黑沙蒿提取物对粘虫的生物活性及有效成分的初步分离
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摘要
本研究以菊科植物黒沙蒿为试验材料,采用多种有机溶剂和不同提取方法对黒沙蒿地上部进行提取,用提取物及其萃取物对危害禾本科的重要害虫——粘虫进行了生物活性测定,同时对黒沙蒿乙酸乙酯提取物中的活性物质进行了初步分离。试验结果如下:
1.以粘虫为试虫,对黒沙蒿等16种植物材料的丙酮提取物进行了室内生物活性筛选,结果发现处理后48h拒食率达到50%以上的有10种植物,浸虫法处理72h后校正死亡率达到50%的有7种植物,黒沙蒿提取物的拒食和触杀活性较高,拒食率和校正死亡率分别为97.83%和80%。
2.选用石油醚、氯仿、乙酸乙酯、正已烷和丙酮等5种不同极性有机溶剂,采用温浸法、索氏法和冷浸法对黒沙蒿地上部进行提取得到15种提取物:3种方法提取以索氏法的产出率最高,乙酸乙酯为溶剂的产出率为17.97%,温浸法次之,为11.41%,冷浸法产出率最低,为5.92%;就所使用的溶剂而言,乙酸乙酯的产出率最高,其次为丙酮、氯仿、正己烷、石油醚。
3.对黒沙蒿的15种提取物进行生物活性测定,结果表明:15种提取物均对粘虫表现出了一定的生物活性,不同提取物的活性存在显著差异,索氏法的乙酸乙酯提取物活性最突出,其主要作用方式为拒食,触杀和生长发育抑制作用,而胃毒作用相对较低。在拒食试验中,24h内拒食中浓度为8.4mg/ml;触杀活性较显著,浸虫法测定72h的校正死亡率达89.29%;有较强的生长发育抑制作用,1d后的生长发育抑制率为91.78%,2d后是86.32%,平均蛹重169.1mg,最终化蛹率为43.33%,且生长发育速度较对照延迟。
4.黒沙蒿乙酸乙酯提取物经液——液萃取,以石油醚萃取物的萃取率最高,其次是氯仿、乙酸乙酯、正丁醇、水萃取物。5种萃取物采用叶碟法和浸虫法进行生物活性测定,以石油醚萃取物的生物活性最强,处理24h后拒食率为91.84%,72h粘虫的死亡率达到86.67%;其次为氯仿萃取物,处理24h后拒食率为77.05%;72h死亡率为63.33%。
5.石油醚萃取物经柱层析分离后,利用TLC法检验,合并相同成分,得到13个流分,采用叶碟法进行生物活性测定,发现拒食活性成分主要集中在流分L3、L4 L5、中,拒食率均在89%以上,其中流分L4对粘虫的效果最好,经TLC检测,发现L4有2个成分,且流分L3只有1个成分。因此推测,活性物质主要是Rf值为0.56的成分,为最终确定有效成分作了前期工作。
根据试验结果,本文就植物材料有效成分的提取、黒沙蒿提取物杀虫生物活性、活性物质的分离、需要解决的问题及应用前景等进行了讨论。
The overground parts of Artemisia ordosica Kraschen, belonging to Compositae, were extracted by different methods and solvents. Biological activities of crude extracts and fractions against Mythimna separata Walker were tested,at the same time, the effective components of ethyl acetate of Artemisia ordosica Kraschen were separated.The results were listed as follows:
1 The acetone extracts of 16 plant species were tested for their biological activities against Mythimna separata Walker. The results showed that the anti-feeding rates of 10 plant species had over 50% after 48h treatment; emendation mortality rates of 7 plant species had over 50% with diping methods after 72h treatment, the acetone extracts of Artemisia ordosica Kraschen possessed the highest activities, the anti-feeding rate and emendation mortality rate were 97.83%and 80%.
2 Three methods and five solvents were used to extract overground parts of Artemisia ordosica Kraschen, 15 extracts were acquired .The results indicated that Sohxlet was the most effective extraction method, the extract rate with ethyl acetate was 17.97%, then warm soaking was 11.41 % , the cold soaking,the last method was 5.92 % respectively . The extract rates with the same extraction methods from heigh to low were ethyl acetate, acetone, chloroform, n-hexane, petroleumether.
