KR20090043657A - Peptide having antioxidant activity from crassostrea gigas - Google Patents

Abstract

Peptides were obtained through digestion of in-vitro gastrointestinal tract from Crasostrea gigas, purified using FPLC and RP-HPLC, and antioxidant activity, hydroxyl radical and superoxide radical (in linoleic acid model system). superoxide radical) A natural antioxidant peptide that can be useful for the development of functional foods by determining the amino acid sequence by selecting the peptide with the highest antioxidant activity after measuring the scavenging activity. Leu-Lys-Gln There is an excellent effect of providing a peptide substance consisting of -Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-Gln-Glu. In addition, the purified peptide of the present invention is easy to mass-produce using proteolytic enzymes using gastrointestinal digestion process, replaces conventional synthetic antioxidants and has higher lipid peroxidation inhibitory activity and free radical scavenging than natural antioxidant α-tocopherol. Because it has the ability, there is no cytotoxicity can be used harmless to the human body has an excellent effect that can be widely used in the food processing industry and functional food field.

Crassostrea gigas, oysters, antioxidant, peptide

Description

Peptide having antioxidant activity from Crassostrea gigas

The present invention relates to an antioxidant peptide isolated from Crassostrea gigas, more specifically to obtain peptides from digestion through in-vitro gastrointestinal digestion, purified using FPLC and RP-HPLC and linoleic acid model system The present invention relates to a natural antioxidant peptide which can be useful for developing functional foods by measuring the antioxidant activity, hydroxyl radical and superoxide radical scavenging activity, and selecting the peptide with the highest antioxidant activity and revealing the amino acid sequence.

Lipid oxidation by reactive oxygen series (ROSs), such as superoxide anions, hydroxyl radicals and hydrogen peroxide, leads to degradation of the nutritional value, safety and appearance of lipids. Moreover, lipid oxidation is a major cause of food deterioration and causes the destruction of biochemical components that play an important role in rancidity, toxicity and physiological metabolism. These radicals are very unstable and easily react with other substances in the body, causing damage to cells or tissues.

Furthermore, free radical involvement modifications to DNA, proteins, lipids and microcellular molecules include atherosclerosis, arthritis, diabetes, cataracts, muscular dystrophy, lung disorders, inflammatory disorders, tissue damage and neurological abnormalities such as Alzheimer's. It is associated with numerous pathological processes (Frlich and Riederer, 1995).

In particular, lipid peroxidation in foods affects nutritional value and may cause disease states after ingestion of potentially toxic reaction products. Thus, nutritional and biochemical research in humans over the past decades has focused on antioxidants derived from food ingredients that can reduce lipid peroxidation.

Free radical scavengers are antioxidants that have a protective function. Antioxidants are therefore known to play an important role in the physical prevention of oxidative stress.

More antioxidants are being used for food preservation to prevent discoloration and deterioration as a result of oxidation. This explains one of the many metabolisms in which oxidative stress causes lipid damage by promoting free radical chain reactions. In this regard, lipid peroxidation is of primary interest to the food industry and consumers because of its potential for unpleasant off-flavor and potential toxic reaction products (Maillard et al., 1996).

Currently, natural antioxidants such as vitamin C and α-tocopherol continue to be used as a means to enhance biological activity and stability of lipids 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) has been severely regulated due to potential health risks (Hettiarachchy et al. , 1996). Therefore, the search for natural antioxidants as an alternative to synthetic antioxidants is of great interest.

There is a growing interest in the study of structural, compositional and sequence properties related to bioactive peptides, and many of the peptides have been described for antioxidant (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 documents show that bioactive peptides produced by enzymatic digestion of food proteins can act as potential physiological metabolic regulators during gastrointestinal digestion.

Bioactive peptides generally have 3 to 20 amino acid residues and their activity varies based on amino acid composition and sequence (Pihlanto-Leppala, 2001).

Recently, several documents have described the in vitro formation of antioxidant peptides from marine foods and their use as alternative antioxidants. Digestion by proteolytic enzymes in the gastrointestinal tract can be used as a production process of antioxidant peptides, the peptide formed has the advantage of inhibiting physiological digestion after oral intake.

The present invention is to investigate the state of in vitro gastrointestinal digestion causing the formation and degradation of antioxidant peptides and to reveal the antioxidant effects of the peptides.

Shellfish and fish sauces are widely used in East and Southeast Asia. It is estimated that 252,000 tonnes of oysters are produced in 2005, and only a few shellfish and salted fish are kept in the province. However, clams and salted fish have recently been revived with increasing consumer interest in taste and aroma.

