银杏、火炬树活性成分及其抗螨性研究
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摘要
银杏(Ginkgo biloba L.)是起源于二叠纪时期的蕨类植物,繁茂于侏罗纪时期,第三纪和第四纪地球的巨变和随后的冰川摧毁了世界上银杏树,凭借我国优越的地理位置和气候条件得以幸免,因此素有“植物活化石”、“植物界熊猫”之美誉。
银杏是闻名遐迩的药用植物,特别对预防和治疗心脑血管疾病、老年性痴呆等具有显著疗效,并相继开发出药品、保健品、化妆品、饮品等,同时银杏还具有抗虫特性。鉴于植物源杀虫剂对环境友好、不易产生抗药性等优点,对银杏抗虫性研究逐渐成为热点,但目前研究的靶标害虫比较少,尤其缺乏对农业危害较为严重的害螨的研究,另外,国内外对银杏的抗虫活性成分也没有一致的观点。
本文旨在明确银杏和火炬树对二斑叶螨Tetranychus urticae Koch和山楂叶螨Tetranychus viennensis Zacher的抗性,进而筛选抗螨性成分及提取工艺。分别采用培养皿法测定叶螨对银杏叶的选择性,以及在限制性条件下银杏叶对两种叶螨生存和产卵的影响;利用固相微萃取(Solid phase microextraction, SPME)采集、GC-MS测定银杏叶挥发性成分,“Y”型嗅觉仪测定它们对叶螨选择行为的影响;分别利用8种银杏单体成分、1种银杏总酸测定它们对叶螨的拒食性和触杀性;另外,比较了微波处理银杏叶水浸液联合乙醇和乙醚提取、溶剂提取与碱性溶液结合提取和乙醚萃取与大孔树脂吸附三种工艺的提取物对山楂叶螨的毒力。结果证明:
1、银杏叶对两种叶螨的拒食率均为100%;在限制性条件下叶螨在银杏叶上的生存时间缩短、产卵被抑制。
2、初步鉴定出壬酸、十四酸、十五酸、十六酸、6,10,14-三甲基-2-十五烷酮、二苯甲酮、丁羟甲苯、4,6-二特丁基-2-甲基苯酚、3-甲基-2-丁烯醇、5,7-二甲基-1,8-萘啶-2氨、1-甲基-9H-吡哆[3,4-b]吲哚-7-醇和1-苯-5-(1-蒎)-4-己烯-2-炔酮等12种银杏叶挥发性成分。
3、在两组气味源中(银杏叶-苹果叶、银杏叶-空气),二斑叶螨和山楂叶螨对银杏叶的选择率分别为22.2%、21.3%和23.5%、24.8%,与对照气味源选择率差异显著;从银杏活性成分中筛选出对二斑叶螨拒食率较高的异鼠李素、山奈素、银杏内酯C,A,B和十三烷基银杏酸,对山楂叶螨拒食率较高的山奈素、异鼠李素、银杏内酯A,B、白果内酯和银杏总酸。银杏内酯A对山楂叶螨48 h的校正死亡率达74.06%,其次为十三烷基银杏酸(52.36%),而其他所测成分均在25%以下。
4、银杏叶粉经乙醚萃取与DM130大孔树脂吸附后的流出液对山楂叶螨的校正死亡率达93.25%,其次为80%乙醇洗脱液(76.4%),而甲醇:乙酸乙酯(80:20 V/V)洗脱液和5% NaOH洗脱液的校正死亡率均在50%以下;银杏叶粉经冷水浸提、70%乙醇处理和乙醚富集后对山楂叶螨的校正死亡率达91.38%,而微波处理银杏叶粉水浸液及有机溶剂与碱性溶液结合提取物对山楂叶螨的触杀效果较差。
5、本文采用与银杏近乎相同的方法研究了火炬树叶对二斑叶螨和山楂叶螨的抗性。结果证明:两种叶螨对火炬叶的选择率在10%以下;在限制性条件下叶螨在火炬树叶上的生存时间明显缩短,产卵被抑制。
6、初步鉴定出十八烯、α-法呢烯、1,12-十三二烯、4-庚醇、4-甲基-1-丙烯-3-醇、3,7,11-三甲基-2,6,10-二十二三烯-1-醇、苯甲酸乙酯、水杨酸甲酯、4,6-二特丁基-2-甲基苯酚、丁基化羟基甲苯和2-甲基-2-丁烯酸等11种火炬叶挥发性成分。
7、在两组气味源中(火炬叶-苹果叶、火炬叶-空气),二斑叶螨和山楂叶螨对火炬叶的选择率分别为9.3%、28.9%和5.6%、28%,与对照气味源选择率差异显著;火炬叶粉经95%乙醇回流提取、DM130树脂柱吸附后的石油醚:乙酸乙酯(65:35 V/V)洗脱液对山楂叶螨24 h的死亡率为100%,初步鉴定其毒性成分为弱极性酸性物质和酚类化合物。
银杏和火炬树均系对二斑叶螨和山楂叶螨具有优良抗性的非寄主植物,银杏叶挥发物和非挥发性次生物质均对叶螨的选择行为造成影响,且拒食性优于触杀性,但银杏内酯A对山楂叶螨的触杀效果尤为突出,并筛选出银杏叶粉经乙醚萃取与DM130大孔树脂吸附提取工艺和火炬叶粉经95%乙醇回流提取、DM130树脂柱吸附后石油醚:乙酸乙酯(65:35 V/V)洗脱提取工艺,必将对银杏、火炬树抗螨性研究和开发利用具有一定的参考作用。
