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Free radical scavenging and angiotensin-I converting enzyme inhibitory peptides from Pacific cod (Gadus macrocephalus) skin gelatin
International Journal of Biological Macromolecules 49 (2011) 1110–1116
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Free radical scavenging and angiotensin-I converting enzyme inhibitory peptides from Pacific cod (Gadus macrocephalus) skin gelatin Dai-Hung Ngo a , BoMi Ryu a , Thanh-Sang Vo a , S.W.A. Himaya a , Isuru Wijesekara a , Se-Kwon Kim a,b,∗ a b
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
a r t i c l e
i n f o
Article history: Received 21 July 2011 Received in revised form 7 September 2011 Accepted 8 September 2011 Available online 16 September 2011 Keywords: Pacific cod (Gadus macrocephalus) skin gelatin Bioactive peptides Antioxidants RAW 264.7 cells Angiotensin-I converting enzyme inhibition
a b s t r a c t Potent antioxidative peptides were purified from Pacific cod (Gadus macrocephalus) skin gelatin using alcalase, neutrase, papain, trypsin, pepsin, and ␣-chymotrypsin. Among them, the papain hydrolysate exhibited the highest antioxidant activity. Therefore, it was further purified and obtained two peptides with amino acid sequences of Thr-Cys-Ser-Pro (388 Da) and Thr-Gly-Gly-Gly-Asn-Val (485.5 Da). The antioxidant activity of the purified peptides was performed by electron spin resonance technique. Moreover, their intracellular free radical scavenging activity using 2 ,7 -dichlorofluorescin diacetate and the protective effect against oxidation-induced DNA damage were evaluated in mouse macrophages (RAW 264.7 cells). Furthermore, both peptides have shown potential angiotensin-I converting enzyme inhibitory effect. The present study demonstrated that the peptides derived from Pacific cod (G. macrocephalus) skin gelatin could be used in the food industry as functional ingredients with potent antioxidative and antihypertensive benefits. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In recent years, cod is one of the important fish-catches of the world and it is used entirely for the cod fillet production. However, the cod skin is largely underutilized and discarded as wastes, which lead to environmental pollution. Recent studies have shown that fish skin provides the best source of gelatin because of its high availability, reducing pollution, no risk of disease transmission, no religious barriers and possibility of higher yields of collagen [1]. High blood pressure is one of the major risk factors for cardiovascular diseases including coronary heart disease, peripheral artery disease and stroke. Angiotensin-I converting enzyme (ACE) plays a critical physiological role in regulation of blood pressure by converting angiotensin-I to angiotensin-II, a potent vasoconstrictor. Therefore, the inhibition of ACE activity is a major target in the prevention of hypertension [2]. Many natural ACE inhibitory peptides have been isolated from different food proteins such as cheese whey, casein, wakame, fermented soymilk, soybean, corn gluten [3], yellowfin sole, Alaska pollack, short-necked clam, pearl oyster, bonito meat and bovine plasma proteins [4] and they could be applied in the prevention of hypertension and in the initial treatment of mildly hypertensive individuals [5].
Antioxidants may have a positive effect on human health since they can protect the human body against deterioration by reactive oxygen species (ROS), including singlet oxygen, hydrogen peroxide, superoxide and hydroxyl radicals. ROS can result in oxidative damage to all the important cellular components and have been linked with developments of various diseases, such as hypertension, neurodegenerative, inflammation, diabetes, cancer and aging [6]. Recently, several studies have been reported on the utilization of fish by-products by enzymatic hydrolysis for the recovery of various valuable components [7]. Free radical scavenging activity in gelatin hydrolysates from various fish species such as Nile tilapia [8], Alaska pollack [9], hoki fish [10], and cobia [11] has been previously reported. However, there are no studies on the protective effect of peptides from Pacific cod (Gadus macrocephalus) skin against free radical-induced oxidative damage. Therefore, the aim of this study was to evaluate the antioxidative activity of peptides derived from enzymatic hydrolysis of Pacific cod (G. macrocephalus) skin gelatin using mouse macrophage RAW 264.7 cells. In addition, ACE inhibitory activity of the purified peptides was also determined. 2. Experimental
∗ Corresponding author at: Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea. Tel.: +82 51 629 7094; fax: +82 51 629 7099. E-mail address: [email protected] (S.-K. Kim). 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.09.009
2.1. Materials Pacific cod (G. macrocephalus) skin was obtained from Jakalchi fish market, Busan, South Korea. The fluorescence probes
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Fig. 1. Radical scavenging activity of enzymatic hydrolysates (alcalase, trypsin, neutrase, papain, pepsin, and ␣-chymotrypsin) derived from Pacific cod (G. macrocephalus) skin gelatin measured by ESR spectroscopy. Values are expressed as mean ± SD of three independent determinations. The significance of differences between two samples was analyzed using the Student’s t-test and P-value of <0.05 was considered significant.
