摘要
本试验以严重为害果树的朱砂叶螨为研究对象,对牵牛子进行了杀螨活性成分研究,并对分离出来的有效物质进行了作用机理初步探讨。
(1)采用玻片浸渍法室内测定了36种中草药提取物对朱砂叶螨的杀螨活性,结果表明:牵牛子石油醚提取物等10种中草药提取物的活性较高,其24h校正死亡率均大于90%;并对上述10种具有较高杀螨活性的中草药提取物对朱砂叶螨的触杀效果也进行了测定,按LC_(50)从低到高依次为:牵牛子石油醚提取物(0.6979±0.0598mg/ml)、马钱子石油醚提取物(0.7670±0.0554mg/ml)、苍耳子石油醚提取物(1.1239±0.0615mg/ml)、苍耳子甲醇提取物(1.3417±0.0773mg/ml)、马钱子甲醇提取物(1.3492±0.0738mg/ml)、百部石油醚提取物(1.3679±0.0810mg/ml)、红藤石油醚提取物(1.4863±0.0921mg/ml)、贯众氯仿提取物(1.6347±0.1060mg/ml)、白鲜皮氯仿提取物(1.7185±0.1003mg/ml)、贯众甲醇提取物(1.8201±0.0934mg/ml),结果表明牵牛子石油醚提取物的杀螨活性最高。
(2)测定牵牛子提取物对朱砂叶螨的生物活性。牵牛子石油醚提取物浓度为2mg/ml时,对朱砂叶螨的雌成螨和卵的校正死亡率分别达到93.7%和77.3%,LC_(50)分别为0.6979mg/ml和1.3037mg/ml;对雌成螨24h后的产卵抑制率和驱避率分别达到82.51%和94.54%。萃取物中石油醚相的活性明显高于甲醇相,浓度为2mg/ml时,石油醚萃取物以朱砂叶螨的雌成螨和卵的校正死亡率分别达到96.2%和82.1%,LC_(50)分别为0.5042mg/ml和1.1016mg/ml;对石油醚萃取物进行柱层析分离最终得到流分12个,其中以流分8杀螨效果最好,浓度为2mg/ml时,24h对雌成螨的校正死亡率达96.4%。流分8对成螨和卵的LC_(50)分别是0.4477mg/ml和1.1971mg/ml,LC_(90)分别是2.2321mg/ml和2.9152mg/ml。
(3)研究牵牛子石油醚提取物中流分8对朱砂叶螨生物活性的作用机理。采用叶片残毒法观察流分8对朱砂叶螨的中毒症状,并采用生化方法测定了谷胱甘肽-S-转移酶、乙酰胆碱酯酶、单胺氧化酶、Ca~(2+)-ATP酶活性的影响,通过透射电镜观察流分8对螨体内亚显微结构的破坏。结果表明,牵牛子流分8处理朱砂叶螨后,螨体内解毒酶谷胱甘肽-S-转移酶被激活,这说明流分8中存在对朱砂叶螨有毒的物质;而乙酰胆碱酯酶、单胺氧化酶、Ca~(2+)-ATP酶均受到不同程度的抑制,这可能引起神经传递的阻断,从而导致螨体的死亡。透射电镜下可见,流分8对螨体内表皮结构、肌纤维、细胞核膜、线粒体、内质网等均有不同程度的破坏。牵牛子可以有效杀死朱砂叶螨,作为新型植物源农药具有良好的开发前景。
Pharbitis purpurea seeds was investigated for its acarocidal activity against Tetranychus cinnabarinus,a serious pest of many crops including fruit trees in china.Action mechanism of effective substance of P. purpurea seeds against T.cinnabarinus was also investigated.
(1)The acaricidic activities of herbal extracts were measured in laboratory by Slide-dip method.The results showed that 24 hours after treatment,the acaricidic activities of 10 herbal extracts were high, with corrected mortality over 90%.The contact toxicities of these 10 herbal extracts against T. cinnabarinus were also tested and the LC_(50) value of the 10 herbal extracts,ranging from low to high, with P.purpurea petroleum ether extract at 0.6979±0.0598mg/ml,followed by Strychnos nux-vomica petroleum ether extract at 0.7670±0.0554 mg/ml,Xanthium sibiricum petroleum ether extract at 1.1239±0.0615mg/ml,Xanthium sibiricum methanol extract at 1.3417±0.0773mg/ml,Strychnos nux-vomica methanol extract at 1.3492±0.0738mg/ml,Stemona japnicaapetroleum ether extract at 1.3679±0.0810mg/ml,Sargentodoxa cuueata,petroleum ether extract at 1.4863±0.0921mg/ml, Osmuncla japonica chloroform extract at 1.6347±0.1060mg/ml,Dictammus dasycarpus chloroform extract at 1.7185±0.1003mg/ml and Osmuncla japonica methanol extract at 1.8201±0.0934mg/ml.
(2)The extracts of P.purpurea seeds investigated for their biological activity against T.cinnabarinus. Slide-dip and Leaf-residue methods were used to measure biological activity.Liquid-liquid distribution and the silica gel column were used to separate and purify active matter.The results indicated that bioactivity(touch kill,oviposit inhibition,repellency)of petroleum ether extract of P.purpurea seeds was the highest with the adjusted mortalities of female mite and egg were 93.7%and 77.3%, Oviposition inhibition and repellency rate were 82.51%and 94.54%respectively at the concentration of 2 mg/ml.LC_(50) is 0.6979mg/ml and 1.3037mg/ml.The petroleum ether extract of P.purpurea seeds was then separated and purified.The bioactivity of petroleum ether purified extracts of P.purpurea seeds was the highest with the adjusted mortalities of female mite and egg were 96.2%and 82.1%,LC_(50) is 0.5042 mg/ml and 1.1016mg/ml.The silica gel column was used to separate and purify active constitutes of the crude purified extracts of petroleum ether.A total of 12 bands were seen by TLC analysis.The isolation fraction 8 appeared a strong acarocidal activity against T.cinnabarinus,the adjusted mortalities of female mite was 96.4%respectively at the concentration of 2 mg/ml.The isolation fraction 8 had strongly acaricidal activities against adult and egg of T.cinnabarinus,the LC_(50) value were 0.4477mg/ml and 1.1971mg/ml,the LC_(90) value were 2.2321mg/ml and 2.9152mg/ml.
(3) Action mechanism of isolation fraction 8 of petroleum ether extract of.purpurea seeds against T. cinnabarinus was investigated.The toxicosis symptom of T.cinnabarinus were measured by Leaf-residue,and several kinds of important enzymes in T.cinnabarinus treated with isolation fraction 8 were tested.The ultrastructure of T.cinnabarinus treated by isolation fraction 8 was observed through transmission electron microscope(TEM).The results showed that the activities of glutathione-S-transferase in T.cinnabarinus were strongly induced,but the activities of acetylcholinease, monoamine oxidase and Ca~(2+)-ATPase were restrained after treating with isolation fraction 8.The result suggested that isolation fraction 8 of P.purpurea seeds was effective against T.cinnabarinus,which may be the cause the transmit block of nerve in mites.Observation tinder TEM revealed that cuticle, muscular filament,nuclear membrane,mitochondria and endoplasmic reticulum were ruptured.
The result from the experiment indicated that the discovery had a good developing prospect for making full use of P.purpurea seeds as new resources of botanical acaricide.
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