Study On The Separation Of Astaxanthin And Astaxanthin From Antarctic

BackgroundThe Antarctic krill (Euphausia superba), or called "big krill‖, as the largestbiomass species on the earth, it’s rich in astaxanthin. Astaxanthin is a kind of strongnatural antioxidant, and it has good effect such as resisting cardiovascular aging,radiation, cancer, alzheimer’s disease, protecting eyes and skin and so on. It has a veryhigh research value. There are mainly three kinds of existence forms of astaxanthin:astaxanthin monoester, astaxanthin diester and free astaxanthin, and the main formamong them is stable de-esterified astaxanthin, namely astaxanthin monoester andastaxanthin diester. As there aren’t standard of astaxanthin monoester and astaxanthindiester, domestic research of the further purification of them from the Antarctic krill isnot mature, research and development is needed.IntentionThe purpose of this study is to establish a method of the further purification ofastaxanthin monoester and astaxanthin diester that extracted from the Antarctic krilland the identification method of isomes and then determine the composition byGC–MS and isomes by HPLC.TechniqueThis study takes the astaxanthin and astaxanthin esters from Antarctic krill oil asthe research object, respectively determines the best futher purification conditions ofastaxanthin monoester and astaxanthin diester by AgNO3high-performance thin layerchromatography (AgNO3HPTLC) H plate separation methods. The separated andpurified astaxanthin monoester and astaxanthin diester are used for carrying outesterification reaction and then detects by GC–MS, respectively analyzes the structureof fatty acids of each chromatographic band. The isomers of astaxanthin, saponifiedastaxanthin monoester and saponified astaxanthin diester are detected and analysed byHPLC. Result1. This study determined the the best condition of separating astaxanthinmonoester and astaxanthin diester by AgNO3HPTLC H plate through a series ofsingle factor experiments such as the concentration of AgNO3methanol solution, soaktime, drying temperature and drying time of the HPTLC H plate, the ratio of thedeveloping solvent. When the concentration of AgNO3methanol solution was2%, thehigh-performance thin layer chromatography H plate soak time was15min, dryingtemperature was60℃, drying time was1h, and the developing solvent waspetroleum ether: triethylamine: acetone=9.5ml:10μl:3ml, astaxanthinmonoester appeared two chromatography bands. When the developing solvent wasn-hexane: acetone=3:1, astaxanthin dester appeared five chromatography bands.2. The two chromatographic bands of astaxanthin monoester and fivechromatographic bands of astaxanthin diester separated by AgNO3HPTLC H platewere detected and analysed by GC–MS. There were6.9%astaxanthin tridecylenicacid ester and48.83%of astaxanthin pentadecylenic acid ester in the firstchromatographic band and100%even fatty acids astaxanthin monoester in the secondchromatographic band of astaxanthin monoester. There were62.03%saturatedastaxanthin diester in the first chromatographic band of astaxanthin diester,73.34%saturated astaxanthin diester in the second chromatographic band of astaxanthindiester,82.26%unsaturated astaxanthin diester in the third chromatographic band ofastaxanthin diester,54.58%unsaturated astaxanthin diester in the fourthchromatographic band of astaxanthin diester, and75.61%unsaturated astaxanthindiester in the fifth chromatographic band of astaxanthin diester.3. The isomers of astaxanthin standard，the astaxanthin obtained from saponifiedastaxanthin monoester and the astaxanthin obtained from saponified astaxanthindiester were analysed and detected by HPLC. There were (3S,3’S),(3S,3’R),(3R,3’R)three kinds of isomers at18.765min and19.938min and18.765min in the staxanthinstandard. There was only a peak at29.971min in the astaxanthin obtained fromsaponified astaxanthin monoester, and compared to standard, it could be concluded that there was only (3R,3’R) isomer in the astaxanthin monoester and astaxanthinobtained from saponified astaxanthin monoester. There were three peaks at18.422min,20.189min and21.281min in the astaxanthin obtained from saponifiedastaxanthin diester, compared to standard, it could be concluded that there were threekinds of isomers in astaxanthin diester:(3S,3’S),(3S,3’R),(3R,3’R).Conclusion and InnovationThis study futher separates astaxanthin monoester and astaxanthin diester fromAntarctic krill by AgNO3HPTLC H plate in domestic and abroad for the first time,and get a certain regular of experiment results. It prepares the odd fatty acidastaxanthin monoester that is rich in tridecylenic acid ester and pentadecylenic acidester for the first time. It will promote the study of further purification, separation andphysiological activity differences of astaxanthin monoester. There is regularity in theresult of the separated astaxanthin diester. It prepares astaxanthin diester that is rich insaturated fatty acids and astaxanthin diester that is rich in unsaturated fatty acids. Itprovides the methods of preparation and detection for the futher separation ofastaxanthin diester and application of different astaxanthin diester. In addition, itfirstly detects that there is only one kind of isomer in astaxanthin monoester fromAntarctic krill, and the astaxanthin that only contains one kind of isome is preparatedat the same time, which will further promote the appraisal work of Antarctic krill oil.