透骨草杀虫活性成分及其作用机理研究

发布时间：2018-06-10 16:48

本文选题：透骨草 + 杀虫活性　；参考：《西北农林科技大学》2014年博士论文

【摘要】：透骨草（Phryma leptostachya L.），为透骨草科透骨草属多年生草本植物，广泛分布于喜马拉雅山脉、北美洲和亚洲东部，在东亚一直被用作传统的杀虫植物。我国主要分布在东北、华北及长江流域各地。本论文以透骨草为研究对象，系统研究了其杀虫活性及活性成分，并对其主要杀虫活性成分双氧木脂素A的作用机理进行了初探。主要研究结果如下： 1.系统评价了石油醚、乙酸乙酯和甲醇超声提取物的杀虫活性。结果表明，三种提取物对供试的粘虫（Mythimna separate）幼虫、家蝇（Musca domestica）成虫及淡色库蚊（Culex pipiens pallens）幼虫有明显的毒杀活性，对3龄粘虫幼虫8h的胃毒麻醉中浓NC50分别为1690μg/mL、6240μg/mL和11390μg/mL，8h的触杀麻醉中量ND50分别为2.26μg/头、7.20μg/头和24.35μg/头；对家蝇成虫24h的胃毒致死中浓LC50分别为1580μg/mL、2940μg/mL和4990μg/mL；对家蝇成虫24h触杀致死中量LD50分别为1.80μg/头、3.32μg/头和5.56μg/头；对4龄淡色库蚊幼虫24h LC50分别为3.23μg/mL、5.24μg/mL和61.86μg/mL。对小菜蛾（Plutella xyllostella）幼虫和豆蚜（Aphis craccivora）无翅成蚜没有明显的毒杀作用。 2.以活性追踪为指导，采用多级硅胶柱层析、凝胶柱层析及RP-HPLC等方法，从透骨草甲醇提取物中分离出5个杀虫活性化合物。采用NMR、UV及HR-ESI/MS等技术对其中4个化合物进行了结构鉴定，4个化合物均是具有二氧双环辛烷骨架的双骈四氢呋喃型木脂素，分别为透骨草灵-I、透骨草灵B、双氧木脂素E及双氧木脂素A，其中透骨草灵B为首次报道的新化合物，双氧木脂素E首次从透骨草中分离得到。 3.杀虫活性测定结果表明，（1）透骨草灵-I对3龄粘虫幼虫只具有麻醉活性，其8h麻醉中浓NC50为2580μg/mL，对4龄淡色库蚊幼虫有一定的毒杀活性，24h毒杀致死中浓LC50为1.21μg/mL。（2）透骨草灵B对3龄粘虫幼虫和4龄淡色库蚊幼虫均有一定的毒杀作用，对3龄粘虫幼虫24h胃毒致死中浓LC50为490μg/mL，对4龄淡色库蚊幼虫24h毒杀LC50为0.69μg/mL。（3）双氧木脂素E对多种供试试虫有一定的杀虫活性，对3龄粘虫幼虫24h胃毒LC50为53μg/mL，24h触杀LD50为0.26μg/头；对3龄槐尺蠖（Semiothisa cinerearia）幼虫24h胃毒LC50为1680μg/mL，24h触杀LD50为1.33μg/头；对家蝇成虫24h胃毒LC50为970μg/mL，24h触杀LD50为1.12μg/头；对4龄淡色库蚊幼虫24h毒杀LC50为0.15μg/mL；对美洲大蠊（Periplaneta americana）成虫48h LD50为7.28μg/头。（4）双氧木脂素A对多种供试试虫有很强的杀虫活性，对3龄粘虫幼虫24h胃毒LC50为35μg/mL，24h触杀LD50为0.09μg/头；对3龄槐尺蠖幼虫24h胃毒LC50为66μg/mL，24h触杀LD50为0.049μg/头；对家蝇成虫24h胃毒LC50为39μg/mL，24h触杀LD50为0.047μg/头；对4龄淡色库蚊幼虫24h毒杀LC50为0.025μg/mL；对美洲大蠊成虫48h LD50为4.98μg/头。4个化合物对小菜蛾幼虫和豆蚜无翅成蚜没有明显的毒杀作用。 4.症状学观察表明，以LD90剂量双氧木脂素A点滴处理6龄粘虫幼虫和家蝇成虫后，粘虫幼虫麻醉、部分试虫不自主肌肉收缩、震颤，直至死亡；家蝇成虫很快兴奋，接着麻醉直至死亡；美洲大蠊成虫先是重心抬高、腹部屈曲，随后麻醉直至死亡。 5.双氧木脂素A对粘虫头部ATP酶的测定结果表明，双氧木脂素A抑制了5龄粘虫头部Na+-K+-ATPase活性，激活了Ca2+-Mg2+-ATPase和Ca2+-ATPase活性。离体条件下双氧木脂素A对Na+-K+-ATPase最大的抑制率为32.77%，活体条件下最大的抑制率为59.72%；离体条件下对Ca2+-Mg2+-ATPase和Ca2+-ATPase最大激活率分别为33.48%、30.34%，活体条件下对Ca2+-Mg2+-ATPase和Ca2+-ATPase最大的激活率分别为29.72%、28.83%。离体条件下对三种ATP酶在某种程度上均具有一定的剂量效应。 6.对果蝇幼虫神经-肌肉接点电位测定结果表明，双氧木脂素A使自发性接点电位（mEJP）释放频率增加，出现了波浪状发放，对兴奋性接点电位（EJPs）没有明显影响。 7.测定了双氧木脂素A对美洲大蠊离体神经细胞胞浆中游离[Ca2+]i动力学的影响。结果表明，10nmol/L到100μmol/L浓度范围双氧木脂素A都可引起胞内游离[Ca2+]i升高，且胞内钙水平升高与双氧木脂素A的浓度正相关。双氧木脂素A作用部位是细胞质膜和内质网膜，，其作用靶标是质膜上的L-型Ca2+通道和内质网膜上的RyR钙通道，引起外钙内流和内钙释放，使得胞内钙水平升高。双氧木脂素A与nifedipine竞争结合于L-型Ca2+通道，与咖啡因在RyR钙通道上存在不同的亲和位点。 8.双氧木脂素A激活L-型Ca2+通道，促进外钙内流；L-型Ca2+通道与RyR在胞浆区的部分相互作用，其构象发生改变并通过蛋白质间的相互作用激活RyR，Ca2+释放通道开放，内质网上的Ca2+释放。外钙内流和内钙释放致使胞浆中游离[Ca2+]i升高，Na+离子流受到抑制，从而抑制Na+-K+-ATPase活性；而突触前膜内Ca2+水平升高，Ca2+结合在囊泡膜上，减少了膜表面的负电荷，使得囊泡之间以及囊泡与突触前膜的粘附易于发生，神经-肌肉自发性接点电位（mEJP）释放频率增加，引起兴奋性神经递质释放，并扩散通过突触间隙，与突触后膜上的受体相结合，引起后膜去极化，形成突触后电位。突触后电位达到Na+的活化阈值，就产生一个新的动作电位，引起中毒试虫的肌肉收缩、震颤直至死亡。