In view of this, the present invention relates to antioxidant peptides derived from Kras-Australian gas by gastrointestinal digestion, and evaluated their antioxidant efficacy by linoleic acid peroxidation and radical scavenging.

Accordingly, an object of the present invention is to isolate and purify peptide components using digestive process in gastrointestinal oyster (Crassostrea gigas, oyster), and to investigate their linoleic acid peroxidation and free radical scavenging activity to set peptides with excellent antioxidant activity And, by revealing its amino acid sequence it is to provide a natural antioxidant that is harmless to the human body that can be used in functional food.

The object of the present invention was achieved by identifying amino acid sequences after isolating and purifying peptides with excellent inhibition of linoleic acid peroxidation and free radical scavenging activity. It was also achieved by examining cytotoxicity experiments and examining the ability of radical scavenging in cells.

The present invention is characterized by providing an antioxidant peptide isolated from the oyster (Crassostrea gigas).

Separation of the peptide in the above may preferably use a protease by applying the digestive process in the stomach. Further separation can be achieved using ion exchange chromatography and RP-HPLC.

Antioxidant effects can be assessed by measuring the degree of peroxidation inhibitory activity, the scavenging activity of hydroxyl radicals and superoxide radicals in the linoleic acid model system.

The present invention also has an amino acid sequence of Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-Gln-Glu and is characterized by providing an antioxidant peptide having a molecular weight of 16,000 Da.

The amino acid sequence and molecular weight analysis can be obtained by mass spectrometry (MS) and tandem mass spectrometry.

In another aspect, the present invention is characterized by providing a natural antioxidant pharmaceutical composition containing the peptide as an active ingredient.

The composition may be prepared in various forms such as oral preparations, and may also be prepared by further containing other conventional pharmaceutically acceptable excipients such as sweetening agents and flavoring agents.

The present invention relates to an antioxidant peptide derived from Crasostrea gigas, in particular Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-Gln-Glu There is an excellent effect of providing the material. In addition, the purified peptide of the present invention is easy to mass-produce using proteolytic enzymes using gastrointestinal digestion process, replaces conventional synthetic antioxidants and has higher lipid peroxidation inhibitory activity and free radical scavenging than natural antioxidant α-tocopherol. Because it has the ability, there is no cytotoxicity can be used harmless to the human body is an excellent invention that can be widely used in the food processing industry and pharmaceutical fields.

Hereinafter, the present invention will be described in detail by way of examples, but the scope of the present invention is not limited only to these examples.

The oysters were obtained from a Chamson food company in Busan, Korea. Proteases for enzyme hydrolysis (pepsin, trypsin and α-chymotrypsin), linoleic acid, ammonium thiocyanate, α-tocopherol and 5,5-dimethyl-1-pyrroline-N-oxide (5,5-dimethyl Radical test compounds such as -1-pyrroline-N-oxide (DMPO), iron sulfide (FeSO ₄ ) and hydrogen peroxide were purchased from Sigma Chemicals. (St. Louis, MO, USA). Cell lines of MRC-5 (human lung fibroblasts) and RAW264.7 (mouse macrophages) were obtained from the American Type of Culture Collection (Manassas, VA, USA). Cell culture media and other materials required for culture were obtained from Gibco BRL, Life Technologies (USA). Other compounds and reagents were commercially available analytical grades.

Example  One : Oyster In in vitro In the stomach  Isolation of Proteins Through Digestion

Digestion of oysters was carried out using a method known by Kapsokefalou and Miller (1991).

100 mL of 4% (w / v) protein separation solution was adjusted to pH indicating gastrointestinal digestion using a mixture of 1 and 10 M sulfuric acid and sodium hydroxide. Pepsin was added at an enzyme to substrate ratio of 1/100 (w / w) and then incubated in a 37 ° C shaker. After 2 hours the pH was set to 2.5 to obtain digestion in the small intestine. In a similar manner, trypsin (EC 3.4.21.4) and α-chymotrypsin (EC 3.4.21.1) were supplemented with an enzyme to substrate ratio of 1/100 (w / w). The solution was then further incubated at 37 ° C. for 2.5 hours. Samples were taken at the beginning and end, with the pH adjusted to 6.5. Samples were centrifuged at 10,000 x g for 15 minutes at 4 ° C and the supernatants were frozen at -80 ° C. The frozen samples were successively lyophilized to obtain dried oyster protein powder.