Ginkgo biloba L.originated from ancient seed ferns during Permian times, and overgrown in Jurassic period. During the Tertiary and Quaternary periods great upheavals of the earth, followed later by an ice age,destroyed the ginkgo trees and other ginkgoales in most of the world. The destruction was least in China for its geographic superiority and climate condition.Therefor Ginkgo biloba tree was called as“a living fossil”and“a panda in the plant field”.
G. biloba L. is reputed for phytopharmaceuticals, specially has an effect on cerebrovascular, cardiovascular disease, senile dementia and so on and has been developped as pharmaceuticals, health products, cosmetics, beverages and the like. At the same time, G. biloba has the characteristic against pests and the studies on its resistance have been a focus due to phytopesticides environment-friendly and resistance-free. However, at present, there are much less pests studied, specially lacking on mites which are serious for agriculture. At the other hand, there are not the same viewpoint for the resistant components from G. biloba.
This paper has systematacially studied the resistance of G. biloba against spider mites according to the general law for studying on phytopesticides. Main contents included that under the nonlimited condition, the selectivity of Tetranychus urticae Koch and Tetranychus viennensis Zacher to G. biloba leaves, and under limited one, the effects of G. biloba on the survival and fecundity of them.Volatile components of G. biloba leaves were identified with GC-MS after volatiles were collected with solid phase microextraction, and antifeedant activity of them were determinated with Y-tube olfactometer. The selective antifeedant activity of spider mites against G. biloba and the contact toxicity of G. biloba against spider mites were bioassayed with nine kinds of monomers in G. biloba at the first in the world. It was compared that the toxicity of extracts against T. viennensis that were extracted from foliar powder of G. biloba with three extraction processes: aqueous solution of powder with microwave treatment followed ethanol and ether extraction, organic solvents and alkaline solution extraction, ether extraction and adsorption with macroporous resin.
