3种蒿属植物杀虫成分提取及制剂配制
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摘要
菊科(Compositae)是被子植物中含有化学组分最多、结构最复杂的科,其中蒿属是该科中杀虫和抑菌活性强、分布广的属之一。研究蒿属植物的杀虫成分、生物活性及其作用机制,将对植物源农药的开发具有重要意义。本研究以3种蒿属植物为材料,对提取技术、组分鉴定、杀虫活性、作用机理和剂型配制进行了研究,旨在为新型植物源农药创制奠定基础。
(1)利用95%乙醇、三氯甲烷、丙酮、石油醚4种溶剂和浸渍、超声波、索氏3种提取方法,对大籽蒿、黄花蒿和猪毛蒿进行了活性成分提取。结果表明,以95%乙醇做溶剂的超声波提取法其提取效果最好。
(2)利用95%乙醇做溶剂对超声波提取条件进行了筛选,结果表明,以大籽蒿、黄花蒿和猪毛蒿3种植物为材料,在提取时间为40min、料液比为1：20、提取次数为3次、粉碎细度60目条件下,3种蒿属植物超声波提取物的杀虫效果最好。
(3)利用GC-MS联用分析技术,对3种蒿属植物的成分及其含量进行分析、鉴定。黄花蒿提取物含有1,8-桉叶油素、α-蒎烯、β-蒎烯等18种组分；大籽蒿提取物含有1,8-桉叶油素、α-蒎烯、β-蒎烯等14种组分；猪毛蒿含有1,8-桉叶油素、樟脑、p-石竹烯等6种组分。
(4)参考植物提取物各组分的含量,以1,8-桉叶油、β-蒎烯、α-蒎烯、α-萜品醇4种萜烯类单体为材料,分别对黄粉虫幼虫的活性进行了测定,其毒力大小依次为1,8-桉叶油>β-蒎烯>α-蒎烯>α-萜品醇。在4种萜烯类单体10个混配组合中,有9组混配组合具有毒力相加作用,占总数的90%,而1,8-桉叶油素、α-蒎烯与α-萜品醇按6：1：1混配对黄粉虫的共毒系数为2.8254,大于2.6,则表现出增效作用。
(5)LC20、LCso浓度的α-萜品醇、α-蒎烯、β-蒎烯和1,8-桉叶油对黄粉虫体内羧酸酯酶(CarE)、酸式磷酸酯酶(ACP)、碱式磷酸酯酶(ALP)、谷胱甘肽-s-转移酶(GSTs)、乙酰胆碱酯酶(AchE)、超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)12h、24h、36h、48h的影响存在差异性,但均表现出具有抑制功能,表明这4种萜烯类单体的毒性机理大致相同。当黄粉虫被4种萜烯类处理后,其体内的CarE、 ACP、 ALP、 GST、 AchE、 SOD、 POD、 CAT都会对这4种萜烯类单体的胁迫做出响应。黄粉虫体内的CarE和AchE均随着4种萜烯类浓度的增加其酶活逐渐下降,抑制作用增强。综合时间-剂量效应及酶受到抑制的程度来看,7种酶对4种萜烯类单体胁迫的敏感性从大到小依次为AchE> GSTs> ALP> ACP> CarE>SOD> POD> CAT。
(6)对4种制剂各种指标的测定结果表明,A和D乳油制剂为单相透明液体,无可见悬浮物或沉淀,有效成分含量≥40%,冷热贮稳定性合格,pH值为6.19-6.31,挥发率为23.79%-23.87%,均符合植物源农药的相关要求。
A乳油稀释100、200、300、400、500倍处理分月扇舟蛾幼虫时,其72h室内死亡率分别为92.22%、83.33%、75.56%、64.44%、57.78%； D乳油稀释100、200、300、400、500倍处理分月扇舟蛾幼虫时,其72h室内死亡率分别为88.89%、85.56%、77.78%、74.44%、64.44%。
A乳油稀释100-300倍喷雾林间防治分月扇舟蛾幼虫,处理1-7d的防效达58.89%-92.22%,防效均随浓度的增加而提高,稀释100倍时防效最好；D乳油稀释100-300倍喷雾防治分月扇舟蛾幼虫,1-7d的防效达58.89%-91.11%,防效均随浓度的增加而提高,稀释100倍时防效最高。
Asteraceae which has the most chemical composition, is the most complex genera in angiosperms. And among them, Artemisia plants are broader distributed and has strong insecticidal and antibacterial activity. It has great significance to study the insecticidal and antibacterial composition, bioactivity and mechanism of action of Artemisia plants. In this paper, we use three Artemisia plants for material, studying the extractive technology, component identification and insecticidal activity. Designed to lay the foundation for the discovery and the creation of new botanical pesticide.
