清除氧自由基

Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin,Rana catesbeiana Shaw
Zhong-Ji Qian a ,Won-Kyo Jung b ,Se-Kwon Kim
a,c,*
a
Department of Chemistry,Pukyong National University,Busan 608-737,Republic of Korea
b
Department of NOAA Sea Grant Development and Food Science,Louisiana State University,Baton Rouge,LA 70803,United States
c
Marine Bioprocess Research Center,Pukyong National University,Busan 608-737,Republic of Korea
Received 28December 2006;received in revised form 3April 2007;accepted 3April 2007
Available online 18May 2007
Abstract
In the present study,a peptide having antioxidant properties was isolated from bullfrog skin protein,Rana catesbeiana Shaw .Bullfrog skin protein was hydrolyzed using alcalase,neutrase,pepsin,papain,a -chymotrypsin and trypsin.Antioxidant activities of respective hydrolysates were evaluated using lipid peroxidation inhibition assay and direct free radical scavenging activity by using electron spin resonance (ESR)spectrometer.Among hydrolysates,alcalase derived hydrolysate exhibited the highest antioxidant activities than those of other enzyme hydrolysates.In order to purity a peptide having potent antioxidant properties,alcalase hydrolysate was separated using consecutive chromatographic methods on a Hiprep 16/10DEAE FF anion exchange column,Superdex Peptide 10/300GL gel filtration column and highan octadecylsilane (ODS)C18reversed phase column.Finally,a potent antioxidative peptide was isolated and its sequence was identified to be LEELEEELEGCE (1487Da)by Q-TOF ESI mass spectroscopy.This antioxidant peptide from bullfrog skin protein (APBSP)inhibited lipid peroxidation higher than that of a -tocopherol as positive control and efficiently quenched different sources of free radicals:DPPH radical (IC 50=16.1l M),hydroxyl radical (IC 50=12.8l M),superoxide radical (IC 50=34.0l M)and peroxyl radical (IC 50=32.6l M).Moreover,MTT assay showed that this peptide does not exert any cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5).
Ó2007Elsevier Ltd.All rights reserved.
Keywords:Antioxidant peptide;Bullfrog skin;Hydrolysate;Lipid peroxidation;Radical scavenging activity
1.Introduction
Free radical-mediated lipid peroxidation,oxidative stress and antioxidants are widely discussed in many cur-rent research areas.Under normal conditions,reactive oxy-gen species (ROS)and free radicals are effectively eliminated by the antioxidant defense systems such as anti-oxidant enzymes and non-enzymatic factors.However,under pathological conditions,the balance between the generation and elimination of ROS is broken,as a result
of these events,biomacromolecules including DNA,mem-brane lipids and proteins are damaged by ROS-mediated oxidative stress.Uncontrolled generation of free radicals that attack membrane lipids,protein and DNA is believed to be involved in many health disorders such as diabetes mellitus,cancer,neurodegenerative and inflammatory dis-eases (Pryor and Ann,1982;Butterfield et al.,2002).Lipid oxidation is of a great concern of the food industry and among consumers because it leads to the development of undesirable off-flavors and potentially toxic reaction prod-ucts (Park et al.,2001).Specially,lipid peroxidation in foods affects the nutritive value and may cause disease con-ditions following consumption of potentially toxic reaction products.Therefore,in research fields of human nutrition and biochemistry an increasing interest is developing to
0960-8524/$-see front matter Ó2007Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2007.04.005
*
Corresponding author.Address:Department of Chemistry,Pukyong National University,Busan 608-737,Republic of Korea.Tel.:+82516206375;fax:+82516288147.
E-mail address:sknkim@pknu.ac.kr (S.-K.Kim).Available online at
Bioresource Technology 99(2008)
1690–1698
identify antioxidant ingredients derived from food ingredi-ents that could retard lipid peroxidation.Antioxidants can act at different levels in an oxidative sequence.This may be illustrated considering one of the many mechanism by which oxidative stress can cause damage by stimulating the free radical chain reaction of lipid peroxidation.Cur-rently,natural antioxidant,a-tocopherol and some syn-thetic antioxidants such as propyl gallate,butylated hydroxyanisole(BHA),and butylated hydroxytoluene (BHT)are commonly used to act against free radicals in food and biological systems.Though the synthetic antiox-idants are effective and cheap compared to natural ones, their applications are restricted due to potential risks related to health.Therefore,new interest has been devel-oped to search natural and safe antioxidative agents from natural sources.
