frequency, 9.41 GHz; amplitude, 1 · 200; sweep time, 4 min. Hydroxyl radical scavenging ability was calculated following equation in which H and H0 were relative peak height of radical signals with and without sample, respectively. 1H Radical scavenging activity ¼ 100% H0 2.4.2. Superoxide anion radical scavenging activity Superoxide anion radicals were generated by UV irradiated riboflavin/EDTA system (Guo et al., 1999). The reaction mixture containing 0.3 mM riboflavin, 1.6 mM EDTA, 800 mM DMPO and indicated concentrations of peptide fraction was irradiated for 1 min under UV lamp at 365 nm. The reaction mixture was then transferred to 100 ll quartz capillary tube of the ESR spectrometer for measurement. The experimental conditions employed were as follows: magnetic field, 336.5 ± 5 mT; power, 10 mW; modulation frequency, 9.41 GHz; amplitude, 1 · 1000; sweep time, 1 min. Superoxide radical scavenging ability was calculated following equation in which H and H0 were relative peak height of radical signals with and without sample, respectively. 1H Radical scavenging activity ¼ 100% H0 2.5. Purification of the antioxidant peptide 2.5.1. Ion exchange chromatography The lyophilized oyster protein (20 mg/ml) was dissolved in 20 mM sodium acetate buffer (pH 4.0), and loaded onto fast protein liquid chromatography (FPLC) on a Hiprep 16/10 DEAE FF anion exchange column equilibrated with
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20 mM sodium acetate buffer (pH 4.0), and eluted with a linear gradient of NaCl (0–1.5 M) in the same buffer at a flow rate of 62 ml/h. Each fraction collected at a volume of 4 ml was monitored at 280 nm, pooled fractions were then concentrated using a rotary evaporator; and antioxidant activities were investigated. The fraction having strong antioxidant properties were lyophilized, and subjected to next separation. 2.5.2. High-performance liquid chromatography (HPLC) The fraction exhibiting highest antioxidative activity was further purified using reversed-phase high performance liquid chromatography (RP-HPLC) on a Primesphere 10 C18 (20 mm · 250 mm) column with a linear gradient of acetonitrile (0–35% in 35 min) containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 1.0 ml/min. Elution peaks were detected at 215 nm, and the active peaks were concentrated using a rotary evaporator. Antioxidant activity was evaluated as described early. The active fraction from analytical column was further applied onto a Synchropak RPP-100 analytical column with a linear gradient of acetonitrile (15% v/v, in 15 min) containing 0.1% TFA at flow rate of 1.0 ml/min. Finally a potent antioxidant peptide was purified and its amino acid sequence was analyzed. 2.6. Determination of amino acid sequence The Accurate molecular mass and amino acid sequence of the purified peptide were determined with a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled with an electrospray ionization (ESI) source. The purified peptide was separately infused into the electrospray source following dissolution in methanol/water (1:1, v/v), and molecular mass was determined by a doubly charged [M+2H]+2 state in the mass spectrum. Following molecular mass determination, the peptide was automatically selected for fragmentation, and sequence information was obtained by tandem mass spectroscopy (MS) analysis. 2.7. Cell culture and viability determination Human lung fibroblast (MRC-5) and mouse macrophage (RAW264.7) cells were cultured and maintained in Dulbecco’s Modification of Eagle’s Medium (DMEM, GIBCO, NY, USA) supplemented with 100 U/ml penicillin, 100 lg/ml streptomycin, 10% fetal bovine serum (FBS) and maintained at 37 C under a humidified atmosphere with 5% CO2. For the cytotoxicity determination studies, the colorimetric (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) MTT assay was performed (Hansen et al., 1989). The cells were cultured in microtiter 96-well plates (1.5 · 105 cell/well) with serum-free media and treated with the different concentrations of purified peptide for 24 h in a humidified 5% (v/v) CO2/air environment at 37 C. Sequentially, 20 ll of MTT dye solution was added to each well. After 4 h incubation, 200 ll of solubilization/stop solution was added for dissolving the
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formazan crystals and the absorbance was read using Genious Multifunction Microplate Reader (Tecan, UK) at 540 nm. 2.8. Cellular ROS determination by DCFH-DA Intracellular formation of ROS was assessed as described previously using oxidation sensitive dye DCFH-DA as the substrate (Englemann et al., 2005). RAW264.7 cells growing in fluorescence microtiter 96-well plates was loaded with 20 lM DCFH-DA in HBSS and incubated in the dark for 20 min. Cells were then treated with different concentrations of peptide and incubated for another 1 h. After washing the cells with PBS for three times, 300 lM H2O2 was added. The formation of 2 0 ,7 0 dichlorofluorescin (DCF) due to oxidation of DCFH in the presence of various ROS was read after every 30 min at the excitation wavelength (Ex) of 485 nm and the emission wavelength (Em) of 535 nm using a GENions fluorescence microplate reader. Following maximum rate of fluorescence increase, each well was normalized to cell numbers using MTT cell viability assay. Dose dependant and time dependant effects of treatment groups were plotted and compared with that of fluorescence intensity from control and blank groups. 2.9. Protective effect of the purified peptide against hydroxyl-radical-induced DNA damage To study the protective effects of the purified peptide on DNA damage induced by hydroxyl radical, the reaction was conducted in an Eppendorf tube at a total volume of 12 ll containing 0.5 ll of PBR 322 DNA in 3 ll of 50 mM phosphate buffer (pH 7.4), 3 ll of 2 mM FeSO4 and 2 ll of the purified peptide at various concentrations. Then, 4 ll of 30% H2O2 was added, and the mixture was incubated at 37 C for 30 min. The mixture was subjected to 0.8% agarose gel electrophoresis. DNA bands (supercoiled, linear and open circular) were stained with ethidium bromide. 2.9.1. Statistical analysis Data were expressed as means ± standard error of the mean after at least three independent experiments. Student’s t-test was used to determine the level of significance (P < 0.05). 3. Results and discussion
Fig. 1. Separation of antioxidant peptides from oyster, Crassostrea gigas by Hiprep 16/10 DEAE FF anion exchange chromatography.
tive portions (Fig. 1). Each fraction was pooled, lyophilized, and measured for antioxidative activity in linoleic acid emulsion system and radical scavenging potencies. Linoleic acid model system was employed to assess antioxidative activity, and fraction I exhibited the highest antioxidative potential to inhibit lipid peroxidation (80.15%) as well as exhibited substantial scavenging potencies on both, hydroxyl and superoxide radicals, respectively Table 1. The lyophilized active fraction III was further separated by RP-HPLC on a Primesphere 10 C18 (20 mm · 250 mm) column with a linear gradient of acetonitrile (0–35%) containing 0.1% trifluoroacetic acid (TFA), and the fraction was divided into three clear fractions (III-1, III-2 and III-3) (data not shown). Active fraction was further pooled, and purified on a Synchropak RPP100 analytical column (10 mm · 250 mm) using a linear gradient of acetonitrile (0–15%) containing 0.1% TFA. Finally, a peptide having potent lipid peroxidation inhibition and radical scavenging potencies was purified. The amino acid sequence of the purified peptide was analysed as Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-GlnGlu and molecular mass was determined to be 1600 Da. The purified peptide determined by ESI/MS spectroscopy was in excellent agreement with theoretical mass calculated from the sequence.
