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水体的富营养化加剧了蓝藻水华的频繁爆发。这不但影响水体景观效果,还会释放藻毒素,严重威胁水生态系统的稳定和公共卫生安全[1]。在众多藻毒素中,微囊藻毒素(microcystins, MCs)是一种具有显著肝脏毒性的七肽环状毒素,是世界各地水体中最为常见且危害最大的藻毒素。微囊藻毒素-LR(MC-LR)是MCs单体中分布最广毒性最强的一类,世界卫生组织(WHO)规定其在饮用水中最高含量不得超过1.0 µg·L−1[2]。鱼腥藻毒素(anatoxin, ANTXs)是一类毒性大、活性高的神经毒素,不慎接触会导致人和动物迅速中毒死亡。鱼腥藻毒素-a(ANTX-a)是鱼腥藻毒素中含量最高的一种单体,饮用水中最大安全剂量为3.0 µg·L−1[3]。在蓝藻水华频发水域,70%以上监测水体中都同时检测到MCs与ANTXs,表明两种毒素时空共存已普遍存在,且MCs或ANTXs浓度通常在1—100 µg·L−1,极端状况下会在短时间内高达1000 µg·L−1以上[4]。这些藻毒素通过灌溉、溢流、打捞堆放、堆沤还田(特别是蓝藻堆肥后施入农田)等途径进入农田系统[5-6]。研究发现,MCs被农作物吸收积累后,不仅影响农作物生长发育,如降低光合效率、破坏激素平衡、抑制抗氧化酶活性等[7-8],也会在植物可食部分积累并通过食物链传递威胁人体健康[9-10]。在调查35种农作物暴露于MCs的结果显示,MCs残留量最高的是生菜(Lactuca sativa)、欧芹(Petroselinum crispum)、茴香(Foeniculum vulgare)和卷心菜(Brassica oleracea)等蔬菜,其次是玉米(Zea mays)、胡萝卜(Daucus carota)和小麦(Hordeum vulgare)等作物[11]。因而关于藻毒素的植物毒理学研究近年来也备受关注。然而已有研究大多集中在MCs对植物生理生化代谢的影响,关于ANTXs的研究多集中在水生植物[12],关于MCs和ANTXs对植物的复合影响却鲜有报道。农作物在人类膳食结构中占有重要地位,如能明确MCs和ANTXs对农作物的复合作用机制,将为客观评价藻毒素的生态风险提供新依据,也为保障食品安全提供新的数据。
活性氧(reactive oxygen species, ROS)是植物有氧代谢中具有很强毒性的副产物,主要包括过氧化氢(H2O2)、超氧阴离子(O2·-)、羟基自由基(·OH)等[13]。作为信号分子,逆境迫使植物细胞中积累大量ROS以诱导防御基因的表达,进而调控植物对逆境的适应性。同时大量积累的ROS会引发氧化损伤,甚至导致细胞死亡[14]。因此,逆境下植物维持胞内ROS稳态是提高植物耐受性的重要机制之一。例如,MCs胁迫下水稻(Oryza sativa)中抗氧化酶清除ROS的能力强于黄瓜(Cucumis sativus),这也是水稻耐受MCs胁迫能力强于黄瓜的主要因素[8]。基于ROS水平与植物耐受环境胁迫机制的密切关系,从活性氧稳态(即产生与清除间的平衡)的角度去理解MCs和ANTXs对植物生长影响的复合作用特征,可为清晰植物耐受蓝藻毒素胁迫的适应机制提供新数据。鉴于此,本文选取广泛种植的生菜(Lactuca sativa)为研究对象,以调控活性氧生成与去除的重要酶类为切入点,探究MCs与ANTXs对生菜中活性氧稳态影响的内在机制,为客观评价蓝藻毒素的生态风险提供新的数据。
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本研究所采用的MCs和ANTXs均提取自蓝藻细胞。用于提取微囊藻毒素(MCs)的新鲜蓝藻打捞自无锡太湖,提取与分析方法参照文献[9]。通过酶联免疫(ELISA)测定总MCs浓度,再通过高效液相色谱(HPLC)确定各MC变体占比(其中MC-LR、MC-RR和MC-YR分别占62.66%、28.48%和0.68%)。产生ANTXs的水华鱼腥藻(FACHB-1255)采购自中国科学院淡水藻种库(FACHB),提取与分析方法参照文献[15]。同样通过ELISA测定总ANTXs浓度,再通过HPLC确定ANTXs各变体占比(ANTX-a: HANTX-a=76.4%:23.6%)。
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将籽粒饱满的生菜(Lactuca sativa L.)种子用0.1% HgCl2去离子水浸泡4 h,在黑暗中发芽(温度20 ℃;相对湿度70%)。待生菜长出两片真叶后移入含有营养液的5 L周转箱进行培养(每箱6株),培养条件为光强为350 µmol·m−2·s−2(日/夜:14 h/10 h),温度为25 ℃/18 ℃(日/夜),相对湿度80%。营养液每7 天更换1次,培养30 d后处理。参考自然水体中MCs与ANTXs出现频率较高的环境浓度范围, 将培养成熟的生菜移至含不同浓度MCs、ANTXs及MCs+ANTXs的营养液中进行处理。实验对照(CK,营养液中无藻毒素),单一MCs处理(营养液中MCs浓度分别为5 µg·L−1和100 µg·L−1),单一ANTXs处理(营养液中ANTXs浓度分别为5 µg·L−1和100 µg·L−1)和MCs+ANTXs复合处理(营养液中MCs和ANTXs浓度为(5+5) µg·L−1和(100+100) µg·L−1。每处理重复3次,处理7 d后收获。
