干旱胁迫对不同品系藜麦内黄酮和抗氧化性的影响
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摘要
利用不同浓度的PEG溶液模拟干旱胁迫,分析了干旱胁迫下不同品系藜麦叶片中黄酮类化合物合成上游的苯丙氨酸解氨酶(PAL)、肉桂酸4-羟基化酶(C4H)及4-香豆酸辅酶A连接酶(4CL)此3个关键酶的活性以及种子内黄酮含量和清除DPPH·能力,探究干旱胁迫对不同藜麦种子内黄酮合成以及体外抗氧化活性的影响。结果表明:(1)随着干旱胁迫增加,三种酶活性均成先增后降趋势,但不同酶之间存在差异,PAL和4CL在5%PEG胁迫下达到最大,藜麦品系NK1、NK2和NK5的C4H活性在10%PEG时达到最大,但NK3和NK4在5%PEG胁迫下达到最大;在CK和5%PEG胁迫下藜麦品系NK3的3种酶活性较高,但在10%PEG和15%PEG胁迫下酶活性迅速降低。(2)随着干旱胁迫加剧,各品系藜麦种子内黄酮含量呈先上升后下降的趋势,在CK和5%PEG胁迫下,藜麦品系NK3种子黄酮含量最高分别为2.94 mg/g和3.61 mg/g,在10%PEG和15%PEG胁迫下,NK5种子黄酮含量最高分别是2.92 mg/g和2.66 mg/g。(3)抗氧化试验表明,各品系藜麦种子内黄酮对DPPH·的清除率在5%PEG胁迫处理时达到最大,NK1、NK2、NK3、NK4和NK5清除率分别是88.52%、87.65%、90.36%、86.53%和89.38%,其中NK3 IC50(半抑制浓度)最大为9.871μg/mL。本研究结果为理解藜麦体内黄酮合成体系和抗氧化剂资源的筛选提供理论依据。
Different con centrations of PEG solution were used to simulate drought stress. The activities of phenylalanine ammonia-lyase(PAL), cinnamate 4-hydroxylase(C4 H) and 4-coumaric acid coenzyme A ligase(4 CL) in leaves of different strains of Chenopodium triplex under drought stress were analyzed. The effects of drought stress on the content of flavonoids in different quinoa seeds were studied and their antioxidant activities in vitro were measured. The results showed that:(1) With the increase of drought stress, the activity of the three enzymes increased first and then decreased, but there were differences among different enzymes. PAL and 4 CL reached the maximum under 5% PEG stress, C4 H activity of quinoa strain NK1, NK2 and NK5 reached the maximum at 10%PEG but NK3 and NK4 reached the maximum under 5% PEG stress. The activity of the three enzymes of NK3 was higher at the concentratio ns of CK and 5% PEG but the enzyme activity was rapidly decreased at the concentrations of 10% PEG and 15% PEG.(2) With the intensification of drought stress, the content of flavonoids in all varieties of quinoa seeds first increased and then decreased. Under the stress of CK and 5% PEG, flavonoids in quinoa seeds increased firstly and then decreased. The highest flavonoids content of quinoa NK3 seeds was2.94 mg/g and 3.61 mg/g, respectively. Under 10% PEG and 15% PEG stress, the highest flavonoids content of NK5 seed was 2.92 mg/g and 2.66 mg/g, separately.(3) The antioxidant experiments showed that the scavenging rate of flavonoids from quinoa seeds to DPPH·reached the maximum in 5% PEG stress treatment, and NK1, NK2,NK3, NK4 and NK5 were 88.52%, 87.65%, 90.36%, 86.53% and 89.38%, respectively. Among them, the NK3 IC50(semi-inhibitory concentration) was 9.871 μg/mL. The results provided theoretical basis for the identification of flavonoids synthesis system and antioxidant resources in quinoa.