Bioactive peptides released by enzymatic proteolysis of various proteins that act as potential physiological modula-tors of metabolism during intestinal digestion are reported in recent reports.These peptides usually contain3–20 amino acid residues,and their activity is depend on their amino acid composition and sequence(Pihlanto-Leppala, 2001).Based on their structural,compositional and sequential properties,they may exhibit different kinds of bioactivities such as antioxidative(Jung et al.,2005;Kim et al.,2001;Rajapakse et al.,2005),antihypertensive(Sue-tsuna,1998;Suetsuna et al.,2004)and immunomodulatory effects(Chen et al.,1995;Tsuruki et al.,2003).
Bullfrog of the genus Rana is belong to Ranidae,an extensive group of amphibians that have proved to be a particularly rich source of peptides.Amphibian skin pep-tides have been the subject of intense research interest for many years in both academic and pharmaceutical groups due to their potential applications in biophysical research, biochemical taxonomy to develop new Pharmaceuticals (Clarke,1997).Here,we report the purification and charac-terization of antioxidative peptide(APBSP)derived from an enzymatic hydrolysate of bullfrog skin protein and assessment of its antioxidant properties base on inhibition of linoleic acid peroxidation and free radical scavenging using electron spin-trapping technique.
2.Methods
2.1.Materials
Bullfrogs(Rana catesbeiana Shaw)were collected from ponds in Yangsan,Korea.The skin were rapidly separated from bullfrog and rinsed with deionized water to eliminate contaminants underÀ4°C,and then stored atÀ20°C until use.a-Chymotrypsin,papain,pepsin and trypsin were pur-chased from Sigma Chemical Co.,USA.alcalase and neu-trase,were purchased from Novo Co.,Denmark.Linoleic acid,ammonium thiocyanate,a-tocopherol,and radical-testing chemicals,including1,1-diphenyl-2-pycryl-hydrazyl (DPPH),5,5-dimethyl-1-pyrroline-N-oxide(DMPO), FeSO4,H2O2,2,2-azobis-(2-amidinopropane)-hydrochlo-ride(AAPH)and a-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN)were purchased from Sigma Chemical Co.(St. Louis,MO,USA).MRC-5(ATCC CCL-171)was obtained from American Type Culture Collection(USA). Cell culture medium and all the other materials required for culturing were obtained from Gibco(Grand Island, NY).All other reagents were of the highest grade available commercially.
2.2.Preparation of bullfrog skin hydrolysates
To extract antioxidant peptide from bullfrog skin,enzy-matic hydrolysis was performed using various enzymes (alcalase,a-chymotrypsin,neutrase,papain,pepsin,and trypsin)with their optimal conditions(Table1).At enzyme/substrate ratio of1/100(w/w),1%substrate and enzyme were mixed.The mixture was incubated for8h at each optimal temperature with stirring and then heated in a boiling water bath for10min to inactivate the enzyme. Degree of hydrolysis was determined by measuring the nitrogen content soluble in10%trichloroacetic acid as dis-cussed by Kim et al.(2001)and lyophilized hydrolysates were stored underÀ80°C until use.
2.3.Lipid peroxidation inhibition assay
The antioxidative activity was measured in a linoleic acid model system according to the methods of Osawa and Namiki(1985).Briefly,a sample(1.3mg)was dis-solved in10ml of50mM phosphate buffer(pH7.0),and added to a solution of0.13ml of linoleic acid and10ml of99.5%ethanol.Then the total volume was adjusted to 25ml with distilled water.The mixture was incubated in a conicalflask with a screw cap at40±1°C in a dark room and the degree of oxidation was evaluated by measuring the ferric thiocyanate values.The ferric thiocyanate value was measured according to the method of Mitsuta et al. (1996).The reaction solution(100l l)incubated in the lin-oleic acid model system was mixed with4.7ml of75%eth-anol,0.1ml of30%ammonium thiocyanate,and0.1ml of 2·10À2M ferrous chloride solution in3.5%HCl.After 3min,the thiocyanate value was measured by reading the absorbance at500nm following color development with FeCl2and thiocyanate at different intervals during the incubation period at40±1°C.
