KR101131223B1 - Antioxidant peptide isolated from Nile tilapia and antioxidant composition comprising of the same - Google Patents

Abstract

The present invention relates to an antioxidant peptide isolated from Nile tilapia (Oreochromis niloticus), and provides a peptide showing an excellent antioxidant activity separated and purified from the gelatin of the scale of Nile tilapia, a by-product of the fishery industry, and an antioxidant composition comprising the same.

Description

Antioxidant peptide isolated from Nile tilapia and antioxidant composition comprising of the same}

The present invention relates to an antioxidant peptide isolated from Nile tilapia (Oreochromis niloticus), and more particularly to an antioxidant peptide isolated from Nile tilapia scale gelatin and an antioxidant composition comprising the same.

Since oxidative stress causes damage to all major cellular components, excessive oxygen in the tissue (reactive oxygen speices, ROS) causes cell death. In addition, ROS have a direct and indirect relationship with the oxidation of cellular biomaterials and are involved in many health disorders such as neurodegenerative, hypertension, inflammation, diabetes, cancer and aging (Calabrese, V., Lodi, R., Tonon, C., D'Agata, V., Sapienza, M., Scapagnini, G., Mangiameli, A., Pennisi, G., Giuffrida Stella, AM, Butterfield, DA (2005) .Oxative stress, mitochondrial dysfunction and cellular stress response in Friedrich's ataxia.Journal of the Neurological Sciences, 233, 145-162; Je, JY, Park, PJ, & Kim, SK (2004) .Free radical scavenging properties of hetero-chitooligosaccharides using an ESR spectroscopy.Food and Chemical Toxicology, 42, 381-387). Thus, many studies have been conducted to discover novel antioxidant compounds using natural sources such as plants, animals, microorganisms and foods to inhibit oxidation with deleterious effects.

Several fish gelatin hydrolysates have been found to exhibit not only antioxidant, antihypertensive, anticancer and antibacterial effects but also inflammation-modulating and cholesterol-lowering effects. Recently, many studies have found that peptides derived from various fish gelatin hydrolysates act as potential antioxidants. Among these studies, lumpfish (Osborne, K., Voight, MN, & Hall, DE (1990) .Utilization of lumpfish (Cyclopterus lumpus) carcasses for production of gelatin.In MN Voight & JK Botta (Eds.) , Advances in fisheries technology and biotechnology for increased profitability (pp. 145-150) .Lancaster, PA, USA: Technomic Publishing Co.), conger eel (Kim, JS, & Cho, SY (1996). for raw material of modified gelatin in marine animal skins caught in coastal offshore water in Korea.Agricultural and Chemical Biotechnology, 39, 134-139, Gudmundsson, M., & Hafsteinsson, H. (1997) .Gelatin from cod skins as affected by chemical treatments.Journal of Food Science, 62, 37-39), Shark (Yoshimura, K., Terashima, M., Hozan, D., Ebato, T., Nomura, Y., Ishii, Y., & Shirai, K. (2000) .Physical properties of shark gelatin compared with pig gelatin.Journal of Agricultural Food Chemistry, 48, 2023-2027), megrim (Sarabia, AI, Gomez-Guillen, M.). C., Solas, M. T., & Montero, P. (2001) .Effect of microbial transglutaminase on the functional properties of megrim (Lepidorhombus boscii) skin gelatin. Journal of the Science of Food and Agriculture, 81, 665-673), tilapia (Jamilah, B., & Harvinder, KG (2002) .Properties of gelatins from skins of fish-black tilapia (Oreochromis mossambicus) and red tilapia (Oreochromis nilotica) .Food Chemistry, 77, 81-84), Pollack (Je, JY, Park, PJ, & Kim, SK (2005) .Antioxidant activity of a peptide isolated from Alaska pollack (Theragra chalcogramma ) frame protein hydrolysate.Food Research International, 38, 45-50) and tuna (Cho, SM, Gu, YS, & Kim, SB (2005) .Extracting optimization and physical properties of yellowfin tuna (Thunnus albacares) skin gelatin compared to Fish gelatin extracted from mammalian gelatins.Food Hydrocolloids (19, 221-229) has been widely studied.

