油茶壳杀黏虫活性成分鉴定及其作用机制

doi:10.3864/j.issn.0578-1752.2025.13.008

摘要/Abstract

摘要：

【目的】明确油茶副产物油茶壳对黏虫（Mythimna separata）的杀虫活性成分及作用机制，为黏虫植物源杀虫剂的开发提供依据。【方法】通过浸虫法和浸叶法测定油茶壳石油醚、三氯甲烷、乙酸乙酯和水提取物对黏虫的杀虫活性；石油醚提取物经硅胶柱层析分离，气相色谱-质谱分析，确定其活性成分；通过非靶向代谢组学技术，对黏虫的差异代谢物进行注释、筛选和分析。【结果】油茶壳石油醚提取物对黏虫的触杀活性最好，其对黏虫1、3、5和7 d触杀活性的LC₅₀分别为9.53、8.00、7.51和7.02 mg·mL^-1。油茶壳石油醚提取物经硅胶柱层析分离，得到20个馏分，馏分6对黏虫的触杀活性最好，1、3、5和7 d触杀活性的LC₅₀分别为3.99、3.85、3.72和3.72 mg·mL^-1。馏分6再分离，得到21个馏分，馏分6.4对黏虫的触杀活性最好；经气相色谱-质谱法分析，正十六烷（19.33%）、1-十六烯（14.21%）、二十八烷（4.03%）、十八烷（9.24%）、二氯菊酸（6.73%）、1-十八烯（7.27%）、二十四烷醇（3.87%）和异植醇（5.92%）为主要化合物，其中二氯菊酸对黏虫的触杀活性最好，1、3、5和7 d触杀活性的LC₅₀分别为91.62、55.61、34.94和24.43 μg·mL^-1，与阳性对照除虫菊酯的杀虫活性相当。通过非靶向代谢组学技术，筛选得到6个差异代谢物，其中云杉苷、大麻素、5-氟大麻素代谢物和己酰甘氨酸显著上调，而4-乙基-2,6-二羟基苯基硫酸氢酯和N-二十五酰甘氨酸显著下调。这些差异代谢物主要富集在辅因子的生物合成、烟酸和烟酰胺代谢、色氨酸代谢、泛醌和其他萜类醌的生物合成4个信号通路中。【结论】油茶壳中对黏虫具有良好杀虫活性的化合物为二氯菊酸，研究结果可为黏虫的生物防治及油茶壳高值化利用提供新方向。

关键词: 油茶壳, 黏虫, 触杀活性, 二氯菊酸, 代谢组学

Abstract:

【Objective】 The objective of this study is to clarify the insecticidal active component and mechanism of Camellia oleifera shell on Mythimna separate, and to provide a basis for the development of botanical insecticides for M. separata. 【Method】 The insecticidal activity of petroleum ether, chloroform, ethyl acetate, and aqueous extracts of C. oleifera shell against 3rd instar larvae of M. separata was evaluated using the immersion and leaf-dipping methods. The petroleum ether extract was subjected to silica gel column chromatography for separation, followed by gas chromatography-mass spectrometry (GC-MS) analysis to identify the active components. Furthermore, a non-targeted metabolomics approach was employed to annotate, screen, and analyze differential metabolites in M. separata. 【Result】 The petroleum ether extract of C. oleifera shell exhibited the highest contact toxicity against M. separata, with LC₅₀values of 9.53, 8.00, 7.51, and 7.02 mg·mL^-1 on 1, 3, 5, and 7 d, respectively. The petroleum ether extract was separated by silica gel column chromatography to obtain 20 fractions, among which fraction 6 showed the best contact toxicity against M. separata, with LC₅₀ values of 3.99, 3.85, 3.72, and 3.72 mg·mL^-1 on 1, 3, 5, and 7 d, respectively. Further separation of fraction 6 yielded 21 subfractions, with subfraction 6.4 demonstrating the highest contact toxicity. GC-MS analysis identified hexadecane (19.33%), 1-hexadecene (14.21%), octacosane (4.03%), octadecane (9.24%), dichlorochrysanthemic acid (6.73%), 1-octadecene (7.27%), 1-tetracosanol (3.87%) and isophytol (5.92%) as the main compounds. Among these, dichlorochrysanthemic acid exhibited the strongest contact toxicity against M. separata, with LC₅₀ values of 91.62, 55.61, 34.94, and 24.43 μg·mL^-1 on 1, 3, 5, and 7 d, respectively. Its insecticidal efficacy was comparable to that of the positive control, pyrethrin. Non-targeted metabolomics analysis identified six differential metabolites, among which piceid, cannabinoid, 5-fluoro-cannabinoid metabolite, and caproylglycine were significantly upregulated, while 4-ethyl-2,6-dihydroxyphenyl sulfate and N-pentacosanoylglycine were significantly downregulated. These differential metabolites were primarily enriched in four metabolic pathways: cofactor biosynthesis, tryptophan metabolism, niacin and nicotinamide metabolism, ubiquinone and terpenoid-quinone biosynthesis. 【Conclusion】 Dichlorochrysanthemic acid from C. oleifera shell extract exhibits insecticidal activity against M. separata, providing a new avenue for the biological control of M. separata and the high-value utilization of C. oleifera shell.

Key words: Camellia oleifera shell, Mythimna separata, contact toxicity, dichlorochrysanthemic acid, metabolomics

詹丽, 梁宗锁, 于靖, 卢俊, 梁倩. 油茶壳杀黏虫活性成分鉴定及其作用机制[J]. 中国农业科学, 2025, 58(13): 2591-2603.