Table 1 Free radical scavenging activity of FPLC and HPLC fractions Free radical scavenging activity (%)
3.1. Isolation of the antioxidant peptide a
The lyophilized oyster protein was dissolved in 20 mM sodium acetate buffer (pH 4.0), and loaded onto a Hiprep 16/10 DEAE FF anion exchange column with the linear gradient of NaCl (0–1.5 M). Elution peaks were monitored at 280 nm, and each fraction was collected as 4 ml and fractionated into one non-adsorptive portion and two adsorp-
Fraction I Fraction IIa Fraction IIIa III-1b III-2b III-3b a b
Hydroxyl radical
Superoxide radical
23.28 ± 1.87 34.75 ± 2.19 55.36 ± 1.24 48.87 ± 2.07 87.78 ± 1.31 69.85 ± 2.13
12.98 ± 2.21 21.36 ± 3.21 35.23 ± 2.04 13.20 ± 1.72 48.36 ± 1.55 35.29 ± 2.16
Scavenging effects were tested at a concentration of 0.5 mg/ml. Scavenging effects were tested at a concentration of 0.1 mg/ml.
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3.2. Antioxidant activities of the purified peptide The antioxidant activities of the purified peptide (III-2) were investigated concerning both lipid peroxidation inhibition activity and direct free radical scavenging effects. As shown in Fig. 2, the purified peptide (III-2) effectively inhibited lipid peroxidation in the linoleic acid emulsion system. The activity of the purified peptide for lipid peroxidation inhibition was higher than that of a-tocopherol, the positive control used in this experiment after 7 days. The purified peptide highly inhibited lipid peroxidation (84.90%) in vitro and it was significantly higher (P < 0.05) compared to a-tocopherol (Fig. 2). Bioactive peptides usually contain 2–20 amino acid residues per molecule (Pihlanto-Leppala, 2001), and the lower the molecular weight, the higher their chance to cross the intestinal barrier and exert biological effects (Roberts et al., 1999). Previous work on antioxidative peptides has shown that peptides with 5–16 amino acid residues could inhibit autoxidation of linoleic acid (Chen et al., 1995). Lipid peroxidation is thought to proceed via radical-mediated abstraction of hydrogen atoms from methylene carbons in polyunsaturated fatty acids (Rajapakse et al., 2005). Hydrophobic amino acid residue Leu composed of about 30% of sequence of the purified peptide. Since hydrophobicity of antioxidants is important for accessibility to the hydrophobic targets (Chen et al., 1996), it is presumed that the presence of hydrophobic amino acids in the purified peptide may have contributed to lipid peroxidation inhibitory activity by increasing solubility of peptides in lipid and thereby facilitating better interaction with radical species. In addition, positioning of hydrophobic amino acid, Leu at the N-terminus of the peptide sequences is thought to be important for the antioxidative activity (Chen et al., 1995; Ranathung et al., 2006) because, it is assumed that Leu can increase interaction between peptides and fatty acids. Additionally, 3.0 III-2 (purified peptide) Control Tocopherol
Absorbance at 500 nm
2.5
2.0
1.5
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presence of Asp seems to play a vital role irrespective to its position as observed in several antioxidative peptide sequences (Rajapakse et al., 2005; Uchida and Kawakishi, 1992). As a whole, the presence of specific amino acids and their specific positioning in the sequence could have been attributed to the antioxidative activity of the purified peptide. The direct free radical scavenging effects of purified peptide were investigated using the ESR spin-trapping technique. Two free radicals were generated in in vitro systems. Hydroxyl radicals were generated in a Fenton reaction and visualized by an ESR spectrometer. The ESR signal was inhibited by the presence of OH scavengers, which compete with DMPO for OH. Superoxide radicals were generated by UV irradiation of a riboflavin/ EDTA solution. Our results showed that purified peptide efficiently quenched toward two free radical sources Table 1. Especially, the purified peptide was more potent for scavenging hydroxyl radicals than for superoxide radicals. The purified peptide was effectively quenched radicals in the order of hydroxyl and superoxide radical, with the IC50 values at 28.76 and 78.79 lM, respectively. Hydroxyl radical and superoxide radicals were scavenged at 87.78% and 48.36%, respectively in the presence of 0.1 mg/ml concentration of the purified peptide Table 1. The IC50 values of purified peptide III-2 to scavenge hydroxyl and superoxide radicals were higher than that of a-tocopherol (hydroxyl, IC50: 179.8uM; superoxide, IC50 > 1000uM), and the purified peptide III-2 could scavenge radicals more effectively. The chemical activity of hydroxyl radical is the strongest among ROS. It easily reacts with biomolecules, such as amino acids, proteins, and DNA (Cacciuttolo et al., 1993). Therefore, the removal of hydroxyl radical is probably one of the most effective defenses of a living body against various diseases. Based on sequence data, the purified peptide consisted of four acidic Leu residues and one Asp residue may have contributed to its higher radical scavenging potential. Suetsuna and Nakano (2000) have reported that the Leu residues from radical-scavenging peptide derived from casein exhibited higher radical scavenging potentials. Moreover, Ranathung et al. (2006) have described the significance of Leu in isolated peptide sequences to possess higher radical scavenging activity. Therefore, it can be expected that Leu residues have contributed for the observed radical scavenging activity of the purified peptide.