Example  2 : Oyster  Protein FPLC Separation and FPLC  Fraction (I, II , III )of Linoleic acid  Measurement of Antioxidant Activity in a Model System

The protein obtained in Example 1 was divided into three fractions using FPLC and each fraction was named I, II, III. FPLC was performed by the following method.

The oyster protein (20 mg / mL) obtained in Example 1 was dissolved in 20 mM sodium acetate buffer (pH 4.0) and then FPLC (fast LC with Hiprep 16/10 DEAE FF anion exchange column equilibrated with 20 mM sodium acetate buffer). protein liquid chromatography) and eluting using a sodium chloride linear gradient on the same buffer at a flow rate of 62 mL per hour.

As shown in Figure 1, as observed in 280nm, three elution peaks were obtained, each fraction was named I, II, III and collected by 4mL. The collected fractions were freeze-dried and the antioxidant activity of the Kraso Australian gaseous protein was measured in a linoleic acid model system according to the method of Osawa and Namiki (1985).

Lyophilized fractions I, II, and III were dissolved in 10 mL of 50 mM phosphate buffer (pH 7.0) at 1.3 mg each, and then added to a solution containing 0.13 mL of linoleic acid and 10 mL of 99.5% ethanol. The total volume was then adjusted to 25 mL using distilled water. The mixture was incubated in conical flasks with screw lids at a temperature of 40 ± 1 ° C. in the dark, and the degree of oxidation was assessed by measuring ferric thiocyanate levels.

Iron thiocyanate levels were determined by the method of Mitsuta et al. (1996). The reaction solution (100 μL) incubated in a linoleic acid model system was transferred to 4.7 mL of 75% ethanol, 0.1 mL of 30% ammonium thiocyanate and 0.1 mL of 2 × 10 in 3.5% hydrochloric acid. It was mixed with an M- ² iron chloride solution. After discoloration of iron chloride and thiocyanate, thiocyanate levels were measured by reading the absorbance at 500 nm after 3 minutes at different time intervals during the incubation period at 40 ± 1 ° C.

Experimental results showed that Fraction III had the highest antioxidant potential to inhibit lipid peroxidation (80.15%).

Example  3: fraction I, II , III of Hydroxyl Radical  Scavenging activity

The hydroxyl radical scavenging activity of fractions I, II, and III obtained by separating oyster protein was examined.

Hydroxy radicals were produced by the iron-based Fenton Haber-Weiss method, which reacted immediately with nitrone spin trap DMPO. The resulting DMPO-OH adduct was detected by ESR spectroscopy. 0.2 mL peptide solution was dissolved in phosphate buffer solution (pH 7.4) in DMPO (0.3 M, 20 μL), FeSO ₄ (10 mM, 20 μL) and hydrogen peroxide (10 mM, 20 μL) and then transferred to a 100 μL quartz capillary tube. After 2 minutes and 30 seconds, the spectral spectrum was recorded using an ESR spectrometer. The experimental conditions applied are as follows. Magnetic field 336.5 ± 5 mT, power 1 mW, modulation frequency 9.41 GHz, amplitude 1 × 200, sweep time 4 minutes

The hydroxyl radical scavenging ability is H and H ₀ Was calculated using the following equations, each representing the relative peak height of the radical signal with and without samples, respectively.

                      1-H

Radical scavenging activity = ---------- × 100%

H ₀

As shown in Table 1, Fraction III showed significant scavenging activity against hydroxyl radicals.

Free radical scavenging activity of FPLC fraction % Free radical scavenging activity Hydroxyl radicals Superoxide radical Fraction I 23.28 ± 1.87 12.98 ± 2.21 Fraction II 34.75 ± 2.19 21.36 ± 3.21 Fraction III 55.36 ± 1.24 35.23 ± 2.04

Scavenging effect was tested at a concentration of 0.5 mg / mL.

Example  4: fraction I, II , III of Superoxide anion Radical  Scavenging activity

The scavenging activity of the superoxide anion radicals of FPLC fractions I, II and III was investigated.

Superoxide anion radicals were generated using a UV emitting riboflavin / EDTA system (Guo et al., 1999).