(1)From the study of four extraction solvents, the95%ethanol, chloroform, acetone, sherwood oil and three extraction methods, soakage extraction method, ultrasonic extraction method, soxhlet extraction technique, the best extraction solvent for the three species of Artemisia was95%ethanol, the best extraction method was ultrasonic extraction method.
(2)The most suitbale extraction sovent of Artemisia sieversiana, Artemisia annua, Artemisia scoparia is95%ethanol. And the most optimum extraction method of Artemisia sieversiana, Artemisia annua, Artemisia scoparia is ultrasonic extraction method. In the condition of40minutes of extraction time,1:20of liquid-solid ratio,3times of extraction,60degree of fineness, the ultrasonic extra of three Artemisia plants have the best insecticidal effect.
(3)By GC-MS technique, we annalyzed and identified the ingredients of three Arte misia species.1,8-cineoie, α-pinene, β-pinene and other18kinds of substances may be the active ingredients of Artemisia annua.1,8-cineole, camphor, β-caryophyllene a nd other6kinds of active substances may be the active ingredient of Artemisia scopari a.
(4)Refer to the content of each component of the plant extracts, use1,8-cineole, β-pinene, α-pinene, α-terpene for material. The activity of the larva were determined respectively. The toxicity followed by1,8-cineole>β-pinene>α-pinene>α-terpene. Among the10kinds of combinations,9of them have the additive effect of toxicity, accounting for90%of the total number.And when the mixture of1,8-cineole, α-pinene, α-terpene is6:1:1, the toxicity coefficient to the larva is2.8254, greater than2.6, showed the additive effect.
(5)Though the LC20and LC50of α-terpineol, α-pinene, α-pinene and1,8-cineole terpenes monomer affect different to the seven kinds of enzymes, carboxylesterase (CarE), acid phosphatase(ACP), alkaline phosphatase(ALP), glutathione s-transferase(GSTs), acetylcholinesterase(AchE), superoxide dismutase(SOD), peroxidase (POD), catalase (CAT) in the tenebrio body in12h，24h，36h,48h. All of them showed inhibition effect, indicating that the toxicity mechanism of these four kins of terpenes monomer is toughly the same. After the experimental treatment of larva, the CarE, ACP, ALP, GSTs, AchE, SOD, POD, CAT in the body of the larva respond to these four kinds of terpenes monomer. With the increase of the concentration of the four kinds of terpenes, the acticity of CarE and AchE of tenebrio body is gradually decreased, the stronger the inhibition. Combining time-does effect and the inhibition degree of the enzyme, we can see that the stress sensitive7biochemical parameters to4kinds of terpenes monomer is AchE>GSTs>ALP>ACP>CarE> SOD>POD>CAT.
(6)Determination results of the various indicators on the four kinds of preparations showed that A and D emulsifiable concentrate preparation is single-phase transparent liquid, no visible suspended matter or sediment, active ingredient content≥40%, hot and cold storage stability is qualified, ph value is6.19-6.31, evaporation rate is23.79%-23.87%. Are in line with the relevant requirements of botanical pesticides.
A EC diluted100,200,300,400,500times, the processing Clostera moth larvae72h indoor mortality were92.22%,83.33%,75.56%,64.44%,57.78%. D EC diluted100,200,300,400times, the processing Clostera moth larvae for72h indoor mortality were88.89%,85.56%,77.78%,74.44%,64.44%.