2 ,7 -dichlorofluorescin diacetate (DCFH-DA) was obtained from Molecular Probes Inc. (Eugene, OR, USA). Papain, ␣-chymotrypsin, pepsin, trypsin, ACE (from rabbit lung), hippuryl-histidyl-leucine (HHL) as a substrate of ACE, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Alcalase and neutrase were purchased from Novozymes Co. (Bagsvaerd, Denmark). Mouse macrophage (RAW 264.7) cell line was obtained from American Type Culture Collection (Manassas, VA, USA). All other chemicals were of the highest grade available commercially. 2.2. Gelatin extraction and hydrolysis The fish skins were initially washed to remove surface slime, cut into small pieces (1 cm × 2 cm), and soaked in 1% Ca(OH)2 with a skin/solution ratio of 1:4 (w/v). The solution was changed every 24 h for 3 times to remove noncollagenous proteins. Alkaline treated skins were then washed with tap water until neutral or slightly basic pH (pH 7–7.5) of wash water was obtained. Thoroughly washed fish skins were soaked in distilled water with a skin/water ratio of 1:4 (w/v) with pH 6 at 60 ◦ C for 30 h with a continuous stirring to extract gelatin from the skin matter. The mixture was then centrifuged at 30,000 × g for 10 min, the supernatant was immediately collected, and freeze-dried using a freeze dryer. The dry matter was referred to as “gelatin powder”. Gelatin was hydrolyzed with various enzymes (alcalase, neutrase, papain, pepsin, ␣-chymotrypsin and trypsin) under optimal conditions to obtain bioactive peptides. At the enzyme to substrate ratio of 1/100 (w/w), 1% substrate and enzyme were mixed. The mixture was then incubated for 4 h at each optimal temperature with stirring and then heated in a boiling water bath for 10 min to inactivate the enzyme. Lyophilized hydrolysates were stored at −80 ◦ C until used.
Fig. 2. (A) FPLC chromatogram of papain hydrolysate by HiPrep 16/10 DEAE FF ion exchange chromatography (lower panel), and their free radicals scavenging activity measured by ESR spectroscopy (upper panel). (B) RP-HPLC chromatogram for further purification of active fraction (fraction I) from FPLC (lower panel), and their free radicals scavenging activity measured by ESR spectroscopy (upper panel).
2.3. Purification of antioxidant peptides 2.3.1. Fast protein liquid chromatography (FPLC) The antioxidant peptides were purified from enzymatic hydrolysates using FPLC (AKTA, Amersham Bioscience Co., Uppsala, Sweden) on a HiPrep 16/10 diethylaminoethyl fast flow (DEAE FF) ion exchange column. The hydrolysate was loaded onto a HiPrep 16/10 DEAE FF ion exchange column equilibrated with a 20 mM
sodium acetate buffer (pH 4.0), and eluted with a linear gradient of NaCl (0–2 M) in the same buffer at a flow rate of 2 ml/min. Each fraction was monitored at 280 nm, collected at a volume of 4 ml and concentrated using a rotary evaporator and antioxidant activity was also determined. The highest antioxidant fraction was lyophilized, and high performance liquid chromatography was used as the next step.
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Fig. 3. Identification of molecular mass and amino acid sequence of the purified peptides using TOF-MS/MS with an ESI source. (A) Fraction I-5 (peptide 5) and (B) fraction I-7 (peptide 7).
2.3.2. High performance liquid chromatography (HPLC) The fraction exhibiting the highest antioxidant activity was further purified using reverse-phase HPLC (RP-HPLC, Dionex Korea Ltd., Sunnyvale, CA, USA) on a Primesphere 10 C18 (10 mm × 250 mm, Phenomenex, Cheshire, England) column with
a linear gradient of acetonitrile (0–40% in 40 min) containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 2 ml/min. Elution peaks were detected at 215 nm, and active peak was concentrated using a rotary evaporator. Potent peaks were collected, their antioxidant activity evaluated, and then lyophilized. The active fraction
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Fig. 4. Cell viability using the MTT assay (A) and DNA protective effect (B) of the purified peptides in RAW 264.7 cells.
from analytical column was further applied onto a SynChropak RP-P-100 column (4.6 mm × 250 mm) with a linear gradient of acetonitrile (15%, v/v, in 20 min) containing 0.1% TFA at the flow rate of 1.2 ml/min. Finally, the purified peptide from papain-digest of Pacific cod (G. macrocephalus) skin gelatin was analyzed for its amino acid sequence. 2.4. Determination of amino acid sequence An accurate molecular mass and amino acid sequence of the purified peptides were determined using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled with an electrospray ionization (ESI) source. The purified peptides were separately infused into the electrospray source after being dissolved in methanol/water (1:1, v/v), and its molecular mass was determined by the 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 spectrometry analysis. 2.5. Electron spin resonance (ESR) spectrometric assay Different radicals were generated and spin adducts were recorded using a JES-FA ESR spectrometer (JEOL Ltd., Tokyo, Japan) at 25 ◦ C. Instrumental settings were as follows: magnetic field 336 ± 5 mT; sweep time 30 s; sweep width 10 mT; modulation width 0.1 mT and modulation frequency 100 kHz. Radical scavenging activity of the test sample was calculated as a scavenging percentage by, S = (H0 − H)/H0 × 100%; where, H and H0 were ESR signal intensity in the presence and the absence of the test sample, respectively. DPPH radical scavenging activity was measured using the method described [12] with slight modifications. A 30 l sample solution (or distilled water itself as control) was added to 30 l of DPPH (60 M) in methanol solution. After mixing vigorously for 10 s, the solution was then transferred into a 50 l quartz capillary tube. After 2 min, the ESR spectrum was recorded at 5 mW microwave power and amplitude 1000.
Fig. 5. Intracellular free radical scavenging activity of the purified peptides in RAW 264.7 cells.