Table1
Conditions for the hydrolysis of bullfrog skin protein
Enzyme Buffer pH Temperature(°C) Alcalase0.1M Na2HPO4–NaH2PO47.050
a-Chymotrypsin0.1M Na2HPO4–NaH2PO48.037
Papain0.1M Na2HPO4–NaH2PO4 6.037
Pepsin0.1M Glycine–HCl 2.037
Neutrase0.1M Na2HPO4–NaH2PO48.050
Trypsin0.1M Na2HPO4–NaH2PO48.037
Z.-J.Qian et al./Bioresource Technology99(2008)1690–16981691
2.4.Assays of electron spin resonance(ESR)spectrometer 2.4.1.Scavenging effect on DPPH radical
DPPH radical scavenging activity was measured using the method described by Nanjo et al.(1995).A30l l pep-tide solution(or ethanol itself as control)was added to 30l l of DPPH(60l M)in ethanol solution.After mixing vigorously for10s,the solution was then transferred into a100l l quartz capillary tube,and the scavenging activity of peptide on DPPH radical was measured using a JES-FA ESR spectrometer(JEOL Ltd.,Tokyo,Japan).The spin adduct was measured on an ESR spectrometer exactly 2min later.Experimental conditions as follows:magnetic field,336.5±5mT;power,5mW;modulation frequency, 9.41GHz;amplitude,1·1000;sweep time,30s.DPPH radical scavenging ability was calculated following equa-tion in which H and H0were relative peak height of radical signals with and without sample,respectively.
Radical scavenging activity¼
1ÀH
H0
Â100%
2.4.2.Hydroxyl radicals scavenging activity
Hydroxyl radicals were generated by iron-catalyzed Fenton Haber–Weiss reaction and the generated hydroxyl radicals rapidly reacted with nitrone spin trap DMPO (Rosen and Rauckman,1984).The resultant DMPO-OH adducts was detectable with an ESR spectrometer.The peptide solution(20l l)was mixed with DMPO(0.3M, 20l l),FeSO4(10mM,20l l)and H2O2(10mM,20l l)in a phosphate buffer solution(pH7.4),and then transferred into a100l l quartz capillary tube.After2.5min,the ESR spectrum was recorded using an ESR spectrometer.Exper-imental conditions as follows:magneticfield, 336.5±5mT;power,1mW;modulation frequency, 9.41GHz;amplitude,1·200;sweep time,4min.Hydroxyl radical scavenging ability was calculated following equa-tion in which H and H0were relative peak height of radical signals with and without sample,respectively.
Radical scavenging activity¼
1ÀH
H0
Â100%
2.4.
3.Superoxide anion radical scavenging activity
Superoxide anion radicals were generated by UV irradi-ated riboflavin/EDTA system(Guo et al.,1999).The reac-tion mixture containing0.3mM riboflavin, 1.6mM EDTA,800mM DMPO and indicated concentration of peptide fraction was irradiated for1min under UV lamp at365nm.The reaction mixture was transferred to100l l quartz capillary tube of the ESR spectrometer for measure-ment.The experimental conditions were as follows:mag-neticfield,336.5±5mT;power,10mW;modulation frequency,9.41GHz;amplitude,1·1000;sweep time, 1min.Superoxide radical scavenging ability was calculated following equation in which H and H0were relative peak height of radical signals with and without sample, respectively.
