| Phryma leptostachya L., a herbaceous perennial plant, is widely distributed in theHimalayas, temperate Asia and northern East America. It has been used as a traditionallynatural insecticide in East Asia. In this paper, the insecticidal components of P. leptostachya L.and its mechanism have been studied. The primary results are as follows:1. The insecticidal activities of crud extracts (extracted by petroleum ether, ethyl acetateand methanol using ultrasonic method) from P. leptostachya L. were evaluated. The resultsshowed that three extracts had stronger stomach toxicity against Mythimna separate andMusca domestica. The NC50values were1690μg/mL,6240μg/mL and11390μg/mL againstthe3rdinstar larvae of M. separate after treated8h, and the LC50values were1580μg/mL,2940μg/mL and4990μg/mL against M. domestica at treated24h. Three extracts also hadobviously contact poisoning toxicity, and the ND50values were2.26μg/larvae,7.20μg/larvaeand24.35μg/larvae against the3rdinstar larvae of M. separate after treated8h, and the LD50values were1.80μg/fly,3.32μg/fly and5.56μg/fly against M. domestica after treated24h,respectively. Additionally, three extracts showed high larvicidal activity against the4thinstarlarvaes of Culex pipiens pallens, and the LC50values were3.23μg/mL,5.24μg/mL and61.86μg/mL at treated24h.2. Five compunds were isolated from P. leptostachya L. by means of gel columnchromatography and pre-HPLC. Four lignans, phrymarolin B and I, haedoxane A and E, with3,7-dioxabicyclo[3.3.0] octanes (furofuran) skeleton, were elucidated mainly based on thedata of NMR and MS. Among them, phrymarolin B,1-hydroxy-2-(3′,4′-methyl-enedioxy)phenox-6-(2″-hydroxy-3″,4″-me-thylene dioxy) phenyl-3,7-dioxabicyclo[3.3.0] octane, was anovel compound and haedoxane E was isolated firstly from the plant.3. The bioassay results showed that phrymarolin-I had obviously narcosis activity againstthe3rdinstar larvae of M. separate with the NC50value of2580μg/mL. Phrymarolin-I hadstronger larvicidal activity against the4thinstar larvaes of C. pipiens pallens with the LC50value of1.21μg/mL. The LC50values of phrymarolin B against M. separate and C. pipienspallens were490μg/mL and0.69μg/mL after treated24h, respectively. Haedoxane Eexhibited significantly insecticidal activity. LC50values (stomach toxicity) of haedoxane Eagainst M. separate, S. cinerearia, M. domestica and C. pipiens pallens were53μg/mL,1680 μg/mL,970μg/mL and0.15μg/mL, respectively. LD50(contact poisoning toxicity) values ofhaedoxane E against M. separate, S. cinerearia and M. domestica were0.26μg/larvae,1.33μg/larvae and1.12μg/fly, respectively. LC50values (stomach toxicity) of haedoxane Aagainst M. separate, S. cinerearia, M. domestica and C. pipiens pallens were35μg/mL,66μg/mL,39μg/mL and0.025μg/mL, respectively. LD50(contact poisoning toxicity) values ofhaedoxane A against M. separate, S. cinerearia and M. domestica and were0.09μg/larvae,0.049μg/larvae and0.047μg/fly, respectively. The LC50values of Haedoxane E and A againstPeriplaneta Americana were7.28μg/adult and4.98μg/adult, respectively.4. The toxicity symptoms of insects treated with haedoxane A were described as follows:The6thinstar larvaes of M. separate gradually anesthesia, some of them had musclecontraction and convulsion until to death. The adults of M. domestica showed excitement andthen anesthesia until to death. The most obvious symptom of the adults of P. Americana waselevating the body and abdominal flexion, and then anesthesia until to death.5. Haedoxane A could decrease the activies of Na+-K+-ATPase and increase the activitiesof Ca2+-Mg2+-ATPase and Ca2+-ATPase from the head of the5thinstar M. separata. Theinhibition rates of Na+-K+-ATPase were32.77%in vitro,59.72%in vivo, the activation ratesof Ca2+-Mg2+-ATPase were33.48%in vitro,30.72%in vivo, and the activation rates ofCa2+-ATPase were30.34%in vitro,28.83%in vivo, respectively.6. The intracellar microelectrode recording was carried out on ventral longitudinalbody-wall muscle fibers of the3rdlarvae of Drosophila melanogaster. The results indicatedthat haedoxane A could increase the spontaneous firing frequency of neuromuscularSpontaneous junction potential (mEJP), and have no difference on neuromuscular excitatoryjunction potentials (EJPs).7. Effects on [Ca2+]iof neurous from the adult of P. Americana were tested. The resultsindicated that haedoxane A could increase the level of [Ca2+]i, it had a positive correlationbetween [Ca2+]i. and the concentration of haedoxane A. The cytomembrane of neurous andthe endoplasmic reticulum membrane might be the sites of action of haedoxane A. L-typeCa2+channels in the cytomembrane and RyRs in the endoplasmic reticulum membrane mightbe the sites of haedoxane A. Haedoxane A and nifedipine would competitively combine withL-type Ca2+channels at the same binding sites. Haedoxane A and caffeine may have differentbinding sites on RyRs.8. Haedoxane A activated L-type Ca2+channels to promote calcium influx. Theinteractions of L-type Ca2+channels and RyR in some cytoplasmic domain, made theconformation change and activated RyR through interaction between proteins, Ca2+channelopened and released Ca2+from endoplasmic reticulum. Calcium influx and intracellular calcium releasing caused cytoplasmic free [Ca2+]iincreasing, Na+ion flow was inhibited, andthen Na+-K+-ATPase activity was inhibited. The [Ca2+]iof presynaptic membrane increased,Ca2+binded in the vesicle membrane, and reduced the negative charge of the membranesurface. The adhesion between the vesicles and the vesicles with the presynaptic membranetended to occur. Neuromuscular junction potentials spontaneously (mEJP) released frequencyincreasing, caused excitatory neurotransmitter quality to release and diffused through thesynaptic cleft. The excitatory neurotransmitter combined with the postsynaptic membranereceptor, membrane depolarization caused the formation of postsynaptic potentials.Postsynaptic potential reached the activation threshold of Na+, it generated a new actionpotential, causing poisoning insects muscle contraction, tremor until to death. |
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