辣木籽抗氧化肽的制备及其对氧化损伤红细胞的保护作用
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摘要
采用酶法制备辣木籽水解物,以抗氧化活性为跟踪指标,通过乙醇分级沉淀和大孔树脂柱层析法定向制备辣木籽抗氧化肽,构建模拟胃、肠道消化模型,评价辣木籽抗氧化肽胃、肠道消化物的氧自由基吸收能力(oxygen radical absorbance capacity,ORAC),利用超高效液相色谱-四极杆-高分辨飞行时间串联质谱技术解析消化物中多肽组成与结构,建立2,2’-偶氮二异丁脒盐酸盐(2,2’-azobis(2-methylpropionamidine) dihydrochloride,AAPH)诱导大鼠红细胞氧化损伤模型,研究经胃、肠道消化后的辣木籽抗氧化肽对氧化损伤红细胞溶血的抑制作用。结果表明:采用20%(体积分数,下同)、40%乙醇分级沉淀辣木籽水解物去除杂质,取60%乙醇分级沉淀组分进行柱层析分离纯化,经水、20%乙醇、40%乙醇洗脱除杂,采用60%乙醇洗脱,定向制备得到富含抗氧化氨基酸、疏水性氨基酸的辣木籽抗氧化肽(蛋白质量分数92.84%,1,1-二苯基-2-三硝基苯肼自由基清除能力、ORAC分别为39.95、1 454.57μmol/g);辣木籽抗氧化肽经模拟胃、肠道消化后,抗氧化活性增强,释放出更多短肽段,包括11条二肽、5条三肽;辣木籽抗氧化肽消化物、Gln-Met、Leu-Phe通过清除胞内活性氧,下调丙二醛含量与超氧化物歧化酶活力,拮抗AAPH所致氧化应激,有效抑制氧化损伤红细胞发生溶血。
An antioxidant peptide was puri?ed from enzymatic hydrolysate of Moringa oleifera seed proteins by antioxidant activity-guided separation using fractional precipitation with 20%, 40% and 60% ethanol and macroporous resin column chromatography. Simulated gastrointestinal digest of the antioxidant peptide was evaluated for oxygen radical absorbance capacity(ORAC). The peptide pro?le and structure of the digest was identi?ed by ultra performance liquid chromatographyquadrupole time of ?ight-tandem mass spectrometry(UPLC-QTOF-MS/MS). The anti-hemolysis activity and protective effect of the digest against 2,2'-azobis(2-methylpropionamidine) dihydrochloride(AAPH)-induced oxidative damage in erythrocytes were studied. The 60% ethanol precipitate was harvested and purified using column chromatography by sequential elution with pure water, 20%, 40% and 60% ethanol, yielding an antioxidant peptide rich in antioxidant and hydrophobic amino acids(which contained 92.84% protein and exhibited 1,1-diphenyl-2-picrylhydrazyl scavenging capacity and ORAC value of 39.95 and 1 454.57 μmol/g, respectively). The antioxidant activity of the peptide was enhanced after gastrointestinal digestion, and short-chain peptides including 11 dipeptides and 5 tripeptides were released. The resulting digest, Gln-Met and Leu-Phe could effectively inhibit oxidative stress-induced hemolysis through scavenging intracellular reactive oxygen species(ROS), down-regulating malondialdehyde(MDA) level and superoxide dismutase(SOD) activity,and inhibiting AAPH-induced oxidative stress.