Radical scavenging activity¼
1ÀH
H0
Â100%
2.4.4.Peroxyl radicals scavenging activity
Alkyl radicals were generated according to the method of Hiramoto et al.(1993).Briefly,20l l of40mM2,20-azo-bis(2-amidinopropane)dihydrochloride(AAPH)was mixed with20l l of phosphate buffered-saline(PBS), 20l l of40mM a-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)and20l l of peptide solution.The mixture was vortexed and incubated at37°C for30min.Subsequently, reaction mixture was transferred to a sealed capillary tube and spin adduct was recorded with controlled spectromet-ric conditions;modulation frequency,100kHz;microwave power,10mW;microwave frequency,9441MHz;magnetic field,336.5±5mT and sweep time,30s.Peroxyl radical scavenging ability was calculated following equation in which H and H0were relative peak height of radical signals with and without sample,respectively.
Radical scavenging activity¼
1ÀH
H0
Â100%
2.5.Purification of antioxidant peptide
2.5.1.Ion exchange chromatography
The lyophilized bullfrog skin protein(20mg/ml)was dissolved in20mM sodium acetate buffer(pH4.0),and loaded onto a Hiprep16/10DEAE FF anion exchange col-umn(16·100mm)equilibrated with20mM sodium ace-tate buffer(pH4.0),and eluted with a linear gradient of NaCl(0–2.0M)in the same buffer at aflow rate of 2.4ml/min.Each fraction was monitored at280nm,col-lected at a volume of4ml and concentrated using a rotary evaporator;antioxidant activity was also investigated.A strong antioxidant fraction was lyophilized,and chroma-tography was used as the next step.
2.5.2.Gelfiltration chromatography
The lyophilized fraction was further purified on Super-dex Peptide10/300GL gelfiltration column (10·300mm)equilibrated with distilled water.The col-umn was eluted with distilled water,and4ml of fractions was collected at aflow rate of0.8ml/min.The fractions were detected at280nm and antioxidant activity was also investigated.A strong antioxidant fraction was lyophilized, and chromatography was used as the next step.
2.5.
3.High-performance liquid chromatography(HPLC)
The fraction exhibiting antioxidative activity was fur-ther purified using reversed-phase high-performance liquid chromatography(RP-HPLC)on a Primesphere10C18 (20·250mm)column with a linear gradient of acetonitrile (0–35%in30min)containing0.1%trifluoroacetic acid
1692Z.-J.Qian et al./Bioresource Technology99(2008)1690–1698
(TFA)at aflow rate of1.0ml/min.Elution peaks were detected at215nm,and active peak was concentrated using a rotary evaporator.Potent peaks were collected,evaluated antioxidant activity,and then lyophilized.The active frac-tion from analytical column was further applied onto a Synchropak RPP-100analytical column with a linear gra-dient of acetonitrile(20%v/v,in15min)containing0.1% TFA atflow rate of1ml/min.Thefinally purified peptide was analyzed amino acid sequence.
2.6.Determination of amino acid sequence
Accurate molecular mass and amino acid sequence of the purified peptide were determined with a Q-TOF mass spectrometer(Micromass,Altrincham,UK)coupled with an electrospray ionization(ESI)source.The purified pep-tide was separately infused into the electrospray source fol-lowing dissolution in methanol/water(1:1,vol/vol),and molecular mass was determined by a doubly charged (M+2H)+2state in the mass spectrum.Following molecu-lar mass determination,the peptide was automatically selected for fragmentation,and sequence information was obtained by tandem mass spectroscopy(MS)analysis. 2.7.Cytotoxicity assay in vitro
To determine the effect of purified peptide on the viabil-ity of normal human lung cells,the colorimetric MTT assay was performed(Hansen et al.,1989).Normal human lungfibroblast cells,MRC-5were seeded at1.3·104cell/ well in96-well microliter plates in DMEM medium con-taining10%FBS for human lung(MRC-5)cell.After 24h of incubation in a humidified5%(v/v)CO2/air envi-ronment at37°C,20l l of MTT dye solution was added to each well.After4h incubation,200l l of solubiliza-tion/stop solution was added to dissolve the formazan crys-tals and incubated the mixture at37°C overnight.The absorbance was read using Genious Multifunction micro-plate reader(Tecan,UK)at540nm.
2.8.Statistical analysis
Results are presented as mean±standard error of the mean(n=3).Student’s t-test was used to determine the level of significance(P<0.05).
