本文選題：透骨草 + 殺蟲(chóng)活性　； 參考：《西北農(nóng)林科技大學(xué)》2014年博士論文

【摘要】：透骨草（Phryma leptostachya L.），為透骨草科透骨草屬多年生草本植物，廣泛分布于喜馬拉雅山脈、北美洲和亞洲東部，在東亞一直被用作傳統(tǒng)的殺蟲(chóng)植物。我國(guó)主要分布在東北、華北及長(zhǎng)江流域各地。本論文以透骨草為研究對(duì)象，系統(tǒng)研究了其殺蟲(chóng)活性及活性成分，并對(duì)其主要?dú)⑾x(chóng)活性成分雙氧木脂素A的作用機(jī)理進(jìn)行了初探。主要研究結(jié)果如下： 1.系統(tǒng)評(píng)價(jià)了石油醚、乙酸乙酯和甲醇超聲提取物的殺蟲(chóng)活性。結(jié)果表明，三種提取物對(duì)供試的粘蟲(chóng)（Mythimna separate）幼蟲(chóng)、家蠅（Musca domestica）成蟲(chóng)及淡色庫(kù)蚊（Culex pipiens pallens）幼蟲(chóng)有明顯的毒殺活性，對(duì)3齡粘蟲(chóng)幼蟲(chóng)8h的胃毒麻醉中濃NC50分別為1690μg/mL、6240μg/mL和11390μg/mL，8h的觸殺麻醉中量ND50分別為2.26μg/頭、7.20μg/頭和24.35μg/頭；對(duì)家蠅成蟲(chóng)24h的胃毒致死中濃LC50分別為1580μg/mL、2940μg/mL和4990μg/mL；對(duì)家蠅成蟲(chóng)24h觸殺致死中量LD50分別為1.80μg/頭、3.32μg/頭和5.56μg/頭；對(duì)4齡淡色庫(kù)蚊幼蟲(chóng)24h LC50分別為3.23μg/mL、5.24μg/mL和61.86μg/mL。對(duì)小菜蛾（Plutella xyllostella）幼蟲(chóng)和豆蚜（Aphis craccivora）無(wú)翅成蚜沒(méi)有明顯的毒殺作用。 2.以活性追蹤為指導(dǎo)，采用多級(jí)硅膠柱層析、凝膠柱層析及RP-HPLC等方法，從透骨草甲醇提取物中分離出5個(gè)殺蟲(chóng)活性化合物。采用NMR、UV及HR-ESI/MS等技術(shù)對(duì)其中4個(gè)化合物進(jìn)行了結(jié)構(gòu)鑒定，4個(gè)化合物均是具有二氧雙環(huán)辛烷骨架的雙駢四氫呋喃型木脂素，分別為透骨草靈-I、透骨草靈B、雙氧木脂素E及雙氧木脂素A，其中透骨草靈B為首次報(bào)道的新化合物，雙氧木脂素E首次從透骨草中分離得到。 3.殺蟲(chóng)活性測(cè)定結(jié)果表明，（1）透骨草靈-I對(duì)3齡粘蟲(chóng)幼蟲(chóng)只具有麻醉活性，其8h麻醉中濃NC50為2580μg/mL，對(duì)4齡淡色庫(kù)蚊幼蟲(chóng)有一定的毒殺活性，24h毒殺致死中濃LC50為1.21μg/mL。（2）透骨草靈B對(duì)3齡粘蟲(chóng)幼蟲(chóng)和4齡淡色庫(kù)蚊幼蟲(chóng)均有一定的毒殺作用，對(duì)3齡粘蟲(chóng)幼蟲(chóng)24h胃毒致死中濃LC50為490μg/mL，對(duì)4齡淡色庫(kù)蚊幼蟲(chóng)24h毒殺LC50為0.69μg/mL。（3）雙氧木脂素E對(duì)多種供試試蟲(chóng)有一定的殺蟲(chóng)活性，對(duì)3齡粘蟲(chóng)幼蟲(chóng)24h胃毒LC50為53μg/mL，24h觸殺LD50為0.26μg/頭；對(duì)3齡槐尺蠖（Semiothisa cinerearia）幼蟲(chóng)24h胃毒LC50為1680μg/mL，24h觸殺LD50為1.33μg/頭；對(duì)家蠅成蟲(chóng)24h胃毒LC50為970μg/mL，24h觸殺LD50為1.12μg/頭；對(duì)4齡淡色庫(kù)蚊幼蟲(chóng)24h毒殺LC50為0.15μg/mL；對(duì)美洲大蠊（Periplaneta americana）成蟲(chóng)48h LD50為7.28μg/頭。