1.0
3.3. Cellular radical scavenging effect of purified peptide
0.5
Cytotoxic effects of purified peptide was evaluated on human lung fibroblast (MRC-5) and mouse macrophage (RAW264.7) cell line, and the results showed that purified peptide (III-2) shows any cytotoxic effects on MRC-5 and RAW264.7 cells (data not shown). The Purified peptide effectively quenched on free radicals generated various methods in this study. Therefore, we were interested in studying the direct effects of this peptide to scavenge
0.0 0
2
4
6
8
Incubation time (day) Fig. 2. Antioxidative activities of purified peptide in a linoleic acid emulsion system measured by the ferric thiocyanate method. The control is defined where no antioxidant is added in the antioxidative activity test.
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cellular radicals. For that, RAW264.7 cells were labeled with fluorescence probe DCFH-DA as described in material and method section. Fluorescent probes have been widely employed to monitor oxidative activity in cells. During labeling, non-fluorescent DCFH-DA dye that freely penetrates into cells gets hydrolyzed by intracellular esterases to DCFH, and traps inside the cells (Veerman et al., 2004). As shown in Fig. 3, fluorescence emitted by DCF following ROS-mediated oxidation of DCFH followed a time course increment up to 2 h. Pre-treatment with the purified peptide decreased the DCF fluorescence doseand time-dependently. Purified peptide exerted a considerable radical scavenging effect at 50 lg/ml concentration after 30 min. More clearly, at the concentration of 100 lg/ml, purified peptide could scavenge radicals significantly throughout the incubation time. These results suggest that the purified peptide can protect cells from oxidative damage by ROS according to those previous studies (Kang et al., 2005), thus it may be developed into a potential bio-molecular candidate to inhibit ROS formation of cellular. 3.4. Protective effect of the purified peptide against hydroxyl-radical-induced DNA damage The antioxidant effect of the purified peptide was evaluated using a method to evaluate the protective effect on free-radical-induced plasmid pBRr 322 DNA damage in vitro. DNA was broken into three forms- supercoiled (SC), open circular (OC) and linear form (Linear) when it was exposed to hydroxyl radical derived from the Fenton reaction. In the present investigation, we investigated the protective effect of purified peptide on hydroxyl-radicalinduced DNA damage by agarose gel electrophoresis Control Blank 10 μg/ml 50 μg/ml 100 μg/ml
DCF fluorescence intensity
3000
2000
1
2
3
4
5
OC DNA Linear DN SC DNA
Fig. 4. Agarose gel electrophoretic patterns of plasmide DNA breaks by OH generated from a Fenton reaction in the presence of purified peptide. An amount of 0.5 ll of PBR 322 DNA was incubated at 37 C for 30 min in FeSO4 and 30% H2O2 with the following additive combinations. Line 1, no addition (plasmide DNA); Line 2, FeSO4 and H2O2 (DNA damage control); Line 3–5, FeSO4 and H2O2 in the presence of purified peptide with concentrations of 45.6, 22.8 and 5.7 lM, respectively.
method. The protective effects of purified peptide on freeradical-induced DNA damage are shown in Fig. 4. Compared with plasmid DNA control (Line 1), the SC form in DNA was completely converted to the OC form due to hydroxyl radical generated from the Fenton reaction (Line 2) when DNA was treated with 2 mM Fe2+ and 30% H2O2 (Line 2). According to the result, DNA treated with the purified peptide at concentrations ranging from 5.7 to 45.6 lM protected hydroxyl-radical-induced DNA damage dose dependently (Lines 3–5), indicating an antioxidant effect. Since a DNA is another major sensitive biotarget of ROS-mediated oxidative damage, these results clearly explain the protective effect of the purified peptide against oxidative damage (Martinez et al., 2003). These observed effects are in line with the observation that the purified peptide efficiently scavenged hydroxyl radical at IC50 28.76 lM in that assessed using spin trapping techniques. This result further strengthened the ability of purified peptide to protect hydroxyl-radical-induced DNA damage. The result of this study suggests that an antioxidant activity peptide derived from oyster could be utilized to develop physiologically functional foods. In addition, it is expected that this will contribute to developing interest in basic research and potential applications of bioactive peptides. 4. Conclusion
1000
0 0
30
60
90
120
150
Incubation time (min)
Fig. 3. Cellular radical scavenging activity of purified peptide. RAW264.7 cells were labeled with non-toxic fluorescence dye, DCFH-DA, and treated with different concentrations of purified peptide. Fluorescence intensities of DCF due to oxidation of DCFH by cellular ROS (generated by H2O2) were detected time-dependently (Ex = 485 nm and Em = 535 nm). Effects of purified peptide on the scavenging of cellular ROS were compared with H2O2 non-stimulated blank and sample nontreated control groups in three independent experiments.
Based on these results, it is suggested that the low molecular weight peptide released from gastrointestinal hydrolysate was also a potent free radical scavenger and effectively inhibited lipid peroxidation, and no any cytotoxic effects on MRC-5 and RAW264.7 cell lines, significantly scavenged cellular ROS and is expected to protect against oxidative damage in living systems in relation to aging and carcinogenesis. These results suggested that antioxidant peptide fractions from gastrointestinal hydrolysate might be useful for food additives, diet nutrients and pharmaceutical agents. However, further detailed studies on peptide fractions with regard to antioxidant activities in vivo are needed.
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Acknowledgements This research was supported by a grant (p-2007-01) from the Marine Bioprocess Research Center of the Marine Bio 21 Center funded by the Ministry of Maritime Affairs and Fisheries, Republic of Korea. References Cacciuttolo, M.A., Trinh, L., Lumpkin, J.A., Rao, G., 1993. Hyperoxia induces DNA damage in mammalian cells. Free Radical Biol. Med. 14, 267–276. Chen, J., Suetsuna, K., Yamauchi, F., 1995. Isolation and characterization of immunostimulative peptides from soybean. J. Nutr. Biochem. 6, 310–313. Chen, H.M., Muramoto, K., Yamauchi, F., Nokihara, K., 1996. Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. J. Agric. Food Chem. 44, 2619–2623. Englemann, J., Volk, J., Leyhausen, G., Geurtsen, W., 2005. ROS formation and glutathione levels in human oral fibroblasts exposed to TEGDMA and camphorquinone. J. Biomed. Mater. Res. Part B, Appl. Biomater. 75B, 272–276. Frlich, I., Riederer, P., 1995. Free radical mechanisms in dementia of Alzheimer type and the potential for antioxidative treatment. Drug Res. 45, 443–449. Guo, Q., Zhao, B., Shen, S., Hou, J., Hu, J., Xin, W., 1999. ESR study on the structure-antioxidant activity relationship of tea catechins and their epimers. Biochem. Biophys. Acta 1427, 13–23. Hansen, M.B., Nielsen, S.E., Berg, K., 1989. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Meth. 119, 203–210. Hettiarachchy, N.S., Glenn, K.C., Gnanasambandam, R., Johnson, M.G., 1996. Natural antioxidant extract from fenugreek (Trigonella foenumgraecum) for ground beef patties. J. Food Sci. 61, 516–519. Je, J.Y., Park, P.J., Byun, H.G., Jung, W.K., Kim, S.K., 2005. Antioxidant activity of a peptide isolated from Alaska Pollack (Theragra chalcogramma) frame protein hydrolysate. Bioresour. Technol. 96, 1624–1629 . Jung, W.K., Rajapakse, N., Kim, S.K., 2005. Antioxidative activity of low molecular peptide derived from the sauce of fermented blue mussel, Mytilus edulis. Eur. Food Res. Technol. 220, 535–539. Jung, W.K., Mendis, E., Je, J.Y., Park, P.J., Son, B.W., Kim, H.C., Choi, Y.K., Kim, S.K., 2006. Angiotensin I-converting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats. Food Chem. 94 (1), 26–32.