3.Results and discussion
3.1.Preparation of bullfrog skin protein hydrolysates and their antioxidant properties
In the present study,bullfrog skin protein was sepa-rately hydrolyzed by alcalase,a-chymotrypsin,neutrase, papain,pepsin,and trypsin,for the extraction of antioxi-dant peptides.The extent of protein degradation by prote-olytic enzymes was estimated by assessing the degree hydrolysis(DH)and it was observed to be58.7%,65.4%and73.9%for alcalase,trypsin and pepsin respectively. Other proteolytic enzymes showed lower DH than50% (Fig.1).The antioxidant activities of the hydrolysates were evaluated using lipid peroxidation inhibition assay and free radical scavenging activity by ESR spin-trapping tech-nique.As shown in Fig.2,the oxidation of linoleic acid was markedly inhibited by hydrolysates derived from bull-frog skin protein with various proteases.Among the hydro-lysate resulting from various enzymes,the highest antioxidative activity was observed in the alcalase hydroly-sate,with exhibited about96.1%inhibition of linoleic acid peroxidation.Other hydrolysates showed lipid peroxida-tion inhibition lower than that of alcalase and a-tocoph-erol.In addition,the antioxidant activity of a substance can be identified more accurately by assessing scavenging activities on free radicals that generate in oxidative sys-tems.The hydrolysates were tested for their free radical scavenging effects on DPPH,hydroxyl,superoxide and per-oxyl radicals,using ESR spin-trapping technique.As
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shown in Table2,scavenging of hydroxyl radicals was more effective than that of DPPH,peroxyl and superoxide species and alcalase hydrolysate was the most potential compared with those of other hydrolysates.However,the scavenging effect of trypsin hydrolysate(73.2%)and pep-tide hydrolysate(48.9%)on hydroxyl and superoxide radi-cals were stronger than that of alcalase hydrolysate(63.8% and45.6%,respectively).But in most cases,alcalase hydro-lysate exposes higher antioxidative activities than other hydrolysates on four radicals.Therefore,alcalase-proteo-lytic hydrolysate was selected for further study.
In the antioxidant assay(Fig.2and Table2),alcalase-proteolytic hydrolysate showed high activities among the other hydrolysates.Many previous reports have come up with thefinding that alcalase is capable of producing bioac-tive peptides when it is incorporated to hydrolyze natural proteins(Park et al.,2001;Li et al.,2006;Heo et al., 2005).In particular,alcalase has been used in the past
Table2
Free radical scavenging effects of various hydrolysates
Hydrolysate Free radical scavenging effects(%)
DPPH radical Hydroxyl radical Superoxide radical Peroxyl radical Alcalase56.3±2.3263.8±1.7845.6±1.7258.4±1.26 Neutrase30.5±1.6442.3±2.5632.8±1.9647.6±2.25
a-Chymotrypsin25.6±1.8243.0±1.3226.8±1.6538.4±2.37 Trypsin35.8±2.5173.2±2.0520.7±2.3850.2±1.78 Papain18.6±1.9834.5±1.6413.5±2.6224.8±1.45 Pepsin43.2±1.6547.6±2.3848.9±2.5657.8±1.36 Scavenging effects were tested at a concentration of1.5mg/ml.Values are means±SD of three determinations.
1694Z.-J.Qian et al./Bioresource Technology99(2008)1690–1698
for the production of antioxidant peptide(Park et al., 2001).When compared with other specific(trypsin,chymo-trypsin)and non-specific(pronase,neutrase)proteases,it affords higher yields in the production of antioxidant pep-tides.In addition,bioactive peptides produced by alcalase are resistant to digestive enzymes such as pepsin,trypsin and chymotrypsin,which would allow for absorption of peptides contained in this sort of hydrolysate(Kim et al., 2001;Park et al.,2001).Moreover,related studies clearly show that alcalase produce shorter peptide sequences as well as terminal amino acid sequences responsible for var-ious bioactivities including antioxidant activity.