An antioxidant peptide was puri?ed from enzymatic hydrolysate of Moringa oleifera seed proteins by antioxidant activity-guided separation using fractional precipitation with 20%, 40% and 60% ethanol and macroporous resin column chromatography. Simulated gastrointestinal digest of the antioxidant peptide was evaluated for oxygen radical absorbance capacity(ORAC). The peptide pro?le and structure of the digest was identi?ed by ultra performance liquid chromatographyquadrupole time of ?ight-tandem mass spectrometry(UPLC-QTOF-MS/MS). The anti-hemolysis activity and protective effect of the digest against 2,2'-azobis(2-methylpropionamidine) dihydrochloride(AAPH)-induced oxidative damage in erythrocytes were studied. The 60% ethanol precipitate was harvested and purified using column chromatography by sequential elution with pure water, 20%, 40% and 60% ethanol, yielding an antioxidant peptide rich in antioxidant and hydrophobic amino acids(which contained 92.84% protein and exhibited 1,1-diphenyl-2-picrylhydrazyl scavenging capacity and ORAC value of 39.95 and 1 454.57 μmol/g, respectively). The antioxidant activity of the peptide was enhanced after gastrointestinal digestion, and short-chain peptides including 11 dipeptides and 5 tripeptides were released. The resulting digest, Gln-Met and Leu-Phe could effectively inhibit oxidative stress-induced hemolysis through scavenging intracellular reactive oxygen species(ROS), down-regulating malondialdehyde(MDA) level and superoxide dismutase(SOD) activity,and inhibiting AAPH-induced oxidative stress.
引文
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[9]OLIVEIRA J T A,SILVEIRA S B,VASCONCELOS I M,et al.Compositional and nutritional attributes of seeds from the multiple purpose tree Moringa oleifera Lamarck[J].Journal of the Science of Food and Agriculture,1999,79(6):815-820.DOI:10.1002/(sici)1097-0010(19990501)79:6<815::aid-jsfa290>3.3.co;2-g.
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[13]ZHAO M M,ZHU D S,SUN-WATERHOUSE D,et al.In vitro and in vivo studies on adlay-derived seed extracts:phenolic profiles,antioxidant activities,serum uric acid suppression,and xanthine oxidase inhibitory effects[J].Journal of Agricultural and Food Chemistry,2014,62(31):7771-7778.DOI:10.1021/jf501952e.
[14]ADEYEYE E I.Amino acid composition of three species of Nigerian fish:Clarias anguillaris,Oreochromis niloticus and Cynoglossus senegalensis[J].Food Chemistry,2009,113(1):43-46.DOI:10.1016/j.foodchem.2008.07.007.
[15]YOU L J,ZHAO M M,REGENSTEIN J M,et al.Changes in the antioxidant activity of loach(Misgurnus anguillicaudatus)protein hydrolysates during a simulated gastrointestinal digestion[J].F o o d C h e m i s t r y,2 0 1 0,1 2 0(3):8 1 0-8 1 6.D O I:1 0.1 0 1 6/j.foodchem.2009.11.018.
[16]ZHAO M M,YANG Q Y,LIN L Z,et al.Intracellular antioxidant activities of selected cereal phenolic extracts and mechanisms underlying the protective effects of adlay phenolic extracts on H2O2-induced oxidative stress in human erythrocytes[J].Journal of Functional Foods,2017,31:160-171.DOI:10.1016/j.jff.2017.01.046.
[17]WIDYARANI R,BOWDEN N A,KOLFSCHOTEN R C,et al.Fractional precipitation of amino acids from agro-industrial residues using ethanol[J].Industrial&Engineering Chemistry Research,2016,55(27):7462-7472.DOI:10.1021/acs.iecr.6b00054.
[18]LIU Y F,LIU J X,CHEN X F,et al.Preparative separation and purification of lycopene from tomato skins extracts by macroporous adsorption resins[J].Food Chemistry,2010,123(4):1027-1034.DOI:10.1016/j.foodchem.2010.05.055.
[19]FU B Q,LIU J,LI H,et al.The application of macroporous resins in the separation of licorice flavonoids and glycyrrhizic acid[J].Journal of Chromatography A,2005,1089(1/2):18-24.DOI:10.1016/j.chroma.2005.06.051.
[20]ZHUANG M Z,ZHAO M M,LIN L Z,et al.Macroporous resin purification of peptides with umami taste from soy sauce[J].Food Chemistry,2016,190:338-344.DOI:10.1016/j.foodchem.2015.05.105.
[21]CHALAMAIAH M,KUMAR B D,HEMALATHA R,et al.Fish protein hydrolysates:proximate composition,amino acid composition,antioxidant activities and applications:a review[J].Food Chemistry,2012,135(4):3020-3038.DOI:10.1016/j.foodchem.2012.06.100.
[22]ZHENG L,DONG H Z,SU G W,et al.Radical scavenging activities of Tyr-,Trp-,Cys-and Met-Gly and their protective effects against AAPH-induced oxidative damage in human erythrocytes[J].Food Chemistry,2016,197(Pt A):807-813.DOI:10.1016/j.foodchem.2015.11.012.