Hydroxyl radicals were generated by Fenton reaction and trapped using a DMPO nitrone spin trap [12] with slight modifications. The resultant DMPO/*OH adducts were detectable with an ESR spectrometer. A sample solution (15 l) was mixed with DMPO (0.3 M, 15 l), FeSO4 (10 mM, 15 l) and H2 O2 (10 mM, 15 l) in a phosphate buffered saline solution (PBS, pH 7.4), and then transferred into a 50 l quartz capillary tube. After 2.5 min, the ESR spectrum was recorded at 1 mW microwave power and amplitude 200. 2.6. Cell cytotoxicity, DNA damage, and intracellular formation of ROS determination RAW 264.7 cell line was cultured and maintained in Dulbecco’s modification of eagle’s medium (DMEM, GIBCO, New York, USA) containing 5% (v/v) fetal bovine serum (FBS), 100 g/ml penicillinstreptomycin and 5% CO2 at 37 ◦ C. Cytotoxicity levels of samples on cells were measured using the MTT (3-(4,5-dimethyl-2-yl)-2,5diphenyltetrazolium bromide) method as described by [13]. Genomic DNA was extracted from RAW 264.7 cells using AccuPrep® genomic DNA extraction kit (Bioneer, Daejeon, South Korea) according to the manufacturer’s protocol. Hydrogen peroxide mediated DNA oxidation was determined according to a previously published procedure [14]. Intracellular formation of ROS was assessed according to a method described previously by employing oxidation sensitive dye DCFH-DA, as the substrate [15]. 2.7. Measurement of ACE inhibitory activity The ACE inhibitory assay was performed according to a previous method [16] with slight modifications. A sample solution (50 l) with 50 l of ACE solution (25 mU/ml) was incubated at 37 ◦ C for
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Table 1 Antioxidant peptides derived from marine organisms: source and amino acid sequence.
Table 2 ACE inhibitory peptides derived from marine organisms: source and amino acid sequence.
Source
Amino acid sequence
Refs.
Source
Amino acid sequence
Refs.
Microalga Rotifer
Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe Leu-Leu-Gly-Pro-Gly-Leu-Thr-Asn-His-Ala Asp-Leu-Gly-Leu-Gly-Leu-Pro-Gly-Ala-His Glu-Ser-Thr-Val-Pro-Glu-Arg-Thr-His-Pro-AlaCys-Pro-Asp-Phe-Asn Val-Lys-Ala-Gly-Phe-Ala-Trp-Thr-Ala-Asn-GluGlu-Leu-Ser Phe-Asp-Ser-Gly-Pro-Ala-Gly-Val-Leu Asn-Ala-Asp-Phe-Gly-Leu-Asn-Gly-Leu-GluGly-Leu-Ala Phe-Gly-His-Pro-Tyr
[22] [23] [23] [24]
Tuna
Gly-Asp-Leu-Gly-Lys-Thr-Thr-Thr-Val-SerAsn-Trp-Ser-Pro-Pro-Lys-Tyr-Lys-Asp-Thr-Pro Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe Met-Glu-Gly-Ala-Gln-Glu-Ala-Gln-Gly-Asp Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-ThrGly-Glu-Ala-Met Ile-Phe-Val-Pro-Ala-Phe Met-Ile-Phe-Pro-Gly-Ala-Gly-Gly-Pro-Glu-Leu Tyr-Asn Val-Ile-Tyr Gly-Pro-Leu
[30]
Hoki Tuna Jumbo squid Giant squid Blue mussel
Microalga Sea cucumber Rotifer
[25] [26] [27] [28]
5 min, and the mixture was pre-incubated with 150 l of substrate (8.3 mM HHL in 50 mM sodium borate buffer containing 0.5 M NaCl at pH 8.3) for 30 min at the same temperature. The reaction was terminated by the addition of 250 l of 1 M HCl. The resulting hippuric acid was extracted with 500 l of ethyl acetate. After centrifugation (30,000 × g, 10 min), 200 l of the upper layer was transferred into a glass tube, and evaporated at 60 ◦ C for 2 h in a vacuum. The hippuric acid was dissolved in 1 ml of distilled water, and the absorbance was measured at 228 nm using UV-spectrophotometer (GENios microplate reader, Tecan Austria GmbH, Grodig/Salzburg, Austria). The IC50 value defined as the concentration of inhibitor required to inhibit 50% of the ACE activity.
Shrimp Yellowfin sole Hard clam Sea bream Alaska pollack
[33] [34] [35] [36] [37] [38] [39] [40]
using electron spin resonance (ESR) technique. As shown in Fig. 1, scavenging effect of the hydrolysates on DPPH radical was more efficient than that of hydroxyl radical. Among the hydrolysates resulting from various enzymes, the papain hydrolysate exhibited the most potential antioxidant activity than other enzymatic hydrolysates. The scavenging effects of papain hydrolysate on DPPH and hydroxyl radicals were found to be about 72% and 56% at 1 mg/ml, respectively. Therefore, papain hydrolysate was selected for further purification.
2.8. Statistical analysis Data were expressed as mean ± standard deviation (SD) of three independent determinations. The significance of differences between two samples was analyzed using the Student’s t-test and P-value of <0.05 was considered as the level of statistical significance. 3. Results and discussion 3.1. Preparation of gelatin hydrolysates and their antioxidant properties The antioxidative activity of the hydrolysates was evaluated for their free radical scavenging effect on hydroxyl and DPPH radicals
Fig. 6. Angiotensin-I converting enzyme inhibitory activity of the purified peptides. Values are expressed as mean ± SD of three independent determinations. The significance of differences between two samples was analyzed using the Student’s t-test and P-value of <0.05 was considered significant.