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采用电子天平测量生菜生物量。参考张清航等[16]的方法测定植物体内MDA含量,采用硫酸钛法测定H2O2含量[17]和羟氨氧化法测定O2·−含量[18]。参照关美艳[19]的方法测定NADPH氧化酶活性。采用Zhang等[20]的方法进行LsRbohA引物设计和扩增程序,基因相对表达量采用2−ΔΔCT法进行计算。采用FRAP法测定植株的总抗氧化能力[21]。SOD和CAT活性的测定参考顾艳芳等[22]的方法并稍作改动。MCs和ANTXs对生菜的复合影响作用使用Abotts公式[23]进行具体评估。
其中,A是单一MCs引起生菜指标的抑制程度;B是单一ANTXs引起生菜指标的抑制程度;C是MCs+ANTXs复合处理引起生菜指标的抑制程度;Cexp是预期抑制率;RI为抑制比率。
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所有数据均以3次独立试验的平均值±标准误差(Mean±SD)表示。采用Levene检验分析各组方差的同质性,组间差异通过LSD检验的单因素方差进行评估(P<0.05)。
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实验中以生物量(FW)表征生菜生长情况,以丙二醛(MDA)含量衡量植物氧化损伤程度[24],结合ROS中重要成员H2O2和O2·−含量变化,探究MCs与ANTXs对生菜中ROS稳态的影响。如表1所示,与CK相比,低浓度(5 µg·L−1)MCs、ANTXs及MCs+ANTXs((5+5) µg·L−1)复合处理分别促进生菜生物量增加75.3%、85.6%和63.3% (P<0.05),其中MCs+ANTXs((5+5) µg·L−1)组生物量低于单一MCs(5 µg·L−1)或ANTXs(5 µg·L−1)。同时,低浓度(5 µg·L−1)MCs或ANTXs对叶片中MDA、H2O2和O2·−含量无显著影响(P>0.05),其中MCs+ANTXs(5+5) µg·L−1复合处理诱导H2O2和O2·−含量小幅上升且MDA含量略高于CK(P<0.05)。综上结果可知,低浓度MCs、ANTXs及MCs+ANTXs复合处理促进生菜生长,呈现Hormesis效应。这种低浓度毒素促进作用可能与刺激植物体内生长促进型激素(生长素IAA、玉米素ZT和赤霉素GA3)含量升高有关[25]。此外,低浓度毒素未引起生菜活性氧大量积累,也未引起脂膜过氧化损伤,表明此时毒素胁迫强度未超出生菜的自调节范围,这也可能是生菜生长仍能正常进行的又一原因。与低浓度处理不同,高浓度(100 µg·L−1)MCs与MCs+ANTXs(100+100) µg·L−1复合处理降低生菜生物量达27.7%、5.1%和16.8%(P<0.05),其中高浓度(100 µg·L−1)ANTXs对生菜生物量无明显影响(P>0.05),这可能与毒素本身的理化性质,如分子量、作用方式以及毒性差异等原因有关[26]。Ariel等[12]发现ANTX-a浓度高达5000 µg·L−1时才会严重抑制品藻的生长。高浓度MCs、ANTXs及MCs+ANTX处理导致叶片中MDA、H2O2和O2·−含量显著增加(P<0.05),其中MDA、H2O2和O2·−含量增幅排序为MCs>MCs+ANTXs>ANTXs。以上结果说明100 µg·L−1MCs或ANTXs诱导了ROS含量升高而导致脂膜损伤,这可能是导致高浓度MCs和MCs+ANTX抑制生菜生长的原因之一。其中高浓度MCs+ANTXs抑制生菜生长的程度低于单一MCs,可能与MCs+ANTXs诱导生菜中产生ROS含量低于单一MCs有关。前期研究也发现MCs对植物细胞中ROS平衡的影响程度受MCs浓度、暴露时间以及植物种类等因素调节,可以反映植物耐受MCs的能力[8]。结合生物量、ROS水平和MDA分析,生菜能够耐受低浓度(5 µg·L−1)毒素胁迫,但不能耐受高浓度(100 µg·L−1)胁迫,且对高浓度MCs+ANTXs复合胁迫的耐受性强于单一MCs却弱于单一ANTXs,这可能与维持细胞内活性氧稳定的能力有关。