Different con centrations of PEG solution were used to simulate drought stress. The activities of phenylalanine ammonia-lyase(PAL), cinnamate 4-hydroxylase(C4 H) and 4-coumaric acid coenzyme A ligase(4 CL) in leaves of different strains of Chenopodium triplex under drought stress were analyzed. The effects of drought stress on the content of flavonoids in different quinoa seeds were studied and their antioxidant activities in vitro were measured. The results showed that:(1) With the increase of drought stress, the activity of the three enzymes increased first and then decreased, but there were differences among different enzymes. PAL and 4 CL reached the maximum under 5% PEG stress, C4 H activity of quinoa strain NK1, NK2 and NK5 reached the maximum at 10%PEG but NK3 and NK4 reached the maximum under 5% PEG stress. The activity of the three enzymes of NK3 was higher at the concentratio ns of CK and 5% PEG but the enzyme activity was rapidly decreased at the concentrations of 10% PEG and 15% PEG.(2) With the intensification of drought stress, the content of flavonoids in all varieties of quinoa seeds first increased and then decreased. Under the stress of CK and 5% PEG, flavonoids in quinoa seeds increased firstly and then decreased. The highest flavonoids content of quinoa NK3 seeds was2.94 mg/g and 3.61 mg/g, respectively. Under 10% PEG and 15% PEG stress, the highest flavonoids content of NK5 seed was 2.92 mg/g and 2.66 mg/g, separately.(3) The antioxidant experiments showed that the scavenging rate of flavonoids from quinoa seeds to DPPH·reached the maximum in 5% PEG stress treatment, and NK1, NK2,NK3, NK4 and NK5 were 88.52%, 87.65%, 90.36%, 86.53% and 89.38%, respectively. Among them, the NK3 IC50(semi-inhibitory concentration) was 9.871 μg/mL. The results provided theoretical basis for the identification of flavonoids synthesis system and antioxidant resources in quinoa.
引文
Aloisi I.,Parrotta L.,Ruiz K.B.,Landi C.,Bini L.,Cai G.,Biond S.,and Duca S.D.,2016,New insight into quinoa seed quality under salinity:changes in proteomic and amino acid profiles,phenolic content,and antioxidant activity of protein extracts,Front.Plant Sci.,7:656
Chen L.,Chang L.,Cao F.L.,Wang G.B.,and Dong X.W.,2013,Effects of temperature and soil water deficit on the flavonoid content and activities of enzymes involved in ginkgo leaves,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),33(4):755-762(陈雷,常丽,曹福亮,汪贵斌,董晓伟,2013,银杏叶黄酮类化合物含量及相关酶活性对温度和干旱胁迫的响应,西北植物学报,33(4):755-762)
Dong S.B.,Ning Y.P.,Yang Z.,Zhang Q.H.,Dong J.Q.,Wang F.J.,and Wang J.Z.,2015,Study on extraction process of quinoa total flavonoids and purification process of macroporous resin,Shipin Gongye Keji(Science&Technology of Food Industry),36(16):272-278(董施彬,宁亚萍,杨喆,张乔会,董洁琼,王丰俊,王建中,2015,藜麦总黄酮提取及大孔树脂纯化工艺的研究,食品工业科技,36(16):272-278)
Fan C.F.,Bi Y.,Wang Y.F.,Ren Y.L.,Yang Z.M.,and Wang Y.,2012,Effect of salicylic acid dipping on postharvest diseases and phenylpropanoid pathway in muskmelon fruits,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),45(3):584-589(范存斐,毕阳,王云飞,任亚琳,杨志敏,王毅,2012,水杨酸对厚皮甜瓜采后病害及苯丙烷代谢的影响,中国农业科学,45(3):584-589)