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Kang, K.A., Lee, K.H., Chae, S., Koh, Y.S., Yoo, B.S., Kim, J.H., Ham, Y.M., Baik, J.S., Lee, N.H., Hyun, J.W., 2005. Triphlorethol-A from Ecklonia cava protects V79-4 lung fibroblast against hydrogen peroxide induced cell damage. Free Radical Res. 39, 883–892. Kapsokefalou, M., Miller, D.D., 1991. Effects of meat and selected food components on the valence of nonheme iron during in vitro digestion. J. Food Sci. 56 (2), 352–355. Maillard, M.N., Soum, M.H., Meydani, S.N., Berset, C., 1996. Antioxidant activity of barley and malt: Relationship with phenolic content. Food Sci. Technol. 29, 238–244. Martinez, G.R., Loureiro, A.P., Marques, S.A., Miyamoto, S., Yamaguchi, L.F., Onuki, J., 2003. Oxidative and alkylating damage in DNA. Mutat. Res. 544, 115–127. Mitsuta, H., Yasumoto, K., Iwami, K., 1996. Antioxidative action of indole compounds during the autoxidation of linoleic acid. Eiyo to Shokuryo. 29, 238–244. Osawa, T., Namiki, M., 1985. Natural antioxidant isolated from eucalyptus leaf waxes. J. Agric. Food Chem. 33, 770–780. Pihlanto-Leppala, A., 2001. Bioactive peptides derived from bovine whey proteins: opioid and ACE-inhibitory peptides. Trends Food Sci. Technol. 11, 347–356. Rajapakse, N., Mendis, E., Jung, W.K., Je, J.Y., Kim, S.K., 2005. Purification of radical scavenging peptide from fermented mussel sauce and its antioxidant properties. Food Res. Int. 38, 175–182. Ranathung, S., Rajapakse, N., Kim, S.K., 2006. Purification and characterization of antioxidative peptide derived from muscle of conger eel (Conger myriaster). Eur. Food Res. Technol. 222, 310–315. Roberts, P.R., Burney, J.D., Black, K.W., Zaloga, G.P., 1999. Effect of chain length on absorption of biologically active peptides from the gastrointestinal tract. Digestion 60, 332–337. Rosen, G.M., Rauckman, E.J., 1984. Spin trapping of superoxide and hydroxyl radicals. Method. Enzymol. 105, 198–209. Suetsuna, K., Maekawa, K., Chen, J., 2004. Antihypertensive effects of Undaria pinnatifida (wakame) peptide on blood pressure in spontaneously hypertensive rats. J. Nutr. Biochem. 15, 267–272. Suetsuna, K., Nakano, T., 2000. Identification of an antihypertensive peptide from peptic digest of wakame (Undaria pinnatifida). J. Nutr. Biochem. 11, 450–454. Tsuruki, T., Kishi, K., Takahashi, M., Tanaka, M., Matsukawa, T., Yoshikawa, M., 2003. Soymetide, an immunostimulating peptide derived from soybean b-conglycinin, is an Fmlp agonist. FEBS Lett. 540, 206–210. Uchida, K., Kawakishi, S., 1992. Sequence-dependant reactivity of histidine-containing peptides with copper(II)/ascorbate. J. Agric. Food Chem. 40, 13–16. Veerman, E.C.I., Nazmi, K., Hof, W.A.T., Bolscher, J.G.M., Hertog, A.L.D., Amerongen, A.V.N., 2004. Reactive oxygen species play no role in the candidacidal activity of the salivary antimicrobial peptide histatin 5. Biochem. J. 381, 447–452.
