黄花蒿对粘虫杀虫活性成分及机制研究

doi:10.13304/j.nykjdb.2023.0589

摘要/Abstract

摘要：

为研究黄花蒿石油醚萃取物对粘虫3龄幼虫杀虫活性成分及初步作用机制，采用硅胶柱色谱进行分离，气相-质谱法分析化学成分，浸虫法测定不同馏分及主要成分的触杀活性，并进一步对活性成分进行粘虫幼虫体内靶标酶和代谢酶活性测定。结果表明，黄花蒿石油醚萃取物经硅胶柱色谱分离，共得到27个馏分，其中馏分8表现出较好的触杀活性。经气相色谱-质谱法检测，十三烷、4-喹啉甲醛、石竹烯氧化物、亚油酸乙酯和亚麻酸乙酯为主要化合物，含量分别为7.9%、72.8%、3.6%、2.4%、3.1%。这5种化合物处理粘虫7 d的半数致死浓度（LC₅₀）分别为7.9、2.5、3.6、5.4和1.5 mg·mL^-1，即亚麻酸乙酯对粘虫的触杀活性最好。扫描电镜结果显示，1.5 mg·mL^-1亚麻酸乙酯处理粘虫7 d，粘虫表皮蜡质层结构排列疏松，蜡质层颗粒明显消失，蜡花间隙增大；在处理过程中，粘虫的乙酰胆碱酯酶和羧酸酯酶表现出先激活后抑制作用；Na⁺-K⁺-ATPase表现出持续抑制作用；过氧化氢酶表现出先抑制后激活作用。综上，亚麻酸乙酯为黄花蒿对粘虫杀虫活性的主要化合物，研究结果为黄花蒿资源的开发利用以及将亚麻酸乙酯作为生物防治粘虫的潜力化合物提供参考。

关键词: 黄花蒿, 粘虫, 亚麻酸乙酯, 作用机制

Abstract:

In order to study the insecticidal activity components and preliminary mechanism of the petroleum ether extract from Artemisia annua against Mythimna separata， the insecticidal activity of different fractions and main components were measured by insect immersion method. The petroleum ether extract was separated by column chromatography and analyzed by gas chromatography-mass spectrometry to determine its chemical components， and the target enzyme and metabolic enzyme activities in the body of Mythimna separata were measured for the active components. The results showed that the petroleum ether extract of Artemisia annua was separated by silica gel column chromatography， and 27 fractions were obtained， of which fraction 8 showed the best contact toxicity. According to gas chromatography-mass spectrometry， the main compounds were tridecane， 4-quinolinecarboxaldehyde， caryophyllene oxide， linoleic acid ethyl ester， linolenic acid ethyl ester with contents of 7.9%， 72.8%， 3.6%， 2.4%， 3.1%，respectively. The lethal concentration of 50% （LC₅₀） of above 5 compounds on the 7th day were 7.9， 2.5， 3.6， 5.4 and 1.5 mg·mL^-1， respectively， which indicated that ethyl linolenic acid had significant contact toxicity against M. separata. On the 7th day of treatment with 1.5 mg·mL^-1 linolenic acid ethyl ester， the scanning electron microscopy results showed that the wax layer structure in the surface of M. separata were loosely， the wax layer particles disappeared significantly， and the wax flower gap increased. And the acetylcholinesterase （AChE） and carboxylesterase （CarE） of M. separata treated with 1.5 mg·mL^-1 linolenic acid ethyl ester showed first activation and then inhibition， Na⁺-K⁺-ATPase showed inhibition over time， and catalase （CAT） showed first inhibition and then activation. To sum up， the linolenic acid ethyl ester was the main active compound of A. annua，which provided reference for the development of new plant-based insecticides and biological control of M. separata and the development and utilization of A.annua resources.

Key words: Artemisia annua, Mythimna separate, linolenic acid ethyl ester, mechanism

中图分类号:

S476

李春燕, 梁倩, 高成香, 梁宗锁, 王芳. 黄花蒿对粘虫杀虫活性成分及机制研究[J]. 中国农业科技导报, 2024, 26(12): 129-137.