[Abstract]:Phryma leptostachya L., perennial perviate perviate perennial herb, widely distributed in the Himalaya mountains, North America and eastern Asia, has been used as a traditional insecticidal plant in East Asia. China is mainly distributed in Northeast, North China and the Yangtze River Basin. This paper is studied systematically in this paper. Insecticidal activity and active ingredients, and the mechanism of its main insecticidal component, A, was studied. The main results are as follows:
1. systematically evaluated the insecticidal activity of the ultrasonic extracts of petroleum ether, ethyl acetate and methanol. The results showed that the three kinds of extracts had obvious toxicity to the larvae of Mythimna separate, the adult Musca domestica and the Culex pipiens pallens (Culex pipiens pallens) larvae, and the medium concentration NC50 scores for the gastric narcotic anesthesia of the 3 age larva 8h. Not 1690 mu g/mL, 6240 g/mL and 11390 u g/mL, the ND50 of 8h was 2.26 mu g/ head, 7.20 mu g/ head and 24.35 mu g/ head respectively, and the gastric poison lethal middle concentration LC50 of housefly adult worms was 1580 mu g/mL, 2940 mu g/mL and 4990 micron respectively. The 24h LC50 of 4 years old Culex pipiens pallens was 3.23 g/mL, 5.24 g/mL and 61.86 mu g/mL., which had no significant toxicity to the larva of the Plutella xylostella (Plutella xyllostella) and the aphis Aphis (Aphis craccivora) without wingless aphids.
2. with the guidance of active tracking, 5 insecticidal active compounds were separated from the methanol extract of DRA drantra by multistage silica gel column chromatography, gel column chromatography and RP-HPLC. The structures of 4 of the compounds were identified by NMR, UV and HR-ESI/MS. The 4 compounds were double parallel hydrogen furrows with two oxygen bicyclic octane skeleton. Mutan type lignans were -I, dioxygen B, dioxygen lignan E and dioxygen lignan A, respectively, of which dioxygen B was first reported as a new compound, and dioxygen lignan E was first isolated from perdiarin.