Fu Q.Q.,Hou P.,and Chen N.,2013,Optimization of extraction process for total flavonoids from Artemisia selengensis Turcz leaves by response surface methodology,Shipin Kexue(Food Science),34(4):94-98(扶庆权,侯佩,陈能,2013,响应面法优化芦蒿叶总黄酮的提取工艺,食品科学,34(4):94-98)
Gamel T.H.,and Abdel-Aal E.M.,2012,Phenolic acids and antioxidant properties of barley wholegrain and pearling fractions,Agr.Food Sci.,21(2):118-131
Jeyaramraja P.R.,Pius P.K.,Kumar R.R.,and Jayakumar D.,2003,Soil moisture stress induced alterations in bioconstituents determining tea quality,J.Sci.Food Agric.,83(12):1187-1191
Jin L.P.,Cui S.M.,Du J.W.,Jin C.X.,Wu Y.F.,and Qi R.G.,2009,Effects of drought stress in different ecological conditions of the Prunus mongolica Maxim almond leaves in PALand C4H activity,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),24(5):118-122(金丽萍,崔世茂,杜金伟,金彩霞,吴玉峰,其日格,2009,干旱胁迫对不同生态条件下蒙古扁桃叶片PAL和C4H活性的影响,华北农学报,24(5):118-122)
Knobloch K.H.,and Hahlbrock K.,1977,4-Coumarate:CoA ligase from cell suspension cultures of Petroselinum hortense Hoffm,partial purification,substrate specificity,and further properties,Arch.Biochem.Biophys.,184(1):237-248
Lamb C.J.,and Rubery P.H.,1975,A spectrophotometric assay for trans-cinnamic acid 4-hydroxylase activity,Analytica Biochemistry,68(2):554-561
Li G.Y.,Luo X.Y.,Sun C.S.,Yu Q.,and Chen G.L.,2017,Effects of progressive drought stress on the accumulation of flavonoids in the growth of Astragalus membranaceus var mongholicus(Bge.)Hsiao,Xibei Zhuwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(1):138-143(李光跃罗晓雅,孙窗舒,于倩,陈贵林,2017,干旱胁迫对黄芪植株生长中黄酮类成分积累的影响,西北植物学报,37(1)138-143)
Li N.N.,Pei Y.T.,Gong Y.C.,Pu Y.Y.,Liu B.M.,Xin F.G.,Liu S.H.,and Ding H.F.,2016,Research status and developmen prospect of Chenopodium quinoa Willd,Shandong Nongye Kexue(Shandong Agricultural Sciences),48(10):145-148(李娜娜,裴艳婷,宫永超,蒲艳艳,刘保民,辛富刚,刘世华,丁汉凤,2016,藜麦研究现状与发展前景,山东农业科学,48(10):145-148)
Liu Q.,Sun Y.,Zheng J.M.,Yan X.L.,Chen H.M.,and Chen JK.,2015,Formononetin sensitizes glioma cells to doxorubicin through preventing EMT via inhibition of histone deacetylase 5,Int.J.Clin.Exp.Pathol.,8(6):6434
Lu X.X.,2012,Research progress in antioxidant mechanism of flavnonids,Shipin Yanjiu Yu Kaifa(Food Research&Development),33(3):220-224(鲁晓翔,2012,黄酮类化合物抗氧化作用机制研究进展,食品研究与开发,33(3):220-224)
Ma L.,Peng L.X.,Cao J.,Zhao J.L.,and Zhao G.,2016,Determination of 3 flavonoid C-glycosides in quinoa by ultrasonic extraction and RP-HPLC,Shipin Gongye(The Food Industry),37(7):192-195(马麟,彭镰心,曹靖,赵江林,赵钢2016,超声波辅助提取RP-HPLC法测定藜麦中的3种碳苷黄酮,食品工业,37(7):192-195)
Ouyang G.C.,and Xue Y.L.,1998,Physiological role and regulation of phenylpropanoid metabolism in plant,Zhiwu Shengl Xuebao(Plant Physiology Communications),(3):11-18(欧阳光察,薛应龙,1998,植物苯丙烷类代谢的生理意义及其调控,植物生理学报,(3):11-18)