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质膜上的NADPH氧化酶是植物细胞活性氧产生的主要酶之一[27],它能够将O2催化成O2·−,因此可用来表征植物ROS生成能力[28]。LsRbohA是编码生菜NADPH氧化酶的关键基因。与CK相比,低浓度(5 µg·L−1)MCs或ANTXs处理对生菜叶片中NADPH氧化酶活性及LsRbohA相对表达量均无显著影响(P>0.05)(图1),MCs+ANTXs复合处理显著提高了NADPH氧化酶活性,LsRbohA的相对表达量上调137.8%(P<0.05)。这表明低浓度MCs或ANTXs对生菜叶中ROS的生成无影响,这是生菜叶片中ROS维持稳态的原因之一。而低浓度(5+5) µg·L−1MCs+ANTXs复合下生菜叶片中LsRbohA表达量的上调可能是NADPH氧化酶活性上升的原因之一。结合生菜生物量、ROS含量和MDA含量(表1)分析可知,低浓度MCs+ANTXs复合处理通过促进NADPH氧化酶活性诱导ROS生成增多,促使防御系统及时对外界环境变化做出适应性应激,避免氧化损伤而不影响植物生长[29]。高浓度MCs、ANTXs和MCs+ANTXs复合处理显著上调LsRbohA表达272%—657%,且诱导NADPH氧化酶活性上升(P<0.05),其中酶活性增幅排序为MCs>MCs+ANTXs>ANTXs。表明高浓度(100 µg·L−1)MCs、ANTXs和MCs+ANTXs复合处理下生菜叶片中LsRbohA表达量大幅上升,合成酶蛋白增多,这可能是NADPH氧化酶活性上升的原因之一。NADPH氧化酶催化生成大量ROS,促发防御系统应激,但仍无法抵御高浓度毒素的伤害,最终引起氧化损伤而抑制生长(表1)。Pérez等[30]也发现,NADPH氧化酶是植物响应非生物胁迫下初始ROS升高的主要原因之一。同时H2O2可以诱导NADPH氧化酶基因Rboh表达[28],这是植物遭受非生物逆境的威胁时,信号分子H2O2启动一系列抗逆相关基因表达来缓解或抵御非生物逆境胁迫对植物造成的危害[31-32]。因而本实验中高浓度MCs+ANTXs复合处理下生菜NADPH氧化酶活性高于单一ANTXs却低于MCs, 这可能是MCs+ANTXs处理组生菜中ROS含量高于单一ANTXs而低于单一MCs的主要因素之一。
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植物细胞中ROS平衡也受抗氧化系统的调节。SOD作为植物抗氧化系统的第一道防线,将胁迫诱导的过量O2·−歧化为H2O2和O2。H2O2在第二道防线CAT的催化下分解为H2O和O2。考虑到植物拥有众多抗氧化酶类和非酶类,将抗氧化酶活性与总抗氧化能力(FRAP)结合能更全面地反映植物细胞清除ROS的能力[21]。如图2所示,与CK相比,低浓度(5 µg·L−1)MCs、ANTXs和MCs+ANTXs(5+5) µg·L−1复合处理显著提高生菜的FRAP (P<0.05),促进SOD活性上升(除单一ANTXs处理组外),而对CAT活性无显著影响(P>0.05)。表明低浓度毒素处理组诱导SOD活性和FRAP上升,维持胞内ROS稳定。低浓度毒素处理下第二道防线的CAT未应激启动再次证实,该强度胁迫未超出生菜耐受范围。高浓度(100 µg·L−1)MCs、ANTXs和MCs+ANTXs显著促进生菜叶片中的SOD和CAT活性上升(P<0.05),增幅排序为MCs>MCs+ANTXs>ANTXs。生菜叶片的FRAP变化却不尽一致,在100 µg·L−1 MCs处理下降低(P<0.05),在100 µg·L−1 ANTXs处理下上升(P<0.05),而在MCs+ANTXs(100+100) µg·L−1处理下无显著变化(P>0.05)。结合FW、MDA和ROS含量变化(表1)分析发现,高浓度(100 µg·L−1)毒素促发SOD和CAT活性升高不足以清除过量生成的ROS,胞内ROS稳态被破坏,氧化损伤发生,这也是生长受抑制的原因之一。不同高浓度毒素处理下生菜FRAP变化不一致,这可能是由于植物体内抗氧化酶类和非酶类对不同毒素胁迫的响应程度有差异。例如酸雨胁迫下水稻幼苗SOD、CAT和脱氢抗坏血酸还原酶(DHAR)活性增加,而抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性降低[33-34]。此外,酚类和类黄酮物质也属于植物的非酶抗氧化剂防御系统的成员,其含量或状态变化也会影响细胞总抗氧化能力[35]。因此,生菜叶中SOD、CAT和FRAP对高浓度(100 µg·L−1)MCs、ANTXs和MCs+ANTXs处理响应存在差异,其中较高的SOD和CAT活性及FRAP有助于缓解高浓度毒素诱导的ROS过量积累,对维持ROS稳态有利,这也许是MCs+ANTXs组ROS含量高于单一ANTXs而低于单一MCs的又一原因。
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两种不同污染物对受试生物的影响可能呈现独立作用、协同作用、加和作用或者拮抗作用。为客观评价MCs和ANTXs对生菜活性氧稳态的复合影响,分析了MCs+ANTXs对生菜ROS稳态相关指标复合影响的特征(表2)。当RI值>1时,呈现协同效应;RI=1,呈现加和效应;RI<1,则呈现拮抗效应[36]。