Bioresource Technology 99 (2008) 3365–3371
Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster, Crassostrea gigas against free radical induced DNA damage Zhong-Ji Qian a, Won-Kyo Jung c, Hee-Guk Byun d, Se-Kwon Kim
a,b,*
a Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea Department of NOAA Sea Grant Development and Food Science, Louisiana State University, Baton Rouge, LA 70803, USA d Faculty of Marine Bioscience and Technology, Kangnung National University, Kangnung 210-702, Republic of Korea b
c
Received 3 July 2007; received in revised form 10 August 2007; accepted 12 August 2007 Available online 27 September 2007
Abstract In this study, in vitro gastrointestinal digestion was employed to obtain potent antioxidative peptide from protein of oyster, Crassostrea gias. The protein was subjected to hydrolysate using consecutive chromatographic methods, on a Hiprep 16/10 diethylaminoethyl fast flow (DEAE FF) anion exchange column and octadecylsilane (ODS) C18 reversed phase column. Finally, the amino acid sequence of the peptide was determined. The peptide, having the amino acid sequence Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-Gln-Glu (1.60 kDa), exhibited the higher activity against polyunsaturated fatty acid (PUFA) peroxidation than that of native antioxidant, atocopherol. The free radical scavenging assay conducted using electron spin resonance (ESR) spectroscopy, clearly exhibited that it scavenged hydroxyl radical and superoxide radical at IC50 values of 28.76 lM and 78.97 lM, respectively. Further, we investigated its antioxidant activities on cellular system, and the results showed that purified peptide significantly scavenged cellular radicals and protective effect on DNA damage caused by hydroxyl radicals generated. Furthermore (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) MTT assay showed no cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5) and mouse macrophages cell (RAW264.7), respectively. These results indicate that this peptide shows potent antioxidant. 2007 Elsevier Ltd. All rights reserved. Keywords: Antioxidant peptide; Oyster Crassostrea gias; In vitro gastrointestinal; Lipid peroxidation; Radical scavenging activity
1. Introduction Lipid oxidation by reactive oxygen species (ROSs) such as superoxide anion, hydroxyl radicals, and hydrogen peroxide causes a decrease in nutritional value of lipids, their safety and appearance. In addition, it is the predominant cause of qualitative decay of foods, which leads to rancidity, toxicity, and destruction of biochemical components that are important in physiologic metabolisms. These rad* Corresponding author. Address: Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea. Tel.: +82 51 620 6375; fax: +82 51 628 8147. E-mail address: [email protected] (S.-K. Kim).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.08.018
icals are very unstable and react rapidly with other groups or substances in the body, leading to cell or tissue injury. Moreover free radicals-mediated modification of DNA, proteins, lipids, and small cellular molecules is associated with a number of pathological processes, including atherosclerosis, arthritis, diabetes, cataractogenesis, muscular dystrophy, pulmonary dysfunction, inflammatory disorders, ischemiareperfusion tissue damage, and neurological disorders such as Alzheimer’s disease (Frlich and Riederer, 1995). Specially, lipid peroxidation in foods affects the nutritive value and may cause disease conditions following consumption of potentially toxic reaction products. Therefore, during last few decade human nutrition and biochemistry research focused on an antioxidant derived from food
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ingredient that could retard lipid peroxidation. Free radical scavenger is a preventive antioxidant. Therefore, antioxidants are suggested to play an important role for bodily protection against oxidative stress. Further antioxidants are used to preserve food products to retard discoloration and deterioration that occur as a result of oxidation. This illustrates one of the many mechanisms which oxidative stress cause damage to lipids by stimulating the free radical chain reaction. In this context lipid peroxidation is of great concern to the food industry and consumers because it leads to the development of undesirable off-flavors and potentially toxic reaction products (Maillard et al., 1996). Presently, natural antioxidants such as vitamin C and atocopherol are continuously used as a means of enhancing biological functions and improving the stability of lipid and lipid-containing products. However, the use of synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), and propyl gallate (PG) is under strict regulation because of the potential health hazards (Hettiarachchy et al., 1996). Therefore, the search for natural antioxidants as alternatives to synthetic ones is of great interest among researchers. Recently a great interest is been developing to study about the structural, compositional and sequential properties of concerning bioactive peptides. They have shown the different kinds of bioactivities such as antioxidative (Jung et al., 2005; Rajapakse et al., 2005), antihypertensive (Jung et al., 2006; Je et al., 2005; Suetsuna et al., 2004) and immunomodulatory effects (Chen et al., 1995; Tsuruki et al., 2003). These reports have confirmed that bioactive peptides released by enzymatic proteolysis of food proteins may act as potential physiological modulators of metabolism during intestinal digestion. Bioactive peptides usually contain 3–20 amino acid residues, and their activities are based on their amino acid composition and sequence (PihlantoLeppala, 2001). Some recent studies have reported the in vitro formation of antioxidant peptides from marine food sources and their possibilities to be used as alternative antioxidants. The digestion by gastrointestinal proteases can be used as a production process for antioxidant peptide, with the advantage that the formed peptides will resist the physiological digestion after oral intake. The scope of this paper is to further investigate the conditions of in vitro gastrointestinal digestion leading to the formation and/or degradation of antioxidant peptides and to elucidate their antioxidative effects. Shellfish and fish sauces are widely used in East and Southeast Asian countries. In Korea, the production of oyster was estimated to be 252,000 ton in 2005, and only a few shellfish and fish sauces have survived in local areas in Korea. However, shellfish and fish sauces have recently been rediscovered because of increased consumer interest in their taste and flavor. From this point of view, the present study intended to an antioxidant peptide derived from oyster Crassostrea gigas by gastrointestinal digestion and their antioxidative
effects on linoleic acid peroxidation and radical scavenging were assessed. 2. Methods 2.1. Materials Oyster (C. gigas) was donated by Chamson Food Co. (Busan, Korea). Proteases for enzymatic hydrolysis (pepsin, trypsin and a-chymotrypsin), Linoleic acid, ammonium thiocyanate, a-tocopherol, and radical-testing chemicals, including 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), FeSO4, and H2O2 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). MRC-5 (human lung fibroblast) and RAW264.7 (mouse macrophage) cell lines were obtained from American Type of Culture Collection (Manassas, VA, USA). Cell culture media and all the other materials required for culturing were obtained from Gibco BRL, Life Technologies (USA). Other chemicals and reagents were of analytical grade commercially available. 2.2. In vitro gastrointestinal digestion The digestion process was carried out using the method described by Kapsokefalou and Miller (1991). A 100 ml of 4% (w/v) oyster protein isolating solution was brought to the desired pH to represent the stomach digestion using 1 and 10 M HCl and NaOH under rigorous mixing. Pepsin (EC 3.4.23.1) was added at the enzyme to substrate ratio of 1/100 (w/w), then incubated at 37 C on a shaker. After 2 h the pH was set to 2.5 to obtain the conditions of small intestine digestion. Similarly trypsin (EC 3.4.21.4) and a-chymotrypsin (EC 3.4.21.1) were supplemented both at the enzyme to substrate ratio of 1/100 (w/w). Then the solution was further incubated at 37 C for 2.5 h. When samples were taken at the start and end of digestion, the pH was adjusted to 6.5. Samples were centrifuged at 10,000g for 15 min at 4 C and the supernatant was frozen and stored at 80 C. The frozen samples were subsequently lyophilized to obtain the dry powder. 2.3. Measurement of the antioxidative activity in linoleic acid model system The antioxidative activity was measured in a linoleic acid model system according to the methods of Osawa and Namiki (1985). Briefly, a sample (1.3 mg) was dissolved in 10 ml of 50 mM phosphate buffer (pH 7.0), and added to a solution containing 0.13 ml of linoleic acid and 10 ml of 99.5% ethanol. Then the total volume was adjusted to 25 ml with distilled water. The mixture was incubated in a conical flask with a screw cap at 40 ± 1 C in a dark room and the degree of oxidation was evaluated by measuring the ferric thiocyanate values. The ferric thiocyanate value was measured according to the method of Mitsuta et al. (1996). The reaction solution (100 ll) incubated in the linoleic acid model system was mixed with