PawelP.,Henryk B.,PawelZ.,Shela G.,Maria F.,and Zofia Z.2009,Anthocyanins,total polyphenols and antioxidant activity in amaranth and quinoa seeds and sprouts during their growth,Food Chem.,115(3):994-998
Qin W.T.,Zhang J.,Wu H.J.,Sun G.Z.,Yang W.Y.,and Liu J.2016,Effects of drought stress on biosynthesis of isoflavones in soybean seedling,Yingyong Shengtai Xuebao(Chinese Journal of Applied Ecology),27(12):3927-3934(秦雯婷,张静,吴海军,孙广泽,杨文钰,刘江,2016,干旱胁迫对苗期大豆异黄酮合成的影响,应用生态学报,27(12):3927-3934)
Sun X.T.,Jiang Y.R.,Yuan J.J.,Wei S.Y.,Lu G.Q.,and Mao Q.2017,Optimization of extraction technology of flavonoids from quinoa seeds by response surface methodology and its antioxidant activities,Zhongguo Shipin Xuebao(Journal of Chinese Institute of Food Science and Technology),17(3):127-135(孙雪婷,蒋玉蓉,袁俊杰,魏述英,陆国权,毛前,2017,响应面法优化提取藜麦种子黄酮及抗氧化活性,中国食品学报,17(3):127-135)
Tang D.,He B.,Zheng Z.G.,Wang R.S.,Gu F.,and Duan T.T.,2011,Inhibitory effects of two major isoflavonoids in Radix astragali on high glucose-induced mesangial cells proliferation and AGEs-induced endothelial cells apoptosis,Planta Med.,77(7):729-732
Wang C.Y.,Chen C.T.,and Wang S.Y.,2009,Changes of flavonoid content and antioxidant capacity in blueberries after illumination with UV-C,Food Chemistry,117(3):426-431
Weisshaar B.,and Jenkins G.I.,1998,Phenylpropanoid biosynthesis and its regulation,Curr.Opin.Plant Biol.,1(3):251-257
Winkel S.B.,1999,Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways,Physiol.Plantarum,107(1):142-149
Xiao Z.C.,and Zhang G.L.,2014,Development and utilization of Chenopodium quinoa Willd.,Zhongguo Yesheng Zhiwu Ziyuan(Chinese Wild Plant Resources),33(2):62-66(肖正春,张广伦,2014,藜麦及其资源开发利用,中国野生植物资源,33(2):62-66)
Yang Y.,Zhao Y.,Ai X.,Cheng B.,and Lu S.,2014,Formononetin suppresses the proliferation of human non-small cell lung cancer through induction of cell cycle arrest and apoptosis,Int.J.Clin.Exp.Pathol.,7(12):8453-8461
Zeng W.M.,2011,Study on extraction and antioxidant activity of antioxidant active substances from oat,Thesis for M.S.,Northwest A&F University,Supervisor:Li J.X.,pp.28-34(曾王旻,2011,燕麦抗氧化活性物质提取及抗氧化活性研究,硕士学位论文,西北农林科技大学,导师:李巨秀,pp.28-34)
Zhang L.M.,Li R.C.,Hao L.M.,Tian A.Y.,Lu J.K.,and Zhang J.C.,2014,Response surface methodology for optimization of extracting total flavonoids from maca leaves and antioxidant evaluation,Xiandai Shipin Keji(Modern Food Science&Technology),30(4):233-239(张黎明,李瑞超,郝利民,田爱莹,鲁吉珂,张建春,2014,响应面优化玛咖叶总黄酮提取工艺及其抗氧化活性研究,现代食品科技,30(4):233-239)
Zhou G.,Li Y.R.,Yang L.T.,Wei X.X.,and Qiu P.L.,2009,Changes in flavonoids content and related enzyme activity of sugarcane leaves under PEG stress,Ganhan Diqu Nongye Yanjiu(Agricultural Research in the Arid Areas),27(6):185-188(周桂,李杨瑞,杨丽涛,韦雪雪,丘佩玲,2009,干旱胁迫下甘蔗叶类黄酮及相关酶活性的变化,干旱地区农业研究,27(6):185-188)
Chen L.,Chang L.,Cao F.L.,Wang G.B.,and Dong X.W.,2013,Effects of temperature and soil water deficit on the flavonoid content and activities of enzymes involved in ginkgo leaves,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),33(4):755-762(陈雷,常丽,曹福亮,汪贵斌,董晓伟,2013,银杏叶黄酮类化合物含量及相关酶活性对温度和干旱胁迫的响应,西北植物学报,33(4):755-762)