如表2所示,MCs+ANTXs(5+5) µg·L−1对MDA含量、H2O2、NADPH氧化酶与CAT活性抑制比率RI值均显著>1,呈现协同效应;而MCs+ANTXs(100+100) µg·L−1对MDA含量、H2O2、NADP氧化酶与CAT活性抑制比率RI值均显著<1,呈现拮抗效应。表明随着MCs和ANTXs浓度升高,两者间复合作用特征由协同作用转为拮抗作用。Wang等[36]研究发现低浓度MC-LR和多环芳烃(菲)对浮萍生长和抗氧化系统产生协同作用,而高浓度呈现拮抗或加和作用。因此,MCs与ANTXs对生菜生长与ROS稳态的复合作用特征受毒素浓度的影响。其中,MCs+ANTXs(100+100) µg·L−1对以上所有参数的抑制比率(RI)均<1,表明高浓度MCs+ANTXs对生菜复合作用特征主要呈拮抗效应,这与Li等研究结果一致[37],他们也发现MC-LR和ANTX-a对苦草生长和蛋白质的复合作用呈现拮抗效应。这与MCs或ANTXs进入细胞存在竞争有关,也可能与铜绿微囊藻与水华鱼腥藻之间存在竞争关系有关。例如在共培养的条件下,水华鱼腥藻对铜绿微囊藻有显著的抑制作用[38]。以上可以推测,水体中一定浓度的ANTXs可以减轻MCs对生菜的氧化伤害。
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(1)低浓度(5 µg·L−1)MCs、ANTXs与MCs+ANTXs诱导生菜中SOD活性和总抗氧化能力(FRAP)上升,利于维持ROS平衡,避免了氧化损伤,促进生物量增加63.3%—85.6%,表明低浓度(5 µg·L−1)MCs、ANTXs和MCs+ANTXs对生菜生长有利。
(2)高浓度(100 µg·L−1)MCs、ANTXs与MCs+ANTXs上调生菜叶片中LsRbohA表达并提高NADPH氧化酶活性,生成大量ROS促发防御系统应激,而SOD和CAT活性的上升不足以清除大量ROS,诱发氧化损伤而抑制生菜生长,降幅为MCs(27.7%)>MCs+ANTXs(16.8%)>ANTXs(5.1%)。
(3)复合特征分析发现,MCs+ANTXs(5+5) µg·L−1对MDA含量、H2O2、NADP氧化酶与CAT活性影响呈现协同效应,而MCs+ANTXs(100+100) µg·L−1则呈现拮抗效应。表明MCs+ANTXs对生菜复合作用受毒素浓度的影响,高浓度ANTXs共存在一定程度上减轻了MCs对生菜的氧化伤害。
微囊藻毒素与鱼腥藻毒素对生菜活性氧稳态的复合影响
Compound effects of microcystin and anatoxin on homeostasis of reactive oxygen species in lettuce
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摘要: 水体富营养化导致水体中蓝藻毒素浓度高于安全值。为客观评价灌溉水中蓝藻毒素的生态风险,本研究探析了微囊藻毒素(MCs)和鱼腥藻毒素(ANTXs)对生菜生长和活性氧(ROS)稳态的复合影响。采用5 µg·L−1或100 µg·L−1MCs、ANTXs和MCs+ANTXs((5+5) µg·L−1或(100+100) µg·L−1)处理生菜7 d后发现,低浓度(5 µg·L−1)MCs、ANTXs或MCs+ANTXs促进生菜鲜重增加63.3%—85.6%,可能是低浓度毒素诱导SOD活性和总抗氧化能力(FRAP)上升以维持H2O2和O2·−平衡进而避免氧化损伤。高浓度(100 µg·L−1)MCs、ANTXs或MCs+ANTXs抑制生菜生长,降幅为MCs(27.7%)>MCs+ANTXs(16.8%)>ANTXs(5.1%)。同时,MDA、H2O2和O2·−含量、NADPH氧化酶活性及LsRbohA表达量、SOD和CAT活性均升高,增幅为MCs>MCs+ANTXs>ANTXs,而FRAP变化不一致。推测高浓度藻毒素上调LsRbohA表达有助于增强NADPH氧化酶活性,生成的ROS增多以刺激防御应激,而SOD和CAT活性上升不足以维持ROS稳定,导致氧化损伤而抑制生长。进一步分析MCs+ANTXs对生菜中ROS稳态复合影响的作用特征发现,MCs+ANTXs(5+5) µg·L−1对MDA、H2O2、NADP氧化酶与CAT活性影响呈现协同效应,在MCs+ANTXs(100+100) µg·L−1处理下呈拮抗效应,表明MCs和ANTXs对生菜ROS稳态的复合影响受毒素浓度的调控,一定浓度的ANTXs共存减轻了MCs对生菜的氧化伤害,为指导灌溉安全提供新的理论依据。