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4.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate, and 0.1 ml of 2 · 102 M ferrous chloride solution in 3.5% HCl. After 3 min, the thiocyanate value was measured by reading the absorbance at 500 nm following color development with FeCl2 and thiocyanate at different intervals during the incubation period at 40 ± 1 C. 2.4. Assays conducted using electron spin resonance (ESR) spectroscopy 2.4.1. Hydroxyl radicals scavenging activity Hydroxyl radicals were generated by iron-catalyzed Fenton Haber–Weiss reaction and the generated hydroxyl radicals were rapidly reacted with nitrone spin trap DMPO (Rosen and Rauckman, 1984). The resultant DMPO-OH adducts was detectable with an ESR spectrometer. The peptide solution (0.2 ml) was mixed with DMPO (0.3 M, 20 ll), FeSO4 (10 mM, 20 ll) and H2O2 (10 mM, 20 ll) in a phosphate buffer solution (pH 7.4), and then transferred into a 100 ll quartz capillary tube. After 2.5 min, the ESR spectrum was recorded using an ESR spectrometer. The experimental conditions employed were as follows: magnetic field, 336.5 ± 5 mT; power, 1 mW; modulation frequency, 9.41 GHz; amplitude, 1 · 200; sweep time, 4 min. Hydroxyl radical scavenging ability was calculated following equation in which H and H0 were relative peak height of radical signals with and without sample, respectively. 1H Radical scavenging activity ¼ 100% H0 2.4.2. Superoxide anion radical scavenging activity Superoxide anion radicals were generated by UV irradiated riboflavin/EDTA system (Guo et al., 1999). The reaction mixture containing 0.3 mM riboflavin, 1.6 mM EDTA, 800 mM DMPO and indicated concentrations of peptide fraction was irradiated for 1 min under UV lamp at 365 nm. The reaction mixture was then transferred to 100 ll quartz capillary tube of the ESR spectrometer for measurement. The experimental conditions employed were as follows: magnetic field, 336.5 ± 5 mT; power, 10 mW; modulation frequency, 9.41 GHz; amplitude, 1 · 1000; sweep time, 1 min. Superoxide radical scavenging ability was calculated following equation in which H and H0 were relative peak height of radical signals with and without sample, respectively. 1H Radical scavenging activity ¼ 100% H0 2.5. Purification of the antioxidant peptide 2.5.1. Ion exchange chromatography The lyophilized oyster protein (20 mg/ml) was dissolved in 20 mM sodium acetate buffer (pH 4.0), and loaded onto fast protein liquid chromatography (FPLC) on a Hiprep 16/10 DEAE FF anion exchange column equilibrated with
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20 mM sodium acetate buffer (pH 4.0), and eluted with a linear gradient of NaCl (0–1.5 M) in the same buffer at a flow rate of 62 ml/h. Each fraction collected at a volume of 4 ml was monitored at 280 nm, pooled fractions were then concentrated using a rotary evaporator; and antioxidant activities were investigated. The fraction having strong antioxidant properties were lyophilized, and subjected to next separation. 2.5.2. High-performance liquid chromatography (HPLC) The fraction exhibiting highest antioxidative activity was further purified using reversed-phase high performance liquid chromatography (RP-HPLC) on a Primesphere 10 C18 (20 mm · 250 mm) column with a linear gradient of acetonitrile (0–35% in 35 min) containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 1.0 ml/min. Elution peaks were detected at 215 nm, and the active peaks were concentrated using a rotary evaporator. Antioxidant activity was evaluated as described early. The active fraction from analytical column was further applied onto a Synchropak RPP-100 analytical column with a linear gradient of acetonitrile (15% v/v, in 15 min) containing 0.1% TFA at flow rate of 1.0 ml/min. Finally a potent antioxidant peptide was purified and its amino acid sequence was analyzed. 2.6. Determination of amino acid sequence The Accurate molecular mass and amino acid sequence of the purified peptide were determined with a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled with an electrospray ionization (ESI) source. The purified peptide was separately infused into the electrospray source following dissolution in methanol/water (1:1, v/v), and molecular mass was determined by a doubly charged [M+2H]+2 state in the mass spectrum. Following molecular mass determination, the peptide was automatically selected for fragmentation, and sequence information was obtained by tandem mass spectroscopy (MS) analysis. 2.7. Cell culture and viability determination Human lung fibroblast (MRC-5) and mouse macrophage (RAW264.7) cells were cultured and maintained in Dulbecco’s Modification of Eagle’s Medium (DMEM, GIBCO, NY, USA) supplemented with 100 U/ml penicillin, 100 lg/ml streptomycin, 10% fetal bovine serum (FBS) and maintained at 37 C under a humidified atmosphere with 5% CO2. For the cytotoxicity determination studies, the colorimetric (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) MTT assay was performed (Hansen et al., 1989). The cells were cultured in microtiter 96-well plates (1.5 · 105 cell/well) with serum-free media and treated with the different concentrations of purified peptide for 24 h in a humidified 5% (v/v) CO2/air environment at 37 C. Sequentially, 20 ll of MTT dye solution was added to each well. After 4 h incubation, 200 ll of solubilization/stop solution was added for dissolving the
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formazan crystals and the absorbance was read using Genious Multifunction Microplate Reader (Tecan, UK) at 540 nm. 2.8. Cellular ROS determination by DCFH-DA Intracellular formation of ROS was assessed as described previously using oxidation sensitive dye DCFH-DA as the substrate (Englemann et al., 2005). RAW264.7 cells growing in fluorescence microtiter 96-well plates was loaded with 20 lM DCFH-DA in HBSS and incubated in the dark for 20 min. Cells were then treated with different concentrations of peptide and incubated for another 1 h. After washing the cells with PBS for three times, 300 lM H2O2 was added. The formation of 2 0 ,7 0 dichlorofluorescin (DCF) due to oxidation of DCFH in the presence of various ROS was read after every 30 min at the excitation wavelength (Ex) of 485 nm and the emission wavelength (Em) of 535 nm using a GENions fluorescence microplate reader. Following maximum rate of fluorescence increase, each well was normalized to cell numbers using MTT cell viability assay. Dose dependant and time dependant effects of treatment groups were plotted and compared with that of fluorescence intensity from control and blank groups. 2.9. Protective effect of the purified peptide against hydroxyl-radical-induced DNA damage To study the protective effects of the purified peptide on DNA damage induced by hydroxyl radical, the reaction was conducted in an Eppendorf tube at a total volume of 12 ll containing 0.5 ll of PBR 322 DNA in 3 ll of 50 mM phosphate buffer (pH 7.4), 3 ll of 2 mM FeSO4 and 2 ll of the purified peptide at various concentrations. Then, 4 ll of 30% H2O2 was added, and the mixture was incubated at 37 C for 30 min. The mixture was subjected to 0.8% agarose gel electrophoresis. DNA bands (supercoiled, linear and open circular) were stained with ethidium bromide. 2.9.1. Statistical analysis Data were expressed as means ± standard error of the mean after at least three independent experiments. Student’s t-test was used to determine the level of significance (P < 0.05). 3. Results and discussion
Fig. 1. Separation of antioxidant peptides from oyster, Crassostrea gigas by Hiprep 16/10 DEAE FF anion exchange chromatography.
