| In recent years, eutrophication of coastal waters is more and moreserious, resulting in "red tide" in China’s coastal areas occured frequently.The direct economic loss in our country caused by the red tide is10billion￥every year, and red tide has becoming a serious marine disasters.Researchers in China and the world have to face up to the problem abouthow to scientificly and effectively control red tide. Methods for theprevention and control of red tide are physical method, chemical methodand biological method. But the physical method is costly, and chemicalmethods will lead to pollution, so biological method is the currentresearch trends. On the other hand, coastal water eutrophication in Chinahas led to the outbreak of the "green tide", Ulva prolifera in coastalfloating and accumulation, threatened the development of fisheries,aquaculture and tourism. However, in recent years, in China’s Jiangsucostal areas，with the increasing outbreaks of Ulva prolifera sea, red tidereduced year by year. How to use the green tide to prevent the red tide isof great significance, and biological control can also protect the marineecological environment. Therefore, this study is to find out theallelochemicals in Ulva prolifera. We finally hope to provide theoreticalbasis for the biological control of red tide. The experiment consists of three parts:(1) The allelopathic effects of green tide alga Ulva proliferaon the proliferation of a red tide algae Skeletonema costatum;(2) Theallelopathic activity tracking in Ulva prolifera methanol extracts;(3) Theextraction, isolation, purification, identification and activity analysis ofallelochemicals.(1) The allelopathic effects of green tide alga Ulva prolifera on theproliferation of a red tide algae Skeletonema costatum were studied. Thefresh tissue, dry powder, culture medium filtrate of U. prolifera were usedfor inhibiting the proliferation of S. costatum. Results showed that thefresh tissue of U. prolifera could inhibit significantly the proliferation ofS. costatum. In all groups with fresh tissue, the inhibition rate was as highas100%after co-culturing for12days. As for the groups with dry powder,the inhibition rate in the1.2g/L group reached50%after co-culturing for12days. It indicated that the inhibition of green tide algae on S. costatumproliferation presented a concentration-response relationship, and therewas not only nutrient competition inhibitory, but also the allelochemicalsinhibition. Further studies showed that there was no significant inhibitionof culture medium filtrate on the proliferation of S. costatum in theexperiment with one-time adding culture medium filtrate of U. prolifera,while the proliferation of S. costatum was significantly inhibited in theexperiment with semi-continuous adding culture medium filtrate of U.prolifera. The inhibition rate for semi-continuous adding culture medium filtrate of U. prolifera was75%after culturing for12days. It indicatedthat allelochemicals in U. prolifera was gradually released toward theenvironment. This research laid a solid foundation for the further researchon the concentration-response relationship between U. prolifera and S.costatum.(2) The allelopathic activity tracking in Ulva prolifera methanolextracts: results of different concentrations of Ulva prolifera extract’saqueous phase and ethyl acetate phase showed a certain degree ofinhibition effect on the growth of red tide algae, the inhibitory activityincreased with concentration. The aqueous phase of methanol extract inUlva prolifera has certain inhibitory effect on Heterosigma akashiwo hascertain inhibitory effect, but not obvious with the concentration change，the inhibitory effect rangs from20%to50%. But the Ulva proliferamethanol extract ethyl acetate phase showed obvious inhibitory effect onred tide algae. At the concentration of0.4mg/ml,0.8mg/ml,1.6mg/ml，all the Heterosigma akashiwo died in the second day, and theexperimental group of0.1mg/ml and0.2mg/ml also showed30%inhibition, we can conclude that the active substances exist in ethylacetate phase. The Ulva prolifera methanol extract ethyl acetate phaseshowed constant inhibition on Nitzschia closterium at the concentrationof1.6mg/ml, and the0.8mg/ml experiment group also showed50%inhibition. Compared with Heterosigma akashiwo, the effect on Nitzschia closterium is significantly lower. Trough our Further follow-up of theactivity, the component of petroleum ether (PE): ethyl acetate (EA)=9:1component showed higher inhibitory effect on red tide algae, theinhibition rate of experimental group of0.8mg/ml on Heterosigmaakashiwo reached95%, while the inhibition rate of0.8mg/ml group onNitzschia closterium was only20%. We guess that there may beresistance material exert on the inhibitory effect in Nitzschia closterium.(3) The extraction, isolation, purification, identification and activityanalysis of allelochemicals: Research methods mainly used in thepurification are extraction, silica gel column chromatography, SephadexLH-20gel column chromatography, thin layer chromatography; themonomer compound structure identification methods mainly include:LC-MS, nuclear magnetic resonance spectroscopy(NMR). Based on theidentification results, palmitic acid and linolenic acid was firstlyidentified from Ulva prolifera. Further analysis showed that the activeidentification, palmitic acid for red tide microalgae had certain degree ofinhibitory effect, the experimental group25μg/ml reached50%, but nolinear relationship; while the alpha linolenic acid on Heterosigmaakashiwo showed obvious inhibitory effect and sustained activity, theeffect of fatty acid increased linearly with concentrations. The halfinhibition concentration is8μg/L. The inhibition rate of Alpha linolenicacid on Nitzschia closterium is very low, the inhibition rate reached30% under the condition of maximum concentration, and concentration didn’texhibit linear relationship. In order to further analysis the activitydifferences of the above two compounds, we also used as the monomercompounds in vitro cell activity analysis. Results showed that palmiticacid on human esophageal cancer cell Eca109’s half inhibitionconcentration is15mg/L; alpha linolenic acid showed good inhibitoryactivity on breast cancer cells4T1, the half inhibitory concentration is1mg/L. The allelopathy results are matched up with the results on red tidealgae. |