Chunyan LI, Qian LIANG, Chengxiang GAO, Zongsuo LIANG, Fang WANGA. Study on Insecticidal Activity Constituents and Mechanism of Artemisia annua Against Mythimna separate[J]. Journal of Agricultural Science and Technology, 2024, 26(12): 129-137.

图/表 5

参考文献 27

1	赵玉玉,李伯辽,李梅梅,等.粘虫对高效氯氰菊酯抗性机制的初步研究[J].应用昆虫学报,2018,55(5):857-864.
	ZHAO Y Y, LI B L, LI M M, et al.. Preliminary research on the resistance mechanism of Mythimna separata to beta-cypermethrin [J]. Chin. J. Appl. Entomol., 2018, 55 (5):857-864.
2	LI H, LIU F F, FU L Q, et al.. Identification of 35 c-type lectins in the oriental armyworm, Mythimna separata (Walker) [J/OL]. Insects, 2021, 12(6):559 [2023-07-10]. .
3	江幸福,张蕾,程云霞,等.我国粘虫发生危害新特点及趋势分析[J].应用昆虫学报,2014,51(6):1444-1449.
	JIANG X F, ZHANG L, CHENG Y X, et al.. Novel features occurrence trends and economic impact of the oriental armyworm, Mythimna separata (Walker) in China [J]. Chin. J. Appl. Entomol., 2014, 51(6):1444-1449.
4	王娟,孔勇,李伯辽,等.5种杀虫剂对粘虫不同发育阶段的室内毒力[J].西北农业学报,2016,25(7):1097-1102.
	WANG J, KONG Y, LI B L, et al.. Indoor toxicity determination of five kinds of insecticides on Mythimna separata (Walker) at different developmental stages [J]. Acta Agric. Bor-Occid. Sin., 2016, 25(7):1097-1102.
5	YAN Y, LIU Q K, ZANG X, et al.. Resistance-gene-directed discovery of a natural-product herbicide with a new mode of action [J]. Nature, 2018, 559:415-418.
6	李晓维,程江辉,韩海斌,等. 植物次生代谢物质对蓟马的行为调控作用[J].昆虫学报,2022, 65(9):1222-1246.
	LI X W, CHENG J H, HAN H B, et al.. Behavioral manipulation of the plant secondary metabolites to thrips and their application in thrips management [J]. Acta Entomol. Sin., 2022, 65(9):1222-1246.
7	中国科学院中国植物志编辑委员会.中国植物志[M].北京:科学出版社,1991:1062.
8	李云寿,邹华英,唐绍宗,等.14种菊科植物提取物对菜青虫的杀虫活性[J].华东昆虫学报,2000,9(2):99-101.
	LI Y S, ZOU H Y, TANG S Z, et al.. Insecticidal activity of extracts from fourteen compositae plants against larvae of cabbage white butterfly Pieris rapae L. [J]. Entomol. J. East China, 2000, 9(2):99-101.
9	MAGGI M E, MANGEAUD A, CARPINELLA M C, et al.. Laboratory evaluation of Artemisia annua L. extract and artemisinin activity against Epilachna paenulata and Spodoptera eridania [J]. J. Chem. Ecol., 2005, 31(7):1527-1536.
10	DEB M, KUMAR D. Bioactivity and efficacy of essential oils extracted from Artemisia annua against Tribolium casteneum(Herbst.1797)(Coleoptera:Tenebrionidae): an eco-friendly approach [J/OL]. Ecotox. Environ. Safe., 2019, 189:109988 [2023-07-10]. .
11	王振吉,杨申明,沈慧,等.黄花蒿提取物对菜蚜的杀虫活性研究[J].西部林业科学,2014,43(4):136-139.
	WANG Z J, YANG S M, SHEN H, et al.. Bioassay of insecticidal effect of extract from Artemisia annua on Lipaphis erysimi [J]. J. West Chin. For. Sci., 2014, 43(4):136-139.
12	张永强,丁伟,赵志模,等.黄花蒿提取物对朱砂叶螨生物活性的研究[J].中国农业科学,2008,41(3):720-726.
	ZHANG Y Q, DING W, ZHAO Z M, et al.. Studies on acarcidal bioactivities of the extracts from Artemisia annua L. against Tetranychus cinnabarinus Bois. (Acari: Tetranychidae) [J]. Sci. Agric. Sin., 2008, 41(3):720-726.
13	朱芬,雷朝亮,王健.黄花蒿粗提物对几种害虫拒食性的初步研究[J].昆虫天敌,2003,5(1):16-19.
	ZHU F, LEI C L, WANG J. Acti-feedant activity of crude extract of Artemisia annua. on some insect pests [J]. Nat. Enemies Insects, 2003, 25(1):16-19.