[23]WU R B,WU C L,LIU D,et al.Overview of antioxidant peptides derived from marine resources:the sources,characteristic,purification,and evaluation methods[J].Applied Biochemistry and Biotechnology,2015,176(7):1815-1833.DOI:10.1007/s12010-015-1689-9.
[24]BERENGUEL O,DE PESS?A G S,ARRUDA M A Z.Total content and in vitro bioaccessibility of tellurium in Brazil nuts[J].Journal of Trace Elements in Medicine and Biology,2018,48:46-51.DOI:10.1016/j.jtemb.2018.02.026.
[25]INTAWONGSE M,DEAN J R.In-vitro testing for assessing oral bioaccessibility of trace metals in soil and food samples[J].TrAC-Trends in Analytical Chemistry,2006,25(9):876-886.DOI:10.1016/j.trac.2006.03.010.
[26]CAMELO-MéNDEZ G A,AGAMA-ACEVEDO E,ROSELL CM,et al.Starch and antioxidant compound release during in vitro gastrointestinal digestion of gluten-free pasta[J].Food Chemistry,2018,263:201-207.DOI:10.1016/j.foodchem.2018.04.075.
[27]HERNáNDEZ-LEDESMA B,DEL MAR CONTRERAS M,RECIO I.Antihypertensive peptides:production,bioavailability and incorporation into foods[J].Advances in Colloid and Interface Science,2011,165(1):23-35.DOI:10.1016/j.cis.2010.11.001.
[28]DAI C H,ZHANG W W,HE R H,et al.Protein breakdown and release of antioxidant peptides during simulated gastrointestinal digestion and the absorption by everted intestinal SAC of rapeseed proteins[J].LWT-Food Science and Technology,2017,86:424-429.DOI:10.1016/j.lwt.2017.08.026.
[29]CIAN R E,GARZóN A G,ANCONA D B,et al.Hydrolyzates from Pyropia columbina seaweed have antiplatelet aggregation,antioxidant and ACE I inhibitory peptides which maintain bioactivity after simulated gastrointestinal digestion[J].LWT-Food Science and Technology,2015,64(2):881-888.DOI:10.1016/j.lwt.2015.06.043.
[30]庞广昌,陈庆森,胡志和,等.蛋白质的消化吸收及其功能评述[J].食品科学,2013,34(9):375-391.DOI:10.7506/spkx1002-6630-201309074.
[31]LIAO W Z,NING Z X,CHEN L Y,et al.Intracellular antioxidant detoxifying effects of diosmetin on 2,2-azobis(2-amidinopropane)dihydrochloride(AAPH)-induced oxidative stress through inhibition of reactive oxygen species generation[J].Journal of Agricultural and Food Chemistry,2014,62(34):8648-8654.DOI:10.1021/jf502359x.
[32]RISTOW M,SCHMEISSER S.Extending life span by increasing oxidative stress[J].Free Radical Biology and Medicine,2011,51(2):327-336.DOI:10.1016/j.freeradbiomed.2011.05.010.
[2]BRILHANTE R S N,SALES J A,PEREIRA V S,et al.Research advances on the multiple uses of Moringa oleifera:a sustainable alternative for socially neglected population[J].Asian Pacific Journal of Tropical Medicine,2017,10(7):621-630.DOI:10.1016/j.apjtm.2017.07.002.
[3]YUSOFF M M,GORDON M H,EZEH O,et al.High pressure pretreatment of Moringa oleifera seed kernels prior to aqueous enzymatic oil extraction[J].Innovative Food Science&Emerging Technologies,2017,39:129-136.DOI:10.1016/j.ifset.2016.11.014.
[4]RAI A,MOHANTY B,BHARGAVA R.Experimental modeling and simulation of supercritical fluid extraction of Moringa oleifera seed oil by carbon dioxide[J].Chemical Engineering Communications,2017,204(8):957-964.DOI:10.1080/00986445.2017.1328415.