3.2.Isolation of antioxidant peptide
The lyophilized bullfrog skin protein hydrolysate by alcalase was dissolved in20mM sodium acetate buffer (pH4.0),and loaded onto a Hiprep16/10DEAE FF col-umn using fast protein liquid chromatography(FPLC) with a linear gradient of NaCl(0–2.0M).Elution peaks were monitored at280nm,and each fraction was collected as4ml and fractionated into four portions(Fig.3a).Each fraction was pooled,lyophilized,and measured for antiox-idative activity in linoleic acid emulsion system and radical scavenging activity.Fractions C exhibited higher antioxi-dative properties to inhibit lipid peroxidation(80.15%)in linoleic acid emulsion system and exhibited substantial scavenging potencies on DPPH radicals,hydroxyl,super-oxide and peroxyl radicals,respectively(Fig.4a).The lyophilized active fraction C(Fig.3a)was further subjected to gel permeation chromatography on a Superdex Peptide 10/300GL gel permeation FPLC column equilibrated with the distilled water and fractionated into three portions (data not shown).The fractions were pooled and lyophi-lized.Among all fractions collected,fraction C-2exhibited the strongest antioxidative activity in linoleic acid emulsion system and radical scavenging activity(Fig.4b).This active fraction was further separated by RP-HPLC on a Prime-sphere10C18(20mm·250mm)column with a linear gradient of acetonitrile(0–35%)containing0.1%trifluoro-acetic acid(TFA),and three main fractions are obtained (Fig.3b).Fraction C-2b showed the most potent antioxida-tive activity in linoleic acid emulsion system as well as rad-ical scavenging activity(Fig.4c).In order to obtain a purified peptide we rechromatographed on a Synchropak RPP-100(10mm·250mm)reversed phase analytical col-
Z.-J.Qian et al./Bioresource Technology99(2008)1690–16981695
umn using a15%acetonitrile concentration containing 0.1%TFA(Fig.3c).Finally,we obtained a purified bull-frog skin protein peptide.The amino acid sequence of the purified peptide having a molecular mass of1487Da was determined to be Leu-Glu-Glu-Leu-Glu-Glu-Glu-Leu-Glu-Gly-Cys-Glu(Fig.5).The molecular mass of the puri-fied peptide determined by ESI/MS spectroscopy was in excellent agreement with theoretical mass calculated from the sequence.
3.3.Antioxidant activities of purified peptide(APBSP)
To obtain a sufficient amount of purified peptide,chro-matographic separations were performed for several times, and its antioxidant activity was investigated using both free radical scavenging effects and lipid peroxidation inhibition assay.
The direct free radical scavenging effects of APBSP were investigated using the ESR spin-trapping technique.DPPH is a stable free radical and accepts an electron or a hydro-gen radical to become a stable diamagnetic molecule. Therefore,DPPH is often used as a substrate to evaluate the antioxidant activity of an antioxidant.Hydroxyl radi-cals were generated in a Fenton reaction and were visual-ized by an ESR spectrometer.The ESR signal is inhibited by the presence of OH scavengers,which compete with DMPO for OH.Superoxide radicals were generated by UV irradiation of a riboflavin/EDTA solution.AAPH can decompose to form Peroxyl radicals that can react swiftly with O2to yield peroxyl radicals to stimulate lipid peroxidation(Halliwell and Gutteridge,1999).As shown in Fig.4c,purified peptide effectively quenched four differ-ent radical sources,and IC50values of APBSP against DPPH,hydroxyl,superoxide and peroxyl radicals.APBSP was effectively quenched in the order of DPPH,hydroxyl, superoxide and peroxyl radical,and IC50values were 16.1,12.8,34.0and32.6l M,respectively.To access lipid peroxidation inhibitory activity,a well known PUFA,lin-oleic acid was incubated to auto-oxidize in a water/ethanol emulsion in a dark room at40±1°C.In this model sys-tem,peroxyl(ROOÅ)and alkoxyl(ROÅ)radicals,derived from the pre-existing lipid peroxide,were employed directly to initiate lipid peroxidation in the emulsified lino-leic acid system(Cheng et al.,2003).As shown in Fig.6, APBSP effectively inhibited lipid peroxidation in linoleic acid emulsion system up to the7days,and the activity was similar to that of a-tocopherol.Cheng et al.(2003) reported that phenolic compounds afforded their protective actions in lipid peroxidation by scavenging the lipid-derived radicals(RÅ,ROÅor ROOÅ)to stop the chain reac-tions in a heterogeneous lipid phase.In another study, Tong et al.(2000)revealed that high molecular weight frac-tion of whey protein was able to inhibit lipid peroxidation via scavenging of free radicals.To exert lipid peroxidation inhibitory activity in this system,the hydrophobic property of APBSP sequence may have played an important role exerting high affinity to linoleic acid.