Abstract: The eutrophication of water leads to the increase of cyanobacteria toxin concentration in water. To objectively evaluate the ecological risk of cyanobacteria toxins in irrigation water, we studied compound effects of microcystin (MCs) and anatoxin (ANTXs) on growth and homeostasis of reactive oxygen species (ROS) in lettuce. After Hydroponic lettuce being treated with MCs (5 µg·L−1 or 100 µg·L−1), ANTXs (5 µg·L−1 or 100 µg·L−1) and MCs+ANTXs (5+5) µg·L−1 or (100+100) µg·L−1 for 7 d, we found that low concentration (5 µg·L−1) of MCs, ANTXs or MCs+ANTXs increased fresh weight of lettuce by 63.3%—85.6%, indicating that the increase of SOD activity and total antioxidant capacity (FRAP) induced by low concentration of toxin can maintain H2O2 and O2·− balance and then avoid oxidative damage. However, high concentration (100 µg·L−1) of MCs, ANTXs or MCs+ANTXs inhibited lettuce growth, and the decreased degree of each treatment was MCs(27.7%)>MCs+ANTXs(16.8%)>ANTXs(5.1%). Meanwhile, MDA, H2O2 and O2·− contents, NADPH oxidase activity and LsRbohA expression, SOD and CAT activities were increased with the order for increased degree as MCs>MCs+ANTXs>ANTXs. In addition, FRAP showed different change among treatments. These results indicate that high concentration treatments up-regulated LsRbohA expression for contributing to enhancing NADPH oxidase activity to produce more ROS for stimulating defense systems. While the increase in SOD and CAT activities was not enough to maintain the stability of ROS, resulting in oxidative damage and inhibition on growth. Furthermore, characteristic analysis of compound effects of MCs+ANTXs on ROS homeostasis in lettuce showed that MCs+ANTXs (5+5) µg·L−1 posed synergistic effects on MDA and H2O2 contents as well as NADPH oxidase and CAT activities in lettuce whereas MCs+ANTXs (100+100) µg·L−1 showed antagonistic effects on them. It can be included that compound effects of MCs and ANTXs on homeostasis of ROS in lettuce was dependent on their concentrations, and the coexistence of ANTXs at a certain concentration could reduce the oxidative damage caused by MCs to lettuce, providing a new theoretical basis for guiding the safety of irrigation water.