The results showed that under the nonlimited condition, two kinds of spider mites had absolutely antifeeded on G. biloba leaves, and under the limited one, the survival time when spider mites were confined in G. biloba leaves reduced and the fecundity of spider mites were suppressed absolutely. Twelve kinds of volatile constituents from G. biloba leaves were identified, and they have a certain effect on the antifeedant activity of spider mites.At the same concentration, nine kinds of active constituents in G. biloba had more antifeedant activity on G. biloba than their contact toxicity against spider mites, and there was interspecific difference between two kinds of spider mites. The toxicity of ginkgolide A against T. viennensis had a prominent effect, the ginkgolic acids, which were generally thought as toxic substance, however, had not a prominent effect on the contact toxicity against spider mites.The effluent after absorption of macroporous resin had 93.25% of corrected mortality against T. viennensis, the eluate that was eluted by ethanol(80%), methanol:ethyl acetate(80:20 V/V) and NaOH (5%), respectively, had much less effect on the mortality of T. viennensis. It was eliminated from extraction processes of acaricides that the aqueous solution of foliar powder of G. biloba were treated with microwave, however, the corresponding extraction process of 70% ethanol and ether treatment had 91.38% of corrected mortality against T. viennensis. There was not an ideal toxicity from extracts of organic solvent and alkaline solution, aqueous solution of G. biloba had a certain toxicity.
Rhus typhina L.(Anacardiaceae), Buck’s horn or Staghorn, which originates from the eastern part of Northern America, is a 5～8 m high shrub or a small arbor, its unpaired serrated leaflets and fruits like torchs, turn bright red in autumn. It was introduced as an ornamental plant from USA by our country in 1959, and so far have been distributed over more twenty provinces and municipalities. R. typhina has been not only used as a plant of enhanced bank and circumscribed sand, but also has been a resistance against some pests. Unfortunately, it has been blamed for its overgrowth and exotic characteristics. Therefor, studies on its resistance against pests have been rare.
