Protective Effect And Mechanism Of Lactones From Elephantopus Scaber Againnst Aeromonas Hydrophila In Zebrafish

Scabrous elephantfoot herb (Elephantopus scaber), one of the most popularlyused traditional Chinese herbal medicine, is abundantly distributed in the world. Thelactones from E. scaber (ESL), as its important active ingredient, have provenantibacterial capacity against Escherichia coli, Staphylococcus aureus, Salmonellaenteritidis, Pasteurella, Bacillus cereus etc. in vitro. The in vitro anti-bacterialmechanism reported is that ESL made the cellular wall of the bactera damaged,changed the bacterial membrane permeability and made the cytoplasm outflowed aswell as the protein denatured and the lysozyme of the bacteria released, resulting inthe bacterial autolysis, during the logarithmic growth phase of the bacteria. However,it is still unknown that whether ESL has in vivo anti-bacterial effect againstAeromonas hydrophila (AH) in aquatic animals and how it works. In this thesis,ultrasonic assisted extraction, pressure column chromatography and thin layerchromatography (TLC) were used and ESL was prepared. Then the protective effectand mechanism of ESL against AH infection in zebrafish were studied. The mainfindings were as follows:1. Three kinds of ESL were isolated by pressure column chromatography on silicagel and were identified as scabertopin, isoscabertopin and deoxyelephantopin, withRfs of0.364,0.316and0.161, respectively, by TLC with the developing agent ofpetroleum ether: chloroform: ether (3:5:2).2. ESL performed significant inhibitory effects on AH in vitro. The MIC and MBCwere12.5mg/ml,15.6mg/ml, respectively.3. Feed with ESL showed certain protective effects against AH infection in zebafish.The protective effects of the high doses of ESL (0.8%,1.6%) were significantly betterthan the lower ones (0.2%,0.4%), respectively. And there were no significantdifferences betweent the infected zebrafish administered with the high doses of ESLand the infected zebrafish administered with0.1%enrofloxacin.4. Feed with high doses of ESL (0.8%,1.6%) increased the survival time of zebrafish in pathogenic extracellular products secreted by AH. It indicated that thehigh dose ESL can enhance the capacity of zebrafish against the pathogenicextracellular products.5. Feed with different doses of ESL had no effect on the head kidney index and thespleen index.6. Feed with ESL (0.8%,1.6%) significantly increased the lysozyme activities in thebody surface mucus, the liver and the gill, respectively. In addition, feed with0.4%ofESL also significantly increased the lysozyme activity in the body surface mucus.7. Feed with ESL (0.8%,1.6%) significantly increased the alkaline phosphataseactivity in the liver.8. Feed with1.6%of ESL significantly increased the total superoxide dismutaseactivity in the gill.9. Feed with0.8%ESL significantly increased the IgM content in the gill.10. Accordingly, the in vivo anti-bacterial mechanism of ESL against AH inzebrafish could be as follows: ESL directly conducted bactericidal effect on AH; ESLenhanced the capacity of zebrafish against pathogenic extracellular products secretedby AH; ESL increased the lysozyme and alkaline phosphatase activities whichhydrolyzed AH; ESL increased the superoxide dismutase activity which indirectlyindicated that AH was destroyed by superoxide free radicals; ESL increased IgMcontent to improve the humoral immune function.