Isolation And Structure Elucidation Of Compounds From Emiliania Huxleyi And Their Biological Activities

Marine microalgae is the primary producer in marine ecosystem and it plays a crucial role in the material cycle and energy flow.Its metabolites contain proteins,lipids,vitamins,pigments,polyphenols,polysaccharides,terpenoids and a variety of bioactive substances.Marine microalgae are among the most promising lipid sources which can be up to 80% of the dry weight.These compounds have good antibacterial,antioxidant,antitumor and other biological function.Coccolithophores is a kind of globally important unicellular marine phytoplankton.It belongs to the member of the Haptophyta,Prymnesiophyceae.Emiliania huxleyi is one of the most important coccolithophores species providing a valuable and rich pool for the bioactive substances,such as docosahexaenoic acid(DHA)and poly ketone compounds,therefore E.huxleyi have broad prospect of application in the field of biotechnology.However there has been very little research reported on the bioactive lipid in E.huxleyi.In this study,we performed all experments using of E.huxleyi BOF92.By optimizing the conditions for extraction for total lipid,we established a highly efficient extraction process of total lipid in E.huxleyi.The antibacterial and antioxidant activity of crude extractions were detected to trace the active components,which were then subjected to the separation,purification and identification.The molecular identification was clarified and the bioactivities of compounds were confirmed further.The experiment results were as follows:(1)The effects of different extraction solvents and pretreatment methods on the extraction rate of total lipid in E.huxleyi were studied.Ethanol was chosen as extraction solvent for common solvent extraction method.By using the single factor and orthogonal test,the optimal extraction condition of total lipid in E.huxleyi were as follows: ethanol concentration was 75%,liquid-material ratio was 10:1,leaching temperature was 65? and leaching time was 2 h.The total lipid extraction rate reached 26.38% under the optimal conditions.(2)The ethanol crude extracts were used for farther extraction with different polarity organic solvents,such as petroleum ether,ethyl acetate and n-butanol to obtain three phase extractions respectively.It was observed that ethyl acetate fraction from E.huxleyi possessed maximum activity against Escherichia coli,Bacillus subtilis,Staphylococcus aureus,and Saccharomyces cerevisiae;the petroleum ether fraction had maximum activity against Vibrioparahemolyticus with an inhibition zone compared to E.coli,B.subtilis and S.aureus;the n-butanol fraction recorded maximum activity against mildew,but it did not show any inhibiting effects on bacteria all above.Antioxidant test in vitro indicated a variable degree of antioxidant activities in three organic solvents phase and the ethyl acetate fraction had strong antioxidant properties.(3)Six monomeric compounds were purified from ethyl acetate fractions in E.huxleyi.Nuclear magnetic resonance spectrum,high resolution mass spectrum and infrared spectrum identification showed that they were palmitic acid,phytol,myristic acid and dibutyl phthalate respectively.(4)There was no antioxidant activities with palmitic acid,myristic acid and dibutyl phthalate,but they all had maximum activity against S.aureus,B.subtilis,V.parahemolyticus.Phytol did not show any inhibiting effects on the growth of bacteria used in this study,but it had the strongest antioxidant activities and a high inhibitory activities against a Hep G2 cell line.