14	BEDINI S, FLAMINI G, COSCI F, et al.. Artemisia spp. essential oils against the disease-carrying blowfly Calliphora vomitoria [J]. Parasite Vector, 2017, 10(1):1-10.
15	程昉,邵亚洲,杨盈盈,等.黄花蒿挥发油对赤拟谷盗成虫和幼虫的生物活性研究[J].中国粮油学报,2020,35(11):119-124.
	CHENG F, SHAO Y Z, YANG Y Y, et al.. Biological activity of Artemisia annua essential oil against adults and larvae of Tribolium castaneum [J]. J. Chin. Cereals Oils Assoc., 2020, 35(11):119-124.
16	TRIPATHI A K, PRAJAPATI V, AGGARWAL K K, et al.. Toxicity, feeding deterrence, and effect of activity of 1,‍8-cineole from Artemisia annua on progeny production of Tribolium castanaeum (Coleoptera: Tenebrionidae) [J]. J. Econ. Entomol., 2001, 94(4):979-983.
17	李春燕,高成香,梁倩.黄花蒿对粘虫的杀虫活性研究[J].现代园艺,2023,46(13):4-9.
	LI C Y, GAO C X, LIANG Q. Study on insecticidal activity of Artemisia annua against M. separata [J]. Mod. Hortic., 2023, 46 (13): 4-9.
18	张新瑞,胡冠芳,刘敏艳,等.红蓼提取物杀虫活性及其对粘虫的作用方式[J].草地学报,2009,17(3):377-382.
	ZHANG X R, HU G F, LIU M Y, et al.. Insecticidal activity of extracts from Polygonum orientale L. and their actionways against Mythimna separate walker [J]. Acta Agric. Sin., 2009, 17(3):377-382.
19	ZHU X Z, SUN M X, ZHANG Z W, et al.. Design synthesis and insecticidal activities of 4-propargyloxybenzene sulfonamide derivatives substituted with amino acids [J]. J. Asian Nat. Prod. Res., 2023 25(4):379-386.
20	LI Y K, WEI J Q, FANG J M, et al.. Insecticidal activity of four lignans isolated from Phryma leptostachya [J/OL]. Molecules, 2019, 24:1976 [2023-07-10]. .
21	於祥,陈娅芳,张艳,等.欧芹酚甲醚腙类衍生物的设计合成及抗乙酰胆碱酯酶活性研究[J].化学研究与应用,2023,35(1):91-97.
	YU X, CHEN Y F, ZHANG Y, et al.. Synthesis and anti-acetylcholinesterase evaluation of osthole esters [J]. Chem. Res. Appl., 2023, 35(1):91-97.
22	陈宇鹏.桔小实蝇不同蛹色品系的抗药性机制[D].广州:华南农业大学,2019.
	CHEN Y P. Insecticide resistance mechanism of pupa strains with different coloration of Bactrocera dorsalis (Hendel) [D]. Guangzhou: South China Agricultural University, 2019.
23	SHEN G M, DOU W, NIU J Z, et al.. Transcriptome analysis of the oriental fruit fly (Bactrocera dorsalis) [J/OL]. PLoS One, 2011, 6 (12): e29127 [2023-07-10]. .
24	YANG S Y, WU H H, XIE J C, et al.. Depressed performance and detoxification enzyme activities of Helicoverpa armigera fed with conventional cotton foliage subjected to methyl jasmonate exposure [J]. Entomol. Exp. Appl., 2013, 147(2):186-195.
25	凌斯全.Itol A对褐飞虱的生理影响及作用机理的初步研究[D].南宁:广西大学,2017.
	LIN S Q. Preliminary study on physiological effect and insecticidal mechanism of Itol a against brown planthopper Nilaparvat alugens (Stal) [D]. Nanning: Guangxi University, 2017.
26	陈义娟.香樟叶提取物中杀螨活性物质的分离鉴定及作用机理研究[D].上海:上海交通大学,2017.
	CHEN Y J. Isolation, identificantion and mechanism of the acaricidal compound from Cinnamomum camphora against Tetranychus cinnabarinus [D]. Shanghai: Shanghai Jiao Tong University, 2017.
27	王召,彭舒,孟立霞.噻虫嗪对鲫鱼的急性毒性和抗氧化酶及乙酰胆碱酯酶活性的影响[J].水产科学,2020,39(6):922-927.
	WANG Z, PENG S, MENG L X. Acute toxicity of thiamethoxam and activities of antioxidant enzymes and acetylcholinesterase in juvenile crucian carp Carassius auratus [J]. Fisheries Sci., 2020, 39(6):922-927.