3.2. Purification and direct free radical scavenging effect of the purified peptides Firstly, papain hydrolysate was dissolved in sodium acetate buffer (20 mM, pH 4.0) at 20 mg/ml, and loaded onto a HiPrep 16/10 DEAE FF ion exchange column using FPLC with a linear gradient of NaCl (0–2.0 M). The elution peaks were monitored at 280 nm, and each fraction was collected as 4 ml and fractionated into three portions. Each fraction was pooled, lyophilized, and measured for its antioxidant activity in direct free radicals scavenging activity (Fig. 2A). Among all fractions, fraction I had the strongest hydroxyl radical scavenging effect by 75% at 500 g/ml. Therefore, fraction I was further purified on an analytical HPLC column using a linear gradient of acetonitrile (0–40%) and its antioxidant activities measured using their potency to scavenge free radicals (Fig. 2B). Amino acid sequences of the purified two peptides were determined to be Thr-Gly-Gly-Gly-Asn-Val (485.5 Da, peptide 5) (Fig. 3A) and Thr-Cys-Ser-Pro (388 Da, peptide 7) (Fig. 3B) by Q-TOF ESI mass spectroscopy. The structure of peptide 5 was composed of residues of one threonine, three glycine, one asparagine and one valine. The structure of peptide 7 was composed of residues of one threonine, one cysteine, one serine and one proline. Further, in the sequence of peptide 5 has hydrophobic amino acid such glycine and valine; in the sequence of peptide 7 has cysteine and hydrophobic amino acid such as proline. Sequence analysis studies revealed that the purified peptides are rich in hydrophobic amino acids (glycine, proline), which contribute substantially for scavenging of free radicals acting as potent electron donors. Cysteine residues are independently important for antioxidant activity as they can directly interact with free radicals. Furthermore, the thiol group of cysteine plays an important role in the protecting cells and cellular biomolecules against oxidative stress. Threonine is an important component in the formation of protein, collagen and elastin. Threonine helps the liver and maintains the body’s proteins in balance. Bioactive peptides usually contain 3–20 amino acids residues and low molecular weight peptides are more potent as bioactive peptides than high molecular weight peptides [17].
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3.3. Cell viability and DNA protective activity of the purified peptides on RAW 264.7 cells RAW 264.7 cells were treated with different concentrations of the purified peptides to determine the cytotoxic effect of the purified peptides. According to the MTT assay, it has shown that no cytotoxicity of the purified peptides on RAW 264.7 cells (Fig. 4A). Consequently, we could use the purified peptides for further intracellular free radical scavenging experiments. The highly reactive hydroxyl radicals can easily react with all components of the DNA molecule and damage DNA. In addition, this damage increases with the increment of the free radicals attacked on cellular DNA, which involved in the occurrence of numerous chronic diseases such as inflammation, cancer, mutagenesis and aging [18–20]. In this study, DNA oxidation was performed by combining effect of 200 M Fe(II) and 2 mM H2 O2 on the integrity of genomic DNA isolated from RAW 264.7 cells. The protective activity of the purified peptides against DNA damage was determined by DNA electrophoresis. After the reaction, almost all DNA was degraded in the control group treated with H2 O2 –Fe(II) alone (Fig. 4B). However, the purified peptides showed a protective effect on free radical-mediated DNA damage by dose-dependently. Even in low concentration (50 g/ml) of the purified peptides, DNA damage was inhibited more than 90% determined based on the intensity of DNA bands. 3.4. Intracellular free radical scavenging activity of the purified peptides on RAW 264.7 cells The intracellular free radical scavenging effect of the purified peptides was carried out on RAW 264.7 cells as these cells are able to release high amounts of ROS following stimulation [21]. For this experiment, intracellular ROS was measured using the fluorescence probe DCFH-DA. During labeling, non-fluorescent DCFH-DA dye that easily penetrates into the cells gets hydrolyzed by intracellular esterase to become DCFH, and this compound is trapped inside the cells and gets oxidized by H2 O2 . The monitoring of DCF fluorescence intensity in every 30 min for 3 h duration that radicalmediated oxidation increased with the incubation time. As shown in Fig. 5, the treatment with the purified peptides reduced the DCF fluorescence intensity in a dose- and time-dependently. The purified two peptides have amino acid residues which are generally present in antioxidant peptides derived from marine organisms. The antioxidant activity of marine-derived bioactive peptides has been summarized in Table 1. These results confirmed that the purified two peptides scavenged free radicals and inhibited radicalmediated oxidation in RAW 264.7 cells. 3.5. ACE inhibitory activity of the purified peptides Bioactive peptides usually contain 3–20 amino acids residues and low molecular weight peptides are more potent as bioactive peptides than high molecular weight peptides [17]. Regarding to the relationship between activity and structure of ACE inhibitory peptides, those peptides had tyrosine, proline, or phenylalanine at the C-terminus and isoleucine or valine at the N-terminal showed highly potential inhibitory activity [29]. Most of the reported ACE inhibitory peptides are usually short peptides with proline residue at the C-terminal end [30,31]. Proline is known to be preventing digestion of enzymes and may pass through the capillary into the circulation of blood in the sequence of short peptides [32]. As shown in Fig. 6, the purified peptide, Thr-Cys-Ser-Pro (388 Da) showed the most potent ACE inhibitory activity (81%) than the other peptide, Thr-Gly-Gly-Gly-Asn-Val (485.5 Da) (68%) at 500 g/ml. Therefore, these peptides can be incorporated into functional foods as novel ACE inhibitors. Amino acid residues in the purified two peptides are
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generally present in ACE inhibitory peptides derived from marine organisms. Marine-derived peptides have been shown potent ACE inhibitory activities (Table 2). 4. Conclusion Marine by-products are natural bioresources that might be useful in health beneficial products, pharmaceuticals and food industries. In this study, two bioactive peptides were purified from Pacific cod (G. macrocephalus) skin gelatin by papain enzymatic hydrolysis. Using consecutive chromatographic methods, two antioxidative and antihypertensive peptides, Thr-Cys-Ser-Pro (388 Da) and Thr-Gly-Gly-Gly-Asn-Val (485.5 Da), were shown to exhibit potent radical scavenging and ACE inhibitory activities. In conclusion, these results suggested that the antioxidant and ACE inhibitory peptides from Pacific cod (G. macrocephalus) skin gelatin could be potential candidates to develop functional foods or nutraceutical products against free radicals, hypertension and related diseases. Acknowledgement This research was supported by a grant from Marine Bioprocess Research Center of the Marine Bio 21 Center funded by the Ministry of Land, Transport and Maritime, Republic of Korea. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34]