Dong S.B.,Ning Y.P.,Yang Z.,Zhang Q.H.,Dong J.Q.,Wang F.J.,and Wang J.Z.,2015,Study on extraction process of quinoa total flavonoids and purification process of macroporous resin,Shipin Gongye Keji(Science&Technology of Food Industry),36(16):272-278(董施彬,宁亚萍,杨喆,张乔会,董洁琼,王丰俊,王建中,2015,藜麦总黄酮提取及大孔树脂纯化工艺的研究,食品工业科技,36(16):272-278)
Fan C.F.,Bi Y.,Wang Y.F.,Ren Y.L.,Yang Z.M.,and Wang Y.,2012,Effect of salicylic acid dipping on postharvest diseases and phenylpropanoid pathway in muskmelon fruits,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),45(3):584-589(范存斐,毕阳,王云飞,任亚琳,杨志敏,王毅,2012,水杨酸对厚皮甜瓜采后病害及苯丙烷代谢的影响,中国农业科学,45(3):584-589)
Fu Q.Q.,Hou P.,and Chen N.,2013,Optimization of extraction process for total flavonoids from Artemisia selengensis Turcz leaves by response surface methodology,Shipin Kexue(Food Science),34(4):94-98(扶庆权,侯佩,陈能,2013,响应面法优化芦蒿叶总黄酮的提取工艺,食品科学,34(4):94-98)
Gamel T.H.,and Abdel-Aal E.M.,2012,Phenolic acids and antioxidant properties of barley wholegrain and pearling fractions,Agr.Food Sci.,21(2):118-131
Jeyaramraja P.R.,Pius P.K.,Kumar R.R.,and Jayakumar D.,2003,Soil moisture stress induced alterations in bioconstituents determining tea quality,J.Sci.Food Agric.,83(12):1187-1191
Jin L.P.,Cui S.M.,Du J.W.,Jin C.X.,Wu Y.F.,and Qi R.G.,2009,Effects of drought stress in different ecological conditions of the Prunus mongolica Maxim almond leaves in PALand C4H activity,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),24(5):118-122(金丽萍,崔世茂,杜金伟,金彩霞,吴玉峰,其日格,2009,干旱胁迫对不同生态条件下蒙古扁桃叶片PAL和C4H活性的影响,华北农学报,24(5):118-122)
Knobloch K.H.,and Hahlbrock K.,1977,4-Coumarate:CoA ligase from cell suspension cultures of Petroselinum hortense Hoffm,partial purification,substrate specificity,and further properties,Arch.Biochem.Biophys.,184(1):237-248
Lamb C.J.,and Rubery P.H.,1975,A spectrophotometric assay for trans-cinnamic acid 4-hydroxylase activity,Analytica Biochemistry,68(2):554-561
Li G.Y.,Luo X.Y.,Sun C.S.,Yu Q.,and Chen G.L.,2017,Effects of progressive drought stress on the accumulation of flavonoids in the growth of Astragalus membranaceus var mongholicus(Bge.)Hsiao,Xibei Zhuwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(1):138-143(李光跃罗晓雅,孙窗舒,于倩,陈贵林,2017,干旱胁迫对黄芪植株生长中黄酮类成分积累的影响,西北植物学报,37(1)138-143)
Li N.N.,Pei Y.T.,Gong Y.C.,Pu Y.Y.,Liu B.M.,Xin F.G.,Liu S.H.,and Ding H.F.,2016,Research status and developmen prospect of Chenopodium quinoa Willd,Shandong Nongye Kexue(Shandong Agricultural Sciences),48(10):145-148(李娜娜,裴艳婷,宫永超,蒲艳艳,刘保民,辛富刚,刘世华,丁汉凤,2016,藜麦研究现状与发展前景,山东农业科学,48(10):145-148)
Liu Q.,Sun Y.,Zheng J.M.,Yan X.L.,Chen H.M.,and Chen JK.,2015,Formononetin sensitizes glioma cells to doxorubicin through preventing EMT via inhibition of histone deacetylase 5,Int.J.Clin.Exp.Pathol.,8(6):6434
Lu X.X.,2012,Research progress in antioxidant mechanism of flavnonids,Shipin Yanjiu Yu Kaifa(Food Research&Development),33(3):220-224(鲁晓翔,2012,黄酮类化合物抗氧化作用机制研究进展,食品研究与开发,33(3):220-224)