Tilapia is the third most important fish species in freshwater aquaculture, after carp and salmon. The tilapia culture increased from 383,654 metric tons in 1990 to 1,505,804 metric tons in 2002, and the value of farmed tilapia increased from US $ 154,000,000 in 1984 to US $ 1,800,000,000 in 2002. As the scale of aquaculture grows, the skin and bones of fish, which are by-products from the pelleting process of fish meat in the fish processing process, are issued in large quantities, and their disposal or disposal is emerging as a new problem. Fish skin and bones contain a lot of collagen, which can be used for gelatin production, and further research is needed to separate new substances such as antioxidants from gelatin.

The present invention has been made in view of the above points, and an object of the present invention is to provide a peptide having an excellent antioxidant activity separated and purified from gelatin of scales of nile tilapia, which is a by-product of the fishery industry, and an antioxidant composition comprising the same.

The object of the present invention was to isolate a novel antioxidant peptide by enzymatically hydrolyzing gelatin of Nile tilapia scales and purifying peptides having high antioxidant activity from ion exchange chromatography and HPLC to determine amino acid sequence. Radical scavenging activity and radical-mediated DNA damage inhibition effect were measured to confirm antioxidant activity.

The present invention separates and produces a peptide having excellent antioxidant activity from gelatin of Nile tilapia scales, which is treated as a by-product in the fish processing industry, thereby having an excellent effect on cost and efficacy.

1 is a graph showing the cytotoxic effects of gelatin hydrolysates on the viability of RAW 264.7 cells (A) and MRC-5 cells (B).
Figure 2 (A) shows the level of intracellular ROS generation of cells measured by DCFH-DA to detect the generated H ₂ O ₂ . The results are expressed as mean ± mean error of three independent experiments. (B) shows protection of DNA oxidative damage by alkalase-derived hydrolysates.
Figure 3 (A) shows the antioxidant activity of free radicals measured by FPLC chromatogram (top panel) and ESR spectroscopy of hydrolyzate (2 ml) loaded on a HiPrep 16/10 DEAE FF ion-exchange column ( Sub-panel). (B) shows the RP-HPLC (Primesphere 10 C ₁₈ column) chromatogram for purification of the antioxidant peptide.
Figure 4 shows the molecular weight and amino acid sequence identification of purified peptides using ESI source and TOF-MS / MS. The active peptide was sequenced in the range of 50-2500 m / z and sequenced using the PepSeq denove sequencing algorithm.
5 (A) shows the cytotoxic effect of purified peptides on the viability of RAW 264.7 cells and MRC-5 cells. The results are expressed as mean ± mean error of three independent experiments. (B) shows the level of intracellular ROS generation of cells measured by DCFH-DA detecting the generated H ₂ O ₂ . The results are expressed as mean ± mean error of three independent experiments. (C) shows DNA oxidative damage protection by purified peptides.

Gelatin was purchased from Geltech Co. (Busan, South Korea). Fluorescent probe 2 ', 7'-dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes Inc. (Eugene, OR, US) and alkalase was purchased from Novozymes Co. (Bagsvaerd, Denmark).

Compounds for radical testing such as 1,1-diphenyl-2-picrylhydrazyl (DPPH), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), FeSO ₄ , H ₂ O ₂ , riboflavin and EDTA And MTT (3- (4,5-dimethyl-2-yl) -2,5-diphenyltetrazolium bromide), agarose, fetal bovine serum (FBS), pronase E, pepsin and trypsin are available from Sigma Chemical Co. (St. Louis, MO, USA).

Mouse macrophage (RAW 264.7) human lung fibroblast (MRC-5) cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Dulbecco's Modified Eagle medium (DMEM), penicillin / streptomycin and other materials required for cell culture were obtained from Gibco BRL, Life Technologies (Grand Island, NY, USA). All other reagents are of the highest grade available.

Example  One. Enzymatic Hydrolyzate  Produce

To extract bioactive peptides from N. niloticus scale gelatin, enzymatic hydrolysis was performed using various commercial enzymes (alkalase, pronase E, pepsin and trypsin) under optimal conditions. The enzyme was mixed in 1% substrate at a rate of 1/100 (w / w). The mixture was incubated for 4 hours with stirring under each optimum temperature and then heated for 10 minutes in a boiling water bath to inactivate the enzyme. Lyophilized hydrolysates were stored at -80 ° C until use.