馏分 Fraction	处理时间Treatment time/d
馏分 Fraction	1	3	5	7
1	4.0±2.5 i	4.0±2.5 j	4.0±2.5 n	4.0±2.5 m
2	32.0±2.0 d	36.0±2.5 e	36.0±2.5 d	36.0±2.5 e
3	32.0±2.0 d	36.0±2.5 e	36.0±2.5 d	36.0±2.5 e
4	4.0±2.5 i	10.0±0.0 i	10.0±0.0 l	10.0±0.0 l
5	18.0±2.0 g	32.0±3.7 f	38.0±0.2 d	56.0±2.5 d
6	28.0±2.0 e	78.0±2.0 b	78.0±2.0 b	78.0±2.0 b
7	46.0±2.5 c	60.0±0.0 d	60.0±0.0 c	70.0±0.0 c
8	100.0±0.0 a	100.0±0.0 a	100.0±0.0 a	100.0±0.0 a
9	66.0±2.5 b	72.0±2.0 c	76.0±2.5 b	76.0±2.5 b
10	6.0±2.5 h	10.0±0.0 i	16.0±4.0 j	20.0±5.5 h
11	4.0±2.5 i	10.0±0.0 i	12.0±2.0 k	14.0±2.5 j
12	12.0±2.0 g	20.0±0.0 g	20.0±0.0 i	24.0±2.5 g
13	2.0±2.0 i	10.0±0.0 i	12.0±2.0 k	12.0±2.0 k
14	34.0±2.5 d	42.0±2.0 e	42.0±2.0 d	42.0±2.0 e
15	34.0±2.5 d	36.0±2.5 e	38.0±2.0 d	40.0±0.0 e
16	2.0±2.0 i	8.0±2.0 j	8.0±2.0 m	8.0±2.0 l
17	12.0±2.0 g	18.0±2.0 g	20.0±0.0 i	20.0±0.0 h
18	12.0±2.0 g	16.0±2.5 h	18.0±2.0 i	18.0±2.0 i
19	28.0±2.0 e	32.0±2.0 f	36.0±2.5 d	36.0±2.5 e
20	16.0±2.5 g	18.0±2.0 g	18.0±2.0 i	18.0±2.0 i
21	4.0±2.5 i	6.0±2.5 j	8.0±2.0 m	8.0±2.0 l
22	36.0±2.5 d	40.0±0.0 e	40.0±0.0 d	40.0±0.0 e
23	14.0±2.5 g	22.0±2.0 g	26.0±2.5 g	26.0±2.5 g
24	18.0±2.0 g	22.0±2.0 g	24.0±2.5 h	24.0±2.5 g
25	18.0±2.0 g	24.0±2.5 g	30.0±0.0 f	30.0±0.0 f
26	26.0±4.0 f	32.0±2.0 f	34.0±2.5 e	38.0±2.0 e
27	6.0±2.5 h	6.0±2.5 j	10.0±0.0	10.0±0.0 l