3 Bioactivity experiments were conducted with Artemisia ordosica Kraschen extracts used Mythimna separata Walker as a test species. The results showed that 15 extracts all had biological activities against Mythimna separata Walker, the activities of different ectracts had distinct difference. Ethyl acetate extracts of Sohxlet of Artemisia ordosica Kraschen showed the highest biological activities mainly in anti-feeding、contacting and inhibiting growth activities, while weak stomach toxicity effect: In anti-feeding test , the AFC50 was 8.4mg/ml. It showed a highest contact efficacy against the Mythimna separata Walker, the 72h emendation mortality with a dipping treatment was 89.29%.Ethyl acetate extracts also possessed significant inhibition growth activities, caused 91.78%and 86.32% inhibition after 24h and 48h treatment, respectively, average pupa weight was 169.1mg , and pupation rate was only 43.33%, the speed of growth and development was delayed . Other extracts also showed biological activity.
4 Ethyl acetate extracts of Sohxlet of Artemisia ordosica Kraschen were successively separated by fluid-fluid distribution, the extracts rates were petroleumether>chloroform >ethyl acetate>n-butanol>water. Bioactivity experiments by using anti-feeding and direct contact methods, petroleumether extract showed the significant activity, anti-feeding rate was 91.84% after 24h treatment, mortality rate was 86.67% after 72h treatment, the chloroform extract also possessed higher activity, anti-feeding rate, mortality rate were 77.05% and 66.33% after 24h and 72h treatment respectively.
5 The petroleumether extracts were isolated and purified by column chromatography and thin-layer chromatography (TLC). Thirteen fractions were got after combination, through direction of bio-assay, the results showed that the activity components of anti-feeding were concentrated in fractions L3, L4, L5, the anti-feeding rates had all over 89%, fraction L4 had the heighest activity, after TLC, the results showed that L4 had two components, L3 had one component, the Rf limit of major component was 0.56, which got to first base for the identification of components.
Based on the result, extraction of plant material and activity of the extraction of the Artemisia ordosica Kraschen, the separation and purification of plant material, the mechanism, the problem and the foreground were discussed in this paper.
1.以粘虫为试虫,对黒沙蒿等16种植物材料的丙酮提取物进行了室内生物活性筛选,结果发现处理后48h拒食率达到50%以上的有10种植物,浸虫法处理72h后校正死亡率达到50%的有7种植物,黒沙蒿提取物的拒食和触杀活性较高,拒食率和校正死亡率分别为97.83%和80%。
2.选用石油醚、氯仿、乙酸乙酯、正已烷和丙酮等5种不同极性有机溶剂,采用温浸法、索氏法和冷浸法对黒沙蒿地上部进行提取得到15种提取物:3种方法提取以索氏法的产出率最高,乙酸乙酯为溶剂的产出率为17.97%,温浸法次之,为11.41%,冷浸法产出率最低,为5.92%;就所使用的溶剂而言,乙酸乙酯的产出率最高,其次为丙酮、氯仿、正己烷、石油醚。
3.对黒沙蒿的15种提取物进行生物活性测定,结果表明:15种提取物均对粘虫表现出了一定的生物活性,不同提取物的活性存在显著差异,索氏法的乙酸乙酯提取物活性最突出,其主要作用方式为拒食,触杀和生长发育抑制作用,而胃毒作用相对较低。在拒食试验中,24h内拒食中浓度为8.4mg/ml;触杀活性较显著,浸虫法测定72h的校正死亡率达89.29%;有较强的生长发育抑制作用,1d后的生长发育抑制率为91.78%,2d后是86.32%,平均蛹重169.1mg,最终化蛹率为43.33%,且生长发育速度较对照延迟。
4.黒沙蒿乙酸乙酯提取物经液——液萃取,以石油醚萃取物的萃取率最高,其次是氯仿、乙酸乙酯、正丁醇、水萃取物。5种萃取物采用叶碟法和浸虫法进行生物活性测定,以石油醚萃取物的生物活性最强,处理24h后拒食率为91.84%,72h粘虫的死亡率达到86.67%;其次为氯仿萃取物,处理24h后拒食率为77.05%;72h死亡率为63.33%。
5.石油醚萃取物经柱层析分离后,利用TLC法检验,合并相同成分,得到13个流分,采用叶碟法进行生物活性测定,发现拒食活性成分主要集中在流分L3、L4 L5、中,拒食率均在89%以上,其中流分L4对粘虫的效果最好,经TLC检测,发现L4有2个成分,且流分L3只有1个成分。因此推测,活性物质主要是Rf值为0.56的成分,为最终确定有效成分作了前期工作。
根据试验结果,本文就植物材料有效成分的提取、黒沙蒿提取物杀虫生物活性、活性物质的分离、需要解决的问题及应用前景等进行了讨论。
The overground parts of Artemisia ordosica Kraschen, belonging to Compositae, were extracted by different methods and solvents. Biological activities of crude extracts and fractions against Mythimna separata Walker were tested,at the same time, the effective components of ethyl acetate of Artemisia ordosica Kraschen were separated.The results were listed as follows:
1 The acetone extracts of 16 plant species were tested for their biological activities against Mythimna separata Walker. The results showed that the anti-feeding rates of 10 plant species had over 50% after 48h treatment; emendation mortality rates of 7 plant species had over 50% with diping methods after 72h treatment, the acetone extracts of Artemisia ordosica Kraschen possessed the highest activities, the anti-feeding rate and emendation mortality rate were 97.83%and 80%.
2 Three methods and five solvents were used to extract overground parts of Artemisia ordosica Kraschen, 15 extracts were acquired .The results indicated that Sohxlet was the most effective extraction method, the extract rate with ethyl acetate was 17.97%, then warm soaking was 11.41 % , the cold soaking,the last method was 5.92 % respectively . The extract rates with the same extraction methods from heigh to low were ethyl acetate, acetone, chloroform, n-hexane, petroleumether.
3 Bioactivity experiments were conducted with Artemisia ordosica Kraschen extracts used Mythimna separata Walker as a test species. The results showed that 15 extracts all had biological activities against Mythimna separata Walker, the activities of different ectracts had distinct difference. Ethyl acetate extracts of Sohxlet of Artemisia ordosica Kraschen showed the highest biological activities mainly in anti-feeding、contacting and inhibiting growth activities, while weak stomach toxicity effect: In anti-feeding test , the AFC50 was 8.4mg/ml. It showed a highest contact efficacy against the Mythimna separata Walker, the 72h emendation mortality with a dipping treatment was 89.29%.Ethyl acetate extracts also possessed significant inhibition growth activities, caused 91.78%and 86.32% inhibition after 24h and 48h treatment, respectively, average pupa weight was 169.1mg , and pupation rate was only 43.33%, the speed of growth and development was delayed . Other extracts also showed biological activity.
4 Ethyl acetate extracts of Sohxlet of Artemisia ordosica Kraschen were successively separated by fluid-fluid distribution, the extracts rates were petroleumether>chloroform >ethyl acetate>n-butanol>water. Bioactivity experiments by using anti-feeding and direct contact methods, petroleumether extract showed the significant activity, anti-feeding rate was 91.84% after 24h treatment, mortality rate was 86.67% after 72h treatment, the chloroform extract also possessed higher activity, anti-feeding rate, mortality rate were 77.05% and 66.33% after 24h and 72h treatment respectively.
5 The petroleumether extracts were isolated and purified by column chromatography and thin-layer chromatography (TLC). Thirteen fractions were got after combination, through direction of bio-assay, the results showed that the activity components of anti-feeding were concentrated in fractions L3, L4, L5, the anti-feeding rates had all over 89%, fraction L4 had the heighest activity, after TLC, the results showed that L4 had two components, L3 had one component, the Rf limit of major component was 0.56, which got to first base for the identification of components.
Based on the result, extraction of plant material and activity of the extraction of the Artemisia ordosica Kraschen, the separation and purification of plant material, the mechanism, the problem and the foreground were discussed in this paper.
引文
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