ZHAN Li, LIANG ZongSuo, YU Jing, LU Jun, LIANG Qian. Insecticidal Active Component Identification of Camellia oleifera Shell Against Mythimna separata and Its Action Mechanism[J]. Scientia Agricultura Sinica, 2025, 58(13): 2591-2603.

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https://www.chinaagrisci.com/CN/Y2025/V58/I13/2591

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时间 Time (min)	流动相Mobile phase A (v%)	流动相B Mobile phase B (v%)
0	95	5
0.5	95	5
5.5	50	50
9.0	5	95
10.5	5	95
10.6	95	5
12.0	95	5

杀虫方式 Insecticidal method	提取物 Extract	校正死亡率Corrected mortality rate (%)
杀虫方式 Insecticidal method	提取物 Extract	1 d	3 d	5 d	7 d
触杀活性 Contact toxicity	石油醚Petroleum ether	84.00±2.45a	84.00±2.45a	84.00±2.45a	84.00±2.45a
	三氯甲烷Chloroform	6.00±2.45c	12.00±3.74c	12.00±3.74d	16.00±5.10d
	乙酸乙酯Ethyl acetate	42.00±12.81b	42.00±12.81b	42.00±12.81bc	46.00±13.27bc
	水Aqueous	4.00±2.45c	6.00±2.45c	10.00±3.16d	14.00±5.10d
胃毒活性 Stomach toxicity	石油醚Petroleum ether	82.00±7.35a	84.00±6.00a	86.00±6.78a	86.00±6.78a
	三氯甲烷Chloroform	52.00±8.60b	54.00±7.48b	58.00±8.00b	58.00±8.00b
	乙酸乙酯Ethyl acetate	14.00±2.45c	18.00±5.83c	18.00±5.83cd	26.00±6.78cd
	水Aqueous	14.00±5.10c	18.00±5.83c	18.00±5.83cd	22.00±3.74cd

杀虫方式 Insecticidal method	处理时间 Treatment time (d)	致死中浓度 LC₅₀ (mg∙mL^-1)	95%置信区间 95% CI	斜率±标准误 Slope±standard error	卡方 χ²	P值 P value
触杀活性 Contact toxicity	1	9.53	5.31-43.85	3.38±0.30	25.00	<0.001
	3	8.00	6.04-11.55	3.00±0.25	6.95	0.073
	5	7.51	6.68-8.55	2.83±0.23	4.87	0.181
	7	7.02	6.24-7.98	2.76±0.23	4.84	0.184
胃毒活性 Stomach toxicity	1	10.34	5.48-125.52	3.68±0.34	27.66	<0.001
	3	9.41	4.36-858.62	2.79±0.24	32.84	<0.001
	5	8.76	4.48-59.47	2.90±0.25	28.16	<0.001
	7	8.76	4.48-59.47	2.90±0.25	28.16	<0.001

馏分 Fraction	校正死亡率Corrected mortality rate (%)
馏分 Fraction	1 d	3 d	5 d	7 d
1	22.00±2.00c	22.00±2.00cd	26.00±2.45cd	26.00±2.45cd
2	—	22.00±2.00cd	30.00±0c	30.00±0c
3	46.00±2.45b	52.00±2.00b	52.00±2.00b	54.00±2.45b
4	10.00±3.16def	10.00±3.16efg	10.00±3.16ghij	10.00±3.16ghij
5	6.00±2.45efgh	8.00±2.00efg	12.00±2.00fghi	12.00±2.00fghi
6	98.00±2.00a	98.00±2.00a	98.00±2.00a	98.00±2.00a
7	6.00±2.45efgh	8.00±2.00efg	10.00±0ghij	10.00±0ghij
8	12.00±2.00de	14.00±2.45def	16.00±2.45efg	16.00±2.45efg
9	8.00±2.00efg	14.00±2.45def	18.00±2.00ef	18.00±2.00ef
10	10.00±0def	16.00±2.45de	22.00±2.00de	22.00±2.00de
11	4.00±2.45fgh	4.00±2.45g	10.00±0ghij	10.00±0ghij
12	16.00±2.45cd	20.00±0cd	22.00±2.00de	22.00±2.00de
13	2.00±2.00gh	4.00±2.45g	8.00±2.00hij	8.00±2.00hij
14	2.00±2.00gh	2.00±2.00g	4.00±2.45j	4.00±2.45j
15	2.00±2.00gh	8.00±2.00efg	8.00±2.00hij	10.00±0ghij
16	2.00±2.00gh	6.00±2.45fg	16.00±2.45efg	16.00±2.45efg
17	52.00±2.00b	56.00±2.45b	56.00±2.45b	56.00±2.45b
18	4.00±2.45fgh	6.00±2.45fg	14.00±2.45fgh	14.00±2.45fgh
19	—	2.00±2.00g	6.00±2.45ij	6.00±2.45ij
20	22.00±2.00c	26.00±2.45c	28.00±2.00cd	28.00±2.00cd

处理时间 Treatment time (d)	致死中浓度 LC₅₀ (mg∙mL^-1)	95%置信区间 95% CI	斜率±标准误 Slope±standard error	卡方 χ²	P值 P value
1	3.99	1.78-19.41	2.71±0.21	32.94	<0.001
3	3.85	1.58-26.52	2.49±0.20	34.31	<0.001
5	3.72	1.36-45.36	2.31±0.18	35.96	<0.001
7	3.72	1.36-45.36	2.31±0.18	35.96	<0.001