馏分 Fraction	处理时间Treatment time/d
馏分 Fraction	1	3	5	7
1	4.0±2.5 i	4.0±2.5 j	4.0±2.5 n	4.0±2.5 m
2	32.0±2.0 d	36.0±2.5 e	36.0±2.5 d	36.0±2.5 e
3	32.0±2.0 d	36.0±2.5 e	36.0±2.5 d	36.0±2.5 e
4	4.0±2.5 i	10.0±0.0 i	10.0±0.0 l	10.0±0.0 l
5	18.0±2.0 g	32.0±3.7 f	38.0±0.2 d	56.0±2.5 d
6	28.0±2.0 e	78.0±2.0 b	78.0±2.0 b	78.0±2.0 b
7	46.0±2.5 c	60.0±0.0 d	60.0±0.0 c	70.0±0.0 c
8	100.0±0.0 a	100.0±0.0 a	100.0±0.0 a	100.0±0.0 a
9	66.0±2.5 b	72.0±2.0 c	76.0±2.5 b	76.0±2.5 b
10	6.0±2.5 h	10.0±0.0 i	16.0±4.0 j	20.0±5.5 h
11	4.0±2.5 i	10.0±0.0 i	12.0±2.0 k	14.0±2.5 j
12	12.0±2.0 g	20.0±0.0 g	20.0±0.0 i	24.0±2.5 g
13	2.0±2.0 i	10.0±0.0 i	12.0±2.0 k	12.0±2.0 k
14	34.0±2.5 d	42.0±2.0 e	42.0±2.0 d	42.0±2.0 e
15	34.0±2.5 d	36.0±2.5 e	38.0±2.0 d	40.0±0.0 e
16	2.0±2.0 i	8.0±2.0 j	8.0±2.0 m	8.0±2.0 l
17	12.0±2.0 g	18.0±2.0 g	20.0±0.0 i	20.0±0.0 h
18	12.0±2.0 g	16.0±2.5 h	18.0±2.0 i	18.0±2.0 i
19	28.0±2.0 e	32.0±2.0 f	36.0±2.5 d	36.0±2.5 e
20	16.0±2.5 g	18.0±2.0 g	18.0±2.0 i	18.0±2.0 i
21	4.0±2.5 i	6.0±2.5 j	8.0±2.0 m	8.0±2.0 l
22	36.0±2.5 d	40.0±0.0 e	40.0±0.0 d	40.0±0.0 e
23	14.0±2.5 g	22.0±2.0 g	26.0±2.5 g	26.0±2.5 g
24	18.0±2.0 g	22.0±2.0 g	24.0±2.5 h	24.0±2.5 g
25	18.0±2.0 g	24.0±2.5 g	30.0±0.0 f	30.0±0.0 f
26	26.0±4.0 f	32.0±2.0 f	34.0±2.5 e	38.0±2.0 e
27	6.0±2.5 h	6.0±2.5 j	10.0±0.0	10.0±0.0 l

序号 No.	保留时间 Retention time/min	化合物 Compound	相对含量 Relative content/%
1	8.7	十三烷Tridecane	7.9
2	10.3	丁香酚Eugenol	1.4
3	15.3	4-喹啉甲醛 4-quinolinecarboxaldehyde	72.8
4	16.6	石竹烯氧化物Caryophyllene oxide	3.6
5	17.9	9，10-脱氢环异龙胆烯 9，10-dehydro-cycloisolongifolene	1.9
6	23.4	6，10，14-三甲基-2-十五烷酮 6，10，14-trimethyl-2-pentadecanone	1.0
7	30.9	亚油酸乙酯 Linoleic acid ethyl ester	2.4
8	31.0	亚麻酸乙酯 Linolenic acid ethyl ester	3.1