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Free radical scavenging and angiotensin-I converting enzyme inhibitory peptides from Pacific cod (Gadus macrocephalus) skin gelatin Dai-Hung Ngo a , BoMi Ryu a , Thanh-Sang Vo a , S.W.A. Himaya a , Isuru Wijesekara a , Se-Kwon Kim a,b,∗ a b
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
a r t i c l e
i n f o
Article history: Received 21 July 2011 Received in revised form 7 September 2011 Accepted 8 September 2011 Available online 16 September 2011 Keywords: Pacific cod (Gadus macrocephalus) skin gelatin Bioactive peptides Antioxidants RAW 264.7 cells Angiotensin-I converting enzyme inhibition
a b s t r a c t Potent antioxidative peptides were purified from Pacific cod (Gadus macrocephalus) skin gelatin using alcalase, neutrase, papain, trypsin, pepsin, and ␣-chymotrypsin. Among them, the papain hydrolysate exhibited the highest antioxidant activity. Therefore, it was further purified and obtained two peptides with amino acid sequences of Thr-Cys-Ser-Pro (388 Da) and Thr-Gly-Gly-Gly-Asn-Val (485.5 Da). The antioxidant activity of the purified peptides was performed by electron spin resonance technique. Moreover, their intracellular free radical scavenging activity using 2 ,7 -dichlorofluorescin diacetate and the protective effect against oxidation-induced DNA damage were evaluated in mouse macrophages (RAW 264.7 cells). Furthermore, both peptides have shown potential angiotensin-I converting enzyme inhibitory effect. The present study demonstrated that the peptides derived from Pacific cod (G. macrocephalus) skin gelatin could be used in the food industry as functional ingredients with potent antioxidative and antihypertensive benefits. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In recent years, cod is one of the important fish-catches of the world and it is used entirely for the cod fillet production. However, the cod skin is largely underutilized and discarded as wastes, which lead to environmental pollution. Recent studies have shown that fish skin provides the best source of gelatin because of its high availability, reducing pollution, no risk of disease transmission, no religious barriers and possibility of higher yields of collagen [1]. High blood pressure is one of the major risk factors for cardiovascular diseases including coronary heart disease, peripheral artery disease and stroke. Angiotensin-I converting enzyme (ACE) plays a critical physiological role in regulation of blood pressure by converting angiotensin-I to angiotensin-II, a potent vasoconstrictor. Therefore, the inhibition of ACE activity is a major target in the prevention of hypertension [2]. Many natural ACE inhibitory peptides have been isolated from different food proteins such as cheese whey, casein, wakame, fermented soymilk, soybean, corn gluten [3], yellowfin sole, Alaska pollack, short-necked clam, pearl oyster, bonito meat and bovine plasma proteins [4] and they could be applied in the prevention of hypertension and in the initial treatment of mildly hypertensive individuals [5].
Antioxidants may have a positive effect on human health since they can protect the human body against deterioration by reactive oxygen species (ROS), including singlet oxygen, hydrogen peroxide, superoxide and hydroxyl radicals. ROS can result in oxidative damage to all the important cellular components and have been linked with developments of various diseases, such as hypertension, neurodegenerative, inflammation, diabetes, cancer and aging [6]. Recently, several studies have been reported on the utilization of fish by-products by enzymatic hydrolysis for the recovery of various valuable components [7]. Free radical scavenging activity in gelatin hydrolysates from various fish species such as Nile tilapia [8], Alaska pollack [9], hoki fish [10], and cobia [11] has been previously reported. However, there are no studies on the protective effect of peptides from Pacific cod (Gadus macrocephalus) skin against free radical-induced oxidative damage. Therefore, the aim of this study was to evaluate the antioxidative activity of peptides derived from enzymatic hydrolysis of Pacific cod (G. macrocephalus) skin gelatin using mouse macrophage RAW 264.7 cells. In addition, ACE inhibitory activity of the purified peptides was also determined. 2. Experimental
∗ Corresponding author at: Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea. Tel.: +82 51 629 7094; fax: +82 51 629 7099. E-mail address: [email protected] (S.-K. Kim). 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.09.009
2.1. Materials Pacific cod (G. macrocephalus) skin was obtained from Jakalchi fish market, Busan, South Korea. The fluorescence probes
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Fig. 1. Radical scavenging activity of enzymatic hydrolysates (alcalase, trypsin, neutrase, papain, pepsin, and ␣-chymotrypsin) derived from Pacific cod (G. macrocephalus) skin gelatin measured by ESR spectroscopy. Values are expressed as mean ± SD of three independent determinations. The significance of differences between two samples was analyzed using the Student’s t-test and P-value of <0.05 was considered significant.
2 ,7 -dichlorofluorescin diacetate (DCFH-DA) was obtained from Molecular Probes Inc. (Eugene, OR, USA). Papain, ␣-chymotrypsin, pepsin, trypsin, ACE (from rabbit lung), hippuryl-histidyl-leucine (HHL) as a substrate of ACE, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Alcalase and neutrase were purchased from Novozymes Co. (Bagsvaerd, Denmark). Mouse macrophage (RAW 264.7) cell line was obtained from American Type Culture Collection (Manassas, VA, USA). All other chemicals were of the highest grade available commercially. 2.2. Gelatin extraction and hydrolysis The fish skins were initially washed to remove surface slime, cut into small pieces (1 cm × 2 cm), and soaked in 1% Ca(OH)2 with a skin/solution ratio of 1:4 (w/v). The solution was changed every 24 h for 3 times to remove noncollagenous proteins. Alkaline treated skins were then washed with tap water until neutral or slightly basic pH (pH 7–7.5) of wash water was obtained. Thoroughly washed fish skins were soaked in distilled water with a skin/water ratio of 1:4 (w/v) with pH 6 at 60 ◦ C for 30 h with a continuous stirring to extract gelatin from the skin matter. The mixture was then centrifuged at 30,000 × g for 10 min, the supernatant was immediately collected, and freeze-dried using a freeze dryer. The dry matter was referred to as “gelatin powder”. Gelatin was hydrolyzed with various enzymes (alcalase, neutrase, papain, pepsin, ␣-chymotrypsin and trypsin) under optimal conditions to obtain bioactive peptides. At the enzyme to substrate ratio of 1/100 (w/w), 1% substrate and enzyme were mixed. The mixture was then incubated for 4 h at each optimal temperature with stirring and then heated in a boiling water bath for 10 min to inactivate the enzyme. Lyophilized hydrolysates were stored at −80 ◦ C until used.