3. the results of insecticidal activity assay showed that (1) kulhakide -I only had anesthetic activity for 3 instar larva, with a medium concentration of 2580 mu NC50 in 8h anesthesia, and a certain toxic activity for the larvae of Culex pipiens pallens at 4 years of age. 24h poisoned and lethal medium concentration LC50 was 1.21 mu g/mL. (2) of kulychulin B for 3 instar and 4 years old Culex pipiens pallens larvae. For 3 instar larvae of 24h, the middle concentration of LC50 was 490 mu g/mL, and 24h poisoned LC50 to 0.69 mu g/mL. (3) for 4 instar larvae of Culex pipiens pallens (3) to a variety of insecticidal activity. The 24h gastric venom of 3 instar larvae was 53 mu g/mL, 24h touch LD50 was 0.26 mu, and 24 larvae of the 3 year old Sophora japonica larva were 24. H LC50 is 1680 mu g/mL, and 24h touch LD50 is 1.33 u g/ head, 24h stomach poison LC50 for housefly adult is 970 u g/mL, 24h touch LD50 is 1.12 mu g/ head, and 4 instar larvae of Culex pipiens pallens are 0.15 mu. (4) dioxygen lignans are strong for a variety of test insects. The insecticidal activity of the 3 instar larvae 24h was 35 g/mL LC50 and 0.09 mu g/ head with 24h touch LD50; 24h poison LC50 of the 3 instar larvae of Sophora japonica was 66 u g/mL and 24h touch LD50 was 0.049 mu g/ head. The 48h LD50 of the Periplaneta Americana is 4.98 mu g/.4 and has no obvious toxic effect on the larvae of the diamondback moth and the aphid of Aphis.
4. symptomatic observation showed that after 6 instar larvae and adult flies were treated with LD90 dose of dioxygen lignan, the larvae of the larvae of the worm were anaesthetized, and some of the insects did not autonomously contractile muscle contraction, tremor, until death. The adult flies were excited quickly, then anaesthetized until death; the adult of Periplaneta americana was first of the center of gravity, abdominal flexion, and then anaesthesia until death. Death.
The results of 5. dioxygen lignan A on the head ATP enzyme showed that dioxygen lignan A inhibited Na+-K+-ATPase activity in the head of 5 age armyworm and activated Ca2+-Mg2+-ATPase and Ca2+-ATPase activity. The maximum inhibition rate of dioxygen lignan A to Na+-K+-ATPase in vitro was 32.77%, and the maximum inhibitory rate under living conditions was 59.72%. The maximum activation rates for Ca2+-Mg2+-ATPase and Ca2+-ATPase were 33.48%, 30.34%, and the maximum activation rate of Ca2+-Mg2+-ATPase and Ca2+-ATPase under living conditions was 29.72% respectively. In vitro, 28.83%. had a certain dose effect on three ATP enzymes.
6. the results of the neuromuscular junction potential of Drosophila larva showed that the spontaneous contact potential (mEJP) release frequency increased, the release of the spontaneous contact potential (mEJP), and no significant effect on the excitatory contact potential (EJPs).
7. the effects of dioxygen lignan A on the dissociative [Ca2+]i kinetics in the cell cytoplasm of Periplaneta americana were measured. The results showed that both 10nmol/L and 100 mol/L concentration of dioxygen lignan A could cause the increase of intracellular free [Ca2+]i, and the increase of intracellular calcium level was positively related to the concentration of dioxygen lignan A. The action site of dioxygen lignin A was fine. The cytoplasmic membrane and endoplasmic reticulum, which target the L- type Ca2+ channel on the plasma membrane and the RyR calcium channel on the endoplasmic reticulum, cause the external calcium influx and internal calcium release and increase the intracellular calcium level. The competition of dioxygen lignan A and nifedipine is combined with L- Ca2+ channel, and there are different affinity sites with caffeine on RyR calcium channel.
8. dioxygen lignan A activates the L- type Ca2+ channel and promotes the external calcium influx; the L- type Ca2+ channel interacts with RyR in the cytoplasm region, and its conformation changes and activates RyR through protein interaction, Ca2+ release channel is open and the endoplasmic reticulum releases Ca2+ release. External calcium influx and internal calcium release lead to the increase of free [Ca2+]i in the cytoplasm, Na+ The ion flow is inhibited and the Na+-K+-ATPase activity is inhibited, while the Ca2+ level in the pre synapse membrane increases and the Ca2+ binding on the vesicle reduces the negative charge on the membrane surface, making the adhesion between the vesicles and the vesicles and the presynaptic membrane easy to occur, and the release frequency of the neural muscle spontaneous contact potential (mEJP) increases and causes the excitatory nerve delivery. The mass release, and the diffusion through the synaptic gap, combined with the receptors on the postsynaptic membrane, causes the depolarization of the posterior membrane to form a postsynaptic potential. The postsynaptic potential reaches the activation threshold of Na+, producing a new action potential, causing muscle contraction, tremor and death of the poisoned insects.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：S482.39

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