瑞香狼毒杀螨成分鉴定及其对草莓螨害的影响
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摘要
植食性害螨是农林生产上的一类重要生物。它具有个体小、繁殖快、密度高、为害重等特点。为满足有机果品栽培的需要,解决化学农药所产生的种种问题,从天然次生代谢产物中开发新型植物源杀虫、杀螨剂已经成为国内外农药研发的热点之一。该研究以瑞香狼毒为材料开发植物源杀螨剂。它具有数量多、分布广、生物量大的优势。对瑞香狼毒进行综合利用,特别是植物源农药领域的开发,能够变害为宝,能够实现生态效益和经济效益的双赢。
该研究借鉴中草药有效成分提取技术,采用中压快速制备色谱仪、制备型高效液相色谱仪,从瑞香狼毒中分离得到一种对朱砂叶螨兼有触杀毒性和内吸毒性的化合物Ⅳ。该化合物经红外波谱分析和核磁共振分析,确定为东莨菪素。此后,针对东莨菪素的杀螨规律,杀螨机理进行了研究,并且评价了东莨菪素对大棚草莓螨害的防治效果,以及东莨菪素对草莓生理反应的影响。研究结果表明：
(1)东莨菪素是瑞香狼毒中重要的杀螨活性成分,采用本研究方法得到的东莨菪素纯度可达到97.756%,天然东莨菪素与东莨菪素标准品的杀螨活性无显著区别；
(2)采用“时间—剂量—死亡率”(TDM)模型和S型生长曲线模型对东莨菪素的杀螨毒力和致死规律进行分析。结果表明,东莨菪素对朱砂叶螨触杀作用的致死中浓度(LC50)为：1.243 mg·mL-1(机率值法,24h)和1.346 mg·mL-1 (TDM法)。内吸作用的LC50为：7.231 mg·mL-1(机率值法,24h)和8.143 mg·mL-1 (TDM法)。东莨菪素对朱砂叶螨的致死速度高于除虫菊素。其中又以东莨菪素触杀作用方式的致死速度最快。
(3)东莨菪素属于神经毒剂,它能够抑制乙酰胆碱酯酶活性,导致乙酰胆碱含量增加,阻断神经传导；抑制Na+-K+-ATP酶活性,使细胞内K+离子积累,导致细胞吸水破裂。同时,对单胺氧化酶、Ca2+-Mg2+-ATP酶活性也有抑制作用。
(4)采用东莨菪素防治朱砂叶螨,施药时间应在发现螨害后的12d内。防治浓度为3 mg·mL-1时即可获得明显的防治效果,浓度达到9 mg·mL-1时防治效果最好。
(5)东莨菪素对草莓生理反应的影响均能恢复至正常水平,属于对草莓安全的杀螨活性物质。浓度为3—9 mg·mL-1的东莨菪素能使叶片相对电导率、MDA含量增加,同时激发SOD和CAT活性明显上升。150 h内,各指标逐渐恢复正常。施药初期叶绿素含量、净光合速率也显著下降,但也可以逐渐恢复。
Phytophagous pest mites are one of the important organisms which seriously harmful to our agricultural and forestral production. In order to meet the need of organic cultivation and to face continued problems of chemical insecticides, the development and preparation of botanical insecticide and acaricide, which base on natural secondary metabolites of plants, has become the hot spot in internal and abroad investigation domains. Stellera chamaejasme L. was selected as potential resources to develop botanical acaricide. In comparison with other plant resources, the advantages of S. chamaejasme are much quantity and broad and higher biomass. The multipurpose utilization of S. chamaejasme, especially in pesticide, is not only its success in ecological reservation, but also its plant-based botanical insecticide development.
In this research, with using the advanced method of extraction and separation in traditional Chinese medicine, a compound IV was obtained from roots of S. chamaejasme by preparative MPLC and HPLC. The structure of compound IV was established as scopoletin by analysis of NMR and IR data. And then the laws of acaricide effect and lethal mechanism of scopoletin were studied. The effects of mites control in strawberries and physiological response of strawberries, which were treated by scopoletin, were also investigated in this study. The main results and conclusion are as follows.
(1) Scopoletin is an important acaricidal substance in Stellera chamaejasme L.. In this study, the purity of scopoletin after purification is up to 97.756%. There was no significant difference of acaricidal effects between natural and standard scopoletin.
(2) The acaricidal toxicity and death dynamic variation of scopoletin treatments anaylized by Time-Dose-Mortality (TDM) model and S-shaped cure showed that the contact toxicity of mean lethal concentrations (LC50) is 1.243 mg·mL-1 (Probit,24 h) or 1.346 mg·mL-1(TDM); the systemic toxicity of LC50 is 7.231 mg·mL-1 (Probit,24 h) or 8.143 mg-mL-1 (TDM). The lethal rate of scopoletin was higher than pyrethrin, and the scopoletin contact treatment had the highest lethal rate.
(3) Scopoletin is a neurotoxin. The mechanism of acaricidal substance is scopoletin can significantly inhibit the activities of AChE, and the inhibition can lead to increasing of ACh, which can block nerve conduction. Scopoletin can also significantly inhibit the activities of Na+-K+-ATPase, and then increase of K+ in cell, which can lead to cell broken caused by excessive absorb water. The MAO and Ca2+-Mg2+-ATPase were also inhibited by scopoletin.
(4) The optimum time of applying acaricide was within 12 days after the spider mites occurrence. The optimal concentration of acaricide was 3 mg·mL-1, while the 9 mg·mL-1 treatment has perfect control.
