| The Eomecon chionantha alkaloids (ECA) extracted from the roots and the rhizomes of Eomecon chionantha Hance, It has been proved to be a good molluscicide and well worthy developing. To Study the killing snails effect of Chelerythrine (CHE) from ECA and the injury mechanism of CHE solution on liver of the snails will provide new experimental evidences for exploitation of Eomecon chionantha Hance as a plant molluscicide.The immerse method was recommended by WHO for molluscicide screening. CHE solution with different concentrations as25mg/L,12.5mg/L,6.25mg/L,3.125mg/L,1.5625mg/L,0.7813mg/L,0.3906mg/L,0.1953mg/L respectively were prepared for immersing the Oncomelania hupensis in the laboratory.The mortality rates of the snails in different immersing time (24h、48h、72h、96h) and different temperature(20℃、25℃、30℃).were tested.The tracks of factors on CHE against Oncomelania hupensis were studied by applying stepwise regression and the method of factors-classifying and values-classifying to analyze data so as to evaluate the trend of the concentrations-to-the mortality rate of Oncomelania hupensis.The snail livers was used as samples to analyse the molluscicide mechanism of CHE on Oncomelania hupensis.Oncomelania hupensis were immersed in different concentration of CHE as20mg/L,10mg/L,5mg/L repectively or clean water for36hours. DNA of snails were extracted from snail liver cells and analyzed by agarose gel electrophoresis. The liver cell suspension was obtained through mashing the liver tissues and apoptosis in liver cells resulted from CHE were analyzed by Flow Cytometry.To identify the differentially expressed proteins in the liver of Oncomelania snails induced by Eomecon chinanthe Chelerythrine, Chelerythrine was extracted and purified from the dry powder of Eomecon chionanthe.The livers were seperated from live snails by immersed in5mg/L Chelerythrine solution or pure water for36hours. Total liver proteins were extracted and separated by two-dimensional gel electrophoresis. The electrophoretogram was analysed with ImageMaster2D5.0software. The differentially expressed proteins between Chelerythrine group and pure water group were selected and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry sequencing of tryptic peptides.The effects of CHE to kill the Oncomelania hupensis and its molluscicide mechanism were observed as follow:(1)CHE showed good effect in controlling the snails. The mortality rates of the snails after72h with the concentrations of0.1953mg/L、30℃and96h、0.7813mg/L、25℃are100%. The effects of the CHE to kill the Oncomelania hupensis when immersed the snails in the0.1953mg/1CHE solution for96h were shown significant differences with control group(P<0.05).(2)Through analysing the experiment data by SPSS17.0, we found that the influence factors of CHE in controlling the snails were the concentration, immersing time and water temperature. The stepwise regression analysis revealed that the concentration, time, and temperature could influence the molluscicidal effect of CHE on snail.With the raising of chemical concentration, temperature, immersing time the results also indicated that the effect of CHE solution killing snails is increased.(3)Agarose gel electrophoresis analysis of DNA from Oncomelania hupensis liver cells treated by CHE solution did not display the typical DNA Ladder map. the percentage of early apoptotic cells in treated group with CHE and control group were5.59%(20mg/L),3.42%(10mg/L),5.71%(5mg/L) and3.85%(water) respectively, the percentage of late apoptotic cells were1.59%(20mg/L),1.31%(10mg/L),2.7%(5mg/L) and0.7%(water) respectively, the total apoptosis rate of was7.18%(20mg/L),4.73%(10mg/L),4.73%(5mg/L) and4.55%(water) respectively, there was on significant difference between these two group (P>0.05).(4)protein spots observed in group and in water group were(405±11)and (385±8) respectively. The four identified differentially expressed proteins were actin,glydenolase-like protein,beta-actin. the proteins were down-regulated in CHE group.From above results we concluded:(1)CHE possesses the effect in killing the Oncomelania hupensis;(2)The influencing factors of the molluscicidal effect of CHE on Oncomelania hupensis include concentration, time and temperature, and the effect is increased with the augmentation of the concentration and the extension of the time;(3)The main way of CHE solution to kill the snails was not cell apoptosis.(4)Expression of liver proteins from Oncomelania hupensis could be changed when immerse in Chelerythrine;... |
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