A,40%homemade emulsifiable concentrate preparation dilute100-300times, the spraying control effect to the clostera anastomosis linnaeus and1-7d in the forest is58.89%-92.22%. Insecticidal effect is best when diluted100times. D,40%homemade emulsifiable concentrate preparation dilute100-300times, the spraying control effect to the clostera anastomosis linnaeus and1-7d in the forest is58.89%-91.11%. Insecticidal effect is best when diluted100times.
(1)利用95%乙醇、三氯甲烷、丙酮、石油醚4种溶剂和浸渍、超声波、索氏3种提取方法,对大籽蒿、黄花蒿和猪毛蒿进行了活性成分提取。结果表明,以95%乙醇做溶剂的超声波提取法其提取效果最好。
(2)利用95%乙醇做溶剂对超声波提取条件进行了筛选,结果表明,以大籽蒿、黄花蒿和猪毛蒿3种植物为材料,在提取时间为40min、料液比为1：20、提取次数为3次、粉碎细度60目条件下,3种蒿属植物超声波提取物的杀虫效果最好。
(3)利用GC-MS联用分析技术,对3种蒿属植物的成分及其含量进行分析、鉴定。黄花蒿提取物含有1,8-桉叶油素、α-蒎烯、β-蒎烯等18种组分；大籽蒿提取物含有1,8-桉叶油素、α-蒎烯、β-蒎烯等14种组分；猪毛蒿含有1,8-桉叶油素、樟脑、p-石竹烯等6种组分。
(4)参考植物提取物各组分的含量,以1,8-桉叶油、β-蒎烯、α-蒎烯、α-萜品醇4种萜烯类单体为材料,分别对黄粉虫幼虫的活性进行了测定,其毒力大小依次为1,8-桉叶油>β-蒎烯>α-蒎烯>α-萜品醇。在4种萜烯类单体10个混配组合中,有9组混配组合具有毒力相加作用,占总数的90%,而1,8-桉叶油素、α-蒎烯与α-萜品醇按6：1：1混配对黄粉虫的共毒系数为2.8254,大于2.6,则表现出增效作用。
(5)LC20、LCso浓度的α-萜品醇、α-蒎烯、β-蒎烯和1,8-桉叶油对黄粉虫体内羧酸酯酶(CarE)、酸式磷酸酯酶(ACP)、碱式磷酸酯酶(ALP)、谷胱甘肽-s-转移酶(GSTs)、乙酰胆碱酯酶(AchE)、超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)12h、24h、36h、48h的影响存在差异性,但均表现出具有抑制功能,表明这4种萜烯类单体的毒性机理大致相同。当黄粉虫被4种萜烯类处理后,其体内的CarE、 ACP、 ALP、 GST、 AchE、 SOD、 POD、 CAT都会对这4种萜烯类单体的胁迫做出响应。黄粉虫体内的CarE和AchE均随着4种萜烯类浓度的增加其酶活逐渐下降,抑制作用增强。综合时间-剂量效应及酶受到抑制的程度来看,7种酶对4种萜烯类单体胁迫的敏感性从大到小依次为AchE> GSTs> ALP> ACP> CarE>SOD> POD> CAT。
(6)对4种制剂各种指标的测定结果表明,A和D乳油制剂为单相透明液体,无可见悬浮物或沉淀,有效成分含量≥40%,冷热贮稳定性合格,pH值为6.19-6.31,挥发率为23.79%-23.87%,均符合植物源农药的相关要求。
A乳油稀释100、200、300、400、500倍处理分月扇舟蛾幼虫时,其72h室内死亡率分别为92.22%、83.33%、75.56%、64.44%、57.78%； D乳油稀释100、200、300、400、500倍处理分月扇舟蛾幼虫时,其72h室内死亡率分别为88.89%、85.56%、77.78%、74.44%、64.44%。
A乳油稀释100-300倍喷雾林间防治分月扇舟蛾幼虫,处理1-7d的防效达58.89%-92.22%,防效均随浓度的增加而提高,稀释100倍时防效最好；D乳油稀释100-300倍喷雾防治分月扇舟蛾幼虫,1-7d的防效达58.89%-91.11%,防效均随浓度的增加而提高,稀释100倍时防效最高。
Asteraceae which has the most chemical composition, is the most complex genera in angiosperms. And among them, Artemisia plants are broader distributed and has strong insecticidal and antibacterial activity. It has great significance to study the insecticidal and antibacterial composition, bioactivity and mechanism of action of Artemisia plants. In this paper, we use three Artemisia plants for material, studying the extractive technology, component identification and insecticidal activity. Designed to lay the foundation for the discovery and the creation of new botanical pesticide.