Studies on R. typhina followed the methods as same as G. biloba. The results showed that under nonlimited condition, selective rate of two kinds of spider mites to staghorn leaves was less 10%, respectively, and under limited one, the survival time when spider mites were confined in the leaves of R. typhina reduced and fecundity was sppressed absolutely. Eleven volatiles from R. typhina leaves were identified as acids, alcohols, phenols and antifeedant activity of spider mites on them were demonstrated with Y-tube olfactometer. Ethyl acetate extract and the eluate which was eluted with petroleum ether:ethyl acetate(65:35 V/V) after adsorption of macroporous resin DM130 from leaves of R. typhina displayed acaricidal properties towards T. viennensis, respectively, and they were preliminarily identified as weak polar acids and phenols.
银杏是闻名遐迩的药用植物,特别对预防和治疗心脑血管疾病、老年性痴呆等具有显著疗效,并相继开发出药品、保健品、化妆品、饮品等,同时银杏还具有抗虫特性。鉴于植物源杀虫剂对环境友好、不易产生抗药性等优点,对银杏抗虫性研究逐渐成为热点,但目前研究的靶标害虫比较少,尤其缺乏对农业危害较为严重的害螨的研究,另外,国内外对银杏的抗虫活性成分也没有一致的观点。
本文旨在明确银杏和火炬树对二斑叶螨Tetranychus urticae Koch和山楂叶螨Tetranychus viennensis Zacher的抗性,进而筛选抗螨性成分及提取工艺。分别采用培养皿法测定叶螨对银杏叶的选择性,以及在限制性条件下银杏叶对两种叶螨生存和产卵的影响;利用固相微萃取(Solid phase microextraction, SPME)采集、GC-MS测定银杏叶挥发性成分,“Y”型嗅觉仪测定它们对叶螨选择行为的影响;分别利用8种银杏单体成分、1种银杏总酸测定它们对叶螨的拒食性和触杀性;另外,比较了微波处理银杏叶水浸液联合乙醇和乙醚提取、溶剂提取与碱性溶液结合提取和乙醚萃取与大孔树脂吸附三种工艺的提取物对山楂叶螨的毒力。结果证明:
1、银杏叶对两种叶螨的拒食率均为100%;在限制性条件下叶螨在银杏叶上的生存时间缩短、产卵被抑制。
2、初步鉴定出壬酸、十四酸、十五酸、十六酸、6,10,14-三甲基-2-十五烷酮、二苯甲酮、丁羟甲苯、4,6-二特丁基-2-甲基苯酚、3-甲基-2-丁烯醇、5,7-二甲基-1,8-萘啶-2氨、1-甲基-9H-吡哆[3,4-b]吲哚-7-醇和1-苯-5-(1-蒎)-4-己烯-2-炔酮等12种银杏叶挥发性成分。
3、在两组气味源中(银杏叶-苹果叶、银杏叶-空气),二斑叶螨和山楂叶螨对银杏叶的选择率分别为22.2%、21.3%和23.5%、24.8%,与对照气味源选择率差异显著;从银杏活性成分中筛选出对二斑叶螨拒食率较高的异鼠李素、山奈素、银杏内酯C,A,B和十三烷基银杏酸,对山楂叶螨拒食率较高的山奈素、异鼠李素、银杏内酯A,B、白果内酯和银杏总酸。银杏内酯A对山楂叶螨48 h的校正死亡率达74.06%,其次为十三烷基银杏酸(52.36%),而其他所测成分均在25%以下。
4、银杏叶粉经乙醚萃取与DM130大孔树脂吸附后的流出液对山楂叶螨的校正死亡率达93.25%,其次为80%乙醇洗脱液(76.4%),而甲醇:乙酸乙酯(80:20 V/V)洗脱液和5% NaOH洗脱液的校正死亡率均在50%以下;银杏叶粉经冷水浸提、70%乙醇处理和乙醚富集后对山楂叶螨的校正死亡率达91.38%,而微波处理银杏叶粉水浸液及有机溶剂与碱性溶液结合提取物对山楂叶螨的触杀效果较差。
5、本文采用与银杏近乎相同的方法研究了火炬树叶对二斑叶螨和山楂叶螨的抗性。结果证明:两种叶螨对火炬叶的选择率在10%以下;在限制性条件下叶螨在火炬树叶上的生存时间明显缩短,产卵被抑制。
6、初步鉴定出十八烯、α-法呢烯、1,12-十三二烯、4-庚醇、4-甲基-1-丙烯-3-醇、3,7,11-三甲基-2,6,10-二十二三烯-1-醇、苯甲酸乙酯、水杨酸甲酯、4,6-二特丁基-2-甲基苯酚、丁基化羟基甲苯和2-甲基-2-丁烯酸等11种火炬叶挥发性成分。
7、在两组气味源中(火炬叶-苹果叶、火炬叶-空气),二斑叶螨和山楂叶螨对火炬叶的选择率分别为9.3%、28.9%和5.6%、28%,与对照气味源选择率差异显著;火炬叶粉经95%乙醇回流提取、DM130树脂柱吸附后的石油醚:乙酸乙酯(65:35 V/V)洗脱液对山楂叶螨24 h的死亡率为100%,初步鉴定其毒性成分为弱极性酸性物质和酚类化合物。
银杏和火炬树均系对二斑叶螨和山楂叶螨具有优良抗性的非寄主植物,银杏叶挥发物和非挥发性次生物质均对叶螨的选择行为造成影响,且拒食性优于触杀性,但银杏内酯A对山楂叶螨的触杀效果尤为突出,并筛选出银杏叶粉经乙醚萃取与DM130大孔树脂吸附提取工艺和火炬叶粉经95%乙醇回流提取、DM130树脂柱吸附后石油醚:乙酸乙酯(65:35 V/V)洗脱提取工艺,必将对银杏、火炬树抗螨性研究和开发利用具有一定的参考作用。
Ginkgo biloba L.originated from ancient seed ferns during Permian times, and overgrown in Jurassic period. During the Tertiary and Quaternary periods great upheavals of the earth, followed later by an ice age,destroyed the ginkgo trees and other ginkgoales in most of the world. The destruction was least in China for its geographic superiority and climate condition.Therefor Ginkgo biloba tree was called as“a living fossil”and“a panda in the plant field”.
