Purification And Activties Of Astaxanthin Esters From Haematococcus Pluvialis And Identification Of Different Astaxanthins

Astaxanthin is a kind of ketone carotenoids that has good coloring and strong biological activity.Different sources of astaxanthin are composed of different isomers and big difference between the activities.Haematococcus pluvialis is a major source of natural astaxanthin,however,mainly in the form of astaxanthin esters.Because of the complexity structure of astaxanthin esters and the limitation of traditional purification methods,the research on purification of astaxanthin esters is still rare now.In this paper astaxanthin esters were separated and purified from Haematococcus pluvialis by using high-speed counter-current chromatography(HSCCC),identified by HPLC and ESI-MS,and the biological activities of astaxanthin monoester were studied further.In addition,a method to identify astaxanthins from different sources was also established.The main results were as follows:(1)The total content of carotenoid was used as index,optimizing the best condition of breaking wall and carotenoid extraction from Haematococcus pluvialis through the single factor and orthogonal experiment design as the follows: the broken wall temperature 50 oC,the broken wall time 30 min,enzyme concentration 0.6 mg/mL,enzyme reaction pH 4.0.The optimized total carotenoid content in the extract was 59.62±0.31 mg/g DW,increased by 25% than before optimization.(2)With two solvent systems of n-hexane-acetonitrile-methanol(4:1:2,v/v/v)and nhexane-methanol(2:1,v/v),canthaxanthin(10.35 mg),astaxanthin hexadecadienoic monoester(8.47 mg),astaxanthin linolenic monoester(15.65 mg),astaxanthin linolic monoester(6.05 mg),?-carotene(15.25 mg)and astaxanthin palmitic-astaxanthin eicosadienoic diester(4.33 mg)were successfully purified from 100 mg Haematococcus pluvialis by high-speed counter-current chromatography(HSCCC)technology for the first time.(3)The antioxidant activity of astaxanthin monoester was studied by contrast with astaxanthin and vitamin E.The results showed that the EC50 values of astaxanthin monoester scavenging ABTS+ and·OH free radicals were 7.09±0.20 ?g/m L and 11.08±0.76 ?g/m L respectively,and the ability was stronger than vitamin E,but weaker than astaxanthin;astaxanthin monoester had good inhibitory effect on oil oxidation;had certain ferric ion reducing power and the FRAP value was 0.236±0.021 mmol/L.Compared with arbutin and orlistat,the inhibitory effect of astaxanthin monoester on mushroom tyrosinase and pancreatic lipase were determined.The results showed that when the concentration of astaxanthin monoester was 800 ?g/m L,the inhibition ratio on mushroom tyrosinase was 39.48%;astaxanthin monoester had strong inhibitory effect on pancreatic lipase,and the IC50 value was 9.85±0.02 ?g/mL.(4)Using YMC Carotenoid C30 chromatographic column,the HPLC analysis of astaxanthin was optimized by single factor experiment.The sample concentration: 5 mg/mL,sample quantity: 20 ?L,flow rate: 1.0 m L/min,column temperature: 35 oC,detection wavelength: 474 nm,mobile phase: methanol,acetonitrile and MTBE.On this basis,a method to identify astaxanthins from different sources was established.The experimental results of this paper provided the theoretical basis and technical supported to largely prepare and utilize astaxanthin esters,and identified astaxanthins from different sources.