tive portions (Fig. 1). Each fraction was pooled, lyophilized, and measured for antioxidative activity in linoleic acid emulsion system and radical scavenging potencies. Linoleic acid model system was employed to assess antioxidative activity, and fraction I exhibited the highest antioxidative potential to inhibit lipid peroxidation (80.15%) as well as exhibited substantial scavenging potencies on both, hydroxyl and superoxide radicals, respectively Table 1. The lyophilized active fraction III was further separated by RP-HPLC on a Primesphere 10 C18 (20 mm · 250 mm) column with a linear gradient of acetonitrile (0–35%) containing 0.1% trifluoroacetic acid (TFA), and the fraction was divided into three clear fractions (III-1, III-2 and III-3) (data not shown). Active fraction was further pooled, and purified on a Synchropak RPP100 analytical column (10 mm · 250 mm) using a linear gradient of acetonitrile (0–15%) containing 0.1% TFA. Finally, a peptide having potent lipid peroxidation inhibition and radical scavenging potencies was purified. The amino acid sequence of the purified peptide was analysed as Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-GlnGlu and molecular mass was determined to be 1600 Da. The purified peptide determined by ESI/MS spectroscopy was in excellent agreement with theoretical mass calculated from the sequence.
Table 1 Free radical scavenging activity of FPLC and HPLC fractions Free radical scavenging activity (%)
3.1. Isolation of the antioxidant peptide a
The lyophilized oyster protein was dissolved in 20 mM sodium acetate buffer (pH 4.0), and loaded onto a Hiprep 16/10 DEAE FF anion exchange column with the linear gradient of NaCl (0–1.5 M). Elution peaks were monitored at 280 nm, and each fraction was collected as 4 ml and fractionated into one non-adsorptive portion and two adsorp-
Fraction I Fraction IIa Fraction IIIa III-1b III-2b III-3b a b
Hydroxyl radical
Superoxide radical
23.28 ± 1.87 34.75 ± 2.19 55.36 ± 1.24 48.87 ± 2.07 87.78 ± 1.31 69.85 ± 2.13
12.98 ± 2.21 21.36 ± 3.21 35.23 ± 2.04 13.20 ± 1.72 48.36 ± 1.55 35.29 ± 2.16
Scavenging effects were tested at a concentration of 0.5 mg/ml. Scavenging effects were tested at a concentration of 0.1 mg/ml.
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3.2. Antioxidant activities of the purified peptide The antioxidant activities of the purified peptide (III-2) were investigated concerning both lipid peroxidation inhibition activity and direct free radical scavenging effects. As shown in Fig. 2, the purified peptide (III-2) effectively inhibited lipid peroxidation in the linoleic acid emulsion system. The activity of the purified peptide for lipid peroxidation inhibition was higher than that of a-tocopherol, the positive control used in this experiment after 7 days. The purified peptide highly inhibited lipid peroxidation (84.90%) in vitro and it was significantly higher (P < 0.05) compared to a-tocopherol (Fig. 2). Bioactive peptides usually contain 2–20 amino acid residues per molecule (Pihlanto-Leppala, 2001), and the lower the molecular weight, the higher their chance to cross the intestinal barrier and exert biological effects (Roberts et al., 1999). Previous work on antioxidative peptides has shown that peptides with 5–16 amino acid residues could inhibit autoxidation of linoleic acid (Chen et al., 1995). Lipid peroxidation is thought to proceed via radical-mediated abstraction of hydrogen atoms from methylene carbons in polyunsaturated fatty acids (Rajapakse et al., 2005). Hydrophobic amino acid residue Leu composed of about 30% of sequence of the purified peptide. Since hydrophobicity of antioxidants is important for accessibility to the hydrophobic targets (Chen et al., 1996), it is presumed that the presence of hydrophobic amino acids in the purified peptide may have contributed to lipid peroxidation inhibitory activity by increasing solubility of peptides in lipid and thereby facilitating better interaction with radical species. In addition, positioning of hydrophobic amino acid, Leu at the N-terminus of the peptide sequences is thought to be important for the antioxidative activity (Chen et al., 1995; Ranathung et al., 2006) because, it is assumed that Leu can increase interaction between peptides and fatty acids. Additionally, 3.0 III-2 (purified peptide) Control Tocopherol
Absorbance at 500 nm
2.5
2.0
1.5
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presence of Asp seems to play a vital role irrespective to its position as observed in several antioxidative peptide sequences (Rajapakse et al., 2005; Uchida and Kawakishi, 1992). As a whole, the presence of specific amino acids and their specific positioning in the sequence could have been attributed to the antioxidative activity of the purified peptide. The direct free radical scavenging effects of purified peptide were investigated using the ESR spin-trapping technique. Two free radicals were generated in in vitro systems. Hydroxyl radicals were generated in a Fenton reaction and visualized by an ESR spectrometer. The ESR signal was inhibited by the presence of OH scavengers, which compete with DMPO for OH. Superoxide radicals were generated by UV irradiation of a riboflavin/ EDTA solution. Our results showed that purified peptide efficiently quenched toward two free radical sources Table 1. Especially, the purified peptide was more potent for scavenging hydroxyl radicals than for superoxide radicals. The purified peptide was effectively quenched radicals in the order of hydroxyl and superoxide radical, with the IC50 values at 28.76 and 78.79 lM, respectively. Hydroxyl radical and superoxide radicals were scavenged at 87.78% and 48.36%, respectively in the presence of 0.1 mg/ml concentration of the purified peptide Table 1. The IC50 values of purified peptide III-2 to scavenge hydroxyl and superoxide radicals were higher than that of a-tocopherol (hydroxyl, IC50: 179.8uM; superoxide, IC50 > 1000uM), and the purified peptide III-2 could scavenge radicals more effectively. The chemical activity of hydroxyl radical is the strongest among ROS. It easily reacts with biomolecules, such as amino acids, proteins, and DNA (Cacciuttolo et al., 1993). Therefore, the removal of hydroxyl radical is probably one of the most effective defenses of a living body against various diseases. Based on sequence data, the purified peptide consisted of four acidic Leu residues and one Asp residue may have contributed to its higher radical scavenging potential. Suetsuna and Nakano (2000) have reported that the Leu residues from radical-scavenging peptide derived from casein exhibited higher radical scavenging potentials. Moreover, Ranathung et al. (2006) have described the significance of Leu in isolated peptide sequences to possess higher radical scavenging activity. Therefore, it can be expected that Leu residues have contributed for the observed radical scavenging activity of the purified peptide.