Fig. 2. (A) FPLC chromatogram of papain hydrolysate by HiPrep 16/10 DEAE FF ion exchange chromatography (lower panel), and their free radicals scavenging activity measured by ESR spectroscopy (upper panel). (B) RP-HPLC chromatogram for further purification of active fraction (fraction I) from FPLC (lower panel), and their free radicals scavenging activity measured by ESR spectroscopy (upper panel).
2.3. Purification of antioxidant peptides 2.3.1. Fast protein liquid chromatography (FPLC) The antioxidant peptides were purified from enzymatic hydrolysates using FPLC (AKTA, Amersham Bioscience Co., Uppsala, Sweden) on a HiPrep 16/10 diethylaminoethyl fast flow (DEAE FF) ion exchange column. The hydrolysate was loaded onto a HiPrep 16/10 DEAE FF ion exchange column equilibrated with a 20 mM
sodium acetate buffer (pH 4.0), and eluted with a linear gradient of NaCl (0–2 M) in the same buffer at a flow rate of 2 ml/min. Each fraction was monitored at 280 nm, collected at a volume of 4 ml and concentrated using a rotary evaporator and antioxidant activity was also determined. The highest antioxidant fraction was lyophilized, and high performance liquid chromatography was used as the next step.
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Fig. 3. Identification of molecular mass and amino acid sequence of the purified peptides using TOF-MS/MS with an ESI source. (A) Fraction I-5 (peptide 5) and (B) fraction I-7 (peptide 7).
2.3.2. High performance liquid chromatography (HPLC) The fraction exhibiting the highest antioxidant activity was further purified using reverse-phase HPLC (RP-HPLC, Dionex Korea Ltd., Sunnyvale, CA, USA) on a Primesphere 10 C18 (10 mm × 250 mm, Phenomenex, Cheshire, England) column with
a linear gradient of acetonitrile (0–40% in 40 min) containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 2 ml/min. Elution peaks were detected at 215 nm, and active peak was concentrated using a rotary evaporator. Potent peaks were collected, their antioxidant activity evaluated, and then lyophilized. The active fraction
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Fig. 4. Cell viability using the MTT assay (A) and DNA protective effect (B) of the purified peptides in RAW 264.7 cells.
from analytical column was further applied onto a SynChropak RP-P-100 column (4.6 mm × 250 mm) with a linear gradient of acetonitrile (15%, v/v, in 20 min) containing 0.1% TFA at the flow rate of 1.2 ml/min. Finally, the purified peptide from papain-digest of Pacific cod (G. macrocephalus) skin gelatin was analyzed for its amino acid sequence. 2.4. Determination of amino acid sequence An accurate molecular mass and amino acid sequence of the purified peptides were determined using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled with an electrospray ionization (ESI) source. The purified peptides were separately infused into the electrospray source after being dissolved in methanol/water (1:1, v/v), and its molecular mass was determined by the 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 spectrometry analysis. 2.5. Electron spin resonance (ESR) spectrometric assay Different radicals were generated and spin adducts were recorded using a JES-FA ESR spectrometer (JEOL Ltd., Tokyo, Japan) at 25 ◦ C. Instrumental settings were as follows: magnetic field 336 ± 5 mT; sweep time 30 s; sweep width 10 mT; modulation width 0.1 mT and modulation frequency 100 kHz. Radical scavenging activity of the test sample was calculated as a scavenging percentage by, S = (H0 − H)/H0 × 100%; where, H and H0 were ESR signal intensity in the presence and the absence of the test sample, respectively. DPPH radical scavenging activity was measured using the method described [12] with slight modifications. A 30 l sample solution (or distilled water itself as control) was added to 30 l of DPPH (60 M) in methanol solution. After mixing vigorously for 10 s, the solution was then transferred into a 50 l quartz capillary tube. After 2 min, the ESR spectrum was recorded at 5 mW microwave power and amplitude 1000.
Fig. 5. Intracellular free radical scavenging activity of the purified peptides in RAW 264.7 cells.