The reaction mixture containing 0.3 mM riboflavin, 1.6 mM EDTA, 800 mM DMPO and each FPLC fraction I, II, III was irradiated with 365 nm UV for 1 minute. The reaction mixture was then transferred to 100 μL quartz capillary tube and measured by ESR spectroscopy. The experimental conditions applied are as follows. Magnetic field 336.5 ± 5 mT, Power 10 mW, Modulation frequency 9.41 GHz, Amplitude 1 × 1,000, Sleep time 1 minute

Superoxide radical scavenging ability is H and H ₀ Was calculated using the following equations, each representing the relative peak height of the radical signal with and without samples, respectively.

                     1-H

Radical scavenging activity = ---------- × 100%

H ₀

Fraction III showed significant scavenging activity against the superoxide radical.

Example  5: fraction III of RP - HPLC Tablet using

Purification was performed for fraction III, which was the most antioxidant active.

Fraction III, which showed the highest antioxidant activity, was further purified using RP-HPLC. RP-HPLC is 10 Primesphere C ₁₈ to a acetonitrile (0-35% in 35 min) containing acetic acid (TFA) in trifluoroacetic 0.1% in flow rate of 1.0mL / min with a linear gradient (20 mm × 250 mm) column was used.

 Elution peaks were observed at 215 nm and each peak was concentrated using a rotary evaporator. The active fraction separated into an analytical column was further purified by Synchropak RPP-100 analytical column with a linear gradient of acetonitrile containing 0.1% TFA at a flow rate of 1.0 mL / min. The active fraction in the process was confirmed by the method shown in Examples 2, 3, 4.

As shown in Table 2, three HPLC fractions were obtained and named III-1, III-2 and III-3, respectively. Among them, the III-2 fraction was found to be the most active.

Free Radical Scavenging Activity of HPLC Fractions (III-1, III-2, III-3) Fraction % Free radical scavenging activity Hydroxyl radicals Superoxide radical III-1 48.87 ± 2.07 13.20 ± 1.72 III-2 87.78 ± 1.31 48.36 ± 1.55 III-3 69.85 ± 2.13 35.29 ± 2.16

The scavenging effect was tested at a concentration of 0.1 mg / mL.

Example  6: Determination of molecular weight and amino acid sequence

The exact molecular weight and amino acid sequence of the III-2 peptide obtained in Example 5 was determined using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) equipped with an electrospray ionization source.

Purified peptides were dissolved in methanol / water (1: 1, v / v) and then injected individually into an electrospray source, and the molecular weight was determined to be ^bivalently charged [M + 2H] ⁺² on the mass spectrometry spectrum. . Peptides for fractions were automatically selected after molecular weight determination and sequence information was obtained by tandem mass spectrometry (MS) analysis.

The amino acid sequence of the purified peptide was Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-Gln-Glu with a molecular weight of 1,600 Da. Purified peptides determined by ESI / MS spectroscopy were surprisingly consistent with the theoretically calculated mass from the sequence.

Example  7: refined Peptide Linoleic acid  Measurement of Antioxidant Activity in a Model System

Antioxidant activity was measured in the same manner as shown in Example 2.

As shown in FIG. 2, the purified peptide (III-2) inhibited lipid peroxidation (84.90%) very high in-vitro and was significantly higher than α-tocopherol (P <0.05).

Bioactive peptides generally contain from 2 to 20 amino acid residues per molecule (Pihlanto-Leppala, 2001), and the lower the molecular weight, the higher the potential for biological effects across the small intestine (Roberts et al., 1999). ). Prior literature on antioxidant peptides has shown that peptides with 5 to 16 amino acid residues can inhibit the automatic oxidation of linoleic acid. (Chen et al., 1995). Lipid peroxidation is thought to occur by the involvement of radicals in polyunsaturated fatty acids to remove hydrogen atoms from methylene carbons (Rajapakse et al., 2005).

About 30% of the purified peptide sequences consist of Leu, a hydrophobic amino acid residue. Since the degree of hydrophobicity of antioxidants is important for the approach of hydrophobic targets (Chen et al., 1996), the presence of hydrophobic amino acids in purified peptides facilitates interaction with radical families by enhancing the solubility of the peptides in lipids. It is estimated to contribute to the lipid oxidation inhibitory activity. It is also thought that the presence of the hydrophobic amino acid Leu at the N-terminus of the peptide sequence is important for antioxidant activity, because it is believed that Leu can increase the interaction between the peptide and the fatty acid (Chen et al., 1995; Ranathung et al., 2006). In addition, as observed in several antioxidant peptide sequences, the presence or absence of Asp seems to play an important role regardless of its location (Rajapakse et al., 2005; Uchida an Kawakishi, 1992).