Example  2. Purification and Sequencing of Antioxidant Peptides

Ion exchange chromatography

Antioxidant peptides were run on a HiPrep 16/10 DEAE FF ion-exchange column (1.6 × 10 cm, Amersham Biosciences, Piscataway, NJ, USA) using fast protein liquid chromatography (FPLC) (FPLC AKTA, Amersham Bioscience Co., Uppsala, Sweden). Purified from enzymatic hydrolysate. The hydrolyzate was loaded on a HiPrep 16/10 DEAE FF ion-exchange column equilibrated with 20 mM sodium acetate buffer (pH 4.0) and linearized with NaCl (0-2 M) in the same buffer at 62 ml / h flow rate. Eluted with a gradient. Each fraction was identified at 280 nm, collected in 4 ml volumes and concentrated using a rotary evaporator; Antioxidant activity was also confirmed. Strong antioxidant fractions were lyophilized and ion exchange chromatography was used as the next step.

HPLC ( High performance liquid chromatography )

Fractions showing antioxidant activity were subjected to Primesphere 10 C18 (10 mm 250 mm, Phenomenex) with a linear gradient (0-40%, 40 minutes) of acetonitrile containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 2 ml / min. , Cheshire, England) and further purified using reverse-phase high performance liquid chromatography (RP-HPLC, Dionex Korea Ltd., Sunnyvale, CA, USA) on a column. Elution peaks were detected at 215 nm and active peaks were concentrated using a rotary evaporator. Activity peaks (target peaks, data not shown) were pooled and immediately lyophilized. The active fraction from the analytical column was converted to a SynChropak RPP-100 column (4.6) with a linear gradient of acetonitrile (15%, v / v, 20 minutes) containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 1.2 ml / min. mm 250 mm, SynChrom, Inc., Lafayette, Indiana, USA). Finally, peptides purified from the alkalase-degradation of O. niloticus scale gelatin were analyzed by their amino acid sequence.

Amino acid sequencing

The exact molecular weight and amino acid sequence of the purified peptides were confirmed using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) with an electrospray ionization (ESI) source. Purified peptides were dissolved in methanol / water (1: 1, v / v) and then injected separately into the electrospray source and their molecular weight was determined by the double charge (M + 2H) +2 state in the mass spectrum. After molecular weight determination, peptides were automatically selected for fragmentation and sequence information was obtained by dual MS analysis.

In view of the free radical scavenging ability, alkalase-derived hydrolysates were used for purification and identification of antioxidant peptides. First, the alkalase-derived hydrolyzate was dissolved in sodium acetate buffer (20 mM, pH 4.0) at 20 mg / ml and HiPrep 16/10 DEAE FF ion with a linear gradient of NaCl (0-2M) using FPLC. -Load on exchange column. After eluting peaks were identified at 280 nm and fractionated into three portions, their antioxidant activity was measured using free radical scavenging ability (FIG. 3A). Fraction 3 restrained hydroxy, DPPH and peroxide radicals at 72.04%, 42.80% and 18.03%, respectively. Fraction 3 with strong radical scavenging ability was lyophilized and RP-HPLC run on a Primesphere 10 C ₁₈ column using a linear gradient of acetonitrile (0-40%). Each fraction was pooled, lyophilized and the antioxidant activity was measured. The active fractions were finally purified on an analytical HPLC column and determined Asp-Pro-Ala-Leu-Ala-Thr-Glu-Pro-Asp-Pro-Met-Pro-Phe (1382.57 Da) by Q-TOF ESI mass spectrometry. (FIG. 4). Proline, alanine and aspartic acid were found 4, 2 and 2 times in the amino acid sequence, respectively.

Example  3. Electronic Spin Resonance Electron spin resonance ; ESR Spectroscopy assay

DPPH At radicals Scatters

DPPH radical scavenging ability is described by Nanjo et al. (Nanjo, F., Goto, K., Seto, R., Suzuki, M., Sakai, M., & Hara, Y. (1996). Scavenging effects of tea catechins and their derivatives on 1,1, -diphenyl-2-picrylhydrazyl radical.Free Radical Biology and Medicine, 21, 895-902). 30 μl of sample solution (or distilled water as a control) was added to 30 μl of DPPH (60 μM) dissolved in methanol solution. After vigorous mixing for 10 seconds, the solution was transferred to 50 μl quartz capillary and the scavenging ability of the hydrolyzate in the DPPH radical was measured using a JES-FA ESR spectrometer (JEOL Ltd., Tokyo, Japan). Spin adducts were measured after exactly 2 minutes with an ESR spectrometer. The spectrometer was set up as follows: magnetic field, 336.5 ± 5 mT; Power, 5 mW; Modulation frequency, 9.41 GHz; Amplitude, 1 x 1000; Modulation width, 0.8 mT; Sweep width, 10 mT; Sweep time 30 seconds. DPPH radical scavenging activity was calculated according to the following formula. H and H ₀ represent the relative peak heights of the radical signals in the presence and absence of the sample, respectively.