Free radicals with major species of ROS are unstable and react readily with other groups or substances in the body,resulting in cell damage and,thus,human disease
1696Z.-J.Qian et al./Bioresource Technology99(2008)1690–1698
(Halliwell and Gutteridge,1989).Therefore,removal of free radicals and ROS is probably one of the most effective defenses of a living body against various diseases.The ben-eficial effects of antioxidants are preventing oxidative dam-age by interrupting the radical chain reaction of lipid peroxidation(Halliwell and Gutteridge,1999).It is gener-ally considered that the inhibition of lipid peroxidation by an antioxidant may be due to free radical scavenging activ-ity.Bioactive peptides usually contain2–20amino acid res-idues per molecule,(Pihlanto-Leppala,2001)and the lower their molecular weight,the higher is their chance to cross the intestinal barrier and exert a biological effect(Roberts et al.,1999).Previous work on antioxidative peptides has shown that peptides with5–16amino acid residues could inhibit auto-oxidation of linoleic acid(Chen et al.,1995). Lipid peroxidation is thought to proceed via radical-medi-ated abstraction of hydrogen atoms from methylene car-bons in polyunsaturated fatty acids(PUFAs)(Rajapakse et al.,2005).Antioxidant peptides derived from different sources have exhibited varying potencies to scavenge free radicals.But,the exact mechanism of scavenging these rad-icals is not clearly understood.In the free radical-mediated lipid peroxidation system,antioxidative activity of peptide or protein is dependent on molecular size and chemical properties such as hydrophobicity and electron transferring ability of amino acid residues in the sequence.Alanine, valine,leucine,praline with non-polar aliphatic groups have high reactivity to hydrophobic PUFAs,and tyrosine,histi-dine,tryptophan,and phenylalanine with aromatic residues can make reactive oxygen species(ROS)stable through direct electron transfer.Moreover hydrogen donors such as,glycine,aspartic acid,glutamate and tyrosine are able to quench unpaired electrons or radicals by supporting protons.The potent antioxidant peptide(APBSP)as the sequence of Leu-Glu-Glu-Leu-Glu-Glu-Glu-Leu-Glu-Gly-Cys-Glu(M w=1487Da)(Fig.5).The structure was com-posed of one cysteine residue,seven acidic residues(Glu)and non-polar residues(Gly,Leu).Further,in the sequence of APBSP has hydrophobic amino acid such leucine,and cysteine.Cysteine residues are independently important for antioxidant action,since they can directly interact with radicals.As reported by Harman et al.(1984),the thiol group of cysteine serves a very important role in protecting cells and cellular biomolecules from oxidative stress.The cytotoxic effect of APBSP was evaluated on human lung fibroblast cell line,and the results showed that APBSP did not show any cytotoxic effects on MRC-5cell(data not shown).
Based on these results,it is suggested that the low molec-ular weight peptide released from bullfrog skin by enzy-matic hydrolysis has potent antioxidant properties.The purified antioxidant peptide also was a potent free radical scavenger and effectively inhibited lipid peroxidation, which would be expected to protect against oxidative dam-age in living systems in relation to aging and carcinogene-sis.Therefore,it can be suggested that bullfrog skin presents a potential nutraceutical and bioactive material. However,further detailed studies on APBSP in regard of antioxidant activities in vivo are needed. Acknowledgements
This research was supported by a grant(p-2004-01) from the Marine Bioprocess Research Center of the Mar-ine Bio21Center funded by the Ministry of Maritime Af-fairs and Fisheries,Republic of Korea.
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