In general, the presence of a specific amino acid and a specific position on the sequence may contribute to the antioxidant activity of the purified peptide.

Example  8 : Of purified peptide (III-2)  Antioxidant activity

The antioxidant activity of the III-2 peptide was measured using the method described in Example 3 and Example 4.

. The free radical direct scavenging effect of the purified peptides was investigated using ESR spin-trapping technique. Two free radicals were produced in vitro. Hydroxyl radicals were generated in the Fenton reaction and visualized through ESR spectroscopy.

The ESR signal was suppressed by the presence of an OH scavenger competing with DMPO for OH. Superoxide radicals were generated by irradiating the riboflavin / EDTA solution with UV.

As shown in Table 2, purified peptides efficiently inhibited two free radical sources. In particular, purified peptides were more potent in eliminating hydroxyl radicals than superoxide radicals. Purified peptides inhibited radicals in the order of hydroxyl radicals and superoxide radicals with IC ₅₀ values of 28.76 and 78.79 μM, respectively.

As shown in Table 2, the hydroxyl and superoxide radicals were eliminated at 87.78% and 48.36%, respectively, in the presence of 0.1 mg / mL of the purified peptide.

IC ₅₀ values of purified peptide III-2 scavenging hydroxyl radicals and superoxide radicals were higher than α-tocopherol (hydroxyl, IC ₅₀ : 179.8 uM; superoxide, IC ₅₀ > 1000 uM), and purified peptide III- 2 could more efficiently eliminate radicals.

The chemical activity of hydroxyl radicals is the strongest among ROS and readily reacts with biomolecules such as amino acids, proteins and DNA (Cacciuttolo et al., 1993). Thus, removing hydroxyl radicals may be one of the most effective defenses of living organisms against various diseases.

Based on sequencing data, the purified peptide consisted of four acidic Leu residues and one Asp residue that could contribute to a strong radical scavenging potential.

Suetsuna and Nakano (2000) reported that the Leu residues of casein-derived radical scavenging peptides exhibit strong radical scavenging potential. Ranathung et al. (2006) also described the importance of Leu to have strong radical scavenging activity in isolated peptide sequences. Thus, it can be expected that Leu residues contribute to the radical scavenging activity of the purified peptide.

Example  9: Of purified peptide (III-2) Intracellular Radical  Elimination effect

Human lung fibroblasts (MRC-5) and mouse macrophages (RAW 264.7) were treated with DMEM medium supplemented with 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% fetal bovine serum (FBS). Dulbecco's Modification of Eagle's medium, GIBCO, NY, USA) was maintained and stored at 37 ℃ under a humid environment of 5% carbon dioxide.

Colorimetric (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) MTT assay was performed for cytotoxicity studies (Hansen et al., 1989).

The cells were incubated in 96 well plates (1.5 × 10 ⁵ cells / well) containing serum-free medium and at different concentrations of purified peptide for 24 hours in a humid atmosphere containing 5% carbon dioxide at 37 ° C. Treated with. 20 μL of MTT staining solution was then added to each well.

 After 4 hours of incubation, 200 μL of the dissolution / stop solution was added to dissolve the formazan crystals and the absorbance was measured at 540 nm using a GENions Multifunction Microplate Reader (Tecan, UK).

Cytotoxicity experiments of purified peptides were carried out on human lung fibroblast (MRC-5) and mouse macrophage (RAW 264.7) cell lines, showing that purified peptide III-2 was isolated against MRC-5 and RAW264.7. There was no cytotoxic effect.

Example  10: DCFH - DA On by Intracellular ROS  decision

The formation of intracellular ROS was assessed using the oxidation sensitive dye DCFH-DA as a fluorescent probe (Englemann et al., 2005). Fluorescent probes have been widely applied to observe oxidation in cells.

During labeling, non-fluorescent DCFH-DA stains are easily permeated into cells, hydrolyzed to DCFH by intracellular esterases and trapped within cells (Veerman et al., 2004).

In the present invention, 20 μM of DCFH-DA was added to RAW264.7 cells incubated in a fluorescent 96-well plate in a 96-well plate and incubated in a dark place for 20 minutes. Cells were then treated with different concentrations of peptide and incubated for another hour. The cells were washed three times with PBS and then 300 μM hydrogen peroxide was added.

 In the presence of various types of ROS, DCF was formed due to oxidation of DCFH, which was measured every 30 minutes at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a GENions fluorescence microplate reader.