G. biloba L. is reputed for phytopharmaceuticals, specially has an effect on cerebrovascular, cardiovascular disease, senile dementia and so on and has been developped as pharmaceuticals, health products, cosmetics, beverages and the like. At the same time, G. biloba has the characteristic against pests and the studies on its resistance have been a focus due to phytopesticides environment-friendly and resistance-free. However, at present, there are much less pests studied, specially lacking on mites which are serious for agriculture. At the other hand, there are not the same viewpoint for the resistant components from G. biloba.
This paper has systematacially studied the resistance of G. biloba against spider mites according to the general law for studying on phytopesticides. Main contents included that under the nonlimited condition, the selectivity of Tetranychus urticae Koch and Tetranychus viennensis Zacher to G. biloba leaves, and under limited one, the effects of G. biloba on the survival and fecundity of them.Volatile components of G. biloba leaves were identified with GC-MS after volatiles were collected with solid phase microextraction, and antifeedant activity of them were determinated with Y-tube olfactometer. The selective antifeedant activity of spider mites against G. biloba and the contact toxicity of G. biloba against spider mites were bioassayed with nine kinds of monomers in G. biloba at the first in the world. It was compared that the toxicity of extracts against T. viennensis that were extracted from foliar powder of G. biloba with three extraction processes: aqueous solution of powder with microwave treatment followed ethanol and ether extraction, organic solvents and alkaline solution extraction, ether extraction and adsorption with macroporous resin.
The results showed that under the nonlimited condition, two kinds of spider mites had absolutely antifeeded on G. biloba leaves, and under the limited one, the survival time when spider mites were confined in G. biloba leaves reduced and the fecundity of spider mites were suppressed absolutely. Twelve kinds of volatile constituents from G. biloba leaves were identified, and they have a certain effect on the antifeedant activity of spider mites.At the same concentration, nine kinds of active constituents in G. biloba had more antifeedant activity on G. biloba than their contact toxicity against spider mites, and there was interspecific difference between two kinds of spider mites. The toxicity of ginkgolide A against T. viennensis had a prominent effect, the ginkgolic acids, which were generally thought as toxic substance, however, had not a prominent effect on the contact toxicity against spider mites.The effluent after absorption of macroporous resin had 93.25% of corrected mortality against T. viennensis, the eluate that was eluted by ethanol(80%), methanol:ethyl acetate(80:20 V/V) and NaOH (5%), respectively, had much less effect on the mortality of T. viennensis. It was eliminated from extraction processes of acaricides that the aqueous solution of foliar powder of G. biloba were treated with microwave, however, the corresponding extraction process of 70% ethanol and ether treatment had 91.38% of corrected mortality against T. viennensis. There was not an ideal toxicity from extracts of organic solvent and alkaline solution, aqueous solution of G. biloba had a certain toxicity.
Rhus typhina L.(Anacardiaceae), Buck’s horn or Staghorn, which originates from the eastern part of Northern America, is a 5～8 m high shrub or a small arbor, its unpaired serrated leaflets and fruits like torchs, turn bright red in autumn. It was introduced as an ornamental plant from USA by our country in 1959, and so far have been distributed over more twenty provinces and municipalities. R. typhina has been not only used as a plant of enhanced bank and circumscribed sand, but also has been a resistance against some pests. Unfortunately, it has been blamed for its overgrowth and exotic characteristics. Therefor, studies on its resistance against pests have been rare.
Studies on R. typhina followed the methods as same as G. biloba. The results showed that under nonlimited condition, selective rate of two kinds of spider mites to staghorn leaves was less 10%, respectively, and under limited one, the survival time when spider mites were confined in the leaves of R. typhina reduced and fecundity was sppressed absolutely. Eleven volatiles from R. typhina leaves were identified as acids, alcohols, phenols and antifeedant activity of spider mites on them were demonstrated with Y-tube olfactometer. Ethyl acetate extract and the eluate which was eluted with petroleum ether:ethyl acetate(65:35 V/V) after adsorption of macroporous resin DM130 from leaves of R. typhina displayed acaricidal properties towards T. viennensis, respectively, and they were preliminarily identified as weak polar acids and phenols.
引文
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