[5]BHUTADA P R,JADHAV A J,PINJARI D V,et al.Solvent assisted extraction of oil from Moringa oleifera Lam.seeds[J].Industrial Crops and Products,2016,82:74-80.DOI:10.1016/j.indcrop.2015.12.004.
[6]MAT YUSOFF M,GORDON M H,EZEH O,et al.Aqueous enzymatic extraction of Moringa oleifera oil[J].Food Chemistry,2016,211:400-408.DOI:10.1016/j.foodchem.2016.05.050.
[7]MUNE M A M,NYOBE E C,BASSOGOG C B,et al.A comparison on the nutritional quality of proteins from Moringa oleifera leaves and seeds[J].Cogent Food&Agriculture,2016,2(1):1-8.DOI:10.1080/23311932.2016.1213618.
[8]IJAROTIMI O S,ADEOTI O A,ARIYO O.Comparative study on nutrient composition,phytochemical,and functional characteristics of raw,germinated,and fermented Moringa oleifera seed flour[J].Food Science&Nutrition,2013,1(6):452-463.DOI:10.1002/fsn3.70.
[9]OLIVEIRA J T A,SILVEIRA S B,VASCONCELOS I M,et al.Compositional and nutritional attributes of seeds from the multiple purpose tree Moringa oleifera Lamarck[J].Journal of the Science of Food and Agriculture,1999,79(6):815-820.DOI:10.1002/(sici)1097-0010(19990501)79:6<815::aid-jsfa290>3.3.co;2-g.
[10]ABEYRATHNE E D N S,HUANG X,AHN D U.Antioxidant,angiotensin-converting enzyme inhibitory activity and other functional properties of egg white proteins and their derived peptides:a review[J].Poultry Science,2018,97(4):1462-1468.DOI:10.3382/ps/pex399.
[11]TAN B L,NORHAIZAN M E,LIEW W,et al.Nutrients and oxidative stress:friend or foe?[J].Oxidative Medicine and Cellular Longevity,2018,2018:1-24.DOI:10.1155/2018/9719584.
[12]LIN L Z,ZHAO H F,DONG Y,et al.Macroporous resin purification behavior of phenolics and rosmarinic acid from Rabdosia serra(MAXIM.)HARA leaf[J].Food Chemistry,2012,130(2):417-424.DOI:10.1016/j.foodchem.2011.07.069.
[13]ZHAO M M,ZHU D S,SUN-WATERHOUSE D,et al.In vitro and in vivo studies on adlay-derived seed extracts:phenolic profiles,antioxidant activities,serum uric acid suppression,and xanthine oxidase inhibitory effects[J].Journal of Agricultural and Food Chemistry,2014,62(31):7771-7778.DOI:10.1021/jf501952e.
[14]ADEYEYE E I.Amino acid composition of three species of Nigerian fish:Clarias anguillaris,Oreochromis niloticus and Cynoglossus senegalensis[J].Food Chemistry,2009,113(1):43-46.DOI:10.1016/j.foodchem.2008.07.007.
[15]YOU L J,ZHAO M M,REGENSTEIN J M,et al.Changes in the antioxidant activity of loach(Misgurnus anguillicaudatus)protein hydrolysates during a simulated gastrointestinal digestion[J].F o o d C h e m i s t r y,2 0 1 0,1 2 0(3):8 1 0-8 1 6.D O I:1 0.1 0 1 6/j.foodchem.2009.11.018.
[16]ZHAO M M,YANG Q Y,LIN L Z,et al.Intracellular antioxidant activities of selected cereal phenolic extracts and mechanisms underlying the protective effects of adlay phenolic extracts on H2O2-induced oxidative stress in human erythrocytes[J].Journal of Functional Foods,2017,31:160-171.DOI:10.1016/j.jff.2017.01.046.
[17]WIDYARANI R,BOWDEN N A,KOLFSCHOTEN R C,et al.Fractional precipitation of amino acids from agro-industrial residues using ethanol[J].Industrial&Engineering Chemistry Research,2016,55(27):7462-7472.DOI:10.1021/acs.iecr.6b00054.
[18]LIU Y F,LIU J X,CHEN X F,et al.Preparative separation and purification of lycopene from tomato skins extracts by macroporous adsorption resins[J].Food Chemistry,2010,123(4):1027-1034.DOI:10.1016/j.foodchem.2010.05.055.