After maximal fluorescence increase, normalization was made to the cell number of each well using MTT cell viability assay. Dose-dependent and time-dependent effects of the treatment groups were graphed and the fluorescence intensity was compared with the control and blank groups.

As a result, as shown in Figure 4, after the oxidation of the ROS-associated DCFH fluorescent material emitted by DCF accumulated over time up to 2 hours. Pretreatment with purified peptide reduced DCF fluorescence in proportion to pretreatment amount and time. The purified peptide showed significant radical scavenging effect after 30 minutes at a concentration of 50 μg / mL. More specifically, peptides purified at a concentration of 100 μg / mL were able to eliminate significant levels of radicals throughout the entire incubation time.

The results show that, as in the literature (Kang et al., 2005), purified peptides can protect cells from oxidative damage by ROS, and thus the purified peptides have potential to inhibit the formation of ROS. It can be developed into biomolecules.

Example  11: refined Peptide Hydroxyl Radical  Judo DNA  Protective effect against damage

The antioxidant effect of the purified peptides in the present invention was carried out using a method for evaluating the protective effect against free radical induced plasmid pBRr 322 DNA damage.

To study the protective effect of purified peptides on DNA damage induced by hydroxyl radicals, 0.5 μL of PBR 322 DNA dissolved in 50 mM phosphate buffer (pH 7.4), 3 μL of 50 mM phosphate buffer (pH 7.4), A total volume of 12 μL containing 3 μL of 2 mM iron sulfide (FeSO ₄ ) and 2 μL of purified peptides of various concentrations was reacted in an Eppendorf tube.

4 μL of 30% hydrogen peroxide was then added and the mixture was incubated at 37 ° C. for 30 minutes. The mixture was subjected to 0.8% agarose gel electrophoresis. DNA bands were stained with ethidium bromide.

After three or more independent experiments, the data were expressed as mean ± standard deviation and significance level was determined using Student's t-test (P <0.05).

DNA exposure to hydroxyl radicals from the Fenton reaction split the DNA into three forms: supercoil (SC), open circle (OC), and linear.

The protective effect of the purified peptide on free radical induced DNA damage is shown in FIG. 4. In comparison with plasmid DNA control (Line 1), DNA of 2 mM Fe ^{2 +,} and 30% hydrogen peroxide (Line 2) in the handling to, DNA due to hydroxyl radicals generated from Fenton reaction supercoiled form of an open circular Completely transformed into.

According to the above results, DNA treated with purified peptide at a concentration of 5.7 to 45.6 μM protected hydroxyl radical-induced DNA damage in proportion to the dose (Line 3-5), indicating antioxidant efficacy.

Since DNA is another major sensitive biological target for ROS involved oxidative damage, these results clearly demonstrate the protective effect of purified peptides on oxidative damage. (Martinez et al., 2003). This observed effect is shown in IC _50, where purified peptides were evaluated using spin trapping techniques. Consistent with what we observed to be effective at scavenging hydroxyl radicals at 28.76 uM. Furthermore, the results of the practice of the present invention support the ability of purified peptides to protect against hydroxyl radical induced DNA damage.

Figure 1 shows the antioxidant activity in the linoleic acid emulsion system at each peak of the fraction separated from the oyster protein using FPLC.

Figure 2 shows the fractionation of the excellent antioxidant activity fraction III using HPLC again to show the sulfate activity in the linoleic acid emulsion system of each fraction.

Figure 3 shows the antioxidant activity of the purified peptide in the linoleic acid emulsion system measured by the iron thiocyanate method. Control means that no antioxidants were added during the antioxidant activity test.

Figure 4 shows the intracellular radical scavenging activity of the purified peptide (III-2).

5 shows agarose gel electrophoresis in which plasmid DNA is cleaved by OH generated in the Fenton reaction in the presence of purified peptide (III-2).

Claims (4)

Peptide having antioxidant activity derived from Crasostrea gigas. The method of claim 1, wherein the peptide has an amino acid sequence of Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-Gln-Glu, and has an antioxidant activity, characterized in that the molecular weight of 16,000 Da. Having a peptide. According to claim 1, wherein the antioxidant composition containing the peptide as an active ingredient, wound, arthritis, inflammation, wrinkles, whitening, diabetes mellitus, gastric ulcers, trauma, burns, periodontal disease or atherosclerosis treatment composition of the disease associated with antioxidant activity . The composition for antioxidant according to claim 1, wherein the composition for antioxidant is powder, tablet, capsule, powder, ointment composition, solution, gel, paste, patch or granule.

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