序号 No.	保留时间 Retention time/min	化合物 Compound	相对含量 Relative content/%
1	8.7	十三烷Tridecane	7.9
2	10.3	丁香酚Eugenol	1.4
3	15.3	4-喹啉甲醛 4-quinolinecarboxaldehyde	72.8
4	16.6	石竹烯氧化物Caryophyllene oxide	3.6
5	17.9	9，10-脱氢环异龙胆烯 9，10-dehydro-cycloisolongifolene	1.9
6	23.4	6，10，14-三甲基-2-十五烷酮 6，10，14-trimethyl-2-pentadecanone	1.0
7	30.9	亚油酸乙酯 Linoleic acid ethyl ester	2.4
8	31.0	亚麻酸乙酯 Linolenic acid ethyl ester	3.1

处理时间/d Treatment time	化合物 Compound	半数致死浓度 LC₅₀/（mg∙mL^-1）	95%置信区间 95% CI	斜率 Slope	卡方 χ²	P值 P value
1	十三烷Tridecane	10.0 d	8.6~15.6	1.8±0.2	3.6	0.3
	4-喹啉甲醛 4-quinolinecarboxaldehyde	9.8 c	8.1~12.9	2.2±0.2	4.1	0.3
	石竹烯氧化物 Caryophyllene oxide	34.6 e	19.4~108.6	1.4±0.3	2.6	0.5
	亚油酸乙酯 Linoleic acid ethyl ester	6.6 b	4.9~10.1	3.5±0.3	8.4	0.0
	亚麻酸乙酯 Linolenic acid ethyl ester	4.6 a	2.4~16.6	2.2±0.2	22.1	0.0
3	十三烷 Tridecane	9.8 d	7.5~14.2	1.5±0.2	0.3	1.0
	4-喹啉甲醛 4-quinolinecarboxaldehyde	5.8 b	4.8~7.4	1.7±0.2	1.4	0.7
	石竹烯氧化物 Caryophyllene oxide	33.6 e	18.0~113.4	1.1±0.2	0.5	0.9
	亚油酸乙酯 Linoleic acid ethyl ester	6.4 c	4.4~12.2	2.5±0.2	9.4	0.0
	亚麻酸乙酯 Linolenic acid ethyl ester	3.1 a	1.6~7.5	1.8±0.2	15.8	0.0
5	十三烷 Tridecane	9.3 e	7.1~13.5	1.4±0.2	1.1	0.8
	4-喹啉甲醛 4-quinolinecarboxaldehyde	2.6 b	0.2~79.2	1.1±0.1	15.2	0.0
	石竹烯氧化物 Caryophyllene oxide	8.8 d	4.6~61.3	0.5±0.1	0.7	0.9
	亚油酸乙酯 Linoleic acid ethyl ester	5.5 c	3.6~10.6	2.1±0.2	9.0	0.0
	亚麻酸乙酯 Linolenic acid ethyl ester	2.0 a	1.2~3.2	1.7±0.2	8.1	0.0
7	十三烷 Tridecane	7.9 e	6.2~10.7	1.5±0.2	2.3	0.5
	4-喹啉甲醛 4-quinolinecarboxaldehyde	2.5 b	0.0~9 134 638.4	1.1±0.1	17.8	0.0
	石竹烯氧化物 Caryophyllene oxide	3.6 c	1.8~13.0	0.4±0.1	0.31	1.0
	亚油酸乙酯 Linoleic acid ethyl ester	5.4 d	3.7~9.6	2.1±0.2	7.2	0.1
	亚麻酸乙酯 Linolenic acid ethyl ester	1.5 a	1.2~1.8	1.6±0.2	4.6	0.2