1.0
3.3. Cellular radical scavenging effect of purified peptide
0.5
Cytotoxic effects of purified peptide was evaluated on human lung fibroblast (MRC-5) and mouse macrophage (RAW264.7) cell line, and the results showed that purified peptide (III-2) shows any cytotoxic effects on MRC-5 and RAW264.7 cells (data not shown). The Purified peptide effectively quenched on free radicals generated various methods in this study. Therefore, we were interested in studying the direct effects of this peptide to scavenge
0.0 0
2
4
6
8
Incubation time (day) Fig. 2. Antioxidative activities of purified peptide in a linoleic acid emulsion system measured by the ferric thiocyanate method. The control is defined where no antioxidant is added in the antioxidative activity test.
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cellular radicals. For that, RAW264.7 cells were labeled with fluorescence probe DCFH-DA as described in material and method section. Fluorescent probes have been widely employed to monitor oxidative activity in cells. During labeling, non-fluorescent DCFH-DA dye that freely penetrates into cells gets hydrolyzed by intracellular esterases to DCFH, and traps inside the cells (Veerman et al., 2004). As shown in Fig. 3, fluorescence emitted by DCF following ROS-mediated oxidation of DCFH followed a time course increment up to 2 h. Pre-treatment with the purified peptide decreased the DCF fluorescence doseand time-dependently. Purified peptide exerted a considerable radical scavenging effect at 50 lg/ml concentration after 30 min. More clearly, at the concentration of 100 lg/ml, purified peptide could scavenge radicals significantly throughout the incubation time. These results suggest that the purified peptide can protect cells from oxidative damage by ROS according to those previous studies (Kang et al., 2005), thus it may be developed into a potential bio-molecular candidate to inhibit ROS formation of cellular. 3.4. Protective effect of the purified peptide against hydroxyl-radical-induced DNA damage The antioxidant effect of the purified peptide was evaluated using a method to evaluate the protective effect on free-radical-induced plasmid pBRr 322 DNA damage in vitro. DNA was broken into three forms- supercoiled (SC), open circular (OC) and linear form (Linear) when it was exposed to hydroxyl radical derived from the Fenton reaction. In the present investigation, we investigated the protective effect of purified peptide on hydroxyl-radicalinduced DNA damage by agarose gel electrophoresis Control Blank 10 μg/ml 50 μg/ml 100 μg/ml
DCF fluorescence intensity
3000
2000
1
2
3
4
5
OC DNA Linear DN SC DNA
Fig. 4. Agarose gel electrophoretic patterns of plasmide DNA breaks by OH generated from a Fenton reaction in the presence of purified peptide. An amount of 0.5 ll of PBR 322 DNA was incubated at 37 C for 30 min in FeSO4 and 30% H2O2 with the following additive combinations. Line 1, no addition (plasmide DNA); Line 2, FeSO4 and H2O2 (DNA damage control); Line 3–5, FeSO4 and H2O2 in the presence of purified peptide with concentrations of 45.6, 22.8 and 5.7 lM, respectively.
method. The protective effects of purified peptide on freeradical-induced DNA damage are shown in Fig. 4. Compared with plasmid DNA control (Line 1), the SC form in DNA was completely converted to the OC form due to hydroxyl radical generated from the Fenton reaction (Line 2) when DNA was treated with 2 mM Fe2+ and 30% H2O2 (Line 2). According to the result, DNA treated with the purified peptide at concentrations ranging from 5.7 to 45.6 lM protected hydroxyl-radical-induced DNA damage dose dependently (Lines 3–5), indicating an antioxidant effect. Since a DNA is another major sensitive biotarget of ROS-mediated oxidative damage, these results clearly explain the protective effect of the purified peptide against oxidative damage (Martinez et al., 2003). These observed effects are in line with the observation that the purified peptide efficiently scavenged hydroxyl radical at IC50 28.76 lM in that assessed using spin trapping techniques. This result further strengthened the ability of purified peptide to protect hydroxyl-radical-induced DNA damage. The result of this study suggests that an antioxidant activity peptide derived from oyster could be utilized to develop physiologically functional foods. In addition, it is expected that this will contribute to developing interest in basic research and potential applications of bioactive peptides. 4. Conclusion
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Fig. 3. Cellular radical scavenging activity of purified peptide. RAW264.7 cells were labeled with non-toxic fluorescence dye, DCFH-DA, and treated with different concentrations of purified peptide. Fluorescence intensities of DCF due to oxidation of DCFH by cellular ROS (generated by H2O2) were detected time-dependently (Ex = 485 nm and Em = 535 nm). Effects of purified peptide on the scavenging of cellular ROS were compared with H2O2 non-stimulated blank and sample nontreated control groups in three independent experiments.
Based on these results, it is suggested that the low molecular weight peptide released from gastrointestinal hydrolysate was also a potent free radical scavenger and effectively inhibited lipid peroxidation, and no any cytotoxic effects on MRC-5 and RAW264.7 cell lines, significantly scavenged cellular ROS and is expected to protect against oxidative damage in living systems in relation to aging and carcinogenesis. These results suggested that antioxidant peptide fractions from gastrointestinal hydrolysate might be useful for food additives, diet nutrients and pharmaceutical agents. However, further detailed studies on peptide fractions with regard to antioxidant activities in vivo are needed.
Z.-J. Qian et al. / Bioresource Technology 99 (2008) 3365–3371
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