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Key words:
- microcystins /
- anatoxin /
- NADPH oxidase /
- antioxidant enzymes /
- reactive oxygen species /
- lettuce
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表 1 MCs和ANTXs对生菜生物量、MDA、H2O2和O2·-含量的复合影响
Table 1. Combined effects of MCs and ANTXs on biomass and contents of MDA, H2O2 and O2·- in lettuce leaves
处理组
Treatments浓度/(µg·L−1)
ConcentrationFW/g MDA/(µmol·g−1 FW) H2O2/(µg·g−1 FW) O2·−/(µg·g−1 FW) CK 0 20.772±1.292c 0.74±0.06d 6.27±0.74e 4.18±0.07c MCs 5 36.415±1.290a 0.75±0.06d 5.50±0.44e 4.25±0.14c 100 15.093±0.791d 1.57±0.13a 15.83±0.66a 10.71±0.98a ANTXs 5 38.552±0.888a 0.73±0.12d 5.47±0.29e 4.86±0.11c 100 19.720±1.764c 1.24±0.10b 10.06±0.61c 8.57±0.53b MCs+ANTX 5+5 33.913±1.158b 0.89±0.03c 8.83±1.00d 4.78±0.26c 100+100 17.283±0.983d 1.37±0.08b 11.31±0.47b 8.97±0.49b 注:不同字母表示各处理组间差异显著(P<0.05). Note: Different letters represent significant differences between treatments (P<0.05). 表 2 MCs和ANTXs对生菜ROS稳态影响的复合作用特征分析
Table 2. Characteristic analysis on combined effects of MCs and ANTXs on homeostasis of ROS in lettuce
指标
IndexMCs/
(µg·L−1)A/% ANTXs/
(µg·L−1)B/% MCs+ANTXs/
(µg·L−1)C/% Cexp/% RI FW 5 −75.31 5 −85.60 5+5 −63.27 −225.36 0.28* 100 27.34 100 5.06 100+100 16.80 31.02 0.54* MDA 5 −2.22 5 1.31 5+5 −28.44 −0.88 32.44* 100 −113.39 100 −68.59 100+100 −85.52 −259.74 0.33* H2O2 5 12.24 5 12.77 5+5 −40.95 23.45 −1.75* 100 −152.58 100 −60.54 100+100 −80.44 −305.49 0.26* O2·− 5 −1.63 5 −16.11 5+5 −14.13 −18.01 0.79* 100 −155.90 100 −104.75 100+100 −114.31 −423.96 0.27* NADPH 5 5.25 5 1.38 5+5 −8.09 6.56 −1.23* 100 −81.90 100 −45.44 100+100 −62.80 −164.55 0.38* SOD 5 −20.59 5 −4.22 5+5 −16.42 −25.67 0.64* 100 −94.40 100 −32.65 100+100 −76.28 −157.86 0.48* CAT 5 2.18 5 1.72 5+5 −4.43 3.87 −1.15* 100 −66.98 100 −18.99 100+100 −49.37 −98.69 0.50* FRAP 5 −70.13 5 −86.76 5+5 −87.78 −217.73 0.40* 100 26.41 100 −17.14 100+100 11.89 13.80 0.86* 注:数据为平均值±标准误差,*代表与1之间的显著性(P<0.05).
Note: The data is denoted as mean±standard deviation, * represents the significance between RI and 1 (P<0.05). -
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