外源草酸对三七皂苷Rg_1自毒效应的缓解作用及机制研究

Effect and mechanism of exogenous oxalic acid on alleviatingautotoxicity of Rg_1 to Panax notoginseng

【目的】明确外源添加草酸缓解皂苷Rg_1对三七根系自毒的缓解效应及作用机理,为缓解三七自毒危害提供依据。【方法】采用外源添加的方式,测定了1.0mg/L皂苷Rg_1溶液单独处理及外源添加质量浓度0.1,1.0,5.0mg/L草酸的混合处理对三七种苗根系萎蔫和鲜质量的影响,同时以体积分数0.1%甲醇为空白对照,分析外源添加草酸对Rg_1引起的三七种苗自毒效应的缓解作用。利用硝基四唑蓝氯化物(NBT)活性氧染色法,比较外源添加草酸后三七根尖活性氧的积累情况,测定抗氧化酶系统中与活性氧清除相关的过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性,以及抗坏血酸-谷胱甘肽(ASC-GSH)循环中抗坏血酸过氧化物酶(APX)和脱氢抗坏血酸还原酶(DHAR)的活性及抗坏血酸(ASC)和脱氢抗坏血酸(DHA)含量,探讨外源添加草酸对Rg_1自毒作用的缓解机制。【结果】1.0mg/L Rg_1单独处理会导致三七植株出现明显萎蔫,萎蔫率达到46.70%,鲜质量减少率达18.10%。外源添加质量浓度为0.1,1.0,5.0mg/L草酸后,三七的萎蔫率显著降低,仅为0～2.50%,鲜质量减少率降低至3.64%～6.17%,明显缓解了由皂苷Rg_1引起的自毒效应。NBT染色结果显示,Rg_1能诱导三七根尖活性氧大量积累,外源添加草酸后根尖积累的活性氧显著减少。酶活性分析表明,Rg_1能降低抗坏血酸-谷胱甘肽(ASC-GSH)循环中的抗坏血酸过氧化物酶(APX)活性和抗坏血酸(ASC)含量,从而降低细胞对氧自由基的清除能力,导致细胞内活性氧的大量积累。外源添加草酸能通过增强过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和脱氢抗坏血酸还原酶(DHAR)的活性及增加抗坏血酸(ASC)的含量来提高根系的抗氧化能力。【结论】外源添加草酸能提高ASC-GSH循环中APX和DHAR的活性及抗氧化剂ASC含量,进而增强三七根系的抗氧化能力,有效缓解皂苷Rg_1对三七生长的自毒活性。

【Objective】The aim of this study was to identify the effect and mechanism of exogenous oxalic acid on alleviating the autotoxicity of Rg1on Panax notoginsengroot and provide evidence for alleviating P.notoginsengautotoxicity damage.【Method】Effect of 1.0mg/L Rg1solution alone or 1.0mg/L Rg1 with 0.1,1.0,and 5.0mg/L oxalic acid on wilt and fresh weight of P.notoginsengseedlings was tested using exogenously application method.The alleviating effect of oxalic acid on the autotoxicity of Rg1was analyzed using 0.1%methanol as blank control.The reactive oxygen species(ROS)accumulation in root tip was observed by NBT reactive oxygen stain.The activities of ROS-related enzymes including superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)and ascorbate-glutathione(ASC-GSH)cycle enzymes including ascorbate peroxidase(APX)and dehydrogenase ascorbate reductase(DHAR),as well as the contents of non-enzymatic antioxidants including ascorbic acid(ASC)and dehydrogenase ascorbic acid(DHA)were determined to explore the alleviating mechanism.【Result】Rg1 with the concentration of 1.0mg/L caused significant wilt and fresh weight reduction of P.notoginsengseedlings.The wilt ratio and fresh weight reduction ratio were 46.70%and 18.10%,respectively.Exogenous oxalic acid with the concentrations of 0.1,1.0,5.0mg/L could significantly reduce the wilt ratio and fresh weight reduction ratio to 0-2.50%and 3.64%-6.17%,which clearly alleviated the toxic influence of Rg1.NBT staining results showed that Rg1could lead to accumulation of reactive oxygen in the root tip of P.notoginseng.After adding oxalic acid,the reactive oxygen accumulation in root tip decreased significantly.Enzyme activity analysis showed that Rg1could inhibit APX in ASC-GSH and decrease the content of ASC,so as to reduce the ability of cells to scavenge oxygen free radicals and lead to accumulation of ROS in cell.Exogenous oxalic acid could help root cell scavenge over-accumulated ROS by promoting activities of SOD,POD,CAT,APX and DHAR and content of ASC.【Conclusion】Exogenous oxalic acid can increase activities of APX and DHAR as well as content of ASC in ASC-GSH cycle,and enhance the anti-oxidative ability of root to effectively relieve the toxicity of Rg1on P.notoginsenggrowth.