Hydroxyl radicals were generated by Fenton reaction and trapped using a DMPO nitrone spin trap [12] with slight modifications. The resultant DMPO/*OH adducts were detectable with an ESR spectrometer. A sample solution (15 l) was mixed with DMPO (0.3 M, 15 l), FeSO4 (10 mM, 15 l) and H2 O2 (10 mM, 15 l) in a phosphate buffered saline solution (PBS, pH 7.4), and then transferred into a 50 l quartz capillary tube. After 2.5 min, the ESR spectrum was recorded at 1 mW microwave power and amplitude 200. 2.6. Cell cytotoxicity, DNA damage, and intracellular formation of ROS determination RAW 264.7 cell line was cultured and maintained in Dulbecco’s modification of eagle’s medium (DMEM, GIBCO, New York, USA) containing 5% (v/v) fetal bovine serum (FBS), 100 g/ml penicillinstreptomycin and 5% CO2 at 37 ◦ C. Cytotoxicity levels of samples on cells were measured using the MTT (3-(4,5-dimethyl-2-yl)-2,5diphenyltetrazolium bromide) method as described by [13]. Genomic DNA was extracted from RAW 264.7 cells using AccuPrep® genomic DNA extraction kit (Bioneer, Daejeon, South Korea) according to the manufacturer’s protocol. Hydrogen peroxide mediated DNA oxidation was determined according to a previously published procedure [14]. Intracellular formation of ROS was assessed according to a method described previously by employing oxidation sensitive dye DCFH-DA, as the substrate [15]. 2.7. Measurement of ACE inhibitory activity The ACE inhibitory assay was performed according to a previous method [16] with slight modifications. A sample solution (50 l) with 50 l of ACE solution (25 mU/ml) was incubated at 37 ◦ C for
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Table 1 Antioxidant peptides derived from marine organisms: source and amino acid sequence.
Table 2 ACE inhibitory peptides derived from marine organisms: source and amino acid sequence.
Source
Amino acid sequence
Refs.
Source
Amino acid sequence
Refs.
Microalga Rotifer
Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe Leu-Leu-Gly-Pro-Gly-Leu-Thr-Asn-His-Ala Asp-Leu-Gly-Leu-Gly-Leu-Pro-Gly-Ala-His Glu-Ser-Thr-Val-Pro-Glu-Arg-Thr-His-Pro-AlaCys-Pro-Asp-Phe-Asn Val-Lys-Ala-Gly-Phe-Ala-Trp-Thr-Ala-Asn-GluGlu-Leu-Ser Phe-Asp-Ser-Gly-Pro-Ala-Gly-Val-Leu Asn-Ala-Asp-Phe-Gly-Leu-Asn-Gly-Leu-GluGly-Leu-Ala Phe-Gly-His-Pro-Tyr
[22] [23] [23] [24]
Tuna
Gly-Asp-Leu-Gly-Lys-Thr-Thr-Thr-Val-SerAsn-Trp-Ser-Pro-Pro-Lys-Tyr-Lys-Asp-Thr-Pro Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe Met-Glu-Gly-Ala-Gln-Glu-Ala-Gln-Gly-Asp Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-ThrGly-Glu-Ala-Met Ile-Phe-Val-Pro-Ala-Phe Met-Ile-Phe-Pro-Gly-Ala-Gly-Gly-Pro-Glu-Leu Tyr-Asn Val-Ile-Tyr Gly-Pro-Leu
[30]
Hoki Tuna Jumbo squid Giant squid Blue mussel
Microalga Sea cucumber Rotifer
[25] [26] [27] [28]
5 min, and the mixture was pre-incubated with 150 l of substrate (8.3 mM HHL in 50 mM sodium borate buffer containing 0.5 M NaCl at pH 8.3) for 30 min at the same temperature. The reaction was terminated by the addition of 250 l of 1 M HCl. The resulting hippuric acid was extracted with 500 l of ethyl acetate. After centrifugation (30,000 × g, 10 min), 200 l of the upper layer was transferred into a glass tube, and evaporated at 60 ◦ C for 2 h in a vacuum. The hippuric acid was dissolved in 1 ml of distilled water, and the absorbance was measured at 228 nm using UV-spectrophotometer (GENios microplate reader, Tecan Austria GmbH, Grodig/Salzburg, Austria). The IC50 value defined as the concentration of inhibitor required to inhibit 50% of the ACE activity.
Shrimp Yellowfin sole Hard clam Sea bream Alaska pollack
[33] [34] [35] [36] [37] [38] [39] [40]
using electron spin resonance (ESR) technique. As shown in Fig. 1, scavenging effect of the hydrolysates on DPPH radical was more efficient than that of hydroxyl radical. Among the hydrolysates resulting from various enzymes, the papain hydrolysate exhibited the most potential antioxidant activity than other enzymatic hydrolysates. The scavenging effects of papain hydrolysate on DPPH and hydroxyl radicals were found to be about 72% and 56% at 1 mg/ml, respectively. Therefore, papain hydrolysate was selected for further purification.
2.8. Statistical analysis Data were expressed as mean ± standard deviation (SD) of three independent determinations. The significance of differences between two samples was analyzed using the Student’s t-test and P-value of <0.05 was considered as the level of statistical significance. 3. Results and discussion 3.1. Preparation of gelatin hydrolysates and their antioxidant properties The antioxidative activity of the hydrolysates was evaluated for their free radical scavenging effect on hydroxyl and DPPH radicals
Fig. 6. Angiotensin-I converting enzyme inhibitory activity of the purified peptides. Values are expressed as mean ± SD of three independent determinations. The significance of differences between two samples was analyzed using the Student’s t-test and P-value of <0.05 was considered significant.