Ma L.,Peng L.X.,Cao J.,Zhao J.L.,and Zhao G.,2016,Determination of 3 flavonoid C-glycosides in quinoa by ultrasonic extraction and RP-HPLC,Shipin Gongye(The Food Industry),37(7):192-195(马麟,彭镰心,曹靖,赵江林,赵钢2016,超声波辅助提取RP-HPLC法测定藜麦中的3种碳苷黄酮,食品工业,37(7):192-195)
Ouyang G.C.,and Xue Y.L.,1998,Physiological role and regulation of phenylpropanoid metabolism in plant,Zhiwu Shengl Xuebao(Plant Physiology Communications),(3):11-18(欧阳光察,薛应龙,1998,植物苯丙烷类代谢的生理意义及其调控,植物生理学报,(3):11-18)
PawelP.,Henryk B.,PawelZ.,Shela G.,Maria F.,and Zofia Z.2009,Anthocyanins,total polyphenols and antioxidant activity in amaranth and quinoa seeds and sprouts during their growth,Food Chem.,115(3):994-998
Qin W.T.,Zhang J.,Wu H.J.,Sun G.Z.,Yang W.Y.,and Liu J.2016,Effects of drought stress on biosynthesis of isoflavones in soybean seedling,Yingyong Shengtai Xuebao(Chinese Journal of Applied Ecology),27(12):3927-3934(秦雯婷,张静,吴海军,孙广泽,杨文钰,刘江,2016,干旱胁迫对苗期大豆异黄酮合成的影响,应用生态学报,27(12):3927-3934)
Sun X.T.,Jiang Y.R.,Yuan J.J.,Wei S.Y.,Lu G.Q.,and Mao Q.2017,Optimization of extraction technology of flavonoids from quinoa seeds by response surface methodology and its antioxidant activities,Zhongguo Shipin Xuebao(Journal of Chinese Institute of Food Science and Technology),17(3):127-135(孙雪婷,蒋玉蓉,袁俊杰,魏述英,陆国权,毛前,2017,响应面法优化提取藜麦种子黄酮及抗氧化活性,中国食品学报,17(3):127-135)
Tang D.,He B.,Zheng Z.G.,Wang R.S.,Gu F.,and Duan T.T.,2011,Inhibitory effects of two major isoflavonoids in Radix astragali on high glucose-induced mesangial cells proliferation and AGEs-induced endothelial cells apoptosis,Planta Med.,77(7):729-732
Wang C.Y.,Chen C.T.,and Wang S.Y.,2009,Changes of flavonoid content and antioxidant capacity in blueberries after illumination with UV-C,Food Chemistry,117(3):426-431
Weisshaar B.,and Jenkins G.I.,1998,Phenylpropanoid biosynthesis and its regulation,Curr.Opin.Plant Biol.,1(3):251-257
Winkel S.B.,1999,Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways,Physiol.Plantarum,107(1):142-149
Xiao Z.C.,and Zhang G.L.,2014,Development and utilization of Chenopodium quinoa Willd.,Zhongguo Yesheng Zhiwu Ziyuan(Chinese Wild Plant Resources),33(2):62-66(肖正春,张广伦,2014,藜麦及其资源开发利用,中国野生植物资源,33(2):62-66)
Yang Y.,Zhao Y.,Ai X.,Cheng B.,and Lu S.,2014,Formononetin suppresses the proliferation of human non-small cell lung cancer through induction of cell cycle arrest and apoptosis,Int.J.Clin.Exp.Pathol.,7(12):8453-8461
Zeng W.M.,2011,Study on extraction and antioxidant activity of antioxidant active substances from oat,Thesis for M.S.,Northwest A&F University,Supervisor:Li J.X.,pp.28-34(曾王旻,2011,燕麦抗氧化活性物质提取及抗氧化活性研究,硕士学位论文,西北农林科技大学,导师:李巨秀,pp.28-34)
Zhang L.M.,Li R.C.,Hao L.M.,Tian A.Y.,Lu J.K.,and Zhang J.C.,2014,Response surface methodology for optimization of extracting total flavonoids from maca leaves and antioxidant evaluation,Xiandai Shipin Keji(Modern Food Science&Technology),30(4):233-239(张黎明,李瑞超,郝利民,田爱莹,鲁吉珂,张建春,2014,响应面优化玛咖叶总黄酮提取工艺及其抗氧化活性研究,现代食品科技,30(4):233-239)
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