(1)From the study of four extraction solvents, the95%ethanol, chloroform, acetone, sherwood oil and three extraction methods, soakage extraction method, ultrasonic extraction method, soxhlet extraction technique, the best extraction solvent for the three species of Artemisia was95%ethanol, the best extraction method was ultrasonic extraction method.
(2)The most suitbale extraction sovent of Artemisia sieversiana, Artemisia annua, Artemisia scoparia is95%ethanol. And the most optimum extraction method of Artemisia sieversiana, Artemisia annua, Artemisia scoparia is ultrasonic extraction method. In the condition of40minutes of extraction time,1:20of liquid-solid ratio,3times of extraction,60degree of fineness, the ultrasonic extra of three Artemisia plants have the best insecticidal effect.
(3)By GC-MS technique, we annalyzed and identified the ingredients of three Arte misia species.1,8-cineoie, α-pinene, β-pinene and other18kinds of substances may be the active ingredients of Artemisia annua.1,8-cineole, camphor, β-caryophyllene a nd other6kinds of active substances may be the active ingredient of Artemisia scopari a.
(4)Refer to the content of each component of the plant extracts, use1,8-cineole, β-pinene, α-pinene, α-terpene for material. The activity of the larva were determined respectively. The toxicity followed by1,8-cineole>β-pinene>α-pinene>α-terpene. Among the10kinds of combinations,9of them have the additive effect of toxicity, accounting for90%of the total number.And when the mixture of1,8-cineole, α-pinene, α-terpene is6:1:1, the toxicity coefficient to the larva is2.8254, greater than2.6, showed the additive effect.
(5)Though the LC20and LC50of α-terpineol, α-pinene, α-pinene and1,8-cineole terpenes monomer affect different to the seven kinds of enzymes, carboxylesterase (CarE), acid phosphatase(ACP), alkaline phosphatase(ALP), glutathione s-transferase(GSTs), acetylcholinesterase(AchE), superoxide dismutase(SOD), peroxidase (POD), catalase (CAT) in the tenebrio body in12h，24h，36h,48h. All of them showed inhibition effect, indicating that the toxicity mechanism of these four kins of terpenes monomer is toughly the same. After the experimental treatment of larva, the CarE, ACP, ALP, GSTs, AchE, SOD, POD, CAT in the body of the larva respond to these four kinds of terpenes monomer. With the increase of the concentration of the four kinds of terpenes, the acticity of CarE and AchE of tenebrio body is gradually decreased, the stronger the inhibition. Combining time-does effect and the inhibition degree of the enzyme, we can see that the stress sensitive7biochemical parameters to4kinds of terpenes monomer is AchE>GSTs>ALP>ACP>CarE> SOD>POD>CAT.
(6)Determination results of the various indicators on the four kinds of preparations showed that A and D emulsifiable concentrate preparation is single-phase transparent liquid, no visible suspended matter or sediment, active ingredient content≥40%, hot and cold storage stability is qualified, ph value is6.19-6.31, evaporation rate is23.79%-23.87%. Are in line with the relevant requirements of botanical pesticides.
A EC diluted100,200,300,400,500times, the processing Clostera moth larvae72h indoor mortality were92.22%,83.33%,75.56%,64.44%,57.78%. D EC diluted100,200,300,400times, the processing Clostera moth larvae for72h indoor mortality were88.89%,85.56%,77.78%,74.44%,64.44%.
A,40%homemade emulsifiable concentrate preparation dilute100-300times, the spraying control effect to the clostera anastomosis linnaeus and1-7d in the forest is58.89%-92.22%. Insecticidal effect is best when diluted100times. D,40%homemade emulsifiable concentrate preparation dilute100-300times, the spraying control effect to the clostera anastomosis linnaeus and1-7d in the forest is58.89%-91.11%. Insecticidal effect is best when diluted100times.
引文
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