[19]FU B Q,LIU J,LI H,et al.The application of macroporous resins in the separation of licorice flavonoids and glycyrrhizic acid[J].Journal of Chromatography A,2005,1089(1/2):18-24.DOI:10.1016/j.chroma.2005.06.051.
[20]ZHUANG M Z,ZHAO M M,LIN L Z,et al.Macroporous resin purification of peptides with umami taste from soy sauce[J].Food Chemistry,2016,190:338-344.DOI:10.1016/j.foodchem.2015.05.105.
[21]CHALAMAIAH M,KUMAR B D,HEMALATHA R,et al.Fish protein hydrolysates:proximate composition,amino acid composition,antioxidant activities and applications:a review[J].Food Chemistry,2012,135(4):3020-3038.DOI:10.1016/j.foodchem.2012.06.100.
[22]ZHENG L,DONG H Z,SU G W,et al.Radical scavenging activities of Tyr-,Trp-,Cys-and Met-Gly and their protective effects against AAPH-induced oxidative damage in human erythrocytes[J].Food Chemistry,2016,197(Pt A):807-813.DOI:10.1016/j.foodchem.2015.11.012.
[23]WU R B,WU C L,LIU D,et al.Overview of antioxidant peptides derived from marine resources:the sources,characteristic,purification,and evaluation methods[J].Applied Biochemistry and Biotechnology,2015,176(7):1815-1833.DOI:10.1007/s12010-015-1689-9.
[24]BERENGUEL O,DE PESS?A G S,ARRUDA M A Z.Total content and in vitro bioaccessibility of tellurium in Brazil nuts[J].Journal of Trace Elements in Medicine and Biology,2018,48:46-51.DOI:10.1016/j.jtemb.2018.02.026.
[25]INTAWONGSE M,DEAN J R.In-vitro testing for assessing oral bioaccessibility of trace metals in soil and food samples[J].TrAC-Trends in Analytical Chemistry,2006,25(9):876-886.DOI:10.1016/j.trac.2006.03.010.
[26]CAMELO-MéNDEZ G A,AGAMA-ACEVEDO E,ROSELL CM,et al.Starch and antioxidant compound release during in vitro gastrointestinal digestion of gluten-free pasta[J].Food Chemistry,2018,263:201-207.DOI:10.1016/j.foodchem.2018.04.075.
[27]HERNáNDEZ-LEDESMA B,DEL MAR CONTRERAS M,RECIO I.Antihypertensive peptides:production,bioavailability and incorporation into foods[J].Advances in Colloid and Interface Science,2011,165(1):23-35.DOI:10.1016/j.cis.2010.11.001.
[28]DAI C H,ZHANG W W,HE R H,et al.Protein breakdown and release of antioxidant peptides during simulated gastrointestinal digestion and the absorption by everted intestinal SAC of rapeseed proteins[J].LWT-Food Science and Technology,2017,86:424-429.DOI:10.1016/j.lwt.2017.08.026.
[29]CIAN R E,GARZóN A G,ANCONA D B,et al.Hydrolyzates from Pyropia columbina seaweed have antiplatelet aggregation,antioxidant and ACE I inhibitory peptides which maintain bioactivity after simulated gastrointestinal digestion[J].LWT-Food Science and Technology,2015,64(2):881-888.DOI:10.1016/j.lwt.2015.06.043.
[30]庞广昌,陈庆森,胡志和,等.蛋白质的消化吸收及其功能评述[J].食品科学,2013,34(9):375-391.DOI:10.7506/spkx1002-6630-201309074.
[31]LIAO W Z,NING Z X,CHEN L Y,et al.Intracellular antioxidant detoxifying effects of diosmetin on 2,2-azobis(2-amidinopropane)dihydrochloride(AAPH)-induced oxidative stress through inhibition of reactive oxygen species generation[J].Journal of Agricultural and Food Chemistry,2014,62(34):8648-8654.DOI:10.1021/jf502359x.
[32]RISTOW M,SCHMEISSER S.Extending life span by increasing oxidative stress[J].Free Radical Biology and Medicine,2011,51(2):327-336.DOI:10.1016/j.freeradbiomed.2011.05.010.