| Jatropha curcas L., a member of the family Euphorbiaceae, is known for its distinguishing toxicity to most species of animal such as human beings, goat, rat, mice, fish and mussels. The toxicity of the plant is mainly concentrated in seeds that are produced every year in large number by J. curcas. In present study, multiple aspect of knowledge in pesticide, phytochemistry, biochemistry, entomology and statistics was using to study the insecticidal property in order to exploit and utilize the resources of the J. curcas in our country. By method of bioassay-guided fractionation, and using various technologies of phytochemistry such as column chromatography, preparation TLC and HPLC, we screened the insecticidal component in J. curcas, and the insecticidal substance, named Jatrophoerol I was isolated and purified. The structure of Jatrophoerol I was identified by comparing and analysis of varied spectrums of UV, IR, NMR and MS. And HPLC was adapted in present study to determinate the Jatrophoerol I in J. curcas seed. We also examined bioactivity of Jatrophoerol I against several kinds of insects and its action modes, and tried to illuminate its toxic action mechanism on individual, tissue, cell and subcell level by evaluation its effect on different system of silkworm.The result showed toxic protein from J. curcas seed had no obvious insecticidal activity to Lipaphis erysimi (Kaltenbach) , Preris rapae, Sitophilus oryzae L. andSitophilus zeamais (Motschulsky) in compare with seed oil and its ethanol extract with high bioactivities against all the pests. Seed oil had strong contact toxicity (LC50=0.7286%) and antifeedant activity (AFC50 =0.2278%) against L. erysimi. No contact toxicity was detected in seed oil to P. rapae, but significant stomach toxicity and antifeedant activity with LC50 and AFC50 of 2.246% and 1.8930% respectively. 5". orzo L. and S. zamias was fed with seed oil treated food, and resulted in high mortalities in 14d with LC50 of 0.4812% and 0.5306% respectively. After extracted with ethanol, the extract of seed oil showed an enhancement in insecticidal activity compared with original seed oil. Evaluated with LC50, contact toxicity of ethanol extract to L. erysimi was 2.76 times of seed oil, while antifeedant activity was 1.26 times. Stomach toxicity of ethanol extract to P. rapae was 5.96 times of seed oil while antifeedant activity was 7.23 times. Field experiments showed that the 0.25% ethanol extract of seed oil was highly effective in control of L erysimi, and the corrected efficacy maintained at 72.11 % when examined at 7th day after treatment.J. curcas seed was extracted with different kind of solvent, the insecticidal active component was mainly concentrated in ethanol extract. Following a procedure of separation on different column chromatography, preparation TLC and HPLC, we isolated two compounds of Ja2 and Ja3 with extraction rates of 0.033% and 0.019% of weight of J. curcas seed. Both Ja2 and Ja3 caused high mortality rate in 3rd instars larvae of silkworm when exposed in food. Ja3 had a stronger toxicity than Ja2 with linear regression equation of Y=2MA2X + 3.7585 (R2 = 0.9817), and LC50 of 0.3649mg/mL. In HPLC, Ja2 showed three to four peaks, while Ja3 had only one peak indicating it was a pure substance. After identified by UV, IR, NMR and MS, the structure was determined of Jatrophoerol I with a molecular formula of Cn^C^.and molecular weight of 322. It was an insecticidal substance we firstly isolated from J. carcas. HPLC method was established for determination Jatrophoerol I in J. carcas seed for first time. Different extraction methods were compared, it was proved ultrasonic extraction was the best method for determination of Jatrophoerol I. After the seeds were treated by ultrasonic, Jatrophoerol I was extracted totally with a concentration of 0.039% of seed weight. This method was simple, sensitive andaccurate which could be used in quality control of J. carcas pesticide in future.Jatrophoerol I showed an insecticidal activity to Bombs mori L., L. erysimi and P. rapae. It acted on B. mori and P. rapae by the same mode of stomach toxicity, antifeedant activity and growth inhibition activity, but not contacts toxicity. Bioactivity of Jatrophoerol I was more significant against B. mori than that against P. rapae. After exposure to Jatrophoerol I for 72h, LC50 in B. mori and P. rapae was 0.2197 and 0.8318mg/mL, respectively, while AFC5owas 0.1451 and 0.5706mg/mL, respectively. Distinguish growth inhibition activity was also observed in B. mori and P. rapae after Jatrophoerol I was ingestioa However, Jatrophoerol I act on L. erysimi by the mode of contact toxicity, antifeedant activity and reproduction inhibition. By the method of dropping and dipping in Jatrophoerol I to examine its contact toxicity against aphid, LC50 were 0.1129 u g/insect and 0.06204mg/mL, respectively. AFC50 was 0.01802mg/ml to L. erysimi. Jatrophoerol I could inhibit the reproduction of L. erysimi with an inhibition rate of 80.06% in 0.05mg/mL of concentration. The oral toxicity of Jatrophoerol I to mice was found to be 82.198mg/kg body weight.The main toxic symptoms of silkworm after ingestion of Jatrophoerol I was sever digestion toxicity such as diarrhea and vomit. It caused a complicate effect on insect detoxification enzymes. Changes of midgut esterase and carboxyliesterase activity after treatment were similar with a decrease in 3h, following a little increase, then a significant decrease at 48h. Analyzing esterase isozymes showed one of isozymes was inhibited obviously while two or three isozymes were induced. Most of isozyme was inhibited at 48h. Similar changes were found in esterase isozymes in hemolymph indicating that esterase might be a key enzyme in insect mediating detoxification reaction against Jatrophoerol I. Changes of activity of midguts glutathion S-transferase were not obvious. Structure of midgut cells in silkworm exposed to Jatrophoerol-I was examined by histological method. Disintegration of fat body and damages in epithelial cells such as breakdown and falling off from base membrane were observed. Ultrastructural studies showed noble pathological changes in endoplasmic reticulum in epithelial cells. Endoplasmic reticulum expanded and resulted in falling off the ribosome. Other injuries in microvilli, mitochondria and nucleus chromatin were alsoobserved by TEM. Exposure to Jatropherol-I in food also significantly depressed midguts protease activity. This inhibition effect was dose and time dependent. The levels of protease were only 32.94% of the control in silkworm treated with 0.25mg/ml of Jatropherol-I for 48h. Protein concentration was reduced in hemolymph and protein synthesis was delayed in treated insect. This result may explain the inhibitory effect of Jatropherol-I in insect growth and reproduction. Jatropherol-I also caused a slight decrease in activity of midgut acethycholinesterase. The effects of Jatropherol-I on ATPase activity were different among different ATPases. It caused significantly depressed activity in Ca-ATPase and Ca-Mg-ATPase with reduction rate of 58.61% and 35.82% at O.lmg/mL of Jatropherol-I treatment, compared with Na-K-ATPase of slightly increasing activity and Mg-ATPase of no significant changed activity.Protein kinase C (PKC) mediates a variety of intracellular signalling events. Activation of PKC always resulted in sever pathological injury in eukaryocytes. Here, PKC was extracted from midgut cell of silkworm, and its activity was determined in vitro and in vivo for the first time. It was found that PKC could be activated by Jatropherol-I not only in vitro but also in vivo. PKC activity was 4.99 times of the control after it was incubated with O.lmg/mL of Jatropherol-I. In vivo experiment, PKC activity and the phosphorylation was enhanced with Jatropherol-I concentration increasing and time developing. It was suggested that PKC was an important intracellular target of Jatropherol-I in insect. Phosphorylation of proteins due to the activatidh of PKC induced by Jatropherol-I might interfere normal message transfer system in midgut cell, and caused a series of physiological and biochemical turbulence that had been stated in front, and finally resulted in death of the insect.
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