（4）雙氧木脂素A對(duì)多種供試試蟲(chóng)有很強(qiáng)的殺蟲(chóng)活性，對(duì)3齡粘蟲(chóng)幼蟲(chóng)24h胃毒LC50為35μg/mL，24h觸殺LD50為0.09μg/頭；對(duì)3齡槐尺蠖幼蟲(chóng)24h胃毒LC50為66μg/mL，24h觸殺LD50為0.049μg/頭；對(duì)家蠅成蟲(chóng)24h胃毒LC50為39μg/mL，24h觸殺LD50為0.047μg/頭；對(duì)4齡淡色庫(kù)蚊幼蟲(chóng)24h毒殺LC50為0.025μg/mL；對(duì)美洲大蠊成蟲(chóng)48h LD50為4.98μg/頭。4個(gè)化合物對(duì)小菜蛾幼蟲(chóng)和豆蚜無(wú)翅成蚜沒(méi)有明顯的毒殺作用。 4.癥狀學(xué)觀察表明，以LD90劑量雙氧木脂素A點(diǎn)滴處理6齡粘蟲(chóng)幼蟲(chóng)和家蠅成蟲(chóng)后，粘蟲(chóng)幼蟲(chóng)麻醉、部分試蟲(chóng)不自主肌肉收縮、震顫，直至死亡；家蠅成蟲(chóng)很快興奮，接著麻醉直至死亡；美洲大蠊成蟲(chóng)先是重心抬高、腹部屈曲，隨后麻醉直至死亡。 5.雙氧木脂素A對(duì)粘蟲(chóng)頭部ATP酶的測(cè)定結(jié)果表明，雙氧木脂素A抑制了5齡粘蟲(chóng)頭部Na+-K+-ATPase活性，激活了Ca2+-Mg2+-ATPase和Ca2+-ATPase活性。離體條件下雙氧木脂素A對(duì)Na+-K+-ATPase最大的抑制率為32.77%，活體條件下最大的抑制率為59.72%；離體條件下對(duì)Ca2+-Mg2+-ATPase和Ca2+-ATPase最大激活率分別為33.48%、30.34%，活體條件下對(duì)Ca2+-Mg2+-ATPase和Ca2+-ATPase最大的激活率分別為29.72%、28.83%。離體條件下對(duì)三種ATP酶在某種程度上均具有一定的劑量效應(yīng)。 6.對(duì)果蠅幼蟲(chóng)神經(jīng)-肌肉接點(diǎn)電位測(cè)定結(jié)果表明，雙氧木脂素A使自發(fā)性接點(diǎn)電位（mEJP）釋放頻率增加，出現(xiàn)了波浪狀發(fā)放，對(duì)興奮性接點(diǎn)電位（EJPs）沒(méi)有明顯影響。 7.測(cè)定了雙氧木脂素A對(duì)美洲大蠊離體神經(jīng)細(xì)胞胞漿中游離[Ca2+]i動(dòng)力學(xué)的影響。結(jié)果表明，10nmol/L到100μmol/L濃度范圍雙氧木脂素A都可引起胞內(nèi)游離[Ca2+]i升高，且胞內(nèi)鈣水平升高與雙氧木脂素A的濃度正相關(guān)。雙氧木脂素A作用部位是細(xì)胞質(zhì)膜和內(nèi)質(zhì)網(wǎng)膜，，其作用靶標(biāo)是質(zhì)膜上的L-型Ca2+通道和內(nèi)質(zhì)網(wǎng)膜上的RyR鈣通道，引起外鈣內(nèi)流和內(nèi)鈣釋放，使得胞內(nèi)鈣水平升高。雙氧木脂素A與nifedipine競(jìng)爭(zhēng)結(jié)合于L-型Ca2+通道，與咖啡因在RyR鈣通道上存在不同的親和位點(diǎn)。 8.雙氧木脂素A激活L-型Ca2+通道，促進(jìn)外鈣內(nèi)流；L-型Ca2+通道與RyR在胞漿區(qū)的部分相互作用，其構(gòu)象發(fā)生改變并通過(guò)蛋白質(zhì)間的相互作用激活RyR，Ca2+釋放通道開(kāi)放，內(nèi)質(zhì)網(wǎng)上的Ca2+釋放。外鈣內(nèi)流和內(nèi)鈣釋放致使胞漿中游離[Ca2+]i升高，Na+離子流受到抑制，從而抑制Na+-K+-ATPase活性；而突觸前膜內(nèi)Ca2+水平升高，Ca2+結(jié)合在囊泡膜上，減少了膜表面的負(fù)電荷，使得囊泡之間以及囊泡與突觸前膜的粘附易于發(fā)生，神經(jīng)-肌肉自發(fā)性接點(diǎn)電位（mEJP）釋放頻率增加，引起興奮性神經(jīng)遞質(zhì)釋放，并擴(kuò)散通過(guò)突觸間隙，與突觸后膜上的受體相結(jié)合，引起后膜去極化，形成突觸后電位。突觸后電位達(dá)到Na+的活化閾值，就產(chǎn)生一個(gè)新的動(dòng)作電位，引起中毒試蟲(chóng)的肌肉收縮、震顫直至死亡。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：S482.39

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