(5) Scopoletin is generally safe to strawberries. The determining quotas of physiological response of strawberries were able to return to the normal level. All different concentration treatments (3 to 9 mg·mL-1) can increase relative conductivity and MDA content of leaves during the initial post-period, while the specific activities of SOD and CAT rose. But all indicators gradually returned to the normal level in 150 h. The dynamic changes of chlorophyll content and net photosynthesis rate were similarly during the whole treatment period.
该研究借鉴中草药有效成分提取技术,采用中压快速制备色谱仪、制备型高效液相色谱仪,从瑞香狼毒中分离得到一种对朱砂叶螨兼有触杀毒性和内吸毒性的化合物Ⅳ。该化合物经红外波谱分析和核磁共振分析,确定为东莨菪素。此后,针对东莨菪素的杀螨规律,杀螨机理进行了研究,并且评价了东莨菪素对大棚草莓螨害的防治效果,以及东莨菪素对草莓生理反应的影响。研究结果表明：
(1)东莨菪素是瑞香狼毒中重要的杀螨活性成分,采用本研究方法得到的东莨菪素纯度可达到97.756%,天然东莨菪素与东莨菪素标准品的杀螨活性无显著区别；
(2)采用“时间—剂量—死亡率”(TDM)模型和S型生长曲线模型对东莨菪素的杀螨毒力和致死规律进行分析。结果表明,东莨菪素对朱砂叶螨触杀作用的致死中浓度(LC50)为：1.243 mg·mL-1(机率值法,24h)和1.346 mg·mL-1 (TDM法)。内吸作用的LC50为：7.231 mg·mL-1(机率值法,24h)和8.143 mg·mL-1 (TDM法)。东莨菪素对朱砂叶螨的致死速度高于除虫菊素。其中又以东莨菪素触杀作用方式的致死速度最快。
(3)东莨菪素属于神经毒剂,它能够抑制乙酰胆碱酯酶活性,导致乙酰胆碱含量增加,阻断神经传导；抑制Na+-K+-ATP酶活性,使细胞内K+离子积累,导致细胞吸水破裂。同时,对单胺氧化酶、Ca2+-Mg2+-ATP酶活性也有抑制作用。
(4)采用东莨菪素防治朱砂叶螨,施药时间应在发现螨害后的12d内。防治浓度为3 mg·mL-1时即可获得明显的防治效果,浓度达到9 mg·mL-1时防治效果最好。
(5)东莨菪素对草莓生理反应的影响均能恢复至正常水平,属于对草莓安全的杀螨活性物质。浓度为3—9 mg·mL-1的东莨菪素能使叶片相对电导率、MDA含量增加,同时激发SOD和CAT活性明显上升。150 h内,各指标逐渐恢复正常。施药初期叶绿素含量、净光合速率也显著下降,但也可以逐渐恢复。
Phytophagous pest mites are one of the important organisms which seriously harmful to our agricultural and forestral production. In order to meet the need of organic cultivation and to face continued problems of chemical insecticides, the development and preparation of botanical insecticide and acaricide, which base on natural secondary metabolites of plants, has become the hot spot in internal and abroad investigation domains. Stellera chamaejasme L. was selected as potential resources to develop botanical acaricide. In comparison with other plant resources, the advantages of S. chamaejasme are much quantity and broad and higher biomass. The multipurpose utilization of S. chamaejasme, especially in pesticide, is not only its success in ecological reservation, but also its plant-based botanical insecticide development.
In this research, with using the advanced method of extraction and separation in traditional Chinese medicine, a compound IV was obtained from roots of S. chamaejasme by preparative MPLC and HPLC. The structure of compound IV was established as scopoletin by analysis of NMR and IR data. And then the laws of acaricide effect and lethal mechanism of scopoletin were studied. The effects of mites control in strawberries and physiological response of strawberries, which were treated by scopoletin, were also investigated in this study. The main results and conclusion are as follows.
(1) Scopoletin is an important acaricidal substance in Stellera chamaejasme L.. In this study, the purity of scopoletin after purification is up to 97.756%. There was no significant difference of acaricidal effects between natural and standard scopoletin.
(2) The acaricidal toxicity and death dynamic variation of scopoletin treatments anaylized by Time-Dose-Mortality (TDM) model and S-shaped cure showed that the contact toxicity of mean lethal concentrations (LC50) is 1.243 mg·mL-1 (Probit,24 h) or 1.346 mg·mL-1(TDM); the systemic toxicity of LC50 is 7.231 mg·mL-1 (Probit,24 h) or 8.143 mg-mL-1 (TDM). The lethal rate of scopoletin was higher than pyrethrin, and the scopoletin contact treatment had the highest lethal rate.
(3) Scopoletin is a neurotoxin. The mechanism of acaricidal substance is scopoletin can significantly inhibit the activities of AChE, and the inhibition can lead to increasing of ACh, which can block nerve conduction. Scopoletin can also significantly inhibit the activities of Na+-K+-ATPase, and then increase of K+ in cell, which can lead to cell broken caused by excessive absorb water. The MAO and Ca2+-Mg2+-ATPase were also inhibited by scopoletin.
(4) The optimum time of applying acaricide was within 12 days after the spider mites occurrence. The optimal concentration of acaricide was 3 mg·mL-1, while the 9 mg·mL-1 treatment has perfect control.
(5) Scopoletin is generally safe to strawberries. The determining quotas of physiological response of strawberries were able to return to the normal level. All different concentration treatments (3 to 9 mg·mL-1) can increase relative conductivity and MDA content of leaves during the initial post-period, while the specific activities of SOD and CAT rose. But all indicators gradually returned to the normal level in 150 h. The dynamic changes of chlorophyll content and net photosynthesis rate were similarly during the whole treatment period.
引文
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