果蠅體內20-羥基蛻皮激素及其類似物萃取法之探討與定量分析之應用

Quantitative Analysis and Extraction of 20-hydroxyecdysone and Its Analogs in Drosophila Melanogaster

紀淑芬(Shu-Fen Ji)

指導教授 ： 曾惠芬

國立暨南國際大學/科技學院/應用化學學系/碩士(2020年)

https://doi.org/10.6837/ncnu202000144

摘要

20-羥基蛻皮激素(20-hydroxyecdysone, 20E)是一種蛻皮類固醇，對於昆蟲來說是一種很重要的固醇類荷爾蒙，因為此荷爾蒙可以用來調控昆蟲蛻皮、生長和生殖；在植物部分因其20E的含量較少，所以推測可能是用來防止害蟲的侵害。然而過去20E的萃取回收率不高且萃取時間過久，因此若需要做這類激素的萃取，仍需要一定數量的樣品以及時間。根據過去的研究顯示兩天大雄性果蠅體內尖葉土杉甾酮(ponasterone A, Pon A)的含量大於五天大的雄性果蠅，而20E的含量則是沒有顯著差異，另外α-蛻皮激素(ecdysone, Ec)以及杯莧甾酮(cyasterone, Cya)都在偵測極限以下。因此本研究參考超音波輔助萃取以及靜置這兩種方法來嘗試最佳化萃取此類的激素，以提高萃取的回收率；並選用簡單和靈敏的高效能液相層析質譜分析法(HPLC/MS)來進行20E及其類似物的定量分析，同時比較零天大雄性果蠅與兩天大雄性果蠅體內20E及其類似物的含量。結果顯示：與短時間的超音波輔助萃取法相較，大部分化合物經長時間震盪後回收率較高，其中Cya不管超音波震盪時間的長短都是低於偵測極限的。而零天大雄性果蠅體內20E及其類似物的含量明顯大於兩天大的雄性果蠅，且Ec與Cya也都偵測的到。

關鍵字

20-羥基蛻皮激素 ； 最佳化萃取法 ； 高效能液相層析質譜儀

並列摘要

20-Hydroxyecdysone (20E) is a steroid hormone, and it plays an important role on the development of insects. For insects, this hormone can regulate molting, reproduction, and growing; for plants, the low amount of this hormone can protect them from the infestation of the pests. However, the extraction recovery of 20E was not high in the past and more time was needed. Previous data showed that the amount of ponasterone A in two-day-old male Drosophila melanogaster was more than that in five-day-old male Drosophila melanogaster; however, the amount of 20E in two-day-old male Drosophila melanogaster was similar to that in five-day-old male Drosophila melanogaster. Besides the other two analogs (cyasterone and α-ecdysone) were below limits of detection. Therefore, this study refers to the two methods, ultrasonic assisted extraction and placing for five days, to improve the recovery of extraction. We choose a simpler and more sensitive instrument, high performance liquid chromatography/mass spectrometry(HPLC/MS), to quantitative 20E and its analogues and compare their amounts in zero-day-old male Drosophila melanogaster to those in two-day-old male Drosophila melanogaster. The results show that the amounts of 20E and its analogues recovered by long-time ultrasonic extraction are large than those from short-time ultrasonic assisted extraction. No matter how long the ultrasonic assisted extraction using, cyasterone is below the detection limit. This result also shows that the amounts of 20E and its analogs in zero-day-old male Drosophila melanogaster are more than those in two-day-old male Drosophila melanogaster, and both cyasterone and α-ecdysone are detected.

並列關鍵字

20-Hydroxyecdysone ； optimized extraction method ； high performance liquid chromatography/mass spectrometry(HPLC/MS)

參考文獻

一、英文部分

(1) Festucci-Buselli, R. A.; Contim, L. A. S.; Barbosa, L. C. A.; Stuart, J.; Otoni, W. C. Botany 2008, 86, 978-987.

(2) Slaidina, M.; Delanoue, R.; Gronke, S.; Partridge, L.; Leopold, P. Dev Cell 2009, 17, 874-884.

(3) Ganter, G. K.; Panaitiu, A. E.; Desilets, J. B.; Davis-Heim, J. A.; Fisher, E. A.; Tan, L. C.; Heinrich, R.; Buchanan, E. B.; Brooks, K. M.; Kenney, M. T.; Verde, M. G.; Downey, J.; Adams, A. M.; Grenier, J. S.; Maddula, S.; Shah, P.; Kincaid, K. M.; O'Brien, J. R. J Insect Physiol 2011, 57, 1179-1184.

(4) Uryu, O.; Ameku, T.; Niwa, R. Zoological Lett 2015, 1, 32.