| Termites are important insect pests in the world. In China, the annual economic losses caused by termite were over3billion RMB. At present, the main termite prevention and control is still the chemical pesticides in domestic and foreign. Because of the forbidden organochlorine and organophosphate insecticides, the termite prevention and control is an embarrassing situation. Therefore, it is very necessary to search new efficiency method. Using the plant essential oil and the bioactivity compound from plant oil to control termite is one of the hotspots in the research fields of termite control. Cryptomeria fortunei Hooibrenk is the most abundant one and unique to China. It is worth to further study its biological activity. Therefore, the study identified the chemical compositions and termiticide activity of essential oil from C. fortunei. Furthermore, we also purified and isolated the activity chemical compound in essential oil, and explored its insecticidal mechanism. At the same time, we analyzed and compared the termiticide activity of analogues of a-terpineol. The results showed as follows:1. Chemical composition and antitermitic activity of C. fortunei essential oil against R. chinensisAiming at to clarify chemistry composition and antitermitic activitie of essential ols from C. fortunei and C. japonica, the dissertation analyzed and compared the chemical composition of essential oil from the C. fortunei and C. japonica with GC-MS, and antitermitic activities of2essential oils against R. chinensis. The results also indicated that the genus of Cryptomeria had a considerable variation in the essential oil compositions, and the antitermitic activity of leaf essential oil of C. fortunei was stronger than that of C. japonica.Furthermore, the chemical compositions of essential oils were analyzed with GC-MS and antitermitic activities were tested against R. chinensis, from9locations in China: Kunming, Yaan, Wuhan, Enshi, Hefei, Nanjing, Mt Tianmu, Fuzhou, Mt Lu, and in different seasons and age, and from4organs:bark, sapwood, heartwood and leaf. The results showed that the essential oil exist a degree of differences in chemical composition from different locations, seasons and age, and organs. However, there were no significant differences in antitermitic activities of essential oils from different locations, seasons and age. However, the leaf essential oil of C. fortunei posses significant antitermitic activity, which promotes the isolation and purification of the main active constituents for further study. The results of this study showed that the leaf essential oil of C. fortunei might be considered as a potential source for fine natural termiticide. Through the identification the composition of C. fortunei leaf essential oil and chemotypes analysis, we can understand the relationship between the composition of C. fortunei leaf essential oil and the provenance of C. fortunei. The results of this study can provide a reference for C. fortunei breeding lines and the C. fortunei leaf essential oil usage or biological activity measurement.2. Bioassay-guided isolation and identification of antitermitic active compound from C. fortuneiIn order to further study the termiticidal components in leaf essential oil of C. fortunei, bioactivity-directed fractionation and purification processes were employed to identify the termiticidal components by silica gel column chromatography. The active compound, a-terpineol with a molecular formula of C10H18O, and molecular weight of154.25, which was responsible for the antitermitic property of C. fortunei leaf, was obtained and identified by IR, MS, UV, and NMR. For a-terpineol, the linear equation Y=0.34+5.05x (R2=0.87) was derived from the regression analysis of probit mortality of R. chinensis bioassay in test solution. The calculated LC50was0.86mg/mL with the95%confidence interval of0.76-0.98mg/mL. The results of this study showed that a-terpineol might be considered as a potent source for the production of environmental safe termiticide. The study layed the foundation for further studying the mechanism of insecticide and the chemical structure--activity relationships and provided a theoretical basis for industrial development.3. Insecticidal mechanism of a-terpineol to R. chinensisTo revealed the insecticidal mechanism of a-terpineol. The effect of a-terpineol on activities of detoxification enzymes and antioxidant enzymes to R. chinensis, were determined. The result showed that the Acetylcholinesterase (AChE), Na+-K+-ATPase, peroxidase (POD) and catalase (CAT) activitives were inhibited by a-terpineol with the prolonging of exposure time at0.86mg/mL. In addition, the activitives of glutathione S-transferase (GST) and superoxide dismutase (SOD) were induced by a-terpineol at the same doage. When R. chinensis work was treated with a-terpineol at0.2,0.4,0.6,0.8,1.0μl/mL doages, the activites of AChE and Na+-K+-ATPase were inhibited at various concentration a-terpineol. The glutathione S-transferase (GST), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activitives were induced with the increasing of α-terpineol dosage at24h. Among, α-terpineol had strong inhibition on activity of Na+-K+-ATPase. With poisoning symptom exacerbating, the inhibition rates were gradually increased. Therefore, Na+-K+-ATPase is likely the target of α-terpineol against R. chinensis.To studied insecticidal mechanism of α-terpineol. The proteomic response of the R. chinensis to α-terpineol was examined using differential proteomics technologies at LC10, LC50, and LC90following α-terpineol treatment. Forty proteins showed differential expression, including seven up-regulated spots, twenty-four down-regulated, of which eight spots newly expressed or disappeared in R. chinensis after exposure to α-terpineol. MS analysis and database searching helped us to successfully identify forty protein spots, including metabolisms, cytoskeletal, and transporter proteins. We found several proteins related with α-terpineol stress proteins, revealing the α-terpineol response in termites was a network response.Treated with α-terpineol, the expression level of some related proteins in R. chinensis were changed. In order to study the gene expression difference in transcription level, using RT-PCR and RACE technology, we cloned the most sequences of ATP synthase-β-subunit in R. chinensis, associated with energy metabolism. From the conserved sequences of ATP synthase in different species we can infer the functions of ATP synthase in different species are conservative.4. Antitermitic activity of analogues of α-terpineol against R.chinensisAiming to clarify relationship between chemical construction and antitermitic activities of analogues of α-terpineol, The fumigation toxicities of7series42compounds, including Hydrocarbons, Alcohols, Phenols, Ketones, Aldehydes, Acetates and Oxides were determined against subterranean termite, R. chinensis. This result indicated that the antitermitic activities of oxygenated hydrocarbon compounds were higher than the hydrocarbon group; the position of hydroxyl (-OH) groups were stronger than those with an acetyl group; the position of the hydroxyl group (-OH) is the only difference in the chemical structures of the two phenols; the position of the hydroxyl group is very important in antifungal and insecticidal activity; the position of the double bond of the propenyl group is also very important for antitermitic activity; some ketones were more effective fumigants than alcohols, the presence of a carbonyl group augments toxicity. The research preliminarily clarified the effects on antitermitic activities when attached different chemical group in monoterpenes, and found a number of active compounds significantly higher than a-terpineol.Our research showed chemical composition of C. fortunei oil from different species, different origin, different seasons, different age or different organs had a certain degree of variation, these oils all had better killing effects against R.chinensis, but the termiticidal activity of them were not significantly different. Our study was first to isolate the termiticidal active substance, a-terpineol, from C. fortunei leaf oil, and explore its termiticidal mechanism. Meanwhile, our study was also carry out the proteomics research on R. chinensis under the stress response of a-terpineol. We found several proteins related with a-terpineol stress proteins, revealing the a-terpineol response in termites was a network response. The research preliminarily clarified the effects on antitermitic activities when attached different chemical group in monoterpenes. The results of our study provided scientific basis for the C. fortunei essential oil application and the new termiticidal drugs development. |
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