3.2. Purification and direct free radical scavenging effect of the purified peptides Firstly, papain hydrolysate was dissolved in sodium acetate buffer (20 mM, pH 4.0) at 20 mg/ml, and loaded onto a HiPrep 16/10 DEAE FF ion exchange column using FPLC with a linear gradient of NaCl (0–2.0 M). The elution peaks were monitored at 280 nm, and each fraction was collected as 4 ml and fractionated into three portions. Each fraction was pooled, lyophilized, and measured for its antioxidant activity in direct free radicals scavenging activity (Fig. 2A). Among all fractions, fraction I had the strongest hydroxyl radical scavenging effect by 75% at 500 g/ml. Therefore, fraction I was further purified on an analytical HPLC column using a linear gradient of acetonitrile (0–40%) and its antioxidant activities measured using their potency to scavenge free radicals (Fig. 2B). Amino acid sequences of the purified two peptides were determined to be Thr-Gly-Gly-Gly-Asn-Val (485.5 Da, peptide 5) (Fig. 3A) and Thr-Cys-Ser-Pro (388 Da, peptide 7) (Fig. 3B) by Q-TOF ESI mass spectroscopy. The structure of peptide 5 was composed of residues of one threonine, three glycine, one asparagine and one valine. The structure of peptide 7 was composed of residues of one threonine, one cysteine, one serine and one proline. Further, in the sequence of peptide 5 has hydrophobic amino acid such glycine and valine; in the sequence of peptide 7 has cysteine and hydrophobic amino acid such as proline. Sequence analysis studies revealed that the purified peptides are rich in hydrophobic amino acids (glycine, proline), which contribute substantially for scavenging of free radicals acting as potent electron donors. Cysteine residues are independently important for antioxidant activity as they can directly interact with free radicals. Furthermore, the thiol group of cysteine plays an important role in the protecting cells and cellular biomolecules against oxidative stress. Threonine is an important component in the formation of protein, collagen and elastin. Threonine helps the liver and maintains the body’s proteins in balance. Bioactive peptides usually contain 3–20 amino acids residues and low molecular weight peptides are more potent as bioactive peptides than high molecular weight peptides [17].
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3.3. Cell viability and DNA protective activity of the purified peptides on RAW 264.7 cells RAW 264.7 cells were treated with different concentrations of the purified peptides to determine the cytotoxic effect of the purified peptides. According to the MTT assay, it has shown that no cytotoxicity of the purified peptides on RAW 264.7 cells (Fig. 4A). Consequently, we could use the purified peptides for further intracellular free radical scavenging experiments. The highly reactive hydroxyl radicals can easily react with all components of the DNA molecule and damage DNA. In addition, this damage increases with the increment of the free radicals attacked on cellular DNA, which involved in the occurrence of numerous chronic diseases such as inflammation, cancer, mutagenesis and aging [18–20]. In this study, DNA oxidation was performed by combining effect of 200 M Fe(II) and 2 mM H2 O2 on the integrity of genomic DNA isolated from RAW 264.7 cells. The protective activity of the purified peptides against DNA damage was determined by DNA electrophoresis. After the reaction, almost all DNA was degraded in the control group treated with H2 O2 –Fe(II) alone (Fig. 4B). However, the purified peptides showed a protective effect on free radical-mediated DNA damage by dose-dependently. Even in low concentration (50 g/ml) of the purified peptides, DNA damage was inhibited more than 90% determined based on the intensity of DNA bands. 3.4. Intracellular free radical scavenging activity of the purified peptides on RAW 264.7 cells The intracellular free radical scavenging effect of the purified peptides was carried out on RAW 264.7 cells as these cells are able to release high amounts of ROS following stimulation [21]. For this experiment, intracellular ROS was measured using the fluorescence probe DCFH-DA. During labeling, non-fluorescent DCFH-DA dye that easily penetrates into the cells gets hydrolyzed by intracellular esterase to become DCFH, and this compound is trapped inside the cells and gets oxidized by H2 O2 . The monitoring of DCF fluorescence intensity in every 30 min for 3 h duration that radicalmediated oxidation increased with the incubation time. As shown in Fig. 5, the treatment with the purified peptides reduced the DCF fluorescence intensity in a dose- and time-dependently. The purified two peptides have amino acid residues which are generally present in antioxidant peptides derived from marine organisms. The antioxidant activity of marine-derived bioactive peptides has been summarized in Table 1. These results confirmed that the purified two peptides scavenged free radicals and inhibited radicalmediated oxidation in RAW 264.7 cells. 3.5. ACE inhibitory activity of the purified peptides Bioactive peptides usually contain 3–20 amino acids residues and low molecular weight peptides are more potent as bioactive peptides than high molecular weight peptides [17]. Regarding to the relationship between activity and structure of ACE inhibitory peptides, those peptides had tyrosine, proline, or phenylalanine at the C-terminus and isoleucine or valine at the N-terminal showed highly potential inhibitory activity [29]. Most of the reported ACE inhibitory peptides are usually short peptides with proline residue at the C-terminal end [30,31]. Proline is known to be preventing digestion of enzymes and may pass through the capillary into the circulation of blood in the sequence of short peptides [32]. As shown in Fig. 6, the purified peptide, Thr-Cys-Ser-Pro (388 Da) showed the most potent ACE inhibitory activity (81%) than the other peptide, Thr-Gly-Gly-Gly-Asn-Val (485.5 Da) (68%) at 500 g/ml. Therefore, these peptides can be incorporated into functional foods as novel ACE inhibitors. Amino acid residues in the purified two peptides are
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generally present in ACE inhibitory peptides derived from marine organisms. Marine-derived peptides have been shown potent ACE inhibitory activities (Table 2). 4. Conclusion Marine by-products are natural bioresources that might be useful in health beneficial products, pharmaceuticals and food industries. In this study, two bioactive peptides were purified from Pacific cod (G. macrocephalus) skin gelatin by papain enzymatic hydrolysis. Using consecutive chromatographic methods, two antioxidative and antihypertensive peptides, Thr-Cys-Ser-Pro (388 Da) and Thr-Gly-Gly-Gly-Asn-Val (485.5 Da), were shown to exhibit potent radical scavenging and ACE inhibitory activities. In conclusion, these results suggested that the antioxidant and ACE inhibitory peptides from Pacific cod (G. macrocephalus) skin gelatin could be potential candidates to develop functional foods or nutraceutical products against free radicals, hypertension and related diseases. Acknowledgement This research was supported by a grant from Marine Bioprocess Research Center of the Marine Bio 21 Center funded by the Ministry of Land, Transport and Maritime, Republic of Korea. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34]
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