Radical scavenging activity (%) = (H ₀ -H) / H0 x 100%

Hydroxy Radical Scatters

Hydroxy radicals were generated by the Fenton reaction and collected using DMPO nitrone spin trap (Rosen & Rauckman, 1984). The resulting DMPO-OH adduct was detected by an ESR spectrometer. Sample solution (15 μl) was mixed with DMPO (0.3 M, 15 μl), FeSO ₄ (10 mM, 15 μl) and H ₂ O ₂ (10 mM, 15 μl) in phosphate buffer (pH 7.4). Transfer to μl quartz capillary. After 2.5 minutes the ESR spectra were measured using an ESR spectrometer. The spectrometer was set up as follows: magnetic field, 336.5 ± 5 mT; Power, 1 mW; Modulation frequency, 9.41 GHz; Amplitude, 1 x 200; Modulation width, 0.1 mT; Sweep width, 10 mT; Sweep time 30 seconds. Hydroxy radical scavenging ability was calculated according to the following formula. H and H ₀ represent the relative peak heights of the radical signals in the presence and absence of the sample, respectively.

Radical scavenging activity (%) = (H ₀ -H) / H0 x 100%

peroxide Radical  Negative ion Scatters

UV radioactive riboflavin / EDTA systems for peroxide radical anions (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.Biochimica et Biophysica Acta, 1427, 13-23). The reaction mixture comprising 0.3 mM riboflavin, 1.6 mM EDTA, 800 mM DMPO, and the sample at the indicated concentration was radioactive for 1 minute under UV lamp at 365 nm. The reaction mixture was transferred to 50 μl quartz capillary on an ESR spectrometer for measurement. The spectrometer was set up as follows: magnetic field, 336.5 ± 5 mT; Power, 10 mW; Modulation frequency, 9.41 GHz; Amplitude, 1 x 1000; Modulation width, 0.1 mT; Sweep width, 10 mT; 1 minute sweep time. Peroxide radical scavenging ability was calculated according to the following formula. H and H ₀ represent the relative peak heights of the radical signals in the presence and absence of the sample, respectively.

Radical scavenging activity (%) = (H ₀ -H) / H0 x 100%

Example  4. Gelatin Hydrolyzate  Preparation and Antioxidant Properties thereof

Gelatin was sequentially hydrolyzed with various enzymes such as alkalase, pronase E, pepsin and trypsin. The antioxidant activity of a substance can be accurately identified by experimenting with the scavenging ability of free radicals in the oxidation system. As a result, the hydrolyzate was evaluated for free radical scavenging ability of DPPH, hydroxy and peroxide radicals using the electron spin resonance technique as in Example 3. As shown in Table 1, the scavenging ability of the hydroxy radicals was more effective than the DPPH and peroxide radicals. Of the hydrolysates, alkalase-induced peroxides showed the highest antioxidant activity compared to other enzymatic hydrolysates. The IC50 (hydrolyzate concentration for 50% radical scavenging capacity) values of the alkalase hydrolyzate in DPPH, hydroxy and peroxide radical scavenging were 660 ± 0.40, 263 ± 0.31 and 720 ± 0.35 μg / ml, respectively. The hydrolysis reaction is due not only to the physical structure of the protein molecule but also to the effectiveness of sensitive peptide bonds where enzyme attacks are concentrated (Diniz and Martin, 1998). Alcalase Bacillus An industrially used alkaline protease made from licheniformis , one of the amino acids at the active site is serine. It has a rather broad range of specificities for peptide binding that can lead to the production of antioxidant peptides when used to hydrolyze native proteins, thereby producing bioactive peptides. Bioactive peptides produced by alcalase are resistant to degradation enzymes such as pepsin, trypsin and pronase E and are absorbed in the presence of this class of hydrolysate. In addition, related studies have clearly indicated that alkalases produce shorter peptides as well as terminal amino acid sequences that respond to a variety of bioactivity including antioxidant activity.

IC ₅₀ values of hydrolyzate scavenging DPPH, hydroxy and peroxide radicals
Hydrolyzate IC50 (μg / mL) ^a
DPPH Hydroxy peroxide Alcalase 660 ± 0.40 263 ± 0.31 720 ± 0.350 Pronase E 1020 ± 0.13 500 ± 0.05 740 ± 0.002 Trypsin 860 ± 0.42 320 ± 0.30 720 ± 0.440 pepsin 920 ± 0.24 420 ± 0.12 1150 ± 0.500

^a concentration of the hydrolyzate required to inhibit 50% of each radical ESR signal strength

Example  5. Freedom of Purified Peptides To radicals  Direct elimination effect

The free radical scavenging effect of the purified peptides on other free radical sources was tested using the same ESR technique as in Example 3. DPPH is a static free radical and accepts an electron or hydrogen radical to become a static diamagnetic molecule. Thus, DPPH is used primarily as a substrate to assess the efficacy of antioxidants. Hydroxy radicals are produced in the Fenton action and visualized by ESR spectrometer. ESR signal was inhibited by the presence of * OH scavenger competing with DMPO for * OH. Peroxide radicals were issued by UV irradiation of the riboflavin / EDTA solution. As shown in Table 2, the peptides eliminated hydroxy, DPPH and peroxide radicals with IC50 values of 7.56, 8.82 and 17.83 μM, respectively. Antioxidant peptides were more dynamic on hydroxy radicals than on other radicals. The chemical reactivity of the hydroxy radicals was the strongest among the ROS. It reacts easily with biomolecules such as amino acids, proteins and DNA. As a result, the removal of hydroxy radicals will be one of the most effective defenses of living organisms against other diseases. Although several amino acids, such as tyrosine, methionine, lysine and tryptophan, exhibit pro-oxidative effects in some cases (Marcuse, R. (1962). The effect of some amino acids on the oxidation of linoleic acid and its methyl ester. Journal of the American Oil Chemists' Society, 39, 97-103; Yamaguchi, N. (1971) .Study on antioxidative activities of amino compounds on fats and oils.Part I. Oxidation of methionine during course of autoxidation of linoleic acid.Nippon Shokuhin Kogyo Gakkaishi, 18, 313-318) is generally accepted as an antioxidant. Many antioxidant peptides are known to contain hydrophobic amino acid residues such as valine or leucine at the N-terminus of the peptide (Chen, HM, Muramoto, K., & Yamauchi, F. (1995). Structural analysis of antioxidative peptides from soybean b -conglycinin.Journal of Agricultural Food and Chemistry, 43, 574-578; Uchida, K., & Kawakishi, S. (1992) .Sequence-dependent reactivity of histidine-containing peptides with copper (II) /ascorbate.Journal of Agricultural and Biological Chemistry, 40, 13-16). Peptides from N. tiloticus scale gelatin are rich in hydrophobic amino acids (> 69%). In fact, small amounts of aromatic amino acids are present in the gelatin peptide sequence and are rich in glycine and proline. Therefore, the observed potent scavenging activity of the N. tiloticus scale gelatin protein against hydroxy, DPPH and peroxide radicals may be due to non-aromatic amino acids such as alanine, proline, valine and leucine (Mendis, E., Rajapakse, N., Byun, HG, & Kim, SK (2005a) .Investigation of jumbo squid (Dosidicus gigas) skin gelatin peptides for their in vitro antioxidant effects.Life Sciences, 77, 2166-2178; Mendis, E , Rajapakse, N., & Kim, SK (2005b) .Antioxidant properties of a radical scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate.Journal of Agricultural and Food Chemistry, 53, 581-587).

Example  6. Confirmation of cell culture and viability

MRC-5 cells and RAW 264.7 cells were incubated in DMEM medium containing 5% (v / v) FBS, 100 μg / ml penicillin-streptomycin and 5% CO 2 at 37 ° C.

The level of cytotoxicity of the sample in cells was determined by Hansen et al. (Hansen, MB, Nielsen, SE, & Berg, K. (1989) .Re-examination and further development of a precise and rapid dye method for measuring cell growth / cell kill. Measurements were made using the MTT method as described in Journal of Immunological Methods, 119, 203-210. Cells were cultured in 96-well plates at a density of 5 × 10 ³ cells / well. After 24 hours the cells were washed with fresh medium and treated with different concentrations of samples. After 48 hours of incubation the cells were rewashed and 50 μl MTT (5 mg / ml) was added and incubated for 4 hours. Finally, the formazan salt formed by dissolving 200 μl DMSO was dissolved and the amount of formazan salt was confirmed by measuring the absorbance (OD) at 540 nm using a GENios microplate reader (TECAN Austria GmbH, Grodig / Salzburg, Austria). .

MRC-5 and RAW 264.7 cells were treated with different concentrations of gelatin hydrolysate to determine non-cytotoxic concentrations for further experiments via this method. Cell viability data confirmed that the gelatin hydrolyzate was non-cytotoxic in both RAW 264.7 cells (FIG. 1A) and MRC-5 cells (FIG. 1B). Accordingly, it was confirmed that this gelatin hydrolyzate can be used for further experiments.

MRC-5 and RAW 264.7 cells were treated with different concentrations of purified peptide to determine non-cytotoxic concentrations for further experiments via this method. Cell viability data confirmed that the purified peptide was non-cytotoxic in both RAW 264.7 cells and MRC-5 cells (FIG. 5A). Therefore, it was confirmed that this gelatin hydrolyzate can be used for further experiments.

Example  7. DCFH - DA  With cover ROS of Intracellular  Confirmation of formation

Intracellular formation of ROS using the oxidation sensitive dye DCFH-DA as substrate, Engelmann et al. (Engelmann, J., Volk, J., Leyhausen, G., & Geurtsen, W. (2005). ROS formation and glutathione levels in human oral fibroblasts exposed to TEGDMA and camphorquinone.Journal of Biomedical Materials Research Part B: Applied Biomaterials, 75B, 272-276). RAW 264.7 cells were incubated in black microtiter 96-well plates, labeled with 20 μM DCFH-DA dissolved in hans balanced salt solution (HBSS) and maintained in the dark for 20 minutes. Non-fluorescent DCFH-DA dyes that are freely permeated into cells are hydrolyzed to 2 ', 7'-dichlorodihydrofluorescein (DCFH) and captured inside the cell by a cell esterase. The cells were then treated with different concentrations of samples and incubated for 1 hour. The cells were washed three times with PBS before 500 μM H ₂ O ₂ (soluble in HBSS) was added. The formation of fluorescent dichlorofluorescein (DCF) by oxidation of DCFH in the presence of various ROS was read every 30 minutes at excitation wavelength of 485 nm and emission wavelength of 528 nm (GENios microplate reader, Tecan Austria GmbH, Grodig). / Salzburg, Austria). The dose-dependent and time-dependent effects of the treatments were plotted and compared to the fluorescence density of the control of the untreated samples.

Gelatin hydrolysates (alkalase, proteinase E, trypsin and pepsin) from other enzymes were cultured for only 30 minutes compared to the control (H ₂ O ₂ treated and non-gelatin hydrolysates) as shown in FIG. 2A. It also shows a free radical scavenging effect. Of the hydrolysates, alkalase-derived hydrolysates exhibit the highest free radical scavenging effect among other enzymatic hydrolysates. These results indicate that their antioxidant capacity is different from gelatin hydrolysates (100, 200, 1000 μg / ml) and other enzyme hydrolysates of varying concentrations. DNA damage at all experimental concentrations of the alkalase treated gelatin hydrolyzate was inhibited by about 80% based on DNA size as shown in FIG. 2B. Therefore, it was confirmed that the alkalase-derived hydrolyzate eliminates free radicals in RAW 264.7 cells in a dose-dependent and time-dependent manner.

The direct scavenging effect of peptides on cellular radicals was confirmed through the ability to scaveng free radicals in the cellular environment. To this end, RAW 264.7 cells were labeled with a DCFH-DA fluorescent probe as described above. DCFH-DA is a specific probe for reactive oxygen species (ROS). The principle of this assay is that DCFH-DA diffuses through the cell membrane and is enzymatically hydrolyzed by esterase to become DCFH and react with ROS to form the fluorescent substance DCF. When cells were labeled with DCFH-DA and allowed to be captured inside the cells and incubated with H ₂ O ₂ for 30 minutes, a sharp increase in DCF fluorescence intensity indicates oxidation of DCFH by intracellular radicals (FIG. 5B). . Confirmation of DCF fluorescence intensity every 30 minutes for 3 hours indicated that radical mediated oxidation increased with incubation time. However, when pretreated with purified peptides, DCF fluorescence decreased over time. Various concentrations of purified peptides (0.2, 2, and 20 μg / ml) showed free radical scavenging effect even after only 30 minutes of incubation compared to the control (H ₂ O ₂ treated and non-peptide) as shown in FIG. 5B. . These results indicate that the antioxidant effect changes with the concentration of the purified peptide and that the free radical scavenging effect of the purified peptide at 20 μg / ml concentration is higher than the other experimental concentrations. Accordingly, it was confirmed that peptides in RAW 264.7 cells eliminated free radicals in a dose and time dependent manner.

Example  8. Genome DNA  detach

High molecular weight genomic DNA was extracted from RAW 264.7 cells using a standard modified phenol / proteinase K method (sambrook & Russell, 2001). Cells cultured in a 10 cm petri dish were washed twice with PBS and scraped into 1 ml PBS containing 10 mM EDTA. After centrifugation the cells were lysed in RNase (0.03 mg / ml), NaOAC (0.175 M), Protanase K (0.25 mg / ml) and SDS (6%). The mixture was incubated at 37 ° C. for 30 minutes and at 55 ° C. for 1 hour. After incubation, phenol / chloroform / isoamyl alcoholal 1: 1 (v / v) was added and the mixture was centrifuged at 6000 g for 5 minutes at 4 ° C. The supernatant was mixed with 100% cold ethanol at a ratio of 1: 1.5 (v / v) and maintained at -20 ° C for 15 minutes. After centrifugation at 12,000 g for 5 minutes, the pellet was dissolved in 10 mM TE buffer (pH 7.5) and DNA purity was confirmed spectrophotometrically at 260/280 nm. In addition, the isolated DNA was qualitatively analyzed using 1% agarose gel electrophoresis in 0.04 M Tris-acetate, 0.001 M EDTA buffer (pH 7.5).

Example  9. Radical -medium DNA  Check for damage

Hydrogen peroxide mediated DNA oxidation is described in Milne et al. (Milne, L., Nicotera, P., Orrenius, S., & Burkitt, MJ (1993) .Effects of glutathione and chelating agents on copper-mediated DNA oxidation: Pro-oxidant and antioxidant properties of glutathione.Archives of Biochemistry and Biophysics, 304, 102-109). 40 μl DNA reaction mixture was subjected to the same sequence of predetermined concentrations of experimental samples (or equivalent volume of distilled water as control), 100 μM final concentration of FeSO ₄ , 0.1 mM final concentration of H ₂ O ₂ and 5 μl of isolated genomic DNA Prepared by addition. The mixture was incubated at room temperature for 10 minutes and the reaction was terminated by addition of 10 mM final concentration of EDTA. Aliquots (20 μl) of the reaction mixture containing about 1 μg of DNA were electrophoresed on a 1% agarose gel at 100 V for 40 minutes. The gel was then stained with 1 mg / ml Et-Br (ethidium bromide) and visualized under UV light using AlphaEase gel image analysis software (Alpha Innotech, St. San Leandro, Calif., USA).

The hydroxy radicals (* OH) produced by the Fenton reaction are known to cause oxidative damage to the DNA strands, making the DNA in open annular or mitigating form. Were isolated from RAW 264.7 cells. The combined effect of 100 μM FeSO 4 and 0.1 mM H ₂ O ₂ on the integrity of genomic DNA in this experiment was determined by DNA electrophoresis in the presence and absence of peptide. After 10 minutes of reaction, almost all DNA was degraded in the control group treated with Fe (II) -H ₂ O ₂ only. This indicates that the peptide significantly inhibits oxidative damage of DNA (FIG. 5C). DNA damage was inhibited by about 70% based on the strength of the DNA band by peptides of all experimental concentrations. These results clearly demonstrate that peptides can prevent oxidative damage of DNA when DNA is exposed to * OH issued by Fe (II) / H ₂ O ₂ . Fe 2+ catalyzes the conversion of H ₂ O ₂ , which is a major route of biosynthesis of * OH in biological systems. * OH reacts with all components of the DNA molecule, including purine and pyrimidine bases, and the deoxyribose backbone, resulting in DNA damage. This damage increases free radical attack on the DNA of the cell, which is involved in mutations, cancer and aging. However, the peptide of the present invention may have an inhibitory effect on DNA oxidation in living cells and may contribute to preventing such diseases.

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Claims (2)

An antioxidant peptide having the amino acid sequence of SEQ ID NO: 1 isolated from Oreochromis niloticus scale gelatin. Antioxidant composition comprising the antioxidant